• SHREEVANI RAJ REDDY

INTRODUCTION

The aim of genetic research is to understand the role of genetic variation. In humans, the most common type of genetic variation involves single DNA bases, and is termed as single nucleotide polymorphism.

DNA polymorphism involves one of two or more variants in a particular DNA sequence and Variation at a single base pair of DNA sequence results in single nucleotide polymorphism (SNPs). SNPs are the most common type of genetic variation among people; each SNPs represents a difference in single nucleotide. On average they occur once in every 300 nucleotide which means there are roughly 10 million SNPs in the human genome. These variations are commonly found in the DNA between genes.

SNPs accounts for much of phenotypic diversity among individuals. In the human genome the half of the known coding region SNPs lead to change in the resulting amino acid sequences and other half do not, these are called synonymous SNPs. Synonymous SNPs encode change in the DNA sequence without altering the resultant protein sequence, these silent SNPs assumed as inconsequential, however these synonymous SNPs represent genetic marker for functional molecular alterations with which they are in linkage disequilibrium. These SNPs alters the function of gene and phenotype by various mechanisms such as altering protein folding, mRNA binding or by affecting splicing of mRNA; stability and expression of mRNA.

These SNPs can act as biological markers (also known as gene marker), therefore helps to locate gene that are associated with disease. Researchers found that SNPs may help to predict an individuals response to certain drugs and susceptibility to environmental factors such as toxins and risk of developing particular disease. SNPs with sufficient technological solutions can enable the mapping of disease genes involved in complex disorders. One of the examples of mapping disease associated with SNPs is Alzheimer’s disease.

GENETIC VARIATION

Genetic Variation is defined as, variation of genomes between groups of species as a result of genetic mutations or genetic drifts.

In all living organisms, the genetic material is made up of same basic components, called nucleotides. Each nucleotide contains one of four nitrogenous bases – A (Adenine), G (Guanine), T (Thymine), C (Cytosine). These 4 building blocks are linked together to form long chains, the sequence of which then codes for various proteins and gene products. The DNA sequence collection and organization is specific for each species, and is called a genome. On average, two humans share 99% genetic identity, although the majority of differences in DNA sequence (genotype) do not result in noticeable physical change (phenotype), the few that account for the diversity in human population are height, eye, skin, & hair color, etc.

Mutation is the process of creating a new genetic variation. Mutation in a gene can arise from natural internal processes such as gene conversions, cell replications, meiotic recombination and also from number of environmental factors such as free radical damage caused by ingestion of toxins and radiations etc.

FROM MUTATION TO POLYMORPHISM:

In humans, every individual has two copies of genome each one copy originating from each parent. So in the genome at a given position, each individual has two copies of a particular sequence. Mutation causes a change in one DNA sequence, resulting in the individual having one copy of original sequence and second new sequence at mutation locus. If mutation occurs in somatic cells, then remains only in the individual in which mutation occurred, and if mutation occurs in germ cells (egg or sperm cells), then these genetic changes passes to offspring and thus are inheritable. This phenomenon termed as genetic drift, can act to either increase or decrease allele frequencies in the population. If mutant allele reaches to a frequency of 1% or more in population, then locus is said to be “polymorphic”.

POLYMORPHISM TYPES

“Polymorphism” is a Greek word meaning “having many shapes”. Therefore genetic polymorphism is easy to imagine that two strands of DNA that differ in sequence rather than shape. In human genome the most common types of polymorphism organized into the 3 classes:-

Repetitive Elements:- In this type of polymorphism DNA sequences found in multiple copies throughout the genome. A classic example is ALU repeat (330 base pair in length), found in over 750,000 copies in genome. Another form of repetitive elements includes simple tandem repeat polymorphism (STRPs) or ‘microsatellites’. In this type of polymorphism, short di-, tri-, or tetra- nucleotide units are repeated consecutively at polymorphic position. Microsatellites are highly polymorphic, having up to 30 alleles and thus shows high allelic diversity and high heterozygosity

Insertion and Deletion:- This type of di-allelic polymorphism is also known as indels. Presence or absence of one or more DNA bases at polymorphic position shows the difference between the allele.

Substitutions:- This type of polymorphism are also most often di- allelic. Alleles of this type of polymorphism are distinguished by replacement of DNA bases, rather than presence or absence as in indels.

SINGLE NUCLEOTIDE POLYMORPHISM

This is a type of polymorphism in which alleles of these involve only single bases (OR type of polymorphism involving variation at a single base pair). SNP alleles can form by insertion or deletion of a single base or by substitution of one base for another. In case of substitution, the SNP alleles is limited to 4; Because DNA is made up of only 4 different nucleotide bases (A, T, G, C ), thus substitution of single nucleotide are at most tetra-allelic. However tetra-allelic or tri-allelic SNPs are very frequent with majority of true documented cases being in the mitochondrial genome, for this reason SNPs are thought of as di-allelic polymorphism.

SNP alleles are created by transition (purine to purine /pyrimidine to pyrimidine) or transversion (purine to pyrimidine / pyrimidine to purine) substitutions. In the human genome 70% of all SNPs involve a Cytosine (C) to Thymine (T) transition. This is due to conversion of 5- methyl cytosine to Thymidine by deamination mechanism.

SNPs are copying Errors:

An existing cell divides in two to make a new cells, first it copies its DNA so the new cell each will have a complete set of genetic instructions. Sometimes cells make mistakes during the coping process, this leads to changes in the DNA sequence at a particular location, called SNPs.

Chromosomal distribution of SNPs:

Although some 3 million SNPs already in databases, this is only a fraction of 11 million SNPs thought to be present in the human genome. By comparing any two randomly chosen chromosomes, number of studies pointed that one SNP occurs in 1-2 kb of sequence in a genome.

However SNPs are not distributed evenly down the length of any chromosome. The human chromosome contains large stretches of non-coding sequences with patches of coding sequences. Roughly the genetic variation is 4 times less in coding sequence than in non-coding sequences. Alteration in the certain sequences such as Exons, promoters, and enhancer sequence could adversely affect biological normal functions; therefore natural selection pressure would act to preserve certain sequences. However, there are few exceptions where coding sequence shows a high degree of polymorphism. For example, there is high sequence variability in and around the HLA genes, which encode for the important components of immune system.

Many SNPs that occur in the exons, are in the wobble position of the reading frame, and thus not alters the protein sequence. This type of changes are thought to have no effect or little effect on the gene product and called as synonymous or silent substitutions. On the other hand non- synonymous variants, cause substitution of one amino acid for another at protein level. The consequence of this type substitution on protein function varies from no effect to total disruption of protein. The changes in most severe single base in the exon regions can produce shifts in open reading frame, or creation of stop codon, either which can cause copy of nonfunctional gene product. These types of non-synonymous variations if reach to a high frequency, then considered as polymorphism. Two additional regions of chromosome are Telomere (end region of chromosome, plays important role in aging) and Centromere (central region of chromosome, which plays a key role in cell division) known to be highly polymorphic.

DETECTION TECHNIQUE FOR SINGLE NUCLEOTIDE POLYMORPHISM

SNPs are the positions in the genome where some individuals have one nucleotide and others have a different nucleotide. There are vast numbers of SNPs in every genome, some of which also gives rise to RFLPs, but many of which do not because the sequence in which they lie is not recognized by any restriction enzyme. In human genome there are at least 1.42 million SNPs, only 100000 of which results in an RFLP.

Although each SNP could, potentially, have four alleles (because there are 4 nucleotides), most exist in just two forms, so these markers suffer from the same drawback as RFLPs with regard to human genetic mapping: there is a high possibility that a SNP does not display any variability in the family that is being studied. The advantages of SNPs are their abundant numbers and the fact that they can be typed by methods that do not involve gel electrophoresis. This is important because gel electrophoresis has proved difficult to automate, so any detection method that uses it will be relatively slow and labor-intensive.

SNP detection is more rapid because it is based on oligonucleotide hybridization analysis. An oligonucleotide is a short single stranded DNA molecule, usually less than 50 nucleotides in length, that is synthesized in the test tube. These synthetic probes are also known as allele specific oligonucleotide (ASO). ASO can identify alleles that differ by single nucleotide. ASOs detect changes of all types of single nucleotide, including those that do not affect the restriction enzyme cutting sites. If the conditions are just right, then an oligonucleotide (ASO) will hybridize with another DNA molecule (with its complementary sequence & not with other sequences) only if the oligonucleotide forms a completely base-paired structure with the second molecule. If there is a single mismatch – a single position within the oligonucleotide that dose not form a base pair, then hybridization does not occur. Oligonucleotide hybridization can therefore discriminate between the two alleles of an SNP. Various screening have strategies have been devised including DNA chip technology and solution hybridization techniques.

SNPs as a genetic marker

A SNP is a type of gene marker (DNA marker) with a single base pair alteration at a particular site in some individuals, that site is SNP locus. These DNA markers are detected by molecular analysis of DNA and can be used in genetic analysis. SNP loci found abundant in the human genome, on average about once in 1000 bp. The presence of abundance of SNP loci allowed researchers to develop detailed maps of location of SNPs on chromosome.

ALZHEIMER’S DISEASE

This is a complex degenerative brain disorder characterized by progressive cognitive decline and memory impairment. This disorder annually, afflicting about 2.5 millions Americans. It is the 4^th leading cause of death among elderly Americans. In 1900 the German neurologist ALIOS ALZHEIMER, found this disease accompanied by organic loss of intellectual function (dementia) as well as memory loss and general incapacitation. Sufferers often cannot speak, walk or tent to do their most basic needs.

In 1987, researchers at several institutions identified a specific gene inducing the brain tissue abnormality, which characterizes the malady. And then simultaneously another research team announced that it was using a DNA probe to locate a genetic marker for the disease on human chromosome 21. But these findings do not suggest that all cases of this disease are genetically linked, they indicate that at least one form of this disease (i.e. familial Alzheimer’s disease (FAD)) may be inheritable.

The gene responsible for FAD abnormality appears for manufacturing a protein called amyloid. Amyloid is a major component of clumps of dead and dying nerve fibers that clog the brains of patients with Alzheimer’s disease. In some individuals this form of dementia acquire before the age of 65 ( refer to as early onset or FAD )but most often occurs late in life.