DataSheet_1_Broad Phenotypes of Disorders/Differences of Sex Development in MAMLD1 Patients Through Oligogenic Disease.pdf

Disorders/differences of sex development (DSD) are the result of a discordance between chromosomal, gonadal, and genital sex. DSD may be due to mutations in any of the genes involved in sex determination and development in general, as well as gonadal and/or genital development specifically. MAMLD1 is one of the recognized DSD genes. However, its role is controversial as some MAMLD1 variants are present in normal individuals, several MAMLD1 mutations have wild-type activity in functional studies, and the Mamld1-knockout male mouse presents with normal genitalia and reproduction. We previously tested nine MAMLD1 variants detected in nine 46,XY DSD patients with broad phenotypes for their functional activity, but none of the mutants, except truncated L210X, had diminished transcriptional activity on known target promoters CYP17A1 and HES3. In addition, protein expression of MAMLD1 variants was similar to wild-type, except for the truncated L210X. We hypothesized that MAMLD1 variants may not be sufficient to explain the phenotype in 46,XY DSD individuals, and that further genetic studies should be performed to search for additional hits explaining the broad phenotypes. We therefore performed whole exome sequencing (WES) in seven of these 46,XY patients with DSD and in one 46,XX patient with ovarian insufficiency, who all carried MAMLD1 variants. WES data were filtered by an algorithm including disease-tailored lists of MAMLD1-related and DSD-related genes. Fifty-five potentially deleterious variants in 41 genes were identified; 16/55 variants were reported in genes in association with hypospadias, 8/55 with cryptorchidism, 5/55 with micropenis, and 13/55 were described in relation with female sex development. Patients carried 1-16 variants in 1-16 genes together with their MAMLD1 variation. Network analysis of the identified genes revealed that 23 genes presented gene/protein interactions with MAMLD1. Thus, our study shows that the broad phenotypes of individual DSD might involve multiple genetic variations contributing towards the complex network of sexual development.

性发育异常与差异（disorders/differences of sex development, DSD）是染色体性别、性腺性别与生殖器性别三者不一致所导致的病症。DSD的致病原因可涉及参与普遍意义上性别决定与发育、以及特定性腺和/或生殖器发育的各类基因发生突变。MAMLD1是已确认的DSD相关基因之一，但其在DSD中的作用尚存争议：部分MAMLD1变异可在正常个体中检出，多项功能研究显示部分MAMLD1突变仍保留野生型活性，且Mamld1基因敲除的雄性小鼠可表现出正常的生殖器发育与生殖功能。我们此前针对9例携带不同MAMLD1变异、表现出广泛表型的46,XY DSD患者的变异体开展了功能活性检测；结果显示，除截短突变体L210X外，其余突变体在已知靶启动子CYP17A1与HES3上均未表现出转录活性降低的情况。此外，除截短突变体L210X外，其余MAMLD1变异体的蛋白表达水平与野生型MAMLD1无显著差异。我们据此提出假设：仅靠MAMLD1变异不足以解释46,XY DSD患者的表型，需开展进一步遗传学研究以探寻可解释其广泛表型的其他致病变异。为此，我们对其中7例携带MAMLD1变异的46,XY DSD患者，以及1例携带MAMLD1变异的46,XX卵巢功能不全患者开展了全外显子组测序（whole exome sequencing, WES）。我们通过一套定制化算法对WES数据进行过滤，该算法整合了MAMLD1相关基因与DSD相关基因的疾病特异性基因列表。最终在41个基因中检出55个潜在致病变异：其中16个变异所在基因与尿道下裂相关，8个与隐睾症相关，5个与小阴茎相关，另有13个变异与女性性别发育相关。所有患者均在1至16个基因中携带1至16个变异，同时携带有MAMLD1变异。对检出基因的网络分析显示，其中23个基因可与MAMLD1发生基因或蛋白层面的相互作用。综上，本研究表明，个体DSD的广泛表型可能由多种遗传变异共同作用所致，这些变异参与构成了复杂的性别发育调控网络。