以改良PCR方法偵測CMT1A基因重組熱點之家族研究

Detection of A Recombinant Hotspot Associated with Charcot-Marie-Tooth Disease Type 1A Duplication in A Large Family by A Improved PCR Method

曾麗華(Zi-Hwa Zeng)

指導教授 ： 游仲逸 ； 鐘育志

高雄醫學大學/醫學院/醫學研究所/碩士(2004年)

摘要

Charcot-Marie-Tooth 1A型 (CMT1A) 是周邊神經系統中最常見的遺傳疾病，約佔CMT1的70 ~ 90%。CMT1A屬於體顯性遺傳，主要是減數分裂重組時，CMT1A repeat sequences (CMT1A-REPs) 在第17對染色體短臂的11.2-12位置發生不相等的互換 (unequal crossing-over) 所導致的1.5-Mb基因重複。這區域包含peripheral myelin protein 22基因 (PMP22)，其兩翼有distal CMT1A-REP 與proximal CMT1A-REP的相似序列。互換中斷點的hotspot就位於CMT1A-REPs中的3.2-kb區域內，此區域界於限制酶EcoRI與SacI之間。目前有許多分子診斷的方法用來偵測CMT1A基因重組，例如：南方墨點法 (southern blot)、脈衝場膠體電泳法 (pulsed-field electrophoresis, PFGE)、螢光原位雜交 (fluorescence in situ hybridization, FISH) 和real-time定量PCR (real-time quantitative polymerase chain reaction)等方法。由於上述方法推廣到臨床上仍然有一些因素的限制，所以本研究目的就是建立一套快速、簡便及準確率高的分子方法來診斷CMT1A基因重組。我們將原本以PCR為基礎的方法（Chang et al., Clin Chem 44: 270, 1998）經過改良，換成只用一組primer及EcoRI就能偵測整個3.2-kb的hotspot區域，然後再將此改良方法應用到一個華人的大家族。結果顯示：43名受檢者血液檢體中，檢測出22名有CMT1A基因重複，其中13名為不自覺有症狀的CMT患者。另外，同樣都有CMT1A基因重複，可是在不同世代，甚至同一個兄弟姊妹間，疾病的表現型，例如 : 疾病的嚴重程度和周邊神經傳導之數值是有所變異的。綜合以上結論，改良式PCR方法比臨床觀察更來得準確，是一個經濟、快速、簡便、且準確性高的方法，能廣泛運用到臨床上懷疑CMT1A基因重複患者之診斷，尤其是有助於早期篩檢出不自覺有症狀的CMT1A患者，來減緩疾病進行。

關鍵字

基因重複 ； 分子診斷 ； 基因重組熱點 ； 不相等互換 ； Charcot-Marie -Tooth 1A疾病

並列摘要

Charcot-Marie-Tooth disease type 1A (CMT1A) also known as hereditary motor and sensory neuropathy (HMSN1A), is common hereditary peripheral neuropathy, almost represented 70 to 90 percent of CMT1 disease. The inheritance of CMT1A is autosomal dominant. It is mainly leading to a tandem DNA duplication of a 1.5-Mb region on chromosome 17p11.2 -12. This region contains the peripheral myelin protein 22 gene (PMP22) which is flanked by homologous proximal and distal CMT1A repeat sequences (CMT1A-REPs). The majority of duplications arise during meiotic recombination following unequal crossing-over between the proximal and distal CMT1A-REPs. The hotspot of crossover breakpoints is most frequently located within a 3.2-kb region between the restriction enzymes EcoRI and SacI. Up to now, several molecular diagnostic methods are currently used for detection of CMT1A duplication, including southern blot, pulsed-field gel electrophoresis (PFGE), fluorescence in situ hybridization (FISH), and real-time quantitative polymerase chain reaction. Above methods still have some limitations for clinical diagnosis. These methods require radioisotope or complicated procedures, and they are time consuming and labor intensive. The aim of this study was to develop a set of quicker, simpler, and accurate molecular method for diagnosis of CMT1A duplication in the recombinant hotspot area. We modified the PCR assay（Chang et al., Clin Chem 44: 270, 1998） by using only a pair of primers and EcoRI to detect the recombination in the whole 3.2-kb hot spot region. Then we applied this assay in a large Chinese family. The results showed that 22 from 43 family members had CMT1A duplication. Duplication was detected in 13 family members who never realize they had the symptom. Besides, the phenotype, such as severity and NCV values, is variable between different generations, even within siblings who had the CMT1A duplication. In summary, the modified PCR method is relatively simple, economic, quick, and highly specific. It can be wildly used for diagnosis of the CMT1A duplication in the recombinant hotspot area, especially for screening whole family members to find the patients who never realize the symptom in order to reduce the disease progression.

並列關鍵字

recombinant hotspot ； unequal crossover ； molecular diagnosis ； gene duplication ； Charcot-Marie-Tooth disease type 1A

參考文獻

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