Genetic

Pedigree drawing
Summary of family medical history

Family medical history
Ask about spontaneus
What is abortions, stillbirths.
important to Ask about living and deceased

members of a family.
know about
Make sure that siblings have
family the same biologic parents. Ask
medical about adoption in family.
history? Ask about consanguineous mating

(mating between close relatives).
Ask about children born thanks to in

Ask not only about those vitro fertilization (IVF).
alive and affected in family
but also about deceased Ask about ethnicity and
siblings. People often tell branches of family.
about only living members.
Korf B.R. 2003. Genetyka człowieka 8-9.
Past records
• medical documentation from hospital medical records’, physicians’

offices, coroners’ records, or other institutions
• the appropriate provincial or state facility
Types of documentation that are most useful include:
- autopsy findings
- reports of surgical procedures
- discharge summaries, which should review all of the pertinent

investigations from that admission
- laboratory investigations, such as chromosome studies
- family photographs
Friedman J.M. i inni 1997 Genetyka
Variation in pedigree drawing
(examples)
Biologic parents and their descendants
x
Recomendations of the PSTF
Bennett R.L., Steinhaus K.A., Uhrich S.B., et al. 1995. Recomendations for standardized human pedigree nomenclature. Pedigree
Standarization Tack Force of National Society of Genetic Counselors. Am. J. Hum. Genet. 56: 745-752.
Stienhaus K.A., Bennett R.L., Resta R.G., Uhrich S.B., Doyle D.L., Marlek D.S., Vincent V.A. 1995. Inconsistencies in pedigree symbols in
human genetics publications: a need for standardization. Am. J. Hum. Genet. 56: 291-295
Healthy person Documented
Disease excluded by a physician
Male
*
female
*
Sex unknown
*
Healthy person Not documented
But no medical documentation
male
fermale
sex unknown
Bennett R.L et al.. J Genet Counsel (2008) 17:424-433 56: 745-752.

Stienhaus K.A.,et al.. Am. J. Hum. Genet. (1995) 56: 291-295
Affected individual Recomendations of the PSTF
male
* # c
female
sex unknown
Deceased individual Recomendations of the PSTF
* male
female
sex unknown
Bennett R.L., Steinhaus K.A., Uhrich S.B., et al. 1995. Recomendations for standardized human pedigree nomenclature. Pedigree Standarization
Tack Force of National Society of Genetic Counselors. Am. J. Hum. Genet. 56: 745-752.
Carrier
male
female
Person in a presymptomatic Recomendations of the PSTF

stage
(high probability of disease in
future for example in male
Huntingtodn disease)
female

Multiple individuals, number known Recomendations of the PSTF
5 2 5 male
4 2 (3)
5 female
sex unknown
5
Multiple individuals, number unknown

n n
n n (n)
n male
n female
n sex unknown

Pregnancy Recomendations of the PSTF
P
P ?
P male
P female
sex unknown
P
Still birth Recomendations of the PSTF
male
SB
female
SB
sex unknown
SB
Spontaneous abortion (SAB) Recomendations of the PSTF
Spontaneous Affected SAB

abortion (SAB)
male
(Sp)
male male
female
SA female female
sex unknown
Termination of pregnancy (TOP) Recomendations of the PSTF

Termination of Affected TOP
pregnancy (TOP)
male
T
male male
female
female
female
sex unknown
Bennett R.L., Steinhaus K.A., Uhrich S.B., et al. 1995. Recomendations for standardized human pedigree nomenclature. Pedigree Standarization
Lack of offspring Recomendations of the PSTF
male
female
Infertility Recomendations of the PSTF
male
female

Proband Recomendations of the PSTF
male
female
Consultant
male
female

Recommended symbol
summary
Pedigree symbols
Adoption by a relative
A break in a relationship
indicates the relationship no
longer exists
Consanguinity Twins Twins Adoption Adoption

monozygotic dizygotic in out
Common pedigree symbols

(Bennett R.L., Steinhaus K.A., Uhrich S.B., et al. Recomendations for standardized human pedigree nomenclature. Pedigree Standarization
ART – Assisted Reproductive Techniqes
Sperm donor
D D
Couple in which woman is carrying
pregnancy using donor sperm. No
or
relationship line is shown between
the woman carrying the pregnancy
and the sperm donor. P P
Ovum donor
Couple in which woman is carrying pregnancy
using a donor egg and partner’s sperm. The D
line of descent from the birthmother is solid
because there is a biologic relationship that
may affect the fetus (e.g., teratogens)
P

Surrogate only
Couple whose gametes are used to S

impregnate a woman (surrogate) who
carries the pregnancy. The line of descent
from the surrogate is solid because there is
a biological relationship that may affect the P
fetus (e.g., teratogens).
Surrogate ovum donor
Couple in which male partner’s

sperm is used to inseminate:
D D
lub
P P
an unrelated woman. a sister who is carrying the

pregnancy for the couple.

Planned adoption
Couple contracts with a woman to carry a D D

pregnancy using ovum of the woman
carrying the pregnancy and donor sperm.

The offspring should be drawn in accordance with the birth order, placing the
oldest child on the left.
in line with the birth order

not in line with the birth order
1985 1990 1955 1960 1965

General rules
I
1 2 3 4
II
1 2 3 4 5 6 7 8 9 10
3
III P
female
1 2 4 5 6 7 8
• If possible, male partner should be to the left of female partner on relationship line
• All individuals of one generation are drawn on horizontal line
• Each generation is marked by Roman numerals
• Individuals in each generation are marked with Arabic numerals (numbers from left to
right)
• Siblings should be listed from left to right in a birth order (oldest to youngest)
PEDIGREE EXAMPLES
Pedigree Example
Kamila Robert Bartek Lidka Maria Krysia Kamil
P
Płeć męska
Zuzia Jarek Kasia Maciej Klaudia Zbyszek Marta

Pedigree Example
Cris July Andrew Rose Betty
Alice John
Pedigree Example
Richard Mary
Paul Anita Eve Harry
Tom Peter Mark Susan Betty

6y 4y
Software for pedigree drawing
• Haplopainter
http://haplopainter.sourceforge.net/
• PEdigree Drawing software

Male Female Sex unknown
Individual
Affected individual
Multiple individuals, 5
5 5
number known
n
Multiple individuals, n n
number unknown
Deceased individual
Stillbirth (SB) SB SB SB
Pregnancy (P) P
P P
Proband
Consultant

Male Female Sex unknown
Spontaneous
abortion (SAB)
Affected SAB
Termination of
pregnancy (TOP)
Affected TOP
Likely to be
affected later
Obligate carrier (will not

manifest disease)
No children by choice or
reason unknown
Infertility

INHERITANCE PATTERN
INHERITANCE PATTERN
Single gene inheritance Multifactorial

(polygenic)
inheritance
Mendelian Mitochondrial
inheritance inheritance
Autosomal X-Linked inheritance
Dominant Recessive Dominant Recessive
Genomic imprinting
Autosomal dominant inheritance
Molecular mechanisms
• Gain of function / overexpression
• Dominant negative effect
(what fraction of homotetramers will be unaffected in heterozygote?)
• Haplotype insufficiency
m m
Mm Mm
M
Mother
mm
mm mm
m
50%
Father
Mm
Diagnostic difficulties: Examples:

germinal mosaicism Marfan syndrome,
de novo mutations Huntington disease
Marfan Syndrome: How is it inherited?
Criteria:
• two sexes are equally affected
• vertical transmission pattern: disease phenotype seen in generation after generation
• father-son transmission of disease gene is possible

• usual recurrence risk: 50%
2
5
Pedigree typical of autosomal dominant inheritance

Autosomal recessive inheritance
C c
CC Cc
C
Mother
C c
Cc cc
Father c
C c
25%
C C
Cc Cc
c
Mother
C C
Cc Cc
c
Father
c c
Features:
Features: Clinical examples 0%
• carrier state • cystic fibrosis
• consanguinity • sickle cell disease
Your Genes, Your Health

Criteria:
• horizontal transmission pattern: disease phenotype seen in multiple siblings, but usually
no earlier generations affected
• both parents of an affected individual are clinically unaffected heterozygotes
• consanguinity is sometimes seen, especially for rare recesive disease
Pedigree typical of recessive inheritance

Recessive inheritance mimicking autosomal dominant
(autosomal pseudodominant inheritance)
c c
Cc Cc
C
Mother
cc cc c c
c
Father
C c
50%
Your Genes, Your Health
X-Linked dominant inheritance
(from mother)
50%
X X*
XX X X*
X
Mather
XY X*Y
X* Y X X*
Y affected
Father Your Genes, Your Health
XY
(from father)
50%
(only daughters)
X X
X*X
X*X X*X
X*X
X*
Mother
XY XY XX
Y

X* Y
Criteria:
• male-male transmission not seen
• vertical transmission pattern: dsease phenotype seen in generation after generation
• all daughters of an affected male are affected
X-Linked recessive inheritance
(from mother)
%
25%
X X* (only sons)
XX X X*
X
Mother
XY X*Y
X* Y X X*
Y carrier
XY
(from father)
50%
50
X X (only daughters)
X*X
X*X X*X
X*X
X*
Mother
XY XY XX
Y

X* Y
Criteria:
• male-male transmission not seen
• only males are affected
• skipped generations may be seen, representing transmission through carrier females
• all daughters of an affected male are healthy (carriers)

Mitochondrial inheritance
Characteristic:
- inheritence exclusively from the mother
- heteroplasmy, i.e. simultaneous ocurrance of mutated and wild mtDNA
- reduced/full penetrance
- most frequent defects:
• point mutations
• large deletions
• depletion, i.e. reduction in number of mtDNA copies
Mitochondrial inheritance
Criteria:
• all children of an affected mother are affected

• only women transmit a trait
• all children of an affected father are unaffected
Typical pedigree
Imprinting disorders
Effect depends on whether
maternal or paternal chromosome is affected.
Specific counselling
Imprinting effects - example of
parental sex-dependent influence
on phenotype
• mare x donkey male = mule,

• stallion x donkey female = hinny (shorter
ears, thicker mane and tail, stronger legs
than the mule)
Imprinting - the differential expression of alleles depending on the parent of origin
An imprinting defect is an abnormality of the parent-of-origin-specific gene regulation, such that a

maternal allele or genomic domain has the resulting pattern of gene expression) of a paternal allele or
genomic domain, or vice versa.
Imprinted genes are often differentially methylated!

EPIGENETICS..
Genomic imprinting - principles of inheritance
Paternal imprinting
Maternal imprinting
Idealized pedigrees for maternal and paternal imprinting. These figures are meant to diagram what a pedigree of human
disease that had imprinting effects might look like. The term "imprinting" implies a modification in expression of a gene or
allele. An "imprintable" allele will be transmitted in a Mendelian manner, but expression will be determined by the sex of the
transmitting parent. In these idealized pedigrees the term "maternal imprinting" is used to imply that there will be no
phenotypic expression of the abnormal allele when transmitted from the mother, and paternal imprinting is used to imply that
there will be no phenotypic expression when transmitted from the father. Because there will be a phenotypic effect only when
the gene in question or chromosome segment in question is transmitted from one or the other parent, there are a number of
nonmanifesting carriers. link
Life cycle of methylated imprints
IC – imprinting centre. Grey color - modification, white color – lack of modification in

corresponding allels. Parental chromosomes: blue – paternal, red - maternal. Arrows
indicate the way of reading imprints (transcriptive interpretation of primary imprints) in
developing embryo. Hum Mol Genet. 1992 Apr;1(1):7-10
Imprinting – mechanisms of inactivation
• methylation
• chromatin condensation
• ncRNA
Paternal alles: unmethylated ICR → ncRNA expression → Ube3a

inhibition
Cell 152, March 14,
2013
Prader-Willi Syndrome:
phenotype
75% of the patients have large
chromosomal deletions of +/- 4 Mb of
15q11-13 region, Always, the deletion
is on the paternally inherited
chromosome.
http://health.allrefer.com/
sou
Am J Med Gen
Angelman Syndrome
• Developmental delay, functionally
severe
• Speech impairment, none or
minimal use of words; receptive
and non-verbal communication
skills higher than verbal ones
• Movement or balance disorder,
usually ataxia of gait and/or
tremulous movement of limbs
• Behavioral uniqueness: any
combination of frequent
laughter/smiling; apparent happy
demeanor; easily excitable
personality, often with hand
flapping movements; hypermotoric
behavior; short attention span
• „HAPPY PUPPET” appearance
70% of the patients have deletions of

+/- 4 Mb in the 15q11-13 region. The
deletion is always on the maternally
inherited chromosome.
http://www.asclepius.com/
Prader-Willi syndrome (PWS)
and Angelman syndrome (AS)
Medical genetics, Jorde, Carey, Bamshad, 4th edition

Causes for Prader-Willi Syndrome (PWS)
and Angelman Syndrome (AS)
Ann Rev 2001
No single gene muations.

A polygenic defect?
Red -maternall chrmosome genes, Blue- paternall chromosome

Imprinting - summary
Form of monoallelic expression in which the

copy from a parent of one sex is always
silenced
Unconsistent nomentclature
Unclear evolutionary origin – may be linked
to maternal-fetal (parental) antagonism
Inactivation is can be caused by DNA
methylation, chromatine condensation or
ncRNA
Probability
Probability – definition
Probability of an event P(A) denotes

the frequency of it’s occurrence
in a long (infinite) series of repeats
| A|
P( A) = ⇔ A∈Ω
|Ω|
0 ≤ P ≤1
It’s represented by a number in the range Probability 0.25 (1/4)

from 0 (event never occurs) indicates that certain event is observed
to 1 (event occurs every time) in 1 from 4 or 25% occurrences
Rules of probability
Rule of addition
If events are mutually exclusive,

then the probability of either event occurring
is a sum
of the individual probabilities
of the occurrence of any of the events
P of giving birth to a boy or girl by a pregnant woman:
1/ + 1/2 = 1
2
P of rolling 2 or 3 on six-sided die:
1/ + 1/6 = 2/6 = 1/3
6
Rule of multiplication
If two or more events are independent,

then their joint probability
is a product
of their individual probabilities
P of rolling a pair of 6’s on two six-sided dice simultaneously :

1/ × 1/6 = 1/36
6
P of giving birth to two boys by a woman in binovular pregnancy:
1/ × 1/2 = 1/4
2
Autosomal recessive diseases
Albinism
Family coming
for an advice
What do we see?
Who is sick?
How many are sick?
Probable mode of
inheritance?
Autosomal recessive pattern of inheritance
Parents – carriers of recessive trait (e.g. cystic fibrosis):
What is P: affected homozygote? healthy carrier (heterozygote)?
1/ 1/ 1/ 1/ 1/ 1/ 1/ 1/
2× 2 2× 2 2× 2 2× 2
1/ 1/ 1/ 1/ = 1/2
4 4 4+ 4
Please pay attention: is the question about a phenotype or a genotype ?!

Parents – carriers of recessive trait:
risk of having an affected child :
1/ × 1/ = 1/
2 2 4
probability of having an unaffected child:
(1/2 × 1/2) + (1/2 × 1/2) + (1/2 × 1/2) = 3/4

healthy heterozygote
homozygote
(sum of two mutually exclusive events)

Mucoviscidosis
A pair of unrelated Caucasians

with negative medical history of cystic fibrosis
Risk of having an affected child?
Frequency of carriers of disease-causing allele in population: 1/25

The risk of having an affected child is
the product
of three (four) independent events :
1/ × 1/ × 1/ × 1/2 = 1/2500
25 2 25
Mr Smith has two children
What's the probability that

both of them are boys?
Two independent events: 1/

2 × 1/2 = 1/4
…or: four possibilities ♀♀ ♂♀ ♀♂ ♂♂ => 1/4
Autosomal dominant pattern of inheritance
A couple of one healthy person

and one affected by a dominant disease (e.g. Marfan syndrome)
A*a A*A* aa (2 normal alleles!)
(statistically 50% of their children would be affected)
Why 50% and not 100%

homozygotes often lethal (not in every disease)
additionally: a simplification for teaching purposes


What is, in fact, P that all three would be affected?

1/
2 × 1/2 × 1/2 = 1/8
What’s the probability that two out of three will be affected?
1/ × 1/ × 1 / = 1/
2 2 2 8 ?
P of any of them being affected = P of any of them being healthy …



What is P that two out of three would be affected?
(1/2 × 1/2 × 1/2)=×1/38 =
? 3/8BUT...
(rule of addition)
Risk of recurrence
One of the most important aspects of genetic counselling

is to assess a genetic risk (risk of recurrence)
Easy in mendelian diseases
Empirical in polygenic diseases and in chromosomal abnormalities

X-linked recessive inheritance pattern
e.g. Duchenne muscular dystrophy
Risk of fourth baby being affected?

Bayes’ theorem
P( A) P( B | A)
P( A | B) =
P( B)
Determination of individual risk of carrier state

by combining the data from the pedigree
with other data after taking into account all modifying factors
Bayes’ theorem
example
Bayes’ theorem
example
Two assumptions
(mutually exclusive)
Bayes’ theorem
example
Two assumptions
II2 is II2 is not

a carrier a carrier
Bayes’ theorem
example
Probability that
II2 is II2 is not

a carrier a carrier
Bayes’ theorem
example
Probability that
II2 is II2 is not

a carrier a carrier
a priori 1/2 1/2

Bayes’ theorem
example
Probability that
II2 is II2 is not

a carrier a carrier
a priori 1/2 1/2
conditional 1/8 1
(3 healthy sons) (1/2 × 1/2 ×1/2) (1)
Bayes’ theorem
example
Probability that
II2 is II2 is not

a carrier a carrier
a priori 1/2 1/2
conditional 1/8 1
(3 healthy sons) (1/2 × 1/2 ×1/2) (1)
joint odds
1/16 1/2
(1/2 × 1/8) (1/2 × 1)
Twierdzenie Bayesa
przykład
Probability that
II2 is II2 is not

a carrier a carrier
a priori 1/2 1/2
conditional 1/8 1
(3 healthy sons) (1/2 × 1/2 ×1/2) (1)
joint odds
1/16 1/2
(1/2 × 1/8) (1/2 × 1)
final risk 1/9 8/9

(joint odds divided by
 116 
the sum of joint odds)  
 116 + 12 
Reduced penetrance
Penetrance - proportion of individuals carrying

dominant allele that also express particular trait
(70% or 0.7)
Penetrance is said to be reduced or incomplete when some

individuals fail to express the trait, even though they carry the
disease-causing allele.
Risk of inheriting a dominant disease:
P = 1/2 × pen
Risk for the child whose parent suffered from retinoblastoma (pen=0,8):
1/ × 0.8 = 0.4
2
Reduced penetrance
Retinoblastoma pen = 0.8
Risk of a baby: A) being affected? B) being sick?

Bayes’ theorem
reduced penetrance
Probability that
II2 is II2 is not a
a carrier carrier
Bayes’ theorem
reduced penetrance
Probability that
II2 is II2 is not a
a carrier carrier
a priori 1/2 1/2

Bayes’ theorem
reduced penetrance
Probability that
II2 is II2 is not a
a carrier carrier
a priori 1/2 1/2
conditional
(healthy)
1 - pen 1
Bayes’ theorem
reduced penetrance
Probability that
II2 is II2 is not a
a carrier carrier
a priori 1/2 1/2
conditional
(healthy)
1 - pen 1
joint odds ½ × (1-pen) 1/2×1

Bayes’ theorem
reduced penetrance
Probability that
II2 is II2 is not a
a carrier carrier
a priori 1/2 1/2
conditional
(healthy)
1 - pen 1
joint odds ½ × (1-pen) 1/2×1
final risk 1
× (1 − pen) 1 − pen
2
=
2 × (1 − pen) + 2 2 − pen
(joint odds divided by 1 1
the sum of joint odds)
1 − pen 1
= ⇔ pen = 0,8
2 − pen 6
Reduced penetrance
Risk of disease for III1 while pen=0,8:
1/6
?
Reduced penetrance
Risk of disease for III1 while pen=0,8:
1 1 1 4 1
PII 1 × × pen = × × =
2 6 2 5 15
1/6
?
Odds
Odds - describe probability as a ratio (proportion)
P( A)
Odd ( A) =
1 − P( A)
Odds take higher values than probability

For example, if probability of getting ill is
1/5, the odds of getting ill is:
1 1
1
odds = 5
= 5
=
1 − 15 4
5 4
Odds vs. probability
Frequency of blue balls:

4/10 = 2/5 (two out of five balls are blue)
Proportion of blue balls vs. yellow balls:
4:6 = 2:3 (2 to 3)
Probability of drawing a blue ball:

2/5 (two blue balls in five attempts)
Odds of drawing a blue ball:
2:3 (in five attempts two ball will be blue and three will be yellow)
Likelihood in pedigrees
and
likelihood ratio
Likelihood (L)
in pedigrees
(initial comments)
Likelihood (L) = probability

Likelihood ratio (LR) = odds
Hardy & Weinberg principle:

for a locus with alleles p and q (with frequencies p and q):
pp pq qp qq
L = p×p + (p×q + q×p) + q×q = p2 + 2×pq + q2 = 1
Likelihood (L) of a pedigree
– an autosomal co-dominant
mode of inheritance
Calculate likelihood of the pedigree below,
assuming the following allele frequencies: 1 – 0,1; 2 – 0,2; 3 – 0,3.
0.1×0.2 [1|2] 12 34
+
0.2×0.1 [2|1]
= 13 11
2×0.1×0.2
11 13
12
– an autosomal co-dominant
mode of inheritance
Calculate likelihood of the pedigree below,
assuming the following allele frequencies: 1 – 0,1; 2 – 0,2; 3 – 0,3.
2×0.1×0.2 12 34 2×0.3×0.4
0.5×0.5 13 11 0.12
0.5×1 11 13 0.5×1
12 0.5×0.2
L = 2×0.1×0.2 × 2×0.3×0.4 × 0.5×0.5 × 0.12 × 0.5×1 × 0.5×1 × 0.5×0.2

– an autosomal dominant
mode of inheritance
Calculate L, assuming: mode of inheritance – autosomal dominant,
penetration – 100%, frequency of a mutated allele – D
2×D×d Dd dd d2
0.5 1
dd
L = 2Dd × d2 × 0.5 × 1
– an autosomal dominant
mode of inheritance
Calculate L, assuming: mode of inheritance – autosomal dominant,
2×D×d Dd dd d2 D2 DD dd d2
0.5 1 1 1
Dd Dd
L = 2Dd × d2 × 0,5 × 1 + D2 × d2 × 1 × 1
– a sex-linked dominant
mode of inheritance
Calculate L, assuming: mode of inheritance – sex-linked dominant,
D×1 DY dd d2
1 Yd Dd1 1
L = D × d2 × 1 × 1
Likelyhood (L)
is usually
a very small number
such as: 0.000 000 001

logarithms of small numbers
look simpler,
0.000 000 001 = 10-9
-9
Log10 10 = -9
L= -9
Likelihood (L)
and its usability
(likelihood ratio, LR)
• A single likelihood calculated as shown above

is not very usefull ...
• ... but when it is calculated for two competing hypotheses,
it allows for their relative comparison !
• Comparison is made by calculating likelihoods
of two considered hypotheses and dividing one by another
• This ratio is called LR – likelihood ratio
• Likelihood ratio (LR) describes odds:
L(z)/L(i) = z/N / i/N = z / i = z/(N-z)

Applications of LR
- 1) verification of kinship (parenthood)
Alleles / markers: 1,2,3,4.
I 12 23
?
II 34
Applications of LR
- 1) verification of kinship (parenthood)
Alleles / markers: 1,2,3,4. Allele / marker frequencies: 1 – 0,1; 2 – 0,2; 3 – 0,3.
I 12 I 3 41 2 I 3142 34
?
II 13 II 1 3 II 13
L1 = 2×0.1×0.2 × 2×0.3×0.4 × 0.5×0.5 L2 = 2×0.1×0.2 × 2×0.3×0.4 × 2×0.1×0.3

LR=L1/L2 = 0.5×0.5 / 2×0.1×0.3 = 4.16
Conclusion:
It is 4.16× more likely that II1 is a child of I1 and I2
than that II1 is not related to I1 and I2
Applications of LR
- 1) verification of parenthood (kinship)
Częstości markera: 1 – 0,1; 2 – 0,2; 3 – 0,3.
I I
2×D×d d2 D×1 d2
12 34 12 34
II 0.5 0.5 II 1 1
13 13
L1 = 2x0,1x0,2 x 2x0,3x0,4 x 0,5x0,5 L2 = 2x0,1x0,2 x 2x0,3x0,4 x 2x0,1x0,3
LR=L1/L2 = 0,5x0,5 / 2x0,1x0,3 = 4,16

Wniosek:
Jest 4,16 razy bardziej prawdopodobne, Ŝe II1 jest dzieckiem I1 i I2
niŜ, Ŝe jest osobą niespokrewnioną z I1 i I2
Applications of LR
- 2) determining the mode of inheritance
Autosomal dominant ? Sex-linked dominant ?
Dd dd DY
DY dd
dd Dd dY Dd
Applications of LR
- 2) determining the mode of inheritance
Autosomal dominant ? Sex-linked dominant ?
2×D×d Dd dd d2 D×1 DY dd d2
0.5 0.5 1 1
dd Dd dY Dd
L1 = 2×D×d × d2 × 0.5 × 0.5 L2 = D×1 × d2 × 1 × 1
L1 / L2 = d×0,5 ≈ 0,5
Applications of LR
- 3a) risk estimation (1)
Huntington disease is an AD disorder with late onset; symptoms usually develop at age of 30 to 60y.
Assuming that 50% of patiens develop symptoms before turning 50,
calculate the risk of inheritance of a mutated allele by the person indicated by an arrow.
hh Hh
Hh
Hh hh
Applications of LR
- 3a) risk estimation (2)
hh Hh hh Hh hh Hh
Hh Hh hh
Hh hh hh
L(carrier)=0.5 × 0.5 × 0.5 = 1/8

L(not a carrier)= 0.5×0.5×0.5 + 0.5×1×1 = 1/8 + 4/8 = 5/8
L1/L2=LR=1/5 (odds: 1-to-5)
P(carrier) = 1/(1+5)=1/6
Applications of LR
- event space (a)
Hh Hh
Hh Hh
1/2
1/2×1/2 1/2×1/2
Hh
hh
1/2
Applications of LR
- event space (b)
Hh Hh Hh Hh
Hh Hh Hh Hh
hh hh Hh Hh
1/2×1/2×1/2=1/8
1/2×1/2 1/2×1/2×1/2=1/8
1/2×1/2 1/2×1/2×1/2=1/8 1/2×1/2×1/2=1/8
Hh Hh
hh hh
hh Hh
1/2
1/2×1=1/2 1/2×0=0
LR=1/8 / (1/8+1/2) = 1/8 / 5/8 = 1/5

LRtotal= LR1 x LR2 x...x LRn
Because:
L1total= L11 x L12 x...x L1n
L2total= L21 x L22 x...x L2n
therefore:
L1/L2total
=
(L11 x L12 x...x L1n)
(L21 x L22 x...x L2n)
=
LR1 x LR2 x...x LRn
Mutations de novo
Introduction to genetics
Department of Medical Genetics

Medical University of Warsaw
De novo mutations
- importance
• so far omitted from our course –

to make things simpler
• very rare indeed, BUT ...
• historical necessity
(population size vs allele heterogeneity vs
consequences of homozygosity)
• suprising frequency in diseases with genetic
background (today’s topic)
• causes of mutations?
Mutations vs allele frequency
(mutation-retromutation equilibrium)
p → q with frequency µ p ← q with frequency ν

(mutation rate) (retromutation rate)
Generation 0: p0=1; q0=0
Generation 1: q1= µp0 =µ; p1=(1-µ)
Generation 2: q2= q1+µp1; p2=(1-µ)2
Generation n: qn=qn-1+µpn-1; pn=(1-µ)n qn=qn-1+µpn-1-νqn-1
equilibrium: qn=qn-1 µpn-1 – νqn-1 = 0 µpn-1 = νqn-1
since p+q=1 p=(1-q) µ(1-q)=vq µ-µq=νq µ=νq+µq=q(µ+ν)

q = µ / (µ+ν)
p = ν / (µ+ν)
Selection & allele frequency
(selection in medical genetics)
For a mutation with a strong phenotypic effect

the frequency of a mutated allele
is determined by selection
rather than retromutation
selection coefficient (S) –

denotes a degree of reproductive deficiency
of a given genotype in relation to a genotype
with the highest reproductive rate in a population
S jest dopełnieniem wartości przystosowawczej (W) genotypu do jedności
(S = 1 – W).
S=1/3
N=9 N=4 (50%->31%)

N=9×3=27 N=4×2=8 (50%->23%)
N=27×3=81 N=8×2=16 (50%->16%)
N=81×3=243 N=16×2=32 (50%->12%)
S=1/3
Genetically lethal disease

either
• kills before puberty
or
• causes sterility S=1
Disease frequency vs
allele frequency
Hardy & Weinberg
Disease frequency F principle
Mutated allele frequency q p2+2pq+q2=1
p+q=1
F F
F = 2pq q= ∩ p ≅1 → q≈
2p 2
Autosomal recesive inheritance
F = q2 q= F
X-linked recessive inheritance

males: Fm= q q = Fm
Autosomal dominant diseases
(genetically lethal)
All defective alleles

are being lost in each generation de novo
and must be replaced via mutations mutation
q=µ
p – wild-type allele frequency

q – mutated allele frequency
selection
µ – mutation frequency
All cases of disease are due to new mutations !!!

Very low recurrence risk apart from germline mosaicism
Autosomal dominant diseases
(all)
Some defective alleles

are being lost in each generation de novo
S×q = µ

selection
S – selection coefficient
All homozygotes are sterile,

their alleles are lost in each generation de novo
• population: N
• disease frequency: q2
• No of individuals not passing their alleles: q2×N
(only q are lost)
• No of alleles lost: 2 × q2×N
• No of all alleles: 2 × N selection
• frequency of alleles being lost: 2 × q2×N / 2 × N = q2
• only q are lost
• lost alleles replaced due to de novo mutations: µ=q2
All homozygotes are sterile,

their alleles are lost in each generation de novo
q2 = µ

selection
example: q=0.01 => µ=0.0001 (µ is very small, ≈0)
Practically in autosomal recessive diseases

mutations de novo need not be considered !
(all)
All homozygotes are less fertile, some

of their alleles are lost in each generation de novo
• population: N
• disease frequency: q2
• No of individuals not passing their alleles: q2×N
(only q are lost)
• No of alleles lost: 2 × S × q2×N
• No of all alleles: 2 × N selection
• frequency of alleles being lost: 2 × S×q2×N / 2 × N = S×q2
• only q are lost
• lost alleles replaced due to de novo mutations: µ=S×q2
(all)
All homozygotes are less fertile, some

of their alleles are lost in each generation de novo
µ = S×q2

selection
S – selection coefficient
example: q=0.01 & S=0.25 => µ=0.000025=2.5×10-5
If an autosomal recessive disease is not genetically lethal,

de novo mutations play even lesser role !
X-linked recessive diseases
If the disease is genetically lethal,

all mutated alleles de novo
carried by hemizygotic males mutation
are lost in each generation
selection
achieving equilibrium is
more complex
XY XX
Frequency of mutations (µ), sicked males (A) and female carriers (H)
A H
A H/2
A=½H+
µ H=½H+2µ
A=½(4µ µ H/2+µ H-½H=2µ
)+µ ½H=2µ //×2
A=2µ+µ H=4µ
A=3µ H/2+ Częstość:
A=3µ
H/2+µ H=4µ A – chorych mężczyzn
µ+µ H – kobiet nosicielek
µ – mutacji
3µ 4µ
3µ 2µ
µ 2µ+µ
2µ+ Częstość:
3µ
2µ+µ 4µ A – chorych mężczyzn
µ+µ H – kobiet nosicielek
µ – mutacji
N = 1000M+1000K
µ = 1/1000
3/1000 4/1000
4/2000
3/1000
(2/1000)
2/1000
1/1000 +
1/1000
2µ+
3/1000
2µ+µ 4/1000
µ+µ
The frequency of female carriers
H=½H+µ+µ
H=4
µ
The frequency of sick males
A=½H+ A=3µ
µ
µ - mutation rate
Sporadic
case 1
Sex-linked recessive disease –
mutation de novo (µf=µm) or inheritance? [Bayes theorem]
Probability of
I2 is I2 isn’t
a carrier a carrier
initial 4µ 1
conditional
1/2 µ
(sick son)
odds 2µ µ
L1: I2 jest nosicielką
final risk 2/3 1/3 L2: mutacja de novo
Sex-linked recessive disease –
mutation de novo (µf=µm) or inheritance? [LR]
~4µ
1/2
L1 = 4µ×0,5 = 2µ
~1
µ L1: I2 is a carrier
L2 = 1×µ = µ L2: mutation de novo
LR=L1/L2=2 (2:1):
Every third sporadic case is due to de novo mutation!!!
Duchenne muscular dystrophy
myopathy due to DMD mutations (1/3500 M)
Results of dystrophin gene mutation in DMD:

Calves pseudohypertrophy
a sample of a muscle of the healthy person (upper)
muscles are replaced with
and the affected one (lower) stained with HE (left) or
fat and connective tissue
immunostained with antibody against dystrophin (right)
in 8-years old boy with
Gowers maneuver: boy with DMD rises from the ground
myopathy due to DMD mutations
• A third of mothers who have

a single son affected will not themselves be
carriers of a mutation in the DMD gene !!!
• Thus they have very low risk (µ)
of subsequent problems
De novo mutation rates of male & female
origin in the diseases studied so far
α - male/female ratio of mutations)
Is evolution male-driven?
Meiosis in females
and in males
Nat Rev Gen 2001

when µF≠µM
A H
A H/2
A=½H+µF H=½H+µM+µF
A=½(µM+ H/2 H-½H=µM+µF
µM
µF)+µF +µF ½H=µM+µF /×2
A=2µF+µM H=2µM+2µF
A= H= Częstość:
H/2+
H/2+µ
2µF+µF 2µF+ A – chorych mężczyzn
µF+µM H – kobiet nosicielek
M 2µM µ – mutacji
X linked recessive inheritance: relationship between µ,
H, and A (cntd.), µ differes between sexes (µf =µm) cntd.
2µf +µm 2(µf +µm)

people
2µf +µm µf +µm

chromosomes
µf +µm+µf
µm gametes =2µf +µm
µf +µm
2µf +µm people
+µf +µm
Relationship between µ, H, and A (cntd.), µ differs between sexes
Nfem.=Nmale.=1000, µm=2/1000, µf=1/1000
4/1000 6/1000
people
4/1000 6/2000
chromosomes
X: (3+1)/1000
X: 2/1000 gametes X: (3+1)/1000
Y: 0/1000
Y: 0/ 1000 X: 4/1000
people
X: 4/1000 X: 2/1000
Sporadic
case 2
X-linked recessive disease –
mutation de novo (µf≠µm) or inherited?
~2×(µf+µm)
1/2
L1= µf+µm
~1
µf L1: I2 is a carrier
L2=1×µf=µf L2: de novo mutation
LR= (µf +µm) / µf.

if µf=0.2µm, then LR=1.2/0.2=6, tzn.:
Only 1/7 of sporadic cases are due to de novo mutations
X-linked recessive disease –
mutation de novo (µf≠µm) or inherited?
0,92
P mother as a carrier
0,87
0,82
0,77
0,72
0,67
1 2 3 4 5 6 7 8 9 10
µm /µf
Hemophilia
Hemophilia A and hemophilia B

are X-linked disorders of
coagulation caused by mutations
in the F8 and F9 genes,
respectively. Mutations of F8
cause deficiency or dysfunction
of clotting factor VIII; mutations of
F9 cause deficiency or
dysfunction of clotting factor IX.
Hemophilia A has an incidence Subcutaneous hematoma of the forehead
of 1 in 5000 to 10,000 newborn in a young heophiliac boy, days after a minor contusion.
males. The appearance of the forehead returned to normal in 6 months
Hemophilia B is far rarer, with an
incidence of 1 in 100,000.
Hemophilia A Factor VIII α ~3 Rosendaal 1990.
Hemophilia B Factor IX α ~4 Sommer 2001.

an exception to the rule?
De novo mutations arise with comparable frequency

during oogenesis and spermatogenesis
The dystrophin gene is a very large (>2mB) and contains
many repetitive regions. Furthermore, a greater than 30-
fold(!) reduction in dystrophin activity must occur before
the DMD phenotype is observed Sommer 2001.
High frequency of large deletions (60% to 65%), large
duplications (5% to 10%), and small deletions, insertions,
or nucleotide changes (25% to 30%).
Most de novo large deletions arise during oogenesis,
whereas most de novo nucleotide changes arise during
spermatogenesis
Cytogenetics
Ideogram of representative G-
banded chromosomes
Cen - centromere
St - stalks
Sa - satellites Human Chromosomes.pdf
Region
Bands
Subbands
Cytogenetic polymorphism
Chromosome polymorphism:
1qh+
9qh+, 9qh- (increase/decrease of ht.
in q-arm), 9ph (ht. in p-arm only),
9phqh (ht. in p and q)
16qh+
Yqh+
Example:
46, XY, 1qh+, 9qh+, 16qh+
Pathogenic changes
Chromosome abnormalities:
1) numerical
a) polyploidy
b) aneuploidy
2) structural
a) deletion
b) insertion
c) duplication
d) translocation
e) ring chromosome
f) isochromosome
Incidence of chromosomal
abnormalities in live births
All chromosome abnormalities 1/160 live births

Chromosomal Syndromes
Frequencies of chromosome
abnormalities
1 in 150 livebirths,
5% of stillbirths,
50% of spontaneous abortions.
The frequency of chromosome abnormalities at fertilization

may be as high as 50%.
Humans are unique with regard to the high frequency of

chromosome abnormalities when compared to other
species. mouse: < 1–2% Saccharomyces cerevisiae:
1/10,000, Drosophila melanogaster: 1/1,700- 6,000
(aneuploidy in fertilized eggs)
Nat Rev Gen 2001
Human Chromosomes.pdf
Numerical abnormalities -
polyploidy
Change in the number of haploid sets of chromosomes
Triploidy (69 chromosomes) is one of the most frequent abnormalities in

spontaneous abortions. Triploids arise when two haploid sperm fertilize a haploid
egg. Triploidy is lethal, with most fetuses dying before birth.
Tetraploidy (two extra sets of chromosomes -92 chromosomes) is more rare and
also lethal.
Numerical abnormalities -
aneuploidy
Loss or gain of a single chromosome(s)
Results from errors in division during meiosis, where a daughter cell receives
both pairs of a particular chromosome (nondisjunction errors).
Addition of an extra chromosome, trisomy, has been described for all the
chromosomes but only three autosomal trisomies survive to birth. Those are
trisomies for chromosomes 21, 18 and 13. The remaining autosomal trisomies
are miscarried.
Trisomy for chromosomes 13 and 18 are much more severe than 21 and those
that survive to term usually die shortly after birth.
Chromosomes 13 and 18 are the two chromosomes composed of the least

amount of GC-rich/gene-rich DNA whereas the chromosome 21 is one of the
smallest autosomes.This may be why they can survive to term when other
autosomal trisomies are miscarried.
Nondisjunction types (meiosisi I or II) can be
diagnosed with the use of polymorphic
genetic markers
Analysis of inheritance of polymorphic DNA markers can be used to determine the

meiotic stage and parent of origin of aneuploidy. In this example, different alleles of
a chromosome 21 centromeric locus are visualized as peaks in sequencing
electrophoretograms. The trisomic offspring (+21) has inherited one paternal allele
and two different maternal alleles; thus, the extra chromosome originated from an
error at maternal meiosis I.
The origin of human trisomy
MI, meiosis I; MII, meiosis II Nat Rev Gen 2001

Meiosis in in males vs.
In male, meiosis begins with
females
puberty and the important
events are sequential: in the
adult testis, cells progress from
prophase to metaphase I and on
to metaphase II without an
intervening delay, and each cell
that enters meiosis produces
four sperm.
In female is extraordinarily
protracted: all oocytes initiate
meiosis during fetal
development, but after
homologous chromosomes
undergo synapsis and initiate
recombination, the oocyte
enters a period of meiotic arrest.
Resumption of meiosis and the
completion of the first division
occur years later, just before the
oocyte is ovulated. After the Nat Rev Gen 2001
completion of MI, the oocyte

arrests at the metaphase of MII
and the second division is
completed only after the egg is
47,XX,+21 female Down syndrome
karyotype (trisomy 21)
Down syndrome: morphology
A palmar simian crease

A protuberant abdomen
and an umbilical hernia
Flat face with

A wide gap
hypertelorism,
between
depressed nasal Small and misshapen auricle
the first and
bridge, protrusion and anomalies of the folds
second toes and Hypodontia
of the tongue
onychomycosis http://www.emedicine.com
http://medgen.genetics.utah.edu
Trisomy 18- Edward’s Syndrome http://www.emedicine.com
1 in 6 000-8 000 live births
clenched hand with the

index finger overriding
the middle finger and the
microphthalmia, micrognathia/retrognathia, 5th finger overriding the
microstomia, low set/malformed ears, short sternum, 4th finger
and abnormal clenched fingers
rocker-bottom foot with
prominent calcaneus
http://medgen.genetics.utah.edu
Patau Syndrome. Trisomy 13
1 case per 8,000-12,000 live births
13 is the largest
autosomal
imbalance that
can be
sustained by
the embryo and
yet allow
survival to term
http://medgen.genetics.utah
Sex chromosome aneuploidy
Much more frequent among live births than autosomal aneuploidy
Reasons:
The Y chromosome is relatively gene poor with the exception of the sex-determining
genes;
All but one X in females and individuals with more than one X, are inactivated.
Klinefelter Syndrome (XXY males):
Pathogenesis
• XXY individuals are slightly feminized. A

small part of the X chromosome near one
telomere the pseudoautosomal region is
not inactivated. In XXY males, this region
is active at twice the level of the
pseudoautosomal region in XY males.
Changes in Chromosome Number.htm

Klinefelter syndrome
Female-type distribution of
Disproportionately Gynecomastia pubic hair and testicular
long arms and legs (increased risk of breast dysgenesis
cancer
though still much less
than in females).
http://www.emedicine.com
45,X Turner syndrome
karyotype (monosomy X)
Monosomy X: Turner Syndrom
1 in 2 500 -3 000 live-born girls
Redundant Nuchal Skin

and Puffiness of the
Hands and Feet
Reduced stature. Broad, "webbed"
neck. Swelling (edema) is in the ankles
and wrists. http://www.emedicine.com/
Advanced maternal age is not associated with an increased incidence Turner's Syndrome.pdf
Turner Syndrome: patogenesis
• Monosomy for the pseudoautosomal
region of the X ?
• Absence of two normal sex chromosomes
before X-chromosome inactivation ?
• Loss of the testis-determining factor (SRY)
gene on the short arm of the Y
chromosome (phenotype of Turner’s
syndrome even without a 45,X cell
population).
Turner's Syndrome.pdf
XYY males and XXX females
• The XYY males are usually fertile. Their meioses are of
the XY type; the extra Y is not transmitted, and their
gametes contain either X or Y, never YY or XY. Attempts
have been made to link the XYY condition with a
predisposition toward violence.
• The XXX individuals are phenotypically normal and
fertile females; In XXX females. Meiosis is of the XX
type, producing eggs bearing only one X. In XXX
females the pseudoautosomal region is active at only 1.5
times the level that it is in XX females. This relatively
lower level of functional aneuploidy, plus the fact that the
pseudoautosomal genes appear to lead to feminization,
may explain the phenotype.
Changes in Chromosome Number.htm

Structural abnormalities
Unbalanced structural rearrangements:
Deletions
Terminal deletion Interstitial deletion

Terminal deletions with
ring chromosome formation
http://www.tokyo-med.ac.jp Chromosomal Syndromes
Duplications
Direct duplication Inverted duplication
Isochromosomes
http://www.tokyo-med.ac.jp
An isochromosome is a chromosome consisting of two

identical copies of one arm of a single chromosome and none of the other.
By cytogenetic techniques impossible to distinquish from homologous ROB
In a person with 46 chromosomes, an isochromosome

results in partial monosomy and partial trisomy. Chromosomal Syndromes
Balanced structural rearrangements:
Inversions
Paracentric inversion Pericentric inversion

Balanced structural rearrangements:
Translocations
Robertsonian
Insertion translocation
(whole arm rearrangements

of the acrocentric
chromosomes, often creating
a dicentric,chromosome)
Reciprocal translocation
Genetic counselling in
Robertsonian translocation carrier
Chromosomal Genetic Disease

Karyotype nomenclature
47, XY,+8 [15] / 46,XY [15]
45,X [85] / 46,XX [15]
45,XY,der(1)t(1;3)(p22;q13),-3
46,XY,dup(12)(q13)
46,XY,der(2)t(2;?)(q37;?)
46,XY,der(1)t(1;3)(p22;q13)
46,XX,del(7)(p11)
45,XY,der(13;21)(p11;p11)
46,XY,der(13;21)(p11;p11) +21
46,XX t (7;14)(q11;p22)
69,XXX
46,XX,inv(3)(q21q26)
46,XY,del(5)(q13q33)
Karyotype nomenclature
Abbrev iat ion Meaning Ex ample
cen centromere
del deletion 46,XX,del(5p)
der derivative chromosome der(1)
dic dicentric chromosome dic(X;Y)
dup duplication
i isochromosome 46,X,i(Xq)
ins insertion
inv inversion inv(3)(p25;q21)
mat maternal origin 47,XY,der(1)mat
p short arm
pat paternal origin
q long arm
r ring chromosome 46,X,r(X)
t translocation 46,XX,t(2;8)(q21;p13)
ter terminus 46, XX, del(Xq21;qter)
upd uniparental disomy
+ gain of a chromosome 47,XX,+21
- loss of a chromosome 45,XY,-14
:: break and join
/ mosaicism 46,XX/47,XX,+8
46,XY,t(4,5)(qter,qter) Chr. 4
Chr. 5
R!
46,XY 46,XY,t(4,5)(qter,qter) 46,XY,der(4)t(4,5)(qter,qter) 46,XY,der(5)t(4,5)(qter,qter)
R!
46,XX
METHODS
Cytogenetic testing
peripheral blood
amniotic fluid
chorion / trophoblast
dermal fibroblasts
bone marrow
umbilical cord blood
fetal tissues
blastomeres
Chromosome banding techniques
Type of
Method Properties of bands
banding
G banding Controlled digestion with trypsin followed by Series of bands along the chromosomes, late
staining with Giemsa replicating, gene poor, condensed, AT rich
R banding Heat denaturation of chromosomes followed by Reverse of G banding, early replicating, gene
staining with Giemsa rich, less condensed, GC rich
C banding Denaturation of chromosomes with BaOH Stains constitutive heterochromatin, esp.

followed by staining with Giemsa centromeres, highly condensed, late replicating,
no genes, not transcribed
NOR Chromosomes are stained with a silver nitrate Stains the active nucleolar organizer regions at
banding solution the stalks
Late replicating, gene poor, condensed, AT rich.

Q banding Fluorescent stain quinacrine dihydrochloride
Similar to G banding
is used which binds preferentially to
AT-rich DNA
Replication Incorporation of the thymidine analogue Stains DNA based on replication timing,
banding BrDU into either late replicating (G bands) highest band levels
or early replicating (R bands) followed by
treatment with UV light to destroy BrDU
incorporated DNA and staining with Giemsa
T banding Controlled heat denaturation followed by Stains the telomere regions, very GC rich, high
staining with Giemsa gene density, early replicating
Preparation and Banding.pdf

BaOH, barium hydroxide; BrDU, bromodeoxyuridine
G banding
Same chromosome prepared six
different times
G-banded HRT normal male
karyotype Human Chromosomes.pdf
Centromeres marked by dashes.

Chromosomes 13, 14, 15, 21 and 22 are acrocentric chromosomes with the secondary
constriction, composed of stalks and satellites, above the centromere.
NOR staining
FISH -fluorescent in situ
Nature Genetics 2001
hybridization
Mid-plane light optical section through a chicken fibroblast nucleus shows mutually
exclusive chromosome territories (CTs) with homologous chromosomes seen in
separate locations. CHROMOSOME TERRITORIES.pdf
FISH examples
Preparation and Banding.pdf
(a) Normal human metaphase

spread showing hybridization of
the centromeric region of
chromosomes 7 (green) and 8 (red)
using α-satellite probes.
Chromosomes counterstained in
blue using DAPI. (b) Human male
metaphase spread with deletion of
the elastin (ELN) locus at the
Williams syndrome critical region
near the centromere on one
homologue of chromosome 7.
Cosmid probe for the ELN locus
and a control probe are visible on
the normal homologue (right);
however, only the control probe
can be seen on the deleted
homologue (left). (c) Normal
human metaphase spread with
whole-chromosome paint probe for
chromosome pair number 15 in red.
(d) Cross-species colour banding
(RX-FISH®) on normal human
male chromosomes arranged in
standard karyotype format.
Chromosome painting with mFISH
(a) Staining of all 46 chromosomes of a human female cell simultaneously in different colors by M-FISH. This
analysis is based on an adaptive spectral classification approach for seven fluorochromes. The
automated analysis results in computer-generated false colors for each chromosome.
(b) Multicolor classified karyogram of the normal female metaphase spread shown in (a).
Multicolor chromosome painting
Interphase FISH
useful in situations where it is difficult to obtain metaphase spreads
for karyotyping,such as cancer tissue, preimplantation embryos and
foetal material after miscarriage.
Applications of fluorescence in situ hybridization
(FISH) to metaphase or interphase preparations
A. A chromosome 1-specific "paint" probe
hybridizes to both normal chromosomes 1 http://harrisons.accessmedicine.com
as well as to a marker chromosome derived
from chromosome 1.
B. A repetitive DNA probe specific for
centromeric -satellite sequences on
chromosome 1 hybridizes to the centromeric
region of both normal chromosomes 1 as
well as to a marker chromosome derived
from chromosome 1.
C. Two-color FISH used to detect a microdeletion
of chromosome 15 associated with Prader-
Willi syndrome. A repetitive classic satellite
probe hybridizes to the short arm of
chromosome 15 (large blue dots) and a
probe for PML (a locus on the distal portion
of chromosome 15, visualized as small black
dots) are observed on both chromosomes.
However, a probe for SNRPN (a locus
within the PWS region of chromosome 15,
small black dots) hybridizes only to the
normal chromosome. The arrow points to
the deleted chromosome.
D. Interphase FISH using chromosome 13 (large
blue dots) and chromosome 21 (small black
dots) unique sequence probes on interphase
cells from direct amniotic fluid
preparations. Three chromosome 21-specific
signals are observed, indicating the presence
of trisomy 21 in the fetus.
CGH - comparative genome
hybridization
Kallioniemi et al. Science 1992
Green-to-red fluorescence ratio profiles of
chromosome 8 (A) and chromosome 2 (B)
after hybridization with COLO 320HSR
and NCI-H69 cell line DNAs, respectively (green).
Normal reference DNA included in the
hybridization is shown in red. The inserts
show the overlaid green and red
fluorescence images of the chromosomes.
(A) the myc locus at 8q24 shows a highly elevated green-

to-red ratio, which is compatible with the known
highlevel amplification of myc in the COLO
320HSR cell line.
(B) 3 regions of amplification are seen on chromosome 2.

The signal at 2p24 corresponds to the s location of
N-myc known to be amplified in the NCI-H69 cell
line. The two other regions with a highly increased
fluorescence ratio, at 2p21 and 2q21, were not
known
Array CGH:
Detailed genomic profiles and FISH validation of copy-number abnormalities identified in cases
with unexplained mental retardation - deletions
• Panels represent individual profiles of the
affected chromosomes for each case, with
clones ordered, for each chromosome, from
pter to qter. The centromeric region is
indicated by a vertical gray dash, the
thresholds for copy-number gain (log2T/R
value 0.3) and copy-number loss (log2 T/R
value −0.3) are indicated by horizontal lines.
• Panels on the right represent the FISH
validation using (one of) the target clone(s)
identified by arrayCGH. Affected
chromosomes are indicated by an asterisk
(*).
• A the deletion on 7q11 with 14 clones in this

region showing an average log2 intensity
ratio of −0.5. FISH validation of this case is
shown in the adjacent panelF, in which one
of the deleted clones on 7q11 is shown in red
and an undeleted control probe is shown in
green.
• B the microdeletion on 2q22 with a total of

three clones crossing the threshold for copy-
number loss, with FISH validation in the
adjacent panel G.
Lisenka et al 2003 AJHG

• C Deletion of a single clone on 1p21
Array CGH: duplications
Lisenka et al 2003 AJHG

The Complete Cytogenetics Arrays
Simplified, streamlined, and all-inclusive

Whole-Genome 2.7M Array
Arrays Reagents
Designed with input from cytogenetic researchers
Chromosome Analysis Suite Instrumentation

(ChAS) Software
THE END
Linkage analysis
Meiosis
Segregation of alleles of two loci –

the closer the loci are located,
the more often they are inherited
together.
Distance between loci can be
described by recombination
fraction θ (theta)
( (0≤θ ≤0,5)
Direct examination of sites of
recombination
A human spermatocyte (obtained from
a testicular biopsy) captured during
the prophase of meiosis I.
Antibodies against SCP3 protein highlight

the synaptonemal complex (red),
the "glue" that tethers homologues
during this phase of meiosis;
CREST antiserum localizes to

centromeric regions of the chromosomes
(blue);
Antibodies against the mismatch-repair

protein MLH1 pinpoint the sites
at which crossovers develop (green).
Nat Rev Gen 2001

Linkage:
joint segregation of two
traits/alleles caused by their
proximity in a chromosome
Linkage analysis is based
on comparison of two likelihoods
by calculating their ratio:
LR– likelihood ratio
L1 = likelihood assuming the linkage
L2 = likelihood assuming a lack of the linkage

Linkage analysis requires
assumtion of a model with
defined parameters
The key parameter is θ (theta)

( – the recombination
fequency between teh studied loci (for example the
marker locus and the disesase locus)
Example 1
Lparents=Lp = 2Dd 2x0.1x0.2 x d2 0.32
D d dd
1 2 33
D d
13
Assuming that D and marker locus are linked

and separated by a distance = θ ,
and in the father ‘1’ and D are located in one chromosome :
L1= Lp x 0.5 x (1- θ) x 1 x 1
L2= Lp x 0.52 x 1 x 1
LR= 0.5(1- θ) / 0.52 = (1- θ) / 0.5
if θ=0 , LR=2
Example 2
D d dd
2 1 33
D d
13
Assuming that D and marker locus are linked and separated by a

distance = θ , and in the father ‘1’ and D are located in different
chromosome:
L1= Lp x 0.5 x θ x 1 x 1
L2= Lp x 0.5 x 0.5 x 1 x 1
LR= 0.5x θ / 0.52 = θ /0.5
if θ=0.1 , LR= 0.2
Example 3
Dd dd
12 33
Dd dd
13 23
L1= 0.5(Lp x 0.5 x (1- θ)x1x1 x 0.5x(1- θ) x 1x1) +

0.5(Lp x 0.5 x θ x1x1 x 0.5x θ x 1x1) =
0.5xLp x 0.52 ((1- θ)2 + θ2 )
L2= Lp x 0.52 x 0.52
LR= 0.5x((1- θ)2 + θ2 ) / 0.52
Mathematically the likelihood assuming
lack of linkage equals likelihood
assuming presence of linkage and θ
=0.5
LR=
General formula for calculating LR
in linkage analysis in AD disease
LR=
Calculating LR
in linkage analysis in AD disease
Concept of phase - phase
known
LR =
Lod score is an usual way of
presenting LR
Lod =log10 LR
Accepted criteria for
evaluation of the Lod score
LOD score >(=)3 (LR>(=) 1000)

proof of the presence of a linkage
LOD score <(=)-2 (LR<(=) 1:100)

proof of the lack of a linkage
LOD score >-2 i <3

Inconclusive result
Calculations are performed for a
range of θ (0≤θ<0,5)
Often multiple families are
investigated
Although in individual families different alleles
cosegregate with disease the resultsing lod scores
can be added up
13 24
23 56
35 25 26 35 25 36 26
LRtotal= LR1 x LR2 x...x LRn
so:
Lodtotal = Lod1 + Lod2 +...+ Lodn
For example: 102 x 101=102+1=103

Application of linkage analysis
• To determine relative position of loci
• Genome mapping
• Localization of disease loci
• „Indirect” diagnosis of the presence of
mutation
The End
Sequencing
Point mutations
(Caused by changing of one or more nucleotides in DNA chain )
SUBSTITUTION
(replacing)
DELETION INSERTION
(falling out) -inserting one or
transitions more nucleotides
- manifested by the loss
into the DNA chain.
- involving the substitution of of one or more
a purine into another purine nucleotides from the
or pyrimidine base to chain,
another pyrimidine,
transversions –
purine base is converted to
the pyrimidine or vice versa,
The effects of
point
mutations in
the coding
region
silent – without any change in protein structure

conservative missense – nucleotide change without changing
protein’s function
nonconservative missense – disfunction of protein followed
by nucleotide change
nonsense mutation – the STOP codon
frame-shift - other codons (triplets) after mutation
Sequencing of the DNA
PCR - Polymerase chain reaction
Consists in determining the sequence of base
pairs in the test DNA molecule.
Currently, the most commonly used is the
Sanger method involving the enzymatic
synthesis of DNA molecules.
„The Gold Stardand”
Insertion dideoxynucleotide to synthesized

chain to prevent further extending because
ddXTP does not have the group-OH at
position 3'dideoxyribose, and it is impossible
to create another phosphodiester bond.
The reaction mixture contains 4 ddNTP

labeled with a fluorochrome and the four
dNTPs, and only one primer (two directions).
Automatic sequencing
Automatic sequencing
Analyzing of the sequencing results
Substitution
HOMOZYGOTE
Sequence without mutation Sequence without mutation (wild)

(wild type) (dzika)
Sequence with mutation Sequence with mutation
Sequnce with Pro Pro Val Val His Glu Asn Pro Ile His
mutation (forward) CCA CCT GTA GTA CAT GAA AAC CCA ATC CAC
CCA CCT GTA GTA CAT AAA AAC CCA ATC CAC
The reference
sequence Pro Pro Val Val His Lys Asn Pro Ile His
Sequence with CCA CCT GTA GTA CAT GAA AAC CCA ATC CAC
mutation (reverse) Pro Pro Val Val His Glu Asn Pro Ile His
What is the mutation at the DNA level? Substitution of adenine into guanine
Homozygote or heterozygote? homozygote
What can we observe at the protein Change of sense - missense muation of Lys into
level? Glu
Substitution
HETEROZYGOTE

(wild type) (dzika) type)
Pro Pro Asp Ala Asp Glu Pro Ala Gly Pro
Sequence with
mutation (forward) CCT CCC GAC GCA G AT GAG CCC GCC GGC CCC
G
CCT CCC GAC GCA GGT GAG CCC GCC GGC CCC
The reference
sequence Pro Pro Asp Ala Gly Glu Pro Ala Gly Pro
Sequence with
mutation (reverse) CCT CCC GAC GCA G AT GAG CCC GCC GGC CCC
G
Pro Pro Asp Ala Asp Glu Pro Ala Gly Pro
What is the mutation at the DNA level? Substitution of adenine into guanine
Homozygote or heterozygote? heterozygote
What can we observe at the protein Change of sense - missense muation of Gly into
level? Asp
Deletion
HOMOZYGOTE
Sequence with Glu Lys Lys Arg Gly Asp Lys Glu Stop
mutation (forward) GAG AAG AAG AGG GGA GAT AAA GAG TGA ATT TAA
GAG AAG AAG AGG AAG TTC ATC AAG GGG GAG ATA
The reference Glu Lys Lys Arg Lys Phe Ile Lys Gly Glu Ile
sequence
Sequence with GAG AAG AAG AGG GGA GAT AAA GAG TGA ATT TAA
mutation (reverse) Glu Lys Lys Arg Gly Asp Lys Glu Stop
What is the mutation at the DNA level? deletion of 14 nucleotides

Homozygote or heterozygote? homozygote
What can we observe at the protein frameshift/ mutation without sense („nonsense”) – codon STOP
level?
Delecja
HETEROZYGOTA

(wild)
Gly Thr Leu Gln Thr Ile Leu Gly Val Stop
GGC ACG CTG CAG ACG ATC CTG GGG GTG TGA AC
Sequence with GG GGA AC
mutation (forward) GGC ACG CTG CAG ACG ATC CTG GGG G TT TTG AA
GGC ACG CTG CAG ACG ATC CTG GGG GGT GTG AAC
The reference Gly Thr Leu Gln Thr Ile Leu Gly Gly Val Asn
sequence
Sequence with GGC ACG CTG CAG ACG ATC CTG GGG GGT GTG AAC
GG GTA A
mutation (reverse) GGC ACG CTG CAG ACG ATC CTG GGG G TT TGG A
GGC ACG CTG CAG ACG ATC CTG GGG GTG TGA AC
Gly Thr Leu Gln Thr Ile Leu Gly Val Stop
What is the mutation at the DNA level? deletion of one guanine
Homozygote or heterozygote? heterozygota
frameshift/ mutation without sense („nonsense”) – codon STOP
What can we observe at the protein
level?
Next generation sequencing
Sanger sequencing NGS sequencing

STEP 1 Preparation of patient’s DNA samples
cBOT
HiSeq sequencer
flow cell
STEP 2 Clusters generating by bridge amplification
Hundreds of millions
ofclusters, each >103
of identical DNA
molecules derived
from one molecule
Density on the Flow

cell
apx. 700-900 C/mm2
STEP 3 Sequencing with reversible terminators
• The four dNTPs for each cycle act as terminators, each
labeled with a different dye - 1 bp elongation
• Washing out of unused terminators and imaging
• Severance dye terminator - the chain ready for the next step
extension
MOVIE
STEP 4 Data ananlyzing
Online IGV
Exercise 1.
Using IGV programme, from genes listed below,
connected with hearing loss (autosomal
recessive), suggest gene where changes are seen:
•GJB 2
•ILDR 1
•TMPRSS 3
Define mutations that You have found.
Odds Ratio vs Relative Risk
Genetic studies
of multifactorial diseases
Department of Medical Genetics

Medical University of Warsaw
 Probability of an event P(A) denotes

the frequency of it’s occurrence in a
long (infinite) series of repeats
| A|
P( A) =  A
||
0  P 1
It’s represented by a number Probability 0.25 (1/4) indicates that certain event is
in the range from 0 (event observed in 1 from 4 or 25% occurrences
never occurs) to 1 (event
occurs every time)
Odds
 Odds - describe probability as a ratio (proportion)
P( A)
Odd ( A) =
1 − P( A)
Odds take higher values than
probability
1 1
1
odds = 5
= 5
=
1 − 15 4
5 4
 Frequency of blue balls:

 Proportion of blue balls vs. yellow balls:
4:6 = 2:3 (2 to 3)
Polygenic diseases
(multifactorial diseases)
• Many genes involved

• Multiple factors (including environmental ones)
+ genetically predetermined susceptibility
to the action of these factors
Influence of
the environment
Polygenic AD AR
Polygenic diseases
(examples)
 atherosclerosis
 hypertension
 diabetes
 neoplasms
 neurodegenerative diseases
 immunological disorders (rheumatoid arthritis,
multiple sclerosis etc.)
 … and many others
Ethiology: single gene
vs polygenic (multifactorial)
Disease: Monogenic Polygenic (multifactorial)
Morbidity Low High
Disease-assoc. Rare Frequent

variants
Loci Single Multiple
Penetration High (~1) Very low (ambiguous net

adverse effect)
Multiplex Frequent Very rare
families
Pathogenicity Clear Unclear
of variants
New methods of analysis needed!

GWAS atherosclerosis polymorphisms
locus Gene Freq SNP OR Other assoc. (incl. with TRF)
1p32.2 PPAP2B 0,91 rs17114036 1,17
1q41 MIA3 0,72 rs17465637 1,14
2q33.1 WDR12 0,14 rs6725887 1,17
3q22.3 MRAS 0,15 rs9818870 1,15
6p21.31 ANKS1A 0,75 rs17609940 1,07
6p24.1 PHACTR1 0,65 rs12526453 1,12
6q23.2 TCF21 0,62 rs12190287 1,08
6q25.3 LPA 0,07 rs10455872 1,70
7q22.3 BCAP29 0,74 rs10953541 1,08
7q32.2 ZC3HC1 0,62 rs11556924 1,09
10q11.21 CXCL12 0,84 rs1746048 1,17
11q22.3 PDGFD 0,22 rs974819 1,07
13q34 COL4A1/2 0,44 rs4773144 1,07
14q32.2 HHIPL1 0,43 rs2895811 1,07
15q25.1 ADAMTS7 0,57 rs3825807 1,08
15q25.1 ADAMTS7 0,65 rs4380028 1,07
17p11.2 SMCR3 0,56 rs12936587 1,07
17q21.32 GIP 0,53 rs46522 1,06
19p13.2 LDLR 0,75 rs1122608 1,15
21q22.11 MRPS6 0,13 rs9982601 1,20
9p21.3 ANRIL 0,56 rs4977574 1,29 aortic/intracranial aneurysm
1p13.3 SORT1 0,81 rs646776 1,19 lipid traits
1p32.3 PCSK9 0,81 rs11206510 1,15 lipid traits
6p21.33 HLA-C 0,56 rs3869109 1,14 lipid traits
6q25.3 LPA 0,02 rs3798220 1,92 lipid traits
11q23.3 APOA5 0,13 rs964184 1,13 lipid traits
10p11.23 KIAA1462 0,43 rs2505083 1,08 non-alcoholic fatty liver disease
9q34.2 ABO 0,21 rs579459 1,10 fytosterol level, venous thromboembolism
10q23.2 LIPA 0,37 rs1412444 1,10 blood pressure
10q24.32 CYP17A1 0,89 rs12413409 1,12 blood pressure, intracranial aneurysm
9p21.3 MTAPe 0,59 rs7865618 1,18 diabetes
17p13.3 SMG6 0,37 rs216172 1,07 diabetes
Genetic background of a disease
– ideal study
 Genotyping:
everybody is genotyped and classified
 Monitoring:
occurrence of the disease is monitored
for a long time
 Comparison:
frequency of the disease in two groups
Genetic background of a disease
– ideal study (an example)
26%
Genotype (+) (n=128)
15%
(n=75)
Genotype (-)
Comparing the risks
- measures
 Risk: % of affected individuals

 Risk difference
 Risk ratio (relative risk, RR)
 Example - a disease occurred:

- in 26% of individuals with given genotype
- in 15% of the persons without the genotype
Risk difference: 26% - 15% = 11%
Risk ratio (RR): 26% / 15% = 1.7
Comparing the risks
- RD vs RR
 If a disease is rare,
the risk difference may be small,
but the risk ratio – large !
 Example:
- risk among those with genotype: R=1.5%
- risk among those without: R =0.3%
Difference = 1.5% - 0.3% = 1.2%
Ratio (RR) = 1.5% / 0.3% = 5.0
Comparing the risks
- RD vs RR – which is better?
 Difference – an absolute measure

- Useful in estimating how many people are ill
because of the genotype
 Ratio – a relative measure

- better for estimating the risk in a given patient
- better for the purpose of basic studies
Comparing the risks
- associations in a table
Affected Healthy
(a+b) a b
observation
Affected Healthy
Genotype(-) (c+d) c d
RR = a/(a+b) / c/(c+d)
Proportion of frequency of affected among those with the genotype
to the frequency of affected among those without the genotype
RR – relative risk
Affected
Yes No
Yes a b a
a+b
RR =
c
No c d c+d
Comparing the odds – odds ratio
(a table)
Affected Healthy
(a+b) a b
observation
Affected Healthy
Genotype(-) (c+d) c d
OR = [a/(a+b) / (1-a/(a+b))] / [c/(c+d) / (1-c/(c+d))]

Proportion of the odds among those with the genotype
to the odds among those without the genotype
OR - odds ratio
Affected
Yes No a
a+b
Yes a b a
1−
a+b
OR =
c
No c d c+d
c
1−
c+d
OR calculated for occurrence of disease
among those with vs. without the genotype
OR calculated for occurrence of genotype
among those with vs. without the disease (cn.)
Comparing the risks
- OR vs RR – rare diseases
 If a disease is rare
(both among those with and without genotype),
RR and OR are very similar !
 Example:
Frequency of RA among
- those with HLA-DR4 - 0.018,
- the rest - 0.003
RR = 0.018/0.003 = 6.0
OR = 0.01833/0.00301 = 6.09
„rare” means <10%

Comparing the risks
- OR vs RR – frequent diseases
 If a disease is frequent,
RR and OR are different !
 Example:
- Risk in the presence of genotype = 0.6
- Risk in the absence of genotype = 0.1 :
RR = 0.6/0.1 = 6.0
OR = 0.6/0.4 / 0.1/0.9 = 13.5
„frequent” means >10%

It is usually easier
to determine OR
than RR !
OR vs RR –
(practical considerations)
Disease
7 493
Genotype (+)
4 496
Genotype (-)
RR=(7/500)/(4/500) the key factor: proportion 7/4

RR=7/4 (considerable error is possible)
The error can be minimized
by enlarging the group of affected
Disease Disease Disease

+ - + + -
7 493 700 493 707 493
+ =
4 496 400 496 404 496
RR=1,76 RR=1,31
OR=1,75 OR=1,75
RR has changed but OR has not !
Conclusions
 RR is the preferred measure of strength of

association between genotype and disease,
esp. in a medical/diagnostic setting
 In practice it is easier to determine OR
 OR is a good approximation of RR,

when the disease is rare (<10%)
both in those with and without the genotype
What to remember?
When to use and how to calculate RR?
When diseases are frequent >10%
When to use and how to calculate OR?

When diseases are rare <10%
Disorders of sex
development (DSD)
JE N NI FER CA STA ŃEDA , M D, P HD JE N CA STANEDA @GM AIL .COM
Clinical features
▪Ambiguous genitalia (in females: enlarged clitoris, labial fusion, in

males: hypospadia, undescended testes)
▪ primary amennorhea
▪ streak gonads, inguinal hernia
▪ delayed development of secondary sex characteristics
▪ signs of virilization in female – enlarged clitoris, acne, hirsutism,
deepening of the voice, increased muscle strength, menstrual
disruption due to anovulation
DSD: definition
▪ Congenital conditions in which development of chromosomal, gonadal or
anatomical sex is atypical
Level 1 – Genetic / chromosomal sex 46 XX vs 46, XY
Level 2 - Gonadal sex – testes / ovaries

Undifferentiated gonad – testicular development dependent on presence of TDF
Level 3 - Phenotypic sex

1. Internal reproductive structures (dependent on Mullerian inhibiting factor)
2. External reproductive structures (dependent on testosterone exposure)
3. Secondary sex characteristics
Embryology of the human genital tract
http://www.glowm.com/section_view/heading/Genetics%20of%20Sexual%20Differentiation/item/346
Important issues
▪ Gender assignment
▪ Risk of gonadal cancer
▪ Infertility
http://synapse.koreamed.org/DOIx.php?id=10.6065/apem.2012.17.3.137&vmode=PUBREADER
https://www.scieeopen.com/document?vid=be86306e-3e81-44d4-8fb5-448b21bdef60nc
Management of disorders of sex development
Hiort O et al., Nature Reviews Endocrinology 10, 520–529 (2014)

Tanner staging
https://en.wikipedia.org/wiki/Tanner_scale http://clinicalgate.com/normal-puberty-and-pubertal-disorders/
Diagnostic procedures
❑Cytogenetic tests (kariotype)

❑ Visualisation of internal sex organs through
ultrasonography or ascending urethrography (ovaries,
uterus)
❑ Hormonal analyses (17-OH progesterone, GC/MS steroid
profile, LH, FSH, testosterone, AMH)
❑ DSD gene chip – around 200 genomic regions
Turner syndrome
Responsible genes: X genes that escape inactivation, SHOX
Proteins: SHOX: Short stature homeobox protein
Cytogenetic locus: SHOX: Xpter-p22.32
Inheritance: sporadic
Clinical Features and Diagnostic Criteria: congenital lymphedema, growth failure, normal intelligence (10%
significant delays), coarctation of the aorta, bicuspid aortic valve, HLHS, hyperlipidemia, gonadal dysgenesis
(10% go into puberty), hypothyroidism, diabetes, strabismus, renal malformation, osteoporosis.
Molecular Tests: Karyotype, FISH SRY
Disease Mechanism: SHOX: thought to act as a transcription regulator with many down-stream targets that
modify growth and stature. SHOX protein has been identified in the growth plate from 12 weeks to late
childhood.
Treatment/Prognosis: GH, HRT, gonadectomy if Y chromosome mosaicism (risk for gonadoblastoma).
Lifelong cardiac follow-up, at risk for aortic dilation and dissection with bicuspid aortic valve.
Klinefelter syndrome
Clinical Features and Diagnostic Criteria: tall stature, slightly delayed motor and language
skills, learning problems, testosterone plateaus age 14, small fibrosed testes, azoospermia
and infertility, gynecomastia, increased cholesterol, slightly increased risk of autoimmune
disorders and mediastinal germ cell tumors (1% risk)
Molecular Tests: karyotype, at least one extra chromosome to a 46,XY
Disease Mechanism: 1st or 2nd meiotic division nondisjunction of either parent.

Maternal>paternal origin. + advanced maternal effect
Treatment/Prognosis: Testosterone in mid-late adolescence for bone density,

development of secondary sex characteristics, muscle mass, cholesterol, improved energy.
Groupwork
▪ Discuss briefly: etiology, diagnosis, clinical symptoms, treatment /
management
Group A: Congenital adrenal hyperplasia (CAH)
Group B: Complete / partial gonadal dysgenesis
Group C: Complete / partial androgen insensitivity syndrome (CAIS /
PAIS)
Ovotesticular DSD
Denys-Drash syndrome
Congenital adrenal hyperplasia (CAH)
https://labtestsonline.org/understanding/analytes/6-17hydroxy/tab/sample/
Congenital adrenal hyperplasia
❑ Autosomal recessive, prevalence of 1/12000-1/15000
❑ Deficiency of one of the 5 enzymes required for cortisol synthesis
❑ 90% of cases: 21-hydroxylase deficiency. CYP21 gene on 6p. Carrier frequency 1/50.
❑ Girls with Classic CAH: virilization
❑ Boys with classic CAH: poor feeding, dehydration, collapse (low plasma Na, high K,
acidosis)
❑Non-classic CAH: asymptomatic or signs of postnatal androgen excess
❑ Treatment if recognized at birth: steroid therapy (hydrocortisone +/- fludrocortisone
and salt
Complete gonadal dysgenesis
❑Kariotype 46,XY; 46,XX
❑ Female phenotype
❑ Vagina, uterus, fallopian tubes present
❑ Streak gonads in the place of ovaries
❑ Absence of secondary sex characteristics
❑ High FSH, low testosterone, low estradiol
Partial gonadal dysgenesis
❑Kariotype 45,X/46,XY or 46,XY
❑ Ambiguous genitalia
❑ Vagina, uterus, vas deferens present
❑ Bilateral dysgenetic testes
❑ Low testosterone, low AMH, low estradiol, high
FSH
https://courses.washington.edu/conj/bess/differentiation/differentiation.htm
Ovotesticular DSD
❑Kariotype 46,XX or 46,XY or mosaics
❑ Ambiguous genitalia
❑ Vagina, uterus, vas deferens present
❑ Presence of ovarian tissue or testicular tissue
❑ Hormonal tests depending on gonadal function
Complex management of DSD
❑ CAH with salt-wasting – life-threatening
❑ Optimal gender assignment/choice, reconstruction of sex organs
❑ Therapy
❑ Risk of gonadal cancer
❑ Hormone secretion
❑ Fertility, sexual issues, psychosocial implications
❑ Genetic counselling
Child with DSD - Consensus of 2006
❑ interdisciplinary diagnosis
❑ Each child should be given a gender.
❑ Parents should take part in decision-making on the child’s
gender.
❑ In doubtful cases, not to do reconstruction operations
prematurely
❑ Gonadectomy in high-risk groups
Gonadectomy in DSD
5th I-DSD Symposium Programme. 11th-13th June 2015,

Ghent, Belgium
Hereditary Cancer Genetics
Krzysztof Szczałuba
2019
Confirmation of ca Dx
1. Pathology report (!)
2. ‘Endometrial ca’ can be any of: ovarian, ca colli

or true endometrial
3. ‘Ascites and ca’ can be any of: abdominal or

thoracic ca
4. ‘Hepatic ca’ can be any ca (!) but most likely a

metastatic one
5. Age at Dx to be considered the starting point

but always check for stage (advanced?)
Cancer surveillance methods - GI surveillance
Colonoscopy – misses 6% of polyps:
- general population: every 10yrs beginning 50yrs (or just once
>50yrs)
- one or more 1st degree relative w/crc >50yrs: every 5 yrs
beginning 40yrs
- one or more 1st degree relative w/crc <50yrs: every 5 yrs
beginning 10yrs before earliest incidence
Flexible sigmoidoscopy – reaches as far as splenic flexure only,
recommended for pop screening, but in cases where adenoma
be found or larger polyps: follow up w/colonoscopy
FOBT – non-invasive; does not replace colonoscopy but
increases awareness of crc in the population
Upper GI endoscopy – every 2-3 yrs in Hereditary Non-Polyposis

Colorectal Cancer
Cancer surveillance methods – Breast & Ovary
Breast:
- general population: US every yr beginning 35yrs →
US+Mammogram: alternately from 40yrs
- one or more 1st degree relative w/brca: begin alternate
screening 10yrs before earliest incidence
MRI – every yr in BRCAmut carriers or in Hereditary Breast (and
Ovarian) ca families. Combine with US/Mammogram.
Ovaries:
Transvaginal US + CA-125/HE4 yearly in high-risk populations
only
What is ‘genetic ca’?
What is ‘hereditary ca’?

Inherited Predisposition to Cancer
Inherited cancers are a minority of the total number
of cases but range from 1-60%.
For most tumor types, e.g. breast, the inherited

fraction fall in the range of 1-10%.
Several rare tumors, adrenocortical carcinoma,

retinoblastoma and optic gliomas have very high
inherited fraction (40-60%).
When to suspect ‘hereditary ca’?
A.Early onset
B.Multiple ca in one individual
C.Bilateral ca for symmetric organs
D.Rare ca type
E. Familial aggregation of ca (What type? How
many? Who in?)
F. Specific pattern of ca in a family (syndrome)
When to suspect ‘Hereditary Predisposition’ –
examples
1. Brca <40
2. Brca receptor triple negative, basal, medullary
3. Any ovca
4. Bilateral brca
5. At least 2x brca in 1st degree relatives (one below 60)
6. Crc <50
7. Crc familial aggregation
8. Any medullary thyroid ca
9. At least 2 cancers in one individual
10.Young age (cancer-location individual)
Chromosomal Abnormalities
Trisomy 21 results in 20 fold increase in leukemia.
Girls with mosaic Turner syndrome or gonadal

dysgenesis are at increased risk for Gonadoblastoma.
Correlates with presence of Y chromosome
containing material (on karyotype; PCR for Y material
unnecessary).
XXY males have an increased risk of breast cancer.

Chromosomal Microrearrangements
A number of disorders where deletions of the Tumour
Suppressor Genes (+/- flanking genes) occurs often
with DD/congenital anomalies
Retinoblastoma - 3% constitutional deletions of 13q14

overlying the RB1 gene.
Neurofibromatosis type 1 - 3-5% of patients = more

severe phenotype
Autosomal Dominant Disorders
Multiple generations affected with cancer.
Transmission through mothers and fathers independent of

tissue type.
Earlier age of onset of cancer compared with sporadic cases.
Increase in multiple and bilateral tumors.
Clustering of specific tumor types within family.
Variable penetrance with unaffected mutation carriers

Tumor Suppressor Genes
Many dominant disorders result from deleterious
mutations in tumor suppressor genes
TSGs normally function:

1. To inhibit proliferation
2. To downregulate the cell cycle
3. To repair DNA
4. At DNA damage checkpoints
For most cancers: hereditary contribution no more
than 10%
>10% Germline Mutations (examples):
Dx Loci
Retinoblastoma RB1
Adrenocortical carcinoma TP53
Pheochromocytoma/VHL, NF1, RET,
paraganglioma SDH genes
Medullary thyroid cancer RET
Acoustic or vestibular schwannomas NF2
Ovarian cancer BRCA1/BRCA2
P53 Tumor Suppressor Gene
Somatic p53 mutations in cancers are very frequent in
osteosarcoma, breast, colon, pancreatic and other
solid tumors.
Majority of mutations are missense mutations that

interfere with functional domains.
Constitutional mutations associated with Li-Fraumeni

Cancer Predisposition Syndrome.
Li-Fraumeni syndrome (LFS)
First identified in children with sarcomas who had family history of
early onset breast cancer.
Autosomal dominant inheritance of cancer susceptibility with

proportion de novo mutations.
Penetrance for women greater then for men even when sex-
specific cancers are eliminated.
Breast cancer risk is very high with average age of diagnosis of 32
Multiple malignancies in one patient is very common.

Li-Fraumeni syndrome (LFS)
Breast Cancer
About 5-10% of breast cancers result from an inherited
predisposition to the disease.
Changes used to predict prognosis.
Biomarkers and gene expression profiles differ between

inherited and non-inherited tumors, e.g. BRCA1 tumors
more likely to be ER-/PR-/HER2- and medullary.
Syndromes that increase the risk of breast cancer
Breast-Ovarian Ca Families – BRCA1>BRCA2
Li-Fraumeni – average diagnosis 32 in P53 carriers.
Peutz-Jeghers Syndrome – 32% by age 60
Cowden’s syndrome – PTEN mutations associated with
thyroid cancer, hamartomas, skin lesions.
PALB2 (variety of truncating mutations)
Moderate risk alleles:

RR - 2.0 CHEK2 (protein trunc muts).
Ataxia telangiectasia (ATM) heterozygotes.
NBN heterozygotes.
BRCA1 Breast/Ovarian Ca
Mutations found throughout the large gene. Most are deleterious:
frameshift or nonsense mutations. Larger rearrangements make up
2-5% of disease alleles. Follows the two hit hypothesis in
hereditary cancers with loss or inactivation of second allele in
tumors. Tumors have basal-like gene expression profile. Triple
negative (ER/PR/HER2) more likely.
Ashkenazi Founder Mutations:

Three founder mutations: Two specific mutations in BRCA1
(185delAG and 5382insC) and one in BRCA2 (6174delT).
Pop carrier frequency of 2.4% for all three mutations (in PL three
founder BRCA1 muts = 60-70% BRCA1 muts). Start testing with
these mutations. If negative for founder mutations, full sequence
mutation analysis for highly affected families
BRCA1/BRCA2 cancer risks
BRCA1 BRCA2
Breast - females 50-80% 50-80%
Breast - males 7%
Ovarian 20-40% 15-30%
Colon 6%
Prostate 8-16% 8-16%
Pancreas 1-2%
BRCA1/BRCA2 contralateral risks
Contralateral ca:
By age 40: BRCA1 33%, BRCA2 18%
By age 70: BRCA1 65%, BRCA2 52%
BRCA genes contribution to breast cancer
Brca 3% vs. 10% Ashk
Brca <45 10% vs. 25%
Brca>45 2% vs. 6%
Ovca 3% vs. 11%
Brca + FH Brca <45 20% vs. 30%
What’s the upper level?

Gudelines for carriers BRCA1/BRCA2
BEGINNING AGE 25:
Breast self-exam monthly
Clinical breast examination every 6 months
US & mammogram yearly
Breast MRI yearly
BEGINNING AGE 30:

Transvaginal US + CA-125/HE4 marker yearly
Prophylactic Surgery BRCA1/BRCA2
Multiple studies demonstrate the efficacy of bilateral
prophylactic salpingo-oopherectomy (BPO):
90% reduction in ovarian cancer risk
It most likely does not reduce breast cancer risk. Surgery
should include resection of the fallopian tubes. Recommend
age 35-40 after completion childbearing.
Bilateral mastectomy does not reduce ovca risk
Chemoprophylaxis w/Tamoxifen (estrogen receptor antagonist)

reduces the recurrence risk
Other genes:
Major: PALB2, TP53
Minor: CHEK2, NBN
BRCA1/2 Treatment
- Mastectomy rather than sparing surgery
- PARP inhibitors as 2nd/3rd line agents for ovca and/or brca
- Cisplatin effective but toxic
TP53 and any other v early brca
- Do not treat w/radiotherapy

Hereditary Non-Polyposis Colon Cancer / Lynch
Syndrome
Autosomal dominant CRC without polyposis (but with a polyp!)
associated with endometrial ca, bile duct, ovarian, ureteral and
gliomas. 70% lifetime risk of CRC and 50-70% endometrial ca in
classic Lynch. Right-sided CRC cancer is more frequent. Better
prognosis of CRC stage for stage. Common to see individuals
with 2 or 3 different primary HNPCC-related tumors.
Bethesda Criteria – based on proband characteristics:
CRC < 50
CRC + HNPCC assoc Ca
1st degree relative with 2 HNPCC cancers
1st degree relative with 2 HNPCC cancers, one < age 50
Two 2nd degree relatives, one with CRC and one with HNPCC
associated cancer
HNPCC- Mutations in Mismatch Repair Genes
Mismatch Repair Genes: MSH2, MLH1, MSH6 and PMS2
MSH2 and MLH1 make up 80-90% of Amsterdam criteria
families.
MSH6 testing: crc risk - 44% for men & 22% for women
HNPCC Surveillance
Screening with colonoscopy every 1-2 years starting at age 25
clearly decreases mortality in mutation carriers.
Prophylactic colectomy only recommended on case-by-case
basis.
Screening for endometrial cancer by endometrial biopsy
recommended.
Familial Adenomatous Polyposis (FAP)
AD with very high penetrance.
15-30% represent new mutation cases.
Adenomatous colonic polyps (tens to hundreds) begin in

childhood to adolescence.
Extracolonic features often called Gardner Syndrome (desmoid

tumours, osteomas, epidermoid cysts, congenital hypertrophy
of retinal pigment epithelium, hepatoblastoma, thyroid cancer)
Colorectal cancer risk: 50% at age 33 (lifetime 100% FAP vs.

lifetime 6% pop risk)
Familial Adenomatous Polyposis (FAP)
Find out another disease where genetic testing is so
indispensable!!!
APC gene testing is standard of care and allows determination of
which family members need surveillance and eventual surgery.
Guidelines typically recommend testing around age 10 – 12:

Surveillance by colonoscopy beginning age 10 – 12; continuing
every 1-2 years.
Colectomy by late teens to early twenties (depending on polyp
load or dysplasia). Restorative proctocolectomy with ileal
pouch-anal anastomosis. Total colectomy with ileorectal
anastomosis with annual surveillance of rectal stump.
Upper GI follow up by endoscopy for risk of gastric adenomas
and duodenal carcinomas
Annual thyroid exam
Example (HOC)
Sanger in founder pops

BRCA1/BRCA2 57%
(Myriad Pro)
BRCA1 3mut: 40%
cost: 300PLN
Sanger BRCA1/BRCA2
22,7% (Myriad Pro)
cost: 4000PLN
Example (HOC) – Ambry Genetics
23 genes
Every ovca
$5000
2-4 wks
Quiz (1)
Female pt aged 33 with 20 polyps (incl six tubular adenomas with
minor dysplasia) in colonoscopy 2015. Family history of
synchronic cecal and rectal cancers at age 40 in the sister.
1. Clinical diagnosis?
2. Genetic testing?
3. Invasive screening procedures in the affected and in the relatives?
Quiz (2)
Female pt aged 55 with brca. Family history of leukemia in in a
deceased son (died at age 15). In the son: microcephaly,
learning difficulties and immune deficiencies.
2. Genetic testing?
Quiz (3)
Female pt aged 43 with low-grade ovarian adenoca. Family
history of ovarian ca in the mother at age 50.
2. Genetic testing?
Quiz (4)
Female pt aged 35 with ovarian sarcoma. Family history of
brca in the mother at age 32.
2. Genetic testing?
Ethical Dilemmas in
Neurogenetics
ANDRZEJ KOCHAŃSKI MD, PhD
Genetic testing concerns a whole
family
• Genetic information involves areas of life
that are very personal, such as one’s
identity and reproductive fitness
• Disclosure of genetic information can
cause financial harm in the form of higher
insurance rates, loss of employement
opportunities, possible loss of insurance,
as well as emotional harm
Ethics in genetic counseling
• Ethic of care (sympathy, compassion, fidelity)
• Autonomy (recognition of the intrinsic value of
each individual, recognition of the patient’s right
to decide what will be done to her body)
• Confidentiality
• Informed consent (patient’s understanding,
authorization)
• Equal care for all
Multiculturalism and prenatal
diagnostics
• Family structure
• Middle Eastern Culture
• Cultural barriers to medical care
• Health and Illness
• Unnatural illnesses in African Americans
Prenatal testing and disability rights
• Prenatal tests have been criticized by the
disability rights community. Prenatal tests
reinforce discrimination against and
misconceptions about people with
disabilities.
Prenatal testing is morally
problematic
• 1. Selective abortion expresses negative
or discriminatory attitudes not merely
about a disabling trait, but about those
who carry it.
• 2. Selective abortion signals intolerance of
diversity in the family and could harm
parental attitudes toward children.
Defiant birth, Women who resist
medical eugenics
• „The counsellor emphasised how horrible and fatal this condition was. We told her
that termination was not an option for us. We got the response „Are you doing this for
religious reasons?” They did not know what to do with a couple who decided to
continue a pregnancy like this in spite of the diagnosis […]. She said there was a lot
of support if we terminated, she offered us nothing to continue with the pregnancy”
• The counsellor emphasised how horrible and fatal this
condition was –genetic counseling should be
nondirective
• Are you doing this for religious reasons?- an ability to
appreciate the values, beliefs, and behavior of cultures
other than our own
• She said there was a lot of support if we terminated, she
offered us nothing to continue with the pregnancy” –
patient support (genetic support groups, diagnostics,
therapy)
Proposed ethical guidelines for
prenatal diagnosis
• Equitable distribution of genetic services
• Prenatal diagnosis may be used to prepare for the birth
of a child with a disorder
• Prenatal diagnosis should be voluntary in nature
• Prenatal diagnosis is not acceptable for paternity testing,
for gender selection
• Prenatal diagnosis for relief of maternal anxiety
• Counseling should precede prenatal diagnosis
• Physicians should disclose all clinically relevant findings
to the pregnant woman or couple
• The woman should be respected and protected. The
couple, not the professional should make the choice
Prenatal diagnosis, McGraw-Hill,

2006
Genetic testing cannot
• Predict age of onset
• Predict type of symptoms
• Predict severity
• Predict disease course
We should know
• Patients understand the benefits, risks,
and limitations of genetic testing and have
thought about the potential and familial
impact of DNA analysis.
What is important
• Patients rights versus relatives rights
• Right to know versus right not to know
• Right to confidentiality
• Right to information
• Use of results of DNA analysis (genetic
discrimination)
Testing of asymptomatic carriers of
mutations
• Amyotrophic lateral sclerosis (ALS)
• Hereditary spastic paraplegia (HSP)
• Spinocerebellar ataxias
• Huntington disease
• Early –onset Alzheimer’s disease
• Frontotemporal dementia
• Parkinsons disease
Testing of children
• Should children be tested for neurogenetic
conditions?
• Testing the child for adoption ?
• Testing the twins
The duty to warn
• Myotonic dystrophy
• Hereditary neuropathies
• Individuals who work in professions where
neurological deficits can jeopardize the
safety of others (airplane pilot, truck driver,
construction worker, heavy equipment
operator)
Should they be tested ?
• Individuals who think they are
asymptomatic
• Individuals who have recently attempted
suicide
• Prenatal testing should be offered for
adult-onset genetic condition?
• Discrimination based on genetic
information
CONGENITAL ANOMALIES,
DYSMORPHOLOGY
Jennifer Castaneda, Krzysztof Szczałuba
WUM 2018
Definitions
 Phenotype: the set of observable characteristics of
an individual resulting from the interaction of its
genotype with the environment.
 Congenital anomalies: structural or functional
anomalies that occur during intrauterine life and can
be identified prenatally, at birth or later in life.
 Major anomalies – abnormalities that have medical,
surgical, or cosmetic significance
 Minor anomalies – cosmetic significance
= dysmorphic features
Causes of 2.68 million deaths during the
neonatal period in 2015, worldwide
Source: adapted from WHO 2000-2015 child causes of death

Congenital anomalies
 2-3% singletons have a major anomaly (e.g. heart
defect)
 10% have a minor anomaly (e.g. polydactyly)
 Causes: localized errors (e.g. clefts), deformation
(by physical force, e.g. oligohydramnios), disruption
(by destruction, e.g. amniotic bands), teratogens
(e.g. FAS), germline errors (syndromes)
Polydactyly
Etiologic heterogeneiety of cleft lip/palate
◼ Teratogens
◼ 22q deletion
◼ Primary mandibular hypoplasia
◼ Trisomy13
◼ Amniotic band syndrome
◼ Kniest dysplasia
◼ Van der Woude syndrome
Causes of congenital anomalies
 Multifactorial : 20-30%
 Monogenic disorders: 10-20%
 Chromosomal aberrations: 15%
 Infection: 2.5%
 Maternal diabetes: 1.5%
 Medication: 1-2%
 Unknown etiology
Empiric Recurrence Risks (%)
for Selected Birth Defects
Condition Affected Relatives(s)
None 1 sib/parent 2 sibs / sib & parent

Cleft lip/palate 0.1 4 10-11
Neural Tube Defect 0.1 3 8
Heart Defect 0.3 4-5 10-11
The risk of having any one major birth defect is

less than 1% but this risk increases significantly if
other relatives have the same birth defect
Causes of recognized development
disorders
 Many genes: chromosomal aneuploidies

 A numer of genes: chromosomal microdeletions /
microduplications
 A single gene: monogenic disorders
Types of morphologic abnormalities
 Malformation
 Deformation
 Disruption
 Dysplasia
Malformation
 Defect of morphogenesis in an organ or structure
due to an intrinsically abnormal problem with
formation, growth, or differentiation of an organ
or structure
 hypoplasia of an organ or structure (microtia),
incomplete closure (NTDs, cleft palate), incomplete
separation (syndactyly)
Deformation
 Abnormal form or position
of a body or region of the
body caused by extrinsic
non-disruptive mechanical
forces on a normally
developing structure (fetal
constraint)
 clubfoot, congenital hip
dislocation, craniofacial Deformity of ear helix due
asymmetry, overfolded ear to uterine compression
Deformations due to
oligohydramnios
Disruption
 Defect resulting from a destructive
breakdown of, or interference
with, a normally developing
structure resulting in death of cells
or tissue destruction.
 May be secondary to mechanical
forces, infections, or vascular Disruption of lip formation
events. due to amniotic bands
 Loss of digit due to amniotic band
constriction, lack of normal limb
development due to intrauterine
vascular accident
http://chicagofootcareclinic.com/footproblems/deformities/
amnioticbandsyndrome.html
Dysplasia
 Error of morphogenesis
causing abnormal cellular
organization or function in
a specific type of tissue,
mostly due to single gene
defects
 Achrondroplasia, ectodermal
dysplasia, osteogenesis
imperfecta
Ectodermal dysplasia
Clouston syndrome – ectodermal
dysplasia (GJB6 gene)
https://www.nfed.org/learn/types/clouston-syndrome/
https://pl.wikipedia.org/wiki/Zesp%C3%B3%C5%82_Cloustona
Diastrophic Dysplasia
Autosomal Recessive
Recognizable Patterns of Anomalies
 Syndromes
 Associations
 Sequences
 Dysplasias
Sequence
• a particular set of developmental anomalies
occurring together in a recognizable and consistent
pattern AND consequent upon a primary defect
(e.g. Pierre Robin sequence = mandibular
hypoplasia → tongue displacement → cleft palate
and upper airway obstruction)
Pierre-Robin sequence
http://slideplayer.com/slide/9674557/
Preoperative frontal and lateral views of an infant with
Pierre Robin sequence.
Sesenna et al. Italian Journal of Pediatrics 2012 38:7 doi:10.1186/1824-
7288-38-7.
http://clinicalgate.com/kidney-and-urinary-tract-disorders/
Potter’s sequence
Syndrome
 From Greek meaning “running together”
 Multiple anomalies in one or more tissues or
structures thought to be pathologically related due
to a specific etiologic mechanism (chromosome
disorder, single gene defect, environmental agent,
or unknown factor)
 Down syndrome, Williams syndrome, FAS,
Turner syndrome
Turner syndrome
Association
 Non-random occurrence of a combination of several
anomalies not yet identified as a specific sequence
or syndrome that occur more often together than by
chance alone.
 VACTERL association
http://www.slideshare.net/nijhum57/genetic-principles-
in-paediatric-surgery
Difficulties in diagnosing syndromes
 Some are very rare disorders - not well described

 Variable expression
 Incomplete penetrance
 Sex-influenced or limited expression
 Pleiotropy
 Etiologic heterogeneity
Etiologic heterogeneity
 Locus heterogeneity: a similar phenotype is
produced by mutations at different loci (Tuberous
Sclerosis, PKD)
 Allelic heterogeneity: a similar phenotype is
produced by different alleles within the same gene
(Craniosynostosis [FGFR3 gene] , CF [CFTR gene])
Variable Expression
 Morphological features expressed at different
degrees of severity in individuals having the
“same” abnormality
 Each individual with a particular syndrome,
sequence, or association will not have every known
feature of that disorder, even within the same
family.
 The degree of variable expression may correlate
with the degree of pleiotropy in single gene
disorders
Shagreen
Patches
Facial
Angiofibromas
Tuberous
Periungual
Fibromas Sclerosis
Penetrance
 proportion of individuals carrying a particular

variant (or allele) of a gene (the genotype) that
also express an associated trait (the phenotype).
 Complete penetrance – neurofibromatosis type 1
 Incomplete penetrance – familial breast cancer
due to BRCA1 gene mutations
Sex-Influenced or Limited Expression
 Some congenital anomalies and/or genetic

syndromes due to autosomal defects are more
easily recognized, or only recognized, in
individuals of a particular gender
 Sex influenced: Genital hypoplasia, hypospadias,
virilization with hypertrophy of the clitoris
 Sex limited: Hereditary prostate cancer
Elements of dysmorphology
Dysmorphology
❑recognition and study of birth defects (congenital

malformations) and syndromes [David Smith, 1960]
Gr. „dys” – abnormal, defective; „morph” – form
"As a medical subspecialty, dysmorphology deals with

people who have congenital abnormalities and with their
families. Whenever any physician is confronted by a
patient with a birth defect, he or she becomes,
for the moment at least, a dysmorphologist.„
(JM Aase: Diagnostic
Dysmorphology, 1990, Plenum, New York).
What does ‘dysmorphic’ mean?
• Children whose physical features are not usually
found in a child of the same age or ethnic
background („be aware of parental looks”)
• Some features are obvious dysmorphisms (e.g.
premature cranial suture fusions) whereas others
insignificant familial traits (e.g. finger syndactyly)
• Not only external variety, but also that of internal
organs
http://www.childrenshospital.org/conditions-and-
treatments/conditions/syndactyly/symptoms-and-causes
Dysmorphology in neonatology and
pediatrics
http://www.medicalnewstoday.com/a http://symptomscausestreatmentpreve
rticles/145554.php ntion.blogspot.com/2014/01/what-is-
turner-syndrome.html
http://www.forgottendiseases.org/ass https://www.hindawi.com/journals/c
ets/ rig/2012/247683/fig1/
Beckwith_Wiedemann_syndrome.ht
ml
Mild congenital anomalies
Hypertelorism/hypotelorism
Epicanthus
Simian crease
Mongoidalne/antymongoidalne
ustawienie Slanted palpebral
fissures
Ear tag, ear pit
Iris coloboma
Fifth finger clinodactyly
Finger syndactyly
Umbilical hernia
Supernumerary nipple
Hypospadia
Bifid uvula Conglomeration of mild anomalies = greater risk of
coexistent major anomaly
Tools in dysmorphology
 Anthropometric measurements
 Dysmorphology databases
 Phenomizer
 POSSUM (Pictures of Standard Syndromes and
Undiagnosed Malformations)
 London Dysmorphology Database (LDDB)
 Face2gene
http://www.fdna.com/hipaa-compliance-declaration/
Facial Gestalt
modelling
(eLife; 3: e02020)
Facial Gestalt (eLife; 3: e02020)
American Journal of Medical Genetics Part A
Copyright © 2014 Wiley Periodicals Inc.
Special Issue: Elements of Morphology: Standard

Terminology
January 2009
Volume 149A, Issue 1
Pages 1–127
Standardization of dysmorphy
terminology
Anatomic reference points
Allanson JE, Cunniff C, Hoyme HE, McGaughran J, Muenke M, Neri G. 2009.

Elements morphology: Standard of terminology for the head and face.
Am J Med Genet Part A 149A:6–28.
Abnormal skull shape
 Brachycephaly –
anterio-posterior
shortening of the skull
 Dolichocephaly –
increased AP
dimension
 Plagiocephaly – skull
assymetry
 Trigonocephaly
Facial dysmorphy
 Flat facial profile
 Coarse facial features

Coarse features in mucopolysaccharydoses
Choroba Morquio (MPS IV)
Choroby Hurlera (MPS IH)
Choroba Hunter (MPS II)
Choroba Sly (MPS VII)
Stowarzyszenie Chorych na MPS

Choroba Sanfilippo (MPS III) http://chorobyrzadkie.pl/?s=5
Choroba Maroteaux-Lamy (MPS VI)
Facial dysmorphy
 Frontal bossing
 Prominent glabella
Facial dysmorphy
 Micrognathia
 Retrognathia
Definition: Apparently reduced length
and width of the mandible when viewed
from the front but not from the side
Comments: This is a bundled term

comprising shortening and narrowing of
the mandible and chin. It is defined here
as it is a term in common usage.
Synonyms: Micrognathism; Jaw, small
Micrognathia
Definition: Excess skin around the neck, often
lying in horizontal folds.
Comments: With age and increased vertical
growth of the neck, excess nuchal skin may
disappear and the neck may become broad or
webbed. If the skin folds are vertical or
paravertical, the term Neck webbing should
be used.
Redundant nuchal skin
Webbed neck
Definition: A fixed reduction in the
vertical distance between the upper and
lower eyelids with short palpebral
fissures.
Comments: This term is based on Saal et
al. 1992. This is an acknowledged bundled
term, though the separate coding of the
components (palpebral fissure absence;
presence of eyelashes) was deemed
Blepharophimosis impractical. This is typically associated
with a rudimentary or small globe.
Frequently, a tuft of hair accompanies the
aberrant skin
Cryptophthalmos
Meeting of the medial eyebrows in the
midline.
Cosmetic hair removal or shaving may

obscure this feature. It is controversial
whether the medial eyebrows must
meet in the midline to warrant this
descriptor, as opposed to eyebrows
that extend markedly toward the
midline but do not meet.
Synophrys
Dysmorphy of the oral region
Thin upper lip
Tented upper lip
Wide mouth
„Cupid bow” mouth

Lip pits
Dysmorphy of ears
Crumpled ears
Attached earlobe Microtia
Cupped ear Upturned lobes Preauricular tag

Definition: Laterally protruding ear that
lacks antihelical folding (including
absence of inferior and superior crura)
Cupped ear
Finger anomalies
Brachydactyly
Finger syndactyly Clenched fist
Tapering fingers
The middle finger is more than 2 SD below the
mean for newborns 27–41 weeks EGA or below
the 3rd centile for children from birth to 16 years
of age AND the five digits retain their normal
length proportions relative to each (i.e., it is not
the case that the middle finger is the only
shortened digit)
This is an acknowledged bundled term as the

definition in most anthropometric sources
assumes that the other fingers are all as relatively
short as is the middle finger. As the determination
of the proportionality of the other four digits is
Short fingers clearly subjective, the term must be regarded as
subjective.
Definition: All digits held completely flexed
at the metacarpophalangeal and
interphalangeal joints
Comment: Is distinguished from

Camptodactyly, as that term may describe
fewer than five digits of a eudactylous hand
and does not involve the MCPJ. The digits
may overlap when they lie flexed in the
palm. It is not necessary to specify the
overlapping fingers finding separately.
Clenched hand
Genetic syndromes with dysmorphic
features
 Groupwork: report on frequency, clinical synopsis
including dysmorphic features, diagnosis,
surveillance
 Gr. A: Noonan syndrome
 Gr. B: Cornelia de Lange syndrome
 Gr. C: Achondroplasia
 Gr. D: Mowat-Wilson syndrome
 Gr. E: Rubinstein-Taybi syndrome
 Gr. F: Kabuki syndrome

Noonan syndrome
https://www.semanticscholar.org/paper/Noonan-syndrome.-Bhambhani-
Muenke/e0bc62192573261a39ac392c3bc887883dfb3149/figure/4
Noonan syndrome
 Frequency 1:1000 – 1:2500
 AD, RAS-MAPK genes: PTPN11, RAF1, KRAS, SOS1,
BRAF, NF1, NRAS
 Short stature, facial dysmorphy (hypertelorism,
downslanted palpebral fissures, ptosis, low-set
ears), cardiac defect (most frequently pulmonary
stenosis (PS)), webbed neck, pectus carinatum /
excavatum
 Clotting disorders
Cornelia de Lange syndrome
https://emedicine.medscape.com/article/942792-
overview
CdLS
 1:10 000 – 1: 200 000
 Gene mutations: NIPBL (50%), SMC1A (5%), SMC3
(1%), unknown etiology (40%)
 Facial dysmorphy: synophrys, long philtrum, wide-
spaced teeth
 Developmental delay, hyperactivity
 Hand/foot anomalies
Achondroplasia
Autosomal Dominant
Achondroplasia
 FGFR3 gene, AD
 Short stature, short limbs (particularly upper arms
and thighs), hyperlordosis, valgus knee, prominent
forehead, midface retrusion
 Normal intellectual development
Mowat Wilson syndrome
MWS
 ZEB2 gene
 Facial dysmorphy (hypertelorism, broad medial
eyebrows, uplifted earlobes, open-mouthed
expression, prominent or pointed chin)
 Moderate to severe ID, seizures
 Congenital anomalies: microphthalmia,
Hirschprung disease, hypospadias, agenesis of
corpus callosum, heart defects
Rubinstein Taybi syndrome
https://jmg.bmj.com/content/39/7/496
https://bjo.bmj.com/content/84/10/1177
RTS
 16p13.3 microdeletion, mutations in CREBBP or
EP300 genes
 Broad, angulated thumbs and toes, short stature,
facial dysmorphy (downslanted palpebral fissures,
beaked nose), ID of varying degrees
 Congenital heart defects, urinary tract
abnormalties, eye defects
Rubinstein Taybi syndrome
https://www.youtube.com/watch?v=rdVIzLogHY0
Kabuki syndrome
 Facial dysmorphy:
everted lower eyelid,
arcuate eyebrows, ear
malformations
 Skeletal abnormalities
 Mild / moderate ID
 Congenital heart
defect
Kabuki syndrome
 KMT2D (formerly MLL2) gene, KDM6A
jencastaneda@gmail.com
POSTNATAL
CYTOGENETICS
Jennifer Castaneda, MD, PhD
Review: chromosome structure
https://thenaturalhistorian.com/2013/02/24/genetic-distance-
humans-chimpanzees-divergence-baraminology/
Classification of chromosomal
aberrations
• Autosomal vs sex chromosome aberrations
• Abnormalities of chromosome number:
• Polyploidy
• Autosomal aneuploidy
• Sex chromosome aneuploidy
• Abnormalities of chromosome structure

• Translocations
• Deletions, microdeletions
• Duplications
• Inversions
• Ring chromosomes
Aneuploidy caused by meiotic
disjunction
http://study.com/academy/lesson/aneuploidy-definition-disorders-quiz.html
Chromosomal aberrations
•1 of 150 live births

•The first chromosomal aberrations
identified (1959):
Trisomy 21 – Down syndrome
XXY – Klinefelter syndrome
Monosomy X – Turner syndrome
Chromosomal aberrations -
symptomatology
• Frequent miscarriage
• Sex chromosome aberrations – short/tall stature,
ambiguous genitalia, infertility, primary amenorrhea,
delayed development of secondary sexual
characteristics
„chromosomal aberration phenotype”:
• Multiple congenital anomalies
• Developmental delay, mental retardation
• Dysmorphic features
Chromosome morphology
http://clinicalgate.com/chromosome-organization/
Cytogenetic analysis methods
• Classic cytogenetics - kariotype
• Molecular cytogenetics
- FISH (Fluorescence in situ hybridization)
- CGH (Comparative genomic
hybridization)
• MLPA (Multiplex ligase-dependent probe
amplification)
FISH
FISH
• Metaphase and interphase

• Detection of identified sequence
• Identification of chromosome
FISH ANALYSIS IN CHRONIC MYELOID LEUKEMIA
Philadelphia chromosome t(9;22)
F
R
G
Ph+
Normalny F
M-FISH, SKY-FISH
MLPA – Multiplex Ligase-Dependent
Probe Amplification
http://mlpa.com/WebForms/WebFormMain.aspx
Methylation-specific MLPA (MS-MLPA)
ME028 Prader-Willi/Angelman
22q11.2 microdeletion – Di George syndrome
http://www.scielo.br/scielo.php?script=sci_arttext&pid=S0066-782X2014002300004
Array CGH
http://www.nggthailand.com/array-comparative-genomic-hybridisation-acgh/
Source of cells for cytogenetic
analyses
•Lymphocytes
•Skin fibroblasts
•Bone marrow
•Amniotic fluid / umbilical blood
- prenatal diagnosis
•Cancer cells from pleural effusion
•Somatic cancer cells
Standard nomenclature
ISCN 2016 - International System for Human Cytogenic Nomenclature
Chromosome, arm, region, band

1q32.1
Chromosome 1, long arm, region 3, band 2, sub-band 1
ISCN symbols
+ Additional chromosome i Izochromosome
Absence of inv Inwersion

-
chromosome
mar marker
cen Centromere
mat Maternal origin
del Deletion
pat Paternal origin
der Derivative
t Translocation
dic Dicentric
? Doubtful or
dup Duplication questionable
OVERVIEW OF
CHROMOSOMAL
DISORDERS
„CHROMOSOME PHENOTYPE””
• developmental delay, behavioral disturbances

• congenital anomalies
• dysmorphic features
FREQUENCY
CZĘSTOŚĆ OFCHROMOSOMOWYCH
ABERRACJI
CHROMOSOMAL ABERRATIONS
• 0,9 % live births, abnormal phenotype in half of

them
• 23 – 34% ID + multiple congenital defects
• 50 - 60% 1st trimester miscarriages
• 6% fetal deaths
MICRODELETIONS/MICRODUPLICATIONS
ZESPOŁY PRZYLEGŁYCH GENÓW (ANG. CONTIGUOUS GENE SYNDROMES)
 Contiguous gene syndromes

 0,7 – 1/1000, mostly de novo, AD
 More severe phenotype effects in microdeletions
than in microduplications
 Microduplications: frequent familial occurence
Groupwork –
microdeletion/microduplication syndromes
On the basis of the given cytogenetic result:
- What is the diagnosis / syndrome?
- Frequency, Clinical symptoms, Health supervision
- Prognosis
Group A: 46,XY,del22q11.2
Group B: 46,XY, del7q11.23
Group C: 46,XY, del17p11.2
Group D: 46,XX, dup22q11.2
Group E: 46,XX, del(5p)
Group F: 46,XX,del22q13.3
www.philipcaruso-story.com
DiGEORGE syndrome
DiGeorge syndrome - Thymic aplasia and

right aortic arch - Anterior view. Neonatal
death by laryngeal atresia
William’s syndrome
http://odlarmed.com/?p=833
WILLIAM’s SYNDROME
• IUGR, postnatal hypotrophy, short stature
• feeding difficulties, gastro-esophageal reflux
• congenital heart defect: SVAS (75%), PPS; hypertension,
arrythmia,
sudden cardiac death, mitral valve prolapse in adults
• connective tissue symptoms: rough voice, inguinal hernia,
hypermobile joints, delicate, elastic skin
• idiopathic hypercalcemia (30%), hypercalciuria (15%)
• constipation
• behavioral phenotype – „cocktail party manner”, empathy,
overfriendly behavior , oversensitivity, anxiety, phobias
• facial dysmorphy
• average IQ or mild ID
• chronić ear infection
• ocular defects (50%)
• renal disorders – stenosis of renal arteries, kidney stones (5%)
SMITH – MAGENIS SYNDROME del 17p11.2
• brachycephaly
• prognatism
• wide face, deepset eyes
• short stature
• short, stubby fingers
• hoarse voice
• frequent ear infections
• hypercholesterolemia
• hypotonia in infancy
• sleep disturbances
• stereotype movements
• autoagression
• ID, severe speech delay
• peripheral neuropathy
16 lat
22q11.2 microduplication s.
Frequent symptoms:
Flat facial profile
Prominent ears
Ear tags
Schooling difficulties, global developmental
delay
Less frequently: Symdrome overlap with

DiGeorge s./VCFS
Palatal, laryngeal insufficiency
hypocalcemia
Autistic behavior Frequent familial cases
Pedigree analysis,
Clinical assessment of relatives
Microduplication syndromes
22q11.2 clinical features similar to microdeletion
(DiGeorge’a/VCFS)
7q11.23 severe speech delay

(WBSCR)
15q11-13 autism, speech delay

(PWS/AS)
Xq28 ID, speech defect, hypotonia, frequent infections

(Rett s.)
MECP2
17p11.2 Potocki – Lupski syndrome

(Smith Magenis s. –del17p11)
dup6q24 - 27 ID, obesity

cri du chat syndrome
• 46, XX, del 5p 46, XY, del 5p
• Round face in newborn, cat’s cry (disappears around age 2),
congenital heart defect, ID
CDC – CONTIGUOUS GENE SYNDROME
Cerruti Mainardi, Paola. (2006). Cri du Chat syndrome. Orphanet journal of

rare diseases. 1. 33. 10.1186/1750-1172-1-33.
PHELAN-MCDERMID SYNDROME
– DEL 22Q13.3
 Developmental delay,
autistic behavior,
increased pain
threshold
 Hypotonia
 Dysmorphic features:
dolichocephaly, long
eyelashes, dysplastic
ears, rounded nose tip,
pointed chin, large
Koolen A, et al (2005). Molecular characterisation of patients with subtelomeric 22q
hands, dysplastic nails
abnormalities using chromosome specific array-based comparative genomic hybridisation.
European journal of human genetics : EJHG. 13. 1019-24. 10.1038/sj.ejhg.5201456.
Phelan, Katy & McDermid, Heather. (2012). The 22q13.3 Deletion Syndrome
(Phelan-McDermid Syndrome). Molecular syndromology. 2. 186-201.
10.1159/000334260.
PMS – SHANK3 GENE
 22q13.3 deletion
(80-85%)
 Ring chromosome
 Unbalanced
translocation
(15-20%)
Phelan, Katy & McDermid, Heather. (2012). The 22q13.3 Deletion Syndrome
(Phelan-McDermid Syndrome). Molecular syndromology. 2. 186-201.
10.1159/000334260.
BECKWITH – WIEDEMANN
syndrome (BWS)
• Duplication / Deletion of 11p15 (IGF2 gene)
• macroglossia, omphalocoele, high birth weight, ear
pits, hemihyperplasia
• hypoglycemia in neonatal period (could cause
developmental delay), high tumor risk (Wilms tumor,
hepatoblastoma, rhabdomyosarcoma, adrenal
tumor)
WOLF-HIRSCHHORN SYNDROME
• del 4p
• Hypertelorism, prominent glabella, wide nose („greek helmet face”),
thick lower lip, iris coloboma, cleft palate, ID
Williams https://www.youtube.com/watch?v=BlexMO
syndrome
https://www.youtube.com/watch?v=BlexMOZCSVQ
ZCSVQ
SUBTELOMERIC
REARRANGEMENTS
TELOMERES
 Protect chromosomes from deterioration during cell

replication
 Chromosomal organization in the nucleus
 Sequence: TTAGGG
https://www.yourgenome.org/facts/what-is-a-telomere
https://en.wikipedia.org/wiki/Subtelomere
SUBTELOMERIC REGION
http://atlasgeneticsoncology.org/Deep/SubTelomereID20025.html
WHAT WE KNOW ON SUBTELOMERES
 Dynamic patchworks of multichromosomal blocks

 Homologous sequences prone to rearrangements
 Subtelomeres include encoding regions
SUBTELOMERIC REARRANGEMENTS
 1p36 deletion
 Fascioscapulohumeral muscular dystrophy (FSHD)
 Wolf-Hirschhorn syndrome (4p-)
 Cri-du-chat syndrome (5p-)
 9q34 deletion
 Miller-Dieker syndrome
 Phelan-McDermid syndrome
SUBTELOMERIC REARRANGEMENTS
 5,1% of children with ID – second most

frequent cause next to Down syndrome
 7,4% of children with moderate or severe ID
 0,5% of children with mild ID
1p36 deletion
Symptoms (~100%)
Severe ID
Absent speech
Hypotonia
Facial dysmorphy
Gait disturbances
Abnormal behavior
Frequent (50 - 80%)

Growth delay
Heart defect
Epilepsy
Deafness
Less frequent (<50%)

Cardiomyopathy
Cleft lip / palate
hypothyroidism
Obesity
Prominent forehead, deepset eyes, flat nose,
pointed chin, horizontal eyebrows
WWW.RARECHROMO.ORG
FACIO-SCAPULO-HUMERAL MUSCULAR
DYSTROPHY (FSHD) – DEL4Q35
 Third most frequent muscular dystrophy, after
DMD/BMD, myotonic dystrophy type I
 Muscle weakness of face, shoulder girdle
 Lower limbs rarely affected
 Hyperlordosis
 Deafness (60% of patients)
 Cardiologic complications - RBBB
https://www.fshsociety.org/facioscapulohumeral-description/
FSHD - DIAGNOSIS
 Typical clinical features

 CK – normal or slightly elevated
 EMG – nonspefic myopathic changes
 Muscle biopsy – dystrophic changes
 MR – evaluation of affected muscles
MILLER-DIEKER SYNDROME – DEL 17P13.3
 Lissencephaly
 Severe ID
 Hypotonia https://prezi.com/hyphafzgrzel/miller-dieker-syndrom/
 Epilepsy before 6 mos

 Microcephaly
 Growth delay
http://www.cytocell.com/probes/123-smithmagenis-
fliimillerdieker-probe-combination
MDS – DYSMORPHIC FEATURES
 Prominent forehead
 Midface hypoplasia
 Short nose
 Lowset, dysplastic ears
 Micrognathia
https://www.ncbi.nlm.nih.gov/books/NBK5189/figure/chrom
17-lis.F2/?report=objectonly
GENES AND SYNDROMES - LISSENCEPHALY
https://www.peds.ufl.edu/divisions/genetics/teaching/brain_malformations
/lissencephalies.htm
GENETIC COUNSELLING
 Estimation of family genetic risks

 Referral to specialists
 Regular visits (new diagnostic tests,
surveillance indications)
 Genetic counselling upon coming of age
 Information on suport groups, family
networks, associations
Dynamic mutations
in neurodegenerative
disorders
JENNIFER CASTANEDA, MD, PHD
Dynamic mutation
 The process by which certain naturally occuring

polymorphic DNA repeat sequences expand
and result in human disease or fragile sites on
chromosomes.
 1-6 nucleotides
 5-100 repeats in a single
locus
Microsatellite  Polymorphic sequence
repeats  Low mutation frequency in
somatic and germline cells
Trinucleotide sequences and disorders
Trinucleotide Location Threshold limits Disorder

CGG 5’ UT 50-200 Fragile X syndrome (FMR1 gene)
GAA Intron 26-50 Friedreich ataxia
CAG (PolyGlu) Exon 29-35 Huntington disease
CAG Exon 6-39 (SCA1) Spinocerebellar atrophy (SCA1,2,3,6,7,17)
CAG Exon 9-36 Spinal bulbar muscular atrophy
(Kennedy’s disease)
CTG 3’ UT 40-250 Myotonic dystrophy 1 (DM1 gene)
https://www.nature.com/articles/nrg.2017.115
https://www.sciencedirect.com/science/article/pii/S0959437X14000756
Anticipation
 Signs and symptoms
become more severe
and appear at an
earlier age as the
disorder is passed from
one generation to the
next.
 Mechanism:
trinucleotide repeat
expansion
 Myotonic dystrophy
pedigree
http://hihg.med.miami.edu/code/http/modules/education/Design/Print.asp
Trinucleotide repeat disorders
 Report on frequency, clinical symptoms, disease mechanism,

health supervision
 Gr. D: Fragile X syndrome, FXTAS

 Gr. E: Huntington disease
 Gr. F: Spinal bulbar muscular atrophy
 Gr. G: Friedreich ataxia
Fragile X syndrome, FXTAS
 Mild to moderate
intellectual disability
 Macrocephaly, long,
narrow face, large ears,
macrorchidism
 Autistic features,
hyperactivity
https://pedclerk.bsd.uchicago.edu/page/fragile-x-syndrome
https://www.researchgate.net/publication/264903258_Mouse_models_of_the_fragile_X_premut
ation_and_fragile_X-associated_tremorataxia_syndrome/figures?lo=1
FraX: Premutation
carriers
 Females:
premature
ovarian failure
(POF), depression
 Males: FXTAS –
tremors, ataxia,
parkinsonism,
autonomic
dysfunction
 HTT gene mutations (protein
huntingtin) – CAG trinucleotide
repeat
 Symptoms: uncontrolled
movements (chorea), cognitive
and emotional problems
Huntington  Adult onset: 30s, 40s. Survival 15-
disease 20 years after onset
 Juvenile form: slow movements,
clumsiness, slurred speech,
decline of school performance,
seizures (30-50%), survival 10-15
yrs after onset of symptoms
http://hihg.med.miami.edu/code/http/modules/education/Design/Print.asp
Spinal bulbar muscular
atrophy
 1:40000 men
 CAG repeats in exon 1 of gene AR (androgen
receptor gene)
 Normal range: 9-36; disease allele: 38-72
 X-linked recessive
 Age of onset: 30-50 yrs
 Early symptoms: tremor, muscle cramps, and
muscle twitching, followed by progressive muscle
weakness and wasting. Dysarthria, dysphonia.
 Other symptoms: gynecomastia, testicular
atrophy and reduced fertility as a result of mild
androgen insensitivity
https://www.mda.org/disease/spinal-bulbar-muscular-atrophy
Friedreich ataxia
 FXN gene (frataxin protein)
 Autosomal recessive
 GAA trinucleotide repeat:
normal range - 5-33, disease
allele – 66-1300
 Gait ataxia, spasticity,
dysarthria
 Hypertrophic cardiomyopathy,
diabetes, impaired
vision, hearing loss, scoliosis https://jamanetwork.com/journals/jamaneurology/fullarticle/796230
Spinocerebellar ataxia
 Frequency 3:100,000
 45 types (OMIM)
 uncoordinated walk (gait), poor hand-
eye coordination, abnormal speech
(dysarthria)
 Progressive, patients require
wheelchair 10-15 yrs after onset of
symptoms
https://ataxia.org/wp-content/uploads/2017/07/SCA-
 Selective neuron loss, intranuclear Making_an_Informed_Choice_About_Genetic_Testing.pdf
inclusions
SCA1 – ATNX1 gene
 Chromosome 6
 Trinucleotide CAG repeats:
 Normal: 6-39, Disease allele:
41-81
 Excessive production of
Ataxin-1 protein causes SCA1
https://en.wikipedia.org/wiki/Ataxin_1
Myotonic dystrophy type 1
 Mild DM1: cataract, mild myotonia, normal life span

 Classic DM1: muscle weakness and wasting,
myotonia, cataract, cardiac conduction
abnormalities, shortened life span
 Congenital DM1: hypotonia, severe generalized
weakness at birth, respiratory insufficiency and
early death, ID
 CTG trinucleotide repeat in the DMPK gene
 Normal: 5-34; Premutation: 35-49; Disease: >50
Intellectual impairment
(II, ID, MR, learning/intellectual
disability/difficulties)
Krzysztof Szczałuba MD PhD
2019
Definition
According to AAMR*:
A. Full-scale Wechsler Intelligence Quotient (IQ) of less than 70
(based on individual psychological assessment)
B. Abnormalities in at least three areas: communicating skills, self-dependence,
social/interpersonal skills, public goods usage, life attitudes, education,
rest/relaxation, health and safety
C. Childhood onset
1. only A
2. A and B
3. A, B and C
*American Association on Mental Retardation. Mental Retardation: Definition, Classification and Systems of Supports,
10th ed, Washington, 2002
Adult individuals with ID?
1. Congenital anomalies syndromes + ID n=26

2. Collagenopathies/Connective tissue disorders n=18
3. Chromosome aberrations n=12
4. ID only n=12
5. Endocrine/Metabolic disorders n=8
6. Bone dysplasias/dysostoses/dysgeneses n=6
7. Others n=6
ID at least 50 of 88
Adults with ID?
I:3 I:4 I:1 I:2
II:4 II:5 II:1 II:2 II:3
III:1 III:2 III:3 III:4
oligophrenia,
encephalopathy, 5p-
II-5 embriopathy,
microcephaly
ID – a clinical symptom
DIVISION BASIS
Borderline IQ 70-85
Mild IQ 50-70
ENVIRONMENTAL
Moderate IQ 35-50
Severe IQ 20-35 GENETIC
Profound IQ<20
INCIDENCE IN PL:
Wald, Zaremba: 0,34% (for more profound ID – incidence
measured for age 7-14yrs)
WORLD: 0,3% for more severe end – 7% for milder end
ID – classification (chosen)
• Constitutional ID: 3%, IQ usually >50, ‘school type’, no CNS

changes, important socioeconomic status
• Pathological ID: 0,3%, IQ usually <50, ‘preschool type’, CNS
changes, no family influence
• According to the causative factor timing:

• prenatal (genetic [1/3], mother’s illness, infections, malnutrition,
dystrophy);
• perinatal (SGA, infections, hypoxia, intracranial hemorrhage);
• postnatal (trauma, infections)
Mild ID– causes (n=439, Bundey JMG 1989)
DS 5,7%
FRAX 4,6%
Other chromosome aberrations 0,6%*
CP, other neonatal disorders, sequelae 2,7%
of intrauterine infections
Postnatal trauma 3,2%
Congenital anomaly and genetic 3,4%**
syndromes
Unknown 80%
* currently likely +5-17% for the whole ID group (telomere screening, CGH, arrayCGH)
** +10-20% after Whole-exome or Whole-genome techniques are applied
Severe ID – causes (Curry AJMG 1997)
Chromosome aberrations 4-28%*

FRAX 2-6%
CNS anomalies 7-17%
Environmental factors, prematurity 5-13%
Congenital anomaly and genetic 10-20%**
syndromes
Unknown 30-50%
* currently likely +5-17% for the whole ID group (telomere screening, CGH, arrayCGH)
** +10-20% after Whole-exome or Whole-genome techniques are applied
ID – chromosome aberrations
mos 45,X/46,X,r(X)
likely clinical picture with ID of variable degree
Down syndrome
• Locus (loci): 21q22.1-q22.2
• Inheritance: 95% de novo, 2% translocation event (50%
familial)
• Clinics: moderate ID, hypotonia, physical delay, strabismus,
cataracts in adulthood, myopia, conductive HL, large tongue,
weak dentition, joint laxity, hypogenitalism, heart defect,
duodenal atresia, Hirschsprung disease, thyroid disorders, early-
onset Alzheimer, ALL
• Diagnostics: PRENATAL: US: NT + NB 80-85%, screening:
 free βHCG, PAPP-A, karyotype from amniocytes, cell-free
fetal DNA 99,7% POSTNATAL: karyotype
Patau syndrome
• Inheritance: 90% de novo, 5-20% translocation event
• Clinics: rarest trisomy in liveborn, holoprosencephaly,
polydactyly, seizures, HL, maicrocephaly, cleft lip and/or
palate, omphalocele, heart and kidney anomalies, ID. In
mosaics: clinial heterogeneity: from typical to milder ID
degree and longer survival
• Diagnostics: PRENATAL: US+biochemical screening,
amnio- karyotype, cell-free fetal DNA 80%,
POSTNATAL: MRI, EEG, audiogram, echocardiogram,
kidney US, karyotype
• 44% does not survive to 1mth, >70% die in the first year.
In others severe ID.
Edwards syndrome
• Inheritance: <1% translocation event
• Clinics: clenched hand, fingers 2&5 overlap 3&4,
IUGR, rocker-bottom feet, micrognathia, prominent
occiput, microphthalmia, VSD, ASD, PDA, kidney
anomalies, ID. In mosaics usually milder picture (to
normal IQ !!)
• Diagnostics: echocardiogram, abdominal US.
PRENATAL screening:  AFP, free βhCG and uE3,
amnio- karyotype, cell-free fetal DNA >99%
POSTNATAL: karyotype
• 50% does not survive to 1mth, 90% die before first
birthday
Cat cry syndrome (5p-)
• Gene and protein: unknown
• Locus: 5p15.2
• Inheritance: 12% translocation event or pericentric inversion in one of
the parents, 85% de novo (80% paternal chromosome)
• Clinics: cat-like cry (abnormal larynx), microcephaly, ID, hypotonia,
strabismus, facial dysmorphism, cat-like cry only when deletion
5p15.32
• Diagnostics: most aberrations identified on routine karyotyping, others
in microdeletion MLPA or CGH (submicroscopic)
• 62 pts with deletions from 5p15.1 to 5p13 showed no correlation with
severity of ID [Cerruti Mainardi, JMG 2001]
Wolf-Hirschhorn syndrome (4p-)
• Genes: WHSCR: WHSC1 and WHSC2 (unknown
function)
• Locus: 4p; critical region165kb
• Inheritance: 87% de novo, 13% translocation event
• Clinics: „Greek helmet” face, microcephaly, pre-
and postnatal short stature, variable degree ID,
seizures, facial asymmetry, ptosis, CNS anomalies,
cleft lip and/or palate, heart defect, renal anomalies
• Diagnostics: EEG, brain MRI, echocardiogram, IgA
level, karyotype HRT 4p16.3 (60-70%),
MLPA/FISH/CGH (>95%)
• Treatment: in 2/3 absence seizures responsive to
valproic acid
ID – chromosome aberrations
What is the most likely diagnosis in a 6yo with

CL/P, TOF, height at 3rd centile for age and ID?
A. Down syndrome
B. Trisomies 13 or 18
C. Velocardiofacial syndrome
D. Otopalatodigital syndrome
E. Cardiofaciocutaneous syndrome
ID in submicroscopic
aberrations – del22q11.2
• Genes: UFDIL, TBX1, VEGF
• Locus: 22q11.2
• Inheritance: 93% de novo
• Clinics: CHD (74%) (esp. TOF, interrupted aortic arch, conotruncal
defects), immunological deficits, palatal abnormalities (69%), feeing
dufficulties, psychomotor retardation, learning difficulties (70-90%),
hypocalcemia (50%), renal abnormalities (37%), psychiatric issues
• Diagnostics: Ca, PTH, lymphocytes T/B , Igs, renal US
videolaryngoscopy, MLPA/FISH/CGH DGSCR (95%)(3 Mb /1,5 Mb)
• Mechanism: branchial arches 3&4 defect
• Treatment: heart defect correction, palatal surgery, Ca
supplementation, in case of an immunological deficit do not administer
live vaccinations
ID in submicroscopic aberrations– del7q11.23
(Williams syndrome)
• Genes: ELN , LIMK
• Locus: 7q11.23
• Inheritance: mostly de novo
• Clinics: arterial stenosis, supravalvular aortic stenosis (75%),
facial dysmorphism, snorring, hernias, joint movement
constriction or joint laxity, ID, specific behaviour,
hypercalcemia, hypercalciuria, hypothyroidism, abnormal
growth in infancy
• Diagnostics: calcium, creatinine, thyroid hormones, audilogic
and ophthalmologic assessment, renal US, echocardiogram,
MLPA/FISH/CGH WBSCR (~99%)
• Mechanism: ELN deletion causes vascular problems, LIMK
deletion causes visuo-spatial coordination and cognitive deficits
• Treatment: adults with bicuspid valve problems, heart
insufficiency, HL, hypothyroidism, diabetes
ID in submicroscopic aberrations – Angelman
syndrome
• Gene: UBE3A
• Protein: ubiquitin E3A ligase
• Locus: 15q11-q13
• Inheritance: functional or total lack of maternally
inherited imprinted allele 15q11.2-q13 AS/PWSCR
• Clinics: severe ID, severe speech delay, ataxia, specific
behaviour, microcephaly, seizures
• Diagnostics: secondary microcephaly, seizures before 4th
birthday, abnormal EEG (high amplitude, slow 2-3Mhz
waves), MS-PCR, methylation MLPA, maternal allele
deletion (4-6 Mb) (65-75%), UBE3A mutations (10-20%),
imprinting center defect (2,5%), paternal UPD (<5%),
unknown
ID in submicroscopic aberrations –Prader-Willi
syndrome
• Genes: SNURF-SNRPN, MKRN3, MAGEL2, NDN

• Locus: 15q11-13
• Inheritance: functional or total lack of paternally inherited
imprinted allele 15q11.2-q13 AS/PWSCR
• Clinics: hypothalamic dysfunction, neonatal hypotonia,
psychomotor delay, hyperphagia and obesity, short stature,
small feet and hands, hypogonadism, ID
• Diagnostics: MS-PCR, methylation MLPA, paternal deletion
3-5 Mb (~70%), maternal UPD (~30%), imprinting center
defect (2%)
• Treatment: GH+diet, beware of feeding problems in
infancy, obesity, OCD, psychoses, scoliosis, diabetes,
osteopenia
ID in submicroscopic aberrations –Rubinstein-Taybi
syndrome
• Genes: CREBBP, EP300
• Locus: 16p13.3, 22q13
• Inheritance: de novo
• Clinics: microcephaly, beaked nose, wide thumbs and big toes,
cryptorchidism, growth delay, severe ID, heart defect, strabismus, ptosis,
tumours (meningioma, pilomatrixoma, leukemia), behaviour problems
• Diagnostics: echocardiogram, MLPA/FISH/CGH CREBBP (~10%),
CREBBP seq (40-60%), EP300 seq (~3%)
• Mechanism: defective histone acetylation
• Treatment: eyesight, hearing, heart defect
Examples of ID in monogenic syndromes
Inborn errors of metabolism: untreated/maltreated

phenylketonuria, galactosemia (speech delay), glutaric
acidemia type I, biotinidase deficit, creatine deficits,
homocystinuria, storage disorders
Can ID be a symptom of monogenic disorders

typically not showing any cognitive problems?
Practically in all cases where microdeletion or

less frequently microduplication of that
particular region is present
ID in monogenic conditions – Tuberous Sclerosis
(TS)
• Genes: TSC1 and TSC2
• Proteins: Hamartin and Tuberin
• Loci: 9q34, 16p13
• Inheritance: 2/3 de novo
• Clinics: hypomelanotic foci, facial angiofibroma, shagreen patches,
periungual fibromas, subependymal tumours, cortical tubers,
astrocytoma, seizures, renal angiomyolipoma, renal epithelial cysts,
<1% cancer risk, cardiac rhabdomyoma (tendency to regress), lung
lymphangiomatosis (TSC2, women 20-40yo), ocular hamartomas;
microdeletion syndrome TSC2/ADPKD1
• Diagnostics: MRI, echocardiogram, renal US, Wood’s lamp, EEG,
TSC1 seq (30% familial) and TSC2 seq (50% familial)
• Mechanism: abnormal activity of tumour suppressor protein
• Treatment: renal US every 1-3yrs, may use CT/MRI (prophylactic
renal artery embolization), thoracic CT if lung symptoms
Seizures and learning difficulties in TS
Dev Child Neurol 1996; 38: 146
ID normal
Seizure onset
<6 mths 41 7
6-24 mths 20 6
2-5 yrs 6 6
>5 yrs 0 13
Seizure type
infantile 33 2
tonic-clonic, absence, partial 18 17
febrile 2 5
Seizure control
insufficient 41 11
good 11 11
ID in monogenic conditions – what disorder is it?
75
I:3 I:4 I:1 I:2

drzenia, zab. równow
50 41 39
II:6 II:5 II:1 II:2 II:3 II:4

POF POF? POF?
20 25
III:3 III:4 III:5 III:1 III:2

nadpobudliwosc ADHD?
POF – premature ovarian failure

Fragile X syndrome (FRAX)
• Gene: FMR-1
• Protein: FMRP
• Locus: Xq27.3
• Inheritance: trinucleotide repeat expansion
• Clinics: developmental delay, ID (moderate/severe in boys, milder degree
in girls), hyperactivity, autistic traits, premutation female carriers: OCD,
depression, 20% POF, premutation male carriers: intention tremour, ataxia,
parkinsonism, autonomic dysfunction (=FXTAS: >30% male carriers and
<5% female carriers; 1,5% ♂ and 3%♀ late-onset ataxia;
1/3000 ♂ two other loci: FraXE: only ID, FraXF: lack of phenotype
• Diagnostics: CGG triplet detection PCR: fast testing, small premutations;
Southern: all mutation classes + normal alleles, mosaics, costly. Normal
allele: 5-44 repeats, median: 45-58 repeats (grey zone), premutation: 59-
200 repeats, mutation: >200 repeats
• Mechanism: >200 repeats = methylation = inactivation = lack of FMRP.
POF and FXTAS (59-200 repeats) gain of function mutation (?)
ID – Fragile X syndrome
The best diagnostic test is:
1. quantitative PCR
2. ASO
large expansions +
3. Southern
methylation status
The likelihood that the healthy mother of an affected son has

normal number of triplet repeats in both alleles is:
1. 50%
2. 25%
3. 10%
4. 5%
5. 0%
Genetic basis of ID
1. Chromosomal aberrations (the most frequent?)
2. Single gene defects
Hypothesis 1
Genes coding for various aspects of cognitive functions are common within the
genome
Hypothesis 2
More such genes are localized on X chromosome than on any other comparable
segment of an autosome
easier identification of hemizygous genes

affected males/ affected females = 1,3/1
X-linked ID (XLMR)
1. XLMR in syndromes (MRXS: Syndromic X-linked Mental Retardation)

2. XLMR non-specific (MRX or NS-XLMR: Non-Specific X-linked Mental
Retardation)
MRXS MRX
?
syndromic association of clinical ID is the only
features including ID and specific characteristic sign
symptoms on clinical exam
Syndromic XLMR - Coffin-Lowry syndrome
• Gene: RPS6KA3
• Locus: Xp22.2-p22.1
• Clinics: severe.profound ID, short soft hands, small
fingertips, short stature, microcephaly, characteristic facial
dysmorphism, normal IQ to severe ID in female carriers
• Diagnostics: X-ray: bone enlargement, abnormal vertebra,
metacarpal pseudoepiphyses, RPS6KA3 seq (35-40%)
• Mechanism: RPS6KA3 is a RAS-MAPK cascade protein
• Treatment: Risperidone in behavioural abnormalities,
echocardiogram
Syndromic XLMR – other phenotypes
1. Autism - NLGN3/4 mutations

2. Cerebellar ataxia – oligophrenin gene mutations
3. High T3- SLC16A2 mutations
4. Dystonia - ARX
Syndromic XLMR – locus heterogeneity
5. Microcephaly - ATRX, MECP2, PQBP1, SMCX

6. Short stature - PQBP1, SMCX
7. Spastic paraplegia - SLC16A2, ATRX, SMCX, MECP2
8. Epilepsy - AGTR2, SYN1, ATRX, SLC6A8, ARX, SMCX
and others
Syndromic XLMR – clinical heterogeneity
dupMECP2 (severe MECP2

ID, infections)
PPM-X (psychosis,
neonatal
pyramidal signs, ID)
encephalopathy Rett syndrome
ARX
myoclonic
epilepsy with West Partington lissencephaly and
spasticity syndrome syndrome ambiguous genitalia
MRX
Same single gene defects lead both to MRXS and

MRX phenotypes
Genetic basis of non-specific X-linked ID (MRX)
FMR2
IL1RAPL1 ARHGEF6
MRX PAK3
TM4SF2
ZNF41
FACL4
DLG3
FTSJ1
GDI J Med Genet 2005
XLMR – diagnostic dilemma
The clinical phenotype in institutionalised adult males with X-linked mental
retardation (XLMR)
Annales de Genetique 44, 2001
436 males
22 males with XLMR
4 = MRX
16 = FRAX 2 = Lujan-Fryns
syndrome
Modern diagnostic techniques
Technique Sensitivity
• FISH >0.04-0.25Mb
• MLPA about 0.04Mb
• HR-CGH >3Mb
• aCGH with BAC probes >1Mb
• aCGH with oligo- probes >0.001Mb
• New Generation Technology unlimited!
/Next Generation Technology (NGS, WGS/WES)
Mb – one milion base pairs

aCGH
Test-DNA Reference-DNA
Patient DNA Cot-1 DNA Control DNA

CGH technique
• Hybridization of two genomic DNAs, reference and
patient’s, fluorescein-stained and mixed 1:1, to normal
chormosomes
patient control spot Cy3 : Cy5

1.0 : 1.0
2 copies 2 copies
0.5 : 1.0
1 copy 2 copies
1.5 : 1.0
3 copies 2 copies
Science 1992; 258: 818-821
aCGH normal Agilent180K
aCGH abnormal
del 10q24.32 ≈317kb
CREBBP
Rubinstein-
Taybi
syndrome Exon 27
(RTS)
EP300
dev delay,
RTS-like
Exons 24-27
FAM58A
multiple
Exon 5
congenital
anomalies
(STAR
syndrome) PTEN Exons 3-5
Cowden
syndrome
Advantages of CGH
• enables identification of genomic copy-
number variants without prior knowledge
(suspicion) of their existence
• analyses the genome in a single run
• array CGH (aCGH): identification of
variants not seen in a standard cytogenetic
analysis (submicroscopic aberrations) with
an unprecedented resolution!
aCGH – clinical validation
• Metaanalysis of 14,000 pts with ID, congenital

anomalies and normal karyotype results: 10%
• (IMiD) in 116 pts with ID and dysmorphism: 11.8%
• aCGH with subtelomeric probes:

1. normal karyotype: 3%
2. abnormal karyotype: 43%
Genet Med 2009; 11: 139-146
Am J Med. Genet 2008; 146: 2361-2369
Am J Med Genet 2008; 146: 2242-2251
Modern diagnostic techniques
Technique Sensitivity
• FISH >0.04-0.25Mb
• MLPA about 0.04Mb
• aCGH with oligo- probes >0.001Mb
• New Generation Technology unlimited!
/Next Generation Technology (NGS, WGS/WES)
Mb – one milion base pairs

Next (new) Generation Sequencing
(NGS) = Massive Parallel Sequencing
(MPS)
NGS = Massive Parallel Sequencing
1. Library creation – random DNA fragmentation, ligation with
linkers
2. Library amplification
3. Direct step-by-step detection of each nucleotide added to seq
reaction
4. Hundreds to hudreds of milions reactions in a single run –
massive parallel seq
- shorter-fragment reads compared to Sanger seq
- digital reads
- paired-end seq, from both ends in parallel reactions
Whole
Target sequence genome
All coding
sequences
(exome)
Chosen
fragments
Number of pts
NGS in medical genetics
Target gene sequencing Exome sequencing
• Known regions/genes • All exons – (only coding

responsible for the sequences) – unknown
disorders genetic basis
- Candidate genes chosen - Monogenic disorders -
based on their putative (dominant and recessive)
role in disease - Multifactorial diseases
pathomechanism - Mitochondrial disorders
NGS in medical genetics
Target gene sequencing Exome sequencing
• Requires selective • Commercially available

„enrichment” „enrichment kits”
• Relatively easy • Much more challenging
interpretation interpretation
• Identification of larger • Structural variants –
structural variants – if suboptimal detection
present in the genome • Still relatively expensive
• Good value for money
NGS – clinical validation
• deLigt et al.: 21,000 genes (exome seq) in 100 pts

with severe ID + so called confirmation series of 765
pts with ID (high-throughput reseq): 16%
• all the muts: de novo, incl: 10 in AD genes, 3 in XLR
genes and 3 in novel genes
• + 19 muts in genes functionally associated with ID
N Engl J Med 2012

Exome sequencing in ID
• ID and WES [Topper, 2010]: in syndromes, sporadic

nonsyndromic, familial
• Rabbani, 2014: review of WES in syndromic ID (30
studies in 20 syndromes)
• Genotype-first: Classen, 2013; Rauch, 2012 (51 trios
with IQ<60): 1.41 de novo mutations/patient
• Schuurs, 2013: familial cases (recessive variants?) –
19 families of 2-5 individuals with ID: 9 pathogenic
mutations
Genome sequencing in ID
• Gilissen, 2013: 21/50 patients with IQ<50 (20

dominant de novo variants and one compound
heterozygote
• What was the selection process like?
1. aCGH: 12%
2. WES: 27%
3. WGS: 60%
NGS panels in ID
Greenwood Genetic Center XLID GeneDx XLID
114 genes >20x Clinician’s decision
$5500 From $3000
Lack of promoter and 3’UTR Lack of assessment of del/dup
assessment
Lack of assessment of trinucleotide Lack of assessment of trinucleotide
expansions expansions
What is the coding sequence coverage Exome slice possible

with median coverage min. 20x?
TAT TAT
Exome slice –exome seq + analysis of genes linked with

phenotype only (choice of genes on customer’s wish)
Diagnostic algorithm in ID
• Careful analysis of three-generation pedigree with detailed
developmental analysis of all the relatives
• Maternal health status assessment from pre-pregnancy era
• Pregnancy-stage assessment
• Birth physical parameters
• Developmental milestones (incl physical development)
• Exclusion of phenylketonuria and hypothyroidism
• IQ and psychological assessment + school progress
• Physical exam with careful assessment of dysmorphism
and neurological symptoms
• Karyotype and/or Fragile X where necessary
• Array CGH / NGS for the rest
• Head MRI (when neurological symptoms/abnormal head
circumference)
• EEG (when seizures) Neurology 2003; 60: 367-380 and further works of
• Metabolic screen Shevell et al.
Treatment of ID
„A child with ID requires a constant and frequently

multidisciplinary care. The choice of therapy depends on
etiological factors causative of cognitive problems as well as an
individual developmental progress of the child.”
1. Optimal development and prevention of developmental regression (stimulation level
depending on the kid’s skills)
2. Medical, psychological and schooling interventions
3. Social care (incl financial issues)
4. Pharmacotherapy only as a supportive tool
5. Early intervention centers, integrative pre-schooling, special groups in mass and
special schools, mass schools with individual therapeutic programs, integrative
classes, special classes in mass schools, classes for kids with severe ID
ID – genetic counselling
ID risk in sibs of the affecteds:
- relative to the level of ID (aberrtion?, monogenic syndrome?)
- when unknown cause, careful estimates: 1-25%
(multifactorial  AR) – beware of MRX families!
- a child with unknown-cause ID = 10x risk for sibs
Greenwood Genetic Center:

- 452 pts with mean IQ=41 and mean head circumference 43c with
idiopathic ID
- next sib: 502 ♂ and 468 ♀
- 21% brothers and 14% sisters with ID
- the higher the IQ, the higher risk in sibs
Taking the Challenge: Finding Recurrence Risks in Idiopathic Mental Retardation. J.S. Collins, A.F.
Nave, G.A. Satten, R.E. Stevenson. Greenwood Genetic Center, Greenwood, S.C., 2002
ID - summary
• A clinical symptom , of genetic disease as well,

which can be the only trait or be part of a syndrome
of congenital anomalies
• The most frequent genetic causes of ID are
chromosome aberrations (at various levels of
detection) and single gene defects. Gene dosage
effect.
• Care for individuals with ID is constant and
multidisciplinary
Mitochondrial Disease
2019
Fishing handles in diagnostics
• Non-specific ID: clinics and genetics
• Mucopolysaccharidoses: clinics,
biochemistry and genetics
• Mitochondrial disorders: clinics,
biochemistry, diagnostic imaging,
histhopathology/histochemistry and
genetics
• Validation of diagnostic methods is critical
Outline
• Characteristics of mitochondrial disorders
• Four main diagnostic levels
• Application of molecular genetics
• Diagnostic algorithm
EXPLANATIONS
NGS – Next-Generation Sequencing
WES – Exome Sequencing (only coding sequences)
NGS panels– chosen and known genes diagnostically relevant
Quiz
Which of the clinical/pedigree scenarios below reflects a
possible mitochondrial disorder?
A. A woman with muscle weakness says that the same problem is present
in one of her sisters and both her brothers. It looks like the disease was
inherited from the maternal side. Pedigree analysis reveals many
mother’s relatives with similar problems, but surprisingly none of the
affected men transmitted the trait to any od their children or
grandchildren.
B. Muscle weakness is present in a brother and a sister. Additionally, in the
sister there is also sensorineuaral deafness. Aside from that the
pedigree is irrelevant.
C. Nystagmus in a 3yo. His brother and two sisters do not show any similar
signs. Pedigree is not relevant.
1. only A
2. A i B
3. all
Case scenario
Proband: del/dup 15q?
2x: lactic acid↑ dup 16p?
MRS planned SNV?
mitochondr?
Genetic defects in
mitochondrial
disease:
¼ mtDNA
¾ nuclear DNA
Clinical level (1)
• Any tissue or organ can be affected at any age
• It can be a single organ (Leber neuropathy - LHON) or a couple
• Main symptoms:
- ocular: ptosis, oculomotor symptoms
- muscular: proximal myopathy, exercise intolerance,
cardiomyopathy
- CNS: seizures, migraine, stroke-like episodes, encephalopathy,
ataxia, spasticity
- sensory organs: retinopathy, sensorineural deafness, optic
nerve atrophy
- other: diabetes, obstetric history of 2nd or 3rd trimester
miscarriages
Nature Genet 1992; 1: 368-371

Arch Neurol 2004; 61: 950-952
Clinical level (2) – criteria
• CLINICAL CRITERIA
• Neuromuscular symptoms
• Progressive extraocular paralysis
• Ptosis
• Exercise intolerance
• Muscle weakness
• Rhabdomyolysis
• Abnormal EMG
• CNS and other organs involvement
• Isoalted CNS involvement
• Isolated single organ involvement
• Involvement of two or more organs
Clinical level (3) – chosen syndromes
• Leigh s.
• Alpers s.
• lethal infantile mitochondrial disease (LIMM)
• Pearson s.
• Kearns-Sayre s. mtDNA deletion
• PEO (ocular muscles) syndromes
• MELAS
• MERRF
• NARP (retinitis)
• MNGIE (gastrointestinal system)
• LHON (2nd nerve)
• Barth (muscular and skeletal systems)
Biochemical and imaging level (1) – criteria
• Metabolic and CNS imaging

• Raised lactate in blood on three occasions
• Raised csf lactate
• Raised alanine in blood
• Raised csf alanine
• Raised Krebs cycle intermediate metabolites in urine
• Raised ethylmalonic, 3-methylglutaconic or dicarboxylic
acids in urine
• Abnormal result of 31P-MRS (spectroscopy) in the
muscle with decrease proportion of phosphocreatine-Pi
• Abnormal signal from basal ganglia in T2
Biochemical and imaging level (2)
MELAS
Leigh
Pathological level – criteria
• Histopathological exam
• Ragged red fibers on microscopy
• Generalized decreased signal of cytochrome
oxidase c in histochemical reaction or the
presence of widely spread fibers without COX
Traditional molecular techniques in
mitochondrial disease - mtDNA
• Common point muts: PCR (PCR-RFLP), ASO and then
qPCR (heteroplasmy)
• Large deletions: Southern (doesn’t provide bkpts or level
of heteroplasmy) or aCGH
• Whole mtDNA: sanger sequencing (no heteroplasmy, no
large deletions)
• Examples:
MELAS: muts m.3243A>G, m.3271T>C and
m.3251A>G, TAT 10-14days, EDTA sample, 420PLN
or
MELAS, MERRF, NARP, LHON: chosen muts (Athena
Diagnostics), 500$
Panel CZD, Warsaw: long PCR and MLPA
mtDNA – 80% MELAS, 80% MERRF, 50%
NARP, 90% LHON (also chosen muts)
KSS, PEO are usually
sporadic cases!!
Quiz
15yo patient with ptosis, limitation of ocular movements and
arrythmia. Clinical suspicion of a mitochondrial disorder. What
molecular defect best explains the phenotype?
MANY
CONDITIONS
A. Dominant de novo mutation of gene encoding subunit of
respiratory chain (nuclear DNA)
B. Deletion of part of mitochondrial DNA KSS, PEO, Pearson
C. Mutation of gene encoding subunit of respiratory chain
(mitochondrial DNA)
D. Mutation of tRNA gene (mitochondrial DNA)
MELAS, MERRF
Modern molecular analysis in mitochondrial
disorders – mtDNA and nDNA (1)
• Genetic heterogenity of mitochondrial disorders caused

by nDNA mutations (>200 known genes; 1500 implicated
genes)
• NGS panels: usually a small numer of genes (a dozen or
so); „narrow” gene panels; best in diagnosing syndromic
forms of mitochondrial disorders
• Panel analysis is usually limited to 1-10% of all Mitome
genes, and to no more than 1% of all genes
NGS „narrow” panels - example
• Isolated complex IV deficit (after COX staining): „narrow”

NGS panel for complex IV genes: Baylor Miraca: 12
genes: COX10, COX15, COX4I1, COX4I2, COX6B1,
COX7A1, FASTKD2, LRPPRC, SCO1, SCO2, SURF1,
TACO1
• Limitations of NGS mtDNA panels:
1. if available, better check muscle bioptate
2. heteroplasmy ≤10% for single nucleotide variations
40%≤ for deletions may not be recognized with modern
techniques
Modern molecular analysis in mitochondrial
disorders – mtDNA and nDNA (2)
• „Broad” NGS panel: e.g. MitoExome

• MitoExome: complex I-V genes (ca 100), „mitochondrial”
nDNA genes (ca 200) and related (ca 500), Mitome
genes (ca 1500)
• MitoExome panel for 1000 nDNA genes: 42 infants with
clinical and biochemical diagnosis of mitochondrial
disorders: 24%(10/42): pathogenic variants in AR
disorders (validation?)
Sci Transl Med. 2012; 4(118): 118ra10

Comparison of „broad” panels - examples
Panel Comprehensive BCM-
Mitochondrial MitomeNGS
Nuclear Gene
Panel
Company GeneDx Baylor
nDNA Y N
mtDNA N Y
Number of genes 319 193+37
Dels/Dups Y N
NGS Panels vs. WES vs. WGS
WGS
?
Target sequence
WES
Dgn
markers
? NGS
panels
(targeted)
Pts
Modern molecular techniques in dgn of
mitochondrial disorders – mtDNA and nDNA
(3)
• WES (only coding sequences) or WGS (so called deep
sequencing)
• WES/WGS: new genes – to Mitome
Algorithm of interpretation of NGS variants
Neurotherapeuthics, 2013: 262-272

Clinical management (1)
• 3yo girl
• Hx: weak sucking reflex, slow increase in growth
parameters, antireflux medication until 9/12; could not sit
unsupported until 9/12
• Head MRI: T2 signal increase – symmetrically in basal
ganglia; MRS: double lactate peak
• Geneticist 2yo: exercise intolerance, hypotonia, slow
increase in growth, „can hear and see well”
• Biochemistry (blood): lactic acid 3mmol/l (n: 2,1),
pyruvate 0,22mmol/l (n: 0,16), proportion 13 (n: 20),
alanine 595mol/l (n: 440)
• Fx irrelevant
Clinical management (2)
• Traditional genetic testing: karyotype, aCGH, most
common mutations of tRNA in mtDNA - normal
• „Narrow” NGS panels: GeneDx (24 nuclear genes)
• WES (nDNA and mtDNA analysis): >100.000 variants,
including 10 mtDNA variants
• mtDNA: MT-ATP6 gene: pathogenic homozygous
p.Leu156Arg variant (10-20% of Leigh syndrome cases)
• No variant has been detected in the mother (WHY?)
• It is very important to confirm the mutation with traditional
Sanger seq!
mtDNA heteroplasmy
• Presence, in a single cel, of two populations of
mitochondria: having normal mtDNA and
mutated mtDNA
• Proportions of mutated and normal mtDNA can
change with age in different tisues
• Lack of guidelines as to clinically significant
heteroplasmy (e.g. 5-10-15%) in a particular
tissue (blood most often tested!)
• NGS panels as a rule have sufficient
coverage but presence of mutation may not
be confirmed with traditional Sanger
sequencing!
Diagnostic algorithm in
mitochondrial disorders (1)
History and PE, including neurological assessment (variable

clinical picture + symptoms typical for mitochondrial
disorders, family history with intrafamilial variable
expressivity)
Labwork: electrolytes, glucose, ammonium, lactic acid,
aminoacidogram, MS/MS, GC/MS
Other tests: ECG, EMG, echocardiogram, audiological and
ocular assessment
Diagnostic imaging: head MRI and MRS
Clinical examination
Syndromic Nonsyndromic
diagnosis diagnosis
Targeted test (single-gene Diagnostic criteria

mutations or „narrow” NGS
panel)
e.g. MELAS, MERRF – mtDNA
NGS („broad" NGS panel
or WES)
Clinical re-analysis and genetic

counselling
Clinical
examination
Syndromic Nonsyndromic
diagnosis diagnosis
Muscle
Targeted test (single-gene biopsy???
mutations or „narrow” NGS
panel)
e.g. MELAS, MERRF – Clinical Diagnostic
mtDNA follow-up criteria
Clinical follow-up and genetic NGS („broad" NGS panel

counselling or WES)
Molecular diagnostics in
neuromuscular disorders
Andrzej Kochański MD, PhD

Distal muscles wasting
Wasting of distal muscles
Small hand muscles
Small hand muscles
Small hand muscles
Small hand muscles
Scoliosis
Scoliosis
Scoliosis
Distal muscles wasting
Distal muscle wasting
Acquired or inherited disease ?
Combination of symptoms
Symmetrical
wasting of
the distal
muscles
Symmetrical
pes cavus
deformity
Hammer
toes
Acquired or inherited disease ?
A phenotype (combination of
symptoms)
Deafness
Mild wasting of
distal muscles
Pes cavus
deformity
Phenotype-genotype correlations
Hearing impairment in
Neuromuscular disorders
• Muscular Dystrophies
• Charcot-Marie-Tooth disease
• The Myotonic dystrophies
• Spinal muscular atrophy
• Periodic Paralyses
Floppy infant
• Prader Willi syndrome
• SMA
• Congenital myotonic dystrophy
• Congenital myopathy
• Congenital muscular dystrophy
SMA
Spinal muscular atrophy (SMA)
• SMA 1- the patients die by two years of age
• SMA 2-the patients achieve the ability to sit
• SMA 3- slowly progressive form of the disease
• All patients with SMA share clinical features:
symmetrical muscle weakness and muscle
atrophy as well as decreased or absent deep
tendon reflexes
Molecular genetics of SMA
• SMA types I, II and III were all mapped to
chromosome 5q11.2-13.3
• Approximately 95% of individuals with
SMA lack both copies of SMN1 exon 7.
• In the remaining 5% of SMA patients
missense, nonsense or splice site
mutations in the SMN1 gene are present.
SMA is inherited in AR trait
Prenatal diagnostics in SMA
• Possible by chorionic villus sampling

(CVS) at 11 weeks gestation
• Carrier testing
Duchenne and Becker Muscular
dystrophies (DMD/BMD)
DMD/BMD
• DMD is a severe X-linked disease
occurring in 1 out of 3500 live male births.
• Disease occurs in early childhood, causing
bilateral weakness in the proximal muscles
of the hip girdle and legs.
• Loss of ambulation in the patients with
DMD at age 11.
DMD/BMD
• DMD is caused by mutations in the
dystrophin gene that change the reading
frame of the transcript (60% of these
mutations are large deletions, 35% of
mutations are point mutations or small
insertions, 5% of mutations are
dupliactions)
• BMD results from mutations maintaining
the reading frame of the transcript.
Molecular diagnostics in Duchenne
dystrophy
• Major deletion testing by multiplex PCR
• DHPLC, DGGE or SSCP to screen for
mutations
• Direct sequencing
• Duplication testing
DMD/BMD is X-linked disease
1 2 4 4
2 4 7 7
.
3 5 8 8
2 ? 1 4 4
4 2 7 7
5 3 8 8
Gonadal mosaicism in Duchenne
dystrophy
Mother of the
proband is not
carrier of theDMD
mutation
Mutations in the
DMD gene are
present in proband
and in the brother of
the proband
An unknown mutation in the DMD
gene
1 2 4 4
2 4 7 7
.
3 5 8 8
2 ? 1 4 4
4 2 7 7
5 3 8 8
Who is carrier of the deletion in the
DMD gene ?
1 0 4 4
2 0 7 7
.
3 0 8 8
1 0 2 ? 1 4 4
2 0 4 2 7 7
3 0 5 3 8 8
The myotonic dystrophies
• Progressive muscle wasting and
weakness
• Myotonia
• Cardiac effects
• The cataract
Genetics of myotonic dystrophies
• The genetic cause of DM1 is mutation
(CTG)n repeat in the 3’-untranslated
region of the protein kinase gene DMPK
CTG number in healthy individuals ranges
from 5 to 37
Premutation-the number of CTG ranges
from 38 to 49
Protomutation CTG ranges from 50 to 80
Mutation- more than 200 CTG
DM family
(CTG)n=45;
premutation
(CTG)n=70;
protomutation
(CTG)n=250;
mutation
Emery-Dreifuss muscular
dystrophy
Emery-Dreifuss muscular
dystrophy
Muscular
dystrophy .
Dilated
cardiomyopathy
Charcot-Marie-Tooth disease
• Autosomal dominant Charcot-Marie-Tooth
disease (CMT1A, CMT1B, CMT1C,
CMT2A)
• CMTX disease
• Autosomal recessive CMT
AR-CMT and consanguinity
Muscular dystrophy and peripheral
neuropathy
Proximal and distal

muscle wasting
Sporadic cases of CMT
hz 715 C>T Leu239Phe
*
AR trait in CMT
Sural nerve biopsy in diagnostics of
CMT
Homozygosity mapping in
Direct sequencing of the gene
Pathogenic effect of DNA
sequence variant
DNA variants are conserved
between species
NEWBORN SCREENING
PROGRAMS
Jennifer Castaneda, MD, PhD
Principles of NBS
 Public health program
 Screening of infants shortly after birth
 Treatable but not clinically evident conditions
 Cost efficient
 Intervention can prevent symptoms
Screening criteria
 JMG Wilson and F. Jungner (1968): Principles and
practice of screening for disease – 10 criteria
 4 criteria for NBS:
1. Acceptable treatment protocol that changes patient
outcome after early diagnosis
2. An understanding of the condition’s natural history
3. Who will be treated as a patient
4. Reliable for both affected and unaffected patients,
acceptable to the public
Newborn screening samples
 Filter paper
 Collection: between 24 hrs and 7 days after birth
 Baby fed at least once
 Samples mailed daily
 Mandatory or with opt-out option
https://www.nhs.uk/conditions/pregnancy-and-
baby/newborn-blood-spot-faqs/
NBS procedures
 Informed consent
 Centralization
 Automized identification
 Efficient contact
 Follow-up: testing coordinated between geneticists
and primary care physicians
NBS methods
 Blood prick
 Metabolite or enzyme activity measurements
 Screening for hearing loss, congenital heart defects
(pulsoxymetry)
Historical context
 USA, 1960s
 Phenyloketonuria
 Robert Guthrie – bacterial inhibitiion assay for
detection of high levels of phenylalanine
Diseases screened by NBS in 2011
 USA – 54
 Germany – 12
 UK – 2 (PKU, MCADD)
 France – 1 (PKU)
 Hongkong – 1 (congenital hypothyroidism)
Tandem Mass Spectrometry (MS/MS)
 Fatty acid oxidation disorders

 Medium chain acyl-CoA dehydrogenase deficiency
(MCADD) – implicated in SIDS
https://www.mdpi.com/2409-515X/4/2/12
Congenital hypothyroidism
 T4 (thyroxin), thyrotropin (TSH) measurement
 Dysgenesis of the thyroid gland
https://www.xpertdox.com/disease-
description/Congenital%20Hypothyroidism
Congenital adrenal hyperplasia
 Deficiency of steroid 21-hydroxylase
 Elevated 17alpha-hydroxyprogesterone (17 -OHP)
https://www.semanticscholar.org/paper/Adrenal-Insuf-fi-ciency-Auron-
Raissouni/8a7816427d0033553be95e7635d7101a2f33c564/figure/2
Organic acidemias
 Urine samples at 3 weeks of age
(Quebec – voluntary second-tier screening)
 Thin layer chromatography
 Tandem MS: propionic acidemia (PA), methylmalonic
acidemia (MMA), isovaleric acidemia (IVA)

Cystic fibrosis (CF)
 Immunoreactive trypsinogen (IRT) in dried blood spots
 High IRT values -> molecular diagnostics to detect
CFTR gene mutations
https://www.sciencedirect.com/science/article/pii
/B9780128038093000129
Hearing loss
 High incidence (1-3 per 1000 live births)

 Serious impacts of late diagnosis
 Transiently evoked otoacoustic emissions, automated
auditory brainstem responses
https://yourhearingcenter.com/audiology-blog-hamilton-
oh/newborn-hearing-screening
Critical congenital heart defects
 Pulsoximetry – bedside screening for CCHD at 24
to 48 hours after birth
 Subsequent examination - echocardiography
https://www.mdpi.com/2409-515X/3/4/28
Other conditions
 Severe combined immunodeficiency (SCID)- PCR,
follow-up with immunologists, treatment with stem
cell transplant
 Duchenne muscular dystrophy (DMD) – elevated
levels of CK in dried blood spots
 Eye and vision screening: strabismus (Hirschberg
test), retinoblastoma screening (red reflex)
Guidelines for NBS
 American Academy of Pediatrics
 American College of Medical Genetics
 Limitationsof NBS – clinical vigilance!

 Education of parents
 Complex health care of child with disease diagnosed

by NBS
 Genetic counseling
Ethical aspects of newborn screening
 Informed consent
 Adequate information on results
 Expanded NBS to Whole Exome
Sequencing?
Probability and likelihood ratio
 Probability of an event P(A) denotes

the frequency of it’s occurrence
in a long (infinite) series of repeats
| A|
P( A) A
| |
0 P 1
It’s represented by a number in the range Probability 0.25 (1/4)

from 0 (event never occurs) indicates that certain event is observed
to 1 (event occurs every time) in 1 from 4 or 25% occurrences
Rule of addition
If events are mutually exclusive,
then the probability of either event occurring
is a sum
of the individual probabilities
of the occurrence of any of the events
P of giving birth to a boy or girl by a pregnant woman:

1/ + 1/2 = 1
2
P of rolling 2 or 3 on six-sided die:
1/ + 1/6 = 2/6 = 1/3
6
Rule of multiplication
If two or more events are independent,

then their joint probability
is a product
of their individual probabilities
P of rolling a pair of 6’s on two six-sided dice simultaneously :

1/
6 × 1/6 = 1/36
P of giving birth to two boys by a woman in binovular pregnancy:
1/
2 × 1/2 = 1/4
Parents – carriers of recessive trait (e.g. cystic fibrosis):
Aa aA
Punnett square
determine the
possible genotypes of
their children and
what’s the
1/ 1/ 1/ 1/ 1/ 1/ 1/ 1/ probability of them
2 2 2 2 2 2 2 2
1/ 1/ 1/ 1 1 | A|
4 4 4 + /4 = /2 P( A)
| |
Parents – carriers of recessive trait:
 risk of having an affected child :
1/
2 × 1/2 = 1/4
 probability of having an unaffected child:
(1/2 × 1/2) + ( 1/2 × 1/2) + ( 1/2 × 1 /2) = 3/4

healthy heterozygote
homozygote
(sum of three mutually exclusive events)

Mucoviscidosis
Unrelated pair of Caucasians with

negative medical history of cystic
fibrosis.
Frequency of carriers of disease-causing allele: 1/25
Aa Aa
4% 4%
? 1/
25 × 1/2 × 1/25 × 1/2= 1/2500
 A couple of one healthy person and one affected by a

dominant disease (e.g. Marfan syndrome)
Aa aa
Aa Aa Aa

What’s the probability that all three children will be affected?
Every child is an independent event (the rule of multiplication)

Aa aa
Aa Aa Aa
1/
2 × 1/ × 1/ = 1/
2 2 8
P of any of them being affected = P of any of them being healthy

What’s the probability that two out of three will be affected?
Aa aa
1/
2 × 1/ × 1/ = 1/
2 2 8 ?
P of any of them being affected = P of any of them being healthy …

Three mutually excluding sequences of events

(rule of addition)
Aa aa Aa aa Aa aa
3 × (1/2 × 1/2 × 1/2)) = 3/8

Odds
 Odds - describe probability as a ratio (proportion)
| A| P( A)
P( A) A Odd( A)
| | 1 P( A)
Odds take higher values than probability

1 1
5 5 1
odds 1 4
1 5 5 4
 Frequency of blue balls:

 Proportion of blue balls vs. yellow balls:
4:6 = 2:3 (2 to 3)
Likelihood in pedigrees
and
likelihood ratio (LR)
Likelihood (L)
in pedigrees
(initial comments)
Likelihood (L) = probability

Likelihood ratio (LR) = odds
Hardy & Weinberg principle:

states that allele and genotype frequencies in a
population will remain constant from generation to
generation in the absence of other evolutionary
influences.
In the simplest case of a single locus with two alleles denoted A and a with
frequencies f(A) = p and f(a) = q, respectively, the expected genotype frequencies
are f(AA) = p2 for the AA homozygotes, f(aa) = q2 for the aa homozygotes, and f(Aa) =
2pq for the heterozygotes
pp pq qp qq
L = p×p + (p×q + q×p) + q×q = p2 + 2×pq + q2 = 1
Applications of LR:
verification of kinship (parenthood)
p2 + 2×pq + q2
Alleles / markers: 1,2,3,4. Allele / marker frequencies: 1 – 0,1; 2 – 0,2; 3 – 0,3.
I 12 I 3 41 2 I 3142 34
?
II 13 II 1 3 II 13
L1 = 2×0.1×0.2 × 2×0.3×0.4 × 0.5×0.5 L2 = 2×0.1×0.2 × 2×0.3×0.4 × 2×0.1×0.3

LR=L1/L2 = 0.5×0.5 / 2×0.1×0.3 = 4.16
Conclusion:
It is 4.16× more likely that II1 is a child of I1 and I2
than that II1 is not related to I1 and I2
Reduced penetrance
 Penetrance - proportion of individuals carrying

dominant allele that also express particular trait
(60% or 0.6)
Penetrance is said to be reduced or incomplete when some

individuals fail to express the trait, even though they carry the
disease-causing allele.
Risk of inheriting a dominant disease:
P = 1/2 × pen
Risk of inheriting a dominant allele while not affected:
P = 1- pen
Applications of LR:
risk estimation (1)
hh Hh hh Hh hh Hh
Hh hh Hh hh hh hh
60y
Hh hh hh
25y
L(carrier)= 0.5 × 0.4 × 0.5 = 1/10 L(not a carrier)= 0.5×0.4×0.5 + 0.5×1×1 = 6/10
LR (odds) = L1/L2 =1/6 (1-to-6)

P(carrier) = 1/(1+6)=1/7
Applications of LR:
risk estimation (2a)
Calculate risk of being a carrier of a recessive X-linked disease for person III5
XY XX
XY X? XY XX
XY XY XY XY
Applications of LR:
XY XX
1.0 0.5
XY X?
XX
1.0×0.5 0.5 0.5 0.5 0.5

XY XY XY XY XX
L1(III5 is a carrier) = 1.0×0.5 × (1.0×0.5)4 × 1.0×0.5 = 1/64

Applications of LR:
XY XX
1.0 0.5
XY X?
XX
1.0×0.5 0.5 0.5 0.5 0.5

XY XY XY XY XX
L1(III5 is a carrier) = 1.0×0.5 × (1.0×0.5)4 × 1.0×0.5 = 1/64

L2(III5 not a carrier)
=
Applications of LR:
risk estimation (2b)
XY XX
XY X?
XY XY XY XY XX
L1(III5 is a carrier) = 1.0×0.5 × (1.0×0.5)4 × 1.0×0.5 = 1/64

L2(III5 not a carrier)
=
Applications of LR:
risk estimation (2b)
XY XX XY XX XY XX
1.0 0.5 1.0 0.5

XY X?
XX XY X? XY XX
X?
1.0 1.0 1.0 1.0 1.0 1.0×0.5 0.5 0.5 0.5 0.5
XY XY XY XY YX
XX YX YX YX XX XY XY XY XY XX
L1(III5 is a carrier) = 1.0×0.5 × (1.0×0.5)4 × 1.0×0.5 = 1/64

L2(III5 not a carier) = (0.5×15) + (0.5×0.55) = 32/64 + 1/64 = 33/64
LR = L1/L2 = 1/33
Pcarrier =1/(1+33)=1/34
What to remember?
 Probalility and rules (addition, multiplication)

 Odds
 Likelihood = probability
 Likelihood ratio = odds
 Hardy – Weinberg principle
Reproductive & Prenatal
Genetics
2019
Qs
What role does infertility play in a couple’s risk for a child with a
genetic disorder?
What modalities exist for screening for genetic disease during

pregnancy?
What are the risks and benefits of prenatal diagnostic studies?

Case Presentation
A couple, in their 30s, are referred for preconception genetic

counseling:
“History is notable for 3 years of unexplained

infertility”
The couple is concerned about a pregnancy complicated by a

genetic or congenital anomaly:
Could the cause of their infertility increase this risk?

Does assisted reproduction technology (ART) increase this risk?
Genetic Evaluation of Infertility
1. Unrecognized disorders of sexual differentiation
2. Genetic conditions associated with impaired fertility
3. Genetic risks of reproductive technologies

1. Disorders of Sexual Differentiation
Level 1 - Genetic sex 46 XX vs 46, XY
Level 2 - Gonadal sex

Undifferentiated gonad – testicular development dependent on
presence of TDF
Level 3 - Phenotypic sex

1. Internal reproductive structures (dependent on Mullerian
inhibiting factor)
2. External reproductive structures (dependent on testosterone
exposure)
3. Secondary sex characteristics at puberty
1. Disorders of Sexual Differentiation
FEMALE MALE INFERTILITY
INFERTILITY
GENETIC SEX 45,X 47,XXY Lack of gonadal

47,XXX 46,XY/45,X development
GONADAL SEX delXp DAZ deletion Lack of gonadal

FRAX premutation support
PHENOTYPIC SEX Late-onset CAH Androgen Effects on

insensitivity secondary sex
characteristics
Karyotype Abnormalities with
Azoospermia or Oligozoospermia
Azoospermia 10-15%
Oligospermia 5 %
Normal population <1%
Unbalanced translocations more common with oligospermia
Sex chromosome anomalies increase with decreasing sperm

count
Turner Syndrome
Responsible genes: X genes that escape inactivation, SHOX
Proteins: SHOX: Short stature homeobox protein
Cytogenetic locus: SHOX: Xpter-p22.32
Inheritance: sporadic
Clinical Features and Diagnostic Criteria: congenital lymphedema, growth failure,
normal intelligence (10% sig delays), coarctation of the aorta, bicuspid aortic valve,
HLHS, hyperlipidemia, gonadal dysgenesis (10% 45,X go into puberty),
hypothyroidism, diabetes, strabismus, recurrent OM, SNHL, Crohns, renal
malformation, osteoporosis.
Clinical Tests: echo, renal US, TFTs, GH testing, FISH SRY
Molecular Tests: Karyotype
Disease Mechanism: SHOX: thought to act as a transcription regulator with many
down-stream targets that modify growth and stature. SHOX protein has been
identified in the growth plate from 12 weeks to late childhood.
Treatment/Prognosis: GH, HRT, gonadectomy if Y chromosome mosaicism (risk for
gonadoblastoma). Need lifelong cardiac follow-up, at risk for aortic dilation and
dissection with bicuspid aortic valve.
Klinefelter Syndrome
Clinical Features and Diagnostic Criteria: tall stature, slightly delayed motor and
language skills, learning problems, testosterone plateaus age 14, small fibrosed
testes, azoospermia and infertility, gynecomastia, increased cholesterol,
slightly increased risk of autoimmune disorders and mediastinal germ cell
tumors (1% risk)
Molecular Tests: karyotype, at least one extra chromosome to a 46,XY
Disease Mechanism: 1st or 2nd meiotic division nondisjunction of either parent.

Maternal>paternal origin. + advanced maternal effect
Treatment/Prognosis: Testosterone in mid-late adolescence for bone density,

Secondary sex characteristics development, muscle mass, cholesterol, increase
libido, improved energy. Can do testicular biopsy and use any retrieved sperm
for ICSI (increased risk sex chrom abnormality so follow with PGD)
2. Genetic conditions associated with
impaired fertility
Cystic fibrosis
Fragile X syndrome
Myotonic dystrophy
Kennedy disease
Kartagener syndrome
impaired fertility – Cystic Fibrosis (CF)
Congenital absence of the vas deferens (CBAVD)
- clinical diagnosis, associated with azoospermia
- 98% of adult males with classic cystic fibrosis
- related to mucosal changes in developing vas
CBAVD - obstructive azoospermia; 1-2% of infertile men

- respiratory assessment supports inclusion of CBAVD in
spectrum of cystic fibrosis
CBAVD with renal abnormalities – normal sweat chloride test,

low rate of CFTR mutations
Congenital absence of the vas deferens (CBAVD)
- clinical diagnosis, associated with azoospermia
- 98% of adult males with classic cystic fibrosis
- related to mucosal changes in developing vas
CBAVD - obstructive azoospermia; 1-2% of infertile men

- respiratory assessment supports inclusion of CBAVD in
spectrum of cystic fibrosis
CBAVD with renal abnormalities – normal sweat chloride test,

low rate of CFTR mutations
MUTATIONS severe/severe severe/mild mild/mild
CLASSIC CF pts 87.8% 11.3% 0.9%
CBAVD pts 0.5% 87.9% 11.6%
Common 23 mutation panel + IVS8-5T

- second mutation is frequently a polyvariant (5T variant of intron 8)
- 47.63% (double heterozygotes); 24.63% (heterozygotes)
Sequencing
- 86.15% have at least one CF mutation
2. Genetic conditions associated with impaired
fertility – Fragile X Syndrome (FRAX)
• Gene: FMR-1 Protein: FMRP Locus: Xq27.3
• Inheritance: trinucleotide repeat expansion
• Clinics: developmental delay, ID (moderate/severe in boys,
milder degree in girls), hyperactivity, autistic traits, premutation
female carriers: OCD, depression, 20% POF, premutation male
carriers: intention tremour, ataxia, parkinsonism, autonomic
dysfunction (=FXTAS: >30% male carriers and <5% female
carriers; 1,5% ♂ and 3%♀ late-onset ataxia;
1/3000 ♂ two other loci: FraXE: only ID, FraXF: lack of phenotype
• Diagnostics: CGG triplet detection PCR: fast testing, small
premutations; Southern: all mutation classes + normal alleles,
mosaics, costly. Normal allele: 5-44 repeats, median: 45-58
repeats (grey zone), premutation: 59-200 repeats, mutation:
>200 repeats
• Mechanism: >200 repeats = methylation = inactivation = lack of
FMRP. POF and FXTAS (59-200 repeats) gain of function mutation
fertility – Myotonic Dystrophy
• Most common form of adult muscular dystrophy
adult myotonia - distal weakness, myotonic grip, frontal
baldness, cardiac arrhythmias, infertility
• Male infertility
oligoazoospermia - sclerosis of seminiferous tubules
• Congenital myotonia – polyhydramnios, neonatal hypotonia,

respiratory dependency, mental retardation
maternal transmission of expanded DNA
dependent on size of expansion
fertility – other disorders
• Kennedy Disease (spinal and bulbar muscular atrophy)

similar to amyotrophic lateral sclerosis
X-linked form of motor neuron disease that affects adult men,
onset mid 40s, slow progression
• Kartagener Syndrome (primary ciliary dyskinesia)

both autosomal recessive and dominant forms
male infertility - immotile cilia
female infertility – fallopian tube motility
Genetic assessment prior to IVF
Causes of Infertility
No genetic causes
CFTR muts
Mendelian
Balanced translocations
Y chromosome abn
3. Genetic risks associated with ART
• Risk of congenital malformations following assisted reproduction

technologies (ART)
What is the evidence of an increased risk ?
What type of genetic conditions are suspected ?
Is the rate of congenital malformations increased among infertile

populations irrespective of route of conception?
Meta-analysis: IVF and congenita anomalies
Highest quality (N=7) Meta-analysis (N=25)
All anomalies OR=1.40 (95%CI: 1.29 (1.21-1.37)

1.28-1.53)
Only major 2.01 (1.49-2.69) 1.32 (1.20-1.45)
Only singletons 1.35 (1.20-1.50) 1.31 (1.17-1.48)
Only following IVF 1.90 (1.41-2.54) 1.94 (1.50-2.50)
Only following ICSI 2.00 (1.30-3.20) 1.28 (1.14-1.43)
Hum Reprod 2005; 20:328-338

3. Genetic risks associated with ART
• New research
• Davies, 2012: Australian study (large groups)
• Current ART outcome vs Current spontaneous but Former
pregnancy ART: no difference
• Current ART outcome vs Infertility but no ART vs Fertile
spontaneous: higher risk ICSI only
Type of Birth Defect Increased in the ART
Population?
• Gametogenesis and preimplantation are important times for

epigenetic mechanisms
• Alterations in DNA structure rather than sequence
- epigenetic states established early in development and passed
intact in mitosis
• Imprinting is one type of epigenetic change
- results in gene transcription from only one parent
- genes of growth and differentiation
- forces biparental contribution to embryo and biologic diversity
Common Imprinting
Ovarian dermoid
46,XX All maternal
Trophoblastic disease –
complete mole
46,XX All paternal
ART and human epigenetic/imprinting disorders?
• Beckwith Wiedemann Syndrome - LGA, macroglossia,

omphalocele
- several causes – about 50% due to imprinting error on
chromosome 15
• Six fold increase in ART among children with BWS (DeBaun,

2003)
• (Maher, 2003; Gicquel, 2003): about 5 % conceived by ART vs <

1.0% general population
Why Worry About Imprinted
Genes ?
• BWS/AS following ART is rare (1/4000)
• Further roles in implantation and early embryo development
• Additional concerns regarding imprinted genes:

- Association with cancer
- Retinoblastoma in children from ART
- Association with neurodevelopment
Genetic Evaluation for Infertile Couple
• Sexual differentiation disorders may present with infertility and

convey specific genetic risks
- oligoazoospermia (translocations) and poor ovarian reserve
(fragile X premutation carrier)
• Some inherited disorders present with infertility with associated
risks to offspring
- cystic fibrosis and CBAVD
• Risk of congenital malformation following assisted reproduction
is increased
- imprinted genes suspected based on animal LOS studies and
human studies of Beckwith-Weidemann Syndrome
The Case
• Couple returns 3 yrs later
• 37yo G3P0 three miscarriages following ART (none karyotyped)
• Balanced translocation carrier?
• What are the numbers:
9.2% if three miscarriages or more
The Case
• 46, XY; 46, XX, t(5q;8q)
• Reciprocal
10-15% abnormal conception (either parent)
• Robertsonian
10-15% abnormal conception maternal origin vs 0-5% paternal
• Correlation with increased size of segments and rate of loss
• 1 year later, she is now 38 yo and pregnant

The Case
• She has her 11 week US obtained and a NL of 4.0 is detected
• How does this happen?
• Should she have the serum testing ?
• Can US look at anything else?
• What are her options for diagnostic testing?
• What other concerns are present?
Additional markers - Nasal Bone (NB)
and Ductus Venosus Flow (DV)
Absent NB 7.05
DV abnormal flow 6.42

Structural Anomalies and Genetic Disorders
• Cardiac anomalies 15% risk if NL>5.5mm

• Increased venous pressures, mediastinal compression
- congenital diaphragmatic hernia
- narrow thorax skeletal dysplasia
• Altered extracellular matrix
- collagen disorders (chondrodysplasias)
• Impaired fetal movement
- fetal akenesia sydromes
• Noonan and Smith Lemli Opitz Syndromes
Do the Serum Screening or Not?
• For NL>4.0 - highly unlikely to screen negative

- 0.09% of population
- 33 % aneuploidy
• For NL 3.0 to 4.0 – 8% have negative serum screen result
- 0.3% of population
- 17 % aneuploidy
Or rather: Do the amnio- or not?

Do the NIPT?
Ultrasound Obstet Gynecol. 2015 Jan 19. doi: 10.1002/uog.14792.
Noninvasive Prenatal Testing for Trisomy 21, 18 and 13 – Clinical Experience from
146,958 Pregnancies. Zhang H et al
NIPT was performed on 146,958 samples, of which outcome data were available in
112,669 (76.7%). 3,213 cases required repeat blood sampling, 145 had no
report. Aneuploidy was confirmed in 720 of 781 T21-positive cases, 167 of 218
T18-positive cases, and 22 of 67 T13-positive cases. There were 9 false negative
identified, including 6 T21 and 3 T18 cases. The overall sensitivity of NIPT was
99.17% for T21, 98.24% for T18, and 100% for T13, and the specificity was
99.95% for T21, 99.95% for T18, and 99.96% for T13. There was no significant
difference in test performance between 72,382 high-risk and 40,287 low-risk
subjects (sensitivity 99.21% vs. 98.97%, p = 0.82; specificity 99.95% vs. 99.95%,
p = 0.98). The major factors contributing to NIPT false positive and false
negative results were maternal copy number variant (CNV) and fetal/placental
mosaicism, but not fetal fraction.
Diagnostic choices in pregnancy
Chorionic villus sampling
Amniocentesis
Percutaneous umbilical blood sampling
Free fetal DNA

Risks involved
I MISCARRIAGE
II FETAL MORBIDITY
Rupture of membranes
Malformation
Infectious
Isoimmunization
III TECHNICAL
No sample obtained
Misdiagnosis
Malformation
Amniocentesis risks of miscarriage
1 in 200
But exact risk probably lower than 1 in 200, but unlikely

as low as 1 in 1600 (randomized control trials not possible)
Focus of counseling on best estimates of loss rate, contributors,

limitations of testing, accuracy of test, disease being tested
ACOG Jan 2007 removed of age 35 as a cutoff “to determine

who is offered screening versus who is offered invasive
testing.”
Amniocentesis – other risks
Premature rupture of membranes: 1% (91% survival rate post

PROM)
Isoimmunization (special attention to isoimmunized patients

and number of invasive procedures)
Hepatitis B carriers
- no increase in HepB carriers, possible increase if HBeAg+
- HIV+ patients – risk low if viral load low
Direct fetal trauma (rare with ultrasound guidance)

Chorionic Villus Sampling – other risks
Miscarriage: 0.6%-1% above standard amniocentesis risk
Limb reduction defects:

5.2 to 5.7/10,000 vs 4.8 to 5.97 general population (NSD)
- recommended CVS > 9 weeks only
- vasoconstrictive with resultant ischemia?
- immunoreactive against fetal cells with apoptosis?
Hemangiomas
3 fold increase (7.4% amniocentesis vs 21.1% CVS) confined
mostly to transcervical, no relationship to sample size, gestation
at sampling, bleeding
Chorionic Villus Sampling –
misdiagnoses
Evaluation of solely direct or cultured cells
Maternal cell contamination
Confined placental mosaicism

- 1-2% of CVS samples
- 1/3 reflect true fetal mosaicism, 2/3 are confined to placenta
- risks of uniparental disomy
- risks of fetal growth restriction
Twins
Early Amniocentesis = before 14wks
Post-procedure loss: 4.2%
Talipes: 1%
Percutaneus Umblical Sampling (PUBS)
Ultrasound guidance, fetal umbilical vessel sampled with 22

gauge needle
Advantages:
full fetal karyotype in 48 hours
all fetal hematology and serology
utility in assessing CVS mosaicism
Disadvantages:
1-2% risk of fetal loss
later in gestation (>18 wks)
Comparison of tests
TIMING RISK OF LOSS FETAL RISKS TECHNICAL
ISSUES
AMNIOCENTE 15WKS AND 0.5% EASIEST

SIS MORE
EARLY 14WKS AND 2-3% INCR INCR

AMNIOCENTE LESS CLUBFOOT CULTURE
SIS FAILURE
CVS 10WKS AND 1% HEMAGIOMA? PLACENTAL
MORE MOSAICISM
PUBS 18WKS AND 1-2% HARDEST

MORE
Free fetal DNA (ffDNA) in the mother
Shorter fragments than maternal free DNA
- detection by 6 -7 weeks from LMP
Increases with gestational age

- 3% of total free DNA in 1st trimester, 6% in 3rd trimester
Sources
- fetal circulation
- placental apoptosis
- fetal cells in maternal circulation
Cleared from maternal circulation in 2 hours

Noninvasive Diagnostics – NGS for
aneuploidy
New work refocusing on massively parallel massively parallel
sequencing (MPS)
“shotgun sequencing”,
“next generation seq”
excess of fragments
chromosome 21 = tris 21
addition of multiplexing
The Case
• She returns with her husband two weeks later. They decided not
to proceed with serum screening and present for CVS
• NL=2.0mm
• “How can this happen?” Unknown currently

• “How often does it happen?” 5/6 trisomy 21 fetuses resolve
increased NT by 2nd trimester
• “Does it change her risk of aneuploidy?” NO since resolution
occurs both in aneuploid and normal fetuses
The Case
• She remains concerned about a problem with the pregnancy.
• What should she do next? What concerns remain?

Risk of a karyotype abnormality
Abnormal second trimester ultrasound
Risk of abnormality detected in newborn or child
Outcomes of NL>4.0mm
• Abnormal karyotype 50%
• Normal karyotype 50%:
- 25% abn 2nd trimester fetal US (cardiac and
other anomalies)
- fetal demise
Always send for fetal echocardiogram!

US in 2nd trimester
• Structural anomalies
- risk of aneuploidy greatest for multiple anomalies (18.8 %)
- smaller but present risk for isolated anomalies (9.3 %)
• Issue of an “isolated” ultrasound anomaly – minor features

and dysmorphia difficult to appreciate
US in 2nd trimester – soft markers
• Individually seen in 1-5% of normal fetuses
• Isolated - increases risk of aneuploidy 1.5 to 3 fold
• >2 present - increases risk 10 fold or more “soft signs”
nuchal fold
echogenic bowel
echogenic cardiac focus
shortened femur/humerus
renal fullness
choroid plexus cysts (trisomy 18)
Array CGH in prenatal diagnostics
aCGH results - example
Normal karyotype + array

3% (53/71)
4% 5%
13% Abn karyotype + array
75% Abn chr morphology

(3/71)
Abn array only (4/71)
Balanced translocations
(2/71)
The Case
• The couple deliver a healthy son.
• They return to the geneticist two years later with about
preimplantation genetic testing (PGD)
Preimplantation Genetic Diagnosis
• Screening modality that decreases the chances of transferring an
“affected” embryo
• Screens only for disorder in question
• Effects of biopsy
• Possible decrease in implantation rate?
Preimplantation Genetic Diagnosis
• PGD for chromosome abnormalities
• Translocation carriers
• No longer advocated for AMA or habitual
• Miscarriage - role for aCGH?
• “More is not better”
• Increased number of probes increases the technical risk and risk
of “false positive” biopsy
Human genome analysis:
potentials for health
care
JENNIFER CASTAŃEDA , MD, PHD
INSTITUTE OF MOTHER AND CHILD, WARSAW
http://hubc.ub.edu/en/hubc/el-projecte/executive-summary
Evolution of genetic diagnostic methods:
chromosomal analysis
Classical FISH array CGH

karyotyping
Evolution of genetic diagnostic methods:
DNA analysis
Sanger Next generation

PCR
sequencing sequencing
Clinical NGS
❑ Whole genome sequencing (WGS)
❑ Whole exome sequencing (WES)
❑ Gene sequencing panels
Personalized / Precision Medicine
❑ Personalization and individual tailoring of therapies and treatment
regimes
Data-intensive Translational
science medicine
❑ Translatabilityof discoveries and techniques from the laboratory

into solutions, drugs and therapies that can be effectively
implemented at the point of healthcare delivery
Hot areas in genomic medicine
❑ Cancer genomics
❑ Pharmacogenomics, drug therapy
❑ Rare, undiagnosed diseases
❑ Preventive medicine
❑ Genomics of pregnancy
Cancer genomics
❑ ER, PR, HER2 receptor markers (Herceptin drug)
❑ Oncotype DX®, MammaPrint®
http://colon-cancer.oncotypedx.com/en-US/Patient/What-Is-A-Genomic-Test/About-ODX-
Colon-Cancer-Test
Pharmacogenomics
❑ interindividual variability in drug
response as a consequence of
genomics, epigenomics,
environment, gender, age,
concommitant medication
❑ additional dynamic omics
activities (transcriptomic,
proteomic, metabolomic,
autoantibody profiles) - highly
complex multiomics data sets
https://mapmygenome.in/blog/2017/11/01/pharmacogenomics-the-exciting-science-of-
personalized-medicine/
Pharmacogenomics – clinical use
❑ Ivacaftor for cystic fibrosis patients bearing the specific
G551D genetic variant in the CFTR gene
❑ Cetuximab – therapeutic benefit in KRAS mutation
carriers with advanced colorectal cancer
❑ Warfarin – optimal starting dose based on CYP2C9 and
VKORC1 genotype
❑ Thiopurines – severe myelotoxicity in carriers of TPMT
gene variants
Development of drug therapy for genetic
disorders
https://cen.acs.org/articles/94/i41/CRISPR-edits-sickle-cell-mutation.html
Rare and undiagnosed diseases
❑ Diagnostic yield:
▪ Gene panel: 40% (8/20 neonates) in the NICU [Daoud H et al., 2016]
▪ Clinical exome sequencing: 26% (213/814) patients with undiagnosed,
suspected genetic conditions [Lee H et al., 2014]
▪ Whole genome sequencing: 45% (45/100 families, 53 of 119 children
affected with neurodevelopmental disorders [Soden SE et al., 2014]
Genomics of pregnancy and childbirth
❑ Fetal-free DNA
❑ Fetal genomic analysis?
❑ Expanded Newborn Screening?
❑ Ethical issues
Preventive medicine
❑ Genome-wideassociation studies on
common diseases
❑ Type 2 diabetes, hypertension,
Crohn’s disease, schizophrenia
❑ Complex biology of common
disorders
❑ So far limited clinical significance
Potential clinical tools
❑ Electronic health records (EHR)
❑ Electronic medical records (EMR)
❑ Biobanks
❑ Smart watches for health
Genomic Medicine: Challenges
❑ Data interpretation: clinical significance of
mutations / variants, proper counselling
❑ Complexity of human biology: interactions
between genes, with the environment, with other
organisms (microbes)
❑ From statistical data to a particular individual
(nonrepresentative patient data)
Genomic Medicine: Challenges
❑ The „clinical eye”: keenness of observation,
combining genetic data with clinical data, pertinent
family history findings
❑ Genomic data as a tool and not as a subsitute for
medical care (humanization of medicine)
❑ Accessibility, distributive equity
❑ Intellectual property
Ethical aspects
❑ Genetic determinism, reductionism
❑ Discrimination, stigmatization
❑ The fetus as patient in prenatal diagnosis:
parental autonomy vs best interest of the child
Conclusions
❑ Diagnostic and therapeutic benefits of genomic
DNA analysis will continue to be discovered.
❑ The translation of genome technology into
clinical use requires close collaboration among
researchers, clinicians, biotech companies, IT
experts, investors, health care policy makers.
❑ Ethical, legal, and social issues need to be
reflected upon and answered.
https://www.pathology.med.umich.edu/quality/quality-patient-centered

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Pedigree drawing

Summary of family medical history

important to Ask about living and deceased

history? Ask about consanguineous mating

Ask about children born thanks to in

• medical documentation from hospital medical records’, physicians’

• the appropriate provincial or state facility

Types of documentation that are most useful include:

- reports of surgical procedures

- discharge summaries, which should review all of the pertinent

- laboratory investigations, such as chromosome studies

Recomendations of the PSTF

Bennett R.L et al.. J Genet Counsel (2008) 17:424-433 56: 745-752.

Deceased individual Recomendations of the PSTF

Person in a presymptomatic Recomendations of the PSTF

Bennett R.L et al.. J Genet Counsel (2008) 17:424-433 56: 745-752.

Multiple individuals, number unknown

Bennett R.L et al.. J Genet Counsel (2008) 17:424-433 56: 745-752.

Still birth Recomendations of the PSTF

Spontaneous Affected SAB

Termination of pregnancy (TOP) Recomendations of the PSTF

Infertility Recomendations of the PSTF

Bennett R.L et al.. J Genet Counsel (2008) 17:424-433 56: 745-752.

Bennett R.L et al.. J Genet Counsel (2008) 17:424-433 56: 745-752.

Consanguinity Twins Twins Adoption Adoption

Common pedigree symbols

Bennett R.L et al.. J Genet Counsel (2008) 17:424-433 56: 745-752.

Couple whose gametes are used to S

Surrogate ovum donor

Couple in which male partner’s

an unrelated woman. a sister who is carrying the

Bennett R.L et al.. J Genet Counsel (2008) 17:424-433 56: 745-752.

Couple contracts with a woman to carry a D D

Bennett R.L et al.. J Genet Counsel (2008) 17:424-433 56: 745-752.

in line with the birth order

1985 1990 1955 1960 1965

Bennett R.L et al.. J Genet Counsel (2008) 17:424-433 56: 745-752.

• All individuals of one generation are drawn on horizontal line

• Each generation is marked by Roman numerals

Kamila Robert Bartek Lidka Maria Krysia Kamil

Zuzia Jarek Kasia Maciej Klaudia Zbyszek Marta

Cris July Andrew Rose Betty

Paul Anita Eve Harry

Tom Peter Mark Susan Betty

• PEdigree Drawing software

Common pedigree symbols

Obligate carrier (will not

Common pedigree symbols

Single gene inheritance Multifactorial

Autosomal X-Linked inheritance

Dominant Recessive Dominant Recessive

Diagnostic difficulties: Examples:

• father-son transmission of disease gene is possible

Pedigree typical of autosomal dominant inheritance

• consanguinity • sickle cell disease

Your Genes, Your Health

• consanguinity is sometimes seen, especially for rare recesive disease

Aa AA* aa (2 normal alleles!)

Aa AA* aa (2 normal alleles!)

Aa AA* aa (2 normal alleles!)