Supplementary Material for: Whole-Exome Sequencing Indicated New Candidate Genes Associated with Unilateral Cryptorchidism in Pigs

Introduction: Cryptorchidism is a hereditary anomaly characterized by the incomplete descent of one or both testicles to the scrotum. One of the challenges of this anomaly is that the retained testicle maintains its endocrine function. As a consequence, cryptorchid animals produce hormone-tainted meat in comparison to castrated animals and are likely to be more aggressive. Cryptorchidism can lead to reduced animal welfare outcomes and cause economic losses. Identifying genetic markers for cryptorchidism is an essential step toward mitigating these negative outcomes and may facilitate genome manipulation to reduce the occurrence of cryptorchidism. Attempts to identify such markers have used genome-wide association studies. Using whole-exome sequencing, we aimed to identify single nucleotide polymorphisms (SNPs) in the coding regions of cryptorchid pigs and to characterize functional pathways concerning these SNPs. Methods: DNA was extracted and sequenced from 5 healthy and 5 cryptorchid animals from the Landrace breed, using the Illumina HiSeq 2500 platform. Data were pre-processed using the SeqyClean tool and further mapped against the swine reference genome (Sus scrofa 11.1) using BWA software. GATK was used to identify polymorphisms (SNPs and InDels), which were annotated using the VEP tool. Network prediction and gene ontology enrichment analysis were conducted using the Cytoscape platform, and STRING software was used for visualization. Results: A total of 63 SNPs were identified across the genes PIGB, CCPG1, COMMD9, LDLRAD3, TRIM44, MYLPF, SEPTIN, ZNF48, TIA1, FAIM2, KRT18, FBP1, FBP2, CTSL, DAPK1, DHX8, GPR179, DEPDC1B, ENSSSCG00000049573, ENSSSCG00000016384, ENSSSCG00000022657, ENSSSCG00000038825, and ENSSSCG00000001229. Using pathway enrichment analyses and network prospection, we have identified the following significant adjusted p value threshold of 0.001 involved with the biological function pathways of estrogen signaling, cytoskeleton organization, and the pentose phosphate pathway. Conclusion: Our data suggest the involvement of new SNPs and genes in developing cryptorchidism in pigs. However, further studies are needed to validate our results in a larger cohort population. Variations in the GPR179 gene, with implications at the protein level, may be associated with the appearance of this anomaly in the swine. Finally, we are showing that the estrogen signaling pathway may be involved in the pathophysiological mechanisms of this congenital anomaly as previously reported in GWAS.引言：隐睾症（Cryptorchidism）是一种遗传性异常，特征为单侧或双侧睾丸未能完全降入阴囊。该异常的核心挑战之一在于，滞留的睾丸仍可维持内分泌功能。由此，与去势动物相比，隐睾动物的肉品会受激素污染，且其攻击性可能更强。隐睾症不仅会降低动物福利水平，还会造成经济损失。鉴定隐睾症的遗传标记是缓解此类负面影响的关键步骤，或可助力基因组编辑以降低隐睾症的发生率。目前针对此类标记的鉴定已采用全基因组关联研究（genome-wide association studies, GWAS）。本研究通过全外显子测序（whole-exome sequencing），旨在鉴定隐睾猪编码区的单核苷酸多态性（single nucleotide polymorphisms, SNPs），并解析这些SNPs相关的功能通路。方法：从长白猪品种中选取5只健康个体与5只隐睾个体，提取其DNA并使用Illumina HiSeq 2500平台完成测序。使用SeqyClean工具对测序数据进行预处理，再通过BWA软件将测序读段比对至猪参考基因组（Sus scrofa 11.1）。采用GATK鉴定多态性位点（SNPs与插入缺失突变InDels），并通过VEP工具完成基因注释。通过Cytoscape平台开展网络预测与基因本体富集分析，使用STRING软件完成可视化。结果：在PIGB、CCPG1、COMMD9、LDLRAD3、TRIM44、MYLPF、SEPTIN、ZNF48、TIA1、FAIM2、KRT18、FBP1、FBP2、CTSL、DAPK1、DHX8、GPR179、DEPDC1B、ENSSSCG00000049573、ENSSSCG00000016384、ENSSSCG00000022657、ENSSSCG00000038825及ENSSSCG00000001229等基因中，共鉴定得到63个SNPs。通过通路富集分析与网络预测，我们在校正后P值阈值0.001的条件下，筛选得到与雌激素信号通路、细胞骨架组织及磷酸戊糖通路生物学功能相关的显著通路。结论：本研究数据表明，新的SNPs与基因参与了猪隐睾症的发生进程。但仍需在更大规模的队列群体中开展研究以验证本研究结果。GPR179基因的变异及其对蛋白水平的影响，或与猪隐睾症的出现相关。最后，本研究证实雌激素信号通路可能参与了该先天性异常的病理生理机制，这与此前GWAS的报道一致。