The Holstein Friesian Lethal Haplotype 5 (HH5) Results from a Complete Deletion of TBF1M and Cholesterol Deficiency (CDH) from an ERV-(LTR) Insertion into the Coding Region of APOB

BackgroundWith the availability of massive SNP data for several economically important cattle breeds, haplotype tests have been performed to identify unknown recessive disorders. A number of so-called lethal haplotypes, have been uncovered in Holstein Friesian cattle and, for at least seven of these, the causative mutations have been identified in candidate genes. However, several lethal haplotypes still remain elusive. Here we report the molecular genetic causes of lethal haplotype 5 (HH5) and cholesterol deficiency (CDH). A targeted enrichment for the known genomic regions, followed by massive parallel sequencing was used to interrogate for causative mutations in a case/control approach.MethodsTargeted enrichment for the known genomic regions, followed by massive parallel sequencing was used in a case/control approach. PCRs for the causing mutations were developed and compared to routine imputing in 2,100 (HH5) and 3,100 (CDH) cattle.ResultsHH5 is caused by a deletion of 138kbp, spanning position 93,233kb to 93,371kb on chromosome 9 (BTA9), harboring only dimethyl-adenosine transferase 1 (TFB1M). The deletion breakpoints are flanked by bovine long interspersed nuclear elements Bov-B (upstream) and L1ME3 (downstream), suggesting a homologous recombination/deletion event. TFB1M di-methylates adenine residues in the hairpin loop at the 3’-end of mitochondrial 12S rRNA, being essential for synthesis and function of the small ribosomal subunit of mitochondria. Homozygous TFB1M-/- mice reportedly exhibit embryonal lethality with developmental defects. A 2.8% allelic frequency was determined for the German HF population. CDH results from a 1.3kbp insertion of an endogenous retrovirus (ERV2-1-LTR_BT) into exon 5 of the APOB gene at BTA11:77,959kb. The insertion is flanked by 6bp target site duplications as described for insertions mediated by retroviral integrases. A premature stop codon in the open reading frame of APOB is generated, resulting in a truncation of the protein to a length of only ConclusionWith the availability of direct genetic tests it will now be possible to more effectively reduce the carrier frequency and ultimately eliminate the disorders from the HF populations. Beside this, the fact that repetitive genomic elements (RE) are involved in both diseases, underline the evolutionary importance of RE, which can be detrimental as here, but also advantageous over generations.

背景 随着多种具有经济重要性的牛品种的大规模单核苷酸多态性（SNP，single nucleotide polymorphism）数据可及，单倍型检测已被用于鉴定未知的隐性遗传病。目前已在荷斯坦弗里西牛（Holstein Friesian cattle）中发现多种所谓的致死单倍型，其中至少7种的致病突变已在候选基因中得到鉴定。然而仍有部分致死单倍型的致病机制尚未明确。本研究阐明了致死单倍型5（HH5）与胆固醇缺乏症（CDH）的分子遗传致病机制。研究采用针对已知基因组区域的靶向富集技术结合大规模平行测序，通过病例-对照研究策略探究致病突变。

方法 针对已知基因组区域的靶向富集结合大规模平行测序，被应用于病例-对照研究策略中。本研究开发了针对致病突变的聚合酶链式反应（PCR，Polymerase Chain Reaction）检测方法，并分别在2100头荷斯坦弗里西牛（HH5群体）与3100头荷斯坦弗里西牛（CDH群体）中，将该检测方法与常规基因型填充进行了对比。

结果 HH5由一段138千碱基对（kbp）的缺失突变所致，该缺失位于牛9号染色体（BTA9）上93233kb至93371kb区域，仅包含二甲基腺苷转移酶1（TFB1M，dimethyl-adenosine transferase 1）基因。该缺失的断裂点两侧分别为牛长散在核元件Bov-B（上游）与L1ME3（下游），提示该突变由同源重组介导的缺失事件产生。TFB1M可对线粒体12S核糖体RNA（rRNA，ribosomal RNA）3'端发夹环中的腺嘌呤残基进行二甲基化修饰，该修饰对于线粒体小核糖体亚基的合成与功能至关重要。已有研究表明，TFB1M基因纯合敲除（TFB1M-/-）的小鼠会出现胚胎致死并伴随发育缺陷。在德国荷斯坦弗里西牛群体中，该缺失突变的等位基因频率为2.8%。CDH由一段1.3kbp的内源性逆转录病毒（ERV2-1-LTR_BT）插入APOB基因的第5外显子所致，插入位点位于BTA11染色体的77959kb位置。该插入序列两侧存在6bp的靶位点重复，这与逆转录病毒整合酶介导的插入事件特征一致。该插入导致APOB基因开放阅读框中出现提前终止密码子，使得编码的蛋白质发生截短，仅剩余极短的片段。

结论 随着直接遗传检测技术的普及，如今可更有效地降低致病基因携带者频率，并最终从荷斯坦弗里西牛群体中根除这两种遗传病。此外，两种疾病均与基因组重复元件（RE，repetitive genomic elements）相关，这一现象凸显了基因组重复元件在进化中的重要性：它们既可能如本研究中那样产生有害影响，也可在世代传承中发挥有益作用。