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性腺发育的相反途径方面发挥了重要作用。在睾丸通路中，SOX9的表达对睾丸诱导至关重要。相反，抑制SOX9的表达对于卵巢的正常发育至关重要。例如，在XX例SOX9重复的DSD患者中，XX个体缺乏y连锁睾丸决定基因SRY。最近对卵巢通路的研究尚未确定长期寻找的SOX9转录在XX性腺中正常消失的机制。这可能是卵巢途径对抗睾丸途径的关键一步。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兄弟姐妹发育卵泡。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性状，其表达仅限于XX个兄弟姐妹，这些兄弟姐妹发育睾丸或卵睾丸。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激活。使用GWAS，我们在模型家系中确定并重新测序了与犬XX DSD显著相关的区域。该区域与SOX9的调控区域重叠。假设：犬XX DSD的致病突变存在于一个基因开关中，该开关通常会抑制XX性腺中SOX9的转录。为了验证这一假设，我们将制作转录组[RNA-seq]来完成一项比较XX DSD胚胎中性腺基因表达与正常XX和XY对照的初步研究。如果我们的假设是正确的，那么SOX9在XX个DSD性腺中的表达将高于XX个对照组，可能接近在XY性腺中观察到的水平。如果我们的假设是不正确的，从这个项目中获得的性腺基因表达数据将对构建替代假设有价值。假设：犬XX DSD的致病突变存在于哺乳动物中保守的遗传开关中。为了验证这一假设，我们将使用比较基因组学和生物信息学相结合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个DSD个体（人类和狗）中发现进化保守的同源基因，但包含与参考基因组不同的核苷酸。这些核苷酸的差异将是我们的候选突变。本项目的结果将通过确定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通路关键元件的合适模型，从而消除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.