建立應用於犬遺傳性疾病－迷你雪納瑞犬慕勒氏管永存症及先天性肌強直症之高解析度融點曲線分析法

Abstract

動物體中,突變的基因可以顯性或隱性的形式遺傳至後代,導致遺傳性疾病的產生。分 子檢測是目前唯一可於臨床症狀發生前篩檢出遺傳性疾病的檢測方法,且已應用於人類。由 於物種的差異性,在犬要執行分子檢測則較為複雜,主因是不同突變可能於不同犬種中造成 相同遺傳疾病,因此目前一種分子檢測方式,只適用於單一或少數犬種的單項遺傳疾病。迷 你雪納瑞犬在美國及臺灣都是常見的犬種,在至今已被證實發生於該犬種的廿五種遺傳性疾 病中,只有少數幾項的致病基因突變已被確認,其中包含慕勒氏管永存症及先天性肌強直 症。目前此兩項疾病的分子檢測皆以限制性片段長度多態性 (restriction fragment length polymorphism, RFLP) 的方式進行,即以聚合酶連鎖反應 (polymerase chain reaction, PCR) 合 併限制酶切割進行檢測;為縮短篩檢時程,本研究除了以RFLP進行臺灣地區迷你雪納瑞慕 勒氏管永存症及先天性肌強直症盛行率之調查外,更進一步發展以高解析度融點曲線分析法 (high resolution melting analysis, HRM analysis) 篩檢該兩項疾病的分子檢測方式。首先,由 各動物醫院收集來自 迷你雪納瑞的血液或口腔拭子樣本中萃取其基因體DNA,透過特定引 子進行PCR後,將其產物以特定限制酶進行消化,再以膠體電泳分析個體的基因型,最後經 由定序確認分析結果。另一方面,本研究架構帶有野生型 (homozygous wild type, WT) 及突 變型 (homozygous mutant type, MT) 的質體,並混合兩種質體作為異形合子突變型 (heterozygous mutant type, HT),以進行HRM analysis條件優化,再透過與RFLP實驗及定序的 結果之比對,以評估HRM analysis篩檢之可行性。以RFLP檢測本研究所收集之106個迷你雪 納瑞樣本顯示,所有樣本均為先天性肌強直症WT,而其中有87個樣本為慕勒氏管永存症 WT,19個為慕勒氏管永存症HT;由所有樣本中隨機挑選34個進行HRM analysis檢測,並將 以HRM analysis與RFLP篩檢結果相異之樣本,以定序確認作為最終判斷依據。根據以上分 子檢測結果判定,106個迷你雪納瑞犬樣本中,85個 (80.2%) 樣本為慕勒氏管永存症WT, 20個 (18.9 %) 為慕勒氏管永存症HT;1個 (0.9%) 為慕勒氏管永存症MT,但並未發現先天 性肌強直症之突變基因,而我們研發的高解析度融點曲線分析法可用於篩檢這兩項犬遺傳疾病。

Animals inherited a mutant gene from their parents (father and/or mother) may cause hereditary diseases. The mutation can be either autosomal recessive or autosomal dominant. Usually, hereditary diseases cannot be noticed until that symptom shows up. Molecular test can identify the mutant gene carriers or hereditary diseases in advance. At present, there are a lot of methods of molecular testing available for human hereditary diseases detection. However, due to the differences of DNA sequence between canine and human, molecular testing of screening hereditary diseases in canine is more complicated than in human because a molecular test can only be used to detect one hereditary disease in one dog breed or in few breeds. Miniature Schnauzer is both popular in America and Taiwan. Among the 25 hereditary diseases discovered in Miniature Schnauzer, only a few mutant genes which are responsible for hereditary diseases of Miniature Schnauzer were identified. Those diseases include PMDS and myotonic congenita. The molecular testing of these diseases is based on RFLP (restriction fragment length polymorphism) which is genomic PCR (polymerase chain reaction) plus specific restriction enzyme digestion. For the purpose of developing a faster method of screening these diseases, the objective of this study is to investigate the prevalence of PMDS and myotonia in Miniature Schnauzer in Taiwan by RFLP and to develop a high resolution melting (HRM) analysis for screening PMDS and myotonia in Miniature Schnauzer. First, we collected whole blood or oral swab from Miniature Schnauzer from animal hospitals. The genomic DNA was extracted from the blood or oral swab. Then polymerase chain reaction (PCR) was performed, and the PCR products were subjected to digestion with the specific restriction enzymes. The digested PCR products were separated on agarose gel stained with commercial safety dye by use of electrophoresis and photographed. The gene mutation was confirmed by Sanger sequencing of the PCR products. On the other hand, plasmid of “WT”, “HT” and “MT” which carried wild type, heterozygous type and mutant type gene was constructed. Then HRM analysis was developed by the use of WT, HT and MT plasmids. To evaluate the HRM analysis by re-confirming the genotypes of the samples screened by RFLP and Sanger sequencing. By the use of RFLP, as a result, in total of 106 samples collected from Miniature Schnauzer, all of them are myotonia WT, 87 of them are PMDS WT, and 19 of them are PMDS HT. We then selected 34 samples randomly from all the samples for HRM analysis. Four samples were Sanger sequenced to confirm the sequences due to the different results obtained by RFLP and HRM analysis. In 106 samples collected from Miniature Schnauzer, genotypes of the samples were finally identified as 85 (80.2%) samples with PMDS WT, 20 (18.9%) samples with PMDS HT,1 (0.9%) sample with PMDS MT, and none of them was myotonia mutant type. In addition, HRM analysis is available for screening PMDS and myotonia in Miniature Schnauzer.