Discovery of Candidate Genes for Stallion Fertility from the Horse Y Chromosome

The genetic component of mammalian male fertility is complex and involves thousands of genes. The majority of these genes are distributed on autosomes and the X chromosome, while a small number are located on the Y chromosome. Human and mouse studies demonstrate that the most critical Y-linked male fertility genes are present in multiple copies, show testis-specific expression and are different between species. In the equine industry, where stallions are selected according to pedigrees and athletic abilities but not for reproductive performance, reduced fertility of many breeding stallions is a recognized problem. Therefore, the aim of the present research was to acquire comprehensive information about the organization of the horse Y chromosome (ECAY), identify Y-linked genes and investigate potential candidate genes regulating stallion fertility. To achieve theses goals, a direct cDNA (complementary DNA) selection procedure was used to isolate Y-linked genes from horse testes and 29 Y-specific genes were identified. All 29 genes were mapped to ECAY and their sequences were used to further expand the existing map. Copy number analysis identified 15 multicopy genes of which 9 were novel transcripts. Gene expression analysis on a panel of selected body tissues showed that some ECAY genes are expressed exclusively in testes while others show ubiquitous or intermediate expression. Quantitative Real-Time PCR using primers for 9 testis-specific multicopy genes revealed 5 genes with statistically significant differential expression in testis of normal fertile stallions and stallions with impaired fertility. Gene copy number analysis showed that the average copy number of 4 such genes was decreased in subfertile/infertile stallions compared to normal animals. Taken together, this research generated the first comprehensive physical gene map for the horse Y chromosome and identified a number of candidate genes for stallion fertility. The findings essentially expand our knowledge about Y chromosome genes in horses, open a new avenue for investigating the potential role of ECAY genes in stallion fertility which contribute to the development of molecular tools for the assessment of fertility in stallions

Discovery of Candidate Genes for Stallion Fertility from the Horse Y Chromosome

The genetic component of mammalian male fertility is complex and involves thousands of genes. The majority of these genes are distributed on autosomes and the X chromosome, while a small number are located on the Y chromosome. Human and mouse studies demonstrate that the most critical Y-linked male fertility genes are present in multiple copies, show testis-specific expression and are different between species. In the equine industry, where stallions are selected according to pedigrees and athletic abilities but not for reproductive performance, reduced fertility of many breeding stallions is a recognized problem. Therefore, the aim of the present research was to acquire comprehensive information about the organization of the horse Y chromosome (ECAY), identify Y-linked genes and investigate potential candidate genes regulating stallion fertility. To achieve theses goals, a direct cDNA (complementary DNA) selection procedure was used to isolate Y-linked genes from horse testes and 29 Y-specific genes were identified. All 29 genes were mapped to ECAY and their sequences were used to further expand the existing map. Copy number analysis identified 15 multicopy genes of which 9 were novel transcripts. Gene expression analysis on a panel of selected body tissues showed that some ECAY genes are expressed exclusively in testes while others show ubiquitous or intermediate expression. Quantitative Real-Time PCR using primers for 9 testis-specific multicopy genes revealed 5 genes with statistically significant differential expression in testis of normal fertile stallions and stallions with impaired fertility. Gene copy number analysis showed that the average copy number of 4 such genes was decreased in subfertile/infertile stallions compared to normal animals. Taken together, this research generated the first comprehensive physical gene map for the horse Y chromosome and identified a number of candidate genes for stallion fertility. The findings essentially expand our knowledge about Y chromosome genes in horses, open a new avenue for investigating the potential role of ECAY genes in stallion fertility which contribute to the development of molecular tools for the assessment of fertility in stallions

Genetic association and characterization of FSTL5 in isolated clubfoot

ACKNOWLEDGEMENTS: The Atherosclerosis Risk in Communities Study is carried out as a collaborative study supported by National Heart, Lung, and Blood Institute contracts (HHSN268201100005C, HHSN268201100006C, HHSN268201100007C, HHSN268201100008C, HHSN268201100009C, HHSN268201100010C, HHSN268201100011C, and HHSN268201100012C). The authors thank the staff and participants of the ARIC study for their important contributions. Funding for GENEVA was provided by National Human Genome Research Institute grant U01HG004402 (E.Boerwinkle). We thank H. Hobbs and J. Cohen for contributing control samples for replication genotyping, Nadav Ahituv for sharing RNA-seq data for both bat and mouse embryonic limb buds, Tommy Hyatt for designing the custom genotyping assay, and members of the UT Southwestern Transgenic Core facility, including John Ritter, Mylinh Nguyen, and Robert Hammer. Publicly available mouse embryonic expression analysis results were provided online at https://oncoscape.v3.sttrcancer.org/atlas.gs.washington.edu.mouse.rna/landing (24). The authors acknowledge the contributions and support of the Center for Excellence in Clubfoot Research at Scottish Rite for Children, including Shawne Faulks and Kristhen Atala. Fstl5 mutant rats were produced by the NIH Mutant Rat Resource at UT Southwestern Medical Center (R24RR03232601, R24OD011108, R01HD036022, and (5R01HD053889). This study was supported by funding from the Scottish Rite for Children Research Fund (J.J.R.), Shriners Hospital for Children (J.T.H), and the National Institutes of Health award R01HD043342 (J.T.H.).Peer reviewedPostprin

DNA Polymerase Epsilon Deficiency Causes IMAGe Syndrome with Variable Immunodeficiency.

During genome replication, polymerase epsilon (Pol ε) acts as the major leading-strand DNA polymerase. Here we report the identification of biallelic mutations in POLE, encoding the Pol ε catalytic subunit POLE1, in 15 individuals from 12 families. Phenotypically, these individuals had clinical features closely resembling IMAGe syndrome (intrauterine growth restriction [IUGR], metaphyseal dysplasia, adrenal hypoplasia congenita, and genitourinary anomalies in males), a disorder previously associated with gain-of-function mutations in CDKN1C. POLE1-deficient individuals also exhibited distinctive facial features and variable immune dysfunction with evidence of lymphocyte deficiency. All subjects shared the same intronic variant (c.1686+32C>G) as part of a common haplotype, in combination with different loss-of-function variants in trans. The intronic variant alters splicing, and together the biallelic mutations lead to cellular deficiency of Pol ε and delayed S-phase progression. In summary, we establish POLE as a second gene in which mutations cause IMAGe syndrome. These findings add to a growing list of disorders due to mutations in DNA replication genes that manifest growth restriction alongside adrenal dysfunction and/or immunodeficiency, consolidating these as replisome phenotypes and highlighting a need for future studies to understand the tissue-specific development roles of the encoded proteins

A Gene Catalogue of the Euchromatic Male-Specific Region of the Horse Y Chromosome: Comparison with Human and Other Mammals

Studies of the Y chromosome in primates, rodents and carnivores provide compelling evidence that the male specific region of Y (MSY) contains functional genes, many of which have specialized roles in spermatogenesis and male-fertility. Little similarity, however, has been found between the gene content and sequence of MSY in different species. This hinders the discovery of species-specific male fertility genes and limits our understanding about MSY evolution in mammals. Here, a detailed MSY gene catalogue was developed for the horse – an odd-toed ungulate. Using direct cDNA selection from horse testis, and sequence analysis of Y-specific BAC clones, 37 horse MSY genes/transcripts were identified. The genes were mapped to the MSY BAC contig map, characterized for copy number, analyzed for transcriptional profiles by RT-PCR, examined for the presence of ORFs, and compared to other mammalian orthologs. We demonstrate that the horse MSY harbors 20 X-degenerate genes with known orthologs in other eutherian species. The remaining 17 genes are acquired or novel and have so far been identified only in the horse or donkey Y chromosomes. Notably, 3 transcripts were found in the heterochromatic part of the Y. We show that despite substantial differences between the sequence, gene content and organization of horse and other mammalian Y chromosomes, the functions of MSY genes are predominantly related to testis and spermatogenesis. Altogether, 10 multicopy genes with testis-specific expression were identified in the horse MSY, and considered likely candidate genes for stallion fertility. The findings establish an important foundation for the study of Y-linked genetic factors governing fertility in stallions, and improve our knowledge about the evolutionary processes that have shaped Y chromosomes in different mammalian lineages

Genome-wide meta-analysis and replication studies in multiple ethnicities identify novel adolescent idiopathic scoliosis susceptibility loci

Adolescent idiopathic scoliosis (AIS) is the most common musculoskeletal disorder of childhood development. The genetic architecture of AIS is complex, and the great majority of risk factors are undiscovered. To identify new AIS susceptibility loci, we conducted the first genome-wide meta-analysis of AIS genome-wide association studies, including 7956 cases and 88 459 controls from 3 ancestral groups. Three novel loci that surpassed genome-wide significance were uncovered in intragenic regions of the CDH13 (P-value_rs4513093 = 1.7E-15), ABO (P-value_ rs687621 = 7.3E-10) and SOX6 (P-value_ rs1455114 = 2.98E-08) genes. Restricting the analysis to females improved the associations at multiple loci, most notably with variants within CDH13 despite the reduction in sample size. Genome-wide gene-functional enrichment analysis identified significant perturbation of pathways involving cartilage and connective tissue development. Expression of both SOX6 and CDH13 was detected in cartilage chondrocytes and chromatin immunoprecipitation sequencing experiments in that tissue revealed multiple HeK27ac-positive peaks overlapping associated loci. Our results further define the genetic architecture of AIS and highlight the importance of vertebral cartilage development in its pathogenesis.Peer reviewe

Structural and functional annotations for mRNAs and ESTs with the highest AC values by RNA-seq.

<p>Structural and functional annotations for mRNAs and ESTs with the highest AC values by RNA-seq.</p

Summary statistics for mapped RNA sequence tags

<p>: (<b>a</b>) Comparison of mapped tags (AC≥1) between the two sperm samples; (<b>b</b>) Proportions of tags with very high (AC≥100), high (10</p

Venn diagram of transcripts detected in stallion sperm and testes by microarray analysis (SNR ≥2).

<p>Venn diagram of transcripts detected in stallion sperm and testes by microarray analysis (SNR ≥2).</p

ECAY transcripts in stallion sperm.

<p>Agarose gel images showing RT-PCR amplicons of 7 ECAY genes and transcripts in stallion sperm.</p