建立次世代定序技術平台應用於新生兒感覺神經性聽損及巨細胞病毒感染篩檢

Abstract

兒童聽損基因異常與CMV病毒感染皆會導致感覺神經性聽力損失。台灣常見導致聽損基因之點位如GJB2 c.109G>A, GJB2 c.235delC, SLC26A4 c.919-2A>G, SLC26A4 c.2168, MTRNR1 m.1555及OTOF c.5098等，近幾年合併CMV納入檢測，兩者皆已運用於目前新生兒聽損基因篩檢中。目前利用即時核酸定量分析反應，作為聽損四個基因六個點位與CMV感染之檢測方法，其優點為高靈敏度及快速偵測，提供個案與醫師進行診斷。由於此方式無法進行多基因多點位之檢測，對於聽損高度遺傳異質性之特性，無法提供有更效率之運用。因此，本研究方法利用次世代定序技術平台之轉換，以設計專一性引子對進行多重聚合酶連鎖反應，配合Adapter接合反應，最後利用次世代定序進行23個聽損變異點位分析，提供一套擴大範圍與更快速的檢測方式，於一次性實驗中能同時獲得兩者之檢測結果。我們發現聽損基因與CMV之次世代定序平台結果，與即時核酸定量方法之結果一致，顯示其平台之準確性。另外，利用此平台發現於不同檢體中，有兩個新突變點位SLC26A4 c.1174A>T與SLC26A4 c.1226G>A，此變異點無法利用傳統即時核酸定量方法發現，對於個案可能有不同之臨床表現型。因此，本研究方法利用次世代定序技術擴大範圍與快速檢測，成功地運用於新生兒聽損基因篩檢，亦發現非常規性篩檢之變異點位，可提供醫師進一步診斷，次世代定序技術平台並可作為個案日後家族遺傳諮詢之新方法。

Genetic hearing loss and cytomegalovirus (CMV) infections can lead to sensorineural hearing loss in children. In recent years, CMV and common genetic loci in Taiwan that lead to hearing loss (such as GJB2 c.109G>A, GJB2 c.235delC, SLC26A4 c.919-2A>G, SLC26A4 c.2168, MTRNR1 m.1555, and OTOF c.5098) were incorporated into screening tests, and these are already part of the current genetic screening test conducted for neonatal hearing loss. At present, real-time polymerase chain reaction (PCR) is used as a method for detecting six loci of four hearing loss genes and CMV infections. The advantage of this method lies in its high sensitivity and quick detection capability, which facilitate the diagnostic process for physicians and patients. However, as this method cannot be used to perform multi-gene, multi-locus detection, it is thus unable to be used effectively to test high levels of genetic heterogeneity of hearing loss. Therefore, next-generation sequencing platforms were utilized in this study to design specific primer pairs that can perform multiplex PCR. These pairs were then used in conjunction with the application of the adapter ligation reaction and next-generation sequencing to conduct an analysis of 23 hearing loss variant loci, so as to provide a detection method that can increase the scope and speed of tests, and concurrently generate test results for the two item types in a single test. The hearing loss gene and CMV detection results of the next-generation sequencing platform were found to be consistent with those generated using the real-time PCR method, which indicated the accuracy of the platform. Furthermore, two new mutation loci (SLC26A4 c.1174A>T and SLC26A4 c.1226G>A) were identified during the use of this platform on two specimens, additionally. These loci were not previously detected using the traditional real-time PCR method. Thus, the next-generation sequencing technique utilized in this study was able to increase the scope and speed of tests, and be successfully applied to genetic screening tests for neonatal hearing loss. This unconventional screening approach also revealed variant loci that can assist physicians in making further diagnoses. This next-generation sequencing technique and platform can be used as a new method for genetic counseling in the future.