CHAPTER 5

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GENETIC DISORDERS

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HUMAN GENETIC ARCHITECTURE
• Coding sequences make up 2% of total genome and >50% of genome represents repetitive blocks of
sequences with unknown functions
• Genome has only 20,000 to 25,000 genes that code for proteins, though alternative splicing can generate
> 100,000 proteins
• Two individuals share 99.5% of DNA sequences
• Most genetic variation arises from single nucleotide polymorphism (SNP) and copy number variation
(CNV)
• SNP's: represent variations at single nucleotides and are mostly balletic
• CNV's: represent variations in large contiguous stretches of DNA from 1000 bp's to millions
• Epigenetics: heritable changes in gene expression that are not caused by specific DNA sequences - are
involved in tissue-specific expression of genes and genetic imprinting
• Proteomics: the analysis of the proteins expressed in a cell
• Bioinformatics: the ability to analyze all the patterns of genetic and protein expression
• MiRNAs (microRNAs): small RNA molecules that inhibit gene expression (are ~1000 in total genome)
!!
GENES AND HUMAN DISEASES
• Can be categorized into 3 large groups:
1. Disorders related to mutations in single genes with large effects
• Follow classic Mendelian patterns and are highly penetrant
• Generally rare, unless maintained in population by strong selective forces
2. Chromosomal disorders
• Uncommon but also highly penetrant
3. Complex multigenic disorders
• Most common category, caused by interactions between multiple variant forms of
genes (polymorphisms) and environmental factors (eg, atherosclerosis, diabetes,
autoimmune diseases)
• There are also single gene disorders with non-classic patterns of inheritance
!
MUTATIONS
• A permanent change in DNA – can be partial or complete deletion of gene or single base – can interfere
with protein synthesis at various levels
1. Point mutation
• Missense mutation
! Conservative: if substituted amino acid causes little change in resulting protein
! Nonconservative: if protein severely altered (eg sickle cell mutation)
• Nonsense mutation: results in stop codon which terminates chain
2. Mutations with noncoding sequences
• Ie. Involving the introns: can affect regulatory areas like promoter and enhancer
sequences
3. Deletions and insertions
• Frameshift mutations: if not multiple of three
4. Trinucleotide repeats
• Characterized by amplification of sequence of three nucleotides
• Dynamic (degree of amplification increases during gametogenesis)
! Eg. Fragile X Syndrome (repeats of GG within FMR1 gene)
! !
• Terminology:
• Hereditary: derived from one’s parents and are transmitted in germ line
• Familial: same as above, only transmitted through generations
• Congenital: implies “born with”
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MENDELIAN DISORDERS
• Result from mutations in single genes that have large effects
• Estimated that each person is a carrier of 5 to 8 deleterious genes
• Whether a given mutation will have a adverse outcome is influenced by compensatory genes and
environmental factors
• Some autosomal mutations produce partial expression in heterozygotes and full expression in
homozygous (eg. Sickle cell disease)
• Can be dominant, recessive, or codominant
• Variable expressivity: variation in the effect caused by a particular mutation (ie: NF1 can range
from brown macules to skin tumors to skeletal deformities)
• Pleiotropism: multiple possible end effects of a single mutant gene (ie. in sickle cell disease,
mutant Hb can cause hemolysis and anemia, as well as vascular occlusion leading to splenic
infarction and bone necrosis)
• Genetic heterogeneity: multiple different mutations leading to the same outcome (ie. there are
multiple AR mutations that can result in childhood deafness
!!
TRANSMISSION PATTERNS OF SINGLE-GENE DISORDERS
Autosomal Dominant Disorders Autosomal Dominant Disorders
• Manifested in heterozygous state
• At least one parent of index case is usually affected System Disorder
Huntington disease
• M = F, both can transmit Neurofibromatosis
• Children have ½ chance Nervous
Myotonic dustrophy

• Clinical features can be modified by variations in penetrance and expressivity Tuberous Sclerosis

• Penetrance: expressed in mathematical terms (ie. 50% penetrance means that 50% GU Polycystic kidney disease
GI Familial polyposis coli
of those who carry the gene express the trait) Hereditary spherocytosis
• Expressivity: the same trait in people carrying the mutant gene can be expressed
Heme
Von Willebrand disease

differently in each person Marfan syndrome

• In many conditions, the age of onset is delayed (eg huntingtons) Skeletal

Ehlers-Danlos syndrome
Osteogenesis imperfecta
• Most mutations lead to reduced production of a gene product or give rise to an inactive Achondroplasia
protein (loss-of-function mutations)
!! Metabolic
Familial
hpercholesterolemia
Acute intermittent

Autosomal Recessive Disorders

• largest category of Mendelian disorders Autosomal Recessive Disorders

• not usually affect parents, but siblings can show disease System Disorder
• sibs have ¼ chance of having the trait Cystic fibrosis

• if mutant gene occurs with low freq in a population, is strong likelihood that the PKU
Galactosemia
proband is product of consangionous marriage Homocystinuria
• expression of defect tends to be more uniform than in AD disorders Metabolic
Lysosomal storage diseases

• complete penetrance is common A1-antitrypsin deficiency

• onset freq early in life

Wilson disease
Hemochromatosis
• new mutations with recessive disorders are rarely detected clinically (b/c Glycogen storage diseases
heterozygotes are asymptomatic and generations can pass before affected offspring Sickle cell anemia

result) Heme
Thalassemias
Congenital adrenal
• many of the mutated genes code enzymes hyperplasia
Ehlers-Danlos syndrome
• includes all inborn errors of metabolism
Skeletal

!!
(some variants)
Alkaptonuria
Neurogenic muscular
atrophoes
Nervous Friedrich ataxia
Spinal muscular atrophy
X-Linked Disorders
• all are sex linked and almost all are recessive X-Linked Recessive Disorders

• no corresponding gene on the Y, therefore the male is hemizygous for all X-linked
System Disease
mutant genes Duchenne muscular dyst
• heterozygous female not usually express full phenotype due to paired normal allele Hemophilia A and B
• females have variable proportion of cells where mutant X chromosome is active due MSK
Chronic granulomatous

to random inactivation of one of the X chroms (so remotely possible for normal dis
G-6-P dehydrogenase def

allele to be inactivated in most cells, allowing expression of heterozygous x-linked Immune

Agammaglobulinemia

condition Wiskott-Aldrich
syndrome
Diabetes insipidus
• transmitted by an affected heterozygous female to half her sons and half her daughters Metabolic Lesch-Nyhan syndrome
but none of his sons if the female is unaffected
! !
Fragile X Syndrome

!
BIOCHEMICAL AND MOLECULAR BASIS OF SINGLE-GENE (MENDELIAN) DISORDERS
• any type of protein can be affected
• mechanisms involved in single gene disorders can be classified into four categories:
!
1. Enzyme Defects and Their Consequences
• mutations may result in synthesis of a defective enzyme with reduced activity or in a
reduced amount of a normal enzyme -- metabolic block

!!
2. Defects in Receptors and Transport Systems
• eg include familial hypercholesterolemia and cystic fibrosis
!
3. Alterations in Structure, Function, or Quality of Nonenzyme Proteins
• eg. Hemogloginopathies: Sickle cell disease
• osteogenesis imperfect
• hereditary spherocytosis and muscular dystrophies
!
4. Genetically Determined Adverse Reactions to Drugs
• eg. Drug induced injuries in those with deficiency of G6PD enzyme
!!
!!
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DISORDERS ASSOCIATED WITH DEFECTS IN STRUCTURAL PROTEINS
!
MARFAN SYNDROME
• a disorder of connective tissues, manifested mainly by changes in skeleton, eyes, and CVS
• 1/5000
• 70-85% familial (AD inheritance)
• Pathogenesis: from defect in extracellular glycoprotein fibrillin-1 (is a major component of microfibrils in
ECM, that provide scaffolding for elastic fibers)
• Abnormal fibrillin results in defective microfibril assembly, resulting in reduced elasticity, as well
as reduced sequestration of TGF-B, which reduces normal vascular smooth muscle development
and matrix production
• >600 distinct mutations (mostly missence) on FBN1 gene in those with Marfan syndrome (therefore
heterogeneous disease with great variation in clinical expression)
• Features:
• Eyes: bilateral subluxation or dislocation of lens (ectopia lentis), retinal detachments from
increased axial length of globe
• Skeleton: unusually tall, long extremities with long, tapering fingers and toes; lax joint ligaments;
spinal deformities (kyphosis, scoliosis, rotation or slipping of dorsal or lumbar vertebrae); pectus
excavatum or pigeon breast deformity; long head with frontal bossing and prominent
supraorbital ridges
• CVS: MVP (most common) and dilation of ascending aorta due to cystic medial necrosis
• Curaneous: striae
!!
EHLERS-DANLOS SYNDROMES (EDS)
• Clinically and genetically heterogeneous group of disorders resulting form defect in synthesis or
structure of fibrillar collagen
• All 3 mendelain patterns are shown in EDS
• Major manifestations involve:
• Skin: hyperenxtensible, extremely fragile and vulnerable to trauma; wound healing is very impaired
due to defectivve collagen synthesis
• Joints: hypermobile and prone to dislocation
• Visceral complications: rupture of colon and large arteries, ocular fragility wit corneal rupture and
retinal detachment
!
• Six major variants (see table)
!!
Classification of Ehlers-Danlos Syndromes

Inheritanc
Clinical Findings Gene Defects
e
Classical (I/II) Skin and joint hpermobility, atrophic scars, easy bruising AD COL5A1, COL5A2
Hypermobility (III) Joint hpermobility, pain, dislocations AD Unknown
Vascular (IV) Thin skin, arterial or uterine rupture, bruising, small joint hyper AD COL3A1
extensibility
Kyphoscoliosis (VI) Hypotonia, joint laxity, congential scoliosis, ocular fragility AR Lysyl hydroxylase
Athrochalasia (VIIa, Severe joint hyper mobility, skin changes (mild), scoliosis, AD COL1A1, COL1A2
b) bruising
Dermatosparaxis Severe skin fragility, cutis laxa, bruising AR Procollagen N-
(VIIc) peptidase
!!
!
DISORDERS ASSOCIATED WITH DEFECTS IN RECEPTOR PROTEINS
!
FAMILIAL HYPERCHOLESTEROLEMIA
• mutation in gene encoding LDL receptor, which is involved in
the transport and metabolism of cholesterol → loss of feedback
control → elevated cholesterol levels → premature
atherosclerosis
• one of most frequent occurring mendelian disorders
• Mutations of other aspects of LDL uptake, metabolism, and
regulation can cause similar phenotype
!
• Heterozygotes (~1/500 individuals) have from birth a two-fold
to three-fold elevation of plasma cholesterol level, leading to
tendinous xanthomas and premature atherosclerosis in adult
life
• Homozygotes are much more severely affected - may have
5-6X elevations in plasma cholesterol levels.
• develop skin xanthomas and coronary, cerebral, and
peripheral vascular atherosclerosis at an early age.
• Myocardial infarction may develop before age 20.
!
Normal Cholesterol Transport and Metabolism:
• ~7% of the body's cholesterol circulates in the plasma, mainly in form of LDL.
• Although most cells possess high-affinity receptors for LDL apoprotein B-100, 70% of plasma LDL is
cleared by liver
• Uptake by other cells (esp macrophages) can occur through distinct scavenger receptors for
chemically altered LDL (eg. Acetylated or oxidized)
!
• amount of plasma cholesterol is influenced by its synthesis and catabolism therefore liver crucial in this:
• Step 1: secretion of VLDL's (rich in TG, lesser amounts of CE) by the liver into bloodstream
• Step 2: VLDL reaches the capillaries of adipose tissue or muscle → cleaved by lipoprotein lipase →
extracts most of TG’s → IDL molecule (lower TG, enriched in CE), but retains B-100 and E
• Step 3: release from the capillary endothelium, IDL particles has one of two fates for removal from
plasma:

A. Transport and metabolism of LDL in liver involves:

• binding to specific LDL pm receptors
• Internalization → dissoc from its receptor in early
endosome → transport to lysosomes
• Lysosomal processing → release of free cholesterol (FC) into
cytoplasm through action of NPC1and 2 proteins (defects
result in NPC, Nieman Pick disease, type C)
• LDL dissociates → degraded enzymatically → protein
hydrolysis → amino acids and CE's → free cholesterol
• Free cholesterol → cytoplasm → used for membrane
synthesis and as regulator of cholesterol homeostasis
• Free cholesterol affects 3 processes:
• suppresses chol synth w/in cell by inhibiting HMG CoA
reductase (rate-limiting enz in the synthetic pathway)
• activates the acyl-coenzyme A : cholesterol
acyltransferase, favoring esterification and storage of
excess cholesterol.
• suppresses the synthesis of LDL receptors, thus
protecting the cells from excessive accumulation of
cholesterol
 B. other by a receptor for oxidized LDL (scavenger receptor)
• in part into the cells of the mononuclear phagocyte system
• Monocytes and macrophages have receptors for chemically altered (e.g., acetylated or oxidized)
LDL.
• Normally the amount of LDL transported along this scavenger receptor pathway is less than that
mediated by the LDL receptor–dependent mechanisms but in hypercholesterolemia there is a
marked increase in the scavenger receptor–mediated traffic of LDL cholesterol into the cells of
the mononuclear phagocyte system and possibly the vascular walls
• results in appearance of xanthomas and contributes to the pathogenesis of premature
atherosclerosis.
!
• complex molecular genetics: >900 mutations, including insertions, deletions, and missense and nonsense
mutations, involving the LDL receptor gene have been identified:
• Class I mutations: inadequate LDL receptor protein synthesis (rare)
• Class II mutations: abnormal LDL receptor folding leading to retention in the ER (common)
• Class III mutations: reduced binding capacity of LDL receptor protein
• Class IV mutations: inability of LDL receptor to internalize
• Class V mutations: inability of LDL and receptor to dissociate, with recycling to cell surface
!!
DISORDERS ASSOCIATED WITH DEFECTS IN ENZYMES
!
LYSOSOMAL STORAGE DISEASES
• result from genetic deficiency of functional lysosomal enzymes or other proteins essential for their
activity
• mutations can also affect the targeting of the enzymes after they are synthesized in the ER (enzymes
destined for the lysosome are tagged by appending a terminal mannose-6-P residue during transit through
the Golgi
• without proper lysosomal processing → accumulation of partially degraded metabolites within lysosomes
→ enlarged lysosomes then interfere with normal cell function
• therapeutic approaches include:
• enzyme replacement
• substrate reduction
• molecular chaperones to assist in normal folding of mutant proteins
!
TAY-SACHS DISEASE (GM2 GANGLIOSIDOSIS: HEXOSAMINIDASE Α-SUBUNIT DEFICIENCY)
• results from mutations in α subunit of hexosaminidase enzyme complex
• most common of the three GM2 gangliosidoses, due to lysosomal GM2-ganglioside accumulation
• most common in Jews of Eastern European (Ashkenazi) origin
• antenatal dx and carrier detection: DNA probe analysis and enzyme assays (amnio)
• neurons most severely affected (b/c rich in gangliosides)
• clinical features: motor and mental deterioration starting (~ 6 mo of age), blindness and death by
age 2-3 years
• Morphology:
• Neuronal ballooning with lipid-filled cytoplasmic vacuoles
• Progressive neuronal destruction with microglial proliferation
• Accumulation of lipids in retinal ganglion cells, rendering them pale in color, therefore
accentuating the normal red color of the macular choroid (cherry-red spot
!
GLYCOGEN STORAGE DISEASES (GLYCOGENOSES)
• Clinical features: affected infants appear normal at birth but begin to manifest signs and symptoms at
about age 6 months:
• relentless motor and mental deterioration (motor incoordination, mental obtundation →
muscular flaccidity, blindness, and increasing dementia)
• early in course the disease → characteristic cherry-red spot in macula of eye
 • Over 1 or 2 years → complete vegetative state, death at age 2 to 3 years
• > 100 mutations in the α-subunit gene; most affect protein folding → triggers “unfolded protein”
response → apoptosis
!
NIEMANN-PICK DISEASE, TYPES A AND B
• related disorders assoc with sphingomyelin deficiency (>100 mutations described)
• sphingomyelin accumulation most prominent in macrophages but can also affect neurons
• also common in Ashkenazi Jews
• acid sphingomyelinase is an imprinted gene expressed on maternal chromosome
• dx established by biochemical assays for sphingomyelinase activity in liver or bone marrow bx
• pts affected with types A and B (and carriers) detected by DNA analysis
Type A:
• more common, severe infantile form with extensive neurologic involvement, marked visceral
accumulations of sphingomyelin, and progressive wasting
• early death within the first 3 years of life
• affected cells engorged with numerous small vacuoles (cytoplasmic foaminess)
• Features:
• Diffuse neuronal involvement → cell death and CNS atrophy; a retinal, cherry spot in 1/2
• Extreme accumulation of lipids in macrophages → massive hepatosplenomegaly and
lymphadenopathy with BM infiltration
• Visceral involvement mainly affecting GI tract and lungs
! Type B:
• organomegaly but not CNS involvement
• usually survive into adulthood.
!
NIEMANN-PICK DISEASE, TYPE C (NPC)
• distinct biochemically and molecularly, more common than types A and B combined
• Mutations in NPC1 (95%) and NPC2 → codes for proteins in chol transport from lysosomes to cytosol
• both cholesterol and gangliosides are accumulated
• can present with hydrops fetalis, neonatal hepatitis, or progressive neurological degeneration (most
common) beginning in childhood with ataxia, vertical supra nuclear gaze palsy, dystonia, dysarthria, and
psychomotor regression
!!
GAUCHER DISEASE
• cluster of AR disorders due to mutations → diminished glucocerebrosidase activity →↓ cleavage of
ceramide (derived from cell membranes of senescent leukocyte and rbc's as well as from turnover of
brain gangliosides)
• most common lysosomal storage disorder
• glucocerebroside accumulates mainly in phagocytes but also in the CNS in some subtypes
• clinical features due to to burden of stored material and macrophage activation + local cytokine
production
Three clinical subtypes:
Type 1 (chronic non-neuronopathic):
• most common form (99%)
• occurs in adults, higher incidence in European Jews
• reduced but detectable levels of glucocerebrosidase activity
• chronic, non-neuronopathic form assoc w/ glucocerebroside storage in mononuclear
phagocytes
• no brain involvement but massive splenomegaly and lymphadenopathy
• marrow involvement → bone erosions → pathologic fractures
• pancytopenia or thrombocytopenia due to hypersplenism
• longevity shortened but not markedly
!
 Type II (acute neuronopathic):
• affects infants, no predilection for Jews
• no detectable glucocerebrosidase activity in tissues
• clinically: hepatosplenomegaly; mainly progressive CNS involvement → death at early age
!
Type III (intermediate between types I and II)
• systemic involvement of macrophages characteristic of type I but progressive CNS disease
(usually begins in adolescence or early adulthood)
! Morphology:
• affected cells (gaucher cells): distended with PAS positive material
• fibrillary appearance resembling "crumbled tissue paper" (composed of elongated
lysosomes containing stored lipid in bilateral stacks)
! Clinical Features:
• Depends on the clinical subtype:
• Type I: sx and signs first appear in adult life → related to splenomegaly or bone involvement
• most commonly pancytopenia or thrombocytopenia due to hypersplenism
• pathologic fractures and bone pain → if extensive expansion of marrow space
• compatible with long life
• Types II and III: CNS dysfunction, convulsions, and progressive mental deterioration mainly
• although liver, spleen, and lymph nodes also affected
• prenatal dx: enzyme assay of amniotic fluid or DNA probe analysis
• replacement tx with recombinant enzymes → main treatment
!!
MUCOPOLYSACCHARIDOSES
• group of closely related syndromes due to inherited deficiencies of enzymes that degrade
glycosaminoglycans (abundant in ECM of connective tissues)
• accumulated substrates include dermatan sulfate, heparan sulfate, keratan sulfate, and chondroitin sulfate
• absence of enzymes → polysaccharide chains accumulate within lysosomes in various tissues and organs
• several clinical variants: from MPS I to MPS VII, based on differing enzyme deficiency
• all a AR except MPS II (Hunter disease - X-linked recessive)
• generally, MPSs are progressive disorders, characterized by:
• coarse facial features
• hepatosplenomegaly
• clouding of the cornea
• valve and sub endothelial arterial thickening
• joint stiffness
• mental retardation
! Morphology: affected cells distended with clear cytoplasm (balloon cells) with PAS positive material
• accumulated MPS in many cell types incl macrophages, fibroblasts, endothelial cells, initial
smooth muscle cells, and neurons
• Urinary excretion of the accumulated mucopolysaccharides is often increased
!!
GLYCOGEN STORAGE DISEASES (GLYCOGENOSES)
• result from hereditary deficiencies in synthesis or catabolism of glycogen
• may be restricted to specific tissues or can be systemic
• based on specific enzyme deficiencies and resultant clinical pictures → divided into 3 major groups:
! 1. Hepatic forms: due to enzyme deficiencies primarily in liver glycogen metabolism
• hypoglycemia and hepatic glycogen accumulation (hepatomegaly)
 • prototype = Von Gierke disease (type 1) due to G6P deficiency (converts G6P to
glucose)
• others include deficiencies in liver phosphorylase or
debranching enzyme
• clinical: hypoglycemia, hyperlipidemia (gout, skin
xanthomas), bleeding (plt dysfunction)
• with tx: most survive and develop late complications
(eg. hepatic adenomas)
! 2. Myopathic form: deficiencies in glycolysis enzymes in striated
muscle
• present with mm weakness and cramping after exercise
w/o exercise-induced rises in blood lactate
• skeletal mm show glycogen accumulation
• McArdle disease (type V): due to deficient muscle
phosphorylase
• Clinical: painful cramps with strenuous exercise;
myoglobinuria in 50%, elevated serum CK; pts have
normal longevity
!
3. Miscellaneous forms: assoc with α-glucosidase (acid maltase)
or lack of branching enzyme (do not fit into hepatic or myopathic categories)
• glycogen overload (lacy cytoplasmic pattern) in many organs and death early in life
• type II glycogenosis, or Pompe disease: due to deficiency of lysosomal acid maltase
• many organs involved but cardiac involvement is most prominent
• Clinical: mild hepatomegaly, massive cardiomegaly, muscle hypotonia and cardioresp
failure within 2 years
• milder adult form with only skeletal mm involvement
!!
ALKAPTONURIA (OCHRONOSIS)
• autosomal recessive disorder
• lack of homogentisic oxidase (homogentisic acid → methylacetoacetic acid in tyrosine degradation
pathway)
• homogentisic acid accumulates in the body:
• urinary excretion → black color to urine if allowed to stand and undergo oxidation
• ochronosis: blue-black pigmentation of ears, nose, and cheeks (due to binding of
homogenistic acid to connective tissue)
• arthropathy: deposition in articulate cartilage; affected cartilage (typically vertebral column,
knee, shoulders, and hips) loses resilience and readily eroded
• metabolic defect present from birth, but degenerative arthropathy not clinically evident until the 30s
• not life-threatening but may be severely crippling
• arthropathy may be as extreme as severe forms of OA in elderly, but occurs at much earlier age
!!
!
COMPLEX MULTIGENIC DISORDERS
• polymorphism: genetic variant that has at least two alleles and occurs in at least 1% of the population
• complex genetic disorders:
• occur when many polymorphisms, each with a modest effect and low penetrance, are inherited
• result from collective inheritances of many polymorphisms
• different polymorphisms vary in significance
• Eg: of the 20–30 genes implicated in type I diabetes, 6–7 are most important, and a few
HLA-alleles contribute >50% of the risk
 • some polymorphisms are common to multiple diseases of the same type; others are disease
specific (eg. In immune-mediated inflammatory disease)
• environmental influences → modify phenotypic expression of complex traits
• Eg. type II diabetes mellitus often becomes clinically apparent after weight gain, so obesity as
well as other environmental influences unmasks the diabetic genetic trait
• range severity levels of a disease →suggests complex multigenic disorder (but variable expressivity and ↓
penetrance of single mutant genes may also account for this phenomenon)
!!
CHROMOSOMAL DISORDERS
!
NORMAL KARYOTYPE
• study of chromosomes = karyotyping
!
NUMERICAL DISORDERS
• euploid: any exact multiple of the haploid number
• aneuploidy: an error in meiosis or mitosis where a cell acquires a chromosome complement that is not
an exact multiple of 23
• usual causes for aneuploidy:
• nondisjunction: occurs during gametogenesis where gametes formed have either an extra
chromosome (n + 1) or one less chromosome (n − 1)
• Fertilization of such gametes by normal gametes results in two types of zygotes →
trisomic (2n + 1) or monosomic (2n − 1)
• anaphase lag: one homologous chromosome in meiosis or one chromatid in mitosis lags
behind and is left out of the cell nucleus
• results in one normal cell and one cell with monosomy
• monosomy or trisomy involving sex chromosomes → compatible with life (usually
assoc with variable degrees of phenotypic abnormalities)
• Monosomy: involves an autosome
• generally too much genetic info lost to permit live birth or even embryogenes
• of those that permit survival → severely handicapped infants with death at early age (except
Trisomy 21)
• Mosaicism: mitotic errors in early development → two or more populations of cells with different
chromosomal complements in the same individual
• can occur from mitotic errors during cleavage of fertilized ovum OR in somatic cells:
• affecting sex chromosomes (relatively common):
• fertilized ovum division → error may lead to one of the daughter cells receiving
three sex chromosomes, whereas other receives only one (eg. 45,X/47,XXX mosaic)
• all descendent cells have either a 47,XXX complement or 45,X complement
• autosomal mosaicism (much less common)
• usually leads to a nonviable mosaic due to autosomal monosomy
• rarely, the nonviable cell population is lost during embryogenesis → viable mosaic
(e.g., 46,XY/47,XY,+21)
!!
STRUCTURAL ABERRATIONS
• Deletion: loss of a portion of a chromosome
• Interstitial deletions: two breaks within a chromosome arm with loss of the chromosomal
material between the breaks and fusion of the broken ends
• Terminal deletions: single break in a chromosome arm → a fragment with no centromere, which
is then lost at the next cell division, and a chromosome bearing a deletion. The end of the
chromosome is protected by acquiring telomeric sequences
• translocation: segment of one chromosome is transferred to another
• balanced reciprocal translocation: single breaks in each of two chromosomes, with exchange of
material with no loss of genetic material → individual is likely phenotypically normal
 • balanced translocation carrier, however, is at ↑ risk to produce abnormal unbalanced
gametes (would not contain the normal complement of genetic material)
• subsequent fertilization by normal gamete → formation of abnormal (unbalanced)
zygote → spontaneous abortion or birth of a malformed child
• robertsonian translocation (or centric fusion): translocation between two acrocentric
chromosomes
• typically breaks occur close to centromeres of each chromosome → transfer of segments
→ one very large chromosome and one extremely small one (usually small product is
lost but since it carries only highly redundant genes (e.g., ribosomal RNA genes) →
compatible with a normal phenotype)
• can result in production of abnormal (unbalanced) gametes (eg. Down syndrome)
• Isochromosome: results when one arm (long or short) is lost and the remaining arm is duplicated → a
chromosome consisting of two short arms only or of two long arm
• has morphologically identical genetic information in both arms
• Inversion: rearrangement that involves two breaks within a single chromosome with reincorporation of
the inverted, intervening segment
• involving only one arm of the chromosome = paracentric
• If breaks are on opposite sides of the centromere = pericentric
• Inversions often fully compatible with normal development
• ring chromosome: deletion affecting both ends followed by fusion of the damaged ends
• ring chromosomes do not behave normally in meiosis or mitosis and usually result in serious
consequences

!!
!
CYTOGENETIC DISORDERS INVOLVING AUTOSOMES
!
TRISOMY 21 (DOWN SYNDROME)
• most common chromosomal disorder
• major cause of mental retardation, ~1 in 700
• ~95% have extra chromosome 21
• of these, 95% result from the extra chromosome begin maternal in origin (secondary to
meiotic nondisjunction, which is influenced by maternal age)
• ~4% have extra chromosomal material derived from a parental chromosome with translocation of
long arm of chrome 21 to chrome 22 or 14
• since fertilized ovum already possesses two normal autosomes 21, the translocated fragment
provides the same tuple-gene dosage as tiresome 21
• often (but not always) familial, b/c parent is a carrier of Robertsonian translocation (maternal
age has no impact)
 • 1% due to mosaic variants: mixture of cells with normal chromosome numbers and cells with an
extra chromosome 21 (maternal age has no impact)
• Sx are variable and milder, depending on the proportion of abnormal cells.
!
• diagnostic clinical features:
• flat facial profile, oblique palpebral fissures, and epicanthic folds; simian hand creases
• severe mental retardation; ~ 80% of those afflicted have an IQ of 25 to 50. .
• some mosaics with Down syndrome have mild phenotypic changes and often even have normal
or near-normal intelligence.
• ~ 40% have congenital heart disease,
most commonly defects of the
endocardial cushion, including
ostium primum, atrial septal defects,
atrioventricular valve malformations,
and ventricular septal defects
• Cardiac problems are responsible
for the majority of the deaths in
infancy and childhood
• 10-fold to 20-fold increased risk of
developing acute leukemia.
• Premature Alzheimer's disease
• abnormal immune responses that
predispose them to serious infections,
particularly of the lungs, and to
thyroid autoimmunity.
• median age at death is 47 years (up from 25
years in 1983).
!
Other Trisomies
• trisomy 18 (Edwards syndrome) and trisomy
13 (Patau syndrome):
• share several karyotypic and clinical
features with trisomy 21
• most due to meiotic nondisjunction and
carry complete extra copy of chrom 18 or
13
• assoc with increased maternal age
• malformations much more severe and
wide-ranging → infants rarely survive past
first year of life (most die within weeks to
months.)
!!
!!
!
CHROMOSOME 22Q11.2 DELETION SYNDROME
• fairly common (1 in 4000 births); due to small deletion of band 11.2 on long arm of chromosome 22, but
often missed because of variable clinical features
• Clinical features: spectrum that includes DiGeorge syndrome (t-cell immunodeficiency and
hypocalcemia) and velocardiofacial syndrome (facial days morphology and cardiac malformations):
• congenital heart defects
• abnormalities of the palate
• facial dysmorphism
• developmental delay
 • variable degrees of T-cell immunodeficiency
• Hypoparathyroidosm
• Increased incidence of psychiatric illnesses (schizophrenia, bipolar, ADHD)
• dx may be suspected on clinical grounds; can be established only by detection of the deletion by FISH
• molecular basis of this syndrome is not fully understood
!!
CYTOGENETIC DISORDERS INVOLVING SEX CHROMOSOMES
• imbalances (excess or loss) of sex chromosomes are much better tolerated than are similar imbalances of
autosomes and more common
• The milder nature of X chromosome-associated aberrations, is related to fact that there is normally
random inactivation of one X chromosome (Lyon hypothesis):
• random inactivation of either paternal or maternal X chromosome occurs early in embryogenesis
and leads to formation of a Barr body
• Normal females are functional mosaics with two cell populations, one with an inactivated
paternal X chromosome and the other with a inactivated maternal X chromosome
• With extra chromosomes, all but one X chromosome is inactivated
• Because numerical aberrations of X chromosomes (extra or missing) are nevertheless associated with
somatic and gonadal abnormalities, the Lyon hypothesis is modified as follows:
• Both X chromosomes are required for normal gamete formation
• X inactivation spaces certain regions of the chromosome necessary for normal growth and
development; up to 20% of the genes on the short arm of any "inactivated" X chromosome
escape inactivation
• The Y chromosome is both necessary and sufficient for male development
• Regardless of the number of X chromosomes, the presence of a single Y drives development toward the
male sex
• Features of sex linked disorders:
• In general, they cause subtle, chronic problems relating to sexual development and fertility
• often difficult to diagnose at birth, and many are first recognized at the time of puberty
• In general, the higher the number of X chromosomes, in both male and female, the greater the
likelihood of mental retardation.
!
KLINEFELTER SYNDROME
• Is male hypogonadism that occurs when there are two or more X chromosomes and at least one Y
chromosome
• one of most frequent forms of genetic disease involving the sex chromosomes and one of most common
causes of hypogonadism in the male
• incidence ~1 in 660 live male births, 47, XXY most common (90%)
• Characteristic features:
• Male infertility and reduced spermatogenesis
• eunuchoid body habitus
• atrophic testes
• Gynecomastia with 20 x increased risk of breast cancer relative to normal males, also of germ
cell tumors and autoimmune diseases
• Female distribution of hair with failure of secondary male characteristics
• mean IQ is somewhat lower than normal, but mental retardation is uncommon
• increased incidence of type 2 diabetes and the metabolic syndrome; and mitral valve prolapse is
seen in about 50% of adults with Klinefelter syndrome
• clinical features of this condition are variable, the only consistent finding being
hypogonadism
• Plasma FSH and estrogen levels elevated; testosterone levels reduced
• Maternal age is increased in the cases associated with errors in oogenesis
• ~15% of patients with Klinefelter syndrome have been found to have a variety of mosaic patterns, most of
them being 46,XY/47,XXY. Other patterns are 47,XXY/48,XXXY and variations on this theme. As is the
case with normal females, all but one X undergoes inactivation in patients with Klinefelter syndrome.
• Hypogonadism and other clinical features explained by pattern of X inactivation
!
TURNER SYNDROME
• is hypogonadism in phenotypic females
• results from complete or partial monosomy of the X chromosome
• most common sex chromosome abnormality in females (~1 in 2000 live-born females).
• three types of karyotypic abnormalities; explaining heterogeneity of phenotype:
• ~57% are missing an entire X chromosome, resulting in a 45,X karyotype.
• Of the remaining 43%:
• 1/3 have structural abnormalities of the X chromosomes (ie. partial delations)
• 2/3 are mosaics

Clinical features:
• lymphedema of neck, hands and feet
• neck webbing (due to lymphatic dilation during development)
• Congenital heart disease (25% to 50%), esp coarctation of aorta
• failure to develop normal secondary sex characteristics.
• mental status of these patients is usually normal, but subtle
defects in nonverbal, visual-spatial information processing have
been noted.
• shortness of stature (rarely exceeding 150 cm in height)
• Turner syndrome is the single most important cause of primary
amenorrhea
• severely atrophic and fibrous ovaries (streak ovaries)
• 50% develop autoantibodies to thyroid gland → 50% develop
hypothyroidism.
• glucose intolerance, obesity, and insulin resistance in a
minority of patients
!
• Hypogonadism and absence of secondary sexual maturation occurs
because both X chromosomes are necessary for normal oogenesis and ovarian development
• Affected pts have an accelerated loss of oocytes and basically undergo menopause before they
experience menarche
• Short stature comes from loss of both (expressed) copies of the short stature homeobox gene (SHOX) on
the X chromosome, effecting height
• Mechanisms for cardiac malformations are unknown
!
HERMAPHRODISM AND PSEUDOHERMAPHRODITISM
• Genetic sex determined by the presence or absence of a Y chromosome
• The initially indifferent gonads of both the male and the female embryos have an inherent tendency to
feminize, unless influenced by Y chromosome–dependent masculinizing factors
• Gonadal sex is based on the histologic characteristics of the gonads
• Ductal sex depends on the presence of derivatives of the müllerian or wolffian ducts
• Phenotypic, or genital, sex is based on the appearance of the external genitalia
• Sexual ambiguity is present whenever there is disagreement among these various criteria for determining
sex
• True hermaphroditism: implies presence of both ovarian and testicular tissue (either combined as an
ovotestis or with one gonad on each side), extremely rare
• karyotype is 46,XX in 50% of patients; of the remaining, most are mosaics with a 46,XX/46,XY and
rarely 46,XY occurs
• Testis in a 46,XX individual implies cryptic chimerism or the SRY gene (dictates testicular
differentiation) or possibly a Y-to-autosome translocation
!
• Female pseudohermaphroditism: have 46,XX, karyotype with normal ovaries and internal genitalia
 • most common cause is androgenic steroid exposure during gestation (eg. congenital adrenal
hyperplasia or androgen secreting maternal tumors
!
• Male pseudohermaphroditism: most complex of all disorders of sexual differentiation
• individuals possess a Y chromosome, and thus their gonads are exclusively testes, but the genital
ducts or the external genitalia are either ambiguous or completely female
• Male pseudohermaphroditism is extremely heterogeneous, with a multiplicity of causes
• results from defective virilization of the male embryo due to reduced androgen synthesis or resistance
to action of androgens
• most common form is complete androgen insensitivity syndrome (testicular feminization), an X-
linked disorder associated with mutations in the androgen receptor gene located at Xq12,
!!
SINGLE-GENE DISORDERS WITH NONCLASSIC INHERITANCE
• certain single-gene disorders does not follow classic mendelian principles. This group of disorders can be
classified into four categories:
• Diseases caused by trinucleotide-repeat mutations
• Disorders caused by mutations in mitochondrial genes
• Disorders associated with genomic imprinting
• Disorders associated with gonadal mosaicism
!
DISEASES CAUSED BY TRINUCLEOTIDE-REPEAT MUTATIONS
• ~40 diseases, associated with the expansion of stretches if nucleotides
• includes Huntington disease, myotonic dystrophy, fragile-x syndrome and multiple types of
spinocerebellar ataxia
• neurodegenerative diseases dominate the clinical picture
• mots of these repeats contain guanine (G) to cytosine (C) nucleotide, and can occur in non-coding
regions (fragile X) or coding regions (Huntington)
• expansion in coding regions (typ CAG trinucleotides) lead to production of poylglutamine tracts in the
proteins and subsequent aberrant folding with aggregation (with large intranuclear inclusions),
mitochondrial dysfunction, an unfolded protein stress response, and apoptosis →characterized by
progressive neurodegeneration, typically striking in midlife
• expansions in non-coding regions suppress the synthesis of the affected protein → these disorders can
affect many systems
• the proclivity for trinulceotide expansion depends on the sex of the transmitting parent; in fragile-X,
expansions occur in oogenesis, vs Huntingtons, occur on spermatogenesis

!
FRAGILE-X SYNDROME
• Fragile-X syndrome is the prototype of diseases in which the mutation is characterized by a long repeating
sequence of three nucleotides
• common cause of familial mental retardation (second most common after Down syndrome
• characterized cytogenetically by a “fragile site” on Xq27.3 visualized as a discontinuity of chromosomal
stain gin when cells are grown in folate-deficient medium
• frequency of 1 in 1550 for affected males and 1 in 8000 for affected female
• the site on Xq has multiple CGG nucleotide repeats in the 5’ untranslated region of the familial mental
retardation-1 (FMR-1) gene.
• normal pts: have ave of 29 (range 6 to 55) repeats vs affected pts have 200 to 4000 repeats
• pts with permutations (clinically solent) have 55 - 200 CGG repeats
• in carrier females, the permutations undergo amplification during oogenesis → full mutations that are
then passed on to offspring
• anticipation: worsening clinical presentation with succeeding generations
!
• since mutations are carried on X chromosome (is X linked recessive disorder) but transmission pattern is
different than classic X-linked patterns since permutations are amplified during oogenesis
• carrier males with permutations do not typically have sx and do not transmit the disease
• but ~50% of daughters of carrier females are affected
• carrier females are also affected at 30-50% freq (higher then typical X-linked disorders)
• expansion of the trinucleotide repeats in FMR-1 beyond 230 copies leads to abnormal gene methylation
and transcriptional suppression
• molecular basis of Fragile X syndrome is related to loss of function of the FMR protein (FMRP), a
cytoplasmic protein abundant in brain and testis
• FMRP is an RNA-binding protein associated with polyribosomes - suppresses the translation of
certain transcripts at synaptic junctions
• loss of FMRP - leads to increased protein translation, and resulting imbalance adversely a fetes
neurone function with permanent changes in synaptic activity
• affected males have severe mental retardation, and 80% have enlarged testes. Other features: elongated
face and large mandible, are inconsistent
• carriers of permutations also develop premature ovarian failure (females) and progressive
neurodegeneration (males)
!!
MUTATIONS IN MITOCHONDRIAL GENES – LEBER HEREDITARY OPTIC NEUROPATHY
• vast majority of genes are located on chromosomes in the cell nucleus and are inherited in classical
Mendelian fashion
• ova contain multiple mitochondria, whereas spermatogonia contain few → the mito contact of zygotes is
derived almost entirely from the ovum (and sperm mito are selectively degraded)
• mtDNA is transmitted entirely by females, and diseases resulting form mutations in mito genes are
maternally inherited
• affected females transmit the disease to all their male and female offspring; daughters and not sons pass
the disease further along to progeny
• expression of disorders resulting from mutations in mito genes is unpredictable:
• when a cell carrying normal and mutant mtDNA divides, the proportion of normal and mutant DNA
in the daughter cells is random and quite variable (heteroplasmy)
• is also a threshold effect related to a minimum number of mutant mtDNA required to see oxidative
dysfunction
• mtDNA encodes 22 tRNAs, 2 rRNAs, and 13 genes for protiens involved in oxidative p-lation ∴
mutations mainly affect organs heavily dependent on mito energy metabolism (neuromusclular system,
liver, heart, kidney)
• prototype → Leber hereditary optic neuropathy:
• progressive bilateral blindness, neurologic dysfunction, and cardiac conduction defects
!!
GENOMIC IMPRINTING
• is an eligenetic process resulting in differential inactivation of either maternal or paternal alleles of certain
genes;
• maternal imprinting refers to transcriptional silencing of the maternal allele
• paternal imprinting implies that the paternal allele is inactivated
• Imprinting occurs in the ovum or the sperm, before fertilization, and then is stably transmitted to all
somatic cells through mitosis
• involves differential DNA methylation or histone H4 deacetylation, leading to selective gene inactivation;
200 to 600 genes are estimated to be imprinted, and although some may occur in isolation, most are
clustered in groups related by common cis-acting elements
!!
PRADER-WILLI SYNDROME AND ANGELMAN SYNDROME
• uncommon genetic disorders caused by deletion of neighbouring regions on chromosome 15 (15q12)
• in this region there are both maternally and paternally imprinted genes (genomic imprinting)
!
Prader-Willi syndrome:
• mental retardation, short stature, hypotonia, profound hyperphagia, obesity, small hands and feet,
and hypogonadism
• occurs when the paternal 15q12 region is deleted, leaving behind only the “silenced” maternal gene
product
• in some cases, an entire paternal chromosome 15 is absent, replaced instead by 2 maternally derived
(ie., silenced) chromosomes (uniparental disomy)
!
Angelman syndrome: born with a deletion of the same chromosomal region derived from their mothers
• mental retardation, short stature, ataxic gait, seizures, and inappropriate laughter ("happy puppets”)
• occurs when the maternal 15q12 region is deleted, leaving behind only the “silenced” paternal gene
product
• can also occur through uniparetnal disomy
• affected paternally imprinted gene is UBE3A - codes for ubiquitin protein ligase with role in directing
proteasomal degradation of variety of intracellular proteins in particular regions of the brain
(converse genes in Prader-Willi are not known)

!!
!
GONADAL MOSAICISM
• results from a mutations that selectively affect cells embryologically destined to form gonads
• since germ cells are affected, one or more offspring can manifest disease even though somatic cells are
uninvolved and the affected individual is phenotypically normal
!!
!!
!!
!
MOLECULAR DIAGNOSIS OF GENETIC DISEASES
• The molecular diagnosis of inherited diseases at the nucleic acid level has distinct advantages over other
surrogate techniques:
• Molecular assays are remarkably sensitive (eg. PCR allows several million-fold amplification of
minute amounts of DNA or RNA.
• DNA-based tests are not dependent on a gene product that may be produced only in certain
specialized cells (e.g., brain) or expression of a gene that may occur late in life. Because the
defective gene responsible for inherited genetic disorders is present in germ line samples, every
postzygotic cell carries the mutation.
!
INDICATIONS FOR ANALYSIS OF GERM LINE GENETIC ALTERATIONS
• dx of germ line diseases involves conventional cytogenetics, fluorescent in situ hybridization (FISH), or
molecular analysis.
• Prenatal genetic analysis: performed on fetal cells obtained by amniocentesis, on chorionic villus
biopsy or from umbilical cord blood, is indicated for:
• advanced maternal age (>35 years)
• parent with a structural chromosomal abnormality (eg. robertsonian translocation, or
inversion)
• previous child with a chromosomal abnormality
• A fetus with ultrasound-detected abnormalities
• carrier of an X-linked genetic disorder (to determine fetal sex)
• Abnormal levels of AFP, βHCG, and estriol performed as the triple test.
!
• Postnatal genetic analysis: performed on peripheral blood lymphocytes, is indicated for:
• Multiple congenital anomalies
• Unexplained mental retardation and/or developmental delay
• Suspected aneuploidy (e.g., features of Down syndrome)
• Suspected unbalanced autosome (e.g., Prader-Willi syndrome)
• Suspected sex chromosomal abnormality (e.g., Turner syndrome)
• Suspected fragile-X syndrome
• Infertility (to rule out sex chromosomal abnormality)
• Multiple spontaneous abortions (to rule out the parents as carriers of balanced translocation;
both partners should be evaluated)
!!
INDICATIONS FOR ANALYSIS OF ACQUIRED GENETIC ALTERATIONS
• Diagnosis and management of malignancy:
• Detection of tumor-specific acquired mutations and cytogenetic alterations that are the hallmarks
of specific tumors (e.g., BCR-ABL1 in chronic myeloid leukemia or CML)
• Determination of clonality as an indicator of malignancy
• identification of specific genetic alterations that can direct therapeutic choices (e.g., HER2/Neu
[official name ERBB2] in breast cancer or EGFR mutations in lung cancer)
• Determination of treatment efficacy (e.g., presence of residual disease)
• Detection of therapy resistant mutants of a given tumor
!
• Diagnosis and management of infectious disease:
• Detection of specific microorganism (e.g., HIV, mycobacteria, HPV, etc)
• identification of specifically drug resistant microbes
• Determination of treatment efficacy (e.g., residual viral loads in HIV and hep C infection)
!!
PCR AND DETECTION OF DNA SEQUENCE ALTERATIONS
• Diagnosis of genetic diseases by recombinant DNA technology can by accomplished by:
• Direct methods involving sequencing of mutant genes
 • Indirect methods without direct sequencing and involving association with other markers, such
as restriction fragment length
!
Direct Detection of DNA Sequence Alterations by Sequencing
• When a causal gene is known or suspected, direct sequencing may be most efficacious approach
• Has identified mutations in multiple genetic disorders
• Recessive diseases usually have only a small number of recurrent mutations
• Dominant disorders can have mutations across the entire coding region
• Speed and cost have previously limited genome-wide sequencing, although newer techniques may soon
enable routine sequencing of entire individual genomes
!!
!
Detection of DNA Mutations by Indirect Methods
• Detection of DNA mutations by indirect methods has the benefits of lower costs and higher throughput
• Altered DNA restriction sites result in different-sized products (visualized on gel electrophoresis) when
DNA from normal or affected individuals is digested by specific restriction enzymes
• Analyses wi fluorescently tagged oligonucleotides that differential.y hybridize with normal or specific
mutant genes can also be extended to fluorescently tagged nucleotides that differential,y incorporate into
PCR-amplified normal or mutant sequences. Tis approach pillows the detection of mutant DNA even in
heterogeneous mixtures
• Real time PCR with oligonucleotide probes induce differential elongation of mutant versus normal
sequences
• Mutations affect the length of DNA (eg., deletions or expansions); these can be detected by restriction
fragment digestion or by PCR analysis (eg., expanded trinucleotide elects in fragile X syndrome)
!!
POLYMORPHIC MARKERS AND MOLECULAR DIAGNOSIS
• When the specific gene related to a particular disease is not known, or of multiple genes contribute to a
phenotype, surrogate markers in the genome can be used instead to identify risk.
• Such linkage analysis assumes that marker loci near disease alleles will be transmitted through the
pedigrees (linkage disequilibrium)
• Eventually a disease "haplotype" can be defined by a panel of marker loci that co-segregate with the
putative disease allele(s)
• This approach assumes a large enough pedigree of normal and affected family members to allow
statistical linkage of markers to disease
• Marker loci in linkage studies are naturally-occurring polymorphisms, that is, normal variants in DNA
sequences. These include:
• Single nucleotide polymorphisms (SNPs): occur at a frequency of ~1 in 1000 base pairs, in both
introns and exons. They serve as a physical landmark in the genome and are stably transmitted
through generations
• Repeat-length polymorphisms: are represented by micro satellite repeats (repeats of 2 to6 base pairs),
usually less than 1 kb in length). The lengths of such repeats is variable in the population but are
stably transmitted across generations, so they can be linked to putative disease alleles. Moreover,
they are easy to analyze by gel electrophoresis with PCR primers that flank the repeat sequences
!
Polymorphisms and Genome-Wide Analysis (GWAS)
• Classical linkage analysis is limited when a disease allele has low penetrance or is only one of several
genes that contribute to a multifactorial phenotype.
• This problem can be circumvented by GWAS that study the linkage of genetic variants (SNPs and repeat
polymorphisms) among large cohorts in the general population with and without disease (rather than
families)
• In GWAS, polymorphisms that are over-represented in a disease population are assumed to link to casual
candidate genes. This approach has been Mae possible by:
 • Haplotype maps (HapMaps) that provide linkage disequilibrium patterns for major ethnic groups,
allowing small numbers of scattered polymorphisms to stand in as markers representing much larger
portions of the genome
• High-density chip-based methods where up to one million SNPs can be sequenced at a time
• Sophisticated low cost compute capacity
!!
MOLECULAR ANALYSIS OF GENOMIC ALTERATIONS
Array-Based Comparative Genomic Hybridization (Array CGH)
• Genetic lesions with large deletions, duplications, or more complex rearrangements typically require
otter diagnostic approaches besides PCR Nadine direct sequence analyses:
!
Southern blotting:
• DNA is digested with restriction enzymes; after electrophoresis, the fragments are hybridized with a
nucleotide probe against the genetic region of interest and compared with patterns from normal
individuals
!
FISH:
• fluorescently labelled nucleotide probes are used to "paint" all or parts of chromosomes, enabling the
detection of aneuploidy or complex genetic re combinations
!
Array-based comparative genomic hybridization (array CGH):
• an approach used when the specific genetic abnormality is not known;
• test DNA and reference (normal) DNA are labelled with different fluorescent dyes (eg. Green and
red) and then hybridized to chip-bound DNA probes that span the human genome (up to 100,000
probes per chip)
• If comparable amounts of DNA are present, then the fluorescent pattern is merged (in this case,
becomes yellow); if there are duplications or deletions, then one or the other colors predominate,
and the signal is green or red
!!
EPIGENETIC ALTERATIONS
Epigenetics: the study of heritable chemical modification of DNA or chromatin that does not alter the DNA
sequence itself.
• Eg: methylation of DNA, and the methylation and acetylation of histones, that do not modify the primary
DNA sequence but impact genetic expression
• epigenetic modifications are critical for normal human development—including the regulation of tissue-
specific gene expression, X chromosome inactivation, and imprinting, as well as for understanding of the
cellular perturbations in the aging process and cancer
• Analysis requires treating DNA with chemicals that convert up methylated nucleotides to a species that
can be uniquely detected or by using antibodies to precipitate modified histones and then sequence the
associated DNA
!
RNA ANALYSIS
• although mRNA is overall less stable than DNA, sequencing mRNA expression patterns can be useful for:
• quantification of RNA viruses such as HIV and hepatitis C virus
• Chromosomal translocation so where the break point is scattered over a large stretch of intronic
sequence; rearrangements may be more readily detected after DNA slicing to make RNA
!!
!!
!!
!
Questions:
!
1. List some features about autosomal dominant disorders:
• Usually (but not always) at least one parent of the "index" person (the person with the disease) is
affected. Sometimes a patient will get the disease from a new mutation in the egg or sperm from the
parents - in which case, the parents would not be affected.
• Males and females are equally affected.
• If an affected person mates with an unaffected person, every child has a 50/50 chance of getting the
disease.
• The clinical features can vary from person to person due to variations in penetrance (some people
can inherit the mutant gene, but not express the disease) and expressivity (people with the mutant
gene can express different clinical symptoms).
• Sometimes, the age at onset is delayed, and symptoms may not appear until adulthood.
!
2. List some AD diseases:
Neurologic diseases:
• Huntington disease
• Neurofibromatosis
• Myotonic dystrophy
• Tuberous sclerosis
Renal diseases:
• Polycystic kidney disease
Gastrointestinal diseases:
• Familial polyposis coli
Hematopoietic diseases:
• Hereditary spherocytosis
• von Willebrand disease
Skeletal diseases:
• Marfan syndrome
• Ehlers-Danlos syndrome
• Osteogenesis imperfecta
• Achondroplasia
Metabolic diseases:
• Familial hypercholesterolemia
• Acute intermittent porphyria
!
3. Fragile X syndrome is one of a group of genetic diseases called trinucleotide repeat diseases. List some
features about fragile X:
• Trinucleotide repeat diseases have mutations consisting of long, repeating DNA sequences of
trinucleotides.
• In fragile X, there is a constriction in the long arm of the X chromosome that makes the chromosome
look broken (it's not really).
• Males with fragile X generally have an IQ in the range of 20-60.
• Males also have a characteristic facies (long face, with a large mandible, and large everted ears),
and macro-orchidism.
• Females have a different clinical syndrome; less than 50% are clinically affected (but many have
premature ovarian failure).
• Fragile X, like other trinucleotide repeat disorders, shows "anticipation" (the clinical features tend to
be worse in each successive generation).
!
4. Epigenetics is the study of inheritable DNA changes that don't alter the actual structure of the DNA
itself. A good example of this is methylation modification.
!
Gene expression is often correlated with the amount of methylation of DNA. Often, if you have too much
methylation, it silences a gene. One example of this is fragile X syndrome, in which hypermethylation
silences the FMR1 gene.
!
People can inherit different propensities for methylation - which would mean that certain genes might be
expressed less in one person, and more in another person.
!
You can't detect methylation by regular DNA sequencing; you have to use special techniques.
!
4.There are all kinds of techniques that can be used to take a look at a patient's chromosomes (or specific
DNA sequences): conventional cytogenetics (karyotyping), fluorescent in-situ hybridization (FISH), and
molecular assays (like PCR). You can order these tests to look for germline abnormalities (like Down
syndrome) or acquired genetic abnormalities (like cancer).
!
For germline abnormalities, the indications for ordering these tests fall into prenatal and postnatal groups:
!
Prenatal analysis (can use cells from amniocentesis, chorionic villus biopsy material, or umbilical cord
blood):
• Moms of advanced age (>35) (because of the risk of trisomies, especially Down syndrome)
• Parent with translocation
• Parent with previous child with chromosomal abnormality
• Fetus with ultrasound-detected abnormalities
• Parent who is a carrier of an X-linked genetic disorder (to determine fetal sex)
• Abnormal triple test (AFP, beta HCG, estriol)
Postnatal analysis (usually use peripheral blood lymphocytes):
• Multiple congenital abnormalities
• Unexplained developmental delay
• Suspected genetic disorder (e.g., features of Down, Prader-Willi, Turner, or fragile X syndrome)
• Infertility (to rule out sex chromosomal abnormality)
• Multiple spontaneous abortions (to rule out parents as carriers of translocations)
!
5.Klinefelter syndrome is a genetic disorder in which patients have two or more X chromosomes and one or
more Y chromosomes (usually, patients have a 47,XXY karyotype). It's one of the most common
causes of hypogonadism in males. Some features of the disease you should know about:
• It's common (1 in 660 live male births).
• It can't usually be diagnosed before puberty (because the testicular abnormality doesn't usually
develop until puberty).
• Most patients have abnormally long legs, small atrophic testes, and lack of secondary male sex
characteristics such as a deep voice, beard, and male distribution of body hair.
• Patients have an increased incidence of type 2 diabetes and mitral valve prolapse.
• Patients have reduced spermatogenesis and may be infertile.
!
6. List some facts about Down syndrome

• Down syndrome is the most common genetic cause of mental impairments and the most common
chromosomal disorder
• The incidence is much higher in moms over 35
• Almost all patients have trisomy 21 (three chromosome 21s), the most common cause being meiotic
non-disjunction (the two chromosomes don't separate properly in one of the two meiotic cell
divisions - usually meiosis 1).
• Characteristic facial features are evident very early (even at birth): a flat facial profile, oblique
palpebral fissures, epicanthic (inner-eye) folds.
• Almost half of patients with Down syndrome have congenital heart disease.
• The risk of leukemia is 10 to 20 times normal.
• Most patients over 40 develop neurologic changes very similar to those in Alzheimer disease.
 • The median age of death is 47 years (it was 25 years in 1983).
!
7. List the 6 main types of Ehlers-Danlos (a genetic disorder caused by a defect in the synthesis or structure
of collagen):
• Classical (I/II): Skin and joint hypermobility, easy bruising
• Hypermobility (III): Joint hypermobility, pain, dislocations
• Vascular (IV): Thin skin, arterial or uterin rupture, bruising
• Kyphoscoliosis (VI): Hypotonia, joint laxity, congenital scoliosis, ocular fragility
• Arthrochalasia (VIIa,b): Severe joint hypermobility, scoliosis, bruising
• Dermatosparaxis (VIIc): Severe skin fragility, cutis laxa, bruising
All are autosomal dominant except Kyphoscoliosis and Dermatosparaxis, which are autosomal recessive.
!
8.Marfan syndrome is a genetic disorder of connective tissue. Most cases are transmitted in an autosomal
dominant fashion, but up to 30% arise from new mutations.
• Incidence: 1 in 5000
• Defect is in the fibrillin-1 (NOT collagen!!) gene
• Fibrillin is the major component of microfibrils, which provide scaffolding for tropoelastin in the
creation of elastic fibers. Microfibrils are particularly numerous in the aorta, ligaments, and lens-
supporting structures in the eye.
• Skeletal abnormalities are the most prominent feature of the disease. Patients are very tall, with long
extremities and long, tapering fingers and toes.
• Other features include spinal deformities, chest deformities, lens dislocation (very uncommon in
patients without Marfan syndrome), mitral valve prolapse, and aortic dilation (leading to aortic
dissection and death in almost half of the patients with Marfan syndrome).
!
9.Almost all X-linked disorders are recessive. Since mutant genes on the X chromosome don't have
corresponding alleles on the Y, X-linked recessive disorders are expressed in males (or in females
with two mutant alleles - though that's pretty dang uncommon).
!
A few things to remember about X-linked recessive disorders:
• An affected male transmits his mutant allele to all his daughters (who are then carriers of the
disease).
• An affected male does not transmit the disorder to his sons (since he gives his sons a Y
chromosome).
• Sons of carrier females have a 50/50 chance of getting the disease.
Some X-linked recessive disorders you should remember:
!
Metabolic diseases:
• Diabetes insipidus
• Lesch-Nyhan syndrome
Hematopoietic diseases:
• Hemophilia A and B
• Chronic granulomatous disease
• Glucose-6-phosphate dehydrogenase deficiency
Musculoskeletal diseases:
• Duchenne muscular dystrophy
Nervous system diseases:
• Fragile-X syndrome
Immune system diseases:
• Agammaglobulinemia
• Wiskott-Aldrich syndrome
!
10. List features of autosomal recessive (AR) disorders:
• The trait doesn't usually affect the parents of the affected person (but siblings may be affected).
• The expression of the defect tends to be more uniform than in autosomal dominant disorders.
 • Complete penetrance is common.
• The onset of disease is usually in childhood.
• New mutations associated with AR disorders may occur - but they are rarely detected clinically (the
person with the new mutation would be asymptomatic; and it may take several generations before a
descendant pairs with another heterozygote and has an affected child).
!
11. List some AR diseases:
! Metabolic diseases:
• Cystic fibrosis
• Phenylketonuria
• Galactosemia
• Homocystinuria
• Lysosomal storage diseases
• Alpha 1 antitrypsin deficiency
• Wilson disease
• Hemochromatosis
• Glycogen storage diseases
Hematopoietic diseases:
• Sickle cell anemia
• Some cases of von Willebrand disease
• Thalassemia
Endocrine diseases:
• Congenital adrenal hyperplasia
Skeletal diseases:
• Ehlers-Danlos syndrome (some cases)
Nervous diseases:
• Friedreich ataxia
• Spinal muscular atrophy