Heteroplasmy in Hair: Study of Mitochondrial DNA Third Hypervariable Region in Hair and Blood Samples*†

Abstract: Mitochondrial DNA (mtDNA) analysis has proved useful for forensic identification especially in cases where nuclear DNA is not available, such as with hair evidence. Heteroplasmy, the presence of more than one type of mtDNA in one individual, is a common situation often reported in the first and second mtDNA hypervariable regions (HV1/HV2), particularly in hair samples. However, there is no data about heteroplasmy frequency in the third mtDNA hypervariable region (HV3). To investigate possible heteroplasmy hotspots, HV3 from hair and blood samples of 100 individuals were sequenced and compared. No point heteroplasmy was observed, but length heteroplasmy was, both in C‐stretch and CA repeat. To observe which CA “alleles” were present in each tissue, PCR products were cloned and re‐sequenced. However, no variation among CA alleles was observed. Regarding forensic practice, we conclude that point heteroplasmy in HV3 is not as frequent as in the HV1/HV2.     

Characterization of the Caucasian haplogroups present in the SWGDAM forensic mtDNA dataset for 1771 human control region sequences. Scientific Working Group on DNA Analysis Methods

Currently, the Scientific Working Group on DNA Analysis Methods (SWGDAM) mtDNA dataset is used to infer the relative rarity of mtDNA profiles (i.e., haplotypes) obtained from evidence samples and for identification of missing persons. The Caucasian haplogroup patterns in this forensic dataset have been characterized using phylogenetic methods. The assessment reveals that the dataset is relevant and representative of U.S. and European Caucasians. The comparisons carried out were both the observation of variable sites within the control region (CR) and the selection of a subset of these sites, which partition the variation within human mtDNA control region sequences into clusters (i.e., haplogroups). The aligned sequence matrix was analyzed to determine both single nucleotide polymorphisms (SNPs) in a phylogenetic context, as well as to check and standardize haplogroup designations with a focus on determining the characters that define these groups. To evaluate the dataset for forensic utility, the haplogroup identifications and frequencies were compared with those reported from other published studies.     

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.     

Detection and quantification of the age-related point mutation A189G in the human mitochondrial DNA

Mutation analysis in the mitochondrial DNA (mtDNA) control region is widely used in population genetic studies as well as in forensic medicine. Among the difficulties linked to the mtDNA analysis, one can find the detection of heteroplasmy, which can be inherited or somatic. Recently, age-related point mutation A189G was described in mtDNA and shown to accumulate with age in muscles. We carried out the detection of this 189 heteroplasmic point mutation using three technologies: automated DNA sequencing, Southern blot hybridization using a digoxigenin-labeled oligonucleotide probe, and peptide nucleic acid (PNA)/real-time PCR combined method on different biological samples. Our results give additional information on the increase in mutation frequency with age in muscle tissue and revealed that the PNA/real-time PCR is a largely more sensitive method than DNA sequencing for heteroplasmy detection. These investigations could be of interest in the detection and interpretation of mtDNA heteroplasmy in anthropological and forensic studies.     

Polymorphic sites in human mitochondrial DNA control region sequences: population data and maternal inheritance

Sequence analysis of the mitochondrial DNA (mtDNA) control region is of central importance for forensic identity testing as well as for studies of human evolution. Here we report the sequencing data of the hypervariable regions I and II from 50 unrelated individuals from a western German population (Rhine area). In regions I and II, 52 and 26 sites of sequence polymorphism, respectively, were noted. Nucleotide substitution rather than insertion/deletion was the majority of variation. The distribution showed a large bias towards transitional changes than transversional changes. Furthermore we investigated uniparental inheritance in seven CEPH families each family with 7–9 maternal descendants. Most maternal relatives shared identical mtDNA sequences. Additionally sequences were compared for father:child pairs and as expected no evidence for paternal transmission of mtDNA was observed. The high variability of mtDNA control region sequences permits utility in forensic identity investigations. The data also indicate that the neomutation rate seems to be very low from one generation to the other.     

Fluorescent SSCP of overlapping fragments (FSSCP-OF): a highly sensitive method for the screening of mitochondrial DNA variation.

The mtDNA analysis (mtDNA) is increasingly being demanded for forensic purposes due to the fact that many times the use of standard nuclear marker fails to analyze degraded samples (such as bones) and specially for the analysis of hair shafts (a common sample in the crime scene). However, analysis of mtDNA sequencing implies a great lab effort when a high number of samples must be analyzed.The present work introduces a novel and reliable method for the screening of mtDNA variation in the first and second hypervariables (HV1 and HV2) regions which we have denominated fluorescent single strand conformation polymorphism (SSCP) of overlapping fragments (FSSCP-OF). FSSCP-OF is based on the basic theory of SSCP analysis and combines two complementary strategies: the use of PCR amplified overlapping fragments and fluorescent detection technology. The overlap region contains a high percentage (50%) of the d-loop mtDNA variation and for this reason, the probability to detect a polymorphic position by SSCP analysis is clearly increased in comparison to conventional SSCP methods due to the fact that the same polymorphic position is usually placed in a different "relative" position in the two overlapped fragments. The use of multicolor fluorescent technology allows also the multiplex amplification of overlapping fragment and its subsequent analysis in an automatic sequencer. We have analyzed 50 samples of unrelated individuals through the FSSCP-OF technique and we have found that using this methodology the probability to distinguish two samples with different sequences is close to 100%. FSSCP-OF has other important advantages with respect to previous screening methods, such as the automation and standardization of the protocols, which is of special interest for the forensic routine.     

Critique of interpretation of high levels of heteroplasmy in the human mitochondrial DNA hypervariable region I from hair

The phenomenon known as heteroplasmy can be operationally observed in some human mitochondrial DNA (mtDNA) samples. Typically, heteroplasmy manifests itself in an individual presenting two mtDNA species that differ at a single base. Heteroplasmy at two, and even possibly three sites, also may occur, but at very low rates. A recent report (Grzybowski, 2000, see ref. [13]) suggests that much higher levels of mtDNA (point substitution) heteroplasmy can occur in hair. This observation is contrary to the experience of the forensic mtDNA community. There are several explanations for the unusual findings of high levels of heteroplasmy. First, the template quantities of DNA are approximately three orders of magnitude higher than required for mtDNA sequencing, and an excessive number of amplification cycles were used. Thus, the protocol used did not follow routine practices by the forensic community. Second, there are misidentifications and tabular errors that call into question the reliability of the findings. Third, by comparing the natural human mtDNA variation with a reference sample population with that observed in the heteroplasmy in hair study, the data are inconsistent with population genetic expectations. The observation of high levels of heteroplasmy may be due to contamination of the samples and/or possibly the amplification of nuclear pseudogenes. The results observed in the heteroplasmy in hair study do not apply to other methods of mtDNA analysis and cannot be used to question the reliability of the current forensic mtDNA practices.     

Phylogeographic investigations: the role of trees in forensic genetics

The human mitochondrial DNA (mtDNA) genome is commonly analyzed in various disciplines, such as population, medical, and forensic genetics, but conceptual and scientific exchange between them is still limited. Here we review several aspects of the mtDNA phylogeny that are particularly--but not exclusively--of interest to the forensic community. Among the issues that arise, we emphasize the importance of integrating evolutionary concepts into the forensic routine. We also discuss topics such as mtDNA mutation-rate heterogeneity and the weight of evidence, ethnic affiliations of mtDNA profiles, and the abuse of reference databases. Finally, we show the usefulness of coding-region variation in a forensic context.     

Microscopical discrimination of human head hairs sharing a mitochondrial haplogroup

In forensic analyses, determining the level of consensus among examiners for hair comparison conclusions and ancestry identifications is important for assessing the scientific validity of microscopical hair examinations. Here, we present data from an interlaboratory study on the accuracy of microscopical hair comparisons among a subset of experienced hair examiners currently analyzing hair in forensic laboratories across the United States. We examined how well microscopical analysis of hair can reliably be used to differentiate hair samples, many of which were macroscopically similar. Using cut hair samples, many sharing similar macroscopic and microscopic features, collected from individuals who share the same mitochondrial haplogroup as an indication of genetic relatedness, we tested multiple aspects that could impact hair comparisons. This research tested the extent to which morphological features related to ancestry and hair length influence conclusions. Microscopical hair examinations yielded accurate assessments of inclusion/exclusion relative to the reference samples among 85% of the pairwise comparisons. We found shorter hairs had reduced levels of accuracy and hairs from populations examiners were not familiar with may have impacted their ability to resolve features. The reliability of ancestry determinations is not yet clear, but we found indications that the existing categories are only somewhat related to current ethnic and genetic variation. Our results provide support for the continued utility of microscopical comparison of hairs within forensic laboratories and to advocate for a combined analytical approach using both microscopical analysis and mtDNA data on all forensic analyses of hair.     

Mitochondrial DNA sequence alterations observed between blood and buccal cells within the same individuals having betel quid (BQ)-chewing habit

There are hundreds of millions of betel quid (BQ) lovers widely spreading around the world. Compositions in BQ may generate reactive oxygen species, which would induce DNA damage. However, oral epithelial cells as well as blood have often been used as reference samples in comparison with the mitochondrial DNA (mtDNA) sequence of hairs. The main purpose of this study was to investigate the extent of mtDNA sequence variation in regular BQ-chewers' oral epithelial cells, and thus to evaluate the forensic availability of the buccal cells from BQ-chewers using the mtDNA markers. The hypervariable segments I and II in the D-loop control region of mtDNA between paired samples of blood and buccal scrape cells from 75 non-BQ-chewers (to be a control group), 60 BQ-chewers, and 67 oral cancerous patients were DNA sequenced and compared. Among the three groups, the alteration rates of 1.3% (1 out of 75), 10% (6 out of 60), and 61% (41 out of 67) were identified from the control, BQ-chewers, and the cancerous group, respectively. In the cancerous group, as expected, high rate of DNA alteration between blood and buccal samples was found. In the BQ-chewers, one and five individuals had the length and point alterations, respectively. Interestingly, most of point alteration sites, e.g., mtDNA positions 153, 16189, 16093 identified from BQ-chewers, were also observed in previous literatures. As for the control subjects, one case with point alteration, and none with length alteration, was identified. For all the three groups, not only the oral cells but also the normal blood samples exhibited high frequency (>55%) of length heteroplasmy at poly-(C)n track. Statistical analyses revealed that significance was observed between the severity of mtDNA alteration in BQ-chewers' oral epithelial cells and the history of BQ-chewing (p = 0.02), with a tendency of positive association. Based on the guidelines by Carracedo et al., we suggest that the interpretation of mtDNA variations between criminal evidences and the oral epithelial cells (as a reference or known sample) from BQ-chewers should be performed with particular caution using the PCR-based mtDNA sequencing. Our findings would be valuable in mtDNA analysis of hair evidence, especially for those countries where the habit of BQ-chewing is popular.     

Variability of the Entire Mitochondrial DNA Control Region in a Human Isolate from the Pas Valley (Northern Spain)

Abstract: In this study, we analyzed the entire mtDNA control region in 61 unrelated individuals from the Pas Valley (Cantabria), a human isolate from northern Spain, to evaluate the suitability of this analysis to increase the power of discrimination of this locus for forensic purposes in human isolates. Low values obtained for the diversity parameters confirmed the relative isolation of this human group. The main findings of this study indicated that even the analysis of the entire mtDNA control region may have important limitations for use in forensic casework when dealing with human isolates: none of the 44 individuals who exhibited identical HVI‐HVII haplotypes could be further differentiated by analysis of segment HVIII. Nevertheless, analysis of the entire mtDNA control region proved to be useful to determine the ancestry of the samples examined, by contributing to the confirmation, and, on occasion, even to the refinement of the haplogroup assignment.     

Forensic genetic analysis of mitochondrial DNA hypervariable region I/II sequences: an expanded Korean population database

We have analyzed variation of the mitochondrial DNA (mtDNA) hypervariable segments I and II (HVS-I and HVS-II) in 185 randomly chosen individuals from Korea to provide an expanded and reliable Korean database. Combined sequence comparison of HVS-I and HVS-II led to the identification of 167 different haplotypes characterized by 154 variable sites. One hundred and fifty-one of the haplotypes were individual-specific, 14 were found in two individuals and 2 were found in three individuals. A pairwise comparison of the 185 HVS-I/II sequences found an average of 10.11 +/- 4.63 differences between individuals. The random match probability and gene diversity for the combined hypervariable regions were estimated at 0.66% and 0.9988, respectively. Analyzing the expanded database including three previously reported data sets and the present data using haplogroup-based comparisons and comparison with closely related sequences allowed errors to be detected and eliminated, thus considerably improving data quality. Sample division comparisons based on PhiST genetic distance measures revealed no significant population differentiation in the distribution of mtDNA sequence variations between the present data set and a database in The Scientific Working Group on DNA Analysis Methods (SWGDAM), but did indicate differences from other sets of data. Based on the results of mtDNA profiles, almost all of the mtDNA types studied here could be classified into subsets of haplogroups common in east Asia, and show that the Koreans possess lineages from both the southern and the northern haplogroup complexes of east Asian populations. The new data, combined with other mtDNA sequences, demonstrate how useful comparison with closely related mtDNA sequences can be for improving database quality, as well as providing haplotype information for forensic and population genetic analyses in the Korean population.     

Heteroplasmy in hair: differences among hair and blood from the same individuals are still a matter of debate

The analysis of mitochondrial DNA (mtDNA) is a useful tool in forensic cases when sample contents too little or degraded nuclear DNA to genotype by autosomal short tandem repeat (STR) loci, but it is especially useful when the only forensic evidence is a hair shaft. Several authors have related differences in mtDNA from different tissues within the same individual, with high frequency of heteroplasmic variants in hair, as also in some other tissues. Is still a matter of debate how the differences influence the interpretation forensic protocols. One difference between two samples supposed to be originated from the same individual are related to an inconclusive result, but depending on the tissue and the position of the difference it should have a different interpretation, based on mutation-rate heterogeneity of mtDNA. In order to investigate it differences in the mtDNA control region from hair shafts and blood in our population, sequences from the hypervariable regions 1 and 2 (HV1 and HV2) from 100 Brazilian unrelated individuals were compared. The frequency of point heteroplasmy observed in hair was 10.5% by sequencing. Our study confirms the results related by other authors that concluded that small differences within tissues should be interpreted with caution especially when analyzing hair samples.     

A DNA microarray system for forensic SNP analysis

Forensic DNA analysis is routinely performed using polymorphic short tandem repeat (STR) markers. However, for degraded or minute DNA samples, analysis of autosomal single nucleotide polymorphisms (SNPs) in short fragments might be more successful. Furthermore, sequencing of mitochondrial DNA (mtDNA) is often performed on highly degraded or scarce samples due to the high copy number of mtDNA in each cell. Due to the increasing number of complete mtDNA genome sequences available, the limited discrimination power of an mtDNA analysis, may be increased by analysis of coding region polymorphisms in addition to the non-coding variation. Since sequence analysis of the coding region would require more material than generally present in forensic samples, an alternative SNP analysis approach is required. We have developed a one-colour microarray-based SNP detection system for limited forensic materials. The method is based on minisequencing in solution prior to hybridisation to universal tag-arrays. In a first outline of a forensic chip, a combination of 12 nuclear and 21 mitochondrial SNP markers are analysed simultaneously. The mitochondrial markers on the chip are polymorphisms within the hypervariable region as well as in the coding region. Even though the number of markers in the current system is limited, it can easily be extended to yield a greater power of discrimination. When fully developed, microarray analysis provides a promising system for efficient sensitive SNP analysis of forensic samples in the future.     

Forensic Analysis of mtDNA Haplotypes from Two Rural Communities in Haiti Reflects Their Population History

Abstract: Very little genetic data exist on Haitians, an estimated 1.2 million of whom, not including illegal immigrants, reside in the United States. The absence of genetic data on a population of this size reduces the discriminatory power of criminal and missing‐person DNA databases in the United States and Caribbean. We present a forensic population study that provides the first genetic data set for Haiti. This study uses hypervariable segment one (HVS‐1) mitochondrial DNA (mtDNA) nucleotide sequences from 291 subjects primarily from rural areas of northern and southern Haiti, where admixture would be minimal. Our results showed that the African maternal genetic component of Haitians had slightly higher West‐Central African admixture than African‐Americans and Dominicans, but considerably less than Afro‐Brazilians. These results lay the foundation for further forensic genetics studies in the Haitian population and serve as a model for forensic mtDNA identification of individuals in other isolated or rural communities.     

Heteroplasmies detected in an amplified mitochondrial DNA control region from a small amount of template

When mitochondrial DNA (mtDNA) heteroplasmies are detected, they often confound forensic identification, especially if they are the result of poor biological sampling. In this study, we determined the ratio of heteroplasmy in samples that were amplified from a very small amount of template mtDNA or a few cells using a highly sensitive nested polymerase chain reaction (PCR) procedure and a direct sequencing analysis. As a result, more than half of the detected sequences (i.e., 17/20, 15/20, and 14/20) showed homoplasmy derived from a variation in the heteroplasmy proportion when only 10 copies of template mtDNA samples were amplified and analyzed. Additionally, with products amplified from one or several white blood cells (WBCs), several previously undetected heteroplasmies were detected. These results indicate the risks associated with using highly sensitive mtDNA techniques in forensic investigations because of the variable proportions of heteroplasmy or nucleotide substitutions that can possibly be detected from a very small biological sample.     

Human and insect mitochondrial DNA analysis from maggots

During the course of our forensic investigations, we have encountered situations where it would have been useful to have evidence, other than direct contact between the two, for concluding that a carrion-fly maggot developed on a particular human victim. If a maggot collected during a death investigation did not develop on the victim, then its age is not relevant to estimating the postmortem interval. In this study we demonstrate that mitochondrial DNA (mtDNA) sequence data can be obtained from the dissected gut of a maggot that had fed on human tissue. These data can be used to identify both the human corpse upon which the maggot had been feeding and the species of the maggot itself.     

基于Ion Torrent PGM~(TM)测序系统的人mtDNA全测序分析

目的应用Ion Torrent PGM~(TM)测序系统对人线粒体DNA(mitochondria DNA,mtDNA)全序列进行分析检测,研究不同组织间mt DNA序列差异情况。方法通过法医尸体检验采集6名无关个体的组织样本,包括胸腔血液、头发、肋软骨、指甲、骨骼肌和口腔上皮。使用4对引物对线粒体全序列进行扩增,应用Ion Shear~(TM)Plus Reagents试剂盒和Ion Plus Fragment Library试剂盒等构建文库,并在Ion Torrent PGM~(TM)测序系统上进行线粒体基因组全序列测序,并针对异质性位点和在HVⅠ区域突变位点,进行Sanger测序验证。结果所有样本的全基因组mtDNA都扩增成功,6名无关个体分属于6种不同的单倍型,同一个体不同组织之间mtDNA存在异质性差异。异质性位点和HVⅠ区域突变位点采用Sanger测序结果均得到验证。通过Kappa统计方法进行一致性检验后发现,相同个体不同组织的mtDNA序列检验结果仍具有较好的一致性。结论本研究所采用的人线粒体基因组全序列的测序检验方法,可以检测出同一个体不同组织间mtDNA的异质性差异,该差异具有较高的一致性,该结果对mtDNA在法庭科学中的应用具有指导作用。