Applications of HGP

1. Applications of HGP Genetic testing Forensics

2. Genetic testing Testing for a pathogenic mutation in a certain gene in an individual that indicate a person’s risk of developing or transmitting a disease PURPOSE • Medical management • Forensics • Research

3. Genetic testing can be done in 3 ways • Directly • Gene tracking • Population screening

4. DIRECT GENETIC TESTING Based on either • MUTATION DETECTION: screening for KNOWN polymorphisms in DNA • MUTATION SCANNING: screening for UNKNOWN polymorphisms in DNA

5. MUTATION DETECTION SNPsby ASOs • Very short specific probes (<21 bp) which hybridize to one allele or other • Such probes are allele-specific oligonucleotides (ASOs) Fig. 11.8

6. Variation in length of DNA sequence (repetitive DNA) MUTATION DETECTION Huntington’s disease -a microsatellite triplet repeat in a coding region Figure 18.12: HMG3

7. RISKY SENSITIVE SPECIFIC PRE REQUISITES Gene loci Size Frequency of known mutations MUTATION SCANNING SCREENING TARGET LOCI FOR UNKNOWN MUTATIONS CFTR mutation frequency F508 79.9% G551D 2.6 % G542X 1.5%

8. MUTATION SCANNING METHODS sequencing • Direct sequencing • Southern blots • dHPLC • Microarrays

9. MUTATION SCANNING Using dHPLC Exon 6 of DMD gene normal affected Fig18.4: HMG3 by Strachan & Read

10. MUTATION SCANNING Using multiplex ARMS test Screening for 29 mutations of the CFTR gene Fig18.10: HMG3 by Strachan & Read

11. GENE TRACKING Analysis of linked markers in families for the inheritance of a high risk chromosome from heterozygous parents. Used when map location of disease locus is known but not the actual disease gene The process has 3 steps 1)  find a closely linked marker for which the parents are heterozygous 2)  work out which chromosome carries the disease allele 3) work out which chromosome the individual has inherited

12. POPULATION SCREENING Genetic Screening programs should meet the following criteria • The condition to be screened for should be serious. • The diagnostic methodology should be accurate and sensitive. • The condition must be sufficiently common to make the program economically feasible. • The individual identified as at risk must have some options, preferably either effective early treatment or prenatal diagnosis e.g. PKU tests /Guthrie (PAH activity) ARMS test (CFTR mutations)

13. Forensics • Identify crime suspects / exonerate innocent • Identify victims • Establish family relationships • Identify endangered species • Detect pollutants • Match organ donor with recipient • Determine seed / livestock pedigree • Authenticate consummables

14. Early markers DNA fingerprinting • Karl Landsteiner’s ABO blood typing • Originally described by Sir Alec Jeffreys (1985) (Nature, 1985, 316: 76-79- Jeffereys et al) • Discovery of hypervariable loci • ‘Differential lysis’ technique in parallel • First conviction using DNA fingerprinting was Colin Pitchfork in 1986

15. Repetitive sequences… Simple sequence repeats (SSRs) • Microsatellites 1-13 bp repeats e.g. (A)n (AC)n Minisatellites 14 - 500 bp repeats 3% of genome (dinucleotides - 0.5%) HUMFES/FPS (ATTT)8-14

16. 1985 technique using hybridisation of Multi locus probes (MLP) • Minisatellite probes consisting of tandem repeats of the myoglobin locus • Number of multiple loci probes (MLP) identified • Core sequence GGAGGTGGGCAGGA • 2 of these used (33.15 and 33.6) hybridised to Southern Blots of restriction-digested genomic DNA • Shared ‘core’ sequences at multiple loci creates hypervariable, multi-band patterns called DNA ‘fingerprints • Together, upto 36 independently inherited bands detected • 2 probes gave a match probability of <5 x 10-19

18. …now superceded by PCR-based methods • Discovery of STR (short tandem repeats) • Use of STR multiplex PCR • Autosomal SNP typing, Y-chromosome / mtDNA markers Advantages • Increased sensitivity • Small sample quantities sufficient • Uses microsatellites, instead of minisatellites

19. How does forensic ID work? • Extract DNA • Analyse specific regions using probes • look for matches between 2 samples at many loci (multilocus) • Scan ~ 10 DNA regions that show locus variability • > 5 matches • Create DNA profile (DNA fingerprint)

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21. Current methods 1) Autosomal STR typing • Needs ~300bp amplicons • SGMPlus database (UK) contains 5 multiplex loci • US FBI CODIS contain 13 STR loci

22. Some STR electropherograms Electropherogram of a second-generation multiplex ‘SGM Plus’ profile from a male Electropherogram profile from a mixture Mixtures can only be identified if the alleles of the minor component are above the background ‘noise’ in an electropherogram (in practice a ratio of ~1:10)

23. Current methods 2) Autosomal SNP typing • Lower heterozygosities compared to STR (0.5) • ~ 50 SNPs need to be typed for low Pm • Difficult to resolve mixtures • ~50bp template sizes enough

24. 3) Mitochondrial DNA typing Current methods Mutation rate ~1/33 generations Heteroplasmy (original and mutated forms co-exist) More stable for forensic analysis • Multicopy • 16.5 kbp • Maternally inherited Highest variation in control region (800bp)

25. 4) Y-chromosome typing Current methods • Haploid • Recombination-deficient (mutations only) • Paternal inheritance • Binary polymorphisms

26. Is DNA effective in casework? • Techniques must be robust and reproducible for sample variability • Only if used intelligently!! • Only regions showing the most variability can be used • Must cover large regions • Must be validated Look for matches ‘beyond a reasonable doubt’

27. Is DNA effective in casework? • evidential weight of a match between crime stain profile and suspect is quantified by the match probability (Pm) • Strength of evidence based on likelihood ratio (LR) • LR = C / C ‘Prosecutor’s fallacy’ or ‘fallacy of the transposed conditional’ ‘The probability of the DNA evidence, if it came from the suspect, is 1 in 50 million’

28. References Hum Mol Gen 3 by Strachan and Read Chapter 18 Hartwell et al – Chapter 11; pages 376-387 DNA profiling in forensics by Peter Gillet al www.els.net