Deep sequencing of a candidate region, harboring the SOX9 gene, for the canine XX disorder of sex development

Disorder of sex development (DSD) in dogs with female sex chromosomes (78, XX), a lack of the SRY gene and the presence of testes or ovotestes is commonly diagnosed in numerous breeds. The molecular background of DSD is not fully recognized but has been linked to the copy number variation (CNV) in the region harboring the SOX9 gene. We applied targeted next generation sequencing (NGS) techniques to compare DSD and normal female dogs. Targeted NGS of a 1.5 Mb region on canine chromosome 9 (CFA9) harboring the SOX9 gene revealed two putatively DSD associated CNVs in 355 kb upstream and 691 kb downstream of the SOX9, four blocks of low polymorphism and two blocks of an elevated heterozygosity. An initial NGS analysis showed the association with 2 SNPs, but validation in larger cohorts did not confirm this result. We identified a large homologous fragment (over 243.8 kb), named hfMAGI2, located upstream of the SOX9, that overlaps a known copy number variation region (CNVR). It shows a high sequence similarity with the 5’-flanking region of the MAGI2 gene located on CFA18, which encodes a protein involved in ovary formation during early embryonic development. Our study showed that the identified CNVR located upstream of the SOX9 gene contains potential regulatory sequences (long non-coding RNA and hfMAGI2) and led to assumption that a multiplication of this elements may alter expression of the SOX9 gene triggering the DSD phenotype.

携带雌性性染色体核型（78, XX）、缺失SRY基因且存在睾丸或卵睾体的犬类性发育异常（Disorder of Sex Development, DSD），在众多犬品种中均有常见诊断报道。该类DSD的分子机制尚未完全阐明，但已被证实与SOX9基因所在区域的拷贝数变异（Copy Number Variation, CNV）相关。本研究采用靶向下一代测序（Next Generation Sequencing, NGS）技术，对DSD患犬与正常雌性犬进行对比分析。对犬9号染色体（CFA9）上包含SOX9基因的1.5 Mb区域进行靶向NGS检测，结果在SOX9基因上游355 kb与下游691 kb处发现两个疑似与DSD相关的CNV，同时检测到4个低多态性区块与2个杂合性升高区块。初步NGS分析显示存在2个单核苷酸多态性（Single Nucleotide Polymorphism, SNP）的关联信号，但在更大样本队列中进行验证后，该结果未得到证实。本研究在SOX9基因上游区域鉴定出一段长逾243.8 kb的同源片段，将其命名为hfMAGI2，该片段与已知的拷贝数变异区域（CNVR）存在重叠。该片段与位于犬18号染色体（CFA18）上的MAGI2基因5’侧翼区域具有高度序列同源性，而MAGI2基因编码的蛋白参与胚胎早期卵巢发育过程。本研究证实，SOX9基因上游的CNV区域包含潜在调控序列（长链非编码RNA与hfMAGI2），据此推测该元件的拷贝数扩增可能会改变SOX9基因的表达，进而诱发DSD表型。