Genetic sexing of subadult skeletal remains.

When subadult skeletons need to be identified, biological sex diagnosis is one of the first steps in the identification process. Sex assessment of subadults using morphological features is unreliable, and molecular genetic methods were applied in this study. Eighty-three ancient skeletons were used as models for poorly preserved DNA. Three sex-informative markers on the Y and X chromosome were used for sex identification: a qPCR test using the PowerQuant Y target included in PowerQuant System (Promega), the amelogenin test included in ESI 17 Fast STR kit (Promega), and a Y-STR amplification test using the PowerPlex Y-23 kit (Promega). Sex was successfully determined in all but five skeletons. Successful PowerQuant Y-target, Y-amelogenin, and Y-chromosomal STR amplifications proved the presence of male DNA in 35 skeletons, and in 43 subadults female sex was established. No match was found between the genetic profiles of subadult skeletons, and the elimination database and negative control samples produced no profiles, indicating no contamination issue. Our study shows that genetic sex identification is a very successful approach for biological sexing of subadult skeletons whose sex cannot be assessed by anthropological methods. The results of this study are applicable for badly preserved subadult skeletons from routine forensic casework.

Y-DNA genetic evidence reveals several different ancient origins in the Brahmin population.

The ancient geographical origins of Brahmins-a prominent ethnic group in the Indian subcontinent-have remained controversial for a long time. This study employed the AMOVA (analysis of molecular variance) test to evaluate genetic affinities of this group with thirty populations of Central Asia and Europe. A domestic comparison was performed with fifty non-Brahmin groups in India. The results showed that Brahmins had genetic affinities with several foreign populations and also shared their genetic heritage with several domestic non-Brahmin groups. The study identified the deep ancient origins of Brahmins by tracing their Y-chromosome haplogroups and genetic markers on the Y-DNA phylogenetic tree. It was confirmed that the progenitors of this group emerged from at least 12 different geographic regions of the world. The study concluded that about 83% of the Brahmins in the dataset belonged to four major haplogroups, of which two emerged from Central Asia, one from the Fertile Crescent, and one was of an indigenous Indian origin.

Analysis of whole Y-chromosome sequences reveals the Japanese population history in the Jomon period.

The Jomon and the Yayoi are considered to be the two major ancestral populations of the modern mainland Japanese. The Jomon people, who inhabited mainland Japan, admixed with Yayoi immigrants from the Asian continent. To investigate the population history in the Jomon period (14,500-2,300 years before present [YBP]), we analyzed whole Y-chromosome sequences of 345 Japanese males living in mainland Japan. A phylogenetic analysis of East Asian Y chromosomes identified a major clade (35.4% of mainland Japanese) consisting of only Japanese Y chromosomes, which seem to have originated from indigenous Jomon people. A Monte Carlo simulation indicated that ~70% of Jomon males had Y chromosomes in this clade. The Bayesian skyline plots of 122 Japanese Y chromosomes in the clade detected a marked decrease followed by a subsequent increase in the male population size from around the end of the Jomon period to the beginning of the Yayoi period (2,300 YBP). The colder climate in the Late to Final Jomon period may have resulted in critical shortages of food for the Jomon people, who were hunter-gatherers, and the rice farming introduced by Yayoi immigrants may have helped the population size of the Jomon people to recover.

Amplification-by-Polymerization in Biosensing for Human Genomic DNA Detection.

A polymerization reaction was employed as a signal amplification method to realize direct visualization of gender-specific DNA extracted from human blood in a polymerase chain reaction (PCR)-free fashion. Clear distinction between X and Y chromosomes was observed by naked eyes for detector-free sensing purposes. The grown polymer films atop X and Y chromosomes were quantitatively measured by ellipsometry for thickness readings. Detection assays have been optimized for genomic DNA recognition to a maximum extent by varying the selection of the proper blocking reagents, the annealing temperature, and the annealing time. Traditional PCR and gel electrophoresis for amplicon identification were conducted in parallel for performance comparison. In the blind test for blood samples examined by the new approach, 25 out of 26 were correct and one was false negative, which was comparable to, if not better than, the PCR results. This is the first time our amplification-by-polymerization technique is being used for chromosome DNA analysis. The potential of adopting the described sensing technique without PCR was demonstrated, which could further promote the development of a portable, PCR-free DNA sensing device for point-of-need applications.

Potentially effective method for fetal gender determination by noninvasive prenatal testing for X-linked disease.

Examination of maternal plasma cell-free DNA (cfDNA) for noninvasive prenatal testing for fetal trisomy is a highly effective method for pregnant women at high risk. This can be also applied to fetal gender determination in female carriers of severe X-linked disease. Polymerase chain reaction (PCR) analysis is a relatively simpler and less expensive method of detecting Y chromosome-specific repeats (Y-specific PCR; YSP), but is limited by the risk of false-negative results. To address this, we have developed a combined strategy incorporating YSP and an estimation of the fetal DNA fraction. Multiplex PCR for 30 single nucleotide polymorphism (SNP) loci selected by high heterozygosity enables the robust detection of the fetal DNA fraction in cfDNA. The cfDNA sample is first subjected to YSP. When the YSP result is positive, the fetus is male and invasive testing for an X-linked mutation is then required. When the YSP result is negative, the cfDNA sample is analyzed using multiplex PCR. If fetal DNA is then found in the cfDNA, invasive testing is not then required. If the multiplex PCR analysis of cfDNA is negative for fetal DNA, the fetal gender cannot be determined and invasive testing is still required. Our technique provides a potentially effective procedure that can help to avoid unnecessary invasive prenatal testing in some female carriers of severe X-linked disease.

200+ Protein Concentrations in Healthy Human Blood Plasma: Targeted Quantitative SRM SIS Screening of Chromosomes 18, 13, Y, and the Mitochondrial Chromosome Encoded Proteome.

This work continues the series of the quantitative measurements of the proteins encoded by different chromosomes in the blood plasma of a healthy person. Selected Reaction Monitoring with Stable Isotope-labeled peptide Standards (SRM SIS) and a gene-centric approach, which is the basis for the implementation of the international Chromosome-centric Human Proteome Project (C-HPP), were applied for the quantitative measurement of proteins in human blood plasma. Analyses were carried out in the frame of C-HPP for each protein-coding gene of the four human chromosomes: 18, 13, Y, and mitochondrial. Concentrations of proteins encoded by 667 genes were measured in 54 blood plasma samples of the volunteers, whose health conditions were consistent with requirements for astronauts. The gene list included 276, 329, 47, and 15 genes of chromosomes 18, 13, Y, and the mitochondrial chromosome, respectively. This paper does not make claims about the detection of missing proteins. Only 205 proteins (30.7%) were detected in the samples. Of them, 84, 106, 10, and 5 belonged to chromosomes 18, 13, and Y and the mitochondrial chromosome, respectively. Each detected protein was found in at least one of the samples analyzed. The SRM SIS raw data are available in the ProteomeXchange repository (PXD004374, PASS01192).

Methodology for Y Chromosome Capture: A complete genome sequence of Y chromosome using flow cytometry, laser microdissection and magnetic streptavidin-beads.

This study is a comparison of the efficiency of three technologies used for Y chromosome capture and the next-generation sequencing (NGS) technologies applied for determining its whole sequence. Our main findings disclose that streptavidin-biotin magnetic particle-based capture methodology offers better and a deeper sequence coverage for Y chromosome capture, compared to chromosome sorting and microdissection procedures. Moreover, this methodology is less time consuming and the most selective for capturing only Y chromosomal material, in contrast with other methodologies that result in considerable background material from other, non-targeted chromosomes. NGS results compared between two platforms, NextSeq 500 and SOLID 5500xl, produce the same coverage results. This is the first study to explore a methodological comparison of Y chromosome capture and genetic analysis. Our results indicate an improved strategy for Y chromosome research with applications in several scientific fields where this chromosome plays an important role, such as forensics, medical sciences, molecular anthropology and cancer sciences.

Human Y-chromosome variation in the genome-sequencing era.

The properties of the human Y chromosome - namely, male specificity, haploidy and escape from crossing over - make it an unusual component of the genome, and have led to its genetic variation becoming a key part of studies of human evolution, population history, genealogy, forensics and male medical genetics. Next-generation sequencing (NGS) technologies have driven recent progress in these areas. In particular, NGS has yielded direct estimates of mutation rates, and an unbiased and calibrated molecular phylogeny that has unprecedented detail. Moreover, the availability of direct-to-consumer NGS services is fuelling a rise of 'citizen scientists', whose interest in resequencing their own Y chromosomes is generating a wealth of new data.

Sex chromosome-dependent differential viability of human spermatozoa during prolonged incubation.

STUDY QUESTION: Are there significant differences in the ability of X chromosome-bearing (X) spermatozoa and Y chromosome-bearing (Y) spermatozoa to survive incubation under stressful conditions? SUMMARY ANSWER: Y spermatozoa are more vulnerable to stress than their X counterparts depending on culture period and temperature, and show higher expression of apoptotic proteins. WHAT IS KNOWN ALREADY: The primary sex ratio is determined by there being an equal number of spermatozoa carrying X and Y chromosomes. This balance can be skewed by exposure to stressful environmental conditions such as changes in pH, pollutants or endocrine disruptors. However, less is known about the ability of sperm carrying either sex chromosome to withstand environmental stress. STUDY DESIGN, SIZE, DURATION: The difference in survival between X and Y spermatozoa was evaluated by measuring motility, viability and Y:X chromosome ratio during incubation for 5 days, at three temperatures (4, 22 and 37°C), and three pH conditions (6.5, 7.5 and 8.5). To identify the critical factors that determine the survival of X and Y bearing spermatozoa, we analysed the expression levels of apoptosis-related proteins (Bcl, Bax and Caspase-3), as well as the extent of DNA damage under a subset of conditions. PARTICIPANTS/MATERIALS, SETTING, METHODS: Semen samples were obtained by masturbation from normozoospermic donors after 3 days of sexual abstinence. Four samples with >60% motility from different donors were mixed to obtain sufficient semen and eliminate sampling-related bias. Data are presented as mean ± SD of three independent experiments. Mean age of donors was 28.7 ± 3.2 years. MAIN RESULTS AND THE ROLE OF CHANCE: In total, 58 489 spermatozoa were scored. The viability of Y spermatozoa was lower after exposure to different temperatures and culture periods than that of X spermatozoa (P < 0.05). Increased expression of apoptotic proteins in live Y spermatozoa was observed, despite the addition of tocopherol to the culture medium (P < 0.05). LIMITATIONS, REASONS FOR CAUTION: Spermatozoa were cultured in vitro during the treatment period. It is difficult to extrapolate the observed lifespan differences to spermatozoa survival in vivo. The experiments were replicated only three times. WIDER IMPLICATIONS OF THE FINDINGS: The prolonged survival of X spermatozoa under stressful conditions might lead to shifts in the ratio of male-to-female births. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by the National Research Foundation of Korea (NRF) grant funded by the Korean government (MEST) (no. NRF-2014R1A2A2A01002706). The authors declare no competing financial interests.

Genetic trail for the early migrations of Aisin Gioro, the imperial house of the Qing dynasty.

The House of Aisin Gioro, the imperial clan of Qing dynasty (1644-1911), affected the history of China and the formation of Manchu ethnicity greatly. However, owing to the lack of historical records and archeological evidences, the origin of the House of Aisin Gioro remains ambiguous. To clarify the origin of Aisin Gioro clan, we conducted whole Y-chromosome sequencing on three samples and Y-single-nucleotide polymorphism (Y-SNP) genotyping on other four samples beside those reported in previous work. We confirmed that the paternal lineage of the Aisin Gioro clan belongs to haplogroup C3b1a3a2-F8951, a brother branch of C3*-Star Cluster (currently named as C3b1a3a1-F3796, once linked to Genghis Khan), which is quite different from the predominant lineage C3c-M48 in other Tungusic-speaking populations. We also determined a series of unique Y-SNP markers for the Aisin Gioro clan. Diversity analyses of haplogroup C3b1a3a2-F8951 revealed the early migration of the ancestors of the Aisin Gioro clan from the middle reaches of Amur River to their later settlement in southeastern Manchuria. Hence, our results suggest that the Aisin Gioro clan may be descendants of ancient populations in Transbaikal region and closely related to origin of current Daur populations. Our research indicated that detailed research of stemma and deep sequencing of Y chromosomes are helpful to explore the prehistoric activities of populations lacking historical records and archeological evidences.

Genetic Variation of 25 Y-Chromosomal and 15 Autosomal STR Loci in the Han Chinese Population of Liaoning Province, Northeast China.

In the present study, we investigated the genetic characteristics of 25 Y-chromosomal and 15 autosomal short tandem repeat (STR) loci in 305 unrelated Han Chinese male individuals from Liaoning Province using AmpFISTR® Yfiler® Plus and IdentifilerTM PCR amplification kits. Population comparison was performed between Liaoning Han population and different ethnic groups to better understand the genetic background of the Liaoning Han population. For Y-STR loci, the overall haplotype diversity was 0.9997 and the discrimination capacity was 0.9607. Gene diversity values ranged from 0.4525 (DYS391) to 0.9617 (DYS385). Rst and two multi-dimensional scaling plots showed that minor differences were observed when the Liaoning Han population was compared to the Jilin Han Chinese, Beijing Han Chinese, Liaoning Manchu, Liaoning Mongolian, Liaoning Xibe, Shandong Han Chinese, Jiangsu Han Chinese, Anhui Han Chinese, Guizhou Han Chinese and Liaoning Hui populations; by contrast, major differences were observed when the Shanxi Han Chinese, Yunnan Bai, Jiangxi Han Chinese, Guangdong Han Chinese, Liaoning Korean, Hunan Tujia, Guangxi Zhuang, Gansu Tibetan, Xishuangbanna Dai, South Korean, Japanese and Hunan Miao populations. For autosomal STR loci, DP ranged from 0.9621 (D2S1338) to 0.8177 (TPOX), with PE distributing from 0.7521 (D18S51) to 0.2988 (TH01). A population comparison was performed and no statistically significant differences were detected at any STR loci between Liaoning Han, China Dong, and Shaanxi Han populations. The results showed that the 25 Y-STR and 15 autosomal STR loci in the Liaoning Han population were valuable for forensic applications and human genetics, and Liaoning Han was an independent endogenous ethnicity with a unique subpopulation structure.

Y-STR genetic screening by high-resolution melting analysis.

Currently, the widely used automated capillary electrophoresis-based short tandem repeat (STR) genotyping method for genetic screening in forensic practice is laborious, time-consuming, expensive, and technically challenging in some cases. Thus, new molecular-based strategies for conclusively identifying forensically relevant biological evidence are required. Here, we used high-resolution melting analysis (HRM) for Y-chromosome STR genotyping for forensic genetic screening. The reproducibility of the melting profile over dilution, sensitivity, discrimination power, and other factors was preliminarily studied in 10 Y-STR loci. The results showed that HRM-based approaches revealed more genotypes (compared to capillary electrophoresis), showed higher uniformity in replicate tests and diluted samples, and enabled successful detection of DNA at concentrations as low as 0.25 ng. For mixed samples, the melting curve profiles discriminated between mixed samples based on reference samples with high efficiency. The triplex Y-chromosome STR HRM assay was performed and provided a foundation for further studies such as a multiplex HRM assay. The HRM approach is a one-step application and the entire procedure can be completed within 2 h at a low cost. In conclusion, our findings demonstrate that the HRM-based Y-STR assay is a useful screening tool that can be used in forensic practice.

Gr/gr deletions on Y-chromosome correlate with male infertility: an original study, meta-analyses, and trial sequential analyses.

We analyzed the AZFc region of the Y-chromosome for complete (b2/b4) and distinct partial deletions (gr/gr, b1/b3, b2/b3) in 822 infertile and 225 proven fertile men. We observed complete AZFc deletions in 0.97% and partial deletions in 6.20% of the cases. Among partial deletions, the frequency of gr/gr deletions was the highest (5.84%). The comparison of partial deletion data between cases and controls suggested a significant association of the gr/gr deletions with infertility (P = 0.0004); however, the other partial deletions did not correlate with infertility. In cohort analysis, men with gr/gr deletions had a relatively poor sperm count (54.20 ± 57.45 million/ml) in comparison to those without deletions (72.49 ± 60.06), though the difference was not statistically significant (p = 0.071). Meta-analysis also suggested that gr/gr deletions are significantly associated with male infertility risk (OR = 1.821, 95% CI = 1.39-2.37, p = 0.000). We also performed trial sequential analyses that strengthened the evidence for an overall significant association of gr/gr deletions with the risk of male infertility. Another meta-analysis suggested a significant association of the gr/gr deletions with low sperm count. In conclusion, the gr/gr deletions show a strong correlation with male infertility risk and low sperm count, particularly in the Caucasian populations.

Total integrated slidable and valveless solid phase extraction-polymerase chain reaction-capillary electrophoresis microdevice for mini Y chromosome short tandem repeat genotyping.

A fully integrated slidable and valveless microsystem, which performs solid phase DNA extraction (SPE), micro-polymerase chain reaction (µPCR) and micro-capillary electrophoresis (µCE) coupled with a portable genetic analyser, has been developed for forensic genotyping. The use of a slidable chip, in which a 1 µL-volume of the PCR chamber was patterned at the center, does not necessitate any microvalves and tubing systems for fluidic control. The functional micro-units of SPE, µPCR, and µCE were fabricated on a single glass wafer by conventional photolithography, and the integrated microdevice consists of three layers: from top to bottom, a slidable chip, a channel wafer in which a SPE chamber, a mixing microchannel, and a CE microchannel were fabricated, and a Ti/Pt resistance temperature detector (RTD) wafer. The channel glass wafer and the RTD glass wafer were thermally bonded, and the slidable chip was placed on the designated functional unit. The entire process from the DNA extraction using whole human blood sample to identification of target Y chromosomal short tandem repeat (STR) loci was serially carried out with simply sliding the slidable chamber from one to another functional unit. Monoplex and multiplex detection of amelogenin and mini Y STR loci were successfully analysed on the integrated slidable SPE-µPCR-µCE microdevice by using 1 µL whole human blood within 60 min. The proposed advanced genetic analysis microsystem is capable of point-of-care DNA testing with sample-in-answer-out capability, more importantly, without use of complicated microvalves and microtubing systems for liquid transfer.

DOES THE PATTERN OF CLONAL EVOLUTION IN THE KARYOTYPE OF PATIENTS WITH ACUTE MYELOID LEUKEMIA AND MYELODYSPLASTIC SYNDROMES DEPEND ON THE TYPE OF THE PRIMARY CHROMOSOMAL ABERRATIONS?

The aim of our study was to define if the type of primary chromosomal aberrations (CA) of the karyotype of patients with Acute myeloid leukemia (AML) and Myelodysplastic syndromes (MDS) determines the way and the rate of karyotype development. Conventional cytogenetic analysis was carried out on 248 AML and 105 MDS patients at diagnosis. Clonal evolution (CE) was found in 40% (51 of 128) of AML patients and in 47.5% (19 of 40) of MDS patients having CA in their karyotype. The first pattern we established was for the most frequent CA which initiate CE in 28 patients with a complex karyotype. These CA were non-balansed rearrangements in the following regions: 5q, 7q, 11q, 3q, monosomy 5, monosomy 7. The second pattern of CE was regarding the most frequent aneuploidias (+8, +11, +21, -Y, and the third pattern concerned balanced CA. We found significant difference in the distribution of karyotypes in different stages of progression between the first and the other two groups (p < 0.001). No statistical difference was found between the patterns in the second and the third group CA (p > 0.5).

[GENE POOL SIMILARITIES AND DIFFERENCES BETWEEN UKRAINIANS AND RUSSIANS OF SLOBOZHANSHINA ON Y-CHROMOSOME DATA].

The results of the study of Y-chromosomal polymorphisms of Russian and Ukrainian population are presented for Slobozhanshina--contemporary border region, former "Wild Field" boundary, which was inhabited in XVII-XVIII centuries by both the Russians from the north and Ukrainians from the west. In general, Ukrainian and Russian populations of Slobozhanshchina genetically are very close, their set and frequency range of Y-chromosome haplogroups are typical for the Eastern Europe. But a detailed analysis of highly informative Y-chromosome markers showed that after 3,5 centuries of coexistence on the same historical territory, the both nations retain the ethnic specificity of their gene pools: Ukrainian populations are similar to the rest of Ukraine, and Russian populations are similar to the south of the European part of Russia. The genetic differences may be due to the spatial characteristics of marriage migration and the predominant ethnic environment.

DDX3Y gene rescue of a Y chromosome AZFa deletion restores germ cell formation and transcriptional programs.

Deletions of the AZFa region (AZoospermia Factor-a) region of the human Y chromosome cause irreversible spermatogenic failure that presents clinically in men as Sertoli-cell only (SCO) pathology of the testis. Deletions of the AZFa region typically encompass two genes: DDX3Y and USP9Y. However, human genetic evidence indicates that SCO is most tightly linked to deletion of DDX3Y and that deletions/mutations of USP9Y can be transmitted from one generation to the next. Here, we generated stable iPSC lines with AZFa deletions, tested complementation via introduction of DDX3Y, and assessed ability to form germ cells in vivo in a xenotransplantation model. We observed a quantifiable improvement in formation of germ cell like cells (GCLCs) from complemented donor iPSCs. Moreover, expression of UTF1, a prospermatogonial protein, was restored in cells complemented by introduction of DDX3Y on the AZFa background. Whole-genome RNA sequencing of purified GCLCs revealed an enrichment of genes involved in translational suppression and transcriptional control in DDX3Y-rescued GCLCs over mutant GCLCs, which maintained a molecular phenotype more similar to undifferentiated iPSCs. This study demonstrates the ability to probe fundamental genetics of human germ cell formation by complementation and indicates that DDX3Y functions in the earliest stages of human germ cell development.

Common AZFc structure may possess the optimal spermatogenesis efficiency relative to the rearranged structures mediated by non-allele homologous recombination.

The azoopsermia factor c (AZFc) region of human Y-chromosome is an essential genomic segment for spermatogenesis with frequent non-allele homologous recombination (NAHR). Recent case-control studies on the association of the NAHR-based AZFc structural mutations with spermatogenic failure produced inconsistent results. To more precisely evaluate their spermatogenesis effects, we investigated the correlation between the subdivided AZFc mutations and sperm production in 3,439 Han Chinese males. Our results showed that both partial AZFc deletion-only and primary duplication mutation presented a significant risk for decreased sperm production. Restoration of the reduced dosage of the AZFc content to the normal level had a milder effect, whereas an overdose of the AZFc content arising from multiple duplications of a partial AZFc-deleted structure produced a more serious consequence compared to the partial deletion-only mutation. Additionally, the AZFc-mutated structures with excessive NAHR-substrate showed a notably negative effect on spermatogenesis. These results suggest that the recurrent NAHR-based AZFc mutations may be associated with decreased spermatogenesis efficiency in present population. More significantly, our finding implies that the overdose of AZFc NAHR-substrate may produce an additional risk for the massive AZFbc deletions during the multi-stage division process of germ cells and thus impair the global spermatogenesis efficiency in the carriers.