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

通过 RNA-seq 和比较基因组学鉴定 XX DSD 突变

• 批准号: 8570646
• 负责人: VICKI N MEYERS-WALLEN
• 金额: $ 7.75万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-08-01 至 2015-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8570646
• 关键词: Affect; Alleles; Animal Model; Bioinformatics; Canis familiaris; Code; Custom; DNA Resequencing; Data; Data Set; Development; Diagnostic tests; Disease; Embryo; Failure; Family Sizes; Gene Duplication; Gene Expression; Gene Expression Profile; Genes; Genetic; Genetic Transcription; Genetic Variation; Genome; Genomics; Goals; Gonadal structure; Histologic; Human; Indium; Individual; Infertility; Ligands; Link; Mammals; Methods; Modeling; Mus; Mutation; Nucleic Acid Regulatory Sequences; Nucleotides; Operative Surgical Procedures; Orthologous Gene; Ovarian; Ovary; Pathway interactions; Patients; Phenotype; Pilot Projects; Play; Regulatory Element; Reporting; Research; Rodent; Rodent Model; Role; Sexual Development; Siblings; Signal Transduction; Social Adjustment; Testing; Testis; Transgenic Organisms; Up-Regulation; WNT4 gene; autosomal recessive trait; beta catenin; candidate identification; comparative genomics; design; genetic linkage analysis; genetic pedigree; genome sequencing; genome wide association study; improved; in vivo; mullerian-inhibiting hormone; mutant; public health relevance; sex; sex determination; transcriptome sequencing

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.

描述（由申请人提供）：性发展疾病的研究（DSD）在理解性别确定的遗传控制和控制性腺发育的相对途径方面发挥了重要作用。在睾丸途径中，Sox9表达对睾丸诱导至关重要。相反，灭绝Sox9表达对于正常的卵巢发育似乎至关重要。例如，在XX DSD重复的XX DSD患者中，缺乏Y连锁睾丸定义基因的XX个体中会出现睾丸。卵巢途径的最新研究尚未确定通常在XX性腺中熄灭SOX9转录的长期寻求机制。这可能是反对睾丸途径的卵巢途径的关键步骤。 我们的目标是在XX DSD的亚型中确定一个因果突变，在XX DSD的亚型中，XX个体在其XX兄弟姐妹发展产量​​时会产生睾丸。这些患者的研究受到阻碍，因为该疾病罕见，家庭大小很小，并且没有啮齿动物模型。犬模型是此XX DSD亚型的唯一模型。在犬科研究的谱系中，XX DSD是一种常染色体隐性特征，表达限于XX兄弟姐妹，它会发展出睾丸或产卵。早期研究表明，在这些性腺中，睾丸途径不完全抑制或不当受到激活。使用GWAS，我们已经鉴定并重新定位了与模型谱系中犬XX DSD显着相关的区域。该区域与Sox9的调节区域重叠。我们的具体目的是：特定目标1假设：犬XX DSD的病因突变位于通常在XX性腺中熄灭Sox9转录的遗传开关。为了检验这一假设，我们将产生转录组[RNA-SEQ]，以完成一项比较XX DSD胚胎中性腺基因表达与正常XX和XY对照组的试验研究。如果我们的假设是正确的，那么在XX DSD性腺中SOX9的表达将比XX对照组更大，也许接近在XY性腺中观察到的水平。如果我们的假设不正确，则该项目的性腺基因表达数据对于构建替代假设将是有价值的。 具体目标2假设：犬XX DSD的病因突变位于哺乳动物中保守的遗传开关内。为了检验这一假设，我们将使用一种组合的比较基因组学和生物信息学方法来比较两个现有数据集，即XX DSD患者的微阵列数据集，并从犬XX DSD模型中重新陈述数据。如果我们的假设是正确的，我们将在进化上保守的XX DSD个体（人类和狗）中识别直系同源物，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鉴定人类患者和犬模型的候选突变来消除XX DSD研究的两个主要障碍，并证实犬XX DSD模型是一种适当的模型，可以在其中进一步表征睾丸和卵巢途径中的关键要素。