Services related to Genetics _ Genetic Testing.pptx

abcd abcd abcd abcd abcd abcd

1. Services related to Genetics : Genetic Testing Dr. Atanu Kumar Dutta Additional Professor Department of Biochemistry AIIMS Kalyani B.Sc. Nursing

2. Definition Genomics  is defined as the study of genes and their functions, and related techniques (WHO). Human Genome: The human genome is the complete set of nucleic acid sequence for humans (Homo sapiens). Human genomes include both protein-coding DNA genes and noncoding DNA. 3,234.83 Mb (Mega-base pairs) per haploid genome. Human Genome Project: First draft  February 12, 2001.

3. What is genetic testing? Genetic testing is a type of medical test that identifies changes in genes and chromosomes 3

4. What are the different types of genetic tests? Targeted single variant Single gene Gene panel Whole exome sequencing/whole genome sequencing 4

5. Targeted mutation analysis-Single mutation Disease shows genetic homogeneity at variant level One or few variants known to be responsible for majority of patients Definitive clinical features or diagnostic tests Examples Sickle cell anemia --- NM_000518.4(HBB):c.20A>T,p.Glu6Val Achondroplasia --- NM_000142(FGFR3):c.1138G>A Apert syndrome--- NM_000141.4(FGFR2):c.755C>G 5

6. Targeted mutation analysis-Single mutation 6

7. Amniocentesis β globin gene Targeted mutation analysis - Small gene Beta thalassemia

8. Next Generation Sequencing Refers to all post-Sanger sequencing technologies that enable massive sequencing at low cost 8 Massive : Millions of DNA fragments are sequenced Parallel : All fragments are sequenced simultaneously Deep : Each fragment is sequenced a number of times Resequencing : Generated sequence is aligned to a reference genome

9. Exome sequencing (WES): Coverage of reads restricted to exons of genes Clinical Exome: covers exons of nearly 7000 genes with reported OMIM phenotype Disease specific panels: usually 50-200 genes specific to the phenotype like deafness, retinitis pigmentosa, hereditary cancer syndromes, etc Whole genome sequencing (WGS): covers the entire genome Trio-Sequencing: Proband + Parents 9 Image : N Engl J Med 2018;379:1353-62 . Clinical NGS

10. What are the uses of genetic testing? Newborn screening Diagnostic testing Carrier testing Prenatal testing Preimplantation testing Predictive and presymptomatic testing Forensic testing 10

11. How is genetic testing done? 11

12. Polymerase chain reaction - PCR 12 PCR-RFLP, qPCR, DNA Sequencing, etc

13. Quantitative PCR As the name suggests used to estimate the amount of nucleic acid material in the sample End Point PCR Signal from the fluorescent probes is measured at the end of PCR reaction Real time PCR Signal from the fluorescent probes is measured during the PCR reaction 13

14. End Point PCR 14 Fluorescent signal is measured after the end of PCR – makes it less sensitive and inaccurate. (Semiquantitative) Modifications in the techniques ( eg - Multiplex ligation-dependent probe amplification) – to ensure accuracy Eg - Aneuploidy testing by QF-PCR, MLPA QF-PCR MLPA

15. Real time PCR Fluorescent signal is plotted against PCR cycles Cycle Threshold ( ct ) - PCR cycle number where the fluorescence from the reporter is greater than threshold level It is inversely proportional to the initial quantity of DNA/RNA Used for Quantitative analysis of gene expression, viral loads CNV validation targeted SNP analysis 15 Sensitive & Accurate Rapid Requires specific equipment and slightly costlier E-gene amplification in a Covid-19 infected individual

16. Sanger sequencing 16 DNA PCR with specific primer PCR product Single primer PCR with dideoxy nucleotides Chromatogram Capillary eletrophoresis

17. Sanger sequencing 17 Homozygous variation Heterozygous variation Heterozygous insertion/deletion Normal

18. NGS www.biorender.com 18

19. What is informed consent? Example 19

20. How can I be sure a genetic test is valid and useful? Analytical validity Clinical validity Clinical utility 20

21. What do the results of genetic tests mean? 21

22. What are the benefits of genetic testing? Early intervention Reducing diagnostic time Family screening Prenatal testing Targeted therapy 22

23. What are the risks and limitations of genetic testing? Medical Emotional Social Financial Discrimination 23

24. Thank You 24