An Unusual Splice Defect in the Mitofusin 2 Gene (<i>MFN2</i>) Is Associated with Degenerative Axonopathy in Tyrolean Grey Cattle

Tyrolean Grey cattle represent a local breed with a population size of ∼5000 registered cows. In 2003, a previously unknown neurological disorder was recognized in Tyrolean Grey cattle. The clinical signs of the disorder are similar to those of bovine progressive degenerative myeloencephalopathy (weaver syndrome) in Brown Swiss cattle but occur much earlier in life. The neuropathological investigation of an affected calf showed axonal degeneration in the central nervous system (CNS) and femoral nerve. The pedigrees of the affected calves suggested a monogenic autosomal recessive inheritance. We localized the responsible mutation to a 1.9 Mb interval on chromosome 16 by genome-wide association and haplotype mapping. The MFN2 gene located in this interval encodes mitofusin 2, a mitochondrial membrane protein. A heritable human axonal neuropathy, Charcot-Marie-Tooth disease-2A2 (CMT2A2), is caused by MFN2 mutations. Therefore, we considered MFN2 a positional and functional candidate gene and performed mutation analysis in affected and control Tyrolean Grey cattle. We did not find any non-synonymous variants. However, we identified a perfectly associated silent SNP in the coding region of exon 20 of the MFN2 gene. This SNP is located within a putative exonic splice enhancer (ESE) and the variant allele leads to partial retention of the entire intron 19 and a premature stop codon in the aberrant MFN2 transcript. Thus we have identified a highly unusual splicing defect, where an exonic single base exchange leads to the retention of the preceding intron. This splicing defect represents a potential explanation for the observed degenerative axonopathy. Marker assisted selection can now be used to eliminate degenerative axonopathy from Tyrolean Grey cattle.蒂罗尔灰牛（Tyrolean Grey cattle）是一个地方品种，注册母牛种群规模约为5000头。2003年，蒂罗尔灰牛中首次发现一种此前未被报道的神经疾病。该病临床症状与瑞士褐牛的牛进行性变性脑脊髓病（韦弗综合征，bovine progressive degenerative myeloencephalopathy (weaver syndrome)）相似，但发病年龄早得多。对患病犊牛的神经病理学检查显示，其中枢神经系统（central nervous system）与股神经存在轴突变性。患病犊牛的谱系分析提示其为单基因常染色体隐性遗传。通过全基因组关联分析与单倍型定位，我们将致病突变定位至16号染色体上1.9 Mb的区间内。该区间内的MFN2基因（MFN2 gene）编码线粒体融合蛋白2（mitofusin 2），一种线粒体膜蛋白。人类的一种遗传性轴索神经病——腓骨肌萎缩症2A型2（Charcot-Marie-Tooth disease-2A2 (CMT2A2)），正是由MFN2基因突变引发。因此，我们将MFN2作为位置功能候选基因，对患病与健康蒂罗尔灰牛开展突变分析。未检测到任何非同义变异，但我们在MFN2基因第20外显子的编码区发现了一个与表型完全连锁的同义单核苷酸多态性（silent SNP）。该SNP位于推定外显子剪接增强子（putative exonic splice enhancer, ESE）区域内，其变异等位基因会导致第19内含子部分滞留，并在异常MFN2转录本中产生提前终止密码子。据此我们发现了一种极不寻常的剪接缺陷：外显子单碱基替换导致其上游内含子滞留。这种剪接缺陷可为本研究观察到的变性轴索病提供合理解释。如今，标记辅助选择（marker assisted selection）可被用于从蒂罗尔灰牛种群中清除变性轴索病。