MBPA 3413

General Pathology III: Genetic

Disorders
Overview Lecture
Scope
• Introduction to Genetic Disorders
• Overview of genetic disorders and their impact on health.
• Molecular Basis of Genetic Disorders
• Genetics and genomics fundamentals.
• Genetic mutations and their consequences.
• Patterns of Inheritance
• Mendelian inheritance (autosomal dominant, autosomal recessive, X-
linked, Y-linked).
• Non-Mendelian inheritance patterns.
Scope
• Single-Gene Disorders
• Disorders caused by mutations in a single gene.
• Chromosomal Disorders
• Disorders caused by abnormalities in chromosomal structure or
number.
• Multifactorial and Polygenic Disorders
• Disorders influenced by both genetic and environmental factors.
• Mitochondrial Disorders
• Disorders caused by mutations in mitochondrial DNA.
Scope
• Genetic Testing and Counselling
• Techniques and methods for genetic testing.
• Genetic counselling and ethical considerations.
• Therapeutic Approaches and Future Directions
• Current and emerging therapies for genetic disorders.
• Advances in gene therapy and genetic engineering.
Thank you
 MBPA 3413
General Pathology III: Genetic
Disorders
INTRODUCTION TO GENETIC DISORDERS
AND MUTATIONS
Scope
• Introduction to Genetic Disorders
• Overview of genetic disorders and their impact on health.
• Application of Medical Genetic
• Molecular basis of human disease
• Production of human biologically active agents
• Gene therapy
• Disease diagnosis
• Mutations
• Types of mutations
• Categories of genetic disorders
INTRODUCTION TO GENETIC DISORDERS
• Lifetime frequency of genetic disease is thought to be higher
than is widely appreciated (670 per 1000)
• “Classic” genetic disorders
• Cardiovascular diseases
• Disorders of immunity
• Cancers etc
• Extremity of genetic disorders varies
• Approx. 50% of early pregnancy miscarriages have demonstrable
chromosomal abnormalities
• Approx. 5% of individuals under 25 years develop a serious disease
with significant genetic component.
• Unknown number of genetic disorders may be mild or remain hidden
• Human beings have about 30,000 genes
• Less than 2% code for proteins
• More than half are repetitive nucleotide codes of mysterious function
• Individuals share 99.9% of their DNA sequence with 0.1%
(about 3 million base pairs) of remaining DNA accounting for the
diversity in the human race.
Applications of Genetics in Medicine
• Molecular basis of human disease
• Functional cloning (classic) approach – A known affected protein (clinical
phenotype) is identified, normal gene is isolated and cloned then molecular
changes affecting the gene determined
• Positional cloning (candidate gene) approach – Mapping disease phenotype
to particular chromosome location and cloning several DNA pieces from the
region in vitro and identification of aberrant proteins from the mutated genes
• Production of human biologically active agents
• Inserting requisite genes into bacteria or other suitable cells in tissue culture
• Soluble TNF receptor, tissue plasminogen activator, growth hormone, erythropoietin etc
• Gene therapy
• Treating genetic diseases by transfer of somatic cells transfected with normal
genes. Do the benefits outweigh the risks?
• Disease diagnosis
• Molecular probes used in diagnosis of both genetic and nongenetic (eg
infectious) diseases
Mutations
• Mutations are permanent changes in the DNA
• Mutations affecting germ cells are transmitted to progeny
• Inherited diseases
• Mutations affecting somatic cells are not transmitted to progeny
• Cancers and some congenital malformations
Examples of Mutations
• Point mutations – substitution of a single nucleotide base by a
different base; resulting in substitution of an amino acid within a
protein e.g. sickle cell anaemia
• Missense mutations
• Nonsense mutations
• Frameshift mutations – insertion or deletion of one/two base pairs
alters the reading frame of the DNA strand
• Trinucleotide repeat mutations – dynamic amplification of a sequence
of three nucleotides (guanine and cytosine involved).
• Dynamic (degree of amplification increases at gametogenesis)
• Fragile X syndrome
Categories of Genetic Disorders
Major Categories
• Diseases related to mutant genes of large effect
• Diseases with multifactorial (polygenic) inheritance
• Diseases arising from chromosomal aberrations

Other Categories
• Diseases resulting from triplet repeat mutations
• Disease resulting from mutations in mitochondrial DNA
• Diseases whose transmission is influenced by genomic imprinting
(epigenetic influences)
Thank you
MBPA 3413 – GENETIC DISORDERS

Mutations, Mendelian Disorders, and Transmission Patterns of

Single Gene Disorders
Objective
• To outline the fundamental aspects regarding mutations,
Mendelian disorders, and transmission patterns of single gene
disorders
Scope
• Mutations
• Definition and types of mutations (e.g., point mutations, insertions,
deletions)
• Mechanisms of mutations
• Effects of mutations on genes and proteins
• Mendelian Disorders
• Introduction to Mendelian inheritance patterns
• Explanation of key concepts like heterozygosity, carriers, and
penetrance
• Transmission Patterns of Single Gene Disorders
• Autosomal Dominant Disorders
• Characteristics and examples
• Penetrance and expressivity in autosomal dominant disorders
• Autosomal Recessive Disorders
• Characteristics and examples
• Consanguinity and prevalence in specific populations
• X-Linked Disorders
• Characteristics and examples
• Carrier status and manifestations in females
Mutations
• Permanent change in the DNA
• Germ cells – transmitted to progeny/inherited diseases
• Somatic cells – Congenital malformations, cancers etc
• Categories of Mutations
• Genome mutations
• Loss/gain of whole chromosomes (monosomies/trisomies)
• Chromosome mutations
• Rearrangement of genetic material resulting in visible structural changes
• Gene mutations
• Partial/complete deletion of a gene or a single base
• Point mutation (substitution)
• Frameshift mutations (insertion/deletion of one or two base pairs leading to alteration of reading
frame
• Gene mutations
• Point mutations within coding sequences
• Missense mutations
• Conservative
• Non-conservative e.g. SCA
• Nonsense mutation
• Change an amino acid codon to a chain terminator
• Mutations within noncoding sequences
• Promoter and enhancer sequences (regulatory sequences)
• Interference with binding of transcription factors leading to reduced or lack of transcription eg some hereditary
haemolytic anaemias
• Deletions and Insertions
• Less than 3 = frameshift mutation
• 3 or 3X = abnormal proteins missing amino acid(s)
• Trinucleotide repeat mutations
• Dynamic amplification of a sequence of 3 nucleotides
Categories of Genetic Disorders
1. Diseases related to mutant genes of large effect
2. Diseases with multifactorial inheritance
3. Chromosomal disorders
Mendelian Disorders
• All Mendelian disorders fall under mutations in single genes of large
effect
• It is estimated that every person carries 5 to 8 deleterious genes –
most being recessive with 80% being familial and the remainder
acquired de novo
• Gene expression may be dominant , recessive or co-dominant
• A single mutant gene may lead to many end effects – pleiotropism
• Mutations at several genetic loci may produce same trait – genetic
heterogeneity
Transmission Patterns of Single-Gene
Disorders
• Patterns of inheritance
1. Autosomal dominant
2. Autosomal recessive
3. X-linked
Autosomal Dominant Disorders
• Typically, at least one parent is affected
• Mutation may occur de novo in unaffected parent gamete (eg relatively older
fathers)
• Affects both males and females
• Both male and females can transmit the disorder
• Clinical features can be modifies by
• Reduced penetrance (% affected, other % phenotypically normal)
• Variable expressivity (trait seen in all with the mutation but expressed differently)
• Many of the conditions have delayed onset
• Mechanisms
• Reduced production of gene product
• Production of inactive protein
Autosomal Recessive
• Largest category of Mendelian disorders
• The trait does not usually affect parents but siblings may show the disease
• 25% recurrence risk for each birth
• Expression of defect tends to be more uniform than in autosomal dominant
disorders
• Complete penetrance is common
• Onset frequently early in life
• De novo mutations may manifest several generations later
• Include almost all inborn errors of metabolism
X-Linked Disorders
• All sex-linked disorders are X-linked, almost all X-linked recessive
• An affected male does not transmit it to sons; daughters are carriers
• Few X-linked dominant conditions e.g. Vitamin D resistant rickets
Biochemical and Molecular Basis of
Mendelian Disorders
• Enzyme defects
• Enzyme inhibitor defects
• Defects in receptors
• Defects in transport systems
• Alterations in structure, function or quantity of non-enzyme proteins
• Defects in haemostasis
• Defects in growth regulation
• Genetically determined adverse reactions to drugs
Thank you
COMPLEX MULTIGENIC
DISORDERS
COMPLEX MULTIGENIC DISORDERS
• Also called multifactorial or polygenic disorders
• They are caused by interactions between variant forms of genes and
environmental factors
• A genetic variant that has at least two alleles and occurs in at least
1% of the population is called a polymorphism
• According to the common disease–common variant hypothesis
complex multigenic disorders occur when many polymorphisms, each
with a modest effect and low penetrance, are co-inherited
• Two additional important facts have emerged from studies of
common complex disorders such as type 1 diabetes:
• While complex disorders result from the collective inheritance of many
polymorphisms, different polymorphisms vary in significance e.g., of the 20 to
30 genes implicated in type 1 diabetes, 6 or 7 are most important, and a few
HLA alleles contribute more than 50% of the risk
• Some polymorphisms are common to multiple diseases of the same type,
while others are disease-specific
• This observation is well illustrated in immune-mediated inflammatory diseases
• Several normal phenotypic characteristics are governed by multigenic
inheritance, such as hair color, eye color, skin color, height, and
intelligence
• These characteristics (also known as quantitative trait loci [QTLs]) show a
continuous variation within, as well as across, all population groups
• Environmental influences significantly modify the phenotypic
expression of complex traits e.g. type 2 diabetes mellitus has many of
the features of a complex multigenic disorder
• Affected persons often first exhibit clinical manifestations of this disease after
weight gain
• Thus, obesity as well as other environmental influences, unmasks the diabetic
genetic trait
• Assigning a disease to this mode of inheritance must be done with
caution
• Such attribution depends on many factors but first on familial
clustering and the exclusion of mendelian and chromosomal modes
of transmission
• A range of levels of severity of a disease is suggestive of a complex
multigenic disorder, but as pointed out earlier, variable expressivity
and reduced penetrance of single mutant genes also may account for
this phenomenon
• Because of these problems, sometimes it is difficult to distinguish between
mendelian and multifactorial disorders
THANK YOU
“Give doctors the honor they deserve, for the Lord gave them their work to do. Their skill came from
the Most High, and kings reward them for it. Their knowledge gives them a position of importance,
and powerful people hold them in high regard.”

Sirach 38:1-3
CYTOGENETIC DISORDERS
INTRODUCTION
• It is estimated 1 in 200 newborn infants has some form of
chromosomal abnormality
• (Approx. 50% of first-trimester spontaneous abortions)

• Cytogenetic disorders may result from alterations in the number or

structure of chromosomes and may affect autosomes or sex
chromosomes
Introduction (cont’d)
• Karyotype is a photographic representation of a stained metaphase
spread in which the chromosomes are arranged in order of
decreasing length
• A variety of techniques for staining chromosomes have been
developed
• With the widely used Giemsa stain (G banding) technique, each chromosome
set can be seen to possess a distinctive pattern of alternating light and dark
bands of variable widths
• The use of banding techniques allows identification of each
chromosome, and can detect and localize structural abnormalities
large enough to produce changes in banding pattern
Numeric Abnormalities
 Euploidy
• Any exact multiple of the haploid number (n) is called euploid
• Chromosome numbers such as 3n and 4n are called polyploid
• Polyploidy generally results in a spontaneous abortion
Aneuploidy
• Any number that is not an exact multiple of n is called aneuploid
• The chief cause of aneuploidy is nondisjunction of a homologous pair
of chromosomes at the first meiotic division or a failure of sister
chromatids to separate during the second meiotic division
• The latter also may occur during mitosis in somatic cells, leading to
the production of two aneuploid cells
• Failure of pairing of homologous chromosomes followed by random
assortment (anaphase lag) can also lead to aneuploidy
• When nondisjunction occurs at the time of meiosis, the gametes
formed have either an extra chromosome (n + 1) or one less
chromosome (n − 1)
• Fertilization of such gametes by normal gametes would result in two types of
zygotes:
• Trisomic, with an extra chromosome (2n + 1) or,
• Monosomic (2n − 1)
• Monosomy involving an autosome is incompatible with life, whereas
trisomies of certain autosomes and monosomy involving sex
chromosomes are compatible with life with variable degrees of
phenotypic abnormality
Mosaicism
• It is the presence of two or more populations of cells with different
complements of chromosomes in the same individual
• In the context of chromosome numbers, postzygotic mitotic
nondisjunction would result in the production of a trisomic and a
monosomic daughter cell
• The descendants of these cells would then produce a mosaic
• Mosaicism affecting sex chromosomes is common, whereas
autosomal mosaicism is not
Structural Abnormalities
• Usually result from chromosomal breakage followed by loss or
rearrangement of material
• Designated using a cytogenetic shorthand in which p (French, petit)
denotes the short arm of a chromosome, and q, the long arm
• Each arm is then divided into numbered regions (1, 2, 3, and so on)
from centromere outward, and within each region the bands are
numerically ordered
• Thus, 2q34 indicates chromosome 2, long arm, region 3, band 4
• The patterns of chromosomal rearrangement after breakage are as
follows:
• Translocations
• Balanced reciprocal
• Robertsonian (centric) fusion
• Isochromosomes
• Deletions
• Inversions
• Ring chromosomes
Translocation
• Implies transfer of a part of one chromosome to another chromosome
• The process is usually reciprocal (i.e., fragments are exchanged between
two chromosomes)
• In genetic shorthand, translocations are indicated by t followed by the
involved chromosomes in numeric order—for example,
46,XX,t(2;5)(q31;p14)
• This notation would indicate a reciprocal translocation involving the long arm (q) of
chromosome 2 at region 3, band 1, and the short arm of chromosome 5, region 1,
band 4

• Balanced reciprocal
• Robertsonian (centric) fusion
• Balanced Reciprocal
• Involves exchange of chromosomal material between two chromosomes with
no net gain or loss of genetic material
• Robertsonian (centric) fusion
• Reciprocal translocation between two acrocentric chromosomes
• Involves the short arm of one and long arm of the other
• The transfer of segments leads to formation of one abnormally large
chromosome and one extremely small one (latter is usually lost)
• Predisposes to the formation of abnormal (unbalanced) gametes
Isochromosomes
• Result when the centromere divides horizontally rather than vertically
• One of the two arms of the chromosome is then lost, and the remaining arm
is duplicated, resulting in a chromosome with two short arms only or two long
arms only
• The most common isochromosome present in live births involves the
long arm of the X chromosome and is designated i(Xq)
• When fertilization occurs by a gamete that contains a normal X chromosome,
the result is monosomy for genes on Xp and trisomy for genes on Xq
Deletion
• Involves loss of a portion of a chromosome
• A single break may delete a terminal segment
• Two interstitial breaks, with reunion of the proximal and distal
segments, may result in loss of an intermediate segment
• The isolated fragment, which lacks a centromere, almost never survives, and
thus many genes are lost
Inversions
• Occur when there are two interstitial breaks in a chromosome, and
the segment reunites after a complete turnaround
Ring Chromosome
• A variant of a deletion
• After loss of segments from each end of the chromosome, the arms
unite to form a ring
General Features of
Chromosomal Disorders
• Chromosomal disorders may be associated with absence (deletion,
monosomy), excess (trisomy), or abnormal rearrangements
(translocations) of chromosomes
• In general, loss of chromosomal material produces more severe
defects than does gain of chromosomal material
• Excess chromosomal material may result from a complete
chromosome (as in trisomy) or from part of a chromosome (as in
robertsonian translocation)
• Imbalances of sex chromosomes (excess or loss) are tolerated much
better than are similar imbalances of autosomes
• Sex chromosomal disorders often produce subtle abnormalities, sometimes
not detected at birth
• Infertility, a common manifestation, cannot be diagnosed until adolescence
• In most cases, chromosomal disorders result from de novo changes
(i.e., parents are normal, and risk of recurrence in siblings is low)
• An uncommon but important exception to this principle is exhibited
by the translocation form of Down syndrome
Thank you
“The work they do holds this world together. When they do their work,
it is the same as offering prayer”
Sirach
Cytogenetic Disorders
Involving Autosomes
Scope
• General Features of Chromosomal Disorders
• Cytogenetic Disorders involving Autosomes
General Features of Chromosomal Disorders
• Chromosomal disorders may be associated with absence (deletion, monosomy), excess
(trisomy), or abnormal rearrangements (translocations) of chromosomes.
• In general, loss of chromosomal material produces more severe defects than does gain of
chromosomal material.
• Excess chromosomal material may result from a complete chromosome (as in trisomy) or
from part of a chromosome (as in robertsonian translocation).
• Imbalances of sex chromosomes (excess or loss) are tolerated much better than are
similar imbalances of autosomes.
• Sex chromosomal disorders often produce subtle abnormalities, sometimes not detected
at birth. Infertility, a common manifestation, cannot be diagnosed until adolescence.
• In most cases, chromosomal disorders result from de novo changes (i.e., parents are
normal, and risk of recurrence in siblings is low). An uncommon but important exception
to this principle is exhibited by the translocation form of Down syndrome
• Three autosomal trisomies (21, 18, and 13) and one deletion
syndrome (cri du chat syndrome), which results from partial deletion
of the short arm of chromosome 5, were the first chromosomal
abnormalities identified.
• Trisomy 21 and 22q11.2 deletion occur with notable frequency
Trisomy 21 (Down Syndrome)
• Most common of the chromosomal disorders
• About 95% of affected persons have trisomy 21, so their chromosome count is 47
• most common cause of trisomy is meiotic nondisjunction
• The parents have a normal karyotype and are normal in all respects
• Maternal age has a strong influence on the incidence of Down syndrome
• It occurs in 1 in 1550 live births in women younger than 20 years and 1 in 25 live births in
women older than 45 years
• The correlation with maternal age suggests that in most cases the meiotic
nondisjunction of chromosome 21 occurs in the ovum
• In 95% of cases the extra chromosome is of maternal origin
• The reason for the increased susceptibility of the ovum to nondisjunction is not fully
understood.
• No effect of paternal age has been found in those cases in which the extra chromosome is
derived from the father.
• In about 4% of all patients with trisomy 21, the extra chromosomal
material is present not as an extra chromosome but as a translocation of
the long arm of chromosome 21 to chromosome 22 or 14
• Such cases frequently (but not always) are familial, and the translocated
chromosome is inherited from one of the parents, who typically is a carrier
of a robertsonian translocation.
• Approximately 1% of patients with trisomy 21 are mosaics, usually having a
mixture of 46- and 47-chromosome cells. These cases result from mitotic
nondisjunction of chromosome 21 during an early stage of embryogenesis.
• Clinical manifestations in such cases are variable and milder, depending on the
proportion of abnormal cells
• The diagnostic clinical features of this condition are usually readily
evident, even at birth
• Flat facial profile
• Oblique palpebral fissures and epicanthic folds etc
• Some mosaics with Down syndrome have mild phenotypic changes
and often even have normal or near normal intelligence
• Down syndrome is a leading cause of severe mental retardation;
approximately 80% of those afflicted have an IQ of 25 to 50.
• Ironically, these severely disadvantaged children may have a gentle, shy
manner
• Approximately 40% of the patients have congenital heart disease, most
commonly defects of the endocardial cushion, including atrial septal
defects, atrioventricular valve malformations, and ventricular septal
defects
• Cardiac problems are responsible for a majority of the deaths in infancy and early
childhood.
• Several other congenital malformations, including atresias of the
esophagus and small bowel, also are common.
• Children with trisomy 21 have a 10- to 20-fold increased risk of developing
acute leukemia
• Both acute lymphoblastic leukemias and acute myeloid leukemias
• Virtually all patients with trisomy 21 older than age 40 develop
neuropathologic changes characteristic of Alzheimer disease
• Patients with Down syndrome demonstrate abnormal immune
responses that predispose them to serious infections, particularly of
the lungs, and to thyroid autoimmunity
• Abnormalities affecting mainly T cell functions, have been reported but the
basis for the immunologic disturbances is not clear
• Improved medical care has increased the longevity of persons with
trisomy 21
• Currently the median age at death is 47 years (up from 25 years in 1983)
Other Trisomies
• Trisomy 13 (Patau syndrome)
• Trisomy 18 (Edwards syndrome)
• Occur much less commonly than trisomy 21
• Are both associated with increased maternal age
• Affected infants have severe malformations and usually die within the
first year of life
22q11.2 Deletion Syndrome
• The 22q11.2 deletion syndrome encompasses a spectrum of disorders
that result from a small interstitial deletion of band 11 on the long
arm of chromosome 22.
• The clinical features of this deletion syndrome include congenital
heart disease affecting the outflow tracts, abnormalities of the palate,
facial dysmorphism, developmental delay, thymic hypoplasia with
impaired T cell immunity, and parathyroid hypoplasia resulting in
hypocalcemia
• Previously, these clinical features were believed to represent two
different disorders: DiGeorge syndrome and velocardiofacial
syndrome. However, it is now known that both are caused by 22q11.2
deletion.
• Variations in the size and position of the deletion are thought to be
responsible for the differing clinical manifestations.
• When T cell immunodeficiency and hypocalcemia are the dominant
features, the patients are said to have DiGeorge syndrome, whereas
patients with the so-called velocardiofacial syndrome have mild
immunodeficiency but pronounced dysmorphology and cardiac
defects.
• In addition to malformations, patients with 22q11.2 deletion are at
particularly high risk for psychoses such as schizophrenia and bipolar
disorder.
• The molecular basis for this syndrome is not fully understood.
• The affected region of chromosome 11 encodes many genes including
a transcription factor gene called TBX1 suspected to be responsible,
since its loss seems to correlate with the occurrence of DiGeorge
syndrome
• The diagnosis of this condition may be suspected on clinical grounds
but can be established only by detection of the deletion by
fluorescence in situ hybridization (FISH)
Thank you
MOLECULAR DIAGNOSIS OF
MENDELIAN AND COMPLEX
DISORDERS
• The field of molecular diagnostics emerged in the latter half of the 20th
century, with the application of low throughput approaches such as
conventional karyotyping for recognition of cytogenetic disorders (e.g.,
Down syndrome) and DNA-based assays such as Southern blotting for the
diagnosis of Huntington disease
• In the era predating the ready availability of molecular diagnostic assays,
rendering the diagnosis of a genetic disorder depended on the
identification of abnormal gene products (e.g., mutant hemoglobin or
abnormal metabolites) or their clinical effects, such as mental retardation
(e.g., in PKU)
• The field of molecular diagnostics has grown tremendously in the recent
past
Several factors have enabled the rapid expansion of molecular
diagnostics
1. The sequencing of the human genome and the deposition of these data in
publicly available databases
2. Thee availability of numerous “off the shelf” polymerase chain reaction
(PCR) kits tailor-made for the identification of specific genetic disorders
3. The availability of high-resolution microarrays (“gene chips”) that can
interrogate both DNA and RNA on a genome-wide scale using a single
platform
4. The emergence of automated and extremely high-throughput, next-
generation (“NextGen”) sequencing technologies
• The last two advances have been especially useful in the context of
new research to elucidate the genetic basis for both mendelian and
complex disorders
• Current estimates for the “worldwide market” runs into tens of
billions of dollars
• Irrespective of the technique used, the genetic aberration being
queried can be either in the germ line (i.e., present in each and every
cell of the affected person, as with the CFTR mutation in a patient
with CF) or somatic (i.e., restricted to specific tissue types or lesions,
as with the NMYC amplification in neuroblastoma cells)
• This consideration will determine the nature of the sample (e.g.,
peripheral blood lymphocytes [PBLs], saliva, tumor tissue) used for
the assay
Molecular Diagnosis of Copy Number
Abnormalities
• Various diseases may occur as a result of copy number abnormalities,
at the level of
• The entire chromosome (trisomy 21)
• Chromosomal segments (22q11 deletion syndrome) or,
• Submicroscopic intragenic deletions (WAGR syndrome)
• Karyotype analysis of chromosomes by G banding remains the classic
approach for identifying changes at the chromosomal level; however,
as might be expected, the resolution with this technique is fairly low
• In order to identify subchromosomal alterations, both focused
analysis of chromosomal regions by FISH and global genomic
approaches such as comparative genomic hybridization (CGH) have
become popular
Fluorescence in Situ Hybridization (FISH)
• FISH utilizes DNA probes that recognize sequences specific to
chromosomal regions of greater than 100 kilobases in size, which
defines the limit of resolution with this technique for identifying
chromosomal changes
• Probes are labeled with fluorescent dyes and applied to metaphase
spreads or interphase nuclei
• The probe hybridizes to its complementary sequence on the chromosome and
thus labels the specific chromosomal region that can then be visualized under
a fluorescence microscope
• The ability of FISH to circumvent the need for dividing cells is invaluable in
its utilization eg
• When a rapid diagnosis is warranted (e.g., in a critically ill infant suspected of having
an underlying genetic disorder)
• Situations where prenatal sampling is required (e.g., cells obtained by amniocentesis,
chorionic villus biopsy, or umbilical cord blood), peripheral blood lymphocytes, and
even archival tissue sections
• The detection of numeric abnormalities of chromosomes (aneuploidy)
• For the demonstration of subtle microdeletions or complex translocations not
detectable by routine karyotyping
• For analysis of gene amplification (e.g., NMYC amplification in neuroblastomas)
• For mapping newly isolated genes of interest to their chromosomal loci
Array-Based Genomic Hybridization
• FISH requires previous knowledge of the one or few specific
chromosomal regions suspected of being altered in the test sample
• However, chromosomal abnormalities may also be detected without
previous knowledge of what these aberrations may be, using a global
strategy known as array based CGH
• Here the test DNA and a reference (normal) DNA are labeled with two
different fluorescent dyes (most commonly, Cy5 and Cy3, which
fluoresce red and green, respectively)
• The differentially labeled samples are then hybridized to an array of
segments of genomic DNA “spotted” on a solid matrix, usually a glass
slide
• These segments of DNA are representations of the human genome at
regularly spaced intervals, and cover all 22 autosomes and the sex
chromosome
• Amplifications and deletions in the test sample produce an increase
or decrease in signal relative to the normal DNA that can be detected
down to a 10-kilobase (kb) resolution
• Newer generations of microarrays using single-nucleotide
polymorphisms (SNPs) provide even higher resolution (with more
than 1 million SNPs from the human genome on a single microarray)
and are currently being used to uncover copy number abnormalities
in a variety of diseases, from cancer to autism
Direct Detection of DNA Mutations by
Polymerase Chain Reaction (PCR) Analysis
• PCR analysis, which involves exponential amplification of DNA, is now
widely used in molecular diagnosis
• If RNA is used as the substrate, it is first reverse-transcribed to obtain cDNA
and then amplified by PCR
• This method involving reverse transcription (RT) often is abbreviated as RT-
PCR
• One prerequisite for direct detection is that the sequence of the
normal gene must be known
• To detect the mutant gene, two primers that bind to the 3′ and 5′
ends of the normal sequence are designed
• By utilizing appropriate DNA polymerases and thermal cycling, the
target DNA is greatly amplified, producing millions of copies of the
DNA sequence between the two primer sites
• The subsequent identification of an abnormal sequence can then be
performed in several ways:
Sanger Sequencing
• The DNA can be sequenced to obtain a readout of the order of nucleotides, and by comparison with a
normal (wild-type) sequence, mutations can be identified
• Most DNA sequencing machines are automated and use a fluorescent dye–based version of a sequencing
technology originally named after its inventor, Frederick Sanger.
• More recently, gene chips (microarrays) also have become available that can be used for sequencing genes
or portions of genes
• Short sequences of DNA (oligonucleotides) that are complementary to the wild-type sequence and to known
mutations are “tiled” adjacent to each other on the gene chip, and the DNA sample to be tested is
hybridized to the array
• Before hybridization, the sample is labeled with fluorescent dyes
• The hybridization (and, consequently, the fluorescent signal emitted) will be strongest at the oligonucleotide
that is complementary to the wild-type sequence if no mutations are present, while the presence of a
mutation will cause hybridization to occur at the complementary mutant oligonucleotide
• Computerized algorithms can then rapidly “decode” the DNA sequence for hundreds of thousands of base
pairs of sequence from the fluorescent hybridization pattern on the chip, to identify potential mutations
• Another approach for identifying mutations at a specific nucleotide position (say,
a codon 12 mutation in the KRAS oncogene that converts glycine [GGT] to
aspartic acid [GAT]) would be to add fluorescently labeled nucleotides C and T to
the PCR mixture, which are complementary to either the wild-type (G) or mutant
(A) sequence, respectively
• Since these two nucleotides are labelled with different fluorophores, the
fluorescence emitted by the resulting PCR product can be of one or another
colour, depending on whether a C or a T becomes incorporated in the process of
primer extension
• The advantage of this allele-specific extension strategy is that it can detect the
presence of mutant DNA even in heterogeneous mixtures of normal and
abnormal cells
• Many variations on this theme have been developed and are being currently used
for mutation detection in the laboratory and clinical settings
NextGen Sequencing
• Next generation (“NextGen”) sequencing technologies are so named because the Sanger sequencing
mentioned earlier is now considered “first generation”
• The availability of NextGen sequencing technology has the potential to alter molecular diagnostics radically,
by the sheer volume of sequencing data (more than 1 giga–base pairs or 1,000,000,000 base pairs of DNA
per day) at relatively cheap costs
• The entire human genome has a little over 3 gigabases, so true “whole genome sequencing” can be performed several times
over in a matter of days
• In contrast with Sanger sequencing, NextGen sequencing technologies utilize platforms where sequencing of
multiple fragments of the human genome (DNA or cDNA) can occur in parallel (“massively parallel
sequencing”), significantly enhancing its speed
• Fluorescently labelled nucleotides are incorporated complementary to the template DNA strands, which are
immobilized on a solid phase, with one nucleotide added per template per cycle
• The cycles are repeated until a sufficient length of “read” is generated that can then be mapped back to the
human genome using sophisticated bioinformatics
• Deep sequencing is now being utilized to sequence somatic mutations in some of the most common tumour
types, while germline sequencing has started identifying the hitherto unknown genetic basis of rare
mendelian disorders
Linkage Analysis and Genome-Wide
Association Studies
• Direct diagnosis of mutations is possible only if the gene responsible
for a genetic disorder is known and its sequence has been identified
• In several diseases that have a genetic basis, including some common
disorders, direct genetic diagnosis is not possible, either because the
causal gene has not been identified or because the disease is
multifactorial (polygenic) and no single gene is involved
• In such cases, two types of analyses can be performed for unbiased
identification of disease-associated gene(s): linkage analysis and
genome-wide association studies (GWASs)
• In both instances, surrogate markers in the genome, also known as marker
loci, must be used to localize the chromosomal regions of interest, based
on their linkage to one or more putative disease-causing genes
• The marker loci utilized are naturally occurring variations in DNA sequences
known as polymorphisms
• The most common DNA polymorphisms—SNPs—occur at a frequency of
approximately one nucleotide in every 1000–base pair stretch and are
found throughout the genome (e.g., in exons and introns and in regulatory
sequences)
• SNPs serve both as a physical landmark within the genome and as a genetic
marker whose transmission can be followed from parent to child
• Two technologic breakthroughs have enabled the application of SNPs to high-
throughput “gene hunting”
1. The completion of the so-called HapMap project, which has provided linkage
disequilibrium patterns in three major ethnoracial groups, based on genome-
wide SNP mapping
• The entire human genome can now be divided into blocks known as “haplotypes,” which
contain varying numbers of contiguous SNPs on the same chromosome that are in linkage
disequilibrium and hence inherited together as a cluster
• As a result, rather than querying every single SNP in the human genome, comparable
information about shared DNA can be obtained simply by looking for shared haplotypes,
using single or a small number of SNPs that “tag” or identify a specific haplotype
2. Second, it is now possible to simultaneously genotype hundreds of thousands
to a million SNPs at one time, in a cost-effective way, using high-density SNP
chip technology
Indications for Genetic Analysis
• In general, genetic testing can be divided into prenatal and postnatal
analysis. It may involve conventional cytogenetics, FISH, molecular
diagnostics, or a combination of these techniques
• For judicious application of genetic analysis it is important to
recognize which persons require genetic testing
• Prenatal genetic analysis can be performed on cells obtained by
amniocentesis, on chorionic villus biopsy material, or on umbilical
cord blood
• Some important indications are the following:
• Advanced maternal age (beyond 34 years), which is associated with greater
risk of trisomies
• Confirmed carrier status for a balanced reciprocal translocation, Robertsonian
translocation, or inversion (in such cases, the gametes may be unbalanced, so
the progeny would be at risk for chromosomal disorders)
• A chromosomal abnormality affecting a previous child
• Determination of foetal sex when the patient or partner is a confirmed carrier
of an X-linked genetic disorder
Postnatal genetic analysis usually is performed on peripheral blood
lymphocytes. Indications are as follows:
• Multiple congenital anomalies
• Unexplained mental retardation and/or developmental delay
• Suspected aneuploidy (e.g., features of Down syndrome)
• Suspected unbalanced autosome (e.g., Prader-Willi syndrome)
• Suspected sex chromosome abnormality (e.g., Turner syndrome)
• Suspected fragile X syndrome
• Infertility (to rule out sex chromosome abnormality)
• Multiple spontaneous abortions (to rule out the parents as carriers of
balanced translocation; both partners should be evaluated)
Thank you