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CRISPR-based genetic engineering tools aimed to bias sex ratios, or drive effector genes into animal populations, often integrate the transgenes into autosomal chromosomes. However, in species with heterogametic sex chromsomes (e.g. XY, ZW), sex linkage of endonucleases could be beneficial to drive the expression in a sex-specific manner to produce genetic sexing systems, sex ratio distorters, or even sex-specific gene drives, for example. To explore this possibility, here we develop a transgenic line of Drosophila melanogaster expressing Cas9 from the Y chromosome. We functionally characterize the utility of this strain for both sex selection and gene drive finding it to be quite effective. To explore its utility for population control, we built mathematical models illustrating its dynamics as compared to other state-of-the-art systems designed for both population modification and suppression. Taken together, our results contribute to the development of current CRISPR genetic control tools and demonstrate the utility of using sex-linked Cas9 strains for genetic control of animals., CRISPR-based engineering can be used to bias sex ratios. Here the authors develop a transgenic line of Drosophila melanogaster expressing Cas9 from the Y chromosome and functionally characterize the utility of this strain for both sex selection and gene drive.

Evolution of genetic mechanisms of sex determination led to two processes causing sex differences in somatic phenotypes: gonadal differentiation and sex chromosome dosage inequality. In species with heteromorphic sex chromosomes, the sex of the individual is established at the time of formation of the zygote, leading to inherent sex differences in expression of sex chromosome genes beginning as soon as the embryonic transcriptome is activated. The inequality of sex chromosome gene expression causes sexual differentiation of the gonads and of non-gonadal tissues. The difference in gonad type in turn causes lifelong differences in gonadal hormones, which interact with unequal effects of X and Y genes acting within cells. Separating the effects of gonadal hormones and sex chromosomes has been possible using mouse models in which gonadal determination is separated from the sex chromosomes, allowing comparison of XX and XY mice with the same type of gonad. Sex differences caused by gonadal hormones and sex chromosomes affect basic physiology and disease mechanisms in most or all tissues.

Allogeneic hematopoietic stem cell transplantation (allo-HCT) is a curative option for blood cancers, but the coupled effects of graft-versus-tumor and graft-versus-host disease (GVHD) limit its broader application. Outcomes improve with matching at HLAs, but other factors are required to explain residual risk of GVHD. In an effort to identify genetic associations outside the major histocompatibility complex, we conducted a genome-wide clinical outcomes study on 205 acute myeloid leukemia patients and their fully HLA-A–, HLA-B–, HLA-C–, HLA-DRB1–, and HLA-DQB1–matched (10/10) unrelated donors. HLA-DPB1 T-cell epitope permissibility mismatches were observed in less than half (45%) of acute GVHD cases, motivating a broader search for genetic factors affecting clinical outcomes. A novel bioinformatics workflow adapted from neoantigen discovery found no associations between acute GVHD and known, HLA-restricted minor histocompatibility antigens (MiHAs). These results were confirmed with microarray data from an additional 988 samples. On the other hand, Y-chromosome–encoded single-nucleotide polymorphisms in 4 genes (PCDH11Y, USP9Y, UTY, and NLGN4Y) did associate with acute GVHD in male patients with female donors. Males in this category with acute GVHD had more Y-encoded variant peptides per patient with higher predicted HLA-binding affinity than males without GVHD who matched X-paralogous alleles in their female donors. Methods and results described here have an immediate impact for allo-HCT, warranting further development and larger genomic studies where MiHAs are clinically relevant, including cancer immunotherapy, solid organ transplant, and pregnancy.

To better define the structure and origin of the Bulgarian paternal gene pool, we have examined the Y-chromosome variation in 808 Bulgarian males. The analysis was performed by high-resolution genotyping of biallelic markers and by analyzing the STR variation within the most informative haplogroups. We found that the Y-chromosome gene pool in modern Bulgarians is primarily represented by Western Eurasian haplogroups with ∼ 40% belonging to haplogroups E-V13 and I-M423, and 20% to R-M17. Haplogroups common in the Middle East (J and G) and in South Western Asia (R-L23*) occur at frequencies of 19% and 5%, respectively. Haplogroups C, N and Q, distinctive for Altaic and Central Asian Turkic-speaking populations, occur at the negligible frequency of only 1.5%. Principal Component analyses group Bulgarians with European populations, apart from Central Asian Turkic-speaking groups and South Western Asia populations. Within the country, the genetic variation is structured in Western, Central and Eastern Bulgaria indicating that the Balkan Mountains have been permeable to human movements. The lineage analysis provided the following interesting results: (i) R-L23* is present in Eastern Bulgaria since the post glacial period; (ii) haplogroup E-V13 has a Mesolithic age in Bulgaria from where it expanded after the arrival of farming; (iii) haplogroup J-M241 probably reflects the Neolithic westward expansion of farmers from the earliest sites along the Black Sea. On the whole, in light of the most recent historical studies, which indicate a substantial proto-Bulgarian input to the contemporary Bulgarian people, our data suggest that a common paternal ancestry between the proto-Bulgarians and the Altaic and Central Asian Turkic-speaking populations either did not exist or was negligible.

Male fertility is an orchestrated interplay of loci on the Y chromosome with a number of genes from across the other chromosomes. In this context, micro-deletions in the Y chromosome have been correlated with spermatogenic failure often leading to infertility. However, causes of infertility in the patients with the normal spermiogram have remained unclear and therefore pose another level of challenge. In the present study, we analyzed 64 STSs, studied different Y-linked genes and loci and conducted single nucleotide variant (SNV) analyses in 31 infertile males with normal spermiogram along with 67 normal fertile males (NFMs) to gain an insight into the organization of their Y chromosome. Further, employing quantitative real-time PCR (qPCR), we studied copy number variation of DYZ1 arrays and three genes and mutational status of SRY by direct sequence analyses. STS analyses of the AZFa, b and c regions in these patients showed known and new mutations. Further, copies of DAZ and BPY2 in the patients were found to be affected (p < 0.001) compared to those in NFMs. All the patients had normal copy number of the SRY however its sequence analysis (in silico) showed mutations in eight patients. In four of these eight patients, SRY mutations resulted into truncated proteins. Similarly, DYZ1 analysis showed micro-deletions and it's much reduced copy number (p < 0.001) as compared to those in NFMs. Present study in males with unexplained infertility revealed deletions similar to those observed in oligospermic and azoospermic patients. Thus, there are some common but still unknown factors underlying infertility in these patients irrespective of their spermatogenic status. This work is envisaged to augment DNA diagnosis, proving beneficial in the context of in vitro fertilization (IVF) and genetic counselling.

Infertility affects around 1 in 10 men and in most cases the cause is unknown. The Y chromosome plays an important role in spermatogenesis and specific deletions of this chromosome, the AZF deletions, are associated with spermatogenic failure. Recently partial AZF deletions have been described but their association with spermatogenic failure is unclear. Here we screened a total of 339 men with idiopathic spermatogenic failure, and 256 normozoospermic ancestry-matched men for chromosome microdeletions including AZFa, AZFb, AZFc, and the AZFc partial deletions (gr/gr, b1/b3 and b2/b3). AZFa and AZFc deletions were identified in men with severe spermatogenic failure at similar frequencies to those reported elsewhere. Gr/gr deletions were identified in case and control populations at 5.83% and 6.25% respectively suggesting that these deletions are not associated with spermatogenic failure. However, b2/b3 deletions were detected only in men with spermatogenic failure and not in the normospermic individuals. Combined with our previous data this shows an association of the b2/b3 deletion (p = 0.0318) with spermatogenic failure in some populations. We recommend screening for this deletion in men with unexplained spermatogenic failure.

Microdeletion of the Azoospermia Factor (AZF) regions in Y chromosome is a well-known genetic cause of male infertility resulting from spermatogenetic impairment. However, the partial deletions of AZFc region related to spermatogenetic impairment are controversial. In this study, we characterized partial deletion of AZFc region in Korean patients with spermatogenetic impairment and assessed whether the DAZ and CDY1 contributes to the phenotype in patients with gr/gr deletions. Total of 377 patients with azoo-/oligozoospermia and 217 controls were analyzed using multiplex polymerase chain reaction (PCR), analysis of DAZ-CDY1 sequence family variants (SFVs), and quantitative fluorescent (QF)-PCR. Of the 377 men with impaired spermatogenesis, 59 cases (15.6%) had partial AZFc deletions, including 32 gr/gr (8.5%), 22 b2/b3 (5.8%), four b1/b3 (1.1%) and one b3/b4 (0.3%) deletion. In comparison, 14 of 217 normozoospermic controls (6.5%) had partial AZFc deletions, including five gr/gr (2.3%) and nine b2/b3 (4.1%) deletions. The frequency of gr/gr deletions was significantly higher in the azoo-/oligozoospermic group than in the normozoospermic control group (p = 0.003; OR = 3.933; 95% CI = 1.509-10.250). Concerning Y haplogroup, we observed no significant differences in the frequency of gr/gr deletions between the case and the control groups in the YAP+ lineages, while gr/gr deletion were significantly higher in azoo-/oligozoospermia than normozoospermia in the YAP- lineage (p = 0.004; OR = 6.341; 95% CI = 1.472-27.312). Our data suggested that gr/gr deletion is associated with impaired spermatogenesis in Koreans with YAP- lineage, regardless of the gr/gr subtypes.

Martínez-Cruz, Begoña et al.-- The Genographic Consortium, Vlad III The Impaler, also known as Dracula, descended from the dynasty of Basarab, the first rulers of independent Wallachia, in present Romania. Whether this dynasty is of Cuman (an admixed Turkic people that reached Wallachia from the East in the 11 th century) or of local Romanian (Vlach) origin is debated among historians. Earlier studies have demonstrated the value of investigating the Y chromosome of men bearing a historical name, in order to identify their genetic origin. We sampled 29 Romanian men carrying the surname Basarab, in addition to four Romanian populations (from counties Dolj, N = 38; Mehedinti, N = 11; Cluj, N = 50; and Brasov, N = 50), and compared the data with the surrounding populations. We typed 131 SNPs and 19 STRs in the non-recombinant part of the Y-chromosome in all the individuals. We computed a PCA to situate the Basarab individuals in the context of Romania and its neighboring populations. Different Y-chromosome haplogroups were found within the individuals bearing the Basarab name. All haplogroups are common in Romania and other Central and Eastern European populations. In a PCA, the Basarab group clusters within other Romanian populations. We found several clusters of Basarab individuals having a common ancestor within the period of the last 600 years. The diversity of haplogroups found shows that not all individuals carrying the surname Basarab can be direct biological descendants of the Basarab dynasty. The absence of Eastern Asian lineages in the Basarab men can be interpreted as a lack of evidence for a Cuman origin of the Basarab dynasty, although it cannot be positively ruled out. It can be therefore concluded that the Basarab dynasty was successful in spreading its name beyond the spread of its genes. © 2012 Martinez-Cruz et al., The present study was supported by the Ministerio de Ciencia e Innovación, Spain (CGL2010-14944), Direcció General de Recerca, Generalitat de Catalunya (2009SGR1101) (both to DC), and a Vici Grant of the Netherlands Organization for Scientific Research (to MGN).

Knowledge of high resolution Y-chromosome haplogroup diversification within Iran provides important geographic context regarding the spread and compartmentalization of male lineages in the Middle East and southwestern Asia. At present, the Iranian population is characterized by an extraordinary mix of different ethnic groups speaking a variety of Indo-Iranian, Semitic and Turkic languages. Despite these features, only few studies have investigated the multiethnic components of the Iranian gene pool. In this survey 938 Iranian male DNAs belonging to 15 ethnic groups from 14 Iranian provinces were analyzed for 84 Y-chromosome biallelic markers and 10 STRs. The results show an autochthonous but non-homogeneous ancient background mainly composed by J2a sub-clades with different external contributions. The phylogeography of the main haplogroups allowed identifying post-glacial and Neolithic expansions toward western Eurasia but also recent movements towards the Iranian region from western Eurasia (R1b-L23), Central Asia (Q-M25), Asia Minor (J2a-M92) and southern Mesopotamia (J1-Page08). In spite of the presence of important geographic barriers (Zagros and Alborz mountain ranges, and the Dasht-e Kavir and Dash-e Lut deserts) which may have limited gene flow, AMOVA analysis revealed that language, in addition to geography, has played an important role in shaping the nowadays Iranian gene pool. Overall, this study provides a portrait of the Y-chromosomal variation in Iran, useful for depicting a more comprehensive history of the peoples of this area as well as for reconstructing ancient migration routes. In addition, our results evidence the important role of the Iranian plateau as source and recipient of gene flow between culturally and genetically distinct populations.

ArunKumar, GaneshPrasad et al.--The Genographic Consortium, Previous studies that pooled Indian populations from a wide variety of geographical locations, have obtained contradictory conclusions about the processes of the establishment of the Varna caste system and its genetic impact on the origins and demographic histories of Indian populations. To further investigate these questions we took advantage that both Y chromosome and caste designation are paternally inherited, and genotyped 1,680 Y chromosomes representing 12 tribal and 19 non-tribal (caste) endogamous populations from the predominantly Dravidian-speaking Tamil Nadu state in the southernmost part of India. Tribes and castes were both characterized by an overwhelming proportion of putatively Indian autochthonous Y-chromosomal haplogroups (H-M69, F-M89, R1a1-M17, L1-M27, R2-M124, and C5-M356; 81% combined) with a shared genetic heritage dating back to the late Pleistocene (10-30 Kya), suggesting that more recent Holocene migrations from western Eurasia contributed, The study is supported by “The Genographic Project” funded by The National Geographic Society, IBM and Waitt Family Foundation. CTS was supported by The Wellcome Trust (Grant number 098051).

One hundred and forty-six previously detected mutations were more precisely positioned in the human Y chromosome phylogeny by the analysis of 51 representative Y chromosome haplogroups and the use of 59 mutations from literature. Twenty-two new mutations were also described and incorporated in the revised phylogeny. This analysis made it possible to identify new haplogroups and to resolve a deep trifurcation within haplogroup B2. Our data provide a highly resolved branching in the African-specific portion of the Y tree and support the hypothesis of an origin in the north-western quadrant of the African continent for the human MSY diversity.

Backgound Y-chromosomal microdeletions (YCMD) are one of the major genetic causes for non-obstructive azoospermia. Genetic testing for YCMD by multiplex polymerase chain reaction (PCR) is an established method for quick and robust screening of deletions in the AZF regions of the Y-chromosome. Multiplex PCRs have the advantage of including a control gene in every reaction and significantly reducing the number of reactions needed to screen the relevant genomic markers. Principal Findings The widely established “EAA/EMQN best practice guidelines for molecular diagnosis of Y-chromosomal microdeletions (2004)” were used as a basis for designing a real-time multiplex PCR system, in which the YCMD can simply be identified by their melting points. For this reason, some AZF primers were substituted by primers for regions in their genomic proximity, and the ZFX/ZFY control primer was exchanged by the AMELX/AMELY control primer. Furthermore, we substituted the classical SybrGreen I dye by the novel and high-performing DNA-binding dye EvaGreen™ and put substantial effort in titrating the primer combinations in respect to optimal melting peak separation and peak size. Significance With these changes, we were able to develop a platform-independent and robust real-time based multiplex PCR, which makes the need for amplicon identification by electrophoretic sizing expendable. By using an open-source system for real-time PCR analysis, we further demonstrate the applicability of automated melting point and YCMD detection.

Chimpanzees and humans are genetically very similar, with the striking exception of their Y chromosomes, which have diverged tremendously. The male-specific region (MSY), representing the greater part of the Y chromosome, is inherited from father to son in a clonal fashion, with natural selection acting on the MSY as a unit. Positive selection might involve the performance of the MSY in spermatogenesis. Chimpanzees have a highly polygamous mating behavior, so that sperm competition is thought to provide a strong selective force acting on the Y chromosome in the chimpanzee lineage. In consequence of evolution of the heterologous sex chromosomes in mammals, meiotic sex chromosome inactivation (MSCI) results in a transcriptionally silenced XY body in male meiotic prophase, and subsequently also in postmeiotic repression of the sex chromosomes in haploid spermatids. This has evolved to a situation where MSCI has become a prerequisite for spermatogenesis. Here, by analysis of microarray testicular expression data representing a small number of male chimpanzees and men, we obtained information indicating that meiotic and postmeiotic X chromosome silencing might be more effective in chimpanzee than in human spermatogenesis. From this, we suggest that the remarkable reorganization of the chimpanzee Y chromosome, compared to the human Y chromosome, might have an impact on its meiotic interactions with the X chromosome and thereby on X chromosome silencing in spermatogenesis. Further studies will be required to address comparative functional aspects of MSCI in chimpanzee, human, and other placental mammals.

The present study was carried out in the Indo-European speaking tribal population groups of Southern Gujarat, India to investigate and reconstruct their paternal population structure and population histories. The role of language, ethnicity and geography in determining the observed pattern of Y haplogroup clustering in the study populations was also examined. A set of 48 bi-allelic markers on the non-recombining region of Y chromosome (NRY) were analysed in 284 males; representing nine Indo-European speaking tribal populations. The genetic structure of the populations revealed that none of these groups was overtly admixed or completely isolated. However, elevated haplogroup diversity and FST value point towards greater diversity and differentiation which suggests the possibility of early demographic expansion of the study groups. The phylogenetic analysis revealed 13 paternal lineages, of which six haplogroups: C5, H1a*, H2, J2, R1a1* and R2 accounted for a major portion of the Y chromosome diversity. The higher frequency of the six haplogroups and the pattern of clustering in the populations indicated overlapping of haplogroups with West and Central Asian populations. Other analyses undertaken on the population affiliations revealed that the Indo-European speaking populations along with the Dravidian speaking groups of southern India have an influence on the tribal groups of Gujarat. The vital role of geography in determining the distribution of Y lineages was also noticed. This implies that although language plays a vital role in determining the distribution of Y lineages, the present day linguistic affiliation of any population in India for reconstructing the demographic history of the country should be considered with caution.

Genetic variants of Y chromosome predispose to hypertension in rodents, whereas in humans the evidence is conflicting. Our purpose was to study the distribution of a panel of Y chromosome markers in a cohort from a cross-sectional population-based study on the prevalence of cardiovascular risk factors in Poland (WOBASZ study). The HindIII, YAP Y chromosome variants, previously shown to influence blood pressure, lipid traits or height, as well as SNPs defining main Y chromosome haplogroups, were typed in 3026, 2783 and 2652 samples, respectively. In addition, 4 subgroups (N∼100 each) representing extremes of LDL concentration or blood pressure (BP) were typed for a panel of 17 STRs. The HindIII and YAP polymorphism were not associated with any of the studied traits. Analysis of the haplogroup distribution showed an association between higher HDL level and hg I-M170 (P = 0.02), higher LDL level and hg F*(xI-M170, J2-M172, K-M9) (P = 0.03) and lower BMI and hg N3-Tat (P = 0.04). Analysis of STRs did not show statistically significant differences. Since all these associations lost statistical significance after Bonferroni correction, we conclude that a major role of Y chromosome genetic variation (defined by HindIII, YAP or main Y chromosome haplogroups) in determining cardiovascular risk in Poles is unlikely.

In a large variety of genetic studies, probabilistic inferences are made based on information available in population databases. The accuracy of the estimates based on population samples are highly dependent on the number of chromosomes being analyzed as well as the correct representation of the reference population. For frequency calculations the size of a database is especially critical for haploid markers, and for countries with complex admixture histories it is important to assess possible substructure effects that can influence the coverage of the database. Aiming to establish a representative Brazilian population database for haplotypes based on 23 Y chromosome STRs, more than 2,500 Y chromosomes belonging to Brazilian, European and African populations were analyzed. No matter the differences in the colonization history of the five geopolitical regions that currently exist in Brazil, for the Y chromosome haplotypes of the 23 studied Y-STRs, a lack of genetic heterogeneity was found, together with a predominance of European male lineages in all regions of the country. Therefore, if we do not consider the diverse Native American or Afro-descendent isolates, which are spread through the country, a single Y chromosome haplotype frequency database will adequately represent the urban populations in Brazil. In comparison to the most commonly studied group of 17 Y-STRs, the 23 markers included in this work allowed a high discrimination capacity between haplotypes from non-related individuals within a population and also increased the capacity to discriminate between paternal relatives. Nevertheless, the expected haplotype mutation rate is still not enough to distinguish the Y chromosome profiles of paternally related individuals. Indeed, even for rapidly mutating Y-STRs, a very large number of markers will be necessary to differentiate male lineages from paternal relatives.

Current familial searching strategies are developed primarily based on autosomal STR loci, since most of the offender profiles in the forensic DNA databases do not contain Y-STR or mitochondrial DNA data. There are generally two familial searching methods, Identity-by-State (IBS) based methods or kinship index (KI) based methods. The KI based method is an analytically superior method because the allele frequency information is considered as opposed to solely allele counting. However, multiple KIs should be calculated if the unknown forensic profile may be attributed to multiple possible relevant populations. An important practical issue is the KI threshold to select for limiting the list of candidates from a search. There are generally three strategies of setting the KI threshold for familial searching: (1) SWGDAM recommendation 6; (2) minimum KI≥KI threshold; and (3) maximum KI≥KI threshold. These strategies were evaluated and compared by using both simulation data and empirical data. The minimum KI will tend to be closer to the KI appropriate for the population of which the forensic profile belongs. The minimum KI≥KI threshold performs better than the maximum KI≥KI threshold. The SWGDAM strategy may be too stringent for familial searching with large databases (e.g., 1 million or more profiles), because its KI thresholds depend on the database size and the KI thresholds of large databases have a higher probability to exclude true relatives than smaller databases. Minimum KI≥KI threshold strategy is a better option, as it provides the flexibility to adjust the KI threshold according to a pre-determined number of candidates or false positive/negative rates. Joint use of both IBS and KI does not significantly reduce the chance of including true relatives in a candidate list, but does provide a higher efficiency of familial searching.