1. Penn State Forensics Science Serving Justice Establishing an mtDNA Forensic Laboratory 11 August 2006 Mitchell M. Holland, Ph.D. Associate Professor Biochemistry and Molecular Biology Pennsylvania State University University Park, PA

2. Penn State Forensics Science Serving Justice 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. Penn State Forensics Science Serving Justice Review Article Mitochondrial DNA sequence analysis – validation and use for forensic casework (1999) For Sci Rev 11: 21-50 www.mitotyping.com

4. Penn State Forensics Science Serving Justice Outline Blend in the science behind mtDNA analysis with … Laboratory Design & Practices mtDNA: Admissibility Issues Contamination? Heteroplasmy? Statistics?

5. Penn State Forensics Science Serving Justice Nucleus Mitochondria Nuclear DNA Mitochondrial DNA Types of DNA in the Cell

6. Penn State Forensics Science Serving Justice MtDNA Characteristics High Copy Number 100s-1000s 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. Penn State Forensics Science Serving Justice 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. Penn State Forensics Science Serving Justice Sources of DNA Blood Semen Saliva Cigarette butts Stamps Envelope flaps Hat bands Latex gloves Bones Tissue Hair Anything with Associated Biological Material Older Shaft

9. Penn State Forensics Science Serving Justice 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. Penn State Forensics Science Serving Justice 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. Penn State Forensics Science Serving Justice Maternal Cousins Great Grandniece Deceased Maternal Inheritance

12. Penn State Forensics Science Serving Justice Maternal Cousins Great Grandniece Brother Defendant Maternal Inheritance

13. Penn State Forensics Science Serving Justice mtDNA Sequence Analysis Hair Sample ExtractionDNA PCR Amplification Automated SequencerComputer Analysis Sequence Data

14. Penn State Forensics Science Serving Justice mtDNA Sequence Data

15. Penn State Forensics Science Serving Justice 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 Standardization??

16. Penn State Forensics Science Serving Justice Human Mitochondrial Genome 0 Displacement Loop or Control region 16000 450 Coding Region HV1 = 16024-16365 = ~342 bps HV2 = 73-340 = ~268 bps HV1HV2 VR3 = ~16366-72 VR4 = ~341-530

17. Penn State Forensics Science Serving Justice AGCTTCAGT Human Published Sequence AACTCCAGC EVIDENCE Sequence Profile *** Established by comparison to a published mtDNA sequence

18. Penn State Forensics Science Serving Justice Typical Reported Profile 16189 C 16319 T 73 G 152 T 263 G 309.1 C 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 HV1 HV2 The hair sample was tested using mtDNA sequence analysis. The following profile was obtained.

19. Penn State Forensics Science Serving Justice AGCTTCAGT PUBLISHED AACTCCAGC EVIDENCE AACTCCAGC REFERENCE *** MATCH Cannot Exclude Match = Corresponds, Agrees or is Consistent With

20. Penn State Forensics Science Serving Justice AGCTTCAGT PUBLISHED AACTCCAGC EVIDENCE AGCTCCAGT REFERENCE *** EXCLUSION Exclusions are (generally) Absolute

21. Penn State Forensics Science Serving Justice AGCT T CAGT PUBLISHED AACT C/T CAGC EVIDENCE AACT C/T CAGC REFERENCE *** MATCH Heteroplasmy = a heterogeneous pool of mtDNA sequences in the cytoplasm of the cell

22. Penn State Forensics Science Serving Justice Heteroplasmy AACT C/T CAGC EVIDENCE AACTCCAGC EVIDENCE AACTTCAGC EVIDENCE + Two separate, different sequences present in the sample

23. Penn State Forensics Science Serving Justice Heteroplasmy v. Contamination How do you tell the difference between heteroplasmy and contamination? Heteroplasmy is generally ONLY seen at one position

24. Penn State Forensics Science Serving Justice Heteroplasmy

25. Penn State Forensics Science Serving Justice 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. Penn State Forensics Science Serving Justice AGCT T CAGT PUBLISHED AACT C CAGC EVIDENCE AACT C/T CAGC REFERENCE *** Match or Exclusion? Heteroplasmy – Variant Ratio Drift

27. Penn State Forensics Science Serving Justice C/T T CONTROL TSAR GEORGIJ ZENIA The Identification of Tsar Nicholas II and His Family T/C

28. Penn State Forensics Science Serving Justice Co-occurrence of Heteroplasmy TSAR GEORGIJ

29. Penn State Forensics Science Serving Justice Bottleneck Theory and Heteroplasmy Position 16185

30. Penn State Forensics Science Serving Justice 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. Penn State Forensics Science Serving Justice Replicates like bacterial cells When they get too large, they undergo fission The mtDNA is replicated prior to division Mitochondrion Replication

32. Penn State Forensics Science Serving Justice mtDNA Variant Drift C/T Heteroplasmy T T T T C C C C Two Mitochondrion 1 with C Homoplasmy 1 with T Homoplasmy

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

34. Penn State Forensics Science Serving Justice Hair Biology Embryonic Development

35. Penn State Forensics Science Serving Justice 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. Penn State Forensics Science Serving Justice AGCTTCAGT PUBLISHED AACTCCAGC EVIDENCE AGCTCCAGC REFERENCE *** Apparent Mutational Events Exclusion or Inconclusive?

37. Penn State Forensics Science Serving Justice 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. Penn State Forensics Science Serving Justice 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. Penn State Forensics Science Serving Justice 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. Penn State Forensics Science Serving Justice 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? Whats the definition of a group? Macro v. Micro differentiation of groups There is so much variability within groups that it isnt surprising that 100 African individuals sampled from Houston will have different sequence types than 100 from NYC

41. Penn State Forensics Science Serving Justice Databases However … Will the observed frequency of your mtDNA sequence in the African populationsignificantly change if you use a database from Houston when compared to a database from NYC … or even Nairobi? The answer is … No

42. Penn State Forensics Science Serving Justice 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. Penn State Forensics Science Serving Justice 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 Statistical Calculations

44. Penn State Forensics Science Serving Justice 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. Penn State Forensics Science Serving Justice 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? When evaluating the results from an established mtDNA lab, whats important?

46. Penn State Forensics Science Serving Justice 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 Recommendations

47. Penn State Forensics Science Serving Justice 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

48. Penn State Forensics Science Serving Justice