Hardy-Weinberg

UNIT 3 GENETIC POLYMORPHISM Equilibrium

Contents
3.1 Introduction
3.2 Balanced Polymorphism
3.3 Transient and Balanced Polymorphisms
3.4 Serological Markers
3.5 Biochemical Polymorphisms
3.6 Molecular Markers
3.6.1 Repetitive DNA Sequence Variants
3.6.2 Non- Repetitive DNA Sequence Variants
3.6.3 Lineage Markers
3.7 Tools for Studying Polymorphisms
3.8 Genetic Markers and Disease
3.9 Genetic Mapping of Disease Gene on Human Chromosome Using
Polymorphic Markers
3.10 Use of Polymorphic Markers in Forensic Testing
3.11 Use of Polymorphic Markers in Population Studies
3.12 Summary
Suggested Reading
Sample Questions

Learning Objectives
&
After reading this unit, you will be able to:
Ø define the concept of genetic polymorphism;
Ø explain genetic polymorphism with respect to serological, biochemical and
molecular markers;
Ø explain the genetic markers in disease association; and
Ø discuss the use of polymorphic markers in population and forensic studies.

3.1 INTRODUCTION
Genetic polymorphism can be defined as the occurrence together in the same
population two or more than two alleles such that the frequency of rare allele is
always >1%, and is maintained in the population not merely by the recurrent
mutation. Polymorphism can be in a coding region (coding region means the
portion of DNA which code for a gene, it may be synonymous or non-
synonymous) or more commonly, in the noncoding regions (which does not code
for functional region), often vary by ethnicity. Basic information about the types,
frequencies and distribution of common polymorphisms are essential not only
for the understanding of pathological entities, but also to know our evolutionary
past and provide guidance about our biological future. The most common
polymorphism in our genome are single base pair sequence variation i.e. SNP
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Human Population Genetics but other types like copy number changes, insertions, deletions, duplications
and rearrangements also occur. The methods to asses this diversity is variable.
Few examples of polymorphic markers are listed in table 3.1.

Table 3.1: Example of Genetic polymorphisms

Type of marker Year No. of loci Properties

Blood groups 1910-1960 ~20 May need fresh blood, rare antisera.
Genotype cannot always be inferred
from phenotype because of dominance.
No easy physical localization.

E l e c t r o p h o r e t i c 1960-1975 ~30 May need fresh serum, specialized

mobility variants of assays, no easy physical localization
serum proteins often limited polymorphisms

Human Leucocyte 1970 1 (multi locus One linked set highly informative. Can
Antigens (HLA) haplotype) only test for linkage to 6p21.3

DNA RFLPs 1975 >105 Two allele markers, maximum

(potentially) heterozygosity 0.5,initially required
Southern blotting, now PCR. Easy
physical localization

DNA VNTRs 1985- >104 Many alleles, highly informative can be

(minisatellites) (potentially) typed by southern blotting easy physical
localization. Tend to cluster near ends
of chromosomes.

DNA VNTRs 1989- 105 (potentially) Many alleles, highly informative

(microsatellites) Can be typed by automated multiplex
(di-,tri-, and PCR, easy physical localization.
tetranucleotide Distributed throughout genome
repeats)

DNA SNPs 1998- 106 (potentially) Less informative than microsatellites.

Can be typed on a very large scale by
automated equipment, without gel
electrophoresis, etc.

3.2 BALANCED POLYMORPHISM

When natural selection favors heterozygotes over both homozygotes, the result
is balanced polymorphism. It accounts for the persistence of an allele even though
it is deleterious when homozygous. Some of the examples are given below:

Sickle Cell Disease

It is an autosomal recessive disorder that causes anemia, joint pain, a swollen
spleen, and frequent, severe infections. It illustrates balanced polymorphism
because carriers are resistant to malaria, an infection by the parasite Plasmodium
falciparum that causes cycles of chills and fever. The parasite spends the first
stage of its life cycle in the salivary glands of the mosquito Anopheles gambiae.
When an infected mosquito bites a human, the malaria parasite enters the red
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blood cells, which transport it to the liver. The red blood cells burst, releasing Genetic Polymorphism
the parasite throughout the body.

It is known since long that malaria is a quite common in the tropical regions of
Africa. Sickle shape red blood cells provide selective advantage as malarial
parasite cannot grow in these cells. Therefore, along with malaria the sickle cell
anemia also increased in these parts of Africa. The sickle cell disease is less
common in Caucasians due to the less frequency of malaria. This shows the
heterozygous advantage of sickle cell as it provides protective effect.

The rise of sickle cell disease goes hand in hand with the cultural development
with the advent of cultivation of crops gave a breeding ground to Anopheles
mosquitoes as the malaria rose the selective pressures gave rise to the change in
the shape of the RBCs from elliptical to sickle shaped and when it occurred in
homozygous condition the disease was caused otherwise it had selective
advantage. The spread of sickle cell disease is associated to the migratory events.
Africa by people migrating from Southern Arabia and India, or it may have arisen
by mutation directly in East Africa.

Settlements with large numbers of sickle cell carriers escaped devastating malaria.
They were therefore strong enough to clear even more land to grow food-and
support the disease-bearing mosquitoes. Even today, sickle cell disease is more
prevalent in agricultural societies than among people who hunt and gather their
food.

G6PD Deficiency
It is a sex-linked enzyme deficiency. It affects 400 million people throughout the
world. It results into hemolytic anemia which is life-threatening. It is under the
influence of certain environmental conditions like eating fava beans, inhaling
certain types of pollen, taking certain drugs, or catching certain infections. It has
been seen in Africa that hemizygous males and heterozygous males for this
enzyme deficiency are at less risk for malaria again revealing a selective advantage
for heterozygotes. Therefore, natural selection acts in two directions hence it
could be one of the example of balanced polymorphism.

3.3 TRANSIENT AND BALANCED

POLYMORPHISMS
Polymorphism occurs when two or more clearly different phenotypes exist in
the same population of a species-in other words, the occurrence of more than
one form or morph.

A transient polymorphism is one that is changing in frequency over time. In

transient polymorphism, one form is gradually being replaced by another. As the
name implies, it represents a temporary situation as a by-product of directional
natural selection.

The phenomenon of industrial melanism occurs in a number of moth species in

Europe and the United States. The British ecological geneticist, E. B.Ford, first
called attention to this phenomenon as a way of demonstrating the effect of
natural selection in nature (as opposed to artificial selection experiments which
have long enjoyed success in the lab). Ford noted that a light colored moth species, 57
Human Population Genetics Biston betularia, occasionally undergoes mutation at a single locus to produce a
dark or melanic individual. Since the mutant allele is dominant, any gamete
containing this mutant will produce a melanic individual upon syngamy. The
first melanic specimen in this species was found in a collection from Manchester,
England dated 1848, but by 1895 about 95% of all collected specimens were
dark morphs, referred to as the form carbonaria. In a series of 12 observations
and mark-recapture experiments during the 1950s, H. B. D. Kettlewell
demonstrated that the two forms (light and dark) were differentially preyed upon
by birds. He found that the birds selectively caught and ate more individuals of
the form that did not match its background as compared to the one that was
masked. In industrialized areas of England where the substrate (walls and tree
trunks) upon which the moths rested were darkened by pollutants in the smoke
poured out by factories, the carbonaria form possessed a selective advantage.
Rural areas, unaffected by pollutants, afforded the light form an adaptive
advantage. The environmental change brought on by the industrial revolution
did not produce the carbonaria form (which presumably appeared from time to
time due to recurrent mutation); it only protected the dark moths from bird
predation (the agent of natural selection). The fact that the light form still exists
in rare numbers in industrialized areas testifies to the amount of time selection
requires to eliminate a recessive allele.

Mendilian Population
A population is a group of individuals who share a common gene pool where the
characters are transmitted in a Mendelian fashion from one generation to the
next generation. A group of individuals within which marriages are performed is
called a Mendelian population. In a given Mendelian population, which is under
Hardy-Weinberg equilibrium, the resultant genotype and phenotype frequencies
are more or less permanently established.

3.4 SEROLOGICAL MARKERS

Blood groups are the best cited examples of serological markers. Both ABO and
Rh are quite important serological markers as they can be used to study population
diversity. These blood groups cause newborn hemolytic diseases. They also have
a role in blood transfusion and also solid organ transplantation. They follow
mendalian inheritance. ABO blood groups were discovered by Landsteiner in
1900 and are cited as a best example of triallelic inheritance. Blood groups can
be tested by using antisera and red blood cells using simple agglutination
techniques. Presently more advanced molecular techniques are also used.

The ranges of phenotypes in humans are a direct result of genetic variations

which act together with environmental and behavioral factors to produce diversity.
The identification of gene polymorphisms, which control the blood group antigen
expression, contributes to the understanding of the biological significance of
blood group systems. In addition to assisting in the characterization of allelic
variations, the identification of gene polymorphisms allows us to estimate the
processes involved in the formation of different populations (the founder effect,
genetic drift, migration, etc.). Thus, blood group gene polymorphisms are valuable
predictors of genomic ethnic ancestry.

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 Genetic Polymorphism
3.5 BIOCHEMICAL POLYMORPHISMS
There is marked difference between individuals on the basis of biochemical
markers like G6PD, human enzymes and proteins etc. This has been explained
in the above section. However, here we would like to throw some light on the
molecular basis of G6PD variants.

Molecular Basis of G6PD Variants

The G6PD gene, located on chromosome Xq28 region, is 18 Kb long consisting
of 13 exons transcribed to a 2.269 Kb messenger RNA with 1.545 Kb of coding
regions. The commonest variant in South China, G6PD Canton, has been
sequenced and was found to be due to a mutation at nucleotide (nt) position
1376 of cDNA, G to T, resulting in a missense mutation in amino acid position
459, Arg to Leu. With improved DNA technology, the whole cDNA sequence
can be amplified and screened for mutation directly. PCR technique and restriction
analysis has been used.

World Incidence and Distribution of G6PD Deficiency

G6PD deficiency in male subjects can be detected easily by a number of screening
tests. The simplest one is the fluorescent spot test developed by Beutler and
Mitchell which relied on the fluorescence of NADPH, generated by an adequate
amount of G6PD enzyme. This test can also be done on blood sample dried on
filter paper similar to the Guthrie cards. In Hong Kong, the routine screening of
newborns have included test for G6PD deficiency.

3.6 MOLECULAR MARKERS

Although ∼99% DNA is known to be similar between individuals but still
sequence differences exist between individuals in non-coding regions of the
genome and such polymorphic regions are useful for various kinds of analyses
in population genetic studies. A genetic marker can be a nucleotide sequence of
variable length, varying from a single base pair to several hundred base pairs.
Selection of markers for any study is dictated by the nature and purpose of the
study. The more commonly used markers in population genetics studies can
broadly be grouped as follows:

3.6.1 Repetitive DNA Sequence Variants

Tandem Repeats
Besides the interspersed repeats (SINEs and LINES), Tandem repeats are the
other kind of repeated elements found in the genome. These are highly variable
tandemly repeated arrays of 2 or more base pair core units in the non-coding
regions of the genome and are located adjacent to each other. On the basis of size
of the core unit, they are categorised into minisatellites (10-60 bp1 ), Short Tandem
Repeats (STRs) or microsatellites (<10 bp). When the number of nucleotides in
the core unit is not known or is variable then it is called Variable Number Tandem
Repeats (VNTRs).

Insertion/ Deletion Polymorphisms

An InDel or Insertion-Deletion polymorphism refers to insertion or deletion of a
DNA sequence of variable length in the genome. The concerned DNA sequence 59
Human Population Genetics may vary in length from a single nucleotide to several hundred nucleotides. They
are widely spread across the genome and constitute around 1.5 million of more
than 10 million polymorphisms known in humans.
Alu InDels – Alu Insertion/ Deletion polymorphisms (Alu InDels) involve Alu
sequences that are characterized by the cleavage action of AluI restriction
endonuclease.
Properties of Alu sequences such as their known ancestral state, identity by
descent, wide occurrence and stability make them ideal markers for human
evolutionary and diversity studies.

3.6.2 Non- Repetitive DNA Sequence Variants

Single Nucleotide Polymorphisms (SNPs or Snips)
SNP or Single Nucleotide Polymorphism is a single nucleotide (base pair) change
in a DNA sequence. As with all polymorphisms, for an alteration to be considered
a snip it must be present in ≥ 1% of the population being considered. They make
up about 90% of all the human gene sequence variation. SNPs may be present in
coding regions (exons) or non-coding regions (introns) or intergenic regions.
Restriction Fragment Length Polymorphisms (RFLPs) are the characteristic
pattern of fragments of DNA produced when a DNA sequence is cleaved by
specific enzymes belonging to endonuclease class of enzymes. The property of
these enzymes that enables them to cleave DNA segment only at specific locations
known as restriction sites have led to their use in detecting genetic differences
on the basis of absence or presence of restriction sites.

3.6.3 Lineage Markers

Mitochondrial Markers
Maternally inherited mitochondrial genome consists of multiple copies of circular
mitochondrial DNA or mtDNA. Markers present on this haploid genome are
primarily used for tracing maternal ancestral lineage(s) in populations because
of their uniparental inheritance.

Y-chromosomal Markers
Like mtDNA, Y- chromosome has a uniparental inheritance but in the male line
and can thus be used for tracing paternal ancestral lineages. In absence of
recombination, Y-chromosome is more or less transmitted unchanged from one
generation to next and the few changes that may occur usually do not have any
effect as around 98% of the DNA is in non-coding region.

Hardy-Weinberg equilibrium
The Hardy-Weinberg equilibrium predicts that in the absence of evolutionary
forces, both allelic and genotypic frequencies remain constant in a population
and that if the equilibrium is disturbed a new equilibrium will be reached within
one generation based on the allelic frequencies of the remaining population. The
conditions that must be met for the predictions of the Hardy-Weinberg equilibrium
to be valid are described below:
1) Random mating: Mating patterns must randomly reflect the entire breeding
population, with no dependence on genotype or closeness of relationship
60 (either positive or negative).
2) No sex bias in allelic frequencies: The distribution of alleles must be the Genetic Polymorphism
same in both sexes.

3) All genotypes equally viable and fertile: There must not be any selective
advantages or disadvantages. This is seldom true in a real population, and
often must be taken into account in terms of evolutionary pressures.

4) Mutation rate too low to alter ratios: The basic assumption is that alleles are
stable through many generations and are not altered or degraded significantly
by mutation. In practice this is generally not a serious problem.

5) Closed population (no in or out migration): The “population” that is being

considered must be a constant one. Introduction of new genes into the
breeding pool or loss of genes from the breeding pool by migration between
“populations” can distort trends.

6) Population must be large: The population must be large enough so that there
are no confounding effects due to genetic drift (random events altering allelic
frequencies by pure chance) or due to “founder” effects, where a recessive
gene becomes fixed in a population because too many of its members are
descendants of a single individual.

The Hardy-Weinberg law can also be applied to multiple alleles and X-linked
alleles. The genotypic frequencies expected under Hardy-Weinberg equilibrium
will differ according to the situation.

3.7 TOOLS FOR STUDYING POLYMORPHISMS

Both conventional and advanced techniques are used to study polymorphisms.
Conventional techniques are blood groups by carrying out simple agglutination
techniques or protein electrophoresis for studying the protein polymorphisms
etc. Under advanced techniques are the tools for studying molecular markers,
and the foremost requirement for carrying out molecular analysis of any kind is
the availability of the genetic material. As mentioned earlier, DNA is the focal
point of human diversity and disease-association studies by virtue of the fact
that it is the blueprint of our existence. There are several techniques for isolating
DNA such as manual methods (like Phenol Chloroform, Salting-out) and kits.
The technique of DNA isolation or extraction varies depending on the starting
material, but, it is the technique of PCR which is the most useful for DNA analysis.

Polymerase Chain Reaction (PCR): It involves cycling of DNA sample through

a series of heating and cooling cycles with the required raw materials and enzymes
to achieve its exponential amplification. The technique has come a long way
since its invention. Instead of having to manually maintain the heating and cooling
cycles, automated thermal cyclers are now available; and instead of having to
add fresh polymerase (earlier derived from E. coli) after every cycle because of
its denaturation due to heating, thermally stable DNA polymerases such as Taq
DNA Polymerase are now made use of.

Amplification of DNA by PCR has found applications in a variety of fields ranging

from forensics to archaeology; study of variation and evolution to mutation
detection; gene mapping and cloning and DNA sequencing to epidemiology
among several others. 61
Human Population Genetics Restriction Digestion: It is the method of cutting DNA sequences into fragments
using restriction endonucleases or enzymes that cut at specific recognition sites.
This generates DNA fragments of varying lengths producing a variation pattern
known as Restriction Fragment Length Polymorphisms (RFLPs). The variation
may be produced in response to absence or presence of particular SNP(s) or an
insertion or deletion event in that region and is recognised in the form of banding
pattern. Resulting fragments are separated according to molecular size using gel
electrophoresis. There are several classes of endonucleases- Type I, Type II, Type
III and Type IV but the most commonly used restriction enzymes are of type II
and they cleave DNA fragment at specific sites within or close to the recognition
sequence. Most of these enzymes cut palindromic sequences.

The technique is useful in detection of mutations/ SNPs. It is also used to detect

VNTRs. The technique has been widely used for constructing physical maps of
the genome, genetic linkage maps; in forensic testing; and in epidemiological
and evolutionary studies.

Electrophoresis: It is one of the few techniques that has been in use since the
beginning of study of classical genetic markers and is still in use for molecular
markers. It is the method of separating macromolecules (both proteins and nucleic
acids) on the basis of size, electric charge or other physical properties under the
influence of electric field.

Sequencing: DNA sequencing refers to establishing the exact sequential

arrangement of bases in a stretch of DNA. Knowledge of exact sequence of
bases in a gene is crucial especially in ascertaining the function of genes. This is
also important as the disease-causing alterations in the genes can then be identified.

The selection of technique and markers depends upon the purpose of study. In
the following section we have discussed the uses of polymorphic markers.

Uses of polymorphisms: All the markers listed in table 3.1 can be used for
population diversity studies. Now a days most extensively studied markers are
Single nucleotide polymorphisms. Genomics and specially SNP research can be
used to improve health care through gene therapy, to yield new targets for drug
discovery, to renew the process of drug development and to discover new
diagnostics.

3.8 GENETIC MARKERS AND DISEASE

Understanding the genetic basis of complex human diseases (like hypertension,
cardiovascular disease, diabetes etc.) has been increasingly emphasized as a means
of achieving insight into disease pathogenesis, with the ultimate goal of improving
preventive strategies, diagnostic tools, and therapies. Genetic approaches to
complex disorders thus offer great potential to improve our understanding of
their pathophysiology, but they also offer significant challenges. These can be
studied either using linkage analysis. In linkage analysis we use families and try
to find out which polymorphic marker is near to the disease gene and then try to
map the gene on the human genome. The other approach is where we study
populations of both types of individuals. One would be those suffering with a
disease and the other would be who are not suffering with the disease. We take
different polymorphic markers and study in these two sets of samples. Then we
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compare both the groups and if both the groups differ significantly at these markers Genetic Polymorphism
we propose that these markers may be associated with the disease.

Association studies can be a very powerful approach for finding genetic

determinants of a complex disorder. It has been suggested that if hundreds of
thousands of single nucleotide polymorphisms (SNPs) were identified across
the genome, then it would be possible to perform genome-wide association studies
to identify the regions of linkage disequilibrium around disease susceptibility
genes. In addition, they noted that much smaller sample sizes would be required
to detect association than to detect linkage. The SNP Consortium is rapidly
identifying single nucleotide polymorphisms, and within next several years,
genome-wide association studies may become a reality.

These association studies can result into positive association or negative

association. Some time they result into false positive or false negative results.
The following general guidelines, summarized in Table 3.2, may be useful for
genetic association studies. First, are the candidate gene(s) under study should
be biologically reasonable. Several factors can determine the appropriateness of
a candidate gene. If human genetic linkage studies have identified a chromosomal
region linked to a disease, or if an animal model for a disease is influenced by a
particular gene or syntenic chromosomal region, positional candidate genes in
such genomic regions warrant strong consideration. In addition, the biologic
plausibility of a candidate gene for involvement in disease pathogenesis is
important. However, obvious limitations of this candidate approach are the large
number of potential candidate genes for complex diseases and the reality that
only known genes can be investigated. Although candidate genes can be selected
for study on this basis, they should not be ruled out on the basis of our current
understanding of disease pathophysiology- important new insights may be missed
if potential candidate genes must fit into current pathophysiologic models.
Table 3.2: Evaluation of candidate gene case-control association studies
Issue Key Questions Possible Solutions

Selection Is candidate gene Demonstration of biologically

of candidate biologically reasonable? functional effect

Gene polymor- Is the candidate gene a Within linked region in man or

phism positional candidate? systemic from animal model

Population Are cases and controls Matching on ethnicity

stratification matched? Family-based association designs
Negative results with multiple
unlinked markers

Hardy-Weinberg Is control group in H-W Calculation of H-W equilibrium

(H-W) equilibrium equilibrium? with goodness-of-fit test (2 alleles)
or simulation (multiple alleles)

Multiple How many alleles were Bonferroni correction

comparisons tested?

How many genetic loci Estimation of empirical P values

were tested?
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Human Population Genetics A second criterion in evaluation of case-control association studies is the careful
selection of cases and control subjects. Do the case subjects meet appropriate
criteria for disease affection? Are control subjects free from symptoms of disease,
associated intermediate phenotypes, and potential confounders? Have control
subjects been exposed to relevant environmental influences involved in disease
pathogenesis while remaining clearly unaffected? Were the cases and controls
matched on demographic and environmental factors? Was consideration of
population stratification included, either by attempting to match ethnicity or by
typing unlinked markers.

A third criterion in the evaluation of case-control studies is assessment of Hardy-

Weinberg equilibrium in the markers studied within the control group. Hardy-
Weinberg equilibrium indicates that the genotype frequencies can be determined
directly from the allele frequencies; failure to demonstrate Hardy-Weinberg
equilibrium could result from genotyping errors, inbreeding, genetic drift,
mutation, or population substructure. Hardy-Weinberg equilibrium can be readily
assessed with a goodness-of-fit chi square test for biallelic markers; for markers
with multiple alleles (such as short-tandem repeat markers), more accurate
determination of Hardy-Weinberg equilibrium can be obtained with Markov
Chain Monte Carlo methods. Significant deviations from the expected proportions
of homozygote and heterozygote classes in a population of case subjects may be
caused by association with the disease allele. Lack of consistency with Hardy-
Weinberg equilibrium among control subjects should prompt investigation for
potential complications, including genotyping errors and population stratification.
A final criterion for evaluation of a case-control study is correction for multiple
comparisons. This remains a problematic topic requiring additional statistical
genetic research. However, an effort to correct for spurious associations, which
can result from testing a large number of alleles, is warranted. The multiple
comparison issue is especially problematic with markers that have multiple alleles
like short-tandem repeat polymorphisms; the conservative Bonferroni approach
to use a corrected significance value calculated by multiplication of the observed
P value by the number of alleles tested. Bonferroni corrections for the total number
of alleles at all loci are probably too conservative because the alleles at one locus
are not independent of each other and closely linked loci are probably not
independent either. A less conservative but more computationally intensive
approach is to estimate empirical significance values using simulation approaches.

Genome Wide Studies

Unlike the direct approach of case-control association with candidate genes,
genome scanning (screening) is an indirect strategy that does not rely on
conjecture. Basically, either affected individuals, usually siblings, from a number
of families or families with two or more affected individuals are genotyped with
polymorphic DNA markers that cover the entire chromosome complement. A
set of about 400 short tandem repeat polymorphic markers that are spaced at
about every 10cM is used for most genome scans. This level of resolution has
been enhanced with the assembly of about 3000 simple sequence repeat
polymorphic markers that are about 1.5cM apart. Single –nucleotide polymorphic
sites (SNPs) are preferred for genome scans because they are uniformly distributed
about every 300 bases throughout the genome and easily identified with automated
equipment. Eventually, sets of SNPs will supersedes short tandem repeat
polymorphic sequence marker systems.
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Furthermore, major landmark attempts that have also been made to study various Genetic Polymorphism
aspects of human genome, and few are listed below.

Human Genome Project (HGP): A National Institute of Health (NIH, US)

initiative started in 1990, HGP was a multinational collaborative project aimed
at identifying all the genes in the human DNA and determining the sequence of
about 3 billion nucleotide pairs that constitute the human DNA to understand
the species’ genetic makeup.

First draft was released in 2001 followed by the complete draft in 2003. Some of
the main findings from the draft sequence are as follows:
• Total number of genes was estimated at 30, 000.
• The average gene was found to consist of 3000 bp but sizes vary greatly.
• Repeated sequences that do not code for proteins (“junk DNA”) make up at
least 50% of the human genome.
• About 1.4 million locations with SNPs were identified.
Findings from HGP are already having profound impact on diverse areas of
research including molecular medicine (improved diagnosis of disease, earlier
detection of genetic predispositions to disease, rational drug design etc.),
bioarchaeology, anthropology, evolution and human migration, DNA forensics
(identification), agriculture, livestock breeding etc.

Human Genome Diversity Project (HGDP): HGDP was formally organised in

1993 under Stanford University’s Morrison Institute, and was aimed at
understanding the diversity patterns worldwide, the contributing factors and the
implications of the observed diversity patterns. Findings from the project could
also shed light on the origins and migration patterns of the entire human species.
HGDP could also aid in understanding the role played by environmental factors
in complex human diseases.

HapMap Project: The International HapMap Consortium is an international

collaborative venture between Japan, the United Kingdom, Canada, China,
Nigeria, and the United States aimed at developing haplotype map of the human
genome in a bid to identify genetic determinants of complex diseases. The
information made available through the HapMap project is helping researchers
find genes that affect health, disease, and individual responses to medications
and environmental factors.

Indian Genome Variation (IGV): IGV was the first large scale effort to document
and understand the genomic structure of enormously varied Indian populations.
The study found high degree of genetic differentiation among the different ethnic
groups.

Genetic Testing and Counseling

Frequently the question may arise as to whether the patient has a certain disease
for which there is a genetic basis. Often among the 10,000 conditions for which
a genetic basis has been identified, the diagnosis can be made from evaluation of
personal and family history, physical examination, and conventional laboratory
tests. A useful database for identifying these conditions is available on Online
Mendelian Inheritance in Man (OMIM) (www.ncbi.nlm.nih.gov/omim). This
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Human Population Genetics catalog is updated regularly and can be searched using multiple terms. The entries
provide information about the clinical signs as well as the genetic basis for the
condition, if known, including mutations that have been found to cause the
condition. To determine whether genetic testing is available for a given condition
and to find a laboratory, a useful link is GeneTests, a free online service
(www.genetests.org). The entries in this catalog indicate the test menus and contact
information for the laboratories, as well as whether the testing is provided on a
routine or research basis. A very useful adjunct in the GeneTests Website is
GeneReviews, which provides succinct summaries about many genetic conditions
and the ways the genetic testing can be used for diagnosing these conditions,
including prediction of natural history.

The clinician is likely to encounter many situations in which a genetic test may
be useful. Sometimes genetic testing is required from diagnosis when it cannot
be made by clinical criteria alone. The fragile X syndrome is the most common
genetic form of mental retardation. Although the diagnosis may be suggested by
the presence of the characteristic signs—large ears, protruding chin, and large
testes—the only way to diagnose fragile X is by genetic testing. For the various
forms of spinocerebellar ataxia, there is considerable overlap. Yet, these can be
readily distinguished by their specific mutations. Patients with atypical forms of
certain diseases may have a negative gold standard test, but positive genetic test.
For most patients with cystic fibrosis, the diagnosis can usually be made by a
sweat chloride test. However, a number of individuals have been described with
pulmonary disease suggestive of this condition for whom the sweat chloride test
is normal. For these patients, the diagnosis has been based on observation of
mutations in both copies of their CFTR genes.

For some conditions, the signs of disease may not yet have developed, yet on the
basis of one’s family history, one may want to know about the risk of developing
disease. This is true for the person whose parent(s) may have died from
Huntington’s disease, a progressive neurodegenerative disease or for the person
whose mother and sister may have died from breast or ovarian cancer, suggesting
a heritable risk. For these individuals, a positive genetic test result will indicate
an increased, although not necessarily absolute, risk for developing the disease.

Genetic testing is used for assessing reproductive risks—by testing the parents
for carrier status and by testing the fetus. Individuals with a positive family history
of genetic disease (usually autosomal recessive or X-linked) or who come from
ethnic groups with an increased prevalence of autosomal recessive or X-linked
diseases are candidates for carrier screening. Currently, carrier screening for cystic
fibrosis, fragile X syndrome, and spinal muscular atrophy is recommended in
the United States. For people of Mediterranean, African, or South Asian ancestry,
hemoglobinopathy screening is recommended. For individuals of Ashkenazi
Jewish ancestry, screening for Tay-Sachs disease, Canavan disease, cystic fibrosis,
Gaucher disease, Bloom syndrome, Fanconi anemia, Niemann-Pick disease,
familial dysau-tonomia, maple syrup urine disease, glycogen storage disease,
and familial hyperinsulinism is available. An individual who is a carrier for a
certain condition may choose not to marry another individual who is a carrier for
the same condition. Alternatively, if a carrier couple is identified, they may choose
to have prenatal diagnosis to determine whether their fetus is affected with this
condition. This can be performed either at 10-11 weeks using the procedure of
chorionic villus sampling where a bit of placenta is obtained under ultrasound
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guidance. As another option, an amniocentesis can be performed at 15-18 weeks Genetic Polymorphism
of pregnancy to obtain cells from the amniotic fluid. These couples might also
choose to have pre- implantation genetic diagnosis with selection implantation
of only those embryos that are deemed unaffected.

Not all genetic testing involves looking for heritable mutations. Sometimes it is
used to look for genetic alterations that are confined to a specific population of
cells. These alterations may cause certain cells to become cancerous, or if
cancerous, to progress to a more aggressive stage. Genetic testing can be used to
identify chromosomal translocations between two non-homologous chromosomal
segments and in the process diagnose a specific form of leukemia. For example,
the translocation between chromosomes 1 and 19 in leukemic cells is diagnostic
of the acute promyelocytic form of this disease and the translocation between
chromosomes 9 and 22 is diagnostic of the chronic myelogenous form. The
expression patterns of RNA transcribed from many genes can be assessed to
predict the natural history of the disease. This approach has been used to predict
breast cancer outcome and whether more or less aggressive therapies should be
used to treat patients.

Individuals might also have genetic tests of identity. These might be voluntary
and selected to test specific questions, such as whether they are members of a
known patrilineal lineage, such a people with a specific surname. These tests
analyze a series of polymorphic genetic markers on the Y chromosome. On the
basis of the general pattern of markers, or “haplogroup,” they may be told of the
geographic region where their Y chromosome originated. According to the number
of markers that match with people who are suspected to be of the same lineage,
individuals may be advised about the common ancestors or other people in that
lineage. Such testing is also possible for matrilineal lineages by testing
mitochondrial DNA markers.

3.9 GENETIC MAPPING OF DISEASE GENE ON

HUMAN CHROMOSOME USING
POLYMORPHIC MARKERS
In genetic mapping the diseased gene polymorphic markers play a very important
role. These markers could be short tandem repeats, variable number of tandem
repeats, blood groups, restriction fragment length polymorphism etc. Mapping
can be done in a step wise manner.

Collect all the pedigrees where the disease is found. Analyse all the members
against various polymorphisms and perform linkage analysis.

Linkage study entails collecting blood cells from members of several two – and
three – generation families or from individuals of a large multiple generation
family with a specific genetic disorder. The blood can be cultured and cell lines
can be maintained large number of polymorphic markers (probes), representing
sites from all parts of all autosomes, are used. A two –point (two – locus) LOD
score is calculated for each polymorphic locus and the site of the genetic disease
from all informative parent offspring combinations and finally the linkage is
established. However, genotyping errors can give –ve or +ve LOD score. Hence
perfect genotyping is must to get the correct results.
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Human Population Genetics
3.10 USE OF POLYMORPHIC MARKERS IN
FORENSIC TESTING
Polymorphic markers have great utility in personal identification. As mentioned
above no two individuals are alike. These differences are at both phenotypic and
genotypic levels. The genetic differences can be identified by testing these
markers. This testing is provided by commercial firms that market directly to
consumers. Identity genetic analysis may also be involuntary and used for paternity
testing of children or fetuses or for identification of forensic samples in murder,
assault or rape cases, in which the perpetrator of the crime left a tissue sample of
blood, semen, hair, or other tissue type from which DNA can be extracted and
the test can be performed. However, it must be kept in mind that there are ethnic
differences in the distribution of these markers. Hence every population should
have its own genetic profile.

3.11 USE OF POLYMORPHIC MARKERS IN

POPULATION STUDIES
Population Diversity Studies
Human genome varies from individual to individual and therefore no two
individuals look alike. This was noted long back. Historically, individual variation
was studied on the basis of conventional somatoscopic markers. However, with
the advancement of technology various genetic markers were discovered and
the gene frequency data for studying the evolution of human races was analyzed
using these markers. Initially, the classical serological and biochemical markers
have played important roles in various types of human population genetic studies.
One of the problems that limited their practical utility results from the limited
number of possible genotypes at each of such loci. The discovery of hyper variable
DNA loci offers the opportunity to ameliorate this problem. It was later realized
that comparison of gene frequencies for one or two loci are not reliable since
each locus has a different geographical distribution, hence the differences observed
may be because of chance factor. Only when a large number of loci are used, the
genetic relationship among populations could be drawn successfully. Recent
analysis based upon polymorphic markers reveal that inter and intragroup genetic
variation may be of a lesser magnitude and may not be of significance if proper
markers are not selected and more so if statistical tools used are not highly
powerful. However, it is important to record population variation because it is
helpful to know the various mechanisms involved in causing variation and it
further enhances our knowledge about the molecular basis of disease
susceptibility.

3.12 SUMMARY
It is difficult to attribute any functional significance to genetic polymorphisms.
However, the non-coding sequences of the genes which are located far away
from the functional region of the gene may affect the function of the gene.
However, these sequences are otherwise useful in studying population diversity,
disease gene mapping, forensic investigations etc. Recently after the advent of
microarray genes for many complex disorders have been found by using genome
68 wide association studies.
Suggested Reading Genetic Polymorphism

Gardner, E.D, Simmons, M.J and Snustad, D.P. 2003. Principles of Genetics, 8th
Edition, New York: John Wiley and Sons.

Simmons, S and Simmons, M.J. 2003. Principles of Genetics, 3rd Edition, New
York: John Wiley and Sons.

Sample Question
1) Define polymorphism with few examples.
2) What are the evolutionary forces that affect gene frequency of polymorphic
markers?
3) Give some uses of polymorphic markers.
4) What is law of Hardy Weinberg?
5) What is genetic testing?
6) Describe the utility of studying molecular markers in anthropological
genetics.

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