台大醫院結節硬化症整合門診病患之基因型鑑定與遺傳諮詢

Abstract

結節硬化症（OMIM 191100）為體染色體顯性遺傳疾病，有極高的穿透率，在腦部、腎臟、肺臟及及其他器官生成缺陷瘤，造成臨床上產生多樣化的症狀，包括癲癇、智能障礙、行為異常及皮膚病變，常造成患者生理與心理上的傷害。結節硬化症患者只有三分之一是遺傳而來，另外三分之二的病人則為新生的突變所致，新生兒發生率為1/6000-1/10000，男女比率相等。目前已知有TSC1(9q34)以及TSC2 (16p13)二個基因突變造成，可確認的突變在結節硬化症約70-80%左右。 基因檢測對結節硬化症的重要性，主要可提供臨床上的確認診斷，了解基因突變的位置，檢測結節硬化症患者家族，分析TSC是否為家族遺傳或是新生的突變，以及提供產前遺傳診斷。基因檢測的結果，透過遺傳諮詢解說，使患者及家屬了解結節硬化症的發生，制訂適合的醫療照護，說明遺傳機率及可能的風險，並提供必要的協助。然而，TSC1及TSC2基因共有64 exons，使得基因檢測時間長，費用又昂貴，常造成患者的負擔，且延誤診治。 臺大醫院於2010年7月成立「結節硬化症整合門診」，整合各醫療專科，以病人為中心，提供專屬結節硬化症病友客製化的「全人醫療」服務，方便病友就醫。 本研究於「臺大醫院結節硬化症整合門診」的病友所提供的血液，利用次世代定序分析(Illumina system)針對47位已確診為結節硬化症及6位可能是結節硬化症的個案，進行TSC1及TSC2基因突變點位檢測。其結果共檢出35位TSC1或TSC2基因序列異常且會影響基因功能的變異位置。然後再從篩檢出可能具有臨床意義之變異的35位TSC患者及其家屬81位，進行聚合酶連鎖反應及標準Sanger sequencing方法做DNA定序進行檢測。其中有16位個案以前做過基因檢測報告，亦列為比對參考。研究期間，共做七例遺傳諮詢紀錄。 本研究結果以次世代定序技術針對TSC1及TSC2基因進行檢測，其檢出率為72%(34/47)，正確率更達97%(34/35)。與16位個案以前做過基因檢測報告比對，符合率達88%(14/16)，且成功地找到以前未能找出的致病基因變異點位。完全不亞於傳統的DNA基因定序檢測方法。由於NGS是採用合成方法進行DNA定序，降低錯誤率；並利用微製程技術，使成本大幅降低，且更有效率。在未來，可快速提供醫師對結節硬化症做診斷，並對病程的預估及治療做規劃。結合專業的遺傳諮詢服務，為結節硬化症的患者及早制訂完善的醫療照護，藉以減緩或解決症狀及併發症，讓患者能過著正常生活，融入社會團體中，有效減輕家庭傷害，大幅降低醫療成本及社會負擔。此基因檢測結果，可提供醫學研究，研發完善的治療方法及預防結節性硬化症的發生。

Tuberous sclerosis complex (TSC[OMIM 191100])is an autosomal dominant disease with high penetrance, characterized by the presence of hamartomas in brain, kidneys, lungs and/or other multiple organ systems. The clinical manifestations include epilepsy, mental retardation, behavioral problems, skin lesions, renal problems, which often cause harm to patients physically and psychologically. Two thirds of patients are sporadic and are thought to represent de novo mutation. Approximately one in 6,000 to one in 10,000 newborns are affected by TSC. Males and females are equally susceptible to have TSC and the chance of passing it on to offspring is 50%. TSC is caused by inactivating genetic variant at either TSC1(9q34) or TSC2 (16p13) gene. About 70% to 80% of individuals who meet definite diagnostic criteria have a small identifiable TSC1 or TSC2 gene causative variant. Genetic testing for TSC patients has many important implications, including, but not limited to, confirmation of the diagnosis, identification of the causative variants of the patient/pedigree, and genetic counseling. The results of genetic testing may be helpful for patients, family members, medical professionals and policymakers. However, there are a total of 64 exons in TSC1 and TSC2 genes, which causative variants genetic testing for TSC to be time-consuming and expensive. It is not uncommon that genetic testing is delayed because the family cannot afford the cost. The Joint TSC Clinics at National Taiwan University Hospital was established in July 2010. A TSC patient can get access to almost all medical professionals related to his/her medical needs at a single visit, which greatly facilitate holistic health care and multi-disciplinary collaboration. The Clinics is a one-stop solution, and provide comprehensive health care for TSC patients. The study was approved by the Institutional Review Board (IRB) of National Taiwan University Hospital. After obtaining informed consent, we collected blood samples from 47 definite and 6 possible TSC patients and their family members at the Joint Tuberous Sclerosis Complex Clinics at National Taiwan University Hospital. We performed genotyping using next-generation sequencing (NGS) (Illumina system) technology for detecting genetic variants at the TSC1 and TSC2 genes. For possible causative variants detected in the NGS experiment, we further verified the results using traditional Sanger sequencing. Sixteen patients were previously genotyped by traditional methods; we therefore made a quick comparison between the genotyping results between the new NGS method and the traditional methods. We also performed genetic counseling for 7 families during the study period. Among the 47 patients with definite TSC diagnosis, we could identify the causative genetic variants for 34 patients (72%), a detection rate comparable with the traditional method. Among the 16 patients previously genotyped, all the results were the same, except for two patients that we identified the causative variants which had been missed using the traditional way. We found NGS to have several advantages as a genotyping tools for TSC diagnosis, including high accuracy, high speed and relatively low cost. In the future, NGS may prove to be a valuable tool for TSC genetic diagnosis. Professional genetic counseling services will also help TSC patients and families through risk prediction and disease understanding. We hope that our comprehensive TSC care system, including the genetic testing modality and genetic counseling, can help early diagnosis and multi-disciplinary treatment of TSC patients, and reduce the number of new TSC patients from known families. The insights gained from genetic study and molecular mechanism might further facilitate the progress of medical research and provide better choices for TSC treatment.