Technical note: multi-alleles at the DYS385ab locus with high frequency in a Han Chinese population from southwestern China.

Y-chromosome short tandem repeat (Y-STR) markers have been widely used in forensic applications and usually show monoallelic or diallelic genotypic patterns at certain double-copied loci. In this study, we have found 13 samples among 703 males with multi-alleles at the DYS385ab locus, including one with five mutant alleles, nine with four, and three with three. The frequency of abnormal DYS385ab genotypes was 1.85% (13/703), which is very high in the Han Chinese population. The percentage of samples with diallelic patterns at DYS385ab was higher than that of monoallelic patterns (80.23% vs. 17.92%). Additionally, the percentage of samples with tetra-allelic patterns at DYS385ab was higher than that of tri-allelic patterns (1.28% vs. 0.43%), suggesting that there are possibly two copies with duplicated events happening frequently on the Y chromosome. Interestingly, the peak height of allele 13 was two to three-folds higher than that of other alleles. The allele 18 peak height was also two-fold higher than others, which could potentially be explained by a duplication event mechanism. We also found that tri-allelic genotypes for alleles 13, 17, and 20, tetra-allelic genotypes for alleles 13, 14, 19, and 20, and tetra-allelic genotypes for alleles 12, 13, 19 and 21 were more common than others. Furthermore, all 13 samples had multi-alleles containing allele 13, implying a founder effect in this particular Chinese-specific ethnic group. Taken together, this study provides new information for this population and will be useful for paternal lineage identification, kinship analysis, and family relationship reconstruction using Y-STR forensic DNA analysis methods.

Profiling of 17 Y-STR loci in Mazandaran and Gilan provinces of Iran

Background/aim: TheY-chromosome mainly consists of heterochromatin regions that have a father-to-son inheritance. Short tandem repeat polymorphic (STRP) markers distributed all over the chromosome provide the opportunity for investigations in forensic medicine and ancestral lineage studies. Due to the existence of wide varieties of geographical and ethnic groups in Iran, studying Y-STRP markers is necessary for further applications. Here we investigated the provinces of Mazandaran and Gilan for the first time. Materials and methods: Samples included 119 and 90 unrelated males from Mazandaran and Gilan, respectively. Using a PCR amplification kit, 17 Y-STRP markers were amplified and genotyping was conducted by capillary electrophoresis. Allele frequency, haplotype diversity (HD), and haplotype discrimination capacity (DC) were calculated. The populations were compared together and to neighboring countries including Afghanistan and Azerbaijan by FST index. Results: A total of 204 unique haplotypes were observed. No uniqueness was observed between the two provinces. HD was 0.9993 and 0.9998 in Mazandaran and Gilan, respectively. DC was 0.9666 and 0.9888 for Mazandaran and Gilan, respectively. DYS385b and DYS391 had the most and least polymorphic content in both provinces, respectively. There was not a significant difference between these two provinces (FST = 0.0006 and P = 0.00) and neighboring countries. Conclusion: The results highlight the effectiveness of these Y-STRP markers for male discrimination in the north of Iran. Using additional markers along with extended sample size would provide a better opportunity for removing matched haplotypes and introducing the best polymorphic markers in this specific population.

Gene conversion violates the stepwise mutation model for microsatellites in y-chromosomal palindromic repeats.

The male-specific region of the human Y chromosome (MSY) contains eight large inverted repeats (palindromes), in which high-sequence similarity between repeat arms is maintained by gene conversion. These palindromes also harbor microsatellites, considered to evolve via a stepwise mutation model (SMM). Here, we ask whether gene conversion between palindrome microsatellites contributes to their mutational dynamics. First, we study the duplicated tetranucleotide microsatellite DYS385a,b lying in palindrome P4. We show, by comparing observed data with simulated data under a SMM within haplogroups, that observed heteroallelic combinations in which the modal repeat number difference between copies was large, can give rise to homoallelic combinations with zero-repeats difference, equivalent to many single-step mutations. These are unlikely to be generated under a strict SMM, suggesting the action of gene conversion. Second, we show that the intercopy repeat number difference for a large set of duplicated microsatellites in all palindromes in the MSY reference sequence is significantly reduced compared with that for nonpalindrome-duplicated microsatellites, suggesting that the former are characterized by unusual evolutionary dynamics. These observations indicate that gene conversion violates the SMM for microsatellites in palindromes, homogenizing copies within individual Y chromosomes, but increasing overall haplotype diversity among chromosomes within related groups.

Y chromosome haplogroup N in a Japanese population is classified into three subclades, and two DYS385 loci, a duplicated Y-STR, are duplicated again in subclade N-M1819.

DYS385 is one of the major Y chromosome short tandem repeats (Y-STRs) in forensic genetics and exists as 2 copies in the human Y chromosome palindrome P4 region. In this study, we found that some samples were estimated to have ≥ 4 copies of DYS385 in Y chromosome haplogroup N in a Japanese population. Y chromosome haplogroup N is distributed widely in eastern/central Asia, Siberia, and eastern/northern Europe, and is also observed in Japan; however, little is known about haplogroup N subclades in the Japanese population. To reveal the link between increased DYS385 copy number and haplogroup N subclades, we sequenced single nucleotide polymorphisms to classify the subclades. As a result, the Japanese Y chromosomes of haplogroup N were classified into three subclades, and an increased DYS385 copy number was specific to subclade N-M1819* (N1b2*). These results are of use in forensic DNA analysis because Y-STR copy number is important for the interpretation of Y-STR typing results of male DNA mixtures and kinship analysis. We also found that DYS458.1 microvariants (DYS458 intermediate alleles with single-nucleotide insertion) were observed only in subclade N-CTS962 (N1b1b∼) samples. Given that previous studies reported that DYS458.1 microvariants are observed in Y chromosomes of haplogroup N in other populations, DYS458.1 might be used to infer haplogroup N subclades without limitation to the Japanese population. The results of this study will be beneficial not only to forensic genetics but also to anthropological studies.

Abnormal detection of Y-STR alleles at DYS385 from female DNA in forensic casework and interchromosomal insertional translocation of P4 palindrome (HSFY/DYS385) from AZFb region.

The unknown origin of DNA samples derived from crime scenes generates a considerable amount of uncertainty, as do unexpected short tandem repeat (STR) results caused by sample mix-ups, contamination, medical interventions, and transgender individuals (broad meaning). Genetic abnormalities such as somatic/germline mutations, mosaicism or chimerism, sex reversal cases, aneuploidies, and chromosomal structural rearrangements are also possible causes of such results. The evidence offered by the present study suggested that additional DYS385 alleles, as seen in mixed stain samples and in the potentially single-source DNA profile of a female, originated from the female DNA source only. For the case reported here, we propose an interchromosomal insertion hypothesis, in which a 768-kb segment including the P4 palindrome of the azoospermia factor (AZFb) region was deleted from the Y chromosome and inserted into the X chromosome or an autosome during male meiosis. Y-SNP data points from the AccuID platform and in-house PCR assays narrowed down the expected length of the target region. Bioinformatics analysis followed by whole genome amplification and whole genome sequencing showed that a 529-kb segment including the P4 palindrome (HSFY/DYS385)/DYS460 region from the female sample mapped to the Y reference sequence (GRCh37). To our knowledge, the interchromosomal insertional translocation event was identified as an unknown type of genomic rearrangement in the forensic genetic field.

Identification of extra alleles in DYS385a/b multi-allelic patterns.

Y chromosome short tandem repeats (Y-STRs) are widely used in forensic DNA analysis. Y-STRs usually genotyped as a monoallelic pattern, or a diallelic pattern at some double-copy loci. Abnormal genotypes with three or four alleles have been reported at DYS385a/b, which is a double-copy locus. However, multi-allelic patterns with more than four alleles have not been reported at this locus. In this study, abnormal five-allelic patterns at DYS385a/b locus were observed in 2 out of 7760 unrelated males from Henan Province, detected by the HG19+14Y fluorescence detection kit; and abnormal six-allelic pattern was observed in 1 out of 4920 unrelated males from Guangdong Province, detected by the AGCU Data Y30 fluorescence kit. The genotypes of these abnormal samples were confirmed again by the Yfiler® Plus and PowerPlex® Y23 kits. In order to understand the genomic structure of the extra alleles at this locus, forward and reverse primers were designed to sequence the three samples. The results showed that the five-banded and six-banded pattern genotype did exist at DYS385a/b locus. These data enriched the knowledge about multi-allelic patterns at DYS385a/b locus, which allowed the use of DYS385a/b profile in forensic analysis, even with multi-allelic patterns.

Y-Short Tandem Repeat Multiplex Systems - Y-PLEX™ 6 and Y-PLEX™ 5.

Y-Chromosome short tandem repeats (Y-STRs) have become a very useful tool in forensic casework, paternity, and male lineage studies. In forensic casework, one can obtain the male profile from a mixture sample containing male and female DNA. Two Y-STR genotyping systems, Y-PLEX™ 6 and Y-PLEX™ 5, have been developed for use in human identification. Y-PLEX™ 6 enables simultaneous amplification of DYS393, DYS19, DYS389II, DYS390, DYS391, and DYS385; Y-PLEX™ 5 enables simultaneous amplification of DYS389I, DYS389II, DYS439, DYS438, and DYS392 loci. The Y-PLEX™ 6 and Y-PLEX™ 5 systems together provide analysis of all nine Y-STR loci generating minimal haplotype and two additional loci, DYS438 and DYS439. These systems also provide analysis for all 11 Y-STR loci recommended by the Scientific Working Group on DNA Analysis Methods (SWGDAM) for forensic casework and population database studies. Both the systems were validated following the Federal Bureau of Investigation (FBI) Director's Quality Assurance Standards. Allelic ladders, which serve as a reference in genotyping, were generated. The nucleotide sequence of alleles in the allelic ladder was determined and the nomenclature is in accord with the recommendations of the International Society of Forensic Genetics (ISFG). The minimum sensitivity of the Y-PLEX™ 6 and Y-PLEX™ 5 systems was 0.2 and 0.1 ng of male DNA, respectively. The nonhuman study revealed that the primers in the Y-PLEX™ 6 and Y-PLEX™ 5 systems were specific for the DNA from humans and some higher primates. Mean stutter values ranged from 3.6 to 11.9%. The Y-PLEX™ 6 and Y-PLEX™ 5 systems were used in several forensic cases. The results from these multiplex systems have been admitted in various U.S. Courts. Thus, Y-PLEX™ 6 and Y-PLEX™ 5 genotyping systems are sensitive, reliable, and robust for use in human forensic and male lineage identification studies.