Project I: Discovery and Annotation of Targets for Gene Therapy of Infertile Men Infertility affects 15% of reproductive-age couples in the US, leading to more than 108,000 new visits to reproductive endocrinology and infertility (REI) clinics per year. Nearly all such clinics offer pre-implantation genetic diagnosis of embryos and genetic testing for known mutations associated with endocrine dysfunction, primary gonadal failure, and recurrent pregnancy loss. However, reproductive medicine specialists rely on old technologies like cytogenetics, sequence-tagged site PCR, and Sanger sequencing for these tests and are not taking advantage of modern whole-genome and RNA sequencing technologies common in clinical genetics of other disease states. New genomic tools, both computational and experimental, promise to revolutionize the way we diagnose and treat infertility. An overall goal of this P50 application is to create a roadmap for how these tools can be used to (a) identify mutations contributing to male infertility, (b) characterize how these mutations may contribute to pathology in somatic tissues, and (c) how gene therapy can be used to treat these pathologies in a safe and targeted manner. In this project, we are recruiting patients of male infertility from three primary sites: Washington University, Weill Cornell Medical School, and Magee-Womens Hospital. We will apply whole- genome sequencing and high-resolution array CGH to map the location of genetic variation in 500 total cases, including 21 large, multiplex families with heritable forms of azoospermia. As part of the patient phenotyping, we will deploy a specialized instrument known as the Charlson Comorbidity index to specifically document the evidence for comorbidity in each case of infertility. We will develop and apply highly sensitive statistical methods, which draw upon the information in massive population control databases, to identify statistically unusual mutations that are likely to confer risk for spermatogenic impairment. We will develop knowledge bases that summarize the evidence from model organisms that these mutations can cause pathology in both gonadal and somatic tissues. And we will attempt to infer the testicular cell type(s) that are the primary sites of pathology for each mutation, to help guide the targeting of gene therapy. The most important long-term outcome of this work will be the publication of analysis tools and knowledge bases that will facilitate the use of genome sequencing in the treatment of infertility. Combined with the results from Project II and Project III, our results will give reproductive medicine specialists a roadmap for the use of modern genetic technologies to investigate and treat infertility.