Genetic Screening,

Genetic counseling

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 Genetic screening
• Targeted Screening
– Screening of populations known to be at risk (families of
an affected individual)
– Screening of high risk ethnic groups (Tay-Sach’s carriers
in Ashkenazi Jewish, thalassemia in people of
Mediterranean origin)
– Identifying persons at risk of having children with a
genetic disease (carriers)

• Population Screening
– Adult Screening (Presymptomatic/ predictive)
• (PAP, breast, prostate)
– Screening all members of a designated population
regardless of family history

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 Targeted screening
• Targeted Screening includes
– Screening for presymptomatic disorders (late
onset or reduced penetrance disorders) in
family members of affected patients:
Huntington’s disease, breast cancer,
hemochromatosis, familial adenomatous
polyposis
– Screening for heterozygotes (carriers)

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 Targeted screening
Population Disease
Ashkenazi Jewish Tay-Sach’s, Gaucher disease
Mediterranean, Asian Thalassemia
African Sickle cell anemia
Caucasian Cystic fibrosis

• Carriers
– Screening of populations known to be at risk (families
of an affected individual)
– Screening of ethnic groups (Tay-Sach’s carriers in
Ashkenazi Jewish (1/29 carrier frequency), thalassemia
in people of Mediterranean origin)

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 Population Screening
• Involves screening all members of a
designated population regardless of family
history
• Prenatal screening
• Newborn Screening

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 Purpose of Prenatal Screening
Typically used to detect 3 abnormalities
• Trisomy 21 (Down Syndrome) ~1/830 live births
• Trisomy 18 ~1/7,500 live births
 Trisomy 13 has similar results to 18 but is ~1/22,700 live births
• Neural tube defects ~1/2,000 live births

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 Prenatal Screening
Non-invasive Optimal time
Maternal Serum Screening 16 weeks
First trimester screen 11-14 weeks + 1 day
Second trimester screen 14 weeks + 2 days-20 weeks
Ultrasound (fetal anomaly scan) 18 weeks
Nuchal translucency 11-14 weeks

Invasive
Amniocentesis 15-18 weeks
Chorionic villus sampling 10-14 weeks
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 Maternal Serum Screening
First Trimester Tests: 11-14 weeks + 1 day
Pregnancy associated plasma protein-A (PAPP-A)
Human chorionic gonadotropin (-hCG)

Second trimester tests: 14 weeks + 2 days to 20 weeks

Triple Test: looks for 3 markers
AFP (alpha fetal protein)
Estriol (unconjugated estriol, or uE3)
Human chorionic gonadotropin (-hCG)

Quad test: Includes another marker

Inhibin A

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AFP Serum Screening

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 AFP Serum Screening

Trisomy 21
“Down”

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11
 Maternal serum screening
• Low serum AFP, estriol and PAPPA with high β-hCG and Inhibin A
indicate a risk for Down Syndrome
• Low serum AFP, estriol, PAPPA and β-hCG and indicate a risk for
trisomy 18 (Edward syndrome) or trisomy 13 (Patau syndrome,
much more rare)
Fetal DNA Fragments “cfDNA” or “Cell free DNA”
• “The fetal DNA fragments (25-30 base pairs long), are matched
to a specific chromosome. The researchers tally how many gene
fragments originated from each chromosome. Women with
Down syndrome pregnancies had more fetal chromosome-21
fragments in their blood than women with normal pregnancies.”
– Analysis of fetal DNA in maternal blood has the advantages of earlier
detection of trisomy and eliminates the risk of abortion by amniocentesis
(non-invasive)
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 Maternal serum screening
• Detection rate
– 80% of Down Syndrome cases
– 80% of trisomy 18 cases
– 80% cases of spina bifida
– >90% of cases of anencephaly
• Results
– Results for trisomy 21 and trisomy 18 are
reported as a risk figure (not a diagnostic test)

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 Ultrasonography
At 18 weeks can detect many different structural abnormalities

Structural
Neural tube defects defect
are best detected by micrognathia
↑maternal AFP (Cri-du-chat)
combined with
Emery’s fig 21.4
ultrasound

Rocker-
Anencephaly bottom foot
(Neural tube Edwards
Syndrome
defect)
(trisomy 18)
Emery’s fig 21.7
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Enlarged Nuchal Translucency (NT) 11-14 weeks
Increased NT thickness is
associated with
chromosome aneuploidy
trisomies 21, 18, 13,
triploidy and Turner
syndrome (45 XO).

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Normal Enlarged
 Chorionic Villus Sampling
• Involves removal of fetal cells by
aspiration from the inner surface of
placenta
• can be done at 11-12 weeks (earlier
than amniocentesis)
• Direct chromosomal analysis by
FISH allows rapid results

Risks
• risk of miscarriage about the same
as amniocentesis (1%)
Emery’s fig. 21.2
• emotional and ethical question of
termination but can be done sooner 16
 Amniocentesis
15-18 weeks
10-20 ml of amniotic fluid is aspirated
trans-abdominally guided by ultrasound
- Fetal cells are pelleted by
centrifugation and used for
chromosome analysis
-supernatant can be used for AFP assay

Risks
- 0.5-1% possibility of miscarriage
- done fairly late in gestation so
emotional and ethical question of late
mid-trimester termination (16 weeks) Emery’s fig. 21.1
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 Percutaneous umbilical blood
sampling
• Usually done after 18 weeks of gestation
• Performed when there is a delayed
suspicion of a chromosomal abnormality
usually detected by ultrasound in 2nd
trimester

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 Prenatal Genetic counseling
Prenatal genetic counselors are involved with women either
during their pregnancy or prior to conception
• History of infertility, miscarriages or stillbirths
• Couples older than 35 years
• Abnormal serum screen for neural tube defects and chromosome
abnormalities
• Abnormal ultrasound findings
• Previous child with birth defect (neural tube defect) or multiple
congenital anomalies
• Previous child with chromosome abnormality or other genetic
disorder
• Specific ethnicity that may have a higher incidence of certain
disorders, such as Tay Sachs disease (Ashkenazi Jewish), Sickle Cell
anemia (Afro Caribbean), Thalassemias (Mediterranean), cystic
fibrosis (Northern European)
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 Preimplantation Genetics
• Preimplantation genetics involves using artificial
reproductive technology (ART).
• This involves fertilizing the egg with sperm by in vitro
fertilization and testing the embryo for a specific
condition before it is implanted into the mother
• polar body blastocyst; 8-16 cell stage

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Preimplantation Genetics available for:
Most common Less Common
Thalassemias Adenomatosis Polyposis Coli (APC)
Charcot-Marie-Tooth Epidermolysis bullosa
Cystic Fibrosis Fanconi Anemia
Huntington Disease Fragile X
Myotonic Dystrophy Gaucher disease
Rh Marfan syndrome
Sickle Cell Anemia Osteogenesis Imperfecta (AD)
Spinal Muscular Atrophy Sanhoff disease
Tay Sachs

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 Newborn screening
Criteria for Undertaking Neonatal Screening
– The disorder produces irreversible damage if
untreated early in life
– there is a treatment available for the disorder
– early intervention is effective
– a reliable lab test for detection (A positive result
should be reconfirmed immediately by retesting)

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 Newborn screening
•Traditional Testing: based on signs, symptoms, or family history
•NBS: done on all to identify those children to treat
•Preclinical: before symptoms, disease or death

Traditional Medicine

Normal Symptoms Diagnosis Treatment

Diagnosis Treatment
Newborn Screening

Normal Symptoms Diagnosis Treatment

Preclinical Stage
Newborn screening – spots of blood

2 MS/MS systems
~120,000 samples/yr at the Mayo Clinic
Rochester, Minnesota 24
 Number of disorders
screened for in
North America
< 10
30-39
40-49
50-52
53
54
57

49 Countries participating in Newborn Screening Quality Improvement Project (2012)

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The first test, Guthrie bacterial
inhibition assay, now

tandem mass
spectroscopy

electrophoresis

Immunoreactive trypsin 26
 Historical:
Guthrie Inhibition Assay (1960’s)

Current:
Tandem Mass Spectrometer (MS/MS)
Amino Acid Profile from Control Blood Spot Amino Acid Profile from PKU Blood Spot
100 100
Pro
Phe

INTERNAL STANDARDS
% Intensity

% Intensity
Ala Phe
50 50
Leu + Ile
Phe
Leu
Tyr
Tyr
Ala ValGln Cit Glu
Gly Ser Val Met Asp Glu
Gly
140 160 180 200 220 240 260 280 300 140 160 180 200 220 240 260 280 300
m/z, amu m/z, amu
PKU Newborn Screening by Tandem MS
(MS/MS)

Molecules (amino acids) are ionized & injected into mass

analyzer of MS/MS
Selected ions are fragmented
Fragments separated by second mass analyzer & amino
acids are detected based on their signature fragment pattern
(see control from previous slide)
 Phenylketonuria
• Newborn screening originally by a bacterial inhibition
test that detects elevated levels of phenylalanine in
circulation
• Today, positive newborn screening test has to be
immediately confirmed by elevated Phe levels in
circulation by a more specific assay (Quantitative mass
spectrometry)
• Management involves the lifelong restriction of dietary
Phe, that results in a marked improvement and normal
mental development in the child
Not Treated
treated Treated from birth,
from 2 y, normal
MR, development
can not
talk
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 Sickle Cell Disease
Point mutation in β-globin resulting in anemia

Detection
• Hemoglobin electrophoresis
• DNA test:
GAG to GTG
Glu to Val
-Southern blot, PCR-RFLP, ASO test

Treatment
•Prevention of sickling crisis, blood transfusions,
use of the drug hydroxyurea
(reactivating fetal hemoglobin)
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 Thalassemia (α and β)
• Both show autosomal recessive inheritance
• Both can be detected by mean corpuscular
hemoglobin and hemoglobin electrophoresis
• Screening of young adults for the β-
thalassemia carrier status has markedly
reduced the birth incidence of β-thalassemia
in Cyprus, Greece

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 Cystic Fibrosis
Mutation in the CF transmembrane conductance regulator (CFTR)
FYI- Incidence: 1 in 3,000 Caucasians or Ashkenazi Jewish, 1 in 8,000
Hispanics, 1 in 15,000 African Americans, 1 in 32,000 Asians.
Many states in U.S.A. routinely screen for CF

Detection
• Immuno-reactive trypsinogen (IRT) in blood followed by a DNA test
• DNA looking for ~32 of the most common mutations
• Sweat chloride test (confirmatory test)

Management
• Antibiotics to combat respiratory infections, gene therapy,
management of associated malabsorption
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SCID- Severe Combined Immunodeficiency
SCID is the result of genetic defects which impair the normal
development of T-cells
SCID infants are phenotypically normal but acquire life-threatening
infections within a few months of life

Detection
•Characterized by failure of T lymphocyte development and absent/low T cell receptor
excision circles (TRECs)
•TRECs are circular DNA fragments (by-products) generated during T-cell receptor
rearrangement.
•In healthy neonates, TRECs are made in large numbers, while barely detectable in
infants with SCID
•TRECs are quantitated by real-time quantitative polymerase chain reaction (RT-qPCR)
on DNA extracted from routine NBS cards
Management
• bone marrow transplant 33
 Extra For your information, not on test

The thymus gland provides an environment for the production of rearranged

diversified populations of T-cell receptors (TCRs) expressed on peripheral T cells.
During TCR rearrangement processes, unused excised DNA fragments create
byproducts termed TCR excision circles (TRECs). Although these byproducts
have no function, their detection in the peripheral blood stream is a clear indication
that a rearrangement process has occurred.
TRECS are quantified by quantitative PCR
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 Carrier screening
• Most couples learn that they are carriers only after the birth
of an affected child
• Carrier screening offers the opportunity to identify couples
at risk of having a child with a genetic disease
• Screening for heterozygotes (carriers) based on
– clinical manifestations
– biochemical abnormalities (Hexosaminidase activity in Tay Sachs
carriers)
– Specific molecular diagnostic tests (ASO array tests) or mutation
analysis
• In a small number of families in whom the mutation cannot
be identified, linkage analysis remains the only method for
the genetic diagnosis of carriers

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 Carrier screening

• Options for a couple in which both partners

are carriers
– Choosing not to have biological children
– Artificial insemination or egg donation
– Prenatal diagnosis and termination of an
affected pregnancy
– Prenatal diagnosis and planning for the care of
an affected child
– No prenatal testing

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Challenges for a carrier screening program
• For diseases like cystic fibrosis and thalassemia, the
occurrence of a wide variety of mutations resulting in
similar phenotype (allelic heterogeneity) has limited
the widespread use of carrier screening
• For cystic fibrosis, the DNA mutation panel can
identify 25 of the most common mutations in the US
population
• A person who has been identified as a “non-carrier” –
has the residual risk of being a carrier of an
infrequent mutant allele, that is not included in the
panel

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 NBS and the Genetic counselor
• “My first patient is a 4-week-old girl coming in due to a positive
newborn screen for cystic fibrosis (CF). Before coming to see me this
afternoon, she will have a sweat test.
• She has no medical history for respiratory or gastrointestinal
problems. Upon her arrival I learn that she has had a negative sweat
test and may be a carrier of cystic fibrosis.
• This is the first thing I explain to the parents, adding that being a
carrier does not have any medical implications for their daughter.
They look very relieved.
• During the remainder of the session, we talk about how CF is
inherited in an autosomal recessive fashion, and most likely that one
of the parents is a CF carrier.
• In addition, when their daughter is planning her family, she may have
a similar consultation with genetics.
• Finally, as it is possible that both parents could be carriers but only
one passed on the mutant CF gene, I suggest that both parents be
tested for carrier status. The parents opt to come back to have their
carrier status tested and an appointment is made for next week.

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 What is genetic counseling???
• Non-directive Counseling
• Genetic counseling is the process of helping people
understand and adapt to the medical, psychological
and familial implications of genetic contributions to
disease.
• This process integrates the following:
– Interpretation of family and medical histories to assess the
chance of disease occurrence or recurrence (in offspring)
– Education about inheritance, testing, management,
prevention, resources and research.
– Counseling to promote informed choices and adaptations to
the risk or condition.

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 Goals of Genetic Counseling
To inform the affected individual AND his/her
family of:
• the characteristics of the disorder
• the probability of developing the disorder
• the risk of passing the disorder on to their
children
• about the options to prevent or ameliorate the
disorder
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 Ethical and legal issues in Medical Genetics
“GENETHICS”
• “Your Genes, Your Choices”
• It is based on the general principles of
Medical Ethics
• The most common issues involve:
– Autonomy
– Informed Choice
– Informed Consent
– Confidentiality

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Autonomy
• The patient should be empowered and should be
allowed to make decisions by himself
• The clinician/ counselor provides appropriate
guidance during the decision making process
• The option of opting out of the testing should be
open at all times of the process, if the person
does not wish to continue (especially important in
predictive testing)
• Eg. Children of a father who has been recently
diagnosed to have Huntington’s disease
(especially as there are no methods to prevent
the disease development, if the mutation is found
to be present)

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Informed choice
• Patient is entitled to full information about the
options available, including opting out of a test at
any stage
• The potential consequences of each decision
should be discussed
• Eg: Testing for familial hypercholesterolemia, in
children (>20years) of a mother diagnosed with
familial hypercholesterolemia. If children have
high serum cholesterol levels, dietary, lifestyle
modifications, and drug therapy can be started at
an early age – which may improve the life
expectancy & quality of life of the affected
children

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 Informed consent
• Before a test/ procedure, a patient is entitled to
know the risks, limitations, implications &
possible outcomes of each procedure
• A signed consent is usually obtained for most of
the procedures
• Challenges:
– Patients undergoing the genetic tests are children
(minors) or are mentally challenged
– Situations where the patient is going to be minimally
benefited by the procedure, but it may be important
for other members of the extended family

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Confidentiality
• A patient (couple) has the right to complete
confidentiality involving the genetic
counseling/ testing
• Challenge in genetics:
– A couple have a son diagnosed with Duchenne
muscular dystrophy. They do not want this to
be disclosed to any of their relatives.
– The child’s maternal aunt is pregnant !!!

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 Ethical dilemmas
• The issue of prenatal diagnosis & subsequent offer of
termination of pregnancy raises many difficult questions
• Discrimination based on genetic tests: For some people,
information about their "problem" genes can bring extra
trouble.
– For example, it can cost them their health insurance. People with
"problem" genes have been refused health insurance or dropped from
their health plans.
• In other cases, they have been told that medical expenses for
their genetic condition will not be covered.
• In still others, they have been told that their children will not
be covered because they are at risk for inheriting genetic
diseases.
• The number of such cases may increase as genetic testing
becomes more common.
Last Slide 46
1. What is the risk of Jan’s sister being a carrier of cystic fibrosis?
2. The incidence of cystic fibrosis in the population is 1/10000. What is
the risk of Jan’s sister having a kid affected with cystic fibrosis?

3. What are the chances of James being a carrier of cystic fibrosis?

“Calculate the risk that a sibling of a known affected for an AR disease will himself be a carrier of the disease allele (2/3)” 47
Jan’s Sister’s Husband… risk of being a carrier…..
For a 2 allele (N and CF ), diploid (two chromosomes)
system,

an individual’s genotype is equal to the

likelihood of inheriting…

the M allele or Cf allele from one parent

and the M allele or Cf allele from the other parent.

(p + q) x (p + q) = (p + q)2 = 1

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Jan’s Sister’s Husband… risk of being a carrier…..
This binomial is expanded to:
p2 + 2pq + q2 = 1, where p + q =1,
and represents the frequency of genotypes in a population
• p2 = incidence of homozygotes for the N allele (N/N)

• 2pq = incidence of heterozygotes (N/ Cf )

• q2 = incidence of homozygotes for the Cf allele (Cf / Cf )
=1/10,000

q = √1/10,000 = 1/100 = 0.01 so q is small and thus p is close enough to 1

• Therefore 2pq = (2)(1)(1/100) = 1/50

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