Polymorphisms: Clinical Implications

By

Amr S. Moustafa, M.D.; Ph.D.

Assistant Prof. & Consultant, Medical Biochemistry Dept.

College of Medicine, KSUamrsm@hotmail.com

Objectives

Polymorphisms Vs mutations

Detection and clinical applications

Polymorphisms at protein level

RFLPs PCR

Polymorphisms at DNA level

Background information: Facts

Human DNA sequence is nearly 99.9% identical

Each new zygote contains ~ 100 bp changes not present in the genome of either parents

Only one bp varies between 2 persons per 1500 bp DNA segment

Human genetic diversity manifested asChanges in DNA sequencesProtein variationsDiseases

Background information: Definitions

Alleles: Different forms of the same gene on a specific locus

Genotype: The set of alleles that make up the genetic constitution

Phenotype: The observable expression of a genotype

Polymorphisms Vs Mutations

Genetic polymorphisms:Common alleles > 1%

Mutations:Rare alleles < 1%

Polymorphisms: Sites

Inter-genes or intronic:Detected by DNA sequence analysis

Gene coding sequences:Different protein variantsDistinct phenotypes (may be)

DNA regulatory regions:may affect phenotypes

Polymorphisms: Detection – 1

At protein levelABO System:

A, B & O alleles: Chromosome 9A & B: 4 nucleotide differencesO: One base deletion and frame-shiftPhenotypes: O, A, B & AB

Rh System:Rh-D: Chromosome 1Phenotypes: Rh-positive and Rh-negativeIncompatibility:Hemolytic disease of newborn

Polymorphisms: Detection – 2At DNA level

DNA sequence analysis

Restriction fragment length polymorphisms (RFLPs)

PCR-based Methods:

Conventional PCR

Real-time PCR

RFLPs

Inherited variations in DNA sequences

Different sizes of DNA fragments

Restriction enzyme

RFLPs: Causes

Single nucleotide polymorphisms (SNPs):Gain or loss of a restriction sitemore frequent than mini- & micro-satellitesVariable number tandem repeat (VNTR):Alteration of number of nucleotides between restriction sites:

Minisatellites: 10 - 100 bpMicrosatellites: 2 - 4 bp

2 unrelated individuals: different patternsIdentical twins: identical pattern

RFLPs: VNTR

For each person a pair ofhomologous chromosomes is shown

RFLPs: Medical applications

Mapping a gene to a particular region of a chromosome

Tissue typing for organ transplantation

Paternity testing and forensic applications

Prenatal diagnosis of genetic diseases

Detection of genetic susceptibility to diseases

RFLPs: Prenatal Diagnosis of sickle cell

anemia - 1

RFLPs: Prenatal Diagnosis of sickle cell

anemia - 2

The Peroxisome Proliferator-Activated Receptors (PPARs)

RFLPs: PPAR-2Pro 12 Ala

Polymorphism

100

200

300

400

295

1 2 3 4bp

178117

Pro AlaPro/Ala

Detection of HgaI RFLP of PPAR-2

Polymorphisms: PCR Detection

ACE gene I/D polymorphisms:

(1) Conventional Method

(2) Real-Time PCR

ACE Gene Insertion/Deletion (I/D)

Polymorphism

Three Genotypes: II, ID, and DD

I or D of 287 bp in intron 16

Two alleles: I and D

PCR for ACE genotypes

1 2 3 4

490 bp

190 bp

II ID DD

ACE gene I/D polymorphism:

Real-Time PCR “1”

Complete amplification

Cool the reaction to 60 °C

Heat slowly to 95 °C

Measure SYBR green fluorescence during heating

Plot signal Vs temperature

Determine melting peaks

Melting (dissociation) curve analysis:

ACE gene I/D polymorphism:

Real-Time PCR “2”

Negative derivative of fluorescence to temperature

Vs temperature (-dF/dT Vs T)

II alleles: 73.9 °C

DD alleles: 76.2 °C

Melting peaks:

ACE gene I/D polymorphism:

Real-Time PCR “3"

70 75 80Temperature (°C)

- d

F/d

T

1

2

3

4

5

II

DD

ID

Conclusions

Polymorphisms:

High degree of biochemical individuality

Different responses of individuals to environment, diet and drugs

Genetic markers: powerful tools in clinical genetics