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.

摘要 到目前为止，特发性男性不育的病因还不完全清楚，激素男性避孕药已经 效果不佳。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，这项建议试图确定新的编码和非编码基因和基因组区域所需的 并通过小鼠模型验证新发现的基因的功能需求。过去时 到目前为止，有限的基于测序的方法只能为1.5%的 出现在临床上的不育男性，随着识别出更多潜在的基因，产量将会提高 男性不育症。过去进展的一个限制是传统的短读测序无法 解析单倍型特定信息：每个基因上的两个或多个突变是相同的还是分开的 等位基因。由于基因功能丧失通常会对两个等位基因造成损害，因此至关重要的是 能够解析此信息。通过长阅读测序(一种新建立和验证的技术) 我们将在单倍型特定分辨率上识别新的破坏性突变，将多个新的破坏性关联起来 我们新测序的患者中存在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，确定对精子发育具有致命性的突变。突变 存在于血液中(代表母本和父本的基因组)，在精子中不存在[具有单倍体 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 实际上可能是有生育能力的男性生育所需的。最后，我们将应用CRISPR/Cas9在小鼠身上进行验证 我们通过测序研究确定的候选基因的功能要求。新奇的男性 在精子或附睾细胞中表达的不育相关基因在进化过程中 在包括老鼠和人类在内的脊椎动物中保守，并在我们的患者中排名最高 确定了验证的优先顺序。我们将评估突变小鼠的生育表型，并进一步探索分子 病理生理学，对于某些基因来说，这可能决定是否有可能成为避孕目标。