Identification of XX DSD Mutations by RNA-seq and Comparative Genomics

描述（由申请人提供）：性发育障碍（DSD）研究在理解性别决定的遗传控制和控制性腺发育的相反途径方面发挥了重要作用。在睾丸途径中，SOX 9表达对睾丸诱导至关重要。相反，消除SOX 9表达似乎对正常卵巢发育至关重要。例如，在具有SOX 9重复的XX DSD患者中，睾丸在缺乏Y连锁睾丸决定基因SRY的XX个体中发育。最近的研究卵巢通路尚未确定长期寻求的机制，SOX 9转录通常是在XX性腺熄灭。这可能是卵巢途径中与睾丸途径相反的关键步骤。 我们的目标是确定XX DSD亚型的致病突变，其中XX个体发育睾丸，而他们的XX兄弟姐妹发育卵睾丸。对这些患者的研究一直受到阻碍，因为这种疾病不常见，家庭规模小，而且没有啮齿动物模型。犬模型是该XX DSD亚型的唯一模型。在犬研究谱系中，XX DSD是一种常染色体隐性遗传性状，表达仅限于发育睾丸或卵睾丸的XX同胞。早期的研究表明，睾丸通路在这些性腺中被不完全抑制或不适当地激活。使用GWAS，我们已经确定并重新测序了与模型谱系中的犬XX DSD显著相关的区域。该区域与SOX 9的调控区域重叠。我们的具体目标是：具体目标1假设：犬XX DSD的致病突变位于一个遗传开关内，该开关通常会破坏XX性腺中的SOX 9转录。为了验证这一假设，我们将产生转录组[RNA-seq]来完成一项初步研究，将XX DSD胚胎中的性腺基因表达与正常XX和XY对照进行比较。如果我们的假设是正确的，SOX 9的表达将是更大的XX DSD性腺比XX控制，也许接近XY性腺中观察到的水平。如果我们的假设是不正确的，从这个项目的性腺基因表达数据将是有价值的构建替代假设。 特定目标2假说：犬XX DSD的致病突变位于哺乳动物中保守的遗传开关内。为了验证这一假设，我们将使用一个组合的比较基因组学和生物信息学的方法来比较两个现有的数据集，一个来自XX DSD患者的微阵列数据集和来自犬XX DSD模型的重测序数据。如果我们的假设是正确的，我们将在XX DSD个体（人和狗）中鉴定出进化上保守的直系同源物，但含有与参考基因组不同的核苷酸。这些核苷酸差异将是我们的候选突变。 该项目的结果将通过识别人类患者和犬模型中的候选突变，并确认犬XX DSD模型是进一步表征睾丸和卵巢途径中关键要素的适当模型，消除XX DSD研究的两个主要障碍。

DESCRIPTION (provided by applicant): Research in Disorders of Sexual Development (DSD) has played a major role in understanding the genetic control of sex determination and the opposing pathways controlling gonadal development. In the testis pathway, SOX9 expression is critical to testis induction. Conversely, extinguishing SOX9 expression appears essential for normal ovarian development. For example, in XX DSD patients with SOX9 duplications, testes develop in XX individuals lacking the Y-linked testis-determining gene, SRY. Recent studies of the ovary pathway have not yet identified the long sought mechanism by which SOX9 transcription is normally extinguished in XX gonads. This could be a key step in the ovary pathway that opposes the testis pathway. Our goal is to identify a causative mutation in the subtype of XX DSD in which XX individuals develop testes while their XX siblings develop ovotestes. Studies in these patients have been impeded because the disorder is uncommon, family sizes are small, and there are no rodent models. The canine model is the only model of this XX DSD subtype. In the canine research pedigree, XX DSD is an autosomal recessive trait with expression limited to XX siblings, which develop testes or ovotestes. Early studies suggested that the testis pathway is incompletely suppressed or inappropriately activated in these gonads. Using GWAS, we have identified and resequenced a region significantly associated with canine XX DSD in the model pedigree. This region overlaps the regulatory region of SOX9. Our Specific Aims are: Specific Aim 1 Hypothesis: The causative mutation for canine XX DSD lies within a genetic switch that normally extinguishes SOX9 transcription in XX gonads. To test this hypothesis, we will produce transcriptomes [RNA-seq] to complete a pilot study comparing gonadal gene expression in XX DSD embryos to that of normal XX and XY controls. If our hypothesis is correct, SOX9 expression will be greater in XX DSD gonads than those of XX controls, perhaps approaching levels observed in XY gonads. If our hypothesis is incorrect, the gonadal gene expression data from this project will be valuable for constructing alternative hypotheses. Specific Aim 2 Hypothesis: The causative mutation for canine XX DSD lies within a genetic switch that is conserved in mammals. To test this hypothesis, we will use a combined comparative genomics and bioinformatics approach to compare two existing datasets, a microarray dataset from XX DSD patients and resequencing data from the canine XX DSD model. If our hypothesis is correct, we will identify orthologs in XX DSD individuals (humans and dogs) that are evolutionarily conserved, yet contain nucleotides that are different from the reference genomes. Those nucleotide differences will be our candidate mutations. Results from this project will remove two major obstacles to the study of XX DSD by identifying candidate mutations in human patients and the canine model, and confirming that the canine XX DSD model is an appropriate model in which to further characterize key elements in the testis and ovary pathways.