PowerPlex~(TM) 16体系在中国人群中罕见等位基因及其类型

目的 分析PowerPlexTM 16体系基因座在中国人群中的罕见等位基因及其类型。方法 应用PCR-STR和DNA序列分析技术,对4650个无关个体在15个STR基因座中的罕见等位基因进行检测。结果 在PowerPlexTM16体系中的D7S820、D16S539、Penta E基因座,检测到2种类型的罕见等位基因,而TH01、D21S11、D5S818、D13S317、Penta D、D8S1179、TPOX、FGA基因座检测出1种类型。其等位基因频率均较低(0.215‰-7.097‰)。结论 超出ladder范围的罕见等位基因序列比相邻等位基因增加(或减少)1个或数个重复单位,因碱基的插入或缺失的罕见等位基因出现在两等位基因之间。     

STR data for the power plex-16 loci in a population from Central Poland

The allele frequencies for the 13 CODIS core loci and two recently developed but not extensively validated in Central European populations pentanucleotide STRs (Penta E and D) were determined using the Power Plex-16 (Promega) kit in a sample of 430 unrelated individuals born in Central Poland. The calculated parameters of polymorphism showed Penta E to be the most valuable marker from the studied set. The obtained distribution of Penta E and D alleles was statistically significantly different from that previously reported for a population of Southern Poland. Analysis of Hardy-Weinberg equilibrium revealed a deviation for the D5S818 locus which may reflect presence of a null allele.     

Characterization of the variant allele 9.2 of Penta D locus

DNA profiles of forensic cases of Córdoba Province, Argentina, typed by PowerPlex 16 kit (Promega), have shown in the Penta D locus few samples with a variant allele migrating as an off ladder between alleles 9 and 10. In order to determine the molecular basis of the new variant allele, three samples were subject to polymerase chain reaction amplification of the Penta D locus by monoplex, and were further purified and sequenced. The sequence analysis revealed that the off ladder allele has ten repeats motifs AAAGA as allele 10, with three nucleotides (TAA) deletion in the 3' flanking region, 128 nucleotides after the last repeat. Therefore, the variant allele could be explained by a deletion of allele 10, and was designated 9.2. Mse I digestion assay allows to corroborate allele 9.2 without sequencing. A population study in Córdoba Province indicates that allele 9.2 of Penta D locus has a frequency of 0.0063.     

Genetic profile of the Madeira Archipelago population using the new PowerPlex16 System kit

Allele and haplotype frequencies of 17 chromosome STR loci, 15 of them included in the kit PowerPlex 16 System from Promega, were determined in a sample of unrelated males from Madeira and Porto Santo Islands. PowerPlex 16 includes STRs not studied before in the Madeira population. The-kit includes two new allele markers (Penta D and Penta E), which proved to be extremely useful for paternity testing (PD = 0.952 and 0.977, respectively). The study revealed that the Madeira population does not differ from that of the north Portugal. Nevertheless, some loci presented alleles found in sub-Saharan and North European populations which were not reported so far in Portugal.     

Genetic diversity at two pentanucleotide STR and thirteen tetranucleotide STR loci by multiplex PCR in four predominant population groups of central India

Genetic diversity study at STR loci in 208 individuals belonging to two backward groups, one caste and one tribal community of Central India called "Chhattisgarh" has been carried out to evaluate significance of Powerplex System loci in human identification and population diversity. Populations are Agharia (72), Satmani (50), Dheria Gond (36) and Teli (50). Fifteen loci (Powerplex 16 Kit) studied are Penta E, D18S51, D21S11, THO1, D3S1358, FGA, TPOX, D8S1179, vWA, Amelogenin, Penta D, CSF1PO, D16S539, D7S820, D13S317 and D5S818. The studied penta nucleotide STR (two) and 13 tetranucleotide (CODIS ) STR are found to be highly polymorphic genetic markers in all studied populations. Most common allele for the four studied population has been found to be same at THO1 (allele 9), D8S1179 (allele 14), CSF1PO (allele 12), Penta E (allele 11) and D16S539 (allele 11). Penta E is found to be most polymorphic (PD=0.89373) among studied 15 STR loci in four populations of Central India.     

用新设计引物调查武汉汉族Penta D和Penta E基因座遗传多态性

目的　研究PentaD和PentaE基因座分型引物设计 ,调查PentaD和PentaE基因座在武汉汉族人群中的遗传多态性。方法　用重新设计的PentaD和PentaE基因座分型引物 ,采用热启动PCR和PAGE技术对 2 81名武汉地区汉族无关个体进行分型调查 ,并将其分型结果与Promega公司的PowerPlexTM16系统荧光标记复合扩增试剂盒分型结果进行比较。结果　新设计引物扩增产物的片段大小范围分别为 15 3～ 198bp和 10 7～ 2 12bp ,其分型结果与PowerPlexTM16系统的分型结果完全一致 ,且用银染法检测的灵敏度显著提高 (由 0 5ng提高到 0 2ng)。PentaD和PentaE基因座在武汉汉族群体分别检出 10个和 2 1个等位基因 ,其基因型频率分布均符合Hardy Weinberg平衡。家系调查证实了其等位基因的传递符合孟德尔遗传规律。两基因座的个体识别能力 (DP)分别为 0 92 62、 0 9860 ,非父排除率 (PE)分别为 0 665 1、 0 83 2 5。结论　新设计引物用于PentaD和PentaE基因座的分型检测准确可靠 ,两基因座多态性程度高 ,在法医学个人识别和亲子鉴定中具有使用价值。     

中国汉族人群15个STR基因座的等位基因频率调查

目的 调查10071名中国汉族无关个体15个STR基因座的等位基因的类型及其频率，并与以往相关文献报道的汉族群体资料进行统计比较。方法 应用PowerPlex~(TM)16荧光标记复合扩增系统，对10071份中国汉族无关个体的血样DNA进行15个STR基因座的复合扩增；用ABI 377或3100遗传分析仪对扩增产物进行分型，统计15个STR基因座的基因频率。结果 15个STR基因座共发现226个等位基因，频率在0.0001～0.5512；除D8S1179基因座外，其它基因座均发现稀有等位基因，数目1～7个不等，共34个。在中国汉族人群，稀有D21S11基因座的等位基因32.1和36.2，D18S51基因座的等位基因15.2和17.2，Penta E基因座的等位基因15.2、17.4、18.4、19.4、26和27，D7S820基因座的等位基因9.2、10.1、11.1和15，Penta D基因座的等位基因18、19和20，TPOX基因座的等位基因14，FGA基因座的等位基因13，以及较常见但欧洲稀有的D21S11基因座的等位基因30.3和D7S820基因座的等位基因9.1和9.2等均为首次报道。结论 大样本基因频率调查有利于观察STR基因座的稀有等位基因；本研究结果与以往相关文献报道的结果有不同程度的差异。     

Mutation in Y STRs: Repeat motif gains vs. losses

Y chromosome specific short tandem repeats (Y-STRs) are widely used in population genetics and forensics. Since these markers do not recombine, mutation is the only source of diversity. The primary mutational mechanism leading to length changes in STRs is thought to be polymerase template slippage, and the most common change is the gain or the loss of one repeat motif. In this work, we aim to study 19 Y-STR alleles’ contraction and expansion. Alleles were grouped into tertiles: short (1^st tertile), intermediate (2^nd tertile) and long alleles (3^rd tertile). Significant differences between repeat gains and losses were found at four markers - DYS19, DYS439 for intermediate alleles, and DYS570 and DYS626 for long alleles. When the average number is computed for the pooled loci, for short alleles, the number of repeat motif gains is higher than of repeat losses, and the opposite happens for long alleles. For intermediate alleles, the proportion between the number of repeat gains and losses is close to one. Generally, the rate of expansion decreases from the first tertile to the third, and conversely, the rate of contraction increases from the first tertile to the third. The pooled loci tertiles’ mutation rate increases from short to long alleles. Our results demonstrate that the mutation direction and rate depend on alleles’ length. The longer the allele the greater the mutation and contraction rates.     

Genetic profile of the Açores Archipelago population using the new PowerPlex 16 system kit

Allele and haplotype frequencies of 16 chromosome STR loci, 15 of them included in the Kit PowerPlex16 System from Promega were determined in a sample of unrelated males from the A?ores Archipelago. All subjects were subjected to an interview in order to make sure that their ancestors belonged to the same island at least back to three generations. This way we intended to look for possible inter-islands differences. PowerPlex16 includes STRs not studied before in the A?ores population. The Kit includes two new allele markers (Penta D and Penta E), which proved to be extremely useful for paternity testing (PD=0.921 and 0.971, respectively). The study revealed that the A?ores population is considerable different from the previous studied Madeira population, but does not differ from that of the north Portugal. Nevertheless, some loci presented alleles not previously reported for Portugal.     

19个STR在人群地域推断中的初步探索

本文汇总了近二十年19个常染色体STR(Short Tandem Repeat)在25个省份汉族人群中的研究报道。统计发现19个STR共有642个等位位点,其中等位位点数目最少的为D8S1179,有20个;数目最多的是D21S11,有60个等位位点。杂合度(He)为0.6203(TPOX)~0.9187(Penta E),多态性指数(PIC)为0.5600(TPOX)~0.9130(Penta E)个体识别率(PD)为0.6279(TPOX)~0.9859(Penta E)。19个STR的CPD、CPE和CMP分别为0.999999999999999999998、0.99999993和1.97×10^-21。通过POPTREE2.0对25个省份汉族19个STR进行聚类分析,发现我国汉族人群的分布有明显南北方地域差异,分为南方省份和北方省份。通过主成分分析也进一步证明了我国汉族人群分布具有南北方地域特点。另外,通过对15个少数民族与其所在省份汉族人群和无关省份汉族人群的STR聚类分析,发现新疆维吾尔族、哈萨克族与新疆汉族聚类在一个亚分支中;广西汉族、云南汉族与该省份的少数民族聚类在一起,这也进一步证明了我国汉族人群和少数民族具有一定的地域分布特性。综上所述,STR不仅可以应用于个体识别、亲子鉴定等,未来还可用于人员地域推断。伴随着STR数量的不断增加和人员STR数据库的不断丰富以及与测序技术的结合,STR技术将会在各类案件中发挥更多作用。     

Genetic polymorphism revealed by 13 tetrameric and 2 pentameric STR loci in four Mongoloid tribal population

The short tandem repeat allelic profiles at to 15 autosomal polymorphic loci were analyzed in four tribal populations of Mizoram (India). The analysis was performed on 354 unrelated healthy individuals belonging to Mongoloid races. All the samples were subjected to sex test (Amelogenin marker) besides the STR typing and in all instances; it has shown no deviation from expectation. The allele frequencies for all the analyzed loci in the studied populations are within expected range in comparison to the populations from same racial background. No significant deviation from the Hardy-Weinberg Equilibrium was observed for all the populations. In no cases the observed heterozygosity is less than that of expected values and it varied from 0.978 (Penta E) to as low as 0.425 (THO1). The discriminatory power and exclusion probability values for all the analyzed markers are significantly high and thus reveal high forensic significance. There is no evidence for association of alleles among the 15 studied loci. This allele frequency data will be useful for human identity testing in Mizo population.     

Significant association of TH01 allele 9.3 and SIDS

Sudden infant death syndrome (SIDS) constitutes a considerable percentage of infant death of unknown etiology. Individual catecholamine response variation is suspected to play a role in SIDS. TH01 is a tetrameric short tandem repeat marker in the tyrosine hydroxylase gene, which regulates gene expression and catecholamine production with allele 9.3 exerting a particularly strong effect on noradrenaline production. We investigated in an age-controlled study the TH01 allele frequencies in 127 cases of SIDS and 406 control cases to assess whether in SIDS cases a distinct TH01 allele distribution could be determined as has been reported by a previous study. We found that genotypes containing one or two 9.3 alleles were significantly more frequent in SIDS patients (58.2%) than in control subjects (48.4%, p=0.038), whereas all other alleles were more frequent in the control subjects. Our findings support the notion that there exists a significant association between TH01 gene configuration and SIDS.     

The short tandem repeat locus VWF2 in Intron 40 of the von Willebrand factor gene consists of two polymorphic sub-loci

This study describes the complex nucleotide sequence structure of the TCTA short tandem repeat (STR) locus, VWF2. Eight alleles of VWF2 were observed in a population of 116 unrelated Caucasian individuals. The alleles ranged in size from 150 to 178 base pairs (bp). Sequence analysis of the isolated alleles revealed two polymorphic regions that were named sub-loci VWF2-a and VWF2-b. VWF2-a is located at the 5' end of the conventional locus, whilst VWF2-b is located at the 3' end. The two sub-loci are joined by a 30-nucleotide non-polymorphic sequence which contains two additional TCTA motif repeats. A semi-nested polymerase chain reaction (PCR) was designed to amplify the VWF2-b region in conjunction with the standard VWF2 amplification. This new amplification method enabled a higher level of allele discrimination than could be achieved by assigning alleles according to size. A cohort of 99 unrelated individuals was tested with this method. VWF2-a expressed five different alleles ranging from zero motif repeats to four motif repeats, while VWF2-b alleles ranged from 8 to 14 motif repeats. Allelic configuration based on the VWF2-a and VWF2-b sub-alleles revealed 23 unique configurations out of a possible 31 for the original eight VWF2 alleles. In conclusion, the VWF2 is a highly polymorphic STR locus with potential application for forensic and parentage testing.     

二组分混合DNA样品STR图谱解释

对混合样品STR图谱的结果进行解释。实验模拟二组分DNA混合样品 ,复合扩增荧光检测 10个基因座 ,比较混合样品谱带 ,计算等位基因峰面积比。结果发现 :二组分DNA混合样品的等位基因数增加 ,样品的混合比例不同就出现峰面积的不平衡。在等位基因峰面积比值与样品组分混合比例接近时 ,可由峰面积比值推断混合样品的混合比例。在混合比例为 1∶2 0时 ,基本上检测不到来自少量混合成分的等位基因 ,表现为单一组分图谱 ;在混合比例为 1∶10时 ,含量低的组分的等位基因峰面积接近与主要组分的“Stutter”峰面积 ,与来自主要组分的等位基因峰面积差异很明显。能检出混合样品中少量成分等位基因的最高混合比例为 1∶10     

Evaluation of the PowerPlex Fusion System in a sample from East Timor

East Timor is a country located in Southeast Asia. In this study, we determined allele frequencies and forensic parameters for 24 STR autosomal loci included in the PowerPlex^® Fusion System. Autosomal STR data was collected from saliva samples of 100 individuals from East Timor. The amplification of the 24 autosomal STRs was performed using PowerPlex^® Fusion System (Promega Corporation) and the amplified products were analysed on 3500 Genetic Analyser using GeneMapper^® ID-X 1.2 Software (Applied Biosystems). All the analysed loci meet Hardy–Weinberg equilibrium after Bonferroni correction. In our samples, we found “off-ladder” alleles (D2S441, Penta E and FGA locus) confirmed by reamplification, amplification with others kits and sequenced when justified.     

Increasing Allele Detection by Altering the Quantity of Internal Lane Standard

Electrokinetic injection (EI) is the primary method used in forensic laboratories to load amplified PCR product in capillary electrophoresis for short tandem repeat (STR) fragment separation. Because all samples subjected to capillary electrophoresis use internal lane standard (ILS), this study investigated the consequence of varying the volume of ILS and its effects on allele peak heights and number of alleles detected. Results demonstrated that when the volume of ILS is reduced, the average peak height and number of alleles increased, thereby increasing the sensitivity of the detection method. Sizing anomalies were observed; however, they did not adversely affect accuracy and precision. The method developed in this study offers a simple and universal procedure to increase the alleles detected in forensic STR analysis. Reducing the volume of ILS to achieve greater sensitivity is applicable to all STR amplification kits and capillary electrophoresis instruments currently used in forensic DNA analysis.     

Structural variations of the VWA locus in humans and comparison with non-human primates

The HUMVWA locus was examined in 160 samples from the Japanese population. A total of 142 fragments were sequenced, and the counterpart sequences were also determined in non-human primates. In humans, 10 different alleles were found; they could be grouped into seven allelic classes based on the total number of repeats. No variation was observed in the alleles 17, 18 and 19, which showed consensus sequence structures and in the allele 14, which showed a different structure. New variation was found in alleles 15, 16, and 20, which had differences occurred in a basic (TCTA)(TCTG)(n) repeat in the 5' side. The counterpart fragments were successfully amplified in three species (chimpanzees, gorilla, and orangutan) out of four kinds of anthropoids, three species (rhesus macaques, Japanese macaques, and green monkey) out of four kinds of old world monkeys, but not in one species of either new world monkey or prosimian. The sizes of the fragments distributed from 92 to 180 bp in non-human primates and showed allelic size differences in four species. The sequence of the 5' flanking region followed by primer sequences in humans and anthropoids, which consisted of 19 bp, was identical in all, but differed from that in old world monkeys. The basic repeat motifs of humans and anthropoids consisted of TCTA, TCTG, and TCCA but that of old world monkeys consisted of TCTG, TCCG and TCCA The structures of humans and anthropoids were essentially similar, but with characteristic difference in each species. Differences in the allelic structures of old world monkeys were complex. Seven different alleles were observed in two rhesus and two Japanese macaques and one type of allele was observed in two green monkeys. Duplication of more than two repeat units of 4 bp was found in an allele of an old world monkey. These data illuminate interesting features of mutational changes in STRs during the long generations and also some insight into evolutional aspects of primates.     

Molecular characterization and Austrian Caucasian population data of the multi-copy Y-chromosomal STR DYS464

DYS464 is a multi-copy STR system with four positions on the Y-chromosome (DYS464a, b, c, and d) which was recently identified and characterized [Forensic Sci. Int. 130 (2002) 97]. The aims of our study were to perform a population study, to estimate the mutation rate and an extensive sequence analysis in order to confirm the nomenclature. Fourteen different alleles were found in an Austrian population sample with an allele length varying from 9 to 19 repeats. All alleles were cloned and sequenced. Alleles 9-19 showed the general repeat structure (CCTT)n...(CCTT)2...(CCTT)3...(CCTT)4...(CCTT)2...(CCTT)2. The nomenclature is based on the number of repeated units of the variable (CCTT)n-stretch only. In 13% of the samples intermediate alleles, namely 14.3A, 14.3B and 15.3 were detected. In these alleles the variable repeat block is interrupted by a CTT motif (14.3A: (CCTT)3CTT(CCTT)11; 14.3B and 15.3: (CCTT)7CTT(CCTT)7/8). A comparison with GenBank entries revealed the existence of a length variant due to a deletion of one cytosine in the 5' flanking region of the first repeat block. We designed an alternative forward primer to circumvent possible ambiguities in the allele designation. A total of 54 different genotypes were identified in 135 men corresponding to a discrimination capacity (DC) of 40% and a gene diversity (GD) of 0.97. These values are much higher than those of other Y-chromosomal short tandem repeats (Y-STRs). DYS464 has the same haplotype diversity (HD) as the combination of the five Y-STR loci with the lowest gene diversities of the Y-STR core set. On the other hand, a combination of the three most diverse loci (DYS464, DYS385 and DYS390) has the same capacity to distinguish between paternal lineages than the complete minimal haplotype (minHT) consisting of eight Y-STR loci. In our population sample the addition of DYS464 to the minHT increases the number of different haplotypes from 110 to 122. The mutation-rate estimate based on the 70 meioses analyzed amounts to 2.86 x 10(-2) (95% confidence interval 3.5 x 10(-3) to 9.95 x 10(-2)). This value is approximately 10 times higher than the average mutation-rate estimate for Y-STRs.     

新Y-STR基因座DYS709在汉族人群中的遗传多态性调查

目的筛选新的Y-STR基因座,调查其在汉族人群中的等位基因频率分布,评价其在法医学及其它方面的应用价值。方法在Y染色体基因组DNA中查找候选基因座,在重复顺序两端设计引物,PCR扩增后用银染法显示结果。结果一个重复单位为CTTT的Y-STR基因座DYS709被发现。在102例汉族无关男性个体血样中共检出了7个等位基因。基因多样性为0.7063,个人识别能力(PD)和非父排除率(PE)均为0.7063。结论新筛选到的DYS709具有较高的遗传多态性,在法医学及人类遗传学方面具有应用价值。     

Sequence variation of new alleles at the short tandem repeat D19S253 locus

This paper reports the sequences of two new alleles identified in a population database study on the short tandem repeat D19S253 locus. A Portuguese Caucasian population and a Portuguese African population were studied. Forty-four selected alleles were sequenced and 11 different alleles were found. All the sequenced alleles shown to possess a simple tetranucleotide GATA repeat region structure. The two new alleles, alleles 6 and 16, follow the simple repeat pattern. During paternity investigation casework, 1028 meiosis were analyzed and five isolated genetic incompatibilities detected. In one case, a non-detectable allele with the used set of primers could be the explanation. In the other four cases, single-step mutations could be considered. The mutation rate obtained for this locus was 3.89 x 10(-3).