Ślaska B, Grzybowska-Szatkowska L, Bugno-Poniewierska M, Gurgul A, Śmiech A, Różańska D, Dudka J
Pol J Vet Sci. 2016 Sep 1;19(3):461-469. doi: 10.1515/pjvs-2016-0058.
The aim of the study was to identify polymorphisms and mutations in the mitochondrial ND4 gene and to analyse the associations between the occurrence of molecular changes in mtDNA and phenotypic traits in tumours in German Shepherd dogs. Fifty samples obtained from blood and tumour tissues of German Shepherd dogs with diagnosed tumours were analysed. DNA extraction, amplification, and sequencing of the mtDNA ND4 gene, and bioinformatics, statistical, and in silico protein coding SNP analyses were performed. ND4 mutations and/or polymorphisms were noted in eleven nucleotide positions in nearly half of the examined dogs. All the changes were substitution mutations. A majority of the changes identified were homoplasmic. In one dog with osteosarcoma, blood heteroplasmy was detected. In two positions of the ND4 gene, presence of non-synonymous mutations leading to amino acid changes in the ND4 protein was reported. Analyses carried out to determine the deleterious effect of mutations indicated an almost 97 and 62% probability that a single amino acid substitution (p.G239V and p.I401T, respectively) in the protein has a negative impact on its function. The results of statistical analyses indicate a significant association between the occurrence of mutations in three loci of the ND4 gene and the location of tumours. The mutations identified may be a result of cell adaptation to the changes in the environment occurring during carcinogenesis. The high frequency of mutations in the tumours may indicate genetic instability of mtDNA, which may also play a role in carcinogenesis.
本研究的目的是鉴定线粒体ND4基因中的多态性和突变，并分析德国牧羊犬肿瘤中线粒体DNA分子变化的发生与表型特征之间的关联。对从患有确诊肿瘤的德国牧羊犬的血液和肿瘤组织中获取的50个样本进行了分析。进行了线粒体DNA ND4基因的DNA提取、扩增和测序，以及生物信息学、统计学和电子蛋白质编码单核苷酸多态性分析。在近一半的受试犬中，在11个核苷酸位置发现了ND4突变和/或多态性。所有变化均为替换突变。所鉴定的大多数变化是同质性的。在一只患有骨肉瘤的犬中，检测到血液中的异质性。据报道，在ND4基因的两个位置存在导致ND4蛋白氨基酸变化的非同义突变。为确定突变的有害作用而进行的分析表明，蛋白中的单个氨基酸替换（分别为p.G239V和p.I401T）对其功能产生负面影响的概率几乎分别为97%和62%。统计分析结果表明，ND4基因三个位点的突变发生与肿瘤位置之间存在显著关联。所鉴定的突变可能是细胞适应致癌过程中发生的环境变化的结果。肿瘤中突变的高频率可能表明线粒体DNA的遗传不稳定性，这也可能在致癌过程中起作用。
