Post on 17-Dec-2015
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