大片段缺失或插入所造成結節硬化症之基因診斷

腫瘤抑制基因中的 TSC1 (MIM#605284)和 TSC2 (MIM#191092)在人類生殖系發生突變時會引起結節硬化症(TSC, MIM#191100)，是屬於體染色體顯性遺傳疾病，並且在身體的多個器官有錯構瘤的產生，包括腦(結節、室管膜下結節和室管膜下巨細胞星形細胞瘤)、皮膚(面部血管纖維瘤和鯊魚斑)、心臟(橫紋肌瘤)、肺(淋巴管肌瘤病)、視網膜(星狀細胞缺陷瘤)和腎臟(血管平滑肌脂肪瘤)。結節硬化症發生率為1/6000~1/10000。大約有2/3的患者屬於散發型，沒有家族遺傳病史，是因為TSC1或TSC2 基因上發生新的突變，或者是父或母親性腺有鑲嵌體影響所引起。 TSC1 和TSC2 基因產物分別為hamartin和 tuberin，在蛋白質複合體的功能，以調節細胞訊號傳遞並且對於調節細胞生長，遷移和增殖是非常重要的。大多數的TSC1和TSC2為點突變和小的插入/缺失。本次研究是將台大醫院結節硬化整合門診病友所提供的血液，53個家族中共62位結節硬化症患者，其中包括 proband 53位，47位有明確診斷的TSC患者及6位可能是TSC患者。這些患者在第一輪的次世代序列(NGS)的實驗和數據分析中，未找出引起變致病變異點或是小的插入或是缺失，本次實驗除了更詳細分析NGS的結果，並且使用多重連接探針擴增（MLPA）技術，針對大的缺失和/或插入進行基因變異篩檢。結果發現在其中1個TSC患者 TSC2 基因發現第16外顯子到第29個外顯子有大片段的缺失，其斷點兩端皆為Alu Y elemen，並且由Sanger定序法證實；而在另1個TSC患者，我們發現不論是在MLPA 或是NGS 中TSC2基因外顯子1的信號雖然皆表現低落，然而卻無法以 Sanger 定序法確認突變。最後我們的結論是NGS和MLPA對於檢測TSC患者的大片段缺失或是插入都屬於十分良好的檢測工具。

關鍵字

結節硬化症；體染色體顯性遺傳；基因檢測；次世代序列； MLPA

並列摘要

In human, germ-line mutations of the TSC1 (MIM#605284) or TSC2 (MIM#191092) tumor suppressor genes cause tuberous sclerosis complex (TSC, MIM#191100), an autosomal dominant disease in which hamartomas and/or hamartias are found in multiple organ systems, including brain (tubers, subependymal nodules and subependymal giant cell astrocytomas), skin (facial angiofibromas, hypomelanotic macules and shagreen patches), heart (cardiac rhabdomyomas), lungs (Lymphangioleiomyomatosis), retina (astrocytic hematoma), and kidney (angiomyolipomas). The prevalence of TSC is estimated as high as 1/6000~1/10000. About two-thirds of TSC patients are sporadic cases with no family history, and appear to represent new mutation occurring in either TSC1 or TSC2, or are transmitted from parents of gonadal mosaicism. The TSC1 and TSC2 gene products (hamartin and tuberin) function in a protein complex to modulate cell signaling pathways that are important for regulation of cell growth, migration and proliferation. Point mutations and small insertions/deletions (indels) account for most TSC1 and TSC2 mutations. This study was carried out at the Joint TSC Clinics of National Taiwan University Hospital. We collected blood samples from 62 TSC patients in 53 pedigrees. Among the 53 probands, 47 individuals had definite diagnosis of TSC, and 6 individuals had possible diagnosis. For those patients that we did not detect causative substitutions or small indels in the first round of next-generation sequencing (NGS) experiments and data analyses, we performed further analyses of the NGS data and also conducted new experiments using the multiplex ligation-dependent probe amplification (MLPA) technology, aiming at identification of large deletions and/or insertions. We found a large deletion spanning exon 16 and exon 29 of the TSC2 gene in one TSC patient and mapped both breakpoints of the deletion to Alu Y elements. The findings were confirmed by Sanger sequencing. In another TSC patient, we found reduced signal at exon 1 of the TSC2 gene by data from both NGS and MLPA; however, we could not confirm this mutation by Sanger sequence yet. We conclude that NGS and MLPA are powerful tools to detect large deletions/insertions for TSC patients.

並列關鍵字

Tuberous sclerosis complex (TSC) ； autosomal dominant disease ； Genetic test ； Next generation sequencing (NGS) ； MLPA

參考文獻

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大片段缺失或插入所造成結節硬化症之基因診斷

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