CHAPTER 5: GENETIC DISORDERS
 Transcription of DNA is initiated and regulated by promoter and enhancer sequences
GENES AND HUMAN DISEASE
 Human genetic disorders can be broadly classified into three categories:
Mutations in  Cause the disease or predispose to the disease (exeption:
single genes w/ hemoglobinopathies)
large effects  Highly penetrant  presence of the mutation is asso. w/ the
disease in a large portion of individuals
 Follow classic Mendelian pattern of inheritance
 Referred to as Mendelial disorders
Chromosomal  Arise from structural or numerical alteration in the
disorders autosomes and sex chromosomes
 Uncommon but asso. w/ high penetrance
Complex  More common
multigenic  Caused by interactions between multiple variant forms of
disorders genes and environmental factors  also called
polymorphism
 Each variant gene confers small increase in disease risk; no
single gene is sufficient to produce a disease
 Several polymorphisms needed to cause a disease 
multigenic or polygenic
 Low penetrance
 Multifactorial disorders
 Atherosclerosis, DM, hypertension, and autoimmune disease
Mutations
 Mutation – permanent change in DNA
o Germ cells affected are transmitted to the progeny and can give rise to
inherited diseases
o Mutations that arise in somatic cells do not cause hereditary diseases;
important in cancer genesis and congenital malformations
 General principles relating to the effects of gene mutation:
Point Mutations within Coding Sequences
 Point mutation – change where a single base is substituted with a different base
 May alter the code in a triplet of bases  lead to replacement of one amino acid 
alter the meaning of sequence of the encoded protein  called missense mutation
 Conservative missense mutation – if substituted amino acid is biochemically
similar to the original and causes little change in the function of the protein
 Nonconservative missense mutation – replaces the normal amino acid w/ a
biochemically different one
o E.g., sickle mutation affecting the β-globin chain of Hb  CTC (glutamic
acid) is replaced by CAC (encodes valine)  alters physicochemical
properties of Hb  sickle cell anemia
 Nonsense mutation – point mutation where amino acid codon is change to a chain
terminator or stop codon
o E.g., CAG creates a stop codon (UAG) is U is substituted for C  β0-
thalassemia
Mutations within Noncoding Sequences
 Point mutations involving these regulatory sequences interfere w/ binding of
transcription factors  lead to marked reduction or total lack of transcription
o Eg., thalassemia
 Point mutations w/in introns may lead to defective splicing of intervening sequences
 interferes w/ normal processing of initial mRNA and results in a failure to form
mature mRNA
 Translation cannot take place  gene product is not synthesized
Deletions and Insertions
 Small deletions or insertions involving coding sequence have two effects.
 If number of base pairs involved is three or multiple of three, the reading frame
will remain intact, and an abnormal protein lacking or gaining one or more amino
acids will be synthesized
 If the number of affected coding bases is not a multiple of three, this will result in
an alteration of the reading frame of the DNA strand, producing a frameshift
mutation
Trinucleotide-repeat Mutations
 Belong to a special category of genetic anomaly
 Cxd by amplification of a sequence of three nucleotides
 Almost all affected sequence share the nucleotides guanine (G) and cytosine (C)
 E.g., fragile X syndrome  familial mental retardation 1 (FMR1)  mental retardation
 Distinguishing feature of trinucleotide-repeat mutations is that they are dynamic
(degree of amplification increases during gamtogenesis)
o Influence the pattern of inheritance and the phenotypic manifestations of
diseases
 Transcription may be suppressed by gene deletions and point mutations involving
promoter sequences
 Abnormal mRNA processing result from mutations affecting introns or splice
junctions or both
 Translation is affected if a nonsense mutation creates a stop codon (chain
termination mutation) within an exon
 Three major categories of genetic disorders:
o Disorders related to mutant genes of large effect
o Diseases with multifactorial inheritance
o Chromosomal disorders
 Single-gene disorders with nonclassic patterns of inheritance – caused by
mutations in single-genes, but they do not follow the Mendelian pattern of inheritance
o Triple-repeat mutations, mutations in mtDNA, genomic imprinting or
gonadal mosaicism
 Hereditary disorders – derived from one’s parentes and transmitted in the germ
line through the generations and therefore are familial
 Congenital – “born with”; some are not genetic (e.g., congenital syphilis)
o Not all genetic diseases are congenital
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MENDELIAN DISORDERS
 Mendelian disorders - result of mutations in single genes that have large effects
o dominant or recessive
o Codominance – both alleles of gene pair are expressed or contribute to the
phenotype; e.g., histocompatibility or blood group Ag
 Sickle cell anemia – caused by substitution of normal Hb (HbA) by HbS
o Homozygote – all Hb is abnormal; disorder is fully expressed
o Heterozygote – proportion of Hb is HbS (the rest is HbA); red cell sickling
occurs only under unusual circumstances (e.g., lowered O2 tension) 
sickle cell trait
 Pleiotropism – single mutant gene may lead to many end effects (sickle cell anemia)
 Genetic heterogenecity – mutations at several genetic loci may produce the same
trait (profound childhood deafness)
2. Key structural proteins, such as collagen and cytoskeletal elements of the red cell
membrane (e.g., spectrin)
o E.g., collagen is a trimer where the three collagen chains are arranged in
helical form  must be normal for assembly and stability  normal
collagen trimers cannot be formed if there is a single mutant collagen chain
o Dominant negative – mutant allele where it impairs the function of a
normal allele
 Gain-of-function mutation – transmission is almost always autosomal dominant;
can take two forms:
o Increase in a protein’s normal function (e.g., excessive enzymatic activity)
o Impart a whole new activity completely unrelated to the affected protein’s
normal function
 Huntington disease – gain of function mutation;
o Huntingtin (abnormal protein) – toxic to neurons; even heterozygotes
develop a neurologic deficit

Transmission Patterns of Single-Gene Disorders Autosomal Dominant Disorders

System Disorder
 Mutations involving single genes typically follow one of three patterns of inheritance: Nervous Huntington disease; Neurofibromatosis; Myotonic dystrophy;
o Autosomal dominant Tuberous sclerosis
o Autosomal recessive Urinary Polycystic kidney disease
o X-linked Gastrointestinal Familial polyposis coll
Hematopoietic Hereditary spherocytosis; von Willebrand disease
Skeletal Marfan syndrome; Ehlers-Danlos syndrome; Osteogenesis
Autosomal Dominant Disdorders
imperfect; Achondroplasia
 Manifested in the heterozygous state, so at least one parent of an index case is
Metabolic Familial hypercholesterolemia
usually affected
Acute intermittent porphyria
 Both males and females are affected; both can transmit the condition
 Characterized by the following:

 With every autosomal dominant disorder, some proportion of patients do

not have affected parents
o New mutations involving egg or sperm from which they were derived 
related to the effect of the disease on reproductive capability
o Siblings are neither affected nor at increased risk
o Many new mutations occur in germ cells of older fathers
 Clinical features can be modified by variations in penetrance and
expressivity
o Incomplete penetrance – inherit the mutant gene but are phenotypically
normal
o Variable expressivity – trait is seen in all individuals carrying the mutant
gene but is expressed differently among individuals ( neurofibromatosis 1)
 The biochemical mechanisms of autosomal dominant disorders depend upon the
nature of the mutation and the type of protein affected
 Most mutations may lead to the reduced production of a gene product or give rise to
dysfunctional or inactive protein
 Many autosomal dominant diseases arising from deleterious mutations fall into one of
a new familiar patterns:
1. Those involved in regulation of complex metabolic pathways that are subject to
feedback inhibition
o E.g., LDL receptors  in familial hypercholesterolemia, 50% loss of LDL
receptors results in secondary elevation of cholesterol
Autosomal Recessive Disorders
 Make up the largest category of Mendelian disorders
 They occur when both alleles at a given gene locus are mutated; characterized by:
1. The trait does not usually affect the parents of the affected individuals; but siblings
may show the disease
2. Siblings have one chance in four of having the trait (25%)
3. If the mutant gene occurs with a low frequency in the population, there is a strong
likelihood that the affected individual (proband) is the product of a consanguineous
marriage
 Ff features distinguish AR from AD diseases:
 Expression of the defect tends to be more uniform than in autosomal dominant
disorders
 Complete penetrance is common
 Onset is frequently early in life
 Rarely detected clinically. (heterozygote + heterozygote = affected offspring)
 Many of the mutated genes encode enzymes. In heterozygotes, equal amounts of
normal and defective enzyme are synthesized
 Autosomal recessive disorders include almost all inborn errors of metabolism
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 Autosomal Recessive Disorders Biochemical and Molecular Basis of Single-Gene (Mendelian) Disorders
System Disorder
Metabolic Cystic fibrosis; Phenylketonuria; Galactosemia; Homocytinuria;  Mendelian disorders result from alterations involving single genes (table 5-4)
Lysosomal storage disease; a1-antitrypsin deficiency; Wilson  Genetic defect may lead to the formation of an abnormal protein or a reduction in the
disease; Hemochromatosis; Glycogen storage disease output of the gene product
Hematopoietic Sickle cell anemia; Thalassemia  Any type of protein may be affected
Endocrine Congenital adrenal hyperplasia
 Mechanisms involved in single-gene disorders are classified into four categories:
Skeletal Ehler-Danlos syndrome; Alkpatonuria
1. Enzyme defects and their consequences
Nervous Neurogenic muscular atrophies; Friedreich ataxia; Spinal muscular
atrophy 2. Defects in membrane receptors and transport systems
3. Alterations in the structure, function, or quantity of nonenzyme proteins
4. Mutations resulting in unsual reactions to drugs

X-Linked Disorders Enzyme Defects and their Consequences

 All sex-linked disorders are X-linked, and almost all are recessive  Mutations may result in the synthesis of an enzyme with reduced activity or a reduced
 Males with mutations affecting the Y-linked genes are usually infertile, and hence amount of a normal enzyme
there is no Y-linked inheritance  Consequence is metabolic block
 X-linked recessive inheritance accounts for small number of well-defined clinical  Biochemical consequences of an enzyme defect in such a reaction may lead to three
conditions major consequences:
 Y chromosome is not homologous to X  male is said to be hemizygous for X-linked
mutant genes  disorders are expressed on males 1. Accumulation of substrate
 Features that characterize these disorders: o May be accompanied by accumulation of intermediates
 Affected male does not transmit the disorder to his sons, but all daughters are o Tissue injury may result if the precursor (intermediates or products) are
carriers. Sons of heterozygous women have one chance in two of receiving the toxic in high concentrations
mutant gene o Ex: galactosemia  deficiency of G1PUT leads to accumulation of
 Heterozygous female does not express the full phenotypic change because of the galactose and tissue damage
paired normal allele; express the disorder partially. o Lysosomal storage disease – excessive accumulation of complex
 X-linked disorders – transmitted by an affected heterozygous female to half her substrates w/in the lysosomes as a result of deficiency of degradative
sons and half her daughters; and by an affected male to all his daughters but none of enzymes
his sons (if the female parent is unaffected) 2. An enzyme defect can lead to a metabolic block and a decreased amount of
o E.g., Vitamin D-resistant rickets end product
o Ex: melanin deficiency may result from lack of tyrosinase  albinism
X-Linked Recessive Disorders o If end product is a feedback inhibitor of enzymes involved in early reactions
System Disease  deficiency of end product may permit overproduction of intermediates
Musculoskeletal Duchenne muscular dystrophy and their catabolic products  toxic at high concentrations
Blood Hemophilia A and B; Chronic granulomatous disease; G6PD deficiency o Ex: Lesch-Nyhan syndrome
Immune Agammaglobulinemia; Wiskott-Aldrich syndrome 3. Failure to inactivate a tissue-damaging substrate
o Ex: a1-antitrypsin deficiency  unable to inactivate neutrophil elastase
Metabolic Diabetes insipidus; Lesch-Nyhan syndrome
in their lungs  destruction of elastin in the wall of lung alveoli 
Nervous Fragile X suyndrome
pulmonary emphysema

Defects in Receptors and Transport System

KEY CONCEPTS: Transmission Patterns of Single-Gene Disorders
 Biologically active substances have to be actively transported across the cell
 Autosomal dominant disorders – cxd by expression in heterozygous state; affect
 Transport is achieved by receptor-mediated enocytosis
males and females equally, and both sexes can transmit the disorder
 Genetic defect in a receptor-mediated transport system
 Enzyme proteins are not affected in autosomal dominant disorders; instead receptors
o Familial hypercholesterolemia  ↓ fxn or synthesis of LDL receptors 
and structural proteins are involved
defective transport of LDL into cells and secondarily to excessive cholesterol
 Autosomal recessive diseases – occur when both copies of a gene are mutated;
synthesis by complex intermediary mechanisms
enzyme proteins are involved; males and females are affected equally
o Cystic fibrosis  transport system for chloride ions in exocrine glands,
 X-linked disorders – transmitted by heterozygous females to their sons, who
sweat ducts, lungs, and pancreas is defective
manifest the disease. Female carriers usually are protected because of random
o Impaired chloride transport leads to serious injury to the lungs and
inactivation of X chromosome
pancreas

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Alterations in Structure, Function, or Quantity of Nonenzyme Proteins Clinical Features
 Genetic defects resulting in alterations of nonenzyme proteins often have widespread
secondary effects (e.g., sickle cell disease, hemoglobinopathies)  Skeletal abnormalities are the most striking feature of Marfan Syndrome
 Hemoglobinopathies – quality defect; defects in structure of globin molecule  Px are unusually tall w/ long extremeties and long, tapering fingers and toes
 Thalassemia – quantity defect; mutations in globin genes that affect the amount of o Joint ligaments are lax  px is double-jointed
globin chains synthesized o Thumb can hyperextend back to the wrist
o Asso. w/ reduced amount of structurally normal a-globin or b-globin chains o Head is dolichocephalic (long-headed) w/ prominent frontal eminence and
 Osteogenesis imperfecta – defective structural collagen
supraorbital ridges
 Hereditary spherocytosis – defective spectrin
 Muscular dystrophies – defective dystrophin  Spinal deformities: kyphosis, scoliosis, or rotation or slipping of the dorsal or
lumbar vertebrae
 Chest is classically deformed  pectus excavatum (deeply depressed sternum) or a
Genetically Defective Adverse Reactions to Drugs pigeon-breast deformity
 Certain genetically determined enzyme deficiencies are unmasked only after exposure  Ocular changes  bilateral subluxation or dislocation of lens (ectopia lentis)
of the affected individual to certain drugs  Cardiovascular lesions  most life-threatening features
 Pharmacogenetics – special area of genetics o Two most common lesions: mitral valve prolapse and dilation of the
 G6PD – classic example of drug-induced injury; asso. w/ deficiency of the enzyme ascending aorta due to cystic medionecrosis
G6PD
 Causes of progressive dilation of the aortic valve ring and the root of aorta that give
o Primaquine – cause severe haemolytic anemia
 Genetic factors have major impact on drug sensitivity and adverse reactions rise to aortic incompetence
o Loss of medial support & Excessive TGF-b signalling in the adventitia
 Weakening of adventitia lead to intimal tear  initiate intramural hematoma that
Disorders Associated with Defects in Structural Proteins cleaves layers of media  produce aortic dissection
o Hemorrhage ruptures through the aortic wall
 Marfan Syndrome and Ehlers-Danlos syndromes (EDS) – affect connective o Cause of death in 30-45% of cases
tissue and involve multiple organs  Mitral valve lesions are more frequent but less important than aortic lesions
 Loss of connective tissue support in mitral valve leaflets makes them soft and billowy
Marfan Syndrome  creates floppy valve
 Disorder of connective tissues manifested principally by changes in the  Valvular lesions w/ lengthening of chordate tendinae  lead to mitral valve
skeleton,eyes and cardiovascular system regurgitation
 Prevalence 1 in 5000; autosomal dominant inheritance
 Great majority of deaths are caused by ruptures of aortic dissections followed by
 Pathogenesis: results from an inherited defect in an extracellular glycoprotein called
cardiac failure
fibrillin-1; two mechanisms where loss of fibrillin leads to clinical manifestations:
o Loss of structural support in microfibril rich connective tissue  Variability in clinical expression is seen w/in a family; interfamilial variability is more
o Excessive activation of TGF-b signalling common and extensive  clinical diagnosis of Marfan syndrome is based on “revised
Ghent criteria”
 Fibrillin – major component of microfibrils found in ECM; provide scaffolding where  Mainstay or medical treatment is administration of beta-blockers which likely act by
tropoelastin is deposited to form elastic fibers; fibrillin-1 and fibrillin-2 reducing heart rate and aortic wall stress
o Microfibrils are widely distributed in aorta, ligaments, and ciliary zonules
that support the lens  affected by Marfan syndrome
Classification of Ehlers-Danlos Syndromes
o Fibrillin 1 – encoded by FBN1 on chromosomes 15q21.1; most common EDS Type Clinical Findings Inheritance Gene Defects
defect; inhibit polymerization of fibrillin fibers (dominant negative effect) Classic (I/II) Skin & joint hypermobility, atrophic Autosomal Dominant COL5A1, COL5A2
o Fibrillin 2 – encoded by FBN2 on chromosomes 5q23.31; mutations are scars, easy bruising
less common and give rise to congenital contractural arachnodactylyl Hypermobility Joint hypermobility, pain, dislocations Autosomal Dominant Unknown
(autosomal dominant disorder) (III)
o Reduction of fibrillin content below a certain threshold weakens the Vascular (IV) Thin skin, arterial or uterine rupture, Autosomal Dominant COL3A1
connective tissue  haploinsufficiency bruising, small joint hyperextensibility
 Bone overgrowth and myxoid changes in mitral valves cannot be attributed to Kyphoscoliosis Hypotonia, joint laxity, congenital Autosomal recessive Lysyl hydroxylase
(VI) scoliosis, ocular fragility
changes in tissue elasticity
Arthrochalasia Severe joint hypermobility, mild skin Autosomal Dominant COL1A1, COL1A2
o Loss of microfibrils give rise to abnormal and excessive activation of TGF-b (VIIa,b) changes, scoliosis, bruising
(normal microfibrils sequester TGF-b and thus control bioavailability) Dermatosparaxis Severe skin fragility, cutis laxa, Autosomal recessive Procollagen N-
o Excess TGF-b has deleterious effects on vascular smooth muscle (VIIc) bruising peptidase
development and increase activity of metalloproteases , causing loss of ECM

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Ehlers-Danlos Syndromes (EDS)
 EDSs comprise a clinically and genetically heterogenous group of disorders that
result from some defect in the synthesis or structure of fibrillar collagen
 Other disorders resulting from mutations affecting collagen synthesis: osteogenesis
imperfect, Alport syndrome, epidermolysis bullosa
 Mode of inheritance encompases all three Mendelian patterns
 Tissues rich in collagen (skin, ligaments, joints) are frequently involved in most
variants of EDS; lack tensile strength
o Skin is hyperextensible; joints are hypermobile
o Bending thumb backward to touch forearm
o Bending knee forward to create right angle
 Skin is extraordinarily stretchable, extremely fragile, and vulnerable to trauma
 The basic defect in connective tissue may lead to serious internal complications
o Rupture of colon and large arteries (vascular EDS)
o Ocular fragility w/ rupture of cornea and retinal detachment (kyphoscoliosis)
o Diaphragmatic hernia (classic EDS)
 Kyphoscoliosis EDS – most common autosomal recessive form of EDS;
o Lysyl hydroxylase – enzyme need for hydroxylation of lysine during
collagen synthesis
 Vascular EDS results from abnormalities of type III collagen
o Genetically heterogeneous; COL3A1
o Affect rate of synthesis of pro-a1 (III) chains; affect secretion of type III
collagen; others lead to the synthesis of structurally abnormal type III
collagen
o Some allele behave as dominant negatives  severe phenotypic defects
o Results from mutations in structural protein rather than enzyme protein 
autosomal dominant inheritance is expected
o Blood vessels and intestines are known to be rich in collagen type III 
consistent with severe structural defects
 Arthrochalasia type and dermatosparaxis type – fundamental defect is in the
conversion of type 1 procollagen to collagen
o This step involves cleavage of noncollagen peptides at the N-terminus and
C-terminus of procollagen molecule  accomplished by N- and C-
terminal-specific peptidases
o Arthrochalasia type – defect in the conversion involve mutations that
affect two type 1 collagens genes, COL1A1 and COL1A2; abnormal pro-a1
or pro-a2 that resist cleavage of N-terminal peptides are formed
o Heterozygotes manifest the disease
o Dermatosparaxis type – caused by mutations in te procollagen-N-
peptidase genes, essential for the cleavage of collagens; caused by enzyme
deficiency and follows an autosomal recessive inheritance
 Classic type of EDS – 30-50% mutations in genes for type V collagen (COL5A1,
COL5A2)
o Genes other than those that encode collagen may also be involved
o Some cases genetic defects that affect the biosynthesis of other ECM
molecules that influence collagen synthesis indirectly may be involved
o Ex: mutation in tenascin-X (EDS-like condition)  affects synthesis and
fibril formation of type VI and type I collagens
 Common thread in EDS is abnormality in collagen  extremely heterogeneous
KEY CONCEPTS
Marfan Syndrome
 Caused by a mutation in the FBN1 gene encoding fibrillin, w/c is required for
structural integrity of connective tissues and regulation of TGF-b signalling
 Major tissues affeccted are the skeleton, eyes, and cardiovascular system
 Clinical features ay include tall stature, long fingers, bilateral subluxation of lens,
mutral valve prolapsed, aortic aneurysm, and aortic dissection
 Clinical trials with drugs that inhibit TGF-b signalling (angiotensin receptor blockers) 
shown to improve aortic and cardiac function in mouse models
Ehlers-Danlos Syndromes
 There are six variants of syndromes, all characterized by defects in collagen synthesis
or assembly. Each variants is caused by a distinct mutation involving one of several
collagen genes or genes that encode other ECM proteins like tenascin-X
 Clinical features: fragile, hyperextensible skin vulnerable to trauma, hypermobile
joints, and ruptures involving colon, cornea, or large arteries. Wound healing is poor
Disorders Associated with Defects in Receptor Proteins
Familial Hpercholesterolemia
 Familial hypercholesterolemia is a “receptor disease” that is the consequence of a
mutation in the gene encoding the receptor for LDL, which is involved in the transport
and metabolism of cholesterol
 There is loss of feedback control and elevated levels of cholesterol that induced
premature atherosclerosis  greatly increased risk of myocardial infarction
 One of the most frequent occurring Mendelian disorders
 Heterozygotes: 2 to 3-fold elevation of plasma cholesterol  tendinous xanthomas
and premature atherosclerosis in adult life
 Homozygotes: 5 to 6-fold elevations in plasma cholesterol  skin xanthomas and
coronary, cerebral, and peripheral vascular atherosclerosis may develop at early age
o Myocardial infarction may occur before age 20 years
o Familial hypercholesterolemia is present in 3-6% of survivors of myocardial
infarction
Normal Process of Cholesterol Metabolism and Transport
 ~7% of body’s cholesterol circulates in plasma; predominantly in the form of LDL
 First step is secretion of VLDL (rich in TAG, less in cholesteryl esters) by the liver into
blood stream
o When VLDL particles reach the capillaries or adipose tissue or muscle, it is
cleaved by lipoprotein lipase  process that extracts most of the TAG
o IDL (resulting molecule)  reduced in TAG content and enriched in
cholesteryl esters but retains 2 of 3 apoproteins (B-100 and E) present in
VLDL particle
 After release from capillary endothelium, IDL particles have one of two fates:
o 50% of newly formed IDL is rapidly taken up by the liver by receptor-
mediated transport (the receptor recognized B-100 and E)  LDL receptor
(also involved in hepatic clearance of LDL)
 In liver cells, IDL is recycled to generate VLDL
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 o IDL particles not taken up by liver are subjected to further metabolic
processing  removes most of the remaining TAG and apo-E  yields
cholesterol-rich LDL particles
 IDL is the immediate and major source of plasma LDL
 Two mechanisms for removal of LDL from plasma:
o Mediated by an LDL-receptor
o Mediated by a receptor for oxidized LDL (scavenger receptor)
 70% of the plasma LDL is cleared by the liver
o First step: binding of LDL to cell surface receptors clustered in coated
pits (specialized regions of the plasma membrane)
o After binding: coated pits w/ receptor-bound LDL are internalized by
invaginating to form coated vesicles then they migrate w/in the cell to fuse
with lysosomes
o LDL dissociates from the receptor, which is recycled to the surface
o In the lysosomes, LDL is enzymatically degraded; apoprotein part is
hydrolyzed to amino acids; cholesteryl esters are broken down to free
cholesterol
o Free cholesterol crosses the lysosomal membrane to enter cytoplasm where
it is used for membrane synthesis and as a regulator of cholesterol
homeostasis
o Exit of cholesterol from lysosomes requires the action of two proteins,
NPC1 and NPC2
 Three separate processes are affected by the released intracellular cholesterol:
1. Cholesterol suppresses cholesterol synthesis w/in the cell by inhibiting the activity of
the enxyme HMG CoA reductase  rate limiting enzyme in the synthetic pathway
2. Cholesterol activates the enzyme acyl-coenzyme A: cholesterol acyltransferase 
favors esterification and storage of excess cholesterol
3. Cholesterol suppresses the synthesis of LDL receptors, thus protecting the cells from
excessive accumulation of cholesterol
 Monocytes and macrophages have receptors for chemically altered (acetylated or
oxidized) LDL
 In hypercholesterolemia, there is marked increase in scavenger receptor-mediated
traffic of LDL cholesterol into the cells of the mononuclear phagocyte system and
vascular walls  responsible for the appearance of xanthomas and contributes to
the pathogenesis of premature atherosclerosis
 Familial hypercholesterolemia is classified into five groups:
Class I Uncommon; lead to complete failure of synthesis of the receptor protein (null allele)
mutations
Class II Common; encode receptor proteins that accumulate in the ER because their folding
mutations defects make it impossible for them to be transported to the Golgi complex
Class III Affect the LDLD-binding domain of receptor; encoded proteins reach the cell surface
mutations but fail to bind LDL or do so poorly
Class IV Encode proteins that are synthesized and transported to the cell surface efficiently;
mutations bind LDL normally but fail to localize in coated pits; bound LDL is not internalized
Class V Encode proteins that are expressed on the cell surface, can bind LDL, and can be
mutations internalized; the pH-dependent dissociation of the receptor and the bound LDL fails
to occur; such receptors are trapped in the endosome, where they are degraded
and fail to recycle to the cell surface
 Statins – suppress intracellular cholesterol synthesis by inhibiting the enzyme HMG
CoA reductase
o Allow greater synthesis of LDL receptors
o Used for secondary prevention of ischemic heart disease
KEY CONCEPTS: Familial Hypercholesterolemia
 Autosomal dominant disorder caused by mutations in the gene encoding the LDL
receptor
 Patients develop hypercholesterolemia as a consequence of impaired transport of LDL
into the cells
 Heterozygotes: elevated serum cholesterol greatly increases the risk of atherosclerosis
and resultant coronary artery disease
 Homozygotes: have greater increase in serum cholesterol and a higher frequency of
ischemic heart disease. Cholesterol also deposits along tendon sheaths to produce
xanthomas
Disorders Associated with Defects in Enzymes
Lysosomal Storage Disease
 Lysosomes – key components of the intracellular digestive tract; contain hydrolytic
enzymes that have two special properties:
o They function in the acidic milieu of the lysosomes
o These enzymes constitue a special category of secretory proteins that are
destined for intracellular organelles (requires special processing in golgi
apparatus)
 Lysosomal enzyme or acid hydrolases – synthesized in the ER and transported to
golgii apparatus
o w/in golgi comlex they undergo posttranslational modifications including
attachment of terminal mannose-6-phosphate groups to oligosaccharide
side chains
o phosphorylated mannose residues serves as “address label” recognized by
receptors on the inner surface of the Golgi membrane
o Lysosomal enzymes bind receptors and segregated from other secretory
proteins
 Genetically determined errors in this remarkable sorting mechanism may give rise to
one form of lysosomal storage disease
 Lysosomal enzymes catalyze breakdown of complex macromolecules
o Autophagy – metabolic turnover of intracellular organelles
o Heterophagy – acquired from outside the cells by phagocytosis
 Inherited deficiency of a functional lysosomal enzyme gives rise to two pathologic
consequences:
 Primary accumulation – catabolism of the substrate of the missing enzyme remains
incomplete, leading to the accumulation of the partially degraded insoluble metabolite
w/in lysosomes
o Lysosomes are stuffed w/ incompletely digested macromolecules; become
large and numerous enough to interfere w/ normal cell functions
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  Secondary accumulation – caused by impaired autophagy from autophagic
substrated such as polyubiquitinated proteins and old effete mitochondria
o Absence of this control mechanism causes accumulation of dysfunctional
mitochondria w/ poor calcium buffering capacity and altered membrane
potentials; trigger generation of free radicals and apoptosis
 Three general approaches to the treatment of lysosomal storage diseases
o Enzyme replacement therapy
o Substrate reduction therapy
o Molecular chaperone therapy
 Distribution of stored material and organs affected is determined by two factors:
o The tissue where most of the material to be degraded is found
o The location where most of the degradation normally occurs
 Lysosomal storage disease can be divided based on the biochemical nature of
accumulated metabolite; creating subgroups: (table 5-6)
o Glycogenoses
o Sphingolipidoses (lipidoses)
o Mucopolysaccharidoses (MPSs)
o Mucolipidoses
Tay-Sachs Disease (GM2 Gangliosidosis: Hexosaminidase α-Subunit Deficiency)
 GM2 gangliosidoses – group of three lysosomal storage diseases caused by an
inability to catabolise GM2 gangliosides
 Degradation of GM2 gangliosides requires three polypeptides encoded by three distinct
genes
 Tay-Sachs disease – the most common form of GM2 gangliosidosis; results from
mutations in the α-subunit locus on chromosome 15; cause severe deficiency in
hexosaminidase A
 Prevalent among Jews (from Eastern European (Ashkenazic) origin); 1 in 30
Morphology
 Hexosaminidase A – absent in all tissues so GM2 ganglioside accumulate in many
tissues (heart, liver, spleen, nervous system)
 Involvement of neurons in the CNS and ANS and retina dominates clinical pic
 Histological: neurons are ballooned w/ cytoplasmic vacuoles  markedly distended
lysosome filled w/ gangliosides; Oil red O and Sudan black B positive
 EM: cytoplasmic inclusions can be seen; prominent whorled configurations w/in
lysosomes composed of onion-skin layers of membranes
 Cherry-red spot thus appears in the macula  represents accentuation of the
normal color of macular choroid
Clinical Features
 Affected infants are normal at birth but begin to manifest signs at 6 months
 Motor and mental deterioration  motor incoordination, mental obtundation leading
to muscular flaccidity, blindness and increasing dementia
 Early course of dx  cxc cherry-red spot appears in macula almost in all px
 1-2 years complete vegetative state; death at age 2-3 years
 100 mutations in α-subunit gene  unfolded protein  apoptosis
 Antenatal diagnosis and carrier detection by enzyme assay and DNA-based analysis
 Chaperone therapy
 **Sandhoff disease - GM2 gangliosidoses β-subunit deficiency
Niemann-Pick Disease Types A and B
 Types A and B are two related disorders that are cxd by lysosomal accumulation of
sphingomyelin due to an inherited deficiency of sphingomyelinase
 Type A – severe infantile form w/ extensive neurologic involvement
o Marked visceral accumulations of sphingomyelin
o Progressive wasting & Early death w/in first 3 years of life
 Type B – px have organomegaly; NO CNS involvement; usually survive into adulthood
 Also common in Ashkenazi Jews
 Gene for acid sphingomyelinase maps to chromosome 11p15.4  one of the
imprinted genes expressed from the maternal chromosome as a result of epigenetic
silencing of the paternal gene
 Typically inherited as an autosomal recessive
 Heterozygotes who inherit the mutant allele from mother can develop Niemann Pick
Disease
Morphology
 Type A – missense mutation cause almost complete deficiency of sphingomyelinase
 Enzyme deficiency blocks degradation of the lipid  accumulates in lysosome (MPS)
 Affected cells become enlarged (90 um in dm)) due to distention of lysosomes w/
sphingomyelin and cholesterol
 Innumerable small vacuoles uniform in size imparts foaminess to cytoplasm
 EM: vacuoles are engorged secondary lysosomes that contains cytoplasmic bodies
resembling concentric lamellated myelin figures ” zebra” bodies
 Lipid-laden phagocytic foam cells are seen in spleen, liver, lymph nodes, BM, tonsilds,
GIT and lungs
 Involvement of spleen generally produces massive enlargement (10x)
 Brain: gyri are shrunken and the sulci are widened
 Neural involvement is diffused  affects all parts of nervous system
 Vacuolation and ballooning of neurons constitute the dominant histologic change
 leads to cell death and loss of brain substance
 Retinal cherry-red spot is present in 1/3 of cases
Clinical Features
 Clinical manifestations in type A are present at birth and become evident by age 6
 Infants typically protuberant abdomen because of hepatosplenomegaly
 Progressive failure to thrive, vominting, fever, and generalized lymphadenopathy as
well as progressive deterioration of psychomotor follows after appearance of
manifestations
 Death at 1st or 2nd year of life
 Diagnosed by biochemical assays for sphingomyelinase activity in liver or BM biopsy
 DNA analysis – detects individuals affected w/ types A and B as well as carriers
Niemann-Pick Disease Type C
 Distinct at biochemical and genetic levels; more common
 Mutations in NPC1 and NPC2 lead to NPC (NPC1 mutations – 95% of cases)
 NPC is due to a primary defect in nonenzymatic lipid transport
 NPC1 – membrane bound; NPC2 – soluble (both are involved in transport of free
cholesterol from the lysosomes to the cytoplasm)
 Clinically heterogenous
 May present as hydrops fetalis and stillbirth, as neonatal sepsis or as a chronic form
cxd by progressive neurologic damage (more common)
 Progressive neurologic damage – marked by ataxia, vertical supranuclear gaze
palsy, dystonia, dysarthria, psychomotor regression
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Gaucher Disease
 Gaucher disease refers to a cluster of autosomal recessive disorders resulting
from mutations in the gene encoding glucocerebrosidase
 Most common lysosomal storage disorder
 Affected gene encodes glucocerebrosidase  enzyme that cleaves the glucose
residue from ceramide
 Glucocerebroside accumulates prinicipally in phagocytes (also in CNS)
 Glucocerebrosides – continually formed from the catabolism of glycolipids derived
from cell membrane of senescent WBC and RBC
 Pathologic changes in GD are caused by:
o Burden of storage material
o Activation of macrophages and consequent secretion of cytokines (IL-1, IL-6
and TNF)
 Three subtypes of Gaucher disease:
Type I  Chronic nonneuropathic form; most common (99% of cases)
 Storage of glucocerebrosides is limited to the mononuclear
phagocytes WITHOUT involving the brain
 Splenic and skeletal involvements dominates the pattern of the dx
 Found principally in Jews of European stock
 Px have reduced but detectable levels of glucocerebrosidase activity
 Longevity is shortened but not markedly
Type II  Acute neuropathic Gaucher disease
 Infantile acute cerebral pattern
 No predilection for Jews
 Px have no detectable glucocerebrosidase activity in tissues
 Hepatosplenomegaly is present; clinical picture is dominated by
progressive CNS involvement leading to death at early age
Type III  Intermediate between types I and II
 Patients have systemic involvement cxc of type I but have
progressive CNS disease that begins in adolescence or early
adulthood
Morphology
 Gaucher cells – distended phagocytic cells; found in spleen, liver, BM, lymph nodes,
tonsils, thymus, Peyer patches
o Rarely appear vacuolated; instead have a fibrillary type of cytoplasm likened
to crumpled tissue paper
o Often enlarged (100um in dm); have one or more dark, eccentrically placed
nuclei
o PAS intensely positive
 EM: the fibrillary cytoplasm can be resolved as elongated, distended lysosomes,
containing the stored lipid in stacks of bilayers
 Type I – enlarged spleen (10kg); lymphadenopathy is mild to moderate (body-wide)
o Accumulation of Gaucher cells in BM (70-100% of type 1 cases) 
produce areas of bone erosions  pathologic fractures
 In px w/ cerebral involvement, Gaucher cells are seen in the Virchow-Robin
spaces, and arterioles are surrounded by swollen adventitial cells
 There no storage of lipids in the neurons, yet neurons appear shrivelled and are
progressively destroyed
 Lipids that accumulate in the phagocytic cells around blood vessels secrete cytokines
that damage nearby neurons
Clinical Features
 Type I – signs and symptoms first appear in adult life and are related to
splenomegaly or bone involvement
o (+) pancytopenia or thrombocytopenia secondary to hypersplenism
o Extensive expansion of marrow space  pathologic fractures and bone pain
o Disease is compatible with long life
 Types II and III – CNS dysfunction, convulsions, and progressive mental
deterioration dominate, although organs such as the liver, spleen, and lymph nodes
are also affected
 Diagnosis of homozygotes can be made by measurement of glucocerebrosidase
activity in peripheral blood WBC or in extracts of cultures skin fibroblasts
 Heterozygotes can be identified by detection of mutations
 Replacement therapy w/ recombinant enzymes is the mainstay for treatment of
gaucher disease
o Effective; type 1 cases can expect normal life (extremely expensive)
 Allogenic hematopoietic stem cell transplantation can be curative  because
fundamental defect resides in mononuclear phagocytic cells from marrow stem cells
Mucopolysaccharidoses (MPS)
 MPSs are a group of closely related syndromes that result from genetically determined
deficiencies of enzymes involved in the degradation of mucopolysaccharides
(glycosaminoglycans); MPS I to MPS VII
 Mucopolysaccharides – long-chain complex carbs that are linked w/ proteins to
form proteoglycans; abundant in the ground substance of CT
 Glycosaminoglycans that accumulate in MPS are:
o Dermatan sulfate
o Heparan sulfate
o Keratan sulfate
o Chondroitin sulfate
 Enzymes involved in degradation cleave terminal sugars from polysaccharide chain
o If absent, these chains accumulate w/in lysosomes
 All MPS except one are inherited as autosomal recessive traits
o Hunter syndrome – X-linked recessive trait
 MPS are progressive disorders, cxd by:
o Coarse facial features
o Clouding of the cornea
o Joint stiffness
o Mental retardation
 Urinary excretion of the accumulated mucopolysaccharides is often increased
Morphology
 Accumulated mucopolysaccharides are generally found in mononuclear phagocytic
cells, endothelial cells, intimal smooth muscle cells and fibroblasts
 Common sites involve are spleen, liver, BM, lymph nodes, blood vessels, heart
 Microscopically: affected cells are distended and have apparent clearing of the
cytoplasm to create so-called balloon cells
 EM: clear cytoplasm can be seen as numerous minute vacuoles  swollen lysosomes
containing finely granular PAS + material (identified as mucopolysaccharide)
 Some of the lysosomes in neurons are replaced by lamellated zebra bodies (similar to
those seen in Niemann-Pick dx)
 Hepatosplenomegaly, skeletal deformities, valvular lesions, and subendothelial arterial
deposits, particularly in the coronary arteries, and lesions in the brain are common
threads that run through all of the MPSs
 Important causes of death: myocardial infarction and cardiac decompensation
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Clinical Features
 Hurler syndrome (MPS I-H) – results from a deficiency of α-1-iduronidase; one of
the most severe form of MPS
o Affected children: normal at birth but develop splenomegaly by age 6-24
months
o Growth is retarded; develop coarse facial features and skeletal deformities
o Death occurs by age 6-10 years; often due to cardiovascular complications
 Hunter syndrome (MPS II) – differs from Hurler syndrome in mode of inheritance
(X-linked), absence of corneal clouding and milder clinical course
KEY CONCEPTS: Lysosomal Storage Diseases
 Lysosomal storage disease – inherited mutations leading to defective lysosomal
enzyme functions gives rise to accumulation and storage of complex substrates in the
lysosomes and defects in autophagy resulting in cellular injury
Tay-Sachs Disease  Caused by an inability to metabolize GM2 gangliosides
due to lack of the α subunit of lysosomal
hexosaminidase.
 GM2 gangliosides accumulate in the CNS and cause
several mental retardation, blindness, motor
weakness, and death by 2-3 years of age
Niemann-Pick Disease  Caused by deficiency of sphingomyelinase
Types A & B  In more severe type A variant, accumulation of
sphingomyelin in the nervous system results in
neuronal damage
 Lipid also is stored in phagocytes w/in the liver,
spleen, BM, and lymph nodes, causing their
enlargement
 Type B – neuronal damage is absent
Niemann-Pick Disease  Caused by a defect in cholesterol transport and
Type C resultant accumulation of cholesterol and
gangliosides in the nervous system
 Affected children most commonly exhibit ataxia,
dysarthria, and psychomotor regression
Gaucher Disease  Results from lack of the lysosomal enzyme
glucocerebrosidase and accumulation of
glucocerebroside in mononuclear phagocytic cells
 Type I – most common; affected phagocytes become
enlarged (Gaucher cells) and accumulate in liver,
spleen, and BM  cause hepatosplenomegaly and
bone erosion
 Types II and III – cxd by variable neuronal
involvement
Mucopolysaccharidoses  Result in accumulation of mucopolysaccharides in
liver, spleen, heart, blood vessels, brain, cornea,
joints.
 Affected px have coarse facial features
 Hurler syndrome – corneal cloudness, coronary
arterial and valvular deposits, and death in childhood
 Hunter syndrome – asso. w/ milder clinical course
Glycogen Storage Disease (Glycogenoses)
 Glycogen storage diseases – result from a hereditary deficiency of one of the
enzyme involved in the synthesis or sequential degradation of glycogen
 Glycogen – storage form of glucose
o Glycogen synthesis begins w/ the conversion of glucose to G6P by
hexokinase (glucokinase)
o Phosphoglucomutase then transforms G6P to G1P then converted to
uridine diphosphoglucose
o Highly branched, large polymer is then built , containing 10,000 glucose
molecules linked together by α-1,4-glucoside bonds
o Glycogen chain & branches continue to elongate by the addition of glucose
molecules mediated by glycogen synthetase
o During degradation, phosphorylases in the liver and muscle pit G1P from
glycogen until about four glucose residues remain in each branch, leaving a
branched oligosaccharide called limit dextrin  can be degraded only by
the debranching enzyme
o Glycogen is also degraded in the lysosomes by acid maltase
o If lysosomes are deficient in this enzyme, the glycogen contained w/in them
is nor accessible to degradation by cytoplasmic enzymes such as
phosphorylases
 On the basis of pathophysiology glycogenoses can be divided into three major
subgroups: (Table 5-7)
Hepatic forms  Liver – key player in glycogen metabolism
 Contains enzymes that synthesize glycogen and break it down
to free glucose
 Inherited deficiency of hepatic enzymes involved in glycogen
degradation leads to storage of glycogen and hypoglycaemia
 Deficiency of G6Pase (von Gierke Dx, or type 1 glycogenosis)
– prime example of hepatic-hypoglycemic form of glycogen
storage disease
 Glycogen is stored in many organs, but hepatic
enlargement and hypoglycaemia dominate in clinical
picture
Myopathic forms  In skeletal muscles, glycogen is used as a source of energy
 Glycolysis  ATP  lactate
 If enzyme that fuel glycolytic pathway are deficient, glycogen
storage occurs in the muscles and asso. w/ muscular
weakness due to impaired energy production
 Ex: deficiencies of muscle phosphorylase (McArdle Dx, or
type V glycogenoses), muscle phosphofructokinase (type VII
GSD)
 Typically, individuals w/ the myopathic forms present w/
muscle cramps after exercise and lactate levels in the blood
fail to rise after exercise due to a block in glycolysis
GSD asso. w/ 1)  Asso. w/ glycogen storage in many organs; death in early life
deficiency of α-  Acid maltase – lysosomal enzyme; deficiency leads to
glucosidase (acid
lysosomal storage of glycogen (type II glycogenosis, or Pompe
maltase) and lack of
branching enzyme
disease) in all organs
 Cardiomegaly is the most prominent feature
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KEY CONCEPTS: Glycogen Storage Disease
 Inherited deficiency of enzymes involved in glycogen metabolism can result in storage
of normal or abnormal forms of glycogen, predominantly in liver or muscles, but also Normal Karyotype
in other tissues as well
Hepatic form  Von Gierke Disease
 Liver cells store glycogen because a lack of hepatic G6Pase
Myopathic  McArdle disease – muscle phosphorylase lacks gives rise to
form storage in skeletal muscles and cramps after exercise
Pompe  There is lack of lysosomal acid maltas; all organs are affected
Disease  Heart involvement is predominant
Disorders Associated with Defects in Proteins That Regulate Cell Growth
 Normal growth and differentiation of cells are regulated by two classes of genes:
o Proto-oncogenes Commonly Used Cytogenetic Terminology
o Tumor suppressor genes
 Mutations in both classes of genes are important in the pathogenesis of tumors
 Cancer-causing mutations affecting somatic cells are not passed in the germ line
 Familial cancers are inherited in an autosomal dominant fashion
COMPLEX MULTIGENIC DISORDERS
 Caused by interactions between variant forms of genes and environmental
factors
 Gene that has at least two alleles, each occurs at a frequency of at least 1% in the
population is polymorphic
o Each variant allele is referred to as a polymorphism
 Complex genetic disorders occur when many polymorphism (each w/ modest effect
and low penetrance) are co-inherited
 Complex disorders result from the collective inheritance of many polymorphism
 Some polymorphisms are common to multiple diseases of the same type, while others Structural Abnormalities of Chromosomes
are disease specific
 Environmental influences significantly modify the phenotypic expression of
complex traits
o Ex: Type 2 DM (multifactorial disorder)  first manifest after weight gain
o Obesity (other environmental influences) unmasks the diabetic genetic trait
 Nutritional influences may cause monozygous twins to achieve different heights
 Difficult to distinguish between Mendelian and multifactorial disease
CHROMOSOMAL DISORDERS
 22 homologous pairs of autosomes; 2 sex chromosomes (XX, XY)
 Karyotyping – study of chromosomes; basic tool of cytogeneticist
o Arrest dividing cells in metaphase w/ mitotic spindle inhibitors (Colcemid)
and stain the chromosomes
o In metaphase spread, individual chromosomes take the form of two
chromatids connected at the centromere
o Stained using Giemsa stain  G banding
o Resolution obtained by banding can be markedly improved by obtaining the
cells in prophase
o 1500 bands per karyotype can be recognized
 Karyotypes are described using a shorthand system of notations in the ff order:
o Total number of chromosomes
o Sex chromosome complement
o Description o abnormalities in ascending numerical order
o E.g., male w/ trisomy 21 47, XY, +21
 p – short arm of a chromosome; p for petit
 q – long arm
 Each arm of the chromosome is divided into two or more regions bordered by
prominent bands
o Regions are numbered from centromere outward
o Each region is further subdivided into bands and sub-bands and are ordered
numerically
o E.g., Xp21.2 – chromosomal segment located on the short-arm of X
chromosome, in region 2, band 1, and sub-band 2
 The aberrations underlying cytogenetic disorders may take the form of an abnormal
number of chromosomes or alterations in the structure of one or more chromosomes
 46,XX or 46,XY – normal chromosome complement
 Euploid – any exact multiple of the haploid number of chromosomes (23)
 Aneuploidy - if an error occurs in mitosis or meiosis and a cell acquires a
chromosome complement that is not an exact multiple of 23; usual cause:
o Nondisjunction
o Anaphase lag
 Nondisjunction – (gamtogenesis) gametes formed have either an extra (n +1) or
less (n – 1) chromosome;
o Fertilization of such gametes results in two types of zygotes:
 Trisomic (2n + 1)
 Monosomic (2n – 1)

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  Anaphase lag – one homologous chromosome in meiosis or one chromatid in mitosis
lags behind and is left out of the cell nucleus
o Result is one normal cell and one cell w/ monosomy
 Monosomy involving an autosome generally causes loss of too much genetic
information to permit live birth or even embryogenesis
o But several autosomal trisomies do permit survival
 Mitotic errors in early development give rise to two or more populations of cells w/
different chromosomal complement  referred to as mosaicism if in the same
individual
 Mosaicism – result from mitotic errors during the cleavage of the fertilized ovum or
in somatic cells
 Mosaicism in sex chromosomes is common  lead to one of the daughter cells
receiving three sex chromosomes , whereas the other receives only one
o E.g., 45,X/47,XXX mosaic  mosaic variant of Turner syndrome (45,X)
 Autosomal mosaicism is less common  leads to nonviable mosaic due to
autosomal monosomy
o Nonviable cell population is lost during embryogenesis, yielding a viable
mosaic (e.g., 46,XY/47,XY, +21)
 Second category of chromosomal aberration is asso. w/ structural changes
 FISH (fluorescence in situ hybridization) – gain higher resolution; can detect
changes as small as kilobases
 Structural changes usually result from chromosome breakage followed by loss or
rearrangement of material
Deletion  Refers to loss of a portion of a chromosome
 Most are interstitial (rarely terminal)
 Interstitial deletions – occur when there are two breaks w/in
a chromosome arm, followed by loss of the chromosomal
material between the breaks and fusion of the broken ends
 E.g., 46,XY,del(16)(p11.2p13.1) – breakpoints in the short arm
of chromosome 16 at 16p11.2 and 16p13.1 w/ loss of material
between breaks
 Terminal deletion – result from a single break in a
chromosome arm; produces fragment w/ no centromere, w/c is
then lost at the next cell division, and a chromosome bearing a
deletion; the end of a chromosome is protected by acquiring
telomeric sequences
Ring  Special form of deletion; produced when a break occurs at both
chromosome ends of a chromosome w/ fusion of the damaged ends
 If genetic material is lost, phenotypic abnormalities result
 E.g., 46,XY,r(14)
 Ring chromosomes do not behave normally in meiosis or mitosis
and usually result in serious consequences
Inversion  Refers to a rearrangement that involves two breaks w/in a single
chromosome w/ reincorporation of the inverted, intervening
segment
 Paracentric – inversion involving only one arm of the
chromosome
 Pericentric – if the breaks are on opposite sides of the
centromere
 Inversion are often compatible w/ normal development
Isochromosome  Results when one arm of a chromosome is lost and the
formation remaining arm is duplicated
 Results in a chromosome consisting of two short arms only or
two long arms
 Has morphologically identical genetic information in both arms
 I(X)(q10) – most common isochrome present in live births;
involves long arm of X
Translocation  Segment of one chromosome is transferred to another
 Balanced reciprocal translocation – there are single breaks
in each of two chromosomes , with exchange of material
o 46,XX,t(2;5)(q31;p14) – translocation between long
arm of chromosome 2 and short arm if chromosome 5
o There is no loss of genetic material  phenotypically
normal
o Carrier is at increased risk for producing abnormal
gametes
 Robertsonian translocation or centric fusion –
translocation between two acrocentric chromosomes
o The breaks occur close to the centromeres
o Transfer of segments leads to one very large
chromosome and one extremely small one
o Usually the small product is lost  still compatible
with normal phenotype
o Significance lies in the production of abnormal
progeny
Cytogenetic Disorders Involving Autosomes
Trisomy 21 (Down Syndrome)
 Most common of the chromosomal disorders and is a major cause of mental
retardation; chromosome count is 47
 FISH reveals extra copy of extra chromosome 21
 Extra chromosomal number is present as a translocation
 Most common cause is meiotic nondisjuntion
 Maternal age has a strong influence on the incidence of trisomy 21
 In 4% of cases of Down syndrome, the extra chromosomal material
derives from the presence of a robertsonian translocation of the long arm of
chromosome 21 to another eccentric chromosome (e.g., 22 or 14)
o Affects chromosome pairing during meiosis resulting to aneuploid
 Approximately 1% of Down syndrome patients are mosaics, having a mixture of cells
w/ 46 or 47 chromosomes
o Results from mitotic nondisjunction of chromosome 21
o Symptoms are variable and milder
 In cases of translocation or mosaic Down syndrome, maternal age is of no importance
 The diagnostic clinical features of this condition are evident even at birth:
o Flat facial profile
o Oblique palpebral fissures
o Epicanthic folds
 Down syndrome is a leading cause of mental retardation  80% has 25-50 IQ
o Children have a gentle, shy manner and often content w/ life
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  40% of the patients have congenital heart disease
o Affects endocardial cushion, including ostium primum, atrial septal defects,
atrioventricular valve malforamtions, and ventricular septal defects
o Cardiac problems are responsible for the majority of deaths in infancy and
early childhood
o Also include atresias of esophagus and small bowel
 Children w/ trisomy 21 have 10-fold to 20-fold increased risk of developing acute
leukemia
o ALL and AML (most commonly acute megakaryoblastic leukemia) occur
 All patients w/ older than age 40 develop neuropathologic changes cxc of Alzheimer
disease
 Patients have abnormal immune response that predisposes them to serious infections
(lungs, thyroid autoimmunity)
Other Trisomies
 Other trisomies: involves chromosomes 8, 9, 13, 18, 22
 Trisomy 18 (Edwards syndrome)
 Trisomy 13 (Patau syndrome)
 Most cases result from meiotic nondisjunction and carry a complete extra copy of
chromosomes
 Association w/ increased maternal age is also noted
 Malformations are much more severe and wide ranging
Chromosome 22q11.2 Deletion Syndrome
 Chromosome 22q11.2 deletion syndrome encompasses a spectrum of disorders that
result from a small deletion of band q11.2 on the long arm of chromosome 22
 Clinical features include:
o Congenital heart defects
o Abnormalities of the palate
o Facial dysmorphism
o Developmental delay
o Variable degrees of T-cell immunodeficiency
o Hypocalcemia
 Represent DiGeorge syndrome and velocardiofacial syndrome
 DiGeorge syndrome – px have thymic hypoplasia w/ T-cell immunodeficiency,
parathyroid hypolasia that lead to hypocalcemia, cardiac malforamtions and mild facial
abnormalities
 Velocardiofacial syndrome – clinical features include: facial dysmorphism
(prominent nose, retrognathia), cleft palate, cardiovascular anomalies, learning
disabilities, immunodeficiency (less frequent)
 Individuals w/ the 22q11.2 deletion syndrome are at high risk for psychotic
illnesses, such as: schizophrenia (25% of adults) and bipolar disorders
 Diagnosis: detection of deletion by FISH
KEY CONCEPTS: Cytogenetic Disorders Involving Autosomes
 Down Syndrome – asso. w/ an extra copy of genes on chromosome 21, most
commonly due to trisomy 21 and less frequently from translocation of extra
chromosomal material from chromosome 21 to other chromosome or from mosaicism
 Patients w/ down syndrome have severe mental retardation, flat facial profile,
epicanthic folds, cardiac malformations, higher risk of leukemia and infections, and
premature development of Alzheimer disease
 Deletion of genes at chromosomal locus 22q11.2 gives rise to malformations affecting
the face, heart, thymus, and parathyroids. The resulting disorders are recognized as:
o DiGeorge syndrome – thymic hypoplasia w/ diminished T-cell immunity
and parathyroid hypoplasia w/ hypocalcemia
o Velocardiofacial syndrome – congenital heart disease involving outflow
tracts, facial dysmorphism, and developmental delay
Cytogenetic Disorders Involving Sex Chromosomes
 Genetic diseases asso. w/ changes involving the sex chromosomes are far more
common than those related to autosomal aberrations.
 Imbalances of sex chromosomes are much better tolerated than are similar
imbalances of autosomes
 Two factors that are peculiar to the sex chromosomes:
1. Lyonization or inactivation of all but one X chromosome
2. The modest amount of genetic material carried by the Y chromosome
 Lyon hypothesis (X-inactivation)
1. Only one of the X chromosomes is genetically active
2. The other X of either maternal or paternal origin undergoes heteropyknosis
and is rendered inactive
3. Inactivation of either the maternal or paternal X occurs at random among all
the cells of the blastocyst on or about day 5.5 of embryonic life
4. Inactivation of the same X chromosome persists in all the cells derived from
each precursor cell
 Normal females  one inactivated maternal X and inactivated paternal X
o Barr body or X chromatin
 XIST – unique gene involve in X inactivation; product is a long noncoding RNA
o XIST allele is switched off in the active X
 Although it was initially thought that all the genes on the inactive X are “switched off”,
more recent studies have revealed that many genes escape X inactivation
 Patients w/ the monosomy of the X chromosome (Turner syndrome: 45,X) have
severe somatic and gonadal deficiency
 Regardless of the # of X chromosomes, presence of a single Y determines
male sex
o Gene that dictates testicular development (SRY gene) is located on its distal
short arm
 Features common to all sex chromosomes disorders:
o Sex chromosome disorders cause subtle, chronic problems relating to sexual
development and fertility
o Often difficult to diagnose at birth, and many are first recognized at the
time of puberty
o The greater the number of X chromosomes, in both male and female, the
greater the likelihood of mental retardation
 Two most important disorders arising in aberrations of sex chromosomes:
o Klinefelter Syndrome
o Turner Syndrome
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Klinefelter Syndrome
 Male hypogonadism that occurs when there are two or more X
chromosomes and one or more y chromosomes
 One of the most frequent form of genetic disease involving sex chromosome; most
common cause of hypogonadism in male; 1 in 660 cases
 Rarely diagnosed before puberty because testicular abnormality does not develop
before early puberty
 Distinctive body habitus; increase in length between the soles and the pubic bone 
body appears elongated
 Eunuchoid body habitus w/ abnormal long legs, small small atrophic testes asso .w/
small penis; lack secondary male characteristics as deep voice, beard and male
distribution of pubic hair.
 Gynecomastia may be present; IQ is lower than normal; mental retardation is
uncommon; increased incidence in DM2 (insulin resistance)
 Mitral valve prolapsed (50% in cases); increased incidence of osteoporosis and
fractures due to sex hormonal imbalance
 Consistent finding  hypogonadism
 ↑ FSH; ↓ testosterone; ↑ estradiol
 Ratio of estrogen and testosterone  determines degree of feminization
 Klinefelter syndrome is an important genetic cause of reduced spermatogenesis and
male infertility
o Testicular tubules are atrophied and replaced by pink, hyaline, collegnous
ghost
 Px have a higher risk for breast cancer, extragonadal germ cell tumors, and
autoimmune diseases such as SLE
 Classic pattern is 47,XXXY karyotype (90% of cases)  results from
nondisjunction during meiotic division in germ cells
 15% of cases  46,XY/47,XXY; 47,XXY/48,XXXY
 In cases of normal females, all but one X chromosome is inactivated in px w/
klinefelter syndrome; px have hypogonadism and same features
o Reason lies in genes on the X chromosome that escape lyonization and in
the pattern of X inactivation
 Uneven dosage compensation during X-inactivation
o 15% of X-linked gene escape inactivation
o Extra dose of genes  overexpression leads to hypogonadism
 Second mechanism involves the gene encoding the androgen receptor,
through w/c testosterone mediates its effect
o Androgen receptor gene maps to X chromosome and contains highly
polymorphic CAG (trinucleotide) repeats  dictates functional response to
particular dose of androgen
o Shorter CAG repeats are more sensitive than w/ long CAG repeats
o In Klinefelter syndrome, X chromosome w/ shortest CAG repeat is
inactivated
o XXY males w/ low testosterone levels  expression of long CAG repeats
exacerbates the hypogonadism  small penis hehehe
Turner Syndrome
 Complete or partial monosomy of the X chromosome; characterized primarily
by hypogonadism in phenotypic females
 Most common sex chromosome abnormality in females (1 in 2500 cases)
 Three karyotypic abnormalities are seen:
1. ~57% are missing an entire X chromosome, resulting in a 45,X karyotype
o 43%  14% have structural abnormalities in X chromosome; 29% are
mosaics
2. Common feature of the structural abnormalities is to produce partial
monosomy of the X chromosome
o Inorder of frequency, the structural abnormalities of the X chromosome
include:
1) Isochrome of the long arm, 46,X,i(X)(q10) resulting in the loss of
the short arm
2) Deletion of portions of both long and short arms resulting in the
formation of a ring chromosome, 46,X,r(X)
3) Deletion of portions of the short or long arm, 46,del(Xq) or
46X,del(Xp)
3. Mosaic patients have a 45,X cell population along with one or more
karyotypically normal or abnormal cell types
o Examples of mosaic Turner females
1) 45,X/46,XX
2) 45,X/46,XY
3) 45,X/47,XXX
4) 45,X/46,X,i(X)(q10)
o In patients w/ high 45,X proportion, phenotypic changes are more severe
than w/ mosaicism w/c only present w/ primary amenorrhea
 5-10% have Y chromosome either as a complete (45,X/46,XY) or as fragments of Y
chromosomes  higher risk for development of gonadal tumor (gonadoblastoma)
 Most severely affected px present during infancy w/ edema of the dorsum of the hand
and foot due to lymph stasis swelling of the nape of the neck
 Latter is related to markedly distended lymphatic channels, producing hygroma
 As infants develop, the swellings subside leaving bilateral neck webbing and
persistent looseness of skin on the back of the neck
 Congenital heart disease is common (25-50%)  most important cause of
increased mortality in children w/ Turner syndrome
 Failure to develop normal secondary characteristics  infantile genitalia, inadequate
breast development, little pubic hair; Mental status is normal but there is subtle
defects in non-verbal, visual-spatial information processing
 Important in establishing the diagnosis is the adult is the shortness of stature
(<150 cm ) and amaenorrhea
 Turner syndrome is the single most important cause of primary amenorrhea
 50% has autoantibodies against thyroid gland  hypothyroidism
 Fetal ovaries develop normally in early embryogenesis, but absence of the second X
chromosome leads to accelerated loss of oocytes (completed by age 2 years) 
“menopause occurs before menarche” and ovaries are reduced to atrophic
strands, devoid of ova and follicles (streak ovaries)
 Short stature homeobox (SHOX) gene at Xp22.33 – gene involve in Turner
phenotype; critical regulator of growth
o Haploinsufficiency gives rise to short stature
o Expressed in fetal life in growth plates (radius, ulna, tibia,, fibula and 1 st and
2nd pharyngeal arches)
o Loss f SHOX  short stature
o Excess copy  tall stature
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Hermaphroditism and Pseudohermaphroditism
 Sexual ambiguity – disagreement among criterias for determining sex
 Genetic sex – determined by the presence or absence of a Y chromosome
 Gonadal sex – based on the histologic cxc of gonads
 Ductal sex – depends on the presence of derivatives of mullerian or wolffian ducts
 Phenotypic or genital sex – based on the appearance of the external genitalia
 Hermaphrodite – presence of both ovarian and testicular tissue
 Pseudohermaphrodite – disagreement between the phenotypic and gonadal sex
(e.g., female pseudohermaphrodite has ovaries but male external genotalia)
KEY CONCEPTS: Cytogenetic Disorders Involving Sex Chromosomes
 Females: one X chromosome is inactivated during development (Lyon hypothesis)
 Klinefelter syndrome – two or more X chromosomes w/ one Y chromosome as a result
of nondisjunction of sex chromosomes; testicular atrophy, sterility, reduced body
hair, gynecomastia, and eunochoid body habitus; most common cause of male sterility
 Turner syndrome – there is partial or comlete monosomy of genes on the short arm of
the X chromosome, most commonly due to the absence of one X chromosome (45,X)
and less commonly from mosaicism, or from deletions involving the short arm of the X
chromosome. Short stature, webbing of the neck, cabitus valgus, cardiovascular
malformations, amenorrhea, lack of secondary sex characteristics, and fibrotic ovaries
are typical clinical features.
SINGLE-GENE DISORDERS WITH NONCLASSIC INHERITANCE
 Transmission of certain single-gene disorders does not follow classic Mendelian
principles
 Can be classified into four categories
o Diseases caused by trinucleotide-repeat mutations
o Disorders caused by mutations in mitochondrial genes
o Disorides asso. w/ genomic imprinting
o Disorders asso. w/ gonadal mosaicism
Diseases Caused by Trinucleotide-Repeat Mutations
 Expansion of trinucleotide repeats is an important genetic cause of human
disease, particularly neurodegenerative disorders (table 5-8)
 General principles that apply to these diseases:
 Causative mutations are asso. w/ the expansion of a stretch of trinucleotides that
usually share the nucleotides G and C
o DNA is unstable and impairs gene function
 Expansion depends strongly on the sex of the transmitting parent
o Fragile X syndrome – expansion occurs during oogenesis
o Huntington disease – expansion occurs during spermatogenesis
 Three key mechanisms w/c unstable repeats cause disease
1) Loss of function of the affected gene
2) A toxic gain of function by alterations of protein structure (Huntington dx)
3) A toxic gain of function mediated by mRNA (fragile X syndrome)
 Polyglutamine diseases – cxd by progressive neurodegeneration in midlife
o Proteins are misfolded and tend to aggregate; aggregates may suppress
transcription of other genes, cause mitochondrial dysfunction, or trigger the
unfolded protein stress response and apoptosis
o Morphologic hallmark  accumulation of aggregated mutant proteins
in large intranuclear inclusions
Fragile X Syndrome and Fragile X Tremor/Ataxia
 Fragile X syndrome is the prototype of diseases in w/c the mutation is cxd by a long
repeating sequence of three nucleotides
 Most affected sequences share the nucleotides guanine (G) and cytosine (C)
 Fragile X Syndrome is the second most common genetic cause of mental
retardation after Down syndrome
o Caused by a trinucleotide mutation in the familial mental retardation-1
(FMR-1) gene; 1 in 1550 males; 1 in 8000 females
o Cytogenetic alteration was seen as a discontinuity of staining or as a
constriction in the long arm of X chromosome in a folate deficient-medium
o Chromosome appears broke ”fragile site”
o Affected males are mentally retarded; 20-60 IQ; long face w. Large
mandible, large everted ears, large testicles (macro-orchidism; most
distinctive feature; 90%)
o Hyperextensible joints, high arched palate, mitral valve prolapsed
 Some patterns of transmission not typically asso. w/ other X-linked recessive
disorders:
 Carrier males  normal transmitting males (20%)
 Affected females  30-50% are affected
 Risk of phenotypic effects
 Anticipation  features worsen w/ each successive generation; becomes
increasingly deleterious as it is transmitted from a man to his grandsons
and great-grandsons
 Xq27.3  where FMR1 gene lies; presence of clinical symptoms and fragile site is
related to the amplification of the CGG repeats
o Normal transmitting males and carrier females carry 55-200 repeats
o Expansion of size is called premutaions
o Px have full mutations (extremely large expansion; 200-400 repeats) 
arise from further amplification of the CGG repeats in permutations
 During the process of oogenesis (but not spermatogenesis) permutations
can be converted to mutations by triple-repeat amplification
o Mental retardation is much higher in grandsons  full mutation
 Molecular basis of mental retardation and somatic changed is related tp loss of
function of FMRP
o When FMR1 exceed 230 (normal is 55 CGG repeats), DNA of entire 5’ region
becomes abnormally methylated
o Methylation results in transcriptional suppression of FMR1
o Absence of FMRP  cause of phenotypic changes
 FMRP – most abundant in brain and testis (two organs most affected by the
disease; functions:
 FMRP selectively binds mRNAs asso. w/ polysomes and regulates their
intracellular transport to dendrites (binds 4% of mammalian brain mRNAs)
 FMRP is a translation regulator
 PCR-based detection of the repeats  method of choice for diagnosis
Fragile X Tremor/Ataxia
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  Although initially assumed to be innocuous, CGG permutations in the FMR1 gene can
cause a disease that is phenotypically different from fragile X syndrome through a
distinct mechanism involving a toxic “gain-of-function”
 Premature ovarian failure; progressive neurodegenerative syndrome
 Cxd by intention tremors and cerebellar ataxia and may progress to
parkinsonism
 FMR1 gene instead of being methylated and silenced continues to be transcribed
 CGG-containing FMR1 mRNAs so formed are “toxic”  accumulate in the nucleus and
form intracellular inclusions
 Aggregated mRNA recruits RNA-binding proteins
KEY CONCEPTS: Fragile X Syndrome
 Pathologic amplification of trinucleotide repeats causes loss-of-function (fragile X
syndrome) or gain-of-function mutations (Huntington disease). Most such mutations
produce neurodegenerative disorders
 Fragile X syndrome results from loss of FMR1 gene function and is cxd by mental
retardation, macro-orchidism and abnormal facial features
 In the normal population, there are about 29-55 CGG repeats in the FMR1 gene. The
genomes of carrier males and females contain permutations w/ 55-200 CGG repeats
that can expand to 4000 repeats (full mutations) during oogenesis. When full
mutations are transmitted to progeny, fragile X syndrome occurs
 Fragile X tremor/ataxia due to expression of a FMR1 gene bearing a permutation
develops in some males and females. The accumulation of corresponding mRNA in the
nucleus binds and sequesteres certain proteins that are essential for normal neuronal
functions
Mutations in Mitochondrial Genes – Leber Hereditary Optic Neuropathy
 A feature unique to mtDNA is maternal inheritance
 mtDNA complement of the zygote is derived entirely from the ovum
 Mothers transmit mtDNA to all their offspring (male and female)
o However, daughters (not sons) transmit the DNA to their progeny
 Features apply to mitochondrial inheritance:
 Human mtDNA contains 37 genes, 22 are transcribed to tRNA and 2 into rRNA
o Remaining 13 genes encode subunits of the respiratory chain enzymes
o Since mtDNA is involved in oxidative phosphorylation, mutations affecting
them has deleterious effects on organs most dependent on oxidative
phosphorylation (CNS, skeletal muscle, cardiac muscle, liver, kidneys)
 Each mitochrondrion contains thousands of copies of mtDNA and deleterious
mutations of mtDNA affect some but not all of these copies
o Heteroplasmy – tissues and individuals may harbour both wild and mutant
mtDNA
o Threshold effect – minimum number of mutant mtDNA must be present
before oxidative dysfunction gives rise to disease
 During cell division, mitochondrion and their contained DNA are randomly distributed
to the daughter cells
 Diseases asso. w/ mitochondrial inheritance are rare and affect the neuromuscular
system
o E.g., Leber hereditary optic neuropathy – manifest a progressive
bilateral loss of central vision (starts at age 15-35) leading to blindness
Genomic Imprinting
 Imprinting – epigenetic process that results in between paternal and maternal allele
o Inactivates either maternal or paternal allele
o Occurs in the ovum or sperm before fertilization and then transmitted to all
somatic cells through mitosis
o Asso.w/ differential patterns of DNA methylation at CG nucleotides, H4
deacytlation and methylation
o Regulated by cis-acting elements called imprinting control regions
o Prader-Willi syndrome and Angelman syndrome
 Maternal imprinting – transcriptional silencing of the maternal allele
 Paternal imprinting – paternal allele is inactivated
Prader-Willi Syndrome and Angelman Syndrome
 Prader-Willi syndrome – cxd by mental retardation, short stature, hypotonia, profound
hyperphagia, obesity, small hands and feet, and hypogonadism
o 65-70% of cases – interstitial deletion of band q12 in the long arm of
chromosome 15, del(15)(q11.2q13)  causes 5-Mb deletion
o It is striking that in all cases the deletion affects the paternally derived
chromosome 15
o In contrast w/ Prader-Willi syndrome, px w/ the phenotypically distinct
Angelman syndrome are born w/ a deletion of the same chromosomal
region derived from their mothers
 Angelman syndrome – mentally retarded, but in addition they present w/ ataxic
gait, seizures, and inappropriate laughter  “happy puppets”
 The comparison of two syndromes demonstrates the “parent-of-origin” effects on
gene function
 Mechanisms involved in the genomic printing of these disease:
o Deletion
 If maternal 15q12 is imprinted, and paternal is functional 
Prader-Willi syndrome
 If paternal is inactivated; maternal is functional  Angelman
syndrome
o Uniparent disomy – inheritance of both chromosome of a pair from one
parent  non-functional set of genes from nonimprinted chromosomes
 Prader-Willi syndrome  if nondeletion, they have two maternal
copies of chromosome 15
 Angelman syndrome – result from uniparental disomy of paternal
chromosome 15
 Second most common mechanism (20-25% of cases)
o Defective imprinting – 1-4% of cases; no functional alleles
 Prader-Willi syndrome – paternal chromosome carries the
maternal imprint
 Angelman Syndrome – maternal chromosome carries paternal
imprint
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Genetic basis of these two imprinting disorders:
Angelman  Affected gene is ubiquitin ligase involved in catalyzing
Syndrome the transfer of activated ubiquitin to target protein
substrates
 UBE3A – gene; maps w/in 15q12 region; imprinted on the
paternal chromosome, and is expressed from maternal
allele primarily in regions of the brain
 Imprinting is tissue specific  UBE3A is expressed from
both alleles in most tissues
Prader-Willi  No single gene has been implicated
syndrome  Series of genes located in the 15q11.2-q13 interval
(imprinted on maternal chromosome and expressed from
paternal chromosome) are involved
 SNORP family of genes – encode small nucleolar RNAs
involved in modification of rRNAs
 Loss of SNORP function  contribute to Prader-Willi syn.
 Molecular diagnosis of these syndrome is based on assessment of methylation of
marker genes and FISH
KEY CONCEPTS: Genomic Imprinting
 Imprinting involves transcriptional silencing of the paternal or maternal copies of
certain genes during gametogenesis. For such genes, only one functional copy exists
in the individual. Loss of the functional (not imprinted) allele by deletion gives rise to
diseases.
 Prader-Willi Syndrome – deletion of band q12 on long arm of paternal
chromosome 15. Genes in this region of maternal chromosome are imprinted so there
is complete loss of their functions. Patients have mental retardation, short stature,
hypotonia, hyperphagia, small hands and feet, hypogonadism
 Angelman syndrome – there is deletion of the same region from the maternal
chromosome. Since genes on the corresponding region of paternal chromosome 15
are imprinted, these patients have mental retardation, ataxia, seizures, and
inappropriate laughter
Gonadal Mosaicism
 In some autosomal dominant disorders, exemplified by oteogenesis imperfect,
phenotypically normal parents have more than one affected child  cause by
gonadal mosaicism
 Results from a mutation that occurs postzygotically during early (embryonic)
development
 If mutation affects only cells to form gonads, the gametes carry the mutation but
somatic cells are normal
 A phenotypically normal parent who has gonadal mosaicism can transmit the disease-
causing mutation to the offspring through their mutated gametes
o More than one child of such parent would be affected
 Occurrence depends on the proportion of the germ cells carrying the mutation
MOLECULAR GENETIC DIAGNOSIS
 Karyotyping for recognition of cytogenetic disorders (e.g., Down syndrome)
 DNA-based assays (e.g., Southernblotting for Huntington disease)
 Regardless of the technique used, human genetic markers can be either
o Constitutional (present in each and every cell of the affected persons;
e.g., CFTR)
o Somatic (restricted to specific tissue types or lesions;e.g., KRAS)
Diagnostic Methods and Indications for Testing
Laboratory Considerations
 Pathologist focus on the sensitivity, specificity, accuracy, and reproducibility of
different methods; and practical factors: cost, labor, reliability, and TAT
 Critical to first understand the spectrum of genetic anomalies responsible for the dx
Indications for Analysis of Inherited Genetic Alterations
 May be required at any age; mostly performed during prenatal or postnatal/childhood
periods
 Prenatal testing should be offered for all fetuses at risk for a cytogenetic
abnormality: indications:
o Advanced maternal age
o A parent known to carry a balanced chromosomal rearrangement (
increased risk of abnormal chromosome segregation and aneuploidy)
o Fetal abnormalities during ultrasound
o Routine maternal blood screening, indicating risk of Down syndrome or
other trisomy
 Prenatal testing may also be considered for children at known risk for genetic
disorders (cystic fibrosis, spinal muscular atrophy) using targeted analysis based on
familial mutations or family history
 Usually performed on cells obtained by amniocentesis, chronic villus biopsy,
umbilical cord blood
 Parents known to be at risk for having a child w/ a genetic disorder can choose to
have genetic testing performed on embryos created in vitro prior to uterine
implantation, eliminating the chance of generational transmission of a familial disease
 Ff birth, testing is done as soon as the possibility of genetic disease arises. Most
commonly performed on peripheral blood DNA. In newborns or children,
indications may be as follows:
o Multiple congenital anomalies
o Suspicion of a metabolic syndrome
o Unexplained mental retardation and/or developmental delay
o Suspected aneuploidy (feat. Of Down syndrome) or other syndromic
chromosomal abnormality (deletions, inversions)
o Suspected monogenic disease (previously described or unknown)
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  In older patients, testing is focused in diseases that manifest at later stages of life;
indications include:
o Inherited cancer syndromes
o Atypical mild monogenic disease
o Neurodegenerative disorders
Indications for Analysis of Acquired Genetic Alterations
 Identify nucleic acid sequences or aberrations specific for acquired diseases;
indication:
o Diagnosis and management of cancer:
 Detection of tumor specific mutations and cytogenetic alterations
that are hallmark of tumors (BCR-ABL)
 Determination of clonality as an indicator of a neoplastic condition
 Identification of specific genetic alterations that can direct
therapeutic choices (HER2)
 Determination of treatment efficacy
 Detection of drug-resistant secondary mutations in malignancies
o Diagnosis and management of infectious disease
 Detection of microorganism-specific genetic material for definitive
diagnosis
 Identification of specific genetic alterations in the genomes of
microbes that are asso. w/ drug resistance
 Determination of treatment efficacy
PCR and Detection of DNA Sequence Alterations
 PCR analysis, w/c involves the synthesis of relatively short DNA fragments from a DNA
template, has been a mainstay of molecular diagnostics for the last few decades
 Uses heat-stable DNA polymerases and thermal cycling  target DNA (<1000
base pairs) lying between primer sites is exponentially amplified
Sanger  The amplified DNA is mixed w/ a DNA polymerase, a DNA
Sequencing primer, nucleotides, and a four dead-end (di-deoxy
terminator) nucleotides (A,T,G,C) labelled w/ fluorescent tags
 Reaction produces series of DNA molecules labelled w/ a tag
corresponding to the base at w/c the reaction stopped due to
addition of terminator nucleotides
 After size separation by capillary electrophoresis, the
sequence can be read and compared w/ normal sequence to
detect mutations
 Application: analysis of large genes or multiple genes
 “gold standard” for sequence determination
Pyrosequencing  There is a release of pyrophosphate when a nucleotide is
incorporated into a growing DNA strand
 Also performed on PCR products using a single sequencing
primer and involves cycling individual nucleotides (A,C,T,G)
 If one or more nucleotides are incorporated into the growing
strand of DNA, pyrophosphate is released and participate in
secondary reaction involving luciferase that produce light
and measured by photo detector
 Most often used when testing for particular sequence and is
more sensitive than Sanger sequencing
 Allows detection for as little as 5% mutated alleles in a
background of normal alleles
 Application: used to analyze DNA obtained from cancer
biopsies, in w/c tumor cells are often “contaminated” w/ large
numbers of stromal cells
Single-base  Useful approach for identifying mutations at a specific
primer nucleotide position (e.g., mutation in codon 600 of the BRAF
extension gene)
 An interrogating sequencing primer is added to the PCR product,
w/c binds just one base upstream of the target
 Differently colored terminator fluorescent nucleotides are also
added and a single base polymerase extension is performed 
fluorescence are then detected
 Very sensitive – 1-2% mutated alleles
 Disadvantage: produce only one base pair of sequence data
Restriction  Involves digestion of DNA w/ endonucleases known as
fragment length restriction enzymes that recognize and cut DNA at specific
analysis sequences
 If specific mutation affects a restriction site, then the amplified
PCR product may be digested, and the normal and mutant PCR
products will yield fragments of different sizes
 Identified as different bands following electrophoresis
 Less comprehensive than direct sequencing but remain useful
for molecular diagnosis when actual mutation occurs at an
invariant nucleotide position
Amplicon length  Mutations that affect the length of DNA (deletion,
analysis expansion) can be detected by PCR
 E.g., Fragile X syndrome asso. w/ alterations in trinucleotide
repeats  two primers flank the region w/ trinucleotide repeats
at the 5’ end of FMR1 gene are used to amplify the intervening
sequence  large differences in the number of repeats  size
of PCR products from normal and permutation individuals are
different
 Easily distinguished by electrophoresis
 This technique will fail if a trinucleotide repeat expansion is
so large (use Southern blot instead)
Real-time PCR  Use fluorophore indicators that can detect and quantify the
presence of a particular nucleic acid sequences in “real time”
 during the exponential phase of NA amplification rather than
post-PCR
 Most often used to monitor the frequency of cancer cells
bearing cxc genetic lesions in the blood or tissues, or the
infectious load of certain viruses
 Can also be used to detect somatic point mutations in
oncogenes such as KRAS and BRAF  an approach that has
advantage of avoiding the need for post-PCR analysis
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Molecular Analysis of Genomic Alterations
 Hybridization-based techniques
Fluorescence in Situ Hybridization (FISH)
 FISH uses DNA probes that recognize sequences specific to particular
chromosomal regions
 Human DNA inserts in clones in order of 100,000-200,000 base pairs  these DNA
clones are labelled w/ fluorescent dyes and applied to metaphase chromosome
spreads to interphase nuclei that are pretreated to “melt” the genomic DNA
 Ability of FISH to circumvent the need for dividing cells is invaluable when a rapid
diagnosis is warranted
 FISH can be performed on: prenatal samples, peripheral blood cells, touch
preparations from cancer biopsies, and even fixed archival tissue sections
 Fish is used to detect numeric abnormalities of chromosomes (aneuploidy),
subtle microdeletions or complex translocations that are not demonstrable by routine
karyotyping , and gene amplification
 Chromosome painting is an extension of FISH
 Number chromosomes that can be detected is limited by the availability of fluorescent
dyes  overcome by introduction of spectral karyotyping or “multicolour FISH” 
powerful that it might called “spectacular karyotyping”
Multiplex Ligation-Dependent Probe Amplification (MLPA)
 MLPA blends DNA hybridization, DNA ligation, and PCR amplification to detect
deletions and duplications of any size, including anomalies that are too large to
be detected by PCR and too small to be identified by FISH
 Each MLPA reaction uses a pair of probes that can hybridize side-by-side to one strand
of the target DNA  probes are joined via ligase reaction
 Probes also contain additional sequences at their ends that can be used as a primer
sequences in a PCR
 Ligated probes create a template that can be amplified by PCR  quantification yields
accurate info regarding the amount of starting material
 MLPA can be performed on very small amounts of genomic DNA
Southern Blotting
 Changes on the structure of specific loci can be detected by Southern blotting 
involves hybridization of radio labeled sequence-specific probes to genomic DNA that
has been first digested w/ a restriction enzyme and separated by gel
electrophoresis
 The probe detect one germ line band in normal individuals and a different size band
depending on the genetic anomaly
 Rarely used; useful in the detection of certain large-trinucleotide-expansion
diseases, including fragile X syndrome
Cytogenomic Array Technology
 Genomic abnormalities can also be detected w/o prior knowledge by using microarray
technology to perform a global genomic survey
Array-Based Comparative Genomic Hybridization (Array CGH)
 The test DNA and a reference (normal) DNA are labelled w/ two different
fluorescent dyes  both are cohybridized to an array spotted w/ DNA probes
(cover all 22 autosomes and sex chromosomes)
 At each chromosomal probe location, binding of the labelled DNA from two
samples is compared
 If two samples are equal  diploid; all spots will fluoresce yellow (green and
red dyes)
 If test sample shows even a focal deletion or duplication, the probe spots
corresponding to it will show skewing toward red or green allowing highly accurate
determinations of copy number variant across the genome
SNP Genotyping Arrays
 Designed to identify single nucleotide polymorphism (SNP) sites genome-
wide
 SNP’s serve as both a physical landmark within the genome and as a genetic
marker whose transmission can be followed from parent to child
 Methods involving SNPs can be used to make copy number variations (CNV)
 This technology is the mainstay of genome wide association studies (GWAS)
 In the clinical laboratory, SNP arrays are routinely used to uncover copy number
abnormalities in pediatric patients when the karyotype is normal but a structural
chromosomal abnormality is still suspected
 Common indications include;
o Congental abnormalities
o Dysmorphic features
o Developmental delay
o autism
Polymorphic Markers and Molecular Diagnosis
 If exact nature of genetic aberration is unknown or if testing for primary disease is
challenging or unfeasible  diagnostic lab uses linkage
 The two types of genetic polymorphism most useful for linkage analysis are SNPs and
repeat-length polymorphism knwon as minisatellite and microsatellite
 Human DNA contains short repetitive sequences of DNA giving rise to “repeat-
length polymorphism”  subdivide into microsatellite and minisatellite repeats
 Microsatellite - <1 kilobase; cxd by a repeat size of 2-6 base pairs
o Scaterred through the human genome; high level of polymorphism
o Ideal for differentiating between two individuals and follow transmission of
the marker from parent to child
o Also use in determining paternity and criminal investigation
 Minisatelllite – larger (1-3 kilobases); repeat motif is usually 15-70 base pairs
 Assays to detect genetic polymorphisms are also important in many other areas of
medicine, including in the determination of relatedness and identity in transplantation,
cancer genetics, paternity testing, and forensic medicine
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Polymorphism and Genome-Wide Analysis
 In GWAS (genome wide association studies), large cohort of patients w/ and
w/o a disease (rather than families) are examined across the entire genome for
common genetic variants with the disease
 GWAS also led to the identification of genetic loci that modulate common quantitative
traits in humans (height, body mass, hair and eye color, and bone density)
Epigenetic Alterations
 Epigenetics – defined as the study of heritable chemical modification of DNA or
chromatin that does not alter the DNA sequence itself
o Ex: methylation of DNA, methylation and acetylation of histones
 Gene expression correlates w/ the level of methylation of DNA, usually of cytosines
specifically in CG dinucleotide-rich promoter regions known as CpG islands
 Increased methylation of these loci is associated with decreased gene expression and
is accompanied by concomitant specific patterns of histone methylation and
acetylation
 Treatment of genomic DNA w/ sodium bisulfite  a chemical that converts
unmethylated cytosine to uracil, w/c acts like thymine in downstream reactions
o Methylated cytosines are protected from modifications and remain
unchanged
o After treatment, it is then straightforward to discriminate the unmethylated
(modified) DNA from the methylated (unmodified) DNA on the basis of
sequence analysis
RNA Analysis
 Mature mRNA – contains the coding sequences of all expressed genes
 RNA can substitute for DNA in a wide range of diagnostic applications
o However, DNA-based diagnosis is preferred because DNA is much more
stable
o Detection and quantification of RNA viruses  HIV and Hep C
o mRNA expression profiling is emerging as an important tool for molecular
stratification
 Cancer cells bearing particular chromosomal translocations are detected w/ greater
sensitivity by analyzing mRNA
o Principal reason for this is that most translocations occur in the scattered
location w/in particular introns, s/c can be very large, beyond the capacity
of conventional PCR amplification
o Since introns are removed by splicing during the formation of mRNA, PCR
analysis is possible if RNA is first converted to cDNA by reverse
transcriptase
 Real-time PCR performed on cDNA is the method of choice for monitoring residual
disease in patients w/ CML and certain other hematologic malignancies
Next-Generation Sequencing
 Next-generation sequencing (NGS) is a term used to describe several newer DNA
sequencing technologies that are capable of producing large amounts of sequence
data in a massively parallel manner
 NGS allows to perform impossible analyses at extremely low relative cost
 The fundamental factor that sets NGC apart from traditional Sanger sequencing is its
input sample requirements
o NGS has no requirements: any DNA from almost any source can be used
o NGS instruments are well suited to heterogenous DNA samples due to the
application of these common basic processes:
Spatial  At the beginning, individual input DNA molecules are physically
separation isolated from each other in space
 Specifics of this process are platform-dependent
Local  After separation, the individual DNA molecules are amplified in
amplification situ using a limited number of PCR cycles
 Amplification is necessary so that sufficient signal can be
generated to ensure detection and accuracy
Parallel  The amplified DNA molecules are sequenced by addition of
sequencing polymerase  yield a “read” that corresponds to its sequence
 Sequence reads from NGS instruments are generally short
(<500bp)
Bioinformatics
 NGS instruments can generate a staggering amount of sequence data  enough to
produce a high-quality sequence spanning an entire human genome
 Basic steps necessary to process this type of data in a generic human DNA context:
o Alignement – process by w/c the sequencing reads from a sample are
mapped to a genome where they can be viewed and interpreted
o Variant calling – process involves “walking” across the reference genome
and evaluating all of the sequence data that mapped to each position and
compare it w/ reference genome
o Variant annotation and interpretation
Clinical Applications of NGS DNA Sequencing
 Basic approaches:
 Targeted sequencing
o Just one gene or a panel of genes minimizes sequencing costs as well as
the time and expense required for manual interpretation and clinical
reporting
o Sample preparation  subselecting relevant clones from a whole genome
library via custom complementary probes or by alternate preparations from
genomic DNA such as multiplex PCR
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  Whole exome sequencing (WES)
o Type of targeted sequencing
o Uses hundreds of thousands of custom probes to pull out 1.5% of the
genome consist of protein-encoding exons
o WES enables a broad survey for protein coding mutations (responsible for
80% of Mendelian disease) at reduced cost
 Whole genome sequencing (WGS)
o Most comprehensive type of DNA analysis
o Current cost and informatics challenges still preclude its routine use in
clinical practice
o Indications: mostly limited to cases where exome sequencing has failed to
provide an answer but the clinical suspicion of genetic disease remains high
o Cancer applications: WGS is the only NGS application that can detect
novel structural rearrangements (insertions, deletions, translocations) that
may be clinically relevant
o WGS is generally performed to lower sequencing depth than either targeted
panels or exomes, and may suffer from a lack of statistical power to detect
low percentage mutations in heterogenous tumor samples

 The choice of approach is mainly a function of sequencing cost and interpretive

workload

Future Applications

 Microbiome analysis and blood screening for early markers of diseases, including
cancer

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