Genetic landscape of the mitochondrial DNA control region in South African populations

A high-quality dataset of the mitochondrial DNA control region sequences was generated with Sanger and next-generation sequencing. The study was aimed at characterizing the maternal genetic ancestry and analyzing the haplogroup distribution of 246 individuals residing in South Africa. The study presents mtDNA sequences in South Africa for forensic applications.     

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.     

Mitochondrial DNA typing screens with control region and coding region SNPs

Mitochondrial DNA (mtDNA) analysis has found an important niche in forensic DNA typing. It is used with highly degraded samples or low-copy number materials such as might be found from shed hair or bones exposed to severe environmental conditions. The primary advantage of mtDNA is that it is present in high copy number within cells and therefore more likely to be recovered from highly degraded specimens. A major disadvantage to traditional forensic mtDNA analysis is that it is time-consuming and labor-intensive to generate and review the 610 nucleotides of sequence information commonly targeted in hypervariable regions I and II (HVI and HVII) of the control region. In addition, common haplotypes exist in HVI/HVII mtDNA sequences that can reduce the ability to differentiate two unrelated samples. In this report we describe the utility of two newly available screening assays for rapid exclusion of non-matching samples. The LINEAR ARRAY mtDNA HVI/HVII Region-Sequencing Typing Kit (Roche Applied Science, Indianapolis, IN) was used to type 666 individuals from U.S. Caucasian, African American, and Hispanic groups. Processing of the LINEAR ARRAY probe panels "mito strips" was automated on a ProfiBlot workstation. Observable variation in 666 individuals is reported and frequencies of the mitotypes within and between populations are presented. Samples exhibiting the most common Caucasian mitotype were subdivided with a multiplexed amplification and detection assay using eleven single nucleotide polymorphisms in the mitochondrial genome. These types of screening assays should enable more rapid evaluation of forensic casework samples such that only samples not excluded would be subjected to further characterization through full HVI/HVII mtDNA sequence analysis.     

脱落毛发线粒体DNA HV1区序列测定的研究

目的　对脱落毛发线粒体DNAHV1区序列测定方法进行研究。方法　嵌合扩增结合末端荧光标记DNA测序。结果　对 2 0例脱落毛发进行分析获得了明确的测序结果 ,与来自同一个体的血液所测得的DNA序列进行比较 ,完全相同。结论　嵌合扩增在对脱落毛发进行线粒体DNA多变区序列分析中是一种有效的方法 ,在法医DNA检验中具有实用价值。     

Individual identification of cats and dogs using mitochondrial DNA tandem repeats?

Cats and dogs are very common animals in the human environment. In Switzerland, one in five households owns a cat or a dog. Their hairs are very easily transferred and could be used as a frequent trace evidence. Using DNA analysis, identification of these animals should be possible as it is in human identification. However, most of the time, no nuclear DNA can be recovered from the hair. It is therefore necessary to rely on mtDNA. Cats and dogs have tandemly repeated sequences in their mtDNA control region. In this study, the authors show that these tandem repeats are very polymorphic but are also the source of a very high level of heteroplasmy. The authors, therefore, examined if this might prevent their use in forensic identification.     

Mitochondrial DNA control region variation in a population sample from Hong Kong, China

Entire mitochondrial control region sequences were generated from 377 unrelated individuals from urban Hong Kong. In line with other control region datasets from China, the sample from Hong Kong exhibited significant genetic diversity that was reflected in a random match probability of 0.19% and a mean pairwise difference of 13.14. A total of 305 haplotypes were identified, of which 262 were unique. These sequences will be made publicly available to serve as forensic mtDNA reference data for China.     

Mitochondrial DNA sequencing of human hair shafts stored for long time

Mitochondrial DNA (mtDNA) sequencing is commonly used for forensic genetic identification of relation and personality identification based on analysis of tooth and skeletal rudiments. We demonstrated the possibility of DNA extraction and subsequent enzymatic amplification of fragments of a hypervariable segment I of mtDNA control region from hair shafts after long storage (up to 75 years). Shed hairs are the most common biological material evidence in forensic investigations. Low content of DNA and its possible degradation in hair shafts without bulbs may cause artifacts in polymerase chain reaction. However comparative analysis of amplified nucleotide sequences of amplified fragments from hair stored for 75 years was identical to the sequence from hairs cut immediately before experiment. This indicates high quality of the resultant matrices, stability of results, and hence, the possibility of using DNA extracted from hair shafts without bulbs stored for a long time for expert genetic analysis. Theoretical and methodological prospects of using mtDNA polymorphism analysis for forensic expert evaluations are discussed.     

Mitochondrial DNA control region polymorphism in the population of Alagoas state, north-eastern Brazil

The sequences of the two hypervariable (HV) segments of the mitochondrial DNA (mtDNA) control region were determined in 167 randomly selected, unrelated individuals living in the state of Alagoas, north-eastern Brazil. One hundred and forty-five different haplotypes, associated with 139 variable positions, were determined. More than 95% of the mtDNA sequences could be allocated to specific mtDNA haplogroups according to the mutational motifs. Length heteroplasmy in the C-stretch HV1 and HV2 regions was observed in 22 and 11%, respectively, of the population sample. The genetic diversity was estimated to be 0.9975 and the probability of two random individuals presenting identical mtDNA haplotypes was 0.0084. The most frequent haplotype was shared by six individuals. All sequences showed high-quality values and phantom mutations were not detected. The diversity revealed in the mitochondrial control region indicates the importance of this locus for forensic casework and population studies within Alagoas, Brazil.     

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.     

A novel method for forensic DNA investigations: repeat-type sequence analysis of tandemly repeated mtDNA in domestic dogs

A highly variable and heteroplasmic tandem repeat region situated in the mitochondrial mt DNA control region (CR) in domestic dogs and wolves was studied to evaluate its suitability as a forensic genetic marker for analysis of single hairs. The tandem repeat array is composed of three 10-bp repeat types that are distributed so that a secondary DNA sequence is formed. Thus, the region presents two levels of variation: variation in the number of repeats and variation in the secondary DNA sequence of repeat types. Two analysis methods were therefore tested; fragment length analysis and analysis of the sequence of repeat types. Fragment analysis produced unique profiles that could be used to discriminate between blood samples from maternally closely related individuals. However, different hairs from one individual did not have the same fragment profile, and the method is, therefore, not suitable for analysis of single hairs. In contrast, analysis of the repeat type sequences (array types) is highly informative. When different hairs from one individual were studied, identical array types were found. The repeat-type sequence variation was studied among individuals having identical nonrepetitive CR mtDNA sequence variants. Seven, six, and two individuals, representing three different sequence variants, respectively, were analyzed. All these individuals had different array types, which implies a very high genetic variation between individuals in this region. The analysis method considerably improves the exclusion capacity of mtDNA analysis of domestic dogs compared with sequence analysis of non-repetitive DNA.     

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.     

中国汉族人mtDNA控制区异质性遗传规律

目的探讨中国汉族人mtDNA控制区异质性分布情况和遗传规律。方法将人mtDNA控制区扩增成6个部分互相重叠的片段,利用已建立的DHPLC技术分析其异质性规律。结果对150例汉族无关个体的多种组织检测,发现异质性个体的发生率达34%(51/150);个体的组织mtDNA异质性检出率最高为脑(50/150)、心肌(48/150)、最低为骨骼(22/150);本组共发现mtDNA控制区异质性位点有36个;同一个体可有多个异质性位点,最多的不超过3个;未发现异质性发生率有性别差异;超过41岁的高年龄组的异质性发生率(27/59)高于低年龄组(24/91);同一个体在2年前后取的血样,异质性检测结果一致;同一母系不同成员的异质性位点相同,但异质性mtDNA的含量有差异。结论DHPLC检测mtDNA控制区异质性具有高分辩力;mtDNA控制区异质性在中国汉族人中广泛存在;上述结果可作为mtDNA控制区多态性作个人认定和亲权鉴定的指导性资料。     

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.     

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.     

人类mtDNA控制区异质性

目的观察mtDNA的点突变异质性和长度异质性。方法运用直接测序法对50名无关个体及16名母系家族成员的血液、口腔上皮细胞、头发的mtDNAHVI、HVII区序列进行分析,并对20例HVI区直接测序失败的无关个体进行克隆后测序分析。结果同一个体的三种检材样本及16名母系家族成员的序列一致,未见异质性存在;同一个体的不同克隆的C延伸区的长度有差异,存在长度异质性。但同一个体的血液和头发具有相似的长度变异类型,即长度异质性在组织间无差异。结论mtDNA碱基序列具有同质性及稳定性,适用于法医学检案。     

中国汉族人群的线粒体DNA控制区多态性研究

探讨mtDNA多态性在法庭科学中个体识别的理论基础。应用PCR扩增产物直接测序方法 ,对 111名中国北方地区汉族人群无血缘关系个体的mtDNA控制区 (HVⅠ和HVⅡ )进行测序分析。在高变区Ⅰ 15 998～ 16 40 0之间发现 10 2处碱基变异 ,10 3个mtDNA单倍型 ;在高变区Ⅱ 0 0 0 35～ 0 0 36 9之间的发现 36处碱基变异 ,6 9个mtDNA单倍型。其可变碱基的变异形式主要为碱基替代 (转换和颠换 )、插入和缺失 ;碱基转换 (78 9% )明显高于颠换(14 3% )、插入 (3 4% ) ,缺失 (3 4% )。分析表明 ,人群个体mtDNA控制区碱基序列 ,基因多样性为 99 9% ,两个无关个体的偶合概率为 0 92 % ,具有高度序列的多态性     

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.     

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。     

中国青海藏族、汉族mtDNA控制区遗传多态性

目的研究中国青海藏族、汉族mtDNA控制区遗传多态性。方法收集69份青海藏族和青海汉族无关人群外周血样本，对其mtDNA控制区进行序列分析，计算多个多态性指标。结合其他民族mtDNA遗传资料，根据Nei法计算包括青海藏族和汉族群体在内的11个群体之间的Fst和Rst遗传距离．进行聚类分析，绘制系统发生树。结果在青海藏族和汉族群体mtDNA控制区中分别发现56和59个多态性位点。Rst遗传距离显示青海藏族人群与各人群之间遗传距离均较远（P〈0．05）；青海汉族人群与西安汉族、蒙古族、长沙汉族等人群之间距离较近（P〉0．05）。结论我国青海藏族和汉族人群mtDNA具有相对独特的遗传特征，其遗传多态性和个体识别力较高，可用于民族起源、迁徙、法医学个体识别等领域研究。