Forensic casework analysis using the HVI/HVII mtDNA linear array assay

The mitochondrial hypervariable regions I and II have proven to be a useful target for analysis of forensic materials, in which the amount of DNA is limited or highly degraded. Conventional mitochondrial DNA (mtDNA) sequencing can be time-consuming and expensive, limitations that can be minimized using a faster and less expensive typing assay. We have evaluated the exclusion capacity of the linear array mtDNA HVI/HVII region-sequence typing assay (Roche Applied Science) in 16 forensic cases comprising 90 samples. Using the HVI/HVII mtDNA linear array, 56% of the samples were excluded and thus less than half of the samples require further sequencing due to a match or inconclusive results. Of all the samples that were excluded by sequence analysis, 79% could be excluded using the HVI/HVII linear array alone. Using the HVI/HVII mtDNA linear array assay, we demonstrate the potential to decrease sequencing efforts substantially and thereby reduce the cost and the turn-around time in casework analysis.     

Typing the 1.1 kb control region of human mitochondrial DNA in Japanese individuals

This study presents a reliable method that uses high-fidelity long-range PCR and optimized primers to assess polymorphism and to genotype human mitochondrial DNA (mtDNA). This method was used to analyze polymorphic sites in the human mtDNA control region, including hypervariable regions I, II, and III (HVI, HVII, and HVIII), from 124 unrelated Japanese individuals. In HVI, HVII, and HVIII, 80, 37, and 14 polymorphic sites were identified, respectively, excluding those in the homopolymeric cytosine stretch (C-stretch) regions. The region between HVI and HVII also contained 15 polymorphic sites. On the other hand, C-stretch length heteroplasmy in HVI or HVII was observed in 66 of 124 Japanese individuals (53%), which is much higher than in Caucasian populations. The variants in the C-stretch regions were characterized by counting the number of heteroplasmic peaks split from the single peak in homoplasmic sequences (i.e., 16244G and 16255G in HVI and 285G in HVII). Including the C-stretch length heteroplasmy, the 124 Japanese mtDNA samples were classified into 116 distinct haplotypes. The random match probability and the genetic diversity were estimated to be 0.95% and 0.998581, respectively, indicating that the method presented here has higher discrimination than the conventional method for mtDNA typing using HVI and HVII. [Correction added after publication 30 January 2007: in the preceding sentence random match probability and genetic diversity estimates were corrected from 0.95 and 0.998581%, respectively, to 0.95% and 0.998581, respectively.] The haplogroups and their frequencies observed in this study (i.e., D4; 13.7%, M7a1; 11.3%, D4a; 9.7% and M7b2; 8.9%) were similar to those observed in other studies of Japanese mtDNA polymorphism. The method described here is suitable for forensic applications, as shown by successful analysis of tissues from highly putrefied remains of an infant, which allowed maternal relationship to be determined via mtDNA haplotyping.     

High‐Resolution Melting (HRM) of Hypervariable Mitochondrial DNA Regions for Forensic Science

Forensic strategies commonly are proceeding by analysis of short tandem repeats (STRs); however, new additional strategies have been proposed for forensic science. Thus, this article standardized the high‐resolution melting (HRM) of DNA for forensic analyzes. For HRM, mitochondrial DNA (mtDNA) from eight individuals were extracted from mucosa swabs by DNAzol reagent, samples were amplified by PCR and submitted to HRM analysis to identify differences in hypervariable (HV) regions I and II. To confirm HRM, all PCR products were DNA sequencing. The data suggest that is possible discriminate DNA from different samples by HRM curves. Also, uncommon dual‐dissociation was identified in a single PCR product, increasing HRM analyzes by evaluation of melting peaks. Thus, HRM is accurate and useful to screening small differences in HVI and HVII regions from mtDNA and increase the efficiency of laboratory routines based on forensic genetics.     

Optimization of a duplex amplification and sequencing strategy for the HVI/HVII regions of human mitochondrial DNA for forensic casework

A duplex primer set for the amplification of mitochondrial DNA HVI and HVII control regions was evaluated for the optimization of a DNA sequencing protocol suitable for forensic casework. HVI and HVII products, with the absence of non-specific products, could be detected by agarose gel electrophoresis when as little as 0.5 and 0.1pg of DNA were amplified for 34 and 38 cycles, respectively. Because HVI and HVII amplicons are co-synthesized in the duplex PCR, fewer steps are required (lessening the risk of cross contamination events) and more frugal use of precious extracted DNA samples is possible, both desirable features for forensic casework. The ABI Prism BigDyetrade mark version 1.1 chemistry provided high quality sequencing data, with little or no background noise and uniform peak heights, outcomes that favored reliable detection of heteroplasmy, particularly at early sequence reads (<40 bases). Optimal compromise between sensitivity and sequence accuracy in the absence of noise was achieved starting at 150 mitochondrial genome copies. The protocol is effective (no sequence errors) with highly degraded DNA (average detectable template size of 200bp). Dual artificial template mixtures with the minor component at 15% suggests that heteroplasmy should be detected at this level with confidence.     

Different informativeness of the three hypervariable mitochondrial DNA regions in the population of Bologna (Italy)

Mitochondrial DNA (mtDNA) sequence variations at hypervariable regions HVI, HVII and HVIII were analysed in 100 unrelated Italians from Bologna. The values of the statistical parameters are in agreement with the range of European populations. We suggest that the less informative HVIII region may be useful to distinguish HVI-HVII identical sequences in forensic analysis especially when nuclear DNA cannot be investigated.     

SSCP法mtDNA分型的法医学应用

目的探讨SSCP法mtDNA分型在法医鉴定实践中的意义。方法PCR扩增mtDNAHV-I和HV-II的序列多态性片段,直接用SSCP技术分型。对70个湖北汉族真三联家系和140例随机个体进行检测,比较家系中mtDNA的单倍型SSCP图谱。统计分析SSCP法用于两个高变区在母系确定、个人识别中的意义和鉴别能力。结果在70个家系中,母亲和孩子的HV-I、HV-IISSCP带型完全相同;家系中父亲与母子的HV-I图谱不同占98.57%;HV-IISSCP图谱不同占97.13%。140例随机个体的HVI、HVII区分别检出21、16种单倍型,GD值分别为0.9556、0.9356。结论mtDNA-SSCP分型在嫌疑人筛查和母系亲缘关系推断中有实际应用价值。     

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.     

Optimization of Human mtDNA Control Region Sequencing for Forensic Applications

Sequencing mitochondrial DNA hypervariable regions I and II (HVI and HVII) is useful in forensic missing person and unidentified remains cases. Improvements in ease and sensitivity of testing will yield results from more samples in a timely fashion. Routinely, amplification of HVI and HVII is followed by Sanger sequencing using the BigDye^® Terminator v3.1 Cycle Sequencing kit (Applied Biosystems) using 4 μL of ready reaction mix (RRM). Each sequencing reaction is then purified through column filtration before capillary electrophoresis. Using lower amounts of RRM (2 μL or 1 μL) and purification using BigDye^® XTerminator^? (Applied Biosystems) instead of columns showed no loss of sequence length and increased the quality and the sensitivity of testing, allowing HVI and HVII typing from mitochondrial genome equivalent to 125 fg of nuclear DNA, or 100 pg of HVI/HVII amplicons. Using this methodology, testing can be completed in 1 day, and the cost of testing is reduced.     

Recommendations for consistent treatment of length variants in the human mitochondrial DNA control region

Human mitochondrial DNA (mtDNA) analysis is a valuable forensic tool, useful in cases where the amount of extracted DNA is low or highly degraded. Population databases are used to determine the relative rarity of a particular profile obtained in a forensic case. Rather than full DNA sequence information, sequence profiles are compared to a reference sequence, and the differences from the reference are recorded in forensic databases. A standard method is proposed for characterizing length variants, and examples are described using actual human control region mtDNA profiles. Consistency in alignment and nomenclature avoids inadvertently describing two sequences as different when in fact they are the same.     

Development of Multiple Assays using 46 SNPs for Comprehensive mtDNA Haplogrouping and Application to Highly Degraded DNA

Six multiplex PCR systems using single‐base extension reactions to analyze 46 mitochondrial DNA (mtDNA)‐coding region single nucleotide polymorphisms (SNPs) that define 42 haplogroups, that is, 24 major mtDNA haplogroups and 18 subclades, were devised. To improve the usefulness of the established systems for the analysis of degraded DNA samples, novel primers to render amplicons with sizes <150 bp were designed. By applying these systems to 214 Japanese individuals, 24 different haplogroups (power of discrimination = 93.4%) were found. To assess the effectiveness of our systems in grouping degraded DNA, an ancient bone sample of a Jomon skeleton was analyzed and then classified as haplogroup N9b. We conclude that the present systems are powerful screening tools for major haplogroups of mtDNA in addition to the prevalent subhaplogroups in the Japanese population and that these systems are capable of analyzing highly degraded DNA samples in forensic studies.     

Detection of sequence variation in the HVII region of the human mitochondrial genome in 689 individuals using immobilized sequence-specific oligonucleotide probes

We have developed a rapid, immobilized probe-based assay for the detection of sequence variation in the hypervariable segment II (HVII) of the mitochondrial DNA (mtDNA) control region. Using a panel of 17 sequence-specific oligonucleotide (SSO) probes immobilized on nylon membrane strips, we typed 689 individuals from four population groups. The genetic diversity value for each population was calculated from the frequency data, and the frequencies of distinct "mitotypes" in each group were determined. We performed DNA sequence analysis of 129 samples to characterize the sequences associated with "blanks" (absence of probe signals) and weak probe signals. Out of 689 samples, we observed five heteroplasmic samples (excluding the variable C-stretch beginning at position 303) using the immobilized SSO probe panel. The SSO probe strips were used for the analysis of shed hairs and bloodstains from several criminal cases in Sweden, one of which is described here. We conclude that this mtDNA typing system is useful for human identification and significantly decreases casework turnaround time.     

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.     

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 C's. 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.     

粪便DNA提取及检验

目的　研究人类粪便DNA的提取和检验方法。方法　 8人份粪便样本 ,磁珠法提取DNA后 ,进行STR复合扩增和mtDNAHVI区测序分析。结果　用 2种方法提取的粪便DNA ,STR复合扩增检验均未获成功 ;方法1提取的粪便DNA有 6个样本、方法 2有 7个样本获得了清晰可读的mtDNAHVI区序列 ,并与唾液对照样本DNA的序列完全一致。结论　用本文建立的方法提取粪便DNA ,不适于STR分析 ,可通过mtDNA测序分析进行检验。     

Characterization of Mitochondrial DNA Sequence Heteroplasmy in Blood Tissue and Hair as a Function of Hair Morphology*,†,‡

Abstract: This study characterizes mitochondrial DNA (mtDNA) sequence heteroplasmy in blood tissue and hair as a function of hair morphology. Bloodstains (127 individuals) and head hairs (128 individuals) were typed using the mtDNA LINEAR ARRAY? assay. A total of 1589 hairs were interpreted: 1478 (93%) were homoplasmic and 111 (7%) exhibited heteroplasmy at one or more positions. Seventy‐one percent (82/116) of individuals were homoplasmic, whereas 29% (34/116) exhibited heteroplasmy in at least one hair. The results demonstrate intra‐ and inter‐tissue differences in heteroplasmy within individuals. Sequence heteroplasmy among hairs from each individual varied from 0 to 90%; the frequency does not differ significantly with population group, cosmetic treatment, age, gender, medulla morphology, region of the scalp, hair growth phase, or, when comparing living and deceased donors. However, the results support a correlation between heteroplasmy and hair pigmentation; typically, lighter‐pigmented hairs exhibit a higher incidence of sequence heteroplasmy compared to darker hairs.     

Improved MtDNA sequence analysis of forensic remains using a "mini-primer set" amplification strategy

Mitochondrial DNA (mtDNA) analysis of highly degraded skeletal remains is often used for forensic identification due largely to the high genome copy number per cell. Literature from the "ancient DNA" field has shown that highly degraded samples contain populations of intact DNA molecules that are severely restricted in size (1-4). Hand et al. have demonstrated the targeting and preferential amplification of authentic human DNA sequences with small amplicon products of 150 bp or less (1,2). Given this understanding of ancient DNA preservation and amplification, we report an improved approach to forensic mtDNA analysis of hypervariable regions 1 and 2 (HV1/HV2) in highly degraded specimens. This "mini-primer set" (MPS) amplification strategy consists of four overlapping products that span each of the HV regions and range from 126 to 170 bp, with an average size of 141 bp. For this study, 11 extracts representing a range of sample quality were prepared from nonprobative forensic specimens. We demonstrate a significant increase in MPS amplification success when compared to testing methods using approximately 250 bp amplicons. Further, 16 of 17 independent amplifications previously "unreported" due to mixed sequences provided potentially reportable sequence data from a single, authentic template with MPS testing.     

A rape case solved by mitochondrial DNA mixture analysis

In cases of stains that contain mixed DNA from different contributors, analyzing mitochondrial DNA (mtDNA) requires the use of cloning techniques. We developed an efficient cloning technique that was applied in a rape case. After a differential lysis-based DNA extraction from vaginal swabs, hypervariable region I and II (HVI, HVII) amplicons obtained from the male fraction were cloned. Although we mainly found the victim's haplotype, we were able to detect the suspect's haplotype in two clones for HVI and in one clone for HVII. As the midpiece of the flagellum, which contains mitochondria, can be lost during the differential lysis, we also investigated the female fraction by cloning to evaluate the proportion of victim/suspect mtDNA. Unfortunately, only clones presenting the victim's haplotype were found. This case highlights the need for an optimal differential lysis protocol to enrich the male fraction not only with nuclear but also mitochondrial DNA.     

The Use of Mitochondrial DNA Single Nucleotide Polymorphisms to Assist in the Resolution of Three Challenging Forensic Cases

Abstract:  Mitochondrial DNA (mtDNA) single nucleotide polymorphisms (SNPs) in an 11-plex assay were typed in three missing person cases involving highly degraded human remains. Unlike the traditional forensic approach to analyzing mtDNA which focuses on sequencing portions of the noncoding Control Region, this assay targets discriminatory SNPs that reside principally in the coding region. In two of the cases, the SNP typing successfully excluded one of two reference families that could not be excluded on the basis of mtDNA hypervariable region sequencing alone, and resulted in the final resolution of both decades-old cases. In a third case, SNP typing confirmed the sorting and reassociation of multiple commingled skeletal elements. The application of a specific mtDNA SNP assay in these cases demonstrates its utility in distinguishing samples when the most common Caucasian hypervariable region type is encountered in forensic casework.     

Mitochondrial DNA amplification success rate as a function of hair morphology

This study examines the amplification success rate of mitochondrial DNA from human head hair with respect to their potential for forensic application. Mitochondrial DNA was isolated using a Chelex-based extraction method and amplified using the LINEAR ARRAY duplex PCR system. The particular focus of this study was to characterize the morphological features of human head hair in order to further the understanding of the factors that influence amplification success rate in hair tissue using the LINEAR ARRAY duplex PCR system. 2554 head hairs from 132 individuals representing four population groups were amplified. The hair samples were characterized as follows: 1251 were identified microscopically as telogen hairs and 1303 were classified as hairs without roots (removed before extraction). Amplification success was assessed as a function of several independent variables: morphological characteristics; telogen root versus no root; donor age; scalp origin; use of cosmetic hair treatments; and race of the donor. The results show that a positive correlation exists between amplification success and the presence of a telogen root. Combining the amplification success with either the original or optimized protocol, telogen hairs result in an overall success rate of 77.5% compared with 65% for hairs with no roots. Controlling for telogen hairs, the findings indicate that the overall success rate is independent of cosmetic hair treatments; medulla structure; shaft length, diameter, and volume; and scalp origin. Conversely, the age of the donor, the race of the donor, and hair pigmentation all contribute to a variation in amplification success rate.     

MtSNP typing before mtDNA sequencing: Why do it?

Analysis of control mitochondrial DNA (mtDNA) hypervariable regions is sometimes the only available method to study hair evidence in forensic casework although being a laborious technique. Nowadays there is a huge interest in new genetic markers such as single nucleotide polymorphisms (SNPs) to type degraded forensic samples. For that purpose, a 10-Plex mitochondrial SNP for haplogroup typing, chosen from several SNP studies and useful to study the most common populations in our laboratory was applied in forensic casework. Hair shafts from three forensic cases with different ethnic backgrounds were studied with mtDNA sequencing and compared with mitochondrial SNPs (mtSNPs) study. Coding mtSNP typing prior to sequencing can allow for a rapid screening in forensic casework, which is emphasized in the first two cases. Moreover, in cases in which mtDNA sequencing fails, mtSNPs can still be detected. This 10 SNP loci multiplex provides a less expensive and simpler method for mitochondrial typing compared to control region mtDNA sequencing, especially when used as a fast screening method.