Sequencing strategy of mitochondrial HV1 and HV2 DNA with length heteroplasmy

We describe a method to obtain reliable mitochondrial DNA (mtDNA) sequences downstream of the homopolymeric stretches with length heteroplasmy in the sequencing direction. The method is based on the use of junction primers that bind to a part of the homopolymeric stretch and the first 2-4 bases downstream of the homopolymeric region. This junction primer method gave clear and unambiguous results using samples from 21 individuals with length heteroplasmy in the hypervariable regions HV1, HV2 or both. The method is of special value for forensic casework, because sequencing of both strands of an mtDNA region is preferable in order to reduce ambiguities in sequence determination.     

Length variation in HV2 of the human mitochondrial DNA control region.

Hair samples were typed from three individuals who exhibited length heteroplasmy in the homopolymeric cytosine stretches (C-stretch) in hypervariable region 2 (HV2). The study demonstrated that for different hairs within an individual, the HV2 C-stretch region can vary with respect to the number of cytosines and/or proportion of C-stretch length variants. Length heteroplasmy may occur regardless of the prominent length variant present in this region. Differences in the number of cytosines at the C-stretch region, or a variation in the relative amounts of heteroplasmic length variants, cannot be used to support an interpretation of exclusion.     

人类线粒体DNA异质性研究进展及在法医学中的应用

线粒体DNA(mtDNA)异质性的存在使其在法医学应用变得复杂。本文对mtDNA异质性形成的可能原因、异质性的分布和遗传特点、异质性的筛查和定量方法、异质性对法医学的影响以及异质性的研究和展望等方面进行综述,探讨异质性在法医学上的应用价值。     

Single-strand conformation polymorphism of hyper-variable regions HV1 and HV2 of human mitochondrial DNA: detection by silver staining

Human mitochondrial DNA from 50 trios consisting of mother (M), child (C) and father (F) was PCR amplified with primers flanking the hyper-variable regions, HVR1 and HVR2. The amplified products were then fractionated under non-denaturing conditions, silver-stained and compared by single-stranded conformational polymorphism (SSCP). In all but one case, mother and child displayed identical patterns, which could be promptly distinguished from that of the father. For the remaining cases, either set of primers was sufficient to resolve the familial ties. In no instance, M displayed alleles different from those of C within each trio, demonstrating that no false exclusions occurred. The SSCP approach proved to be a robust technique suitable as a preliminary screening in cases requiring identification of multiple samples.     

用PCR-DGGE技术检测人外周血mtDNA HVⅠ单倍型及异质性

目的建立简单、有效的m tDNA单倍型检测及异质性筛查技术,并获取其相应的汉族人群频率分布。方法用PCR-DGGE技术对200例武汉汉族无关个体外周血m tDNA HVⅠ15997~16174nt和16208~16401nt区域进行分型检测。结果200例汉族无关个体中,15997~16174nt和16208~16401nt区域分别检出20种和22种单倍型,其单倍型多样性(HD值)分别为0.8159和0.8844;m tDNA HVⅠ组合单倍型共90种,其HD值达0.9803。两区域分别有4名和2名个体观察到异质性,其发生率为3%。结论PCR-DGGE是一种简单、灵敏、高效的m tDNA多态性及异质性检测技术,可应用于法医学实践。     

Results of a collaborative study of the EDNAP group regarding mitochondrial DNA heteroplasmy and segregation in hair shafts

A collaborative exercise was carried out by the European DNA Profiling Group (EDNAP) in order to evaluate the distribution of mitochondrial DNA (mtDNA) heteroplasmy amongst the hairs of an individual who displays point heteroplasmy in blood and buccal cells. A second aim of the exercise was to study reproducibility of mtDNA sequencing of hairs between laboratories using differing chemistries, further to the first mtDNA reproducibility study carried out by the EDNAP group. Laboratories were asked to type 2 sections from each of 10 hairs, such that each hair was typed by at least two laboratories. Ten laboratories participated in the study, and a total of 55 hairs were typed. The results showed that the C/T point heteroplasmy observed in blood and buccal cells at position 16234 segregated differentially between hairs, such that some hairs showed only C, others only T and the remainder, C/T heteroplasmy at varying ratios. Additionally, differential segregation of heteroplasmic variants was confirmed in independent extracts at positions 16093 and the poly(C) tract at 302-309, whilst a complete A-G transition was confirmed at position 16129 in one hair. Heteroplasmy was observed at position 16195 on both strands of a single extract from one hair segment, but was not observed in the extracts from any other segment of the same hair. Similarly, heteroplasmy at position 16304 was observed on both strands of a single extract from one hair. Additional variants at positions 73, 249 and the HVII poly(C) region were reported by one laboratory; as these were not confirmed in independent extracts, the possibility of contamination cannot be excluded. Additionally, the electrophoresis and detection equipment used by this laboratory was different to those of the other laboratories, and the discrepancies at position 249 and the HVII poly(C) region appear to be due to reading errors that may be associated with this technology. The results, and their implications for forensic mtDNA typing, are discussed in the light of the biology of hair formation.     

PCR结合变性梯度凝胶电泳法检测人血mtDNA HVⅡ

目的建立简单、有效的mtDNA单倍型检测及异质性筛查技术,并获取其相应的汉族人群频率分布。方法用PCR结合变性梯度凝胶电泳(DGGE)技术对200例武汉汉族无关个体外周血mtDNA HVⅡ29～290nt区域进行分型检测。结果200例汉族无关个体中,检出17种单倍型,其单倍型多样性(HD值)为0.8826;有4名个体观察到异质性,其发生率为2%。结论PCR-DGGE是一种简单、灵敏、高效的mtDNA多态性及异质性检测技术,可应用于法医学实践。     

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.     

Comparative Analysis of the HV1 and HV2 Regions of Human Mitochondrial DNA by Denaturing High-Performance Liquid Chromatography

Abstract:  Denaturing high-performance liquid chromatography (DHPLC) was evaluated as a sequencing-independent means of detecting the presence of sequence differences in pair-wise mixtures of nonconcordant amplicons of human mitochondrial DNA (mtDNA). A total of 920 pair-wise combinations of HV1 and HV2 mtDNA amplicons from 95 individuals were assayed by DHPLC for sequence concordance/nonconcordance. For the 72 combinations of amplicons from different individuals who shared identical DNA sequences, DHPLC assays consistently indicated sequence concordance between the samples. This was in 100% agreement with sequencing data. For the 849 combinations of amplicons which differed in sequence, DHPLC detected the presence of sequence nonconcordance in all but 13 assays to yield 98.5% concordance with sequencing. Thus, DHPLC can be used to detect a diversity of sequence differences (transitions, transversions, insertions, and deletions) in the mtDNA D-loop. Accordingly, DHPLC may have utility as a presumptive indicator of mtDNA sequence concordance samples, as a screen for heteroplasmy/situational mixtures, and as a means for the physical fractionation of the individual contributors to an mtDNA mixture prior to sequencing.     

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.     

The analysis of coercive chains between children,mothers, and siblings

The study examines coercive episodes observed between young children, siblings, and their mothers in two samples of families. One sample consisted of aggressive children, the other of nonaggressive children. Aggressive children initiated more coercive chains, especially when the mother and the sibling had been acting neutrally, and engaged in longer chains than normal children. The longest coercive chain occurred when sibling conflict took place in the aggressive group. Findings showed the level of coercive behavior within chains was a function of the initiator of the chain. The initiator had substantially higher levels of negative behavior throughout the chain, compared to the level shown by the recipients of the chains. This held across both samples. Moreover, the initiator was more likely than the recipient to terminate the chain by displaying the last negative response.     

人外周血白细胞端粒DNA长度与年龄的定量关系

目的探讨人外周血白细胞端粒DNA长度变化规律及其与年龄的关系,为法医学推断年龄提供理论依据。方法抽取西藏那曲地区0~81岁健康人外周血样本105例,其中男性53例,女性52例,采用Southern杂交法检测其端粒限制性片断平均长度。各年龄组性别差异作t检验,端粒长度与年龄的关系作相关及回归分析。结果人外周血白细胞端粒长度随年龄增长逐渐缩短,且存在性别差异;得到推断年龄回归方程:Y=-16.539X+236.287±9.832。结论人外周血白细胞端粒DNA长度与年龄存在明显相关性,为法医学在分子水平上推断个体年龄提供了可能。     

Advantages of dental mitochondrial DNA for detection and classification of the sequence variation using restriction fragment length polymorphisms.

After amplification by polymerase chain reaction (PCR), the nucleotide sequences of a 452-bp section of the D-loop region of human mitochondrial DNA (mtDNA) were determined in 40 teeth extracted from patients living in Kanagawa prefecture, Japan. Dental DNA was extracted separately from the dental pulp and dentin (i.e., the attached pulp cells from the most superficial layer of the pulp cavity wall) of the same tooth. Comparison of the nucleotide sequences of the 452-bp region of the D-loop demonstrated that nucleotide substitutions and insertion/deletion events were identical in material from both sources. Thus, dentin produces equivalent results when the dental pulp of a tooth is unsuitable for mtDNA analysis. To establish the reliability of the screening procedure for the sequence analysis, we identified restriction sites for the enzymes KpnI and MnlI in the 452-bp region of the D-loop. Thirteen of 14 patterns of four polymorphisms analyzed using the mtDNA from the 40 tooth samples were identifiable by an initial screening procedure involving restriction fragment length polymorphism (RFLP) analysis. Combined use of sequence analysis and RFLP analysis proved extremely efficient in analyzing mtDNA polymorphisms, allowing identification of individuals.     

线粒体DNA编码区的多态性

目的研究线粒体DNA(mtDNA)编码区单核苷酸多态性,建立mtDNA编码区多态性在法庭科学中应用的理论基础。方法针对mtDNA编码区nt8162-8483以及nt13070-13299两段序列设计引物,应用直接测序技术研究其多态性。结果两对引物扩增片段长分别为322bp和230bp,共检测到21种变异,24种单倍型,基因多样性为0.7511,两个无关个体的偶合概率为0.2564。结论线粒体DNA编码区多态性位点作为线粒体DNA控制区多态性位点的补充,联合应用可以提高线粒体DNA在法医学应用中的个体识别能力。     

线粒体DNA在年龄推断中的应用

线粒体是机体ATP产生的场所,近年来研究发现线粒体DNA(MitochondrialDNA)的损伤程度会随着年龄的增长而增加。深入研究并将mtDNA的损伤程度应用在法医学软组织的年龄推断中将有重要意义。     

Correlation of microscopic and mitochondrial DNA hair comparisons

Expert opinions regarding the microscopic comparison of human hairs have been accepted routinely in courts for decades. However, with the advent of mitochondrial DNA (mtDNA) sequencing, an assessment can be made of the association by microscopic hair comparisons in casework between a questioned hair and reference hairs from an individual. While each method can be used separately, the two analytical methods can be complementary and together can provide additional information regarding source association. Human hairs submitted to the FBI Laboratory for analysis between 1996 and 2000 were reviewed. Of 170 hair examinations, there were 80 microscopic associations; of these, only nine were excluded by mtDNA. Importantly, 66 hairs that were considered either unsuitable for microscopic examinations or yielded inconclusive microscopic associations provided mtDNA results. Only six hairs did not provide sufficient mtDNA, and only three yielded inconclusive results. Consistency was observed in exculpatory results with the two procedures. This study demonstrates the utility of microscopic hair examinations and the strength of combining microscopic analysis with mtDNA sequencing.     

Diversity and heterogeneity in mitochondrial DNA of North American populations

Variation in the mitochondrial DNA (mtDNA) control region as detected by sequence-specific oligonucleotide (SSO) probes is described for 2282 individuals from African-American, European-American, and Hispanic subpopulations from five broadly defined regions of North America (Northeast, Southeast, Central, Northwest, Southwest). Population diversity estimates were uniformly high for all subpopulations and for each major ethnic group. Only the Pennsylvania Hispanic group was remarkable with respect to its mitochondrial DNA types, having both six low frequency population specific types (ranging from 1.2-8.6%) and three high frequency shared types (10-20% each). There was no statistically significant subpopulation heterogeneity present within any of the three major groups at either the subpopulation level or the regional level (p > 0.01). However, statistically significant heterogeneity was measured when comparing the three major groups to each other, with the variance component attributable to this large division accounting for 18.60% of the total variance (p < 0.001). Overall mtDNA is a satisfactory forensic typing locus within broadly defined African-American, European-American, and Hispanic groups from North America, based on the high diversity estimates and absence of heterogeneity, as characterized by SSO typing.     

DNA length polymorphism of the apo B 3' region: frequency distribution of the alleles in the German population.

A hypervariable region has been described 3' to the human apolipoprotein B (apo B) gene. Using the polymerase chain reaction amplification followed by agarose gel electrophoresis, at least 16 different alleles can be distinguished. In order to introduce this system into forensic DNA analysis detailed knowledge of the allele frequency is one of the most important prerequisites. For this reason we studied the allele distribution of 340 unrelated individuals originating predominantly from Southern Germany.