Mitchell M. Holland

DNA Commission of the International Society for Forensic Genetics: guidelines for mitochondrial DNA typing

Sequence analysis of human mitochondrial DNA (mtDNA) is being used widely to characterize forensic biological specimens, particularly when there is insufficient nuclear DNA in samples for typing. Hair shafts, bones, teeth and other samples that are severely decomposed may be subjected to mtDNA analysis, e.g. [1–5]. Although many of the quality assurance, quality control and interpretational guidelines used for PCR-based nuclear DNA analyses apply to mtDNA analysis, there are some features of mtDNA that warrant specific consideration: (1) mtDNA is maternally inherited; (2) heteroplasmy; and (3) the greater sensitivity of detection of mtDNA typing. It is imperative that guidelines consider the features of mtDNA and that practices do not exceed the state-of-knowledge on mtDNA. In a effort to refine previously published guidelines [6] and to assist those currently using mtDNA protocols and those considering implementing mtDNA analysis, the DNA Commission of the ISFG met on 16th August 1999 in San Francisco to develop current guidelines. The following are the recommendations by the DNA Commission on the use of mtDNA analysis.

Mitochondrial DNA Sequence Analysis of Human Skeletal Remains: Identification of Remains from the Vietnam War

Deoxyribonucleic acid (DNA) sequence analysis of the control region of the mitochondrial DNA (mtDNA) genome was used to identify human skeletal remains returned to the United States government by the Vietnamese government in 1984. The postmortem interval was thought to be 24 years at the time of testing, and the remains presumed to be an American service member. DNA typing methods using nuclear genomic DNA, HLA-DQ alpha and the variable number of tandem repeat (VNTR) locus D1S80, were unsuccessful using the polymerase chain reaction (PCR). Amplification of a portion of the mtDNA control region was performed, and the resulting PCR product subjected to DNA sequence analysis. The DNA sequence generated from the skeletal remains was identical to the maternal reference sequence, as well as the sequence generated from two siblings (sisters). The sequence was unique when compared to more than 650 DNA sequences found both in the literature and provided by personal communications. The individual sequence polymorphisms were present in only 23 of the more than 1300 nucleotide positions analyzed. These results support the observation that in cases where conventional DNA typing is unavailable, mtDNA sequencing can be used for human remains identification.

Population data for 101 Austrian Caucasian mitochondrial DNA d-loop sequences: application of mtDNA sequence analysis to a forensic case.

Abstract The sequence of the two hypervariable segments of the mitochondrial DNA (mtDNA) control region was generated for 101 random Austrian Caucasians. A total of 86 different mtDNA sequences was observed, where 11 sequences were shared by more than 1 individual, 7 sequences were shared by 2 individuals and 4 sequences were shared by 3 individuals. One of the four most common mtDNA sequences in Austrians is also the most common sequence in both U.S. and British Caucasians, found in approximately 3.0% of Austrians, 4.0% of British, and 3.9% of U.S. Caucasians. Of the remaining three common Austrian sequences, one was not observed in either U.S. or British Caucasians. However, three British Caucasians exhibited a similar sequence type. Therefore, this particular cluster of sequence polymorphisms may represent a common “European” mtDNA sequence type. In general, Austrian Caucasians show little deviation from other Caucasian databases of European descent. Finally, mtDNA sequence analysis was applied to a forensic case, where hairs found at a crime scene matched the control hairs from the suspect.

Mitochondrial DNA regions HVI and HVII population data

Data from 1393 unrelated individuals have been compiled from eight population groups: African Americans, Africans (Sierra Leone), U.S. Caucasians, Austrians, French, Hispanics, Japanese, and Asian Americans. The majority of the mtDNA sequences were observed only once within each population group (i.e., ranging from a low of 60.3% (35/58) of the Asian American sequences to a high of 85.3% (93/109) of the French sequences). Genetic diversity ranged from 0.990 in the African sample to 0.998 in African Americans. Random match probability ranged from 2.50% in the Asian American sample to 0.52% in U.S. Caucasians. The average number of nucleotide differences between individuals in a database is greatest for the African American and African samples (14.1 and 13.1, respectively), and the least variable are the Caucasians (ranging from 7.2 to 8.4). Substitutions are the predominate polymorphism, and at least 92% of the substitutions are transitions. The most prevalent transversions are As substituted for Cs and Cs substituted for As. For most population groups these transversions occurred predominately in the HVI region; however, the African, African American, and Hispanic samples also demonstrated a large portion of their C to A and A to C transversions in the HVII region (at sites 186 and/or 189). Most insertions occur in the HVII region at sites 309.1 and 315.1, within a stretch of Cs. Insertions of an additional C are common in all population groups. The sequence data were converted to SSO mtDNA types and compared with population data on Caucasians, Africans, Asians, Japanese, and Mexicans described by Stoneking et al. [M. Stoneking, D. Hedgecock, R.G. Higuchi, L. Vigilant, H.A. Erlich, Population variation of human mtDNA control region sequences detected by enzymatic amplification and sequence-specific oligonucleotide probes, Am. J. Hum. Genet. 48 (1991) 370-382] using an R x C contingency table test. Differences between major population groups (i.e., between African, Caucasian, and Asian) are quite evident, and similar ethnic population groups carried similar SSO polymorphism frequencies. There were only a few SSO types that showed significant differences between subpopulation groups. The SSO data alone can not be used to describe the population genetics with complete sequence data. However, the results of the SSO comparisons are similar to other analyses, and differences in sequence data in regions HVI and HVII are greater between major population groups than between subgroups.

DNA Commission of the International Society for Forensic Genetics: guidelines for mitochondrial DNA typing.

Sequence analysis of human mitochondrial DNA (mtDNA) is being used widely to characterize forensic biological specimens, particularly when there is insufficient nuclear DNA in samples for typing. Hair shafts, bones, teeth and other samples that are severely decomposed may be subjected to mtDNA analysis, e.g. [1–5]. Although many of the quality assurance, quality control and interpretational guidelines used for PCR-based nuclear DNA analyses apply to mtDNA analysis, there are some features of mtDNA that warrant specific consideration: (1) mtDNA is maternally inherited; (2) heteroplasmy; and (3) the greater sensitivity of detection of mtDNA typing. It is imperative that guidelines consider the features of mtDNA and that practices do not exceed the state-of-knowledge on mtDNA. In a effort to refine previously published guidelines [6] and to assist those currently using mtDNA protocols and those considering implementing mtDNA analysis, the DNA Commission of the ISFG met on 16th August 1999 in San Francisco to develop current guidelines. The following are the recommendations by the DNA Commission on the use of mtDNA analysis.

Expanding the forensic German mitochondrial DNA control region database: genetic diversity as a function of sample size and microgeography.

Abstract Mitochondrial DNA control region sequences were determined in 109 unrelated German Caucasoid individuals from north west Germany for both hypervariable regions 1 (HV1) and 2 (HV2) and 100 polymorphic nucleotide positions (nps) were found, 63 in HV1 and 37 in HV2. A total of 100 different mtDNA lineages was revealed, of which 7 were shared by 2 individuals and 1 by 3 individuals. The probability of drawing a HV1 sequence match within the north west Germans or within published sets of south Germans and west Austrians is similar (within a factor of 2) to drawing a sequence match between any two of these three population samples. Furthermore, HV1 sequences of 700 male inhabitants of one village in Lower Saxony were generated and these showed a nearly linear increase of the number of different haplotypes with increasing number of individuals, demonstrating that the commonly used haplotype diversity measure (Nei 1987) for population samples tends to underestimate mtDNA diversity in the actual population.

Extraction, Evaluation, and Amplification of DNA from Decalcified and Undecalcified United States Civil War Bone

Deoxyribonucleic acid (DNA) was extracted from documented skeletal specimens of U.S. Civil War soldiers to determine the need for decalcification prior to extraction. The polymerase chain reaction (PCR) was performed to determine if the calcification state had an effect on the ability to amplify the extracts and to determine how successful amplification would be with these aged specimens. Bone samples were pulverized to a fine powder and divided into two sets. One set of samples was decalcified and the other set left undecalcified. Both sets were extracted using an organic procedure. The results demonstrate that decalcification is not a necessary step in the extraction process and that the yield of DNA is generally two times greater when decalcification is omitted. Furthermore, the calcification state had no effect on the ability to perform the PCR. Although the extracted DNA was very degraded, a 410 base pair (bp) segment of the mitochondrial DNA (mtDNA) control region was amplified. These results suggest that DNA can be extracted and amplified from 125 year old bone without decalcification, which may assist in the identity of modern and historic forensic specimens.

Human hair histogenesis for the mitochondrial DNA forensic scientist.

Analysis of mitochondrial DNA (mtDNA) sequence from human hairs has proven to be a valuable complement to traditional hair comparison microscopy in forensic cases when nuclear DNA typing is not possible. However, while much is known about the specialties of hair biology and mtDNA sequence analysis, there has been little correlation of individual information. Hair microscopy and hair embryogenesis are subjects that are sometimes unfamiliar to the forensic DNA scientist. The continual growth and replacement of human hairs involves complex cellular transformation and regeneration events. In turn, the analysis of mtDNA sequence data can involve complex questions of interpretation (e.g., heteroplasmy and the sequence variation it may cause within an individual, or between related individuals. In this paper we review the details of hair developmental histology, including the migration of mitochondria in the growing hair, and the related interpretation issues regarding the analysis of mtDNA data in hair. Macroscopic and microscopic hair specimen classifications are provided as a possible guide to help forensic scientists better associate mtDNA sequence heteroplasmy data with the physical characteristics of a hair. These same hair specimen classifications may also be useful when evaluating the relative success in sequencing different types and/or forms of human hairs. The ultimate goal of this review is to bring the hair microscopist and forensic DNA scientist closer together, as the use of mtDNA sequence analysis continues to expand.

Second generation sequencing allows for mtDNA mixture deconvolution and high resolution detection of heteroplasmy

Aim To use parallel array pyrosequencing to deconvolute mixtures of mitochondrial DNA (mtDNA) sequence and provide high resolution analysis of mtDNA heteroplasmy. Methods The hypervariable segment 1 (HV1) of the mtDNA control region was analyzed from 30 individuals using the 454 GS Junior instrument. Mock mixtures were used to evaluate the system’s ability to deconvolute mixtures and to reliably detect heteroplasmy, including heteroplasmic differences between 5 family members of the same maternal lineage. Amplicon sequencing was performed on polymerase chain reaction (PCR) products generated with primers that included multiplex identifiers (MID) and adaptors for pyrosequencing. Data analysis was performed using NextGENe® software. The analysis of an autosomal short tandem repeat (STR) locus (D18S51) and a Y-STR locus (DYS389 I/II) was performed simultaneously with a portion of HV1 to illustrate that multiplexing can encompass different markers of forensic interest. Results Mixtures, including heteroplasmic variants, can be detected routinely down to a component ratio of 1:250 (20 minor variant copies with a coverage rate of 5000 sequences) and can be readily detected down to 1:1000 (0.1%) with expanded coverage. Amplicon sequences from D18S51, DYS389 I/II, and the second half of HV1 were successfully partitioned and analyzed. Conclusions The ability to routinely deconvolute mtDNA mixtures down to a level of 1:250 allows for high resolution analysis of mtDNA heteroplasmy, and for differentiation of individuals from the same maternal lineage. The pyrosequencing approach results in poor resolution of homopolymeric sequences, and PCR/sequencing artifacts require a filtering mechanism similar to that for STR stutter and spectral bleed through. In addition, chimeric sequences from jumping PCR must be addressed to make the method operational.

Maternal age effect and severe germ-line bottleneck in the inheritance of human mitochondrial DNA.

Significance The frequency of intraindividual mitochondrial DNA (mtDNA) polymorphisms—heteroplasmies—can change dramatically from mother to child owing to the mitochondrial bottleneck at oogenesis. For deleterious heteroplasmies such a change may transform alleles that are benign at low frequency in a mother into disease-causing alleles when at a high frequency in her child. Our study estimates the mtDNA germ-line bottleneck to be small (30–35) and documents a positive association between the number of child heteroplasmies and maternal age at fertilization, enabling prediction of transmission of disease-causing variants and informing mtDNA evolution. The manifestation of mitochondrial DNA (mtDNA) diseases depends on the frequency of heteroplasmy (the presence of several alleles in an individual), yet its transmission across generations cannot be readily predicted owing to a lack of data on the size of the mtDNA bottleneck during oogenesis. For deleterious heteroplasmies, a severe bottleneck may abruptly transform a benign (low) frequency in a mother into a disease-causing (high) frequency in her child. Here we present a high-resolution study of heteroplasmy transmission conducted on blood and buccal mtDNA of 39 healthy mother–child pairs of European ancestry (a total of 156 samples, each sequenced at ∼20,000× per site). On average, each individual carried one heteroplasmy, and one in eight individuals carried a disease-associated heteroplasmy, with minor allele frequency ≥1%. We observed frequent drastic heteroplasmy frequency shifts between generations and estimated the effective size of the germ-line mtDNA bottleneck at only ∼30–35 (interquartile range from 9 to 141). Accounting for heteroplasmies, we estimated the mtDNA germ-line mutation rate at 1.3 × 10−8 (interquartile range from 4.2 × 10−9 to 4.1 × 10−8) mutations per site per year, an order of magnitude higher than for nuclear DNA. Notably, we found a positive association between the number of heteroplasmies in a child and maternal age at fertilization, likely attributable to oocyte aging. This study also took advantage of droplet digital PCR (ddPCR) to validate heteroplasmies and confirm a de novo mutation. Our results can be used to predict the transmission of disease-causing mtDNA variants and illuminate evolutionary dynamics of the mitochondrial genome.