Allelic alterations at the STR markers in the buccal tissue cells of oral cancer patients and the oral epithelial cells of healthy betel quid-chewers: an evaluation of forensic applicability

Although cancerous specimens are usually not used in forensic DNA typing, they might be forcibly employed under certain instances. On the other hand, though the oral epithelial samples have been applied to forensic identification, the great popularity of betel quid (BQ)-chewing in Taiwan, which is known to be a risk factor leading to an oral cancer, makes this application questionable. The DNA stability of nine short tandem repeat (STR) markers (the AmpFlSTR kit) was first investigated and then used to evaluate the forensic appropriateness of the oral samples of both healthy BQ-chewers and the archived clinical specimens from oral cancer patients. The analyses were performed on buccal samples from 100 BQ-chewers and 100 oral cancer patients, as well as their paired peripheral blood samples, and a group of 100 non-BQ-chewers were used for the control. In the group of 100 oral cancer patients, two types of DNA instability were found. They were major allelic imbalance, and allelic alterations including the expansion, the contraction and the un-classified type (i.e. can not be confirmed as the expansion or the contraction). The overall percentage of the cancerous subjects demonstrating DNA instability was 33% (five patients possessing both types of DNA instability). Both types of DNA instability showed a tendency of increasing with the severity of the pathological stage of oral cancer. Forty-four occurrences of major allelic imbalance were found from 21 cancer patients. The statistical result revealed that there was no significant difference in the allelic imbalanced occurrence among the nine STR loci. Allelic alterations were found in 17 patients, within which 12 individuals had the expansion, five had the contraction, and three were the un-classified type. Further, among these 17 patients, three were found to acquire multiple allelic alterations at multiple loci. In the group of 100 unrelated healthy BQ-chewers, two loci with major allelic imbalance were detected. However, the two imbalanced alleles were virtually half lost, and could still be recognized as heterozygous alleles. The statistical results of ANOVA, chi(2), and Scheffe tests indicated that the means of allelic imbalance at the nine STR loci of the oral cancerous group revealed a significant difference from those in the control group. Our results suggest that oral cancer tissues cannot be used as references for forensic purposes using the PCR-based STR systems, whereas the oral swabs from healthy BQ-chewers can be employed, but should be done with caution.     

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.     

Allelic distribution of nine short tandem repeat (STR), HLA-DQA1, and polymarker loci in an Omani sample population

Allele frequency distributions of nine short tandem repeat (STR) loci, D3S1358, vWA, FGA, D8S1179, D21S11, D18S51, D5S818, D7S820, and D13S317, HLA-DQA1 and polymarker (PM) loci were studied in a sample population from Sultanate of Oman, Middle East. Blood samples were collected from 162 unrelated individuals. For all STR, HLA-DQA1 and PM loci, no deviations from Hardy-Weinberg equilibrium, based on the exact test, were observed. The most discriminating loci were D18S51 (PD=0.966) and FGA (PD=0.964), while the least informative locus is GYPA (PD=0.585). The allele frequency data may be useful in forensic case work.     

Recovery of genomic DNA from archived PCR product mixes for subsequent multiplex amplification and typing of additional loci: forensic significance for older unsolved criminal cases

A method for genomic DNA recovery from different types of PCR product mixes suitable for multiplex amplification and typing using the Profiler Plus STR typing system has been investigated. The application of this method is of significance in cases where the original DNA samples have been exhausted due to repeated typing analyses in an effort to maximize their evidentiary value. Such cases typically involve samples analyzed using the available DNA typing systems of the time which gave a markedly lower power of discrimination, either alone or in combination, compared to that of modern multiplex STR typing systems. It was found that an effective method for recovering genomic DNA from HLA-DQA1 +PM and CTT triplex amplification mixes, suitable for reproducible achievement of the complete Profiler Plus profile, involved the use of Amicon Microcon-100 microconcentrators. Interestingly, this method was not required to achieve the complete nine STR profile using D1S80 amplification mixes.     

微量口腔脱落细胞检材的DNA检验

目的寻求提高微量口腔脱落细胞检材的DNA检验成功率的简便有效的提取方法。方法对不同载体上的100份微量口腔脱落细胞检材采用小体积Chelex-100法提取DNA,在ABI7500型荧光定量PCR仪上进行定量,同时用IdentifilerTM复合扩增系统扩增,在ABI3130遗传分析仪上进行STR分型。结果从25根饮料吸管上提取的DNA量在0.72～116.7.8ng,16个水杯杯缘提取的DNA量在2.15-142.5ng,31个饮料瓶(罐)口提取的DNA量在1～34.65ng,10根筷子上提取的DNA量在3.35～26.6ng,12个果核中提取的DNA量在0.294～21.4ng,6份吃剩的骨头中提取的DNA量在0.88～5.88ng。100份检材性别及9个以上STR位点分型成功率平均为59.38%。除了使用者的个人原因外,检材的提取送检方式、检材的质地、饮料的性质对提取的DNA量有显著影响,是否加蛋白酶K对提取的DNA量无显著影响。结论采用小体积Chelex-100法可对60%左右的微量口腔脱落细胞检材提取DNA进行STR分型。     

激光捕获显微分离技术在DNA检验中的应用研究

目的建立一套显微细胞捕获技术用于法医学生物检材DNA检验方法。方法使用VeritasTM LCM激光捕获仪,采用紫外加红外的捕获方式,对框架覆膜玻片上经苏木素染色口腔上皮细胞进行捕获,采用改良硅珠法提取细胞DNA,使用Identifiler TM试剂盒在5μL体系中进行PCR扩增。结果成功获得20个口腔上皮细胞的13个以上完整STR基因座分型谱带。结论本研究建立的方法适合法医学生物检材制成的染色涂片上细胞的DNA检验。     

World population data for the HLA-DQA1, PM and D1S80 loci with least and most common profile frequencies for combinations of loci estimated following NRC II guidelines

All published and unpublished gene frequency data for the PCR-based loci HLA-DQA1, LDLR, GYPA, HBGG, D7S8, GC, and D1S80 that could be located are presented in summary tables. These gene frequencies provide the data necessary for estimating probabilities of chance match according to NRC II guidelines for any DNA profile that includes any combination of these loci for any of the populations. To illustrate the range of polymorphism for combined locus profiles, least and most common profile frequencies were estimated following NRC II guidelines for: the PM loci for all populations for which PM data were available; and for combinations of HLA-DQA1/PM, HLA-DQA1/D1S80, PM/D1S80, and HLA-DQA1/ PM/D1S80 for populations for which data were available for the relevant combinations. The profile frequencies were calculated at theta values of zero and 0.01. Minimum allele frequencies (MAF) were calculated, and are shown, for each data set for which the MAF was greater than the lowest observed allele frequency. Least common profile frequencies were calculated using MAF in those cases to illustrate a conservative estimate. The effect of using MAF versus lowest observed allele frequency in estimating least common profile frequencies is briefly illustrated as well. We finally show that aggregate U.S. gene frequency data for the classical MN and GC polymorphisms for both Caucasian and African-American populations is fully in accord with the DNA-based gene frequency data obtained from PM reverse dot-blot strips for GYPA and GC, respectively.     

激光显微捕获口腔上皮细胞的DNA分型

目的探索激光显微技术(lasercapturemicrodissectionsystem,LCM)捕获口腔上皮细胞,并进行STR-DNA分型检测的方法。方法用VERITAS显微切割仪红外低能激光显微捕获一定数量口腔上皮细胞,进行ProfilerPlus试剂盒STR复合扩增,检测DNA基因型。结果7～8个口腔上皮细胞能成功获得STR-DNA分型。3～4个口腔上皮细胞不能成功获得STR-DNA分型。结论激光显微捕获作为一种分离单个细胞的新技术,对于微量口腔上皮细胞的STR-DNA分型是可行的。     

The application of minisatellite variant repeat mapping by PCR (MVR-PCR) in a paternity case showing false exclusion due to STR mutation

A boy and a girl with their mother brought a paternity suit against an alleged but deceased father. We tested six conventional genetic markers, the AmpliType PM+ DQA1 and twelve STR loci the children and mother together with the alleged paternal grandparents. We also DNA typed the bloodstain found later in the alleged father's medical record. Only the result at D3S1358 in a nineplex STR system excluded the alleged father from parentage of the boy, whereas all markers were inclusive for the girl. Accordingly, we performed sequence analysis at D3S1358 to confirm the presence of a paternal exclusion or mutation. The sequence analysis indicated that the boy's allele 17 could have originated from either of the alleged father's allele 16 or 18 by a single-step mutation associated with slippage mutation in STR loci. We carried out minisatellite variant repeat mapping by PCR (MVR-PCR) at loci D1S8 (MS32) and D7S21 (MS31A) and mapped allele haplotypes of all individuals except the deceased alleged father. The MVR-PCR analysis showed that the boy has no inconsistency with the relationship between the alleged grandparents, and was very effective at increasing the paternity index (PI) value. We conclude that there is biological relationship between not only the girl but also the boy and the alleged father.     

签字笔上附着的脱落上皮细胞STR分型

目的 探讨遗留在签字笔上微量脱落细胞DNA分型的可行性以及保存时间对分型的影响.方法 17名志愿者每人使用7支签字笔,每支笔每天使用20 min,为期1个月,分别保存1、3、5、7、14、21和28 d,运用硅珠法提取签字笔上微量脱落细胞中的DNA,应用荧光标记PCR-STR技术进行DNA分型,同时采集上述17名志愿者口腔拭子作为对照,分析签字笔作为检材进行DNA分型的可行性以及保存时间对DNA分型的影响. 结果以基因座检出个数为指标,签字笔脱落细胞和口腔拭子的DNA分型结果随保存时间变化而产生的差异具有统计学意义(P＜0.01).签字笔保存1、3、5、7、14、21和28 d后进行DNA分型检出的基因座个数与对应的口腔拭子DNA分型检出的基因座个数相比差异均有统计学意义(P＜0.01).签字笔使用后保存1 d进行DNA分型.可明确判读12个以上基因座的占41.2%. 结论签字笔上附着的微量手指脱落细胞可作为一种法庭生物检材进行DNA分型,但其保存时间会影响DNA分型.     

浙江汉族人群6个STR基因座的遗传多态性的调查

目的进一步完善浙江省汉族人群STR基因座遗传多态性的调查,为其应用提供基础数据。方法采用AmpF1STRSGMplus和AmpF1STRCoficer反应试剂盒,使用ABI310型基因分析仪对浙江汉族人群200名无关个体血样进行了D16S539、D2S1338、D19S433、TH01、TPOX和CSF1PO6个STR基因座遗传学分析。结果分别发现了9、15、15、11、8、10个等位基因,发现的基因型分别为23、42、35、19、16、17个,其分布经X2检验均符合Hardy-Weinberg平衡定律,并分别统计了6个STR基因座的H、DP、PM、PE及PIC参数。结论6个STR基因座适合法医学应用。     

犬11个STR基因座的遗传多态性

目的调查犬11个STR基因座的群体遗传多态性。方法应用自主构建的犬11个STR基因座（PEZ1、PEZ2、PEZ3、PEZ5、PEZ6、PEZ8、PEZ12、FH2010、FH2054、FH2132和FH2611）荧光复合扩增体系，扩增105只犬的样本，统计各基因座扩增结果，并分析其群体遗传参数。结果11个STR基因座的累积非父排除率和累积个体识别率分别为0．9330621和0．9999999．平均杂和度和平均多态信息含量分别为0．502和0．640。结论该11个犬STR基因座的遗传多态性较好，可以有效用于犬的个体识别和亲权鉴定。     

Inferring recent human phylogenies using forensic STR technology

STR loci are characterized by extremely high mutation rates and thus, high levels of length polymorphism both within and among populations. In addition, much of the observed variation is believed to be nearly selectively neutral. Because of these features, STRs are ideal markers for genetic mapping, intra-species phylogenetic reconstructions and forensic analysis. In the present study, we investigate the application of five STR loci (CS1PO, TH01, TPOX, FGA and vWA) routinely used in forensic analysis for delineating the phylogenetic relationships of 10 human populations representing the three major racial groups (African-Caribbean, Croatian from the island of Hvar, East Asian, Han Chinese, Italian, Japanese, Portuguese, UK Caucasian, US Caucasian and Zimbabwe). The resulting tree topology exhibited strong geographic and racial partitioning consistent with that obtained with mtDNA haplotypes, Y-chromosome markers, SNPs, PAIs (polymorphic Alu insertions) as well as classic genetic polymorphisms. These findings suggest that forensic STR loci may be particularly powerful tools and provide the necessary fine resolution for the reconstruction of recent human evolutionary history.     

茚三酮薰显法在人体接触细胞发现采集中的应用

目的 研究茚三酮薰显法在人体接触细胞发现采集中的应用价值.方法 对衣服、口罩、棍棒等可疑人体接触细胞检材258份,采用1％茚三酮溶液进行显色反应,然后用磁珠法提取DNA,并进行DNA定量和STR检测.结果 可疑人体接触细胞检材中有172份显色反应阳性,其中115份检出的DNA浓度在0.02ng/μL以上并检出6个以上STR基因座的基因型,检出率为66.86％;显色反应阴性的86份检材均未检出DNA浓度或成功进行STR基因型.结论 茚三酮薰显技术可用于指导案件人体接触细胞的发现采集.     

汉族12个STR位点群体遗传学调查

目的240个汉族无关个体12个STR位点基因频率调查及其法医学应用;方法采用12位点复合扩增及变性聚丙烯酰胺凝胶电泳基因分型;结果该系统12个STR基因位点在汉族人群中均为高识别率位点,特别适合于陈旧血痕检验;结论12位点STR-PCR复合扩增系统检测方法简便,经济实用,在法医个体识别和亲子鉴定中具有应用价值。     

Using hydrophilic adhesive tape for collection of evidence for forensic DNA analysis

Known exemplar samples of human DNA have traditionally been body fluids, such as blood, saliva, and semen. In each case, the presence of water is a risk for the bacterial growth, which may degrade the DNA evidence. In this study, the authors have developed a method that employed a hydrophilic adhesive tape (HAT) for collecting DNA evidence. The HAT method was used to remove surface cells from relatively hairless areas on the body. The area examined were ankle, arm, behind the ear, between fingers and back of the neck. The HAT was then dissolved in the extraction buffer. DNA typing was performed at vWA, THo1, F13A1, and FES loci using the short tandem repeat (STR) analysis. Our results show that the samples collected from ear give the best results with a success rate of 100%. All subjects tested by this method had known STR genotypes established from buccal swabs. The authors' results suggest that the HAT method can be used as a less invasive method for collecting biological evidence for forensic DNA analysis. In addition, this collection method should reduce the risk of DNA degradation due to the moisture, which is encountered using conventional collecting methods.     

甲醛固定及石蜡包埋组织的DNA分型及其应用

目的：建立甲醛固定及石蜡包埋组织ＤＮＡ的基因分型方法并使之应用于实际检案。方法：用Ｃｈｅｌｅｘ和有机溶剂提取法提取ＤＮＡ,以ＰＣＲ与反向探针杂交技术作ＰＭ和ＨＬＡ－ＤＱＡ１位点分型。结果：２６份包埋组织块有２４份能得到理想的分型结果。结论：本法可用于法医学个人识别和亲子鉴定     

荧光标记复合扩增检测4个STR基因座多态性

目的建立荧光标记复合扩增D1S2142,D13S1492,D14S306,D15S659基因座检测分型方法,并对成都汉族群体4个基因座的遗传多态性进行调查。方法用6-FAM标记D1S2142和D15S659引物,HEX、TMR分别标记D14S306和D13S1492引物,PCR复合扩增,310基因分析仪电泳自动收集电泳结果数据,GeneScan Analysis Software3.7NT软件计算扩增产物片段相对大小,Genotyper(3.7NT软件进行样本基因型分型,建立了荧光标记复合扩增检测4个STR基因座基因型的方法,对145名成都汉族无关个体样本进行分型。结果荧光标记复合扩增D1S2142,D13S1492,D14S306,D15S659基因座,每个STR基因座都获得了清晰的基因型分型结果。145份样本,4个STR基因座分别检出10,14,7,12个等位基因和22,54,21,39种基因型,其基因型分布均符合Hardy-W e inberg平衡。4个基因座在成都汉族群体的杂合度分别依次为0.7793,0.8345,0.7793和0.8345;多态信息含量分别依次为:0.7656,0.8730,0.7470和0.8312。累计非父排除率为0.9783,累计个人识别机率为0.9999 917。结论荧光标记复合扩增D1S2142,D13S1492,D14S306,D15S659基因座,可实现对每个基因座准确分型;成都汉族群体该4个基因座的遗传学数据,可为群体遗传学和法医学研究与应用提供基础资料。     

Evaluation of gastrointestinal cancer tissues as a source of genetic information for forensic investigations by using STRs

Malignant tissue samples may sometimes be the only source of biological material for forensic investigations, including identification of individuals or paternity testing. However, in use of such samples, uncertainties due to microsatellite instability (MSI) and loss of heterozygosity (LOH) often associated with neoplasias may be encountered. In this study, we have analysed the applicability of autosomal tetranucleotide short tandem repeat (STR) markers, which are routinely used in forensic analysis, to gain genetic information. MSI and LOH were analysed in 41 surgically removed gastrointestinal cancer specimens and the adjascent non-cancerous tissue marginals. The cancer specimens showed great variability in their genetic phenotypes due to MSI or LOH, with only 32% being microsatellite-stable. Of the 15 autosomal STR loci analysed, only TH01 had no MSI-type alteration in these samples. The loci most frequently affected by MSI were D8S1179, D21S11, D18S51 and D19S433 (MSI in 15-17% of cases). LOH-type alterations were observed at all of the loci, including the amelogenin locus used for sex determination. The highest LOH frequency was found at locus D18S51 (27%). The genetic alterations at the marker loci may indicate false homozygosity or heterozygosity, and false gender may result from erroneous deduction of DNA profiles. Therefore, typing of autosomal STRs from malignant tissues in forensic settings warrants careful interpretation of MSI and LOH results together with microscopic analysis of a tissue specimen. Results by two commercially available and widely used forensic DNA profiling kits used here were comparable.     

ABO基因分型与多重STR联合检测在法医学中的应用

目的建立ABO基因型和Goldeneye16A试剂盒联合检测的方法,并评价其在法医学实践中的应用价值。方法将6种ABO基因型(A/A,A/O,B/B,B/O,A/B,O/O)的序列特异性引物(PCR-SSP)检测方法与Goldeneye16A试剂盒相整合进行同步分型。通过对460份男性个体血痕样本、9947A DNA及90份案件样本进行检测,考察方法的一致性、灵敏度及对法庭科学检材的适用性。结果应用本文方法可同时检出6种ABO基因型和15个常染色体STR基因座及性别决定基因座,检测灵敏度为125pg,其中ABO基因检测灵敏度达63pg。460份男性血痕和90份案件检材证实该联合分型方法用于各类检材结果准确、稳定。结论本文ABO基因分型与多重STR联合检测方法,适用于各类含有核细胞的生物检材,在法庭科学DNA鉴定中有较好的应用前景。