Search for new genes involved in male infertility through novel approaches

通过新方法寻找与男性不育相关的新基因

• 批准号: 10577839
• 负责人: Thomas Garcia
• 金额: $ 55万
• 依托单位: BAYLOR COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-02-22 至 2026-11-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10577839
• 关键词: Affect; Alleles; Animal Model; Area; Bioinformatics; Biological Process; Biological Specimen Banks; Biology; Blood; CRISPR/Cas technology; Candidate Disease Gene; Cells; Child; Chromosomes; Classification; Clinic; Clinical Management; Code; Complex; Contraceptive Agents; Contraceptive methods; Copy Number Polymorphism; Couples; Development; Diagnosis; Diagnostic; Etiology; Evaluation; FDA approved; Family; Fertility; Functional disorder; Future; Genes; Genetic; Genetic Counseling; Genome; Genomic Segment; Goals; Haploid Cells; Haploidy; Haplotypes; Heterozygote; Hormonal; Human; Impairment; Individual; Infertility; Intervention; Investigation; Knowledge; Laboratories; Link; Male Contraceptive Agents; Male Infertility; Mediating; Meiosis; Methods; Modeling; Molecular; Molecular Abnormality; Molecular Diagnostic Testing; Mus; Mutant Strains Mice; Mutation; Pathway interactions; Patients; Pharmaceutical Preparations; Phenotype; Population; Research; Resolution; Role; Sampling; Spermiogenesis; Therapeutic; Third Generation Sequencing; Time; Translating; United States National Institutes of Health; Untranslated RNA; Validation; Variant; Vertebrates; Whole Blood; Woman; Work; analysis pipeline; candidate identification; clinical infrastructure; cohort; contraceptive target; empowerment; gene product; gene synthesis; gene therapy; genetic testing; genetic variant; genome sequencing; improved; large datasets; loss of function; male; male fertility; men; mouse model; next generation; novel; novel strategies; reproductive; small molecule inhibitor; sperm cell; standard of care; subfertility; technology validation; transmission process; whole genome

SUMMARY To date, the etiology of idiopathic male infertility is not fully understood, and hormonal male contraceptives have not been effective. Through novel approaches to sequencing and sequencing analysis of male infertile patients and controls, this proposal seeks to identify new coding and non-coding genes and genomic regions required for male fertility and to validate the functional requirement of newly identified genes through mouse models. Past limited sequencing-based approaches have so far only been able to provide diagnostic variants to 1.5% of infertile men who present to the clinic, a yield that will improve with identification of more genes underlying infertility in men. One limitation to past progress has been the inability of conventional short-read sequencing to resolve haplotype-specific information: whether two or more mutations in each gene are on the same or separate alleles. Since gene loss-of-function typically requires damage to both alleles, it is of paramount importance to be able to resolve this information. Through long-read sequencing (a newly established and validated technology) we will identify novel damaging mutations at haplotype-specific resolution, correlating multiple novel damaging mutations with potential loss-of-function in our newly sequenced patients. Additionally, since sperm are haploid and previous studies have demonstrated a requirement for newly synthesized genes after meiosis, we will identify mutations lethal to sperm development through sequencing of both whole blood and sperm. Mutations present in blood (representing both maternal and paternal genomes) that are absent in sperm [with haploid genomes representing 8 million (2^23) chromosome combinations] demonstrate a requirement for those genes in the later stages of sperm development. Thus, through our proposed work we will identify for the first-time genes required for male fertility in men who may in fact be fertile. Lastly, we will apply CRISPR/Cas9 in mice to validate the functional requirement of candidate genes that we identify through our sequencing studies. Novel male infertility associated genes that are found to be expressed in sperm or epididymal cells, are evolutionarily conserved in vertebrates, including mice and humans, and rank among the highest in our patients will be prioritized for validation. We will assess the fertility phenotype of mutant mice and further explore molecular pathophysiology, which for some genes may determine candidacy as a contraceptive target.

总结 迄今为止，特发性男性不育症的病因尚未完全了解，激素男性避孕药已 不是有效的。通过新的方法对男性不育患者进行测序和测序分析 和控制，这项建议旨在确定新的编码和非编码基因和基因组区域所需的 雄性生育力，并通过小鼠模型验证新鉴定的基因的功能要求。过去 有限的基于测序的方法到目前为止只能为1.5%的人提供诊断性变体。 不育男性谁提出的诊所，产量将提高与更多的基因识别的基础上， 男性不育症过去进展的一个局限性是传统的短读测序无法 解析单倍型特异性信息：每个基因中的两个或多个突变是相同的还是不同的 等位基因由于基因功能丧失通常需要对两个等位基因都造成损害，因此， 能够解决这些信息。通过长读序测序（一种新建立和验证的技术） 我们将在单倍型特异性分辨率下识别新的破坏性突变， 在我们新测序的患者中有潜在功能丧失的突变。此外，由于精子是单倍体， 以前的研究已经证明了减数分裂后需要新合成的基因，我们将 通过对全血和精子进行测序，确定对精子发育致命的突变。突变 存在于血液中（代表母亲和父亲的基因组），在精子中不存在[与单倍体 代表800万（2^23）个染色体组合的基因组]证明了这些基因在 精子发育的后期阶段因此，通过我们提出的工作，我们将首次确定基因 男性生育力所需的蛋白质。最后，我们将在小鼠中应用CRISPR/Cas9来验证 我们通过测序研究确定的候选基因的功能要求。一种新型公 不育相关基因在精子或附睾细胞中表达， 在脊椎动物中保守，包括小鼠和人类，并在我们的患者中排名最高， 优先进行验证。我们将评估突变小鼠的生育表型，并进一步探索其分子生物学机制。 病理生理学，这对某些基因可能决定候选人作为避孕目标。