肢帶型肌肉失養症的基因研究

肢帶型肌肉失養症是排除目前已知、特異性的退化性肌肉疾病後，一群由表現特徵為漸進式肩帶和腰帶等近端肌肉無力，以及肌肉萎縮為臨床表現的疾病總稱。它的臨床及基因遺傳表現具有多樣性，顯示是由不同的遺傳方式和病因所造成的疾病。我們研究一個四代、以體染色體顯性形式遺傳的漸進性肌肉無力家庭。首先，排除已知會造成體染色體顯性遺傳的肢帶型肌肉失養症基因的五個染色體位置所在，其中包含5q31 (1A)，1q11-21 (1B)，3p25 (1C)，6q23 (1D)和7q (1E)。在論文裡，我進一步澄清這個家族與其他體顯肢帶型肌肉失養症家族臨床表現不同的，並且試圖找出造成這個四代家族肌肉無力的基因所在位置。用392個短重覆序列(STR)標誌去掃瞄整體染色體的資料做連鎖分析，嘗試獲得造成這種遺傳病的可能區域。然而，這個最早的分析並沒有任何勝算比對數分數(LOD score)大於3，導致任何有意義決定性的結果。最高的勝算比對數分數1.59，是位於染色體8p23的標誌D8S277；我們接著在這個區域附近，作細部基因定位分析，結果並不支持候選基因在這個位置。在疾病基因定位方面，我們應用基因整體染色體掃瞄技術製圖晶片Affymetrix 10K SNP單一核苷酸多型性的資料做連鎖分析，繼續尋找可能的候選基因。同時，再次追蹤整個家庭，並且透過完整的病史問診、理學以及神經學檢查，以了解目前他們發病的狀態；期望在收集這些臨床表現型後，能夠有助於正確完整的判斷疾病發病與否；並且藉由臨床表徵的差異性，試圖找出在遺傳上更加同質的子群，將肢帶型肌肉失養症做精確的子群分類，使得增加找到致病基因的可能性。其中在第三代和第四代的四個家庭成員，早先報告為未知或未受影響者，經過多年後臨床的重新評估，被認為為發病者; 另一名早先認為受影響的成員，現在已被排除是發病者，並且因為年紀輕，而被嚴格歸類為未知的狀態。根據更新後的家譜資料的Affymetrix 10K SNP數據，執行非參數多點連鎖分析(multipoint linkage analysis)，顯示最高的勝算比對數分數4.6出現在染色體14q31和21q22。根據更新後的家譜，早先392個短重覆序列標誌整體染色體掃瞄的數據也重新被分析；雖然所有勝算比對數分數都沒有大於3，最高的勝算比對數分數約在2附近，出現在染色體8，14和21區域；同時比較以Affymetrix 10K SNP數據做的連鎖分析資料的結果，勝算比對數分數在染色體14和21，以兩種不同方法分析同時都升高。我們繼續在這三個可能的區域 (染色體8，14和21)，選擇更多的短重覆序列標誌，作更細部基因定位，結果顯示最高的勝算比對數分數最後落在染色體14q31。經由不同的整體染色體掃瞄方法和一再重複的統計分析策略，其結果都沒有很大的變化，顯示造成這個漸進性肌肉無力家族的疾病致病基因，高度可能是在14q31這個區域。在這個染色體的地區內，我們檢視了目前已知功能的基因，並且由其功能和在骨骼肌組織高度的表現，懷疑SNW1 和NRXN3是致病的候選基因，但是還需進一步做直接DNA序列分析確認。總而言之，這個研究澄清了一個以體顯形式遺傳的肢帶型肌肉失養症家庭的臨床表現型，並且鑒別一個新的基因座所在地。我們期待透過臨床特徵的辨識，和致病基因的了解，使肢帶型肌肉失養症有更好分類，而且進一步對疾病和基本的肌肉生理的發病原理有更深入的了解。

關鍵字

肢帶型肌肉失養症；臨床及基因多樣性；短重覆序列；單一核苷酸多型性； 14q31

並列摘要

Limb girdle muscular dystrophy (LGMD) is comprised of a clinically and genetically heterogeneous group of muscle disorders characterized by progressive destruction of the shoulder and hip girdle muscles. We identified four generations of a family group apparently with the autosomal dominant form of LGMD. Preliminary gene mapping studies excluded five chromosomal loci 5q31 (1A), 1q11-21 (1B), 3p25 (1C), 6q23 (1D), and 7q (1E) which had been previously implicated for the autosomal dominant LGMD (AD-LGMD). The aim of my thesis was to further clarify the clinical presentations and attempt to localize the genetic loci for this four-generation family with AD-LGMD. A whole genome scan with 392 STR markers followed by linkage analysis was conducted as the initial attempt to obtain candidate regions for this genetic disease. However, the initial analysis did not produce any conclusive results with LOD score greater than 3. The greatest LOD score was 1.59 which was associated with marker D8S277 located at chromosome 8p23. Further fine mapping analysis did not support the linkage to this locus. The advent of whole genome scan technology such as Affymetrix 10K SNP mapping chip provided another platform where linkage analysis could be carried out. At the same time, I reexamined the entire family thoroughly for their affected status to update and confirm the pedigree information. Four family members, previously reported as unknown or unaffected, are now considered affected; while one previously affected member is now reassigned as unknown status with respect to this AD-LGMD. Non parametric multipoint linkage analysis with Affymetrix 10K SNP mapping data based on the new pedigree information indicated the highest peaks rendering NPL score of 4.6 at chromosomes14q31 and 21q22. Previous whole genome scan data with 392 STR markers were re-analyzed with the updated pedigree information. Although no LOD score greater than 3 was obtained, the highest LOD scores around 2 were found on chromosomes 8, 14 and 21, where the regions on chromosomes 14 and 21 echoed that obtained from linkage analysis with Affymetrix 10K SNP mapping data. Further fine-mapping of these candidate regions with more STR markers generated a LOD score near 3 on chromosome 14q31. It is highly probable that the disease gene lies in this region at 14q31, and is now subjected to candidate gene analysis. Within this chromosomal region, SNW domain containing 1, and neurexin 3 genes are potential candidate genes. In conclusion, I further clarified the clinical phenotypes of an autosomal dominant form of the LGMD family and identified a novel locus for this AD-LGMD. I hope by identifying the gene responsible for this novel form of AD-LGMD that we are not only able to categorize LGMD better, but also to enhance our understanding of the pathogenesis of the disease and the basic muscle physiology.

並列關鍵字

Limb girdle muscular dystrophy ； gene mapping ； STR markers ； Affymetrix 10K SNP mapping ； 14q31

參考文獻

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肢帶型肌肉失養症的基因研究

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