GENE TRACKING

Speaker : Dr. Thomas Alex Kodiatte

Moderator : Dr. Kalyani R.

OUTLINE

• Introduction

• Gene Tracking

• Principal Strategies of Diagnosis of Genetic Diseases

• Methods

Introduction • Terminologies

• Genetic Linkage

• Molecular Basis of Inherited Genetic Diseases

DNA – Basis of Life and Disease

DNA - Sequence

• Nucleotide: Composed of three parts: Base, Pentose Sugar and Phosphate group

• Nucleoside : Base + Pentose Sugar

• Sequence: A DNA sequence or genetic sequence is a succession of letters representing the primary structure of a real or hypothetical DNA molecule or strand, with the capacity to carry information as described by the central dogma of molecular biology - The possible letters are A, C, G, and T

Chromosome

What is a Locus

• Locus: A unique chromosomal location defining the position of an individual gene or DNA sequence

Basic Definitions • Allele: One of several alternative forms of a gene or DNA

sequence at a specific chromosomal location (locus). At each Autosomal locus an individual possesses two alleles, one inherited from the father and one from the mother

• Phenotype: The observable characteristics of a cell or organism, including the result of any test that is not a direct test of the genotype

• Genotype: The genetic constitution of an individual, either overall or at a specific locus

Basic Definitions

• Heterozygous: An individual is heterozygous at a locus if

(s)he has two different alleles at that locus

• Homozygous: An individual is homozygous at a locus if

(s)he has two identical alleles at that locus

• Haplotype: A series of alleles found at linked loci on a

single chromosome

Basic Definitions

• Primer : A short oligonucleotide, often 15–25 bases long pairs specifically to a target sequence to allow a polymerase to initiate synthesis of a complementary strand.

• Suitable primers are crucial for PCR, RT-PCR and DNA sequencing

Mutations Change the structure and function of Genes

Introduction • Difference in DNA sequences between any 2

individuals = 0.1 %

• Sequence Variant/Alteration : Any sequence change

• Polymorphism : If a sequence alteration is present in at least 1 % of a population

• Egs of intergenic polymorphic sequences – SNP, SSR, RFLP etc..

SNP = Single Nucleotide Polymorphism ; SSR = Simple Sequence Repeats

Single Nucleotide Polymorphism

• The most common sequence variations are single base changes – SNP

• < 1% of SNPs occur in coding regions and alter the genetic product Disease

• Marker that is co-inherited with a disease causing gene due to physical proximity

Simple Sequence Repeats(SSR)

• Microsatellites / Short Tandem Repeats (STR): Short segments of DNA (2 to 6 bp long) that are repeated end to end

• Minisatellites / Variable Number of Tandem Repeats (VNTRs) : Repeated segments of DNA that are 15 to 70 bp long

• Critical markers in Genetic linkage studies and in forensic or medical identity testing

• Highly pleomorphic between individuals

• On average , one SSR occurs every 2000 bases * * = Fundamentals of Molecular

Diagnostics

What is DNA Marker

• DNA marker: A Polymorphic locus on the gene that is easily assayed yielding reproducible results

Uses of Genetic/DNA Markers 1. To identify the chromosomal location of mutant genes associated

with hereditary diseases

2. DNA fingerprinting and Individual Identification

3. Determination of relatedness and identity in transplantation

4. Cancer genetics

5. Paternity testing

6. Detection & quantification of Transplant chimerism in allogeneic BM transplants

7. Epidemiology and food safety science

8. Human Population History/ Anthropology

9. Improvement of domesticated plants and animals

10. Ecological indicators

11. Evolutionary genetics

Genetic Linkage • According to Mendel’s 3rd

law – States that the alleles of genes at different loci segregate independently – True for genes on different chromosomes

• If 2 loci are positioned close together on the same chromosome, so that alleles at these loci are inherited together more often than not, these loci are said to be LINKED

Genetic Linkage

• Recombination fraction (θ): Measure of the distance separating 2 loci

• Centimorgan (cM) / Map unit = Unit of measurement for genetic linkage

• Eg: If 2 loci are 1 cM apart, recombination frequency is 1 in every 100 meiosis ,i.e. θ = 0.01

• So greater the θ value greater is the chance of recombination and lesser is the chance of being linked

• If 2 loci are not linked , then θ = 0.5

Linkage Analysis • The basic methodology

involves study of the segregation of the disease in large families with polymorphic markers from each chromosome

• Eventually, a marker will be identified that co-segregates with the disease more often than would be expected by chance ,i.e. the marker and disease loci are linked

Genetic Linkage

• Each DNA polymorphism serves as a genetic marker for its own location in the chromosome

• The importance of genetic linkage is that DNA markers that are sufficiently close to the disease gene will tend to be inherited together with the disease gene in pedigrees

DNA - polymorphisms

D

Close enough to Disease gene Genetically Linked Inherited together

DNA Polymorphisms along the chromosome

Too far from Disease gene not linked not inherited in pedigrees

Genetic mapping

• So the initial approach to the identification of a disease gene is to find DNA markers that are genetically linked with the disease gene in order to identify its chromosomal location – GENETIC MAPPING

Linkage Studies • A method using DNA markers physically adjacent

(i.e. linked) to a disease gene ↓

• This indirect analysis allows the disease gene to be tracked through a family

↓ • This way, the genetic status of at-risk individuals

can be determined even when the identity of the disease-causing DNA sequence variant is unknown

Molecular Basis of Inherited Diseases

• 2 strategies to isolate and characterise involved genes :

1. Functional Cloning or Classic Approach –

- To study variety of inborn errors – Eg : PKU,

disorders of Hb synthesis

- The knowledge of the abnormal gene

product and the corresponding affected

protein is known

Molecular Basis….contd..

2. Positional Cloning / The Candidate Gene Approach :

- No clue to the nature of the defective gene product

- Relies on mapping the disease phenotype to

a particular chromosome location

Accomplished if the disease is associated with a

distinctive cytogenetic change or by

Linkage analysis - Used successfully in Cystic

fibrosis, NF, DMD, PKD, Huntington Disease

GENE TRACKING

GENE TRACKING • A method for determining the inheritance of a

particular gene in a family

• It is used in the diagnosis of genetic diseases - cystic fibrosis and Huntington's chorea

• Restriction fragment length polymorphisms (RFLPs) situated in or near the locus of interest are identified using gene probes, and suitable marker RFLPs selected

• These can then be traced through members of the family and used to detect the presence or absence of the disease locus prenatally in future at-risk pregnancies

Defining Gene Tacking

• Following a gene through a family by observing the inheritance of a marker which is being inherited alongside the gene.

Knowledge on Genes helps in Exploration

• How much information a genetic test can give depends on the state of knowledge about the gene(s) involved, but in principle, laboratory genetic diagnosis can be made in two essentially different ways.

Testing the Genetic basis

1. Direct testing: A sample (DNA, RNA, protein, etc.) from a consultand is tested to see whether or not he has a certain genotype - typically, a pathogenic mutation in a certain gene. The test is of an individual and gives information about that individual

2. Gene tracking (Indirect Testing): Linked markers are used in family studies to discover whether or not the consultand inherited the high-risk chromosome from a heterozygous parent. The test is of a family and gives information about the segregation of a chromosomal segment in the family

Consultand : The individual (not necessarily affected) who presents for genetic

counselling and through whom a family with an inherited disorder comes to medical attention

History of Gene Tracking

• First type of DNA diagnostic method to be widely used

• Most Mendelian Diseases went through a phase of gene tracking, then moved on to direct tests, once the genes were cloned

• Gene tracking using linked markers still has its place in modern molecular diagnosis*

Gene tracking shrinks and Direct testing Growing

• With each year that passes, the role of gene tracking shrinks and the applications of direct testing grow. However, direct testing is not always possible and even when it is scientifically possible, it may not always be practical in the context of a routine diagnostic service

Essential Logic of Gene Tracking

1. Distinguish the 2 chromosomes in the relevant parent(s) – i.e. find a closely linked marker for which they are heterozygous

2. Determine PHASE – i.e. work out which chromosome carries the disease allele and work out which chromosome the consultant received

Essential Logic of Gene Tracking

• Phase: of linked markers – the relation (coupling or repulsion) between alleles at two linked loci

• If allele A1 is on the same physical chromosome as allele B1, they are in coupling; if they are on different parental homologs they are in repulsion

Steps in Identification • A lady who is pregnant, wishes a pre-symptomatic

test to show whether she has inherited the disease allele.

1. The first step is to tell the parent's two chromosomes apart. A marker, closely linked to the disease locus, is found for which her mother is heterozygous.

2. Next establish phase - that is, work out which marker allele in the mother is segregating with the disease allele.

• Note that it is the segregation pattern in the family, and not the actual marker genotype, that is important

Pre-requisites for Gene Tracking

1. The disease should be adequately mapped, so that markers can be used that are known to be tightly linked to the disease locus.

2. The pedigree structure and sample availability must allow determination of PHASE.

• This emphasizes the need for both an appropriate pedigree structure (DNA must be available from the affected child) and informative marker types.

Recombination sets a fundamental limit on the accuracy of gene tracking

• The recombination fraction and ,hence the error rate, can be estimated from family studies by standard linkage analysis

• With almost any disease, there should be a good choice of markers showing less than 1 % recombination with the disease locus

• Recombination b/w marker and disease can never be ruled out, even for very tightly linked markers, but the error rate can be greatly reduced by using 2 marker loci, situated on opposite sides of the disease locus

Calculating risks in gene tracking

• Factors in assessing the final risk :

1. Probability of disease-marker and marker-marker recombination

2. Uncertainty due to imperfect pedigree structure or limited information of the markers

3. Uncertainty as to if somebody in the pedigree is carrying a newly mutant disease allele

Genetic Risk Calculation Bayes’ Theorem

… Bayes’ Theorem

1. Linkage Analysis Program

Linkage Analysis Program

Summary of Gene Tracking • Once a disease gene has been located using

linkage analysis, DNA markers can be used to track the disease gene through families to predict the genetic state of individuals at risk.

• Prior to identifying specific gene mutations, this can provide information about carrier risk and enable prenatal diagnosis in certain situations.

Summary of Gene Tracking

• Before gene tracking can be used to provide a predictive test, family members known to be affected or unaffected must be tested to find an informative DNA marker within the family and to identify which allele is segregating with the disease gene in that particular kindred.

• Because recombination occurs between homologous chromosomes at meiosis, a DNA marker that is not very close to a gene on a particular chromosome will sometimes be inherited independently of the gene.

Summary of Gene Tracking

• The closer the marker is to a gene, the less likely it is that recombination will occur.

• In practice, markers that have shown less than 5% recombination with a disease gene, have been useful in detecting carriers and in prenatal diagnosis.

• There is always a margin of error with this type of test and results are quoted as a probability of carrying the gene and not as a definitive result.

Summary of Gene Tracking

• Linkage studies using intragenic markers provide much more accurate prediction of genetic state, but this approach is only used now when mutation analysis is not possible, as in some cases of Duchenne muscular dystrophy, Marfan syndrome and neurofibromatosis type 1.

DIAGNOSIS OF GENETIC DISEASES

Diagnosis of Genetic Diseases

• Cytogenetic Analysis – Prenatal and Postnatal

• Molecular Analysis

Advantages of Recombinant DNA Technology in Diagnosis of Inherited

Diseases

• Remarkably sensitive

• Not dependent on a gene product or gene expression that may occur late in life

Approaches to the Diagnosis of Single-Gene diseases

1. Direct Detection of Mutations

2. Indirect Detection – Based on linkage of disease gene with a harmless “ marker ” gene

Direct Gene Diagnosis

• Diagnostic Biopsy of the Human Genome

• Diagnosis depends on the detection of an important qualitative change in the DNA

• The knowledge of the abnormal gene product and the corresponding affected protein is known

• Based on PCR Analysis

• Direct sequencing is the method of choice

INDIRECT DNA DIAGNOSIS : LINKAGE ANALYSIS

• In many genetic diseases, information about the gene sequence is lacking

↓

• Therefore, alternate strategies must be employed to track the mutant gene on the basis of its linkage to detectable genetic markers

• Basically one has to determine if a given foetus or family member has inherited the same relevant chromosomal region(s) as a previously affected member

Use of Polymorphic markers with Linkage Studies in Diagnosis of

Genetic Diseases 1. When the causal gene has not been identified

2. When the disease is multifactorial and no single gene is involved ( HTN, DM, Cancer)

3. If the disease originates from several different mutations in a given gene (e.g., fibrillin-1), and gene sequencing is either not practical or negative but there is very strong clinical suspicion, linkage analysis can be useful

Surrogate Marker can locate chromosomal regions

• In such cases, surrogate markers in the genome, also known as marker loci, can be used to localize the chromosomal regions of interest, on the basis of their linkage to one or more putative disease-causing genes.

Linkage analysis identifies are of Interest

• Linkage analysis deals with assessing these marker loci in family members having the disease or trait of interest.

• With time it becomes possible to define a “disease haplotype” based on a panel of marker loci, all of which co-segregate with the putative disease allele.

• Eventually, linkage analysis facilitates localization and cloning of the disease allele.

Haplotype : A series of alleles found at linked loci on a single chromosome

Polymorphisms/Marker Loci in Linkage Studies

• 2 general categories:

SITE Polymorphism – RFLP

LENGTH Polymorphism – SNPs, Micro & Mini satellites

Restriction Fragment Length Polymorphism

• Refers to variation in fragment length between individuals that results from DNA sequence polymorphisms

• Usually this variation in DNA sequences : 1 nucleotide in every 200 – 500 bp stretches

• Most variations occur in non-coding regions

• Single base pair changes may abolish/create recognition sites for restriction enzymes, thereby altering the length of DNA fragments produced after digestion with certain restriction enzymes

RFLP….

• Using appropriate DNA probes that hybridize with sequences in the vicinity of the polymorphic sites, it is possible to detect DNA fragments of different lengths by Southern Blot Analysis

• Can be used in Gene Tracking

RFLP…. • When DNA from such an individual is digested

with the appropriate restriction enzyme and probed with a cloned DNA fragment that hybridizes with a stretch of sequences between the restriction sites, the normal chromosome yields a 7.6 kb band whereas the other chromosome produces a smaller 6.8 kb band

• Possible by this technique to distinguish family members who have inherited both normal chromosomes from those who are heterozygous or homozygous for the mutant gene

Length Polymorphisms

• Microsatellites and minisatellites

• Helpful in linkage studies in human chromosomes

SNP –genetic marker

• SNP – physical landmark within genome and as genetic marker whose transmission can be followed from parent to child

• B’cos of their prevalence in human genome and their stability, SNPs can be used in linkage analysis for identifying haplotypes associated with diseases gene discovery and mapping

• Genetic marker of choice – Study of complex genetic traits

Limitations of Linkage Studies

• Mutant gene itself is not identified

• For diagnosis, several relevant family members must be available for testing

• Key family members must be heterozygous for the polymorphism

• Normal exchange of chromosomal material between homologous chromosomes may lead to “separation” of the mutant gene from the polymorphism pattern with which it had been previously co-inherited

Uses of Linkage Analysis in Inherited Genetic Disorders

• Antenatal or pre-symptomatic diagnosis of disorders – Huntington’s Disease, Cystic Fibrosis and Adult Polycystic Kidney Disease

METHODS OF DNA SEQUENCE ANALYSIS

Methods of DNA Sequence Analysis

• Methods of SCANNING a gene for mutations – Used to check whether a gene carries a mutation or not

• Methods of TESTING a gene for a specified mutation

Mutation Scanning Methods

Mutation Scanning Methods….

Mutation Scanning Methods….

RT-PCR as Diagnostic Tool

• Sequencing , particularly of RT-PCR product, is the method of choice for mutation scanning

• Other popular methods – Heteroduplex methods and SSCP Analysis

• Methods of the Future : Oligonucleotide arrays and DNA chips

Heteroduplex : A double-stranded DNA in which there is some mismatch between the two strands.

Methods of testing for a Specified Mutation

Direct Methods METHOD ADVANTAGES DISADVANTAGES

DIDEOXY ENZYMATIC SEQUENCING

Detects and fully characterizes all changes; inexpensive and quick; can be automated

Computer-generated inferred base sequence can contain errors

OLIGONUCLEOTIDE ARRAYS - Hybridization based - Mini Sequencing based

Provide quick , high-throughput analysis

Require expensive equipment; Available for a limited no of genes; Do not characterise all changes

MASS SPECTROMETRY Quick; High throughput

Still under development; Requires expensive instrumentation

Indirect Methods METHODS ADVANTAGES DISADVANTAGES

ALLELIC DISCRIMINATION Based on Size

Quick; simple; high throughput

Detects insertions and deletions only; Doesn’t reveal position

ALLELIC DISCRIMINATION based on RFLP Analysis

Quick; simple; high throughput

Only detects targeted change

ALLELE-Specific PCR Quick; simple; useful for screening

Only detects targeted change

HETERODUPLEX ANALYSIS Simple; Inexpensive Limited sensitivity; limited to < 200bp long; Doesn’t reveal position or type of change

DENATURING GRADIENT GEL ELECTROPHORESIS

High sensitivity; Useful for screening

Requires optimization for each individual DNA region; Doesn’t reveal position or type of change

ADVANTAGES Quick; simple; high throughput Quick; simple; high throughput

Quick; simple; useful for screening Simple; Inexpensive

High sensitivity; Useful for screening

METHODS ADVANTAGES DISADVANTAGES

SINGLE STRAND CONFORMATIONAL POLYMORPHISM ANALYSIS

Simple; useful for screening

Limited sensitivity; limited to < 200bp long; Doesn’t reveal position or type of change

LIGASE CHAIN REACTION Simple; useful for screening

Only detects targeted changes

PROTEIN TRUNCATION TEST

Highly sensitive for truncating mutations; Indicates general position of change

Only detects terminating mutations; technically difficult

CLEAVAGE OF MISMATCHED NUCLEOTIDES

Chemical and Enzymatic - Indicates general position of change

Toxic chemicals

Basic Principles of the Mutation Tests

• DNA Sequencing by the Dideoxy-Mediated Chain Termination: Priming of enzymatic DNA synthesis by the chain termination method of DNA sequencing is achieved by the use of synthetic oligonucleotide primers complimentary to a known sequence of the template strand to be analysed and the method is simplified by the use of chain-terminating dideoxynucleoside triphosphates

Allele discrimination by Size :

• Variant and normal alleles that vary by small insertions or deletions can be distinguished based on the size of the PCR product after gel electrophoresis

Allele discrimination based on susceptibility to a Restriction enzyme

(RFLP): • Using RFLP analysis technique, mutations

that either create or destroy a restriction endonuclease site can be easily distinguished by a 2-step process that involves DNA digestion with the restriction endonuclease followed by gel electrophoresis to size-fractionate the digested DNA

Allele-Specific PCR/ARMS

• ARMS (Amplification refractory mutation system) employs oligonucleotide primers designed to discriminate between target sequences that differ by a single base

Single-Strand Conformational Polymorphism Analysis

• Under non-denaturing conditions, single stranded DNA molecules fold into complex 3-dal structures that are stabilised primarily by base-pairing hydrogen bonds and that alter the DNA’s mobility during non-denaturing gel electrophoresis

• Because the 3-d structure is dictated by the DNA sequence, wild-type and mutant molecules of same length differing by even a single nucleotide will likely adopt different 3-d structures and exhibit different electophoretic mobility

Heteroduplex Analysis

• Method of identifying DNA sequence variation that is based on gel electrophoresis of double-stranded DNA

Denaturing Gradient Gel Electrophoresis

• The denaturation of ds-DNA, whether by heat or by chemical reagents like urea does not occur in a single step

• Melting occurs in a series of steps and contains many melting domains. The behaviour of each melting domain is a function of its base sequence

• Changes in the base sequence usually alters the melting profile

Chemical Cleavage of Mismatched Nucleotides

• Based on the fact that pyrimidines in DNA that are incorrectly paired, are much more reactive with specific modifying chemicals – Osmium tetraoxide (T – G/C)) and hydroxylamine (C – A/T)

Ribonuclease Cleavage Assay

• Takes advantage of the fact that ribonucleases can specifically cleave RNA at the site of sequence mismatches in RNA-DNA or RNA-RNA duplexes

Allele-Specific Oligonucleotide(ASO) Hybridization

• Under suitable stringent hybridization conditions, the ASO synthetic probes hybridize only to a perfectly matched sequence

Oligonucleotide Ligation Assay

• 2 oligonucleotides are constructed that hybridize to adjacent structures in the target, with the join sited at the position of the mutation

• DNA ligase will covalently join the 2 oligonucleotides only if they are perfectly hybridized

Ligase Chain Reaction

• DNA ligases catalyze the formation of a phosphodiester bond between 2 adjacent oligonucleotides when there is perfect complementary base pairing b/w the target and the 2 hybridized probes, whereas a nucleotide mismatch within the 2 bases of the probe joining site will inhibit the reaction

Protein Truncation Test

• Used to detect frame shift mutations, splice site mutations and nonsense mutations that truncate a protein product

• Useful test for diseases – DMD, Familial Adenomatous Polyposis, NF-1, BRCA-related Breast cancer

• A truncated polypeptide after PCR and gel electrophoresis points to the presence of a premature stop codon

Protein Truncation Test

• Coding sequence without introns (cDNA or large exons in genomic DNA) is PCR amplified using a special forward primer that includes a T7 promoter, a eukaryotic translation initiator with an ATG start codon and a gene-specific 3′ sequence designed so that the sequence amplified reads in-frame from the ATG. A coupled transcription-translation system is used to produce polypeptide from the PCR product, and the protein is checked for size by SDS-PAGE gel electrophoresis. A truncated polypeptide points to the presence of a premature stop codon

DNA Microarray Technology

• Hybridization-based approach

• Mini-sequencing-based Assay

DNA Microarray Technology

Hybridization-Based Approach

• Loss of Hybridization signal Approach: Patient samples and wild-type reference samples are hybridized to the oligonucleotide array & sequence variations are scored by quantitation of relative losses in the signal between the 2 samples

• Gain of Hybridization signal Approach: Permits a partial scan of a DNA region for all possible sequence variations through the use of oligonucleotide probes complementary to each of the 4 possible nucleotides present on a DNA strand at a particular location

References 1. Fundamentals of Molecular Diagnostics –

Bruns, Ashwood

2. Robbins’ Pathologic Basis Of Disease – 7th , 8th Edition

3. Human Molecular Genetics 2 - Strachan, Tom and Read, Andrew P. by Garland Science - 1999

Gene Tracking A Topic of Academic Interest for Postgraduates in Basic Sciences