背景:性发育差异(DSD)是先天条件,性腺,或表型性别不典型。在超过50%的人类DSD病例中,分子诊断是不可用的。在密集养殖的猪种群中,XXDSD猪的发病率相对较高,给养猪人带来经济损失。有趣的是,在大多数38只XXDSD猪中,尽管没有睾丸决定基因(SRY),性腺仍发育成睾丸样结构或卵细胞。然而,目前对XXDSD猪的分子背景的了解仍然有限.
方法:采用尸检和HE染色对XXDSD猪的解剖和组织学特征进行分析。我们采用全基因组测序(WGS)和10×基因组学技术,并使用从头组装方法研究正常雌性和XXDSD猪。最后,在32只XXDSD猪中验证了鉴定的变体,并进一步检查了XXDSD猪性腺中候选变体的表达水平。
结果:XXDSD猪的特征是性交生殖器官和性腺的精细管中没有生殖细胞。我们从XXDSD猪的非同义突变中鉴定了4,950个单核苷酸多态性(SNP)。队列验证结果突出了两个特定的SNP,“干扰素诱导的跨膜蛋白1基因(IFITM1)”中的“c.218T>C”和“新生儿卵巢同源盒基因(NOBOX)”中的“c.1043C>G”,仅在XXDSD猪中发现。此外,我们从1,474SV中验证了14个候选结构变体(SV),在62.5%的XXDSD猪中鉴定了含WW结构域的氧化还原酶基因(WWOX)内含子5中的70bp缺失片段。这三个候选基因在XXDSD猪性腺中的表达水平与正常母猪有显著差异。
结论:IFITM1的核苷酸变化(c.218T>C),NOBOX(c.1043C>G),WWOX的70bp缺失片段是XXDSD猪中最主要的变异。本研究为更好地了解XXDSD猪的分子背景提供了理论依据。DSD是影响性腺或生殖器发育的条件。这些疾病可以发生在许多不同类型的动物中,包括猪,山羊,狗,和人。在人们中,DSD发生在大约0.02-0.13%的新生儿中,在猪身上,率在0.08%至0.75%之间。猪有一种常见的DSD类型,动物有雌性染色体(38,XX),但没有SRY基因,通常在男性的Y染色体上发现。XXDSD猪的外表看起来既像雄性,也像雌性,里面有睾丸样或卵睾丸(卵巢和睾丸的混合物)性腺。XXDSD猪经常导致不能有仔猪,增长较慢,较低的生存机会,和较差的肉质。这里,我们使用全基因组从头测序的方法来寻找XXDSD猪DNA中的变异体.然后我们在更大的一组猪中检查了这些差异。我们的结果揭示了IFITM1的核苷酸变化(c.218T>C),NOBOX(c.1043C>G),和WWOX内含子5中的70bp缺失片段,都与XXDSD猪有关。与正常母猪相比,这三个基因在XXDSD猪的性腺中的表达水平也不同。这些变体有望作为XXDSD猪的有价值的分子标记。因为猪的基因很像人类,生理学,和身体结构,这项研究可以帮助我们更多地了解人类DSD的原因。
BACKGROUND: Differences of sex development (DSD) are congenital conditions in which chromosomal, gonadal, or phenotypic sex is atypical. In more than 50% of human DSD cases, a molecular diagnosis is not available. In intensively farmed pig populations, the incidence of XX DSD pigs is relatively high, leading to economic losses for pig breeders. Interestingly, in the majority of 38, XX DSD pigs, gonads still develop into testis-like structures or ovotestes despite the absence of the testis-determining gene (SRY). However, the current understanding of the molecular background of XX DSD pigs remains limited.
METHODS: Anatomical and histological characteristics of XX DSD pigs were analysed using necropsy and HE staining. We employed whole-genome sequencing (WGS) with 10× Genomics technology and used de novo assembly methodology to study normal female and XX DSD pigs. Finally, the identified variants were validated in 32 XX DSD pigs, and the expression levels of the candidate variants in the gonads of XX DSD pigs were further examined.
RESULTS: XX DSD pigs are characterised by the intersex reproductive organs and the absence of germ cells in the seminiferous tubules of the gonads. We identified 4,950 single-nucleotide polymorphisms (SNPs) from non-synonymous mutations in XX DSD pigs. Cohort validation results highlighted two specific SNPs, \"c.218T > C\" in the \"Interferon-induced transmembrane protein 1 gene (IFITM1)\" and \"c.1043C > G\" in the \"Newborn ovary homeobox gene (NOBOX)\", which were found exclusively in XX DSD pigs. Moreover, we verified 14 candidate structural variants (SVs) from 1,474 SVs, identifying a 70 bp deletion fragment in intron 5 of the WW domain-containing oxidoreductase gene (WWOX) in 62.5% of XX DSD pigs. The expression levels of these three candidate genes in the gonads of XX DSD pigs were significantly different from those of normal female pigs.
CONCLUSIONS: The nucleotide changes of IFITM1 (c.218T > C), NOBOX (c.1043 C > G), and a 70 bp deletion fragment of the WWOX were the most dominant variants among XX DSD pigs. This study provides a theoretical basis for better understanding the molecular background of XX DSD pigs. DSD are conditions affecting development of the gonads or genitalia. These disorders can happen in many different types of animals, including pigs, goats, dogs, and people. In people, DSD happens in about 0.02-0.13% of births, and in pigs, the rate is between 0.08% and 0.75%. Pigs have a common type of DSD where the animal has female chromosomes (38, XX) but no SRY gene, which is usually found on the Y chromosome in males. XX DSD pigs may look like both males and females on the outside and have testis-like or ovotestis (a mix of ovary and testis) gonads inside. XX DSD pigs often lead to not being able to have piglets, slower growth, lower chance of survival, and poorer meat quality. Here, we used a method called whole-genome de novo sequencing to look for variants in the DNA of XX DSD pigs. We then checked these differences in a larger group of pigs. Our results reveal the nucleotide changes in IFITM1 (c.218T > C), NOBOX (c.1043 C > G), and a 70 bp deletion fragment in intron 5 of the WWOX, all linked to XX DSD pigs. The expression levels of these three genes were also different in the gonads of XX DSD pigs compared to normal female pigs. These variants are expected to serve as valuable molecular markers for XX DSD pigs. Because pigs are a lot like humans in their genes, physiology, and body structure, this research could help us learn more about what causes DSD in people.