Establishing an mtDNA Forensic Laboratory 11 August 2006

1. Establishing an mtDNA Forensic Laboratory11 August 2006 Mitchell M. Holland, Ph.D. Associate Professor Biochemistry and Molecular Biology Pennsylvania State University University Park, PA

2. Experience • Started the Armed Forces DNA Identification Laboratory (AFDIL) in 1991 • Developed methods for mtDNA analysis that have been used for the past 15 years at AFDIL and have been transferred to labs around the world • Developed laboratory methods for the analysis of evidence such as hairs, bone, teeth, and finger nails • Studied various aspects of mtDNA: including apparent mutation rates, heteroplasmy, variant drift, and population diversity

3. Review Article Mitochondrial DNA sequence analysis – validation and use for forensic casework (1999) For Sci Rev 11: 21-50 www.mitotyping.com

4. Outline Blend in the science behind mtDNA analysis with … • Laboratory Design & Practices • mtDNA: Admissibility Issues • Contamination? • Heteroplasmy? • Statistics?

5. Types of DNA in the Cell Nucleus Mitochondria Mitochondrial DNA Nuclear DNA

6. MtDNA Characteristics • High Copy Number • 100’s-1000’s of copies per cell • More sensitive test • Works on samples that give no STR results • Maternally Inherited • Good for historical cases • Maternal relatives share your mtDNA profile

7. Laboratory Practices for mtDNA Analysis • The sensitivity of mtDNA sequence analysis requires special attention to laboratory design and practices • Physical separation for the analysis of evidence samples (with low quantities of DNA) and reference samples (with high levels of DNA) • Use of hood space • Careful handling of evidence • Cleaning the surface of the evidence, if possible

8. Blood Semen Saliva Cigarette butts Stamps Envelope flaps Older Shaft Sources of DNA • Hat bands • Latex gloves • Bones • Tissue • Hair Anything with Associated Biological Material

9. Hairs • Hairs are the sample most often tested for mtDNA analysis in criminal cases • Root contains nuclear DNA for STR analysis • Shaft only contains enough mtDNA for analysis

10. Cleaning Hairs • Hairs are an excellent sample type for mtDNA analysis • The surface of the hair can be “cleaned” with biological detergents • Blood, semen, saliva encrusted hairs or hairs that have been handled can be cleaned to completely remove the surface contributor

11. Maternal Inheritance Deceased Maternal Cousins Great Grandniece

12. Maternal Inheritance Defendant Brother Maternal Cousins Great Grandniece

13. mtDNA Sequence Analysis Hair Sample Extraction DNA PCR Amplification Automated Sequencer Computer Analysis Sequence Data

14. mtDNA Sequence Data

15. Standardization?? • mtDNA analysis can be performed using different … • extraction methods • amplification conditions • sequencing conditions • instruments • analysis software • However, when possible the laboratory should use the same general amp/seq methods on both evidence and references

16. HV1 HV2 Human Mitochondrial Genome Displacement Loop or Control region 450 16000 0 HV1 = 16024-16365 = ~342 bps HV2 = 73-340 = ~268 bps Coding Region VR3 = ~16366-72 VR4 = ~341-530

17. * * * Established by comparison to a published mtDNA sequence Sequence “Profile” AGCTTCAGTHuman Published Sequence AACTCCAGCEVIDENCE

18. HV1 HV2 Typical Reported Profile What Does This Mean? The “C” means that the genetic code has changed at position 16189 to a “C” The “309.1 C” means that an additional “C” is present after position 309 16189 C 16319 T 73 G 152 T 263 G 309.1 C The hair sample was tested using mtDNA sequence analysis. The following profile was obtained.

19. MATCH AGCTTCAGT PUBLISHED AACTCCAGC EVIDENCE AACTCCAGC REFERENCE * * * “Cannot Exclude” Match = Corresponds, Agrees or is Consistent With

20. EXCLUSION AGCTTCAGT PUBLISHED AACTCCAGC EVIDENCE AGCTCCAGT REFERENCE * * * “Exclusions are (generally) Absolute”

21. MATCH AGCT T CAGT PUBLISHED AACT C/T CAGCEVIDENCE AACT C/T CAGCREFERENCE * * * Heteroplasmy = a heterogeneous pool of mtDNA sequences in the cytoplasm of the cell

22. AACTCCAGCEVIDENCE + AACTTCAGCEVIDENCE Two separate, different sequences present in the sample Heteroplasmy AACT C/T CAGCEVIDENCE

23. Heteroplasmy v. Contamination • How do you tell the difference between heteroplasmy and contamination? • Heteroplasmy is generally ONLY seen at one position

24. Heteroplasmy

25. Heteroplasmy v. Contamination • How do you tell the difference between heteroplasmy and contamination? • Heteroplasmy is generally ONLY seen at one position • Heteroplasmy should be reproducible • HOWEVER may show slight drift in variant ratio due to sampling

26. Match or Exclusion? AGCT T CAGT PUBLISHED AACT CCAGCEVIDENCE AACT C/T CAGCREFERENCE * * * Heteroplasmy – “Variant Ratio” Drift

27. The Identification of Tsar Nicholas II and His Family CONTROL C/T TSAR GEORGIJ ZENIA T/C T

28. Co-occurrence of Heteroplasmy TSAR GEORGIJ

29. Bottleneck Theory and Heteroplasmy Position 16185

30. Understanding Heteroplasmy • What is it? • How can you tell the difference between heteroplasmy and contamination? … and show experimentally that you have it? • How is it passed from one generation to the next? … or from one cell to the next?? • Which types of samples may have more heteroplasmy than others? … AND WHY??

31. Mitochondrion Replication • Replicates like bacterial cells • When they get too large, they undergo “fission” • The mtDNA is replicated prior to division

32. mtDNA Variant Drift C C C/T Heteroplasmy T T C Two Mitochondrion 1 with C Homoplasmy 1 with T Homoplasmy C T T

33. Lasts for 0.5-7 years Hair Biology Growth Stages 150 Hairs are shed each day Human hair histogenesis for the mitochondrial DNA forensic scientist (2001) JFS 46: 844-853

34. Hair Biology Embryonic Development

35. Hair Biology and mtDNA • Different hairs may have different ratios of heteroplasmy • Hair mtDNA profiles may or may not share the same ratio of heteroplasmic variants as blood reference samples • Nonetheless, we are usually able to interpret the results and make conclusions

36. Exclusion or Inconclusive? AGCTTCAGT PUBLISHED AACTCCAGC EVIDENCE AGCTCCAGC REFERENCE * * * “Apparent” Mutational Events

37. Heteroplasmy Interpretation • At what site does the heteroplasmy occur? • How uncommon is the mtDNA sequence in the population (excluding the position of heteroplasmy)? • How much sequence data do you have? • Are there other reference/exemplar samples that can be tested?

38. Results of mtDNA Analysis • In general, an mtDNA result can provide strong circumstantial evidence to associate an evidence sample to an individual • However … issues surrounding the possibility that maternal relatives are associated with the same case should be resolved • mtDNA analysis DOES NOT provide a positive means of identification

39. Statistics • What question are you asking? • What question is important to ask in the context of the forensic case? • What databases are you using and are they sufficient to answer the important/forensically relevant questions being asked?

40. Databases • Exist for the major population groups • As the databases grow in size, more weight can be placed on the meaning of the match • Are there population groups that are under-represented? • What’s the definition of a “group”? • Macro v. Micro differentiation of groups • There is so much variability within groups that it isn’t surprising that 100 African individuals sampled from Houston will have different sequence types than 100 from NYC

41. Databases • However … • Will the “observed frequency” of your mtDNA sequence in the African population “significantly change” if you use a database from Houston when compared to a database from NYC … or even Nairobi? • The answer is … No

42. Statistical Calculations • “Practical” Stats • ~6,000,000,000 People in the World • ~100,000 mtDNA Profiles Possible • Therefore, 1/60,000 • Conservative to say … “Can exclude 99% of the population as the source of a sample using mtDNA” • What about “common sequences”? • May want to take into consideration the number of maternal relatives living in the same area of the crime who had an opportunity to commit the crime

43. Statistical Calculations • Statistical Methods • Confidence Limits from Zero Proportion • For sequences not seen in the database • As the database size grows, so does the CLZP calculation – 99.5% for databases of 500 or more • Normal Approximation of the Binomial • For sequences seen multiple time in the database • Bootstrapping • For sequence seen very few times in the database

44. Courtroom Issues • Did the expert present the results correctly or did the expert mislead the jury/judge? • Interpretation of the data • Weight of the evidence • Are there issues with respect to the racial background of the defendant in relation to where the crime occurred? • Were the “right” samples tested … or could other samples have been tested?

45. When evaluating the results from an established mtDNA lab, what’s important? • Did the laboratory doing the analysis perform the tests correctly? • Does the laboratory have the experience necessary to understand the subtleties of mtDNA analysis? • Practices for handling foreign sources of DNA • Interpretation of complex data • Heteroplasmy – understanding, proper interpretation • Was replicate testing performed? … or could it be performed?

46. Recommendations • Recommend independent review on a case-by-case basis by an outside expert … however, the expert should • Be someone who has a strong understanding of the scientific principles • AND, someone who has considerable practical experience with forensic samples/results

47. Thanks for Your Attention • Contact Information • mholland@forensicdnaconsultants.com www.forensicdnaconsultants.com • University Address: Penn State University 107 Whitmore Laboratory University Park, PA 16802 mmh20@psu.edu