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.     

A new allele of the short tandem repeat (STR) locus, CSF1PO.

CSF1PO is one of the thirteen core loci used for the CODIS database, and alleles reported for this short tandem repeat (STR) locus contain from 6 to 15 repeats of the tetranucleotide AGAT. Screening of DNA from 76 individuals by gel electrophoresis and silver stain detection yielded one sample that contained a rare, off-ladder CSF1PO allele; an allele larger than CSF1PO15 was detected in a heterozygote that also contained a CSF1PO10 allele. Capillary electrophoresis analysis using GeneScan software demonstrated that the variant allele contained four bases more than CSF1PO15. Following agarose gel electrophoresis to separate the two alleles of the heterozygote and cycle sequencing using dye terminators, sequence analysis showed that the variant, which was otherwise identical to the CSF1PO GenBank sequence, contained exactly 16 AGAT repeats. These results demonstrate the existence of an additional CSF1PO allele, a previously unreported size variant, CSF1PO16.     

Understanding the behavior of stutter through the sequencing of STR alleles

This work explores the influence of several variables on stutter formation across sequenced autosomal STR loci (simple, compound, and complex motifs) and different alleles within each locus. The variables are sequence variations within the repeating motifs and flanking region [1,2]; longest uninterrupted stretch (LUS) [3]; parental allele length [3]; and base pair content and length value of each repeating motif from which the stutter has generated [3,4]. Over six hundred unrelated individuals from different populations were amplified with the prototype PowerSeq 46GY System and sequenced on the Illumina MiSeq platform. Raw FASTQ files were analyzed with STRait Razor v3 [5]. Stutter ratio was calculated for motifs that exhibited stutter using the ratio of the observed coverage of the stutter sequence at (N-1) position to the observed coverage of the allelic sequence. Understanding the behavior (abundance, reproducibility, sequence context) of non-allelic artifacts will help in establishing probabilistic models for the prediction of stutter rate and interpretation of sequence-based STR profiles.     

PCR typing of DNA fragments of the two short tandem repeat (STR) systems upstream of the human myelin basic protein (MBP) gene in Danes and Greenland Eskimos

DNA from the double short tandem repeat (STR) system MBP (locus 18q23-pter) was amplified by the polymerase chain reaction (PCR) and the two polymorphic repeat systems were separated by cutting with the restriction enzyme NlaIII. The lengths of the DNA fragments of the two MBP STR systems MBP-A and MBP-B were analyzed by vertical electrophoresis in polyacrylamide gels followed by silver staining. DNA samples from 112 unrelated Danes, 140 unrelated Greenland Eskimos, and 88 Danish mother/child pairs were analyzed. The distributions of MBP phenotypes were in Hardy-Weinberg equilibrium in both the Eskimo and Danish populations. Significant differences were observed between the distribution of fragments (‘alleles’) in Greenland Eskimos and in Danes. The allele MBP-A7 was considerably more frequent in Eskimos (0.2214) than in Danes (0.0775) and also the allele MBP-B9 was considerably more frequent in Eskimos (0.225) than in Danes (0.06). Strong gametic associations were found between fragments from the MBP-A and MBP-B series in both Danes and Eskimos. Some of the associations were different in Danes and Eskimos. In the 88 Danish mother/child pairs, the segregation of the MBP genotypes were in accordance with a genetic model of co-dominant inheritance and no mutation was found. Two MBP STR regions with irregular structures were sequenced. One fragment had a single base G to A transition at position 124 in the primer binding region between the MBP-A and MBP-B regions. In the other fragment, a deletion starting at position 117 and including the primer binding region between the MBP-A and MBP-B regions was found.     

Amplification of a variable number of tandem repeats (VNTR) locus (pMCT118) by the polymerase chain reaction (PCR) and its application to forensic science

A genetic locus (D1S58, defined by DNA probe pMCT118) that contains a variable number of tandem repeats (VNTR) has been successfully amplified from a very small amount of genomic deoxyribonucleic acid (DNA) by the polymerase chain reaction (PCR). The DNA sequence of the locus was determined and was found to consist of a 16-base consensus sequence and flanking sequences. Oligonucleotide primers complementary to the flanking sequences were synthesized to serve as primers for amplification of MCT118 by the PCR method. Human genomic DNA isolated from blood (2 ng from each sample) was successfully amplified at the MCT118 locus, and polymorphic bands were detectable by ethidium bromide staining after electrophoresis on polyacrylamide gels. Determination of genotypes at this VNTR locus can now be routinely achieved within 24 h, without the need for Southern blots or radioactive materials. Furthermore, the small size (387 to 723 base pairs) of the DNA fragments produced in the PCR amplification permits good resolution of individual alleles that differ by only one repeat unit. The precise specification of the number of tandem repeats present in each allelic fragment is reproducible from one analysis to another.     

KCNQ1内含子1a中STR基因座遗传多态性及PIA分型

目的调查汉族群体KCNQ1基因内含子1a中STR基因座的遗传多态性,并采用PIA分型技术确定等位基因的亲代来源。方法用PCR-STR分型技术对230例武汉汉族无关个体样本进行KCNQ1基因内含子1a中STR基因座分型检测;同时选用两种甲基化敏感的限制酶（msRE）HhaI和HpaⅡ对家系中孩子的基因组DNA进行消化后,采用PIA分型技术检测父源等位基因。结果KCNQ1内含子1a中STR基因座在汉族人群中检出10个等位基因、24种基因型,其个体识别能力（PD）、多态性信息含量（PIC）和非父排除率（PE）分别为0．852、0．66和0．484。HhaI和HpaII可消化个体的母源等位基因,PIA分型仅能检测出单一的父源等位基因。结论KCNQ1内含子1a中STR基因座在汉族群体具有较高的遗传多态性,PIA分型技术可以确定个体等位基因的亲代来源,具有较高的法医学应用价值。     

A proposal for standardization in forensic canine DNA typing: allele nomenclature of six canine-specific STR loci

In this study a proposal for the allele nomenclature of six polymorphic short tandem repeat (STR) loci (PEZ3, PEZ6, PEZ8, PEZ10, FHC2161, and FHC2328) for canine genotyping (Canis lupus familiaris) is presented. The nomenclature is based on the sequence data of the polymorphic region of the microsatellite markers as recommended by the DNA commission of the International Society of Forensic Haemogenetics (ISFH) in 1994 for human DNA typing. To cover commonly and rarely occurring alleles, a selection of homozygous and heterozygous animals were analyzed and subjected to sequence studies. The alleles consisted of simple tri- and tetra-nucleotide repeat patterns as well as compound and highly complex repeat patterns. Several alleles revealing the same fragment size but different repeat structures were found. The allele designation described here was adopted to the number of repeats, including all variable regions within the amplified fragment. In a second step the most commonly occurring alleles were added to an allelic ladder for each marker allowing a reliable typing of all alleles differing in size. A total number of 142 unrelated dogs from surrounding municipal animal homes, private households, and canines in police duty were analyzed. The data were added to a population database providing allele frequencies for each marker.     

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.     

Subtyping of D20S85 STR alleles by single-strand conformation polymorphism (SSCP) analysis

During a population study of STR locus D20S85, we discovered two types of sequence variations by direct sequencing of the alleles: two transitions each of G to A and A to G occur in the 5′ flanking region in the individuals possessing allele 6 and some of those possessing allele 7 [1]. Using single-strand conformation polymorphism (SSCP) analysis, we were able to distinguish two subtypes of allele 7 from each other. This analysis method enables rapid screening for STR alleles of the same length with different sequences, and should find application to other complex STR loci because of the practical advantage of simplicity in comparison to sequencing.     

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.     

The analysis of three short tandem repeat (STR) loci in the Slovene population by multiplex PCR

Allele frequencies for three tetrameric short tandem repeat (STR) loci D3S1358, HUMVWA, and HUMFGA were determined in a Slovene Caucasian population sample. DNA samples from a total of 221 Slovenes were amplified by multiplex PCR using the commercial kit AmpFISTR Blue (Perkin-Elmer). Separation and detection of the amplified STR fragments were carried out using a 377 automated genetic analyzer (Applied Biosystem Division/Perkin Elmer). Seven alleles at the D3S1358 locus, 8 alleles at the HUMVWA31A locus, and 13 alleles at the HUMFGA locus were observed. A deviation from Hardy-Weinberg equilibrium was observed, only at the HUMVWA31A locus (p = 0.045, exact test). The departure at this locus was not significant after Bonferroni correction. There were no detectable departures between pairwise comparisons of the loci. The combined power of discrimination for all three loci is 0.9998, and the power of exclusion is 0.9526. The observed allele frequencies for the loci D3S1358, HUMVWA31A, and HUMFGA are similar to those in European and U.S. Caucasian populations.     

印记基因KCNQ1的遗传多态性及在亲权鉴定中的应用

目的为了调查印记基因KCNQ1的STR位点在中国汉族人群中的遗传多态性，利用亲源印记等位基因（parentally imprinting allele，PIA）分型法确定孩子的等位基因亲代来源，为亲权鉴定提供新的侯选STR位点。方法应用Chelex法提取153例佳木斯地区汉族健康无血缘关系个体DNA，用QIAamp Blood Kit（Qiagen）法提取3个家庭10个个体DNA，PCR扩增，凝胶电泳分型，ABIPRISM^TM 3730XL DNA测序仪测序；甲基化敏感性限制性内切酶消化孩子基因组DNA，PCR扩增，确定孩子等位基因的亲代来源。结果发现在中国佳木斯地区汉族人群中KCNQ1基因的STR有7个等位基因，多态信息含量为0．662，且KCNQ1基因的STR位点呈父源印记。结论印记基因KCNQ1的STR位点有很好的多态性，可为亲权鉴定提供新的侯选遗传标记，其亲源特异性甲基化标记有望应用于单亲鉴定中。     

Alternative primers for DYS391 typing: advantages of their application to forensic genetics

The amplification of the STR DYS391, using the primers described in the Genome Data Base (GDB: G00-365-251), shows not only an additional band to the Y-specific one in males with a size range of 26 bp less than those of DYS391 locus alleles, but also a polymorphic pattern in females in the same size range as the additional band observed in males. The DYS391 pattern in families reflects a Y-specific linked locus and also a polymorphic X locus with an X-linked pattern of inheritance. A first screening in the X homologous locus allowed the identification of five different alleles. Allele frequencies were explored in different population groups for both the Y locus and the homologous locus in the X chromosome showing a similar allele distribution pattern in the X and Y homologous loci. An alternative reverse primer was designed to amplify the Y-chromosome specific STR in order to improve the specificity and applicability of this system to forensic genetics. Comparative results of the amplification with the new and the previously described primers proved that with this new primer there is a substantial increase in the specificity of the amplification. Moreover, a smaller fragment is amplified with a size out of the range of the alleles of the other Y-STRs usually used in forensic applications, therefore simplifying its inclusion in multiplex systems.     

成都汉族、甘肃东乡族群体D10S1432和D10S1213 位点的遗传多态性分析

目的本研究的目的是了解人类基因组中D10S1432及D10S1213两个STR位点在成都汉族和甘肃东乡族群体中的遗传多态性分布及两个群体之间的关系。方法采用PCR、聚丙烯酰胺凝胶电泳及银染技术,共调查了209例样本。结果在D10S1432位点上观察到5个等位基因,15种基因型。在D10S1213位点上观察到9个等位基因,31种基因型。两位点的基因型频率在调查的两个群体中的分布符合Hardy-Weinberg平衡定律（P>0.05）。经统计,D10S1432在这两个群体中的杂合度为0.664和0.737,个人识别几率为0.827和0.820。D10S1213的杂合度为0.664和0.657,个人识别几率为0.836和0.882。结论结果表明,D10S1432和D10S1213两个位点在法医学个人识别和亲子鉴定中有较高应用价值。     

Additional variability at the D12S391 STR locus in an Austrian population sample: sequencing data and allele distribution

The highly polymorphic STR locus D12S391 was investigated in an Austrian population sample (N=150) by PCR-amplification, comparative detection on native and denaturing polyacrylamide gels and solid phase single stranded sequencing of three size variant alleles and several additional alleles. A total of 15 alleles, distinguishable by size under denaturing conditions, could be detected. No deviations from Hardy-Weinberg equilibrium were observed in the population investigated (P=0.52). Sequencing of size variants designated 17.3 and 18.3 showed an incomplete (GAT) repeat unit at position two of the tandem region. Additional new sequence variants due to varying compositions of the number of (AGAT) and (AGAC) repeats could be identified. Due to distinct electrophoretical mobilities of alleles of the same size but different sequence structures, denaturing detection conditions should be employed when the aim is standardization.     

Analysis of eight STR loci in two Hungarian populations

A collection of eight STR loci (D3S1358, FGA, D8S1179, D21S11, D18S51, D5S818, D13S317, D7S820) was used to generate allele frequency databases for two Hungarian population samples, Caucasians from the Budapest area and Romanies from Baranya county. During the analysis two intermediate sized alleles and a sequence variant allele were observed at the D7S820 locus. All three types of allelic variants were found to have modification (deletion, insertion, transversion) in the same block of a (T)(9) stretch located within the 3' flanking region of each allele, which may indicate a possible higher mutation rate of this (T)(9) block. For the loci D3S1358 and D7S820 the Romany population database showed departures from Hardy-Weinberg equilibrium. The forensic efficiency values for the Romany population were slightly different from those found in the Hungarian Caucasian population. Comparing the allele frequency values by G-statistic, calculating the F(st) indices and with the pairwise comparisons of inter-population variance, the two Hungarian populations could be distinguished using data of the eight STR loci.     

Characterization of deletions in the DYS385 flanking region and null alleles associated with AZFc microdeletions in Koreans

Eight DYS385 allele size discrepancies and six DYS448 null types were detected among 708 Korean men when results of three in-house multiplex short tandem repeat (STR) systems were compared. The systems included both ordinary and reduced size amplicons. Sequence analysis revealed deletion mutations at two sites upstream of the DYS385 core repeats and deletion of the entire DYS448 locus. At DYS385, allele size differences were one or two repeats and were dependent on the primer set used for polymerase chain reaction (PCR) amplification. Location of the primer target sequence in a flanking region of the STR, distal or proximal to the deletion, determined allele size. Two widely used commercial kits amplify DYS385 so as to include the mutable sites. Arrangement analysis of sequence tagged sites demonstrated that the deletion patterns at DYS448 (and DYS464) were associated with arrangements of the azoospermia factor c gene (AZFc). The DYS448 deletion appears relatively frequent in Asians.     

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.     

Y染色体OSU49基因座遗传多态性分析

目的调查Y染色体OSU49基因座在河南汉族群体中的遗传多态性,评价其法医学应用参数。方法知情同意情况下,采集300名河南汉族男性个体的血样本,荧光标记PCR,扩增产物采用ABI 3130遗传分析仪检测。根据分型检测结果,对不同等位基因进行序列分析(测序)。结果 OSU49基因座包含五核苷酸和四核苷酸两种核心序列。在河南汉族群体中基序表现为:(CTTTC)pCTT(CCCT)7T(CTTTC)1(TCTT)5(TCCT)m(TCTT)n TCT(TCCT)4,五核苷酸核心序列的重复次数为12~17,四核苷酸核心序列的重复次数为20~30,按其片段长度命名等位基因,共发现34个等位基因,GD值为0.918 6,DP值为0.915 5。结论 Y染色体OSU49基因座序列结构复杂,在河南汉族群体中具有较高的遗传多态性,可应用于法医学和人类遗传学研究中。