以SYBR Green I染劑進行即時定量聚合酶連鎖反應作為恰克-馬利-杜斯氏症以及遺傳性壓力易感性神經病變的分子診斷

Molecular Diagnosis of Charcot-Marie-Tooth Disease Type 1A and Hereditary Neuropathy with Liability to Pressure Palsies by Real-time Quantitative PCR using SYBR Green I dye

郭弘昌(Hung-Chang Kuo)

指導教授 ： 鐘育志

高雄醫學大學/醫學院/醫學研究所/碩士(2009年)

摘要

恰克-馬利-杜斯氏症 (Charcot-Marie-Tooth disease，CMT) 是最常見的遺傳性神經病變，依電生理的特質、遺傳方式、與不同的基因變異可分為許多亞型，其中以CMT1A最常見。CMT1A是自體顯性遺傳的脫髓鞘型神經病變，基因上的變異主要是發生在chromosome 17p11.2-12，內含peripheral myelin protein 22 (PMP22) gene，因為這一段基因發生基因複製 (duplication)，造成患者的DNA之中含有三套的PMP22 gene。同時，若是在同一段基因產生基因缺失 (deletion)，則會造成另一種遺傳性的神經病變，稱之為遺傳性壓力易感性神經病變 (Hereditary neuropathy with liability to pressure palsies，HNPP)。
在診斷方法上，過去主要是依靠臨床的表現，加上家族遺傳史，神經電生理檢查，以及神經切片來做大致上的診斷與分類，然而，上述方法無法提供疾病的基因變異，因此各種分子生物學的方法便孕育而生。
早期所使用的分子生物學的方法各有其優缺點，有的很準確，但是耗時或者比較昂貴，有些方法則敏感性比較不夠。即時定量聚合酶連鎖反應 (real-time quantitative PCR) 具有快速、準確等優點，有相當好的敏感度與特異性，相對節省成本，而且具有很好的再現性，是很好的實驗方法。本實驗即是利用SYBR Green I dye進行即時定量聚合酶連鎖反應的方法來做分子診斷。這個方法的操作簡易、快速，無需設計專用的probes，相對費用較低，很適合拿來作為篩檢之用。利用comparative threshold cycle method來計算PMP22 的基因套數比例 (gene copy number ratio)，我們的方法可以正確的分辨出CMT1A (平均基因套數比例為1.47)、HNPP (平均基因套數比例為0.52)、與control (平均基因套數比例為0.99) 三組檢體，作出正確的分子診斷。
藉由分子診斷，我們可以對患有遺傳性神經病變的患者，或者雖然沒有明確家族史，但是臨床上為脫髓鞘型神經病變的患者作出正確的診斷，讓患者以及家屬可以對疾病有正確的認識以及得到醫療上的協助，對於沒有症狀的家族成員，也可以提供疾病的篩檢與遺傳上的諮詢。

關鍵字

恰克-馬利-杜斯氏症 ； 遺傳性壓力易感性神經病變 ； 即時定量聚合酶連鎖反應

並列摘要

Charcot-Marie-Tooth disease (CMT) is one of the most common inherited peripheral neuropathies. According to the hereditary pattern, clinical manifestations and electrophysiologic findings, CMT could be divided into many subgroups. CMT type 1 is an autosomal dominant demyelinating motor and sensory neuropathy. The most common subtype of CMT type 1 is CMT1A, which is caused by the duplication of a 1.5 Mb region containing the peripheral myelin protein 22 (PMP22) gene on chromosome 17p11.2-12. Hereditary neuropathy with liability to pressure palsies (HNPP), another autosomal dominant peripheral neuropathy, is caused by deletion of the PMP22 gene.
The diagnosis of CMT in the past was mainly dependent on clinical manifestations, family history, electrophysiologic findings and/or sural nerve biopsies. However, these methods could not give information about the underlying genetic defects, so molecular methods were developed for genetic diagnosis. Some molecular diagnostic methods have been used for the diagnosis of CMT. Some methods are accurate, but have the disadvantage of labor-intensive and time-consuming. Some methods are relatively expensive and require specific equipments. We performed real-time quantitative PCR using SYBR Green I dye with our own designed primers for the diagnosis of CMT1A and HNPP. We use comparative threshold cycle method to calculate the estimated PMP22 gene copy number ratio. The estimated PMP22 gene copy number ratio is 1.47 for CMT1A, 0.52 for HNPP and 0.99 for control. Our method could separate the three groups of samples accurately without overlapping.
Real-time quantitative PCR using SYBR Green I dye is a sensitive, specific, accurate and reproducible method for the diagnosis of PMP22 gene duplication or deletion. By this method, we could make diagnosis for patients with CMT1A, HNPP, and other types of hereditary neuropathy and could provide them useful information about medical resources and therapeutic developments of the disease. For asymptomatic and high risk families, this method could also provide genetic screening and counseling.

並列關鍵字

CMT ； HNPP ； PMP22 ； real-time quantitative PCR

參考文獻

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