利用次世代定序進行困難疾病之基因檢測，以先天性腎上腺增生症及神經纖維瘤症第一型為例

Abstract

先天性腎上腺增生症為參與合成皮質醇的酵素缺乏導致異常之體染色體隱性遺傳疾病。其症狀包含皮質醇缺乏造成低血糖、雄性素過多導致女嬰外生殖器外觀異常、醛固酮不足引發體內鹽類失衡，出現高血鉀、低血鈉、脫水與代謝性酸中毒等病症。超過九成個案為CYP21A2基因異常，導致身體無法有效合成類固醇-21羥化酶，引發體內荷爾蒙生成異常。CYP21A2位於重組頻率極高的MHC 區域，又有與其相似性極高的假性基因CYP21A1P，造成臨床上分子檢測極大的困難。 神經纖維瘤第一型是一種常見的自體顯性遺傳疾病。主要致病原因為NF1基因突變引起神經纖維細胞不正常增生，致使病患身體各處長出大小不一的腫瘤，皮膚出現淺咖啡色的斑點，屬於全身性的疾病。NF1目前已知超過3000個變異位置，臨床進行基因檢測需要耗費龐大的人力與時間成本。 有鑑於此，本研究建立以探針擷取的次世代定序方法，應用於先天性腎上腺增生症與神經纖維瘤第一型之基因檢測，以期解決CYP21A2與NF1目前分子檢測上之難題，並達到快速篩檢成效。 結果顯示，利用次世代定序平台確實檢驗出先天性腎上腺增生症與神經纖維瘤第一型之變異位置，並且有效鑑別不同變異形式，包含假性基因突變點小片段交換、大片段缺失、單一位點變異以及嵌合型等複雜型態，此外更有效降低時間及人力成本，達到高效率高準確性之篩檢成果。於臨床醫學輔助鑑別診斷，於遺傳諮詢則可提供適切的衛教資訊，協助個案做生涯規劃及風險管理。

Congenital adrenal hyperplasia (CAH) is an autosomal recessive disorder caused by defects in one of several steroidogenic enzymes involved in the synthesis of cortisol from cholesterol in the adrenal glands. More than 90% of cases are caused by 21-hydroxylase deficiency due to disease-causing variants in the CYP21A2 gene. CYP21A2 is part of the RCCX module inside human major histocompatibility complex (MHC), a chromosomal region highly prone to genetic recombination events that can result in a wide variety of complex rearrangements. Besides, a pseudogene, CYP21A1P, shares 98% sequence homology with CYP21A2 but contains several alterations rendering it inactive, which make it technically very challenging to detect variation of CYP21A2. Neurofibromatosis type I (NF1) is a common autosomal dominant disease, caused by mutations in the NF1 gene. Clinically, NF1 patients are prone to develop café-au-lait macules, neurofibromas, lisch nodules and skinfold freckling. There are over 3000 disease-causing variants, including structural variations, of NF1. Patients can show extreme variability in their clinical features, even in individuals from the same family with an identi­cal germline NF1 mutation. All of these make it difficult to confirm NF1 patient by genetic testing. In this study, we established a pipeline taking advantage of capture-based target enrichment followed by next-generation sequencing (NGS)-based genetic testing to detect the disease-causing variants of CAH and NF1. The result showed probe-captured NGS-based genetic testing represents a rapid and sensitive tool for detecting various kinds of disease-causing variants of CAH and NF1, including gene conversion, large deletion, single nucleotide variation, small indels and mosaicism.