3. Polymorphisms: Clinical Implications By Amr S. Moustafa, M.D.; Ph.D. Assistant Prof. & Consultant, Medical Biochemistry Dept. College of Medicine, KSU amrsm@hotmail.com

4. Objectives  Polymorphisms Vs mutations  Detection and clinical applications  Polymorphisms at protein level  RFLPs  PCR  Polymorphisms at DNA level

5. 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 as Changes in DNA sequences Protein variations Diseases

6. 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

7. Polymorphisms Vs Mutations Genetic polymorphisms: Common alleles > 1% Mutations: Rare alleles < 1%

8. Polymorphisms: Sites Inter-genes or intronic: Detected by DNA sequence analysis Gene coding sequences: Different protein variants Distinct phenotypes (may be) DNA regulatory regions: may affect phenotypes

9. Polymorphisms: Detection – 1 At protein level ABO System: A, B & O alleles: Chromosome 9 A & B: 4 nucleotide differences O: One base deletion and frame-shift Phenotypes: O, A, B & AB Rh System: Rh-D: Chromosome 1 Phenotypes: Rh-positive and Rh-negative Incompatibility:Hemolytic disease of newborn

10. Polymorphisms: Detection – 2 At DNA level  DNA sequence analysis  Restriction fragment length polymorphisms (RFLPs)  PCR-based Methods: Conventional PCR Real-time PCR

11. RFLPs Inherited variations in DNA sequences Different sizes of DNA fragments Restriction enzyme

12. RFLPs: Causes Single nucleotide polymorphisms (SNPs): Gain or loss of a restriction site more frequent than mini- & micro-satellites Variable number tandem repeat (VNTR): Alteration of number of nucleotides between restriction sites: Minisatellites: 10 - 100 bp Microsatellites: 2 - 4 bp 2 unrelated individuals: different patterns Identical twins: identical pattern

13. RFLPs: VNTR For each person a pair of homologous chromosomes is shown

14. 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

15. RFLPs: Prenatal Diagnosis of sickle cell anemia - 1

16. RFLPs: Prenatal Diagnosis of sickle cell anemia - 2

17. The Peroxisome Proliferator- Activated Receptors (PPARs)

18. RFLPs: PPAR-  2 Pro 12 Ala Polymorphism 100 200 300 400 295 1 2 3 4 bp 178 117 Pro Ala Pro/Ala Detection of HgaI RFLP of PPAR-  2

19. Polymorphisms: PCR Detection ACE gene I/D polymorphisms: (1) Conventional Method (2) Real-Time PCR

20. 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

21. PCR for ACE genotypes 1 2 3 4 490 bp 190 bp II ID DD

22. 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:

23. 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:

24. ACE gene I/D polymorphism: Real-Time PCR “3" 70 75 80 Temperature (°C) - dF/dT 1 2 3 4 5 II DD ID

25. Conclusions Polymorphisms: High degree of biochemical individuality Different responses of individuals to environment, diet and drugs Genetic markers: powerful tools in clinical genetics