Robustness of the Y STRs DYS19, DYS389 I and II, DYS390 and DYS393: optimization of a PCR pentaplex

Various technical methods were investigated with the aim of developing a multiplex system to amplify five Y-chromosome STR loci in the same PCR reaction: DYS393, DYS19, DYS390, DYS389 I and DYS389 II. A sequenced allelic ladder was constructed with previously sequenced alleles including the most common ones. A number of reamplification conditions of the allelic ladders were tested. The pentaplex was evaluated for typing using two different platforms (ABI and ALF) with promising results. However, in degraded samples non-specific artifacts were observed in the DYS393 system in the same range of sizes as the real alleles. This system can also be typed in females under relatively low stringency conditions in the PCR amplification, making this system prone to errors in critical samples. This lack of specificity can be reduced by increasing the stringency of the PCR conditions. The DYS19 ladder cannot be reamplified as stutters appear after a few reamplifications. These stutters are probably due to a 2 bp slippage induced by the presence of a TA repeat stretch in the PCR amplified fragments. Non-specific products were also noted in the DYS389 I and DYS389 II amplification, although out of the range of other alleles in this pentaplex. This newly constructed pentaplex has proved to be very useful in population genetic studies because all five Y STR markers can be loaded in the same lane of a gel with other Y STR singleplex or multiplexes. The usefulness of Y-chromosome STRs in criminal casework is especially evident in analyzing azoospermic individuals.     

Population studies of the Y-chromosome specific polymorphisms DYS19, DYS389 I + II, DYS390 and DYS393 in a western German population (Bonn area).

Population studies were carried out on the Y-specific short tandem repeat (STR) systems DYS19, DYS389I + II, DYS390 and DYS393 in a Western German population sample. Determination of the allele frequencies revealed for all these systems, unimodal distribution. The number of observed alleles varied: five for DYS19, six for DYS390, three for DYS389I, seven for DYS389II and six for DYS393. In 102 unrelated male individuals, 56 different haplotypes were found. The haplotype diversity values were similar to those of other European populations.     

DYS385检测方法的改进

目的建立检测DYS385的新方法。方法比较基因数据库(GDB)中推荐的引物和Schneider所设计的引物扩增DYS385的效果;在此基础上,以GDB推荐的引物作为外引物,Schneider所设计的引物作为内引物,通过调整内、外引物的浓度,优化扩增条件,建立DYS385的半巢式PCR体系。结果与常规方法相比,采用Schneider所设计的引物扩增DYS385,扩增片段缩短112bp,电泳分离的效果好,灵敏度提高2倍,达500pg;建立的半巢式扩增方法,能特异性扩增短片段,灵敏度提高20倍,达50pg。结论建立的DYS385半巢式扩增方法具有更高的特异性和灵敏度,适合法医学应用。     

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.     

Results of the GEP-ISFG collaborative study on the Y chromosome STRs GATA A10, GATA C4, GATA H4, DYS437, DYS438, DYS439, DYS460 and DYS461: population data

The Spanish and Portuguese ISFG Working Group (GEP-ISFG) carried out a collaborative exercise in order to asses the performance of two Y chromosome STR tetraplexes, which include the loci DYS461, GATA C4, DYS437 and DYS438 (GEPY I), and DYS460, GATA A10, GATA H4 and DYS439 (GEPY II). The groups that reported correct results in all the systems were also asked to analyse a population sample in order to evaluate the informative content of these STRs in different populations. A total of 1020 males out of 13 population samples from Argentina, Brazil, Costa Rica, Macao, Mozambique, Portugal and Spain were analysed for all the loci included in the present study. Haplotype and allele frequencies of these eight Y-STRs were estimated in all samples. The lowest haplotype diversity was found in the Lara (Argentina) population (95.44%) and the highest (99.90%) in Macao (China). Pairwise haplotype analysis showed the relative homogeneity of the Iberian origin samples, in accordance with what was previously found in the European populations for other Y-STR haplotypes (http://www.ystr.org). As expected, the four non-Caucasian samples, Macao (Chinese), Mozambique (Africans), Costa Rica (Africans) and Argentina (Lara, Amerindians), show highly significant Phist values in the pairwise comparisons with all the Caucasian samples.     

武汉汉族群体3个多拷贝Y-STR基因座的遗传多态性

目的提供DYS385、DYS459和DYS464基因座的群体遗传学资料。方法用荧光标记引物及ABI 3100型基因分析仪对武汉地区176名汉族男性无关个体的DYS385、DYS459和DYS464 3个多拷贝Y-STR基因座进行分型。结果在DYS385和DYS459基因座的个体,可观察到1~2个不同长度的扩增产物;DYS464基因座个体,可观察到1~4个不同长度的扩增产物。DYS385基因座检出14个等位基因及47种单倍型,DYS459检出4个等位基因及7种单倍型,DYS464检出9个等位基因及51种单倍型,其单倍型多样性分别为0.9591、0.6047和0.9560。3个基因座构成的联合单倍型共有133种,其多样性值达0.9909。结论3个多拷贝Y-STR基因座均为高多态性的遗传标记,联合应用具有较高的个体分辨能力。     

Results of the GEP-ISFG collaborative study on two Y-STRs tetraplexes: GEPY I (DYS461, GATA C4, DYS437 and DYS438) and GEPY II (DYS460, GATA A10, GATA H4 and DYS439)

A collaborative exercise was carried out by the Spanish and Portuguese ISFG Working Group (GEP-ISFG) in order to evaluate the performance of two Y-chromosome STR PCR tetraplexes, which include the loci DYS461, GATA C4, DYS437 and DYS438 (GEPY I), and DYS460, GATA A10, GATA H4 and DYS439 (GEPY II). The participating laboratories were asked to type three samples for the eight markers, using a specific amplification protocol. In addition, two control samples, with known haplotypes, were provided. The results obtained by the 13 different participating laboratories were identical, except for two laboratories that failed to type correctly the same two samples for GATA C4. By sequence analyses, two different GATA C4 allele structures were found. One control sample (allele 21) and two questioned samples (allele 22, correctly typed by all the laboratories, and allele 25) presented the following repeat structure: (TCTA)4(TGTA)2(TCTA)2(TGTA)2(TCTA)n, but different from the one found for allele 26 in one sample included in this exercise, as well as in the second control sample (allele 23), namely (TCTA)4(TGTA)2(TCTA)2(TGTA)2(TCTA)2(TGTA)2(TCTA)n. The collaborative exercise results proved that both Y-tetraplexes produce good amplification results, with the advantage of being efficiently typed using different separation and detection methodologies. However, since GATA C4 repeat presents a complex structure, with alleles differing in sequence structure, efficient denaturing conditions should be followed in order to avoid typing errors due to sizing problems.     

DXYS267: DYS393 and its X chromosome counterpart

The GATA repeat DYS393 was reported in 1987 among other Y-specific short tandem repeats. It has since been used for forensic and evolutionary studies. We decided to test its Y-specificity when we found that female DNA gave amplicons, in agreement with recent GDB-recorded experiences on radiation hybrids. Parent-child triplets revealed that heterozygous daughters always carried the same paternally derived amplicon which, however, was not amplified in their fathers' DNAs. The X-assignment was verified in larger families. A half-new primer set with a new reverse DYS393 primer, outside the old one, resulted in X amplicons in females as well as Y and X amplicons in males. This new primer set defines the new DXYS267 (GDB Data Curation). DNA-sequencing revealed four base pair differences between the Y- and the X-sequences. Two are within the reverse primer site sequence, thus probably causing preferential hybridization to the Y sequence when using the conventional primers. The two others are within the repeat array, giving the regular repeat GATA in the Y-sequence, and TATA and GACA, respectively, in the X-sequence. Allele frequency distribution in DYS393 was studied in 300 unrelated Norwegian males, allele distribution in the X-locus in 48 Norwegian women. Even if allele repeat numbers are overlapping between the loci, leading to identical fragment lengths, the allele distribution is different between DYS393 and the X-chromosome locus. The differences between the two homologous loci on the Y and X indicate a considerable lap of time since common ancestry. To avoid co-amplification of the X-locus in DYS393 typing, primer A was elongated to include one of the sequence differences between the two loci. This to a considerable extent improved the specificity of the DYS393 primers.     

Results of a collaborative study of the EDNAP group regarding the reproducibility and robustness of the Y-chromosome STRs DYS19, DYS389 I and II, DYS390 and DYS393 in a PCR pentaplex format

A collaborative exercise was carried out by the European DNA Profiling Group (EDNAP) in the frame work of the STADNAP program, i.e. standardization of DNA profiling in Europe, in order to evaluate the performance of a Y-chromosome STR pentaplex, which includes the loci DYS19, DYS389 I and II, DYS390 and DYS393 and to determine whether uniformity of results could be achieved among different European laboratories.Laboratories were asked to analyze the five Y-STRs using singleplex and multiplex conditions in three bloodstains and one mixed stain (95% female and 5% male).All the laboratories reported the same results even for the mixed stain included in the exercise. This demonstrates the reproducibility and robustness of Y-chromosome STR typing even with multiplex formats and proves the usefulness of Y-STR systems for analyzing mixed stains with a male component.A total of 930 male samples from 10 different populations from Europe were also analysed for all the loci included in the pentaplex. Eight of these ten populations also included haplotype data.As for single gene analysis, haplotype diversity was higher in Germany and Italy and lower in Western European countries and Finland.Pairwise haplotype analysis shows the Finnish departure from the rest of the populations and a relatively homogeneity in the other European populations with F(ST) estimates lower than 0.05.UPGMA analysis shows an association of Western European population (Ireland, UK, Portugal and Galicia) on the one hand and central European populations on the other.     

Population data of new Y-chromosome STRs GATA C4, DYS438, DYS437, GATA A7.2, GATA H4, DYS439 and GATA A10 in males from Colombia

Two hundred twenty-five unrelated males were typed for 7 over 8 loci Y-chromosome STRs proposed in a collaborative study by The Spanish and Portuguese ISFG Working Group. The markers amplification were in two multiplex reactions GEPY I with GATA C4, DYS438, DYS437, DYS461 (GATA A7.2) and GEPY II with GATA H4, DYS439, GATA A10 and DYS460 (GATA A7.1). All gene diversities were upper 0.5 with the highest value in DYS439 with 0.64. Furthermore, 152 haplotypes from 7 loci Y-chromosome STRs were found within studied population and a high haplotype diversity 0.9902 was found. The DYS460 (GATA A7.1) marker can not be studied because its diverse alleles were not able for interpret.     

Sequence characterization of microvariant alleles at DYS627 and DYS458

Y chromosome short tandem repeats (Y-STRs) have been widely used in genetic applications and forensic casework. Recently, we found two intermediate alleles, the DYS627 allele 24.1 and the DYS458 allele 15.3, from Chinese Han population. The two allelic variants have not been recorded by the YHRD database. We have examined the molecular structure of these allelic variants by Sanger sequencing. The results showed that this intermediate allele at DYS627 was confirmed as 24.1, the sequence of which showed a base “A” insertion in the 13th repeat unit, and the intermediate allele at DYS458 was confirmed as 15.3, the sequence of which showed a base “G” deletion in the 12th repeat unit. This may be important for individual identification and paternal kinship testing. Besides, more allelic variants detected can be enriched in the Y-STR database.     

Population data of Galicia (NW Spain) on the new Y-STRs DYS437, DYS438, DYS439, GATA A10, GATA A7.1, GATA A7.2, GATA C4 and GATA H4

Haplotype, allele frequencies and population data of eight Y-chromosome STR loci, DYS437, DYS438, DYS439, GATA A10, GATA A7.1, GATA A7.2, GATA C4 and GATA H4, were determined from a sample of 212 unrelated male individuals from Galicia (NW of Spain).     

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.     

DYS391、DYS393基因座X染色体扩增对法医鉴定影响的探讨

目的探讨提高DYS391和DYS393基因座特异性扩增方法及两基因座的X染色体扩增产物对法医学鉴定结论的影响。方法DYS391和DYS393基因座采用PCR扩增、聚丙烯酰胺凝胶电泳结合银染进行分析。结果稀释模板DNA浓度和提高退火温度对提高此两Y染色体基因座特异性扩增并不明显。DYS391和DYS393的X染色体扩增产物对法医学鉴定结论有误导可能性。结论在法医学鉴定尤其是性别鉴定中应谨慎应用DYS391和DYS393基因座。     

DYS393多态性分析及其法医学应用

探讨了Y-DNASTR基因座DYS393的多态性及其法医学应用价值,用变性PAGE结合荧光DNA自动测序仪分析及非变性PAGE,结合银染显示两种PCR扩增产物的方法,并调查了广州地区120例汉族无关男性个体DYS393等位基因分布状况;在此基础上,建立了DYS393和HumARA两基因座的复合扩增体系,结果显示DYS393基因座共检出4种等位基因,复合扩增体系可同时提供性别鉴定和个体识别的信息.     

DYS STR analysis with epithelial cells in a rape case

We report here the application of Y-chromosomal DNA analysis in a rape case, which occurred in Stuttgart, Germany. Microscopic examination of the victim's vaginal swabs and her underwear showed no sperm cells. DNA was extracted from vaginal and epithelial cells and analysed with the autosomal systems SE33, THO1 (singleplex) and with the multiplex Profiler Plus (Applied Biosystems, Foster City, USA). The results of these autosomal STR analysis gave no hint at a mixed sample and failed to identify a male profile. DYS STR analysis with the systems DYS391, DYS392, DYS393, DYS19 and DYS389 I/II showed the same characteristic features as the suspect. We used this incomplete haplotype to search in the Y-STR Haplotype Reference Database via Internet. In a Caucasian population sample of 3589 minimal haplotypes we found 71 matches. The suspect confessed the crime and was finally condemned to 4 years imprisonment.     

Polymorphism data at DYS385 locus in five ethnic groups from Kerala in Southern West India

POPULATION: We have studied the DNA polymorphism at DYS385, a Y-chromosomal tetranucleotide repeat locus among five anthropologically distinct ethnic groups of Kerala state in Southern West India. The ethnic groups were Ezhavas, Muslims, Nairs, Arayas and Thandans and they speak "Malayalam." an Indo-Dravidian language. Peripheral blood samples were collected from 72 random, healthy and normal male volunteers for this study.     

A DYS438 null allele observed in two generations of a large family

Typing of Y-chromosomal STR loci using the AmpFISTR^® Yfiler kit showed a DNA profile lacking the DYS438 allele in an Austrian Caucasoid brother pair.Buccal swabs were collected from additional males of two generations and different branches of the family. All family members investigated did not show a DYS438 allele in their Yfiler profile. Using the Powerplex^® Y system an allele with dramatically reduced peak height was amplified. Subsequent sequencing of the DYS438 locus exhibited a transition upstream of the repetitive region.     

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.