Molecular mechanisms in disorders of gonadal development

Abstract: “Hur många barn får jag när jag blir stor?” Controlling fertility, or rather infertility, is a common problem for couples nowadays where approximately 10% experience infertility. This thesis focuses on the rare conditions leading to infertility in which the gonads have either failed to develop properly, or prematurely lost their function. The overall aim of this thesis was to identify and study candidate genes in gonadal disorders of sex development (DSD) and primary ovarian insufficiency (POI) to achieve a better understanding of the underlying molecular mechanisms and to improve available diagnostic tools and genetic counseling for affected patients and family members. The process of sex determination is sensitive to gene dosage, and genes affecting gonadal development are located on sex chromosomes and autosomes. Attempting to identify new candidate regions for testicular development, nine patients with 46,XY complete gonadal dysgenesis were investigated with a customized 1M array-CGH (comparative genomic hybridization) platform with whole-genome coverage and probe enrichment targeting 78 genes involved in DSD. Aberrations affecting SUPT3H and C2ORF80 were identified and characterized in two affected sisters. Sequence analysis of these genes in all patients revealed no additional mutations. PIP5K1B and PRKACG were also identified as candidate genes in another patient. All four genes are expressed in testicular tissues, and one is shown to cause gonadal DSD in mice (Paper I). Based on the report of one patient with 46,XY ovarian DSD due to CBX2 mutations and the corresponding knock out mouse model, we considered CBX2 as a candidate gene for 46,XY/46,XX gonadal DSD and POI. By sequencing and MLPA a cohort of 47 patients with gonadal DSD or POI was investigated for sequence alterations and copy number changes in CBX2. No causative mutation was detected. Our study does therefore not support CBX2 mutations as a common cause of gonadal DSD (Paper II). DNA samples from 26 patients with POI were analyzed by our customized array-CGH platform to identify novel candidate genes in POI. Eleven unique copy number changes were identified in a total of 13 patients. We identified the first mutation affecting the regulatory region of GDF9, a 475bp duplication containing three NOBOX binding elements and a regulatory E-box. In addition, we found the second DNAH6 deletion, corroborating its potential role in causing in POI. Also TSPYL6, SMARCC1, CSPG5, SH3GL3 and ZFR2 were identified as candidate genes in POI (Paper III). PSMC3IP mutations have been described causing autosomal recessive POI in a consanguineous family with several affected members. We are the first to have investigated the PSMC3IP gene in a group of unrelated patients with POI. No mutation was detected and we therefore conclude that PSMC3IP mutations are not a common cause of POI in a Swedish patient cohort (Paper IV). In conclusion, there is accumulating evidence that gonadal DSD and POI are highly genetically heterogeneous. We recommend that the genetic investigation of patients with gonadal DSD should be centralized to a specialized DSD unit. Patients with POI should be handled by subspecialists within reproductive endocrinology and clinical genetic investigation can require genetic counseling. A suggested clinical investigation procedure for POI is presented. Ideally, genetic investigation of patients with gonadal DSD or POI should include investigation for gene dosage aberrations as well sequencing of several candidate genes.