Tetradysmelia is a rare genetic disorder that is characterized by an extremely severe reduction of all limb parts distal of the scapula and pelvic girdle. We studied a Holstein Friesian backcross family with 24 offspring, among which six calves displayed autosomal recessive tetradysmelia. In order to identify the genetic basis of the disorder, we genotyped three affected calves, five dams and nine unaffected siblings using a Bovine Illumina 50 k BeadChip and sequenced the whole genome of the sire. Pathological examination of four tetradysmelia cases revealed a uniform and severe dysmelia of all limbs. Applying a homozygosity mapping approach, we identified a homozygous region of 10.54 Mb on chromosome 14 (Bos taurus BTA14). Only calves that were diagnosed with tetradysmelia shared a distinct homozygous haplotype for this region. We sequenced the whole genome of the cases’ sire and searched for heterozygous single nucleotide polymorphisms (SNPs) and small variants on BTA14 that were uniquely present in the sire and absent from 3102 control whole-genome sequences of the 1000 Bull Genomes Project, but none were identified in the 10.54-Mb candidate region on BTA14. Therefore, we subsequently performed a more comprehensive analysis by also considering structural variants and detected a 50-kb deletion in the targeted chromosomal region that was in the heterozygous state in the cases’ sire. Using PCR, we confirmed that this detected deletion segregated perfectly within the family with tetradysmelia. The deletion spanned three exons of the bovine R-spondin 2 (RSPO2) gene, which encode three domains of the respective protein. R-spondin 2 is a secreted ligand of leucine-rich repeats containing G protein-coupled receptors that enhance Wnt signalling and is involved in a broad range of developmental processes during embryogenesis. We identified a 50-kb deletion on BTA14 that disrupts the coding sequence of the RSPO2 gene and is associated with bovine tetradysmelia. To our knowledge, this is the first reported candidate causal mutation for tetradysmelia in a large animal model. Since signalling pathways involved in limb development are conserved across species, the observed inherited defect may serve as a model to further elucidate fundamental pathways of limb development.

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破坏RSPO2基因的50 kb删除与荷斯坦黑白花牛的四肢抽搐相关

四肢抽动症是一种罕见的遗传性疾病，其特征是肩severe骨和骨盆带远端的所有肢体部位严重减少。我们研究了具有24个后代的Holstein Friesian回交家族，其中有6个犊牛表现出常染色体隐性四体发育不良。为了鉴定该疾病的遗传基础，我们使用牛Illumina 50 k BeadChip对三只受影响的小牛，五个水坝和九个未受影响的兄弟姐妹进行了基因分型，并对整个父亲的基因组进行了测序。对四例四肢痛症患者进行病理检查，发现四肢均匀且严重的肢体痛觉异常。应用纯合性作图方法，我们在14号染色体（Bos taurus BTA14）上鉴定了10.54 Mb的纯合区域。只有被诊断患有四鞭毛病的小牛对该区域具有独特的纯合单倍型。我们对病例父亲的整个基因组进行了测序，并在其父亲中独特存在的BTA14上搜索了杂合的单核苷酸多态性（SNP）和小变异，但在1000 Bull基因组计划的3102个对照全基因组序列中却没有在BTA14的10.54-Mb候选区域中被鉴定出来。因此，我们随后还通过考虑结构变异来进行了更全面的分析，并在病例父亲的杂合状态的目标染色体区域中检测到50 kb的缺失。使用PCR，我们证实了这种检测到的缺失在四鞭毛家族中完全隔离。该缺失跨越了牛R-spondin 2（RSPO2）基因的三个外显子，它们编码各自蛋白质的三个结构域。R-spondin 2是富含亮氨酸的重复序列的分泌配体，其中含有增强Wnt信号传导的G蛋白偶联受体，并参与胚胎发生过程中的广泛发育过程。我们在BTA14上发现了一个50 kb的缺失，该缺失破坏了RSPO2基因的编码序列，并与牛四肢抽搐有关。据我们所知，这是在大型动物模型中首次报道的四鞭毛病候选因果突变。由于跨物种保守涉及肢体发育的信号传导途径，因此观察到的遗传缺陷可作为进一步阐明肢体发育的基本途径的模型。我们在BTA14上发现了一个50 kb的缺失，该缺失破坏了RSPO2基因的编码序列，并与牛四肢抽搐有关。据我们所知，这是在大型动物模型中首次报道的四鞭毛病候选因果突变。由于跨物种保守涉及肢体发育的信号传导途径，因此观察到的遗传缺陷可作为进一步阐明肢体发育的基本途径的模型。我们在BTA14上发现了一个50 kb的缺失，该缺失破坏了RSPO2基因的编码序列，并与牛四肢抽搐有关。据我们所知，这是在大型动物模型中首次报道的四鞭毛病候选因果突变。由于与肢体发育有关的信号通路在整个物种中都是保守的，因此观察到的遗传缺陷可作为进一步阐明肢体发育基本通路的模型。