penn state forensics science serving justice establishing an mtdna forensic laboratory 11 august...

Penn State Forensics

Science Serving Justice

Establishing an mtDNA Forensic Laboratory

11 August 2006

Mitchell M. Holland, Ph.D.Associate Professor

Biochemistry and Molecular BiologyPennsylvania State University

University Park, PA



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



Review Article

Mitochondrial DNA sequence analysis – validation and use for

forensic casework (1999) For Sci Rev 11: 21-50

www.mitotyping.com



Outline

Blend in the science behind mtDNA analysis with …

Laboratory Design & Practices

mtDNA: Admissibility Issues Contamination? Heteroplasmy? Statistics?



Nucleus

Mitochondria

Nuclear DNA

Mitochondrial DNA

Types of DNA in the Cell



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



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



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



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



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



Maternal Cousins

Great Grandniece

Deceased

Maternal Inheritance



Maternal Cousins

Great Grandniece

BrotherDefendant

Maternal Inheritance



mtDNA Sequence Analysis

Hair Sample

Extraction DNA PCR Amplification

Automated Sequencer Computer Analysis

Sequence Data



mtDNA Sequence Data



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??



Human Mitochondrial Genome

0

Displacement Loop or Control region

16000 450

Coding Region

HV1 = 16024-16365 = ~342 bps

HV2 = 73-340 = ~268 bps

HV1 HV2

VR3 = ~16366-72

VR4 = ~341-530



AGCTTCAGT Human Published Sequence

AACTCCAGC EVIDENCE

Sequence “Profile”

* * *

Established by comparison to apublished mtDNA sequence



Typical Reported Profile

16189 C16319 T73 G152 T263 G309.1 C

What Does This Mean?

The “C” means that thegenetic code has changedat position 16189 to a “C”

The “309.1 C” means thatan additional “C” is present

after position 309

HV1

HV2

The hair sample was tested usingmtDNA sequence analysis. Thefollowing profile was obtained.



AGCTTCAGT PUBLISHED

AACTCCAGC EVIDENCE

AACTCCAGC REFERENCE

* * *

MATCH

“Cannot Exclude”Match = Corresponds, Agrees or is Consistent With



AGCTTCAGT PUBLISHED

AACTCCAGC EVIDENCE

AGCTCCAGT REFERENCE

* **

EXCLUSION

“Exclusions are (generally) Absolute”



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



Heteroplasmy

AACT C/T CAGC EVIDENCE

AACTCCAGC EVIDENCE

AACTTCAGC EVIDENCE+

Two separate, different sequences present in the sample



Heteroplasmy v. Contamination

How do you tell the difference between heteroplasmy and contamination?

Heteroplasmy is generally ONLY seen at one position



Heteroplasmy



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



AGCT T CAGT PUBLISHED

AACT C CAGC EVIDENCE

AACT C/T CAGC REFERENCE

* **

Match or Exclusion?

Heteroplasmy – “Variant Ratio” Drift


Science Serving Justice C/T

T

CONTROLTSAR

GEORGIJ ZENIA

The Identification of Tsar Nicholas II and His Family

T/C



Co-occurrence of Heteroplasmy

TSAR

GEORGIJ



Bottleneck Theory and Heteroplasmy

Position 16185



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??



Replicates like bacterial cells

When they get too large, they undergo “fission”

The mtDNA is replicated prior to division

Mitochondrion Replication



mtDNA Variant Drift

C/THeteroplasmy

TT

T

T

C

C

CC Two Mitochondrion

1 with C Homoplasmy

1 with T Homoplasmy



Hair Biology

GrowthStages

Lasts for0.5-7 years

150 Hairs areshed each day

Human hair histogenesis for the mitochondrial DNA forensic scientist (2001) JFS 46: 844-853



Hair Biology

Embryonic Development



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



AGCTTCAGT PUBLISHED

AACTCCAGC EVIDENCE

AGCTCCAGC REFERENCE

* **

“Apparent” Mutational Events

Exclusion or Inconclusive?



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?



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



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?



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



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



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



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



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?



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, what’s important?



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



Thanks for Your Attention

Contact Information

[email protected]

www.forensicdnaconsultants.com

University Address:Penn State University

107 Whitmore Laboratory

University Park, PA 16802

[email protected]

penn state forensics science serving justice establishing an mtdna forensic laboratory 11 august...

Documents

penn state forensics

analysis slide

justice mtdna analysis

mtdna profile slide

sample slide

published mtdna sequence

cell slide

possible slide