Supplementary Material for: Mutations in the <i>RSPO1</i> Coding Region Are Not the Main Cause of Canine <i>SRY</i>-Negative XX Sex Reversal in Several Breeds

This report details a case of SRY-negative XX sex reversal in a mixed breed dog and surveys affected dogs of several breeds for mutations in RSPO1 coding regions. Genomic DNA from the mixed breed case was evaluated for mutations in candidate genes. Sequencing identified a homozygous G to A transition in RSPO1 exon 4 that changes a highly conserved amino acid codon in the thrombospondin domain. The possibility that this was a single nucleotide polymorphism (SNP) could not be excluded by genotyping family members. Therefore, the coding region of RSPO1 was sequenced in a survey of affected dogs, which identified a T to C transition (exon 3) in some, the above G to A transition (exon 4) in others, and no change in the remaining affected dogs. Genotypes at these base pair positions were not uniquely associated with the affected phenotype in any breed, indicating the identified transitions are most likely SNPs, not causative mutations for this canine disorder. However, the possibility that polymorphisms play a modifier role, such as changing threshold or severity of phenotypic expression in a mixed breed dog, cannot be excluded. This study emphasizes the importance of canine pedigree, breed, and population studies in evaluating candidate mutations.本报告详述了1例混血犬SRY（Sex-determining Region Y）阴性XX性逆转病例，并针对多个品种的患病犬开展RSPO1（R-spondin 1）编码区突变筛查。对该混血病例的基因组DNA开展候选基因突变检测，测序结果显示，RSPO1第4外显子存在一处纯合G→A转换突变，该突变会改变血小板反应蛋白结构域中一个高度保守的氨基酸密码子。通过对家系成员进行基因分型，无法排除该突变为单核苷酸多态性（single nucleotide polymorphism, SNP）的可能性。因此，本研究对一批患病犬开展RSPO1编码区测序，结果显示部分个体存在第3外显子的T→C转换突变，部分个体存在前述第4外显子的G→A转换突变，其余患病犬则未出现上述突变。在所有品种中，上述碱基位点的基因型均未与患病表型呈现唯一关联，这表明本次发现的转换突变极大概率为单核苷酸多态性，而非该犬类疾病的致病突变。但仍无法排除多态性发挥修饰作用的可能性，例如改变混血犬的表型表达阈值或严重程度。本研究强调了犬类系谱、品种及群体研究在候选基因突变评估中的重要性。