Novel rapid genotyping assays for neuronal ceroid lipofuscinosis in Border Collie dogs and high frequency of the mutant allele in Japan
Novel rapid genotyping assays for neuronal ceroid lipofuscinosis in Border Collie dogs and high frequency of the mutant allele in Japan
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DOI：
10.1177/1040638711425590
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发表时间：
2011-11-01
影响因子：
1.5
通讯作者：
Yamato, Osamu
中科院分区：
文献类型：
--
作者：
Mizukami, Keijiro;Chang, Hye-Sook;Yamato, Osamu
神经性蜡样脂褐素沉积症(NCL)是一组主要影响神经细胞的隐性遗传性溶酶体贮存性疾病。这些疾病在人类和动物身上都有一定的临床和病理特征。80年代在澳大利亚首次发现边界牧羊犬的神经元性蜡样脂褐素沉着症，其致病突变为犬CLN5基因外显子4的无义突变(C.619C>；T)。本研究建立了聚合酶链式反应-限制性片段长度多态性、聚合酶链式反应诱导限制性内切酶分析、突变分离聚合酶链式反应和TaqMan小沟槽结合探针实时定量聚合酶链式反应等新的快速基因分型方法。并对芯片电泳法的实用性进行了评价。此外，利用这些方法对日本边境牧羊犬群体进行了基因分型调查，以确定日本目前的等位基因频率，为下一阶段控制和预防这种疾病提供信息。本研究中开发的所有检测方法都可用于区分这些基因型别，微芯片电泳法显示出比琼脂糖凝胶电泳法更省时的优势。在所有检测方法中，实时荧光定量聚合酶链式反应因其高通量而最适合大规模检测。基因分型调查表明，携带者频率为8.1%。这一发现表明，在日本，边境牧羊犬NCL的突变等位基因频率足够高，因此有必要采取措施控制和预防这种疾病。本研究建立的基因分型方法可用于边境牧羊犬NCL的预防。
Neuronal ceroid lipofuscinosis (NCL) constitutes a group of recessively inherited lysosomal storage diseases that primarily affect neuronal cells. Such diseases share certain clinical and pathologic features in human beings and animals. Neuronal ceroid lipofuscinosis in Border Collie dogs was first detected in Australia in the 1980s, and the pathogenic mutation was shown to be a nonsense mutation (c.619C>T) in exon 4 in canine CLN5 gene. In the present study, novel rapid genotyping assays including polymerase chain reaction (PCR)-restriction fragment length polymorphism, PCR primer-induced restriction analysis, mutagenically separated PCR, and real-time PCR with TaqMan minor groove binder probes, were developed. The utility of microchip electrophoresis was also evaluated. Furthermore, a genotyping survey was carried out in a population of Border Collies in Japan using these assays to determine the current allele frequency in Japan, providing information to control and prevent this disease in the next stage. All assays developed in the current study are available to discriminate these genotypes, and microchip electrophoresis showed a timesaving advantage over agarose gel electrophoresis. Of all assays, real-time PCR was the most suitable for large-scale examination because of its high throughput. The genotyping survey demonstrated that the carrier frequency was 8.1%. This finding suggested that the mutant allele frequency of NCL in Border Collies is high enough in Japan that measures to control and prevent the disease would be warranted. The genotyping assays developed in the present study could contribute to the prevention of NCL in Border Collies.