Allelic structure and distribution of two STR loci, D8S580 and D22S442, in the Japanese population

The allelic frequency and structural characteristics of two STR loci D8S580 and D22S442 were investigated using blood samples from 143 unrelated healthy Japanese individuals. Thirty-eight alleles in D8S580 locus and 13 alleles in D22S442 locus were identified. The discrimination power, heterozygosity, and the polymorphic information content of those loci displayed high values (0.98, 0.88, and 0.87 in D8S580 and 0.97, 0.86 and 0.85 in D22S442), and their frequency distributions met Hardy-Weinberg equilibrium expectations. The allelic pattern of D8S580 was complex and differentiated into three groups (group I: alleles 184-194bp; group II: alleles 203-223, 235, 239, 243, 252 and 255bp; group III: alleles 227-286bp). Most of their alleles contained five categories of repeat units (A: aaaag; B: aaag; C: aagg; D: caag; E: agaa). On the other hand, D22S442 contained only two types of repeat units (A: agga; B: aggg). The present study, hence, proves that both D8S580 and D22S442 are highly polymorphic and represent stable genetic markers applicable to forensic investigations.     

Hypervariable locus of the 3'-flanking region of the neurotensin receptor gene: an effective region for personal identification in forensic practice

We examined the complex short tandem repeat (STR) locus at the 3'-flanking region of the neurotensin receptor (NTR) gene. The polymorphism of this locus was first reported as a simple tetranucleotide repeat variation by Le et al., but it also offers a surprisingly informative variation, that permits reliable individual identification by two complementary strategies: fluorescent-labelled polymerase chain reaction (PCR)/electrophoresis and direct sequencing of the PCR products. We determined the alleles in 203 Japanese by fluorescent-labelled PCR/electrophoresis. Determination was based on their length with a reliability of +/-1 bp, and the frequency of each allele was very low. Sequencing analysis further grouped these alleles in detail. Sequencing demonstrated that the locus varied by six repetitive units and three insertion/deletion positions of nucleotide fragments. We detected multiple alleles having different structures even in the same allele length. We found structural differences in homozygous alleles having the same base pair size. We also determined that apparently homozygous alleles were heterozygous from sequencing electropherograms showing an overlap of nucleotides or +/-1 bp difference. These results indicate that this locus is structurally hypervariable in addition to having allelic length variations, promising a great advance in individual identification in forensic practice.     

中国汉族与日本群体DYFl55S1基因座的遗传多态性

目的探讨Y染色体DYF155S1基因座的遗传多态性及群体间差异。方法 应用MVR-PCR、荧光显谱及DNA序列分析技术,对来自中国群体(北方汉族,64例)和日本群体(43例)男性个体的DYF155S1基因座进行初步分析。结果107例样本共检出了5种重复序列类型,包括新的命名为6型的重复序列,它是在1型的基础上T22A置换所形成,仅存在于日本群体,可作为民族特征性遗传标记。2群体重复序列的排列方式以3134顺序为主,在中国和日本群体中各占73.44％和67.44％,是黄种人的特点。134顺序在中国群体中占第二位,为17.19％,6134排列占日本群体的16.28％。3’端的4型重复序列的平均数目在日本群体为8.8条,明显低于中国群体的12.5条。结论DYF155S1基因座具有非常高的遗传多态性和明显的群体差异。     

Genetic variation and population genetic data of the short tandem repeat locus D8S320

The locus D8S320 is an STR system first described in 1993 as a simple (AAAG) repeat. Sequencing data revealed that the D8S320 locus is a complex STR system consisting of (AAAG)- and (AAAC)-repeat units. A total of 22 different alleles were found in a population survey of 210 unrelated individuals from the Rhine area with frequencies ranging from <0.01 to 0.198. The population data revealed the existence of variant alleles differing by 1 and 2bp from the consensus allele. Due to the complex repeat structure consisting of three variable regions and one constant region, electrophoresis under denaturing conditions is strongly recommended. The statistical values were calculated to be 0.89 (observed heterozygosity rate), 0.96 (discrimination index) and 0.71 (mean exclusion chance). No deviation from Hardy-Weinberg equilibrium (HWE) was observed.     

Sequence determination of an allelic ladder for the STR polymorphism at the CD4 locus and application of the ladder in testing an Austrian Caucasian population sample

The short tandem repeat (STR) polymorphism at the CD4 locus, designated HUMCD4. was examined by PCR, native polyacrylamide electrophoresis and subsequent silver staining using an allelic ladder of eight distinguishable alleles occurring in an Austrian Caucasian population sample as a standard size marker. The ladder was produced by pooling equal concentrations of eluted, separately amplified and sequenced alleles, which were previously identified by their different electrophoretical migration. Components of the ladder are in regular intervals of five basepairs. Alleles 4 to 8 were designated according to the number of AAAAG repeat units. The four longer alleles 8′ to 11 showed a stable A to G transition in one of the repeat units and were designated counting the AAAGG unit for a AAAAG. Allele 8′ was not included in the ladder because it showed the same electrophoretic mobility as allele 8. This ladder proved to be a precise and reliable tool in the analysis of 600 chromosomes of the Austrian population. The population investigated showed no deviation from Hardy-Weinberg equilibrium (P = 0.23).     

A D19S433 primer binding site mutation and the frequency in Japanese of the silent allele it causes

Short tandem repeat studies are powerful tools for parentage analysis and for identification of missing persons, victims of murder, and victims of mass fatalities when reference samples are unavailable. The primer in the Identifiler kit failed to amplify an allele at the D19S433 locus, producing a silent ("null") allele. The causal mutation is a base change (G>A) 32 nucleotides downstream from the 3' end of the AAGG repeats. The silent alleles are problematical in parentage analysis because when transmitted, they can cause a parent-child inconsistency that is unrelated to Mendelian genetics. The inconsistency is sometimes termed an "apparent opposite homozygosity" and it produces false evidence of nonparentage. Alternative primers were designed to amplify the D19S433 locus alleles and they detect the silent allele. Frequencies of the (no longer) silent allele were determined to be 0.0114 in 176 people from Shizuoka (Honshu) and 0.0128 in 156 people from Okinawa.     

Genetic diversity at 15 fluorescent-labeled short tandem repeat loci in the Patel and other communities of Gujarat, India

Thirteen tetranucleotide and 2 pentanucleotide repeat units were analyzed in 120 unrelated individuals of Patel and other communities of Gujarat, India. Allele frequency data obtained from the analysis of 15 short tandem repeat markers of the population were found to be satisfying Hardy-Weinberg equilibrium, with marginal deviations. Departures from Hardy-Weinberg equilibrium were observed in Patel communities at locus vWA and for that of the other communities at locus D7S820 and at locus TPOX. The power of discrimination values on an average fall within the range of 0.718 and 0.870, with deviations at locus D3S1358 showing a value of 0.400 for Patels. The value ranged between 0.709 and 0.869, with slight variations among the studied alleles in the other group. Thus, the 15 markers selected for this study were found to be highly suitable in human identification and for providing information on genetic polymorphism of the population of Gujarat.     

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.     

A highly polymorphic STR locus in Cannabis sativa

We report on the first short tandem repeat (STR) locus to be isolated from the plant Cannabis sativa. The STR locus, isolated by a hybrid-capture enrichment procedure, was found to contain a simple sequence repeat motif of 6 bp. This 6 bp repeat motif showed no variation in repeat length but with minor variations in repeat unit sequences. The data show the locus to be highly polymorphic with the number of repeat units ranging from 3 to 40 in 108 screened samples. The observed heterozygosity was approximately 87.04%. The forward and reverse primers (CS1F and CS1R) produced no PCR products in cross-reaction study from 20 species of plants, including highly related species such as Humulus japonicus and Nicotiana tabacum. This hexanucleotide repeat DNA locus could be used to identify cannabis samples and predict their genetic relationship as the test is specific to C. sativa and is highly reproducible.     

Sequence variation in humans and other primates at six short tandem repeat loci used in forensic identity testing

A large number of alleles from the six different short tandem repeat (STR) loci FGA, D3S1358, vWA, CSF1PO, TPOX and TH01, used in human identity testing were sequenced to provide support for the robustness of fluorescent STR DNA typing by allele size. Sequence information for some of these loci (FGA, vWA, TH01) is an extension of published work, whereas no extensive sequence information is available with respect to the D3S1358, CSF1PO, and TPOX loci. Sequencing of alleles at each locus has provided quantitative data with respect to the true nucleotide length of common alleles, and of alleles that vary in length from the common alleles. All alleles that were identified as "off-ladder" alleles through fluorescent typing at these STR loci have proven to be true length variant alleles. Sequencing at the D3S1358 and CSF1PO loci allowed for the establishment of a common nomenclature for these loci. A correlation between percent stutter and the length of the core tandem repeat is demonstrated at the FGA locus. Alleles in which the core tandem repeat is interrupted by a repeat unit of different sequence have a reduced percent stutter. DNA samples from three non-human primates (chimpanzee, orangutan, and gorilla) were compared to the human sequences, and shown to differ markedly across loci with respect to their homology. The effects of primer binding site mutations on the amplification efficiency at a particular locus, and methods used to interpret amplification imbalance of heterozygous alleles at a locus is also addressed.     

Importance of storing emergency serum samples for uncovering murder with insulin

Using the polymerase chain reaction (PCR), we studied the short tandem repeat (STR) polymorphism observed at the D12S391 locus. In 350 Japanese examined, 14 different alleles ranging from 209 bp to 261 bp were detected. Allele 18 (221 bp) showed the highest frequency at 0.30. Observed and expected values of respective genotypes satisfied the Hardy-Weinberg equilibrium (chi 2 = 24.08, P = 0.24, df = 20). In addition, 18 additional sequence structures (suballeles), were detected in this study. Within the suballeles, sequence variants, in which the initial repeat of (AGAT) was replaced with (AGGT), was found in five samples. It was found that the analysis of single-strand conformation polymorphism (SSCP) before sequence analysis was useful for distinguishing these suballeles.     

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).     

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.     

PowerPlex^TM16体系OL等位基因序列分析及命名探讨

目的观察中国汉族人群PowerPlexTM16体系STR基因座分型标准物外等位基因(OL等位基因)的序列组成,探讨其类型及命名。方法应用PowerPlexTM16体系和ABI377或3100遗传分析仪,对10071名中国汉族无关个体的血样DNA进行15个STR基因座的分型,筛选出OL等位基因样本;对该样本进行单基因座扩增、聚丙稀酰胺凝胶电泳、银染显色,获取等位基因条带并再次扩增和测序。结果在11个基因座检见OL等位基因,共32个,频率0.05‰￣4.02‰,各基因座OL等位基因数目1￣9个不等。按其组成分为四类:(1)重复单位完整重复,但重复次数在ladder范围外;(2)不完整重复;(3)侧翼序列个别碱基的插入或缺失;(4)较大片断的缺失。结论OL等位基因类型不一,既有重复次数的变化,也有侧翼序列或核心序列的变化,现有命名原则尚不能反映其组成类型。     

Sequence variation at three X chromosomal short tandem repeats in Caucasian and African populations

Sequence variation for the X chromosome short tandem repeats (X-STRs) DXS9898, DXS6789 and GATA172D05 was studied in two major population groups, namely Caucasians and Africans. DXS6789 revealed two different subtype sequence polymorphisms: for shorter alleles, with less than 17 repeats, results showed a simple composition with the following structure: (TATG)_m-(TATC)_n. For longer alleles, a constant TATC insertion was observed at the beginning of the variable repeat unit. Additionally, alleles identical in size revealed structural variations regarding the TATG/TATC proportion. Africans showed a higher intra-allelic variation at this locus than the Caucasian population group. For all three loci, DXS9898, DXS6789 and GATA172D05, no unique structure was found among Africans and Caucasians.     

Frequencies of D8S384 alleles and genotypes in European, African-American, Chinese, and Japanese populations

D8S384 is a tetranucleotide tandem repeat locus. In order to evaluate the forensic validation of D8S384, the genotype distributions and allele frequencies in ten populations from three main ethnic groups were investigated, including Germans, Slovakians, African Americans, Japanese, and Chinese (Jilin, Guangzhou, Nanning, Hailaer, Dali, and Chengdu). A total of 1011 unrelated individuals, 41 pedigrees, 30 disputed paternity trios and three personal identification cases were analyzed for D8S384 by Amp-FLP technique. Many kinds of tissues, body fluids, secreta and stains have been tested. The alleles were determined by comparison with a human allele ladder. The results showed that D8S384 typing was both precise and reliable. There were eight alleles in these populations. The genotype distributions conformed to Hardy-Weinberg equilibrium predictions. No mutation events were observed. With a maximum likelihood method, the mutation rate was indirectly estimated as 2.14 x 10(-5). The heterozygosity was 0.704 +/- 0.014 at D8S384 locus. All these results suggest that D8S384 locus is a useful marker for forensic identification and paternity analysis.     

Sequence analysis of STR polymorphisms at locus ACTBP2 in the Taiwanese population

A highly polymorphic sequence structure is reported in the human beta-actin related pseudogene 2 (ACTBP2) (SE33) locus in members of the Taiwanese Han population. A total of 100 unrelated members of the Taiwanese Han population were used in the study. Alleles that shared the same size but differ in their sequence are described to allow for inter laboratory sharing of data. PCR products amplified from this locus were separated by single-strand conformation polymorphism electrophoresis, the single-stranded DNA bands were excised from the gels, a second amplification performed, and then the PCR products were sequenced. All the alleles differed by either 2 or 4 bp. Sequence variations were observed as deletions or insertions in the repeat units AG (or AA) and AAAG. Additionally, transitions in the flanking regions were recorded. A total of 27 alleles with 71 associated genotypes were recorded if the alleles were defined by size, but 68 alleles with 88 associated genotypes were noted with the alleles were scored on the basis of sequence variation. The power of discrimination (Pd) of this single locus was 0.9874 making the human ACTBP2 a good alternative marker for individual identification and paternity testing.     

Variant alleles on the penta E locus in the PowerPlex 16 kit

Penta E in the PowerPlex 16 kit is a pentanucleotide tandem repeat marker located on Chromosome 15, containing an AAAGA repeat motif. Variant alleles (18.4 and 19.4) were found in the Japanese population. A sequence analysis revealed that both the variant alleles had a partial repeat motif of AAAA, resulting in one-base-shorter alleles compared to known alleles. Despite the relatively large amplicon sizes (379 to 474 bp) of Penta E, an accurate allele assignment can be reliably made by capillary electrophoresis. However, alleles differing in size by only one base (e.g., 18.4 and 19) were not separated and appeared as a single broad peak. The Genotyper software assigned one of the component alleles to this peak. Therefore, such broad peaks require careful interpretation so as to not overlook the other component allele contained by the peak. As an index to recognize a peak containing two alleles, the ratio of peak area to peak height was found to be useful.     

Sequence analysis of STR polymorphisms at locus ACTBP2 in the Taiwanese population

A highly polymorphic sequence structure is reported in the human beta-actin related pseudogene 2 (ACTBP2) (SE33) locus in members of the Taiwanese Han population. A total of 100 unrelated members of the Taiwanese Han population were used in the study. Alleles that shared the same size but differ in their sequence are described to allow for inter laboratory sharing of data. PCR products amplified from this locus were separated by single-strand conformation polymorphism electrophoresis, the single-stranded DNA bands were excised from the gels, a second amplification performed, and then the PCR products were sequenced. All the alleles differed by either 2 or 4 bp. Sequence variations were observed as deletions or insertions in the repeat units AG (or AA) and AAAG. Additionally, transitions in the flanking regions were recorded. A total of 27 alleles with 71 associated genotypes were recorded if the alleles were defined by size, but 68 alleles with 88 associated genotypes were noted with the alleles were scored on the basis of sequence variation. The power of discrimination (Pd) of this single locus was 0.9874 making the human ACTBP2 a good alternative marker for individual identification and paternity testing.     

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。