曾焙枰，許紅恩，毛璐，湯文學(xué)

遺傳性耳聾分子診斷及梯級檢測策略應(yīng)用

曾焙枰1，許紅恩2，毛璐2，湯文學(xué)3

1. 鄭州大學(xué)華大基因?qū)W院與醫(yī)藥科學(xué)研究院，鄭州 450052 2. 鄭州大學(xué)醫(yī)學(xué)科學(xué)院精準醫(yī)學(xué)中心，鄭州 450052 3. 鄭州大學(xué)第二附屬醫(yī)院精準醫(yī)學(xué)研究應(yīng)用中心，鄭州 450014

遺傳性耳聾是人類最常見的感覺障礙之一，具有高度遺傳異質(zhì)性。目前常用的遺傳性耳聾分子診斷方法包括基因芯片、Sanger測序、靶向富集測序和全外顯子組測序等，診斷率可達33.5%～56.67%，但還有相當一部分患者不能進行及時有效的分子病因診斷。考慮到患者家庭的經(jīng)濟負擔及目前全外顯子組/全基因組測序仍相對昂貴，根據(jù)患者情況提供包含多種檢測手段的梯級診斷策略至關(guān)重要。因此，本文對遺傳性耳聾分子診斷現(xiàn)狀以及梯級檢測在遺傳性耳聾分子診斷中的應(yīng)用進行綜述，以期為診斷策略的選擇提供參考。

遺傳性耳聾；遺傳異質(zhì)性；分子診斷；高通量檢測；梯級檢測

聽力損失是人類最常見的感覺障礙之一，據(jù)2021年世界衛(wèi)生組織數(shù)據(jù)統(tǒng)計，世界上超過5%的人口(約4.66億人)患有聽力殘疾[1]。聽力損失病因復(fù)雜，可歸為環(huán)境因素和遺傳因素兩大類，其中遺傳因素的貢獻率超過60%[2,3]。遺傳性耳聾具有高度遺傳異質(zhì)性[4]，遺傳模式包括常染色體隱性遺傳(80%)，常染色體顯性遺傳(15%～20%)，性染色體連鎖遺傳(1%)和線粒體遺傳(1%)[5,6]。根據(jù)是否伴隨有其他系統(tǒng)或器官異常，遺傳性耳聾可分為綜合征型耳聾(syndromic hearing loss, SHL)(約30%)和非綜合征型耳聾(non-syndromic hearing loss, NSHL)(約70%)[7,8]。到目前為止，發(fā)現(xiàn)有400多種綜合征表現(xiàn)出聽力損 失[9]，已鑒定出120多種非綜合征型耳聾基因(https:// heredi-taryhearingloss.org/)。

在我國，、和是常見的耳聾基因，占先天性聽力損失病例的30～50%[10～15]。是我國最常見的耳聾致病基因，2002年對中國非綜合征型聽力損失患者的研究中發(fā)現(xiàn)突變攜帶率為20.5%(43/210)[16～18]，包括12.9%(27/210)的純合突變及7.6%(16/210)的單雜合突變，其最常見的突變是c.235delC、c.299_300delAT、c.109G>A和c.176_191del等。在中國非綜合征型耳聾患者中，攜帶編碼區(qū)單等位基因變異的比例為6.1%[19]。2010年，Yuan等[20]發(fā)現(xiàn)在1.89%(4/212)的單雜合突變患者中檢出c.–23+1G>A(NM_004004.6)變異，這表明基因的第一個外顯子應(yīng)納入常規(guī)檢測。是非綜合征型耳聾第二大常見致病基因，約占東亞耳聾患者的5.5%～19.39%[21～25]，中國耳聾人群中的熱點突變有c.919-2A>G和c.2168A> G等。突變遵循母系遺傳模式，其常見突變?yōu)閙.1555A>G和m.1494C>T，與氨基糖苷類藥物造成的耳聾有關(guān)[26]。

除單核苷酸變異及小插入缺失突變外，拷貝數(shù)變異(copy number variation, CNV)也是非綜合征型耳聾的常見原因[27～29]。分子病因明確的聽力損失患者中，有18.7%(50/267)因CNV導(dǎo)致[27]；在突變導(dǎo)致的聽力損失患者中CNVs占2%(14/700)[30]；CNVs對非綜合征型耳聾患者的貢獻率約5.5%(16/288)[31]。最近，有研究表明基因是輕中度聽力損失的重要責(zé)任基因[32～35]。2020年，Kim等[35]發(fā)現(xiàn)基因解釋了34.9%(29/83)的輕中度聽力損失病例，1/3(27/83, 32.5%)的病例檢測到至少一個CNV。由于假基因的存在，應(yīng)采用多重連接依賴探針擴增技術(shù)(multiplex ligation-dependent probe amplification, MLPA)來檢測基因CNV。

隨著基因突變檢測技術(shù)的發(fā)展，高通量檢測技術(shù)逐漸成為遺傳病診斷的主力。目前遺傳性耳聾基因診斷的主流方法有基因芯片檢測、多重PCR聯(lián)合高通量測序、基因靶向富集測序和全外顯子組測序(whole exome sequencing, WES)等[36]，甚至也會使用全基因組測序(whole genome sequencing, WGS)、RNA-seq或三代測序等技術(shù)來探索耳聾患者潛在遺傳病因，這些高通量檢測技術(shù)的原理及步驟見圖1。高通量檢測技術(shù)極大地提高了篩查已知耳聾基因以及發(fā)現(xiàn)新的耳聾基因的效率[37～39]?，F(xiàn)階段耳聾基因檢測方法診斷率可達33.5%～56.67%[25,37,40～46]，但還有相當一部分患者未能得到及時有效診斷。因此，為患者制定合適的分子診斷策略成為必然趨勢。有學(xué)者提出WES一步式檢測策略[40,45～47]，但分析和解釋W(xué)ES數(shù)據(jù)通常耗時費力?，F(xiàn)階段，已有多個研究提出逐步進行分子診斷——梯級檢測的思路，并 證明了梯級檢測策略在遺傳性耳聾分子診斷上的優(yōu) 勢[48～53]。針對患者的不同表型采用合適的分子診斷策略可以做到既有效又節(jié)約成本。因此，本文討論了高通量檢測技術(shù)與梯級檢測策略在遺傳性耳聾分子診斷中的應(yīng)用現(xiàn)狀。

1 高通量檢測技術(shù)在遺傳性耳聾分子診斷中的應(yīng)用

1.1 基因芯片檢測

耳聾分子流行病學(xué)研究表明我國耳聾群體有熱點基因及突變，如c.235delC、c.919-2A>G和線粒體m.1555A>G等。2007年Li等[54]首次利用多重等位基因特異性PCR進行等位基因鑒別反應(yīng)，用帶有不同探針標簽的通用固相芯片來顯示PCR結(jié)果。由此，基因芯片開始用于遺傳性耳聾的分子檢測。耳聾基因診斷芯片主要適用于四個基因(、、和)的熱點突變檢測。2017年He等[13]利用包含4個基因9個熱點突變的基因芯片檢測了2500個新生兒的突變情況，其中101名(4.04%)檢測到突變，9名(0.36%)得到診斷。2019年Dai等[12]利用基因芯片篩查了180 469名新生兒的突變情況，結(jié)果顯示8136名(4.508%)檢測到突變，449名(0.25%)得到診斷。該方法的優(yōu)點與局限性見表1。

圖1 高通量檢測技術(shù)示意圖

A：基因芯片技術(shù)。針對不同耳聾基因突變位點設(shè)計帶有不同標簽的上游探針和下游帶熒光標記的通用引物，進行多重等位基因特異性引物延伸PCR。PCR產(chǎn)物變性后，與固定有標簽互補寡核苷酸序列的芯片雜交，通過激光掃描檢測出突變位點。B: 多重PCR技術(shù)。第一輪PCR中使用的引物包含位點特異性引物及通用序列，第二輪PCR中使用的引物包含通用接頭序列及通用序列對應(yīng)引物。第二輪PCR產(chǎn)物經(jīng)過純化后可直接進行測序。C: 耳聾基因靶向富集測序和全外顯子組測序。將基因組DNA片段化及末端修復(fù)后加上接頭序列構(gòu)建文庫，然后采用芯片雜交捕獲或液相捕獲等方法對目標基因區(qū)域進行富集并測序，全外顯子組測序需要對全部外顯子區(qū)域進行捕獲并測序。D：全基因組測序。先將基因組進行DNA片段化處理，文庫構(gòu)建完成后直接測序，無需捕獲步驟。E：RNA-seq。mRNA逆轉(zhuǎn)錄為cDNA后，片段化、建庫及測序。F：三代測序?；蚪MDNA片段化后，連接接頭序列構(gòu)建文庫，純化后上機測序。

1.2 耳聾基因靶向富集測序

目標區(qū)域富集方法可分為多重PCR(multiplex PCR)和雜交捕獲。多重PCR是在同一PCR反應(yīng)體系里加上兩對以上引物，同時擴增出多個核酸片段的PCR反應(yīng)。因此，多重PCR可以同時對多個耳聾基因的全部外顯子進行檢測[55]。本研究團隊自主研發(fā)的高通量測序耳聾基因檢測試劑盒可同時檢測五個耳聾常見基因，分別是與基因的編碼外顯子，基因，以及和的熱點突變[56]。多重PCR檢測相較于基因芯片擴大了檢測范圍，提高了診斷率[56, 57]，但對于高度遺傳異質(zhì)性的耳聾，該方法的診斷率仍有待提高。

在單基因遺傳疾病的診斷中，基因靶向富集測序是常用的檢測方法。2010年Smith等[58]首次發(fā)表了通過耳聾基因靶向富集測序?qū)β犃p失患者進行分子診斷的研究。隨后，Tang等[59]提出了一種基于cDNA探針捕獲已知耳聾基因外顯子，并進行高通量測序從而實現(xiàn)耳聾分子診斷的方法。此類方法具有低成本、高特異性的優(yōu)點，可以對目前已鑒定的一百多個耳聾基因的全部外顯子進行檢測，適合于高通量測序，克服了臨床應(yīng)用的主要障礙。2016年Sloan-Heggen等[37]對1119名聽力損失患者進行耳聾基因靶向富集測序，結(jié)果確定了440名聽力損失患者(39%)的遺傳原因。2018年Cabanillas等[60]對50個聽力損失患者進行了包含199個耳聾相關(guān)基因的panel檢測，診斷率為42%(21/50)。2020年Yuan等[25]在433名散發(fā)性耳聾患者與30個耳聾家系中進行了129個耳聾基因的panel檢測，結(jié)果得出中國散發(fā)性非綜合征型聽力損失患者的診斷率為52.19%，中國耳聾家系的診斷率為56.67%。目前，在散發(fā)性聽力損失患者中，耳聾基因panel檢測診斷率大概在39%～52.19%，在耳聾家系中的診斷率會略高一些，最高可達56.67%。該方法的優(yōu)點與局限性見表1。

表1 高通量檢測技術(shù)在遺傳性耳聾分子診斷中的應(yīng)用

1.3 全外顯子組測序

全外顯子組測序(WES)是對基因組中所有編碼蛋白質(zhì)的基因外顯子區(qū)域進行測序。這些蛋白質(zhì)編碼區(qū)域約為6000萬個堿基對，約占人類參考基因組的2%[61]。WES可以實現(xiàn)對所有外顯子(>95%)進行測序，理論上可以涵蓋人類孟德爾疾病相關(guān)的大多數(shù)(85%)遺傳變異[62,63]。

全外顯子組僅占全基因組的2%，相較于全基因組測序的成本和分析復(fù)雜性更低[58]，已成為鑒定孟德爾遺傳疾病致病基因的有效手段[64]。2017年Zazo Seco等[40]利用WES對200名耳聾患者進行分子診斷，結(jié)果在67名(33.5%)耳聾患者中發(fā)現(xiàn)了引起聽力損失的變異；在10名患者(5.0%)中發(fā)現(xiàn)了意義不明的變異。2018年Sheppard等[45]對43例聽力損失兒童患者進行WES分子診斷，診斷率為37.2%。2020年Downie等[42]對106名中度至重度聽力損失的嬰兒進行WES分子診斷，診斷率為56%(59/106)。2019年Feng等[46]利用WES對33個常染色體隱性非綜合征型聽力損失家系進行分子診斷，48.5% (16/33)的家系確定了遺傳病因?，F(xiàn)階段WES檢測診斷率大概在33.5%～56%。該方法的優(yōu)點與局限性見表1。

1.4 全基因組測序

相比于WES，全基因組測序(WGS)是更全面的檢測手段，能夠識別外顯子區(qū)域和WES不能捕獲的98%的非編碼區(qū)域中的變異，也可以用來分析結(jié)構(gòu)變異[65]。以前的研究認為非編碼(內(nèi)含子)區(qū)域DNA是無用的[66]，然而多個研究發(fā)現(xiàn)非編碼區(qū)域變異可能導(dǎo)致疾病[67, 68]，這表明非編碼區(qū)域具有重要作用。在實驗步驟上，WGS相較于WES和耳聾基因靶向測序跳過了目標基因捕獲步驟，上機測序更快速便捷[69]。WGS已在聽力損失相關(guān)研究中得到初步應(yīng)用。2018年Vuckovic等[70]為了尋找年齡相關(guān)性聽力損失潛在的遺傳因素，對年齡均超過50歲的78個聽力損失患者和78個正常對照進行了WGS，通過比較分析確定了21個與年齡性聽力損失相關(guān)的基因。2021年Le Nabec等[71]對9例攜帶單雜合突變和1例攜帶del (-D13S1830)突變的DFNB1患者進行了全基因組測序，在4名患者中發(fā)現(xiàn)了的另一個變異。WGS也存在一些局限性(表1)，使得其不如WES和耳聾基因靶向富集測序應(yīng)用廣泛，但相信在不久的將來，隨著生物信息學(xué)的發(fā)展及變異解讀能力的提高，WGS會成為遺傳性耳聾的常規(guī)檢測手段。

1.5 三代測序的發(fā)展及潛在用途

第三代測序技術(shù)又稱為單分子測序，不需要經(jīng)過PCR擴增，直接對單個DNA分子進行實時測序。目前比較知名的是Oxford Naropore公司的單納米孔測序[72]和Pacific Biosciences的單分子實時測序[73]兩種平臺。與二代測序相比，三代測序的主要優(yōu)勢有以下幾點：真正實現(xiàn)了單分子測序，無GC偏好性[74]；超長的測序讀長，平均測序讀長達到10～15 kb，最長讀長可達40 kb甚至更長[75]，這使得對基因組結(jié)構(gòu)變異的分析得到極大改善[76～80]；可直接檢測堿基修飾，如DNA甲基化[81,82]；除DNA測序外，三代測序還可應(yīng)用于轉(zhuǎn)錄組研究，識別新的異構(gòu)體和基因融合[83]，這些都是二代短讀長測序做不到的[84]。

某些遺傳性耳聾基因的致病變異多以結(jié)構(gòu)變異為主，三代測序因其出色的識別結(jié)構(gòu)變異的能力，具有重要的應(yīng)用價值，比如在和等基因會檢測出較多CNVs[30,35,85]。三代測序應(yīng)用于遺傳性耳聾結(jié)構(gòu)變異鑒定的研究相對較少，2021年Dai等[85]利用三代測序?qū)θ丛\斷的內(nèi)耳畸形(IP-III)患者進行檢測，在兩名患者中發(fā)現(xiàn)了基因的結(jié)構(gòu)變異。三代測序也存在一些局限性(表1)，還在不斷發(fā)展，相信將來三代測序在遺傳性耳聾分子診斷上的應(yīng)用會越來越普遍。

1.6 RNA-seq結(jié)合WES以期提高遺傳性耳聾診斷率

轉(zhuǎn)錄組測序(RNA-seq)是指利用第二代高通量測序技術(shù)對組織或細胞中所有RNA 反轉(zhuǎn)錄而成的cDNA進行測序，常用于差異基因表達分析及可變剪切分析。現(xiàn)階段遺傳性耳聾分子診斷主要以耳聾基因靶向測序和WES為主，耳聾基因的剪接位點變異和可能影響基因表達量的錯義突變可以被檢測到，但卻無法確定其功能和致病性。因此，在現(xiàn)階段遺傳性耳聾分子診斷的背景下，RNA-seq與WES的結(jié)合可能實現(xiàn)優(yōu)勢互補，并將有助于提高遺傳性耳聾診斷率。

近年來RNA-seq逐步應(yīng)用于孟德爾遺傳病病因診斷。2017年Cummings等[86]利用RNA-seq對50例罕見肌肉疾病患者進行了基因診斷的隊列研究，并得到35%的診斷率，也首次闡釋了RNA-seq在孟德爾遺傳病臨床診斷中具有重要意義。2019年Gonorazky等[87]利用RNA-seq在先用基因panel或WES鑒定為陰性病例的25個遺傳性神經(jīng)肌肉疾病家族中新檢測出36% (9/25)的病例。2019年Frésard 等[88]利用血液轉(zhuǎn)錄組分析了94名罕見遺傳病患者，診斷率為7.5%，并為16.7%的患者篩選出強候選致病基因。另外，該研究使用RNA-seq檢測目前人類孟德爾遺傳數(shù)據(jù)庫(Online Mendelian Inheritance in Man, OMIM)中已知的主要罕見病致病基因在血液中的表達情況，發(fā)現(xiàn)70.6%的基因和50%的RNA剪接事件都可在血液轉(zhuǎn)錄組中獲得相關(guān)信息。這也為利用血液樣本RNA-seq來進行遺傳性耳聾分子診斷提供了可行性，然而已知耳聾基因在血液中的表達情況目前尚不明確。

2 梯級檢測在遺傳性耳聾分子診斷中的應(yīng)用

如前所述，遺傳性耳聾分子診斷的手段有很多，常用的包括基因芯片、Sanger測序、MLPA、靶向富集測序、WES和WGS等，通過多種檢測方法的組合使用可以更為經(jīng)濟有效地實現(xiàn)診斷。目前已有多篇梯級檢測非綜合征型聽力損失患者隊列的研究報道，它們的檢測策略主要分為以下兩種：

第一種檢測策略主要分為常見耳聾基因檢測和耳聾基因靶向富集測序或WES兩個步驟。第一步檢測的常見耳聾基因主要有和線粒體基因等，檢測方式包括僅對檢測、和線粒體基因Sanger測序加上和大片段缺失重復(fù)分析、或者多重PCR應(yīng)用于、和的檢測。2017年Baux等[49]對207個法國非綜合征型聽力損失家庭進行分子診斷，首先對兩個外顯子及其側(cè)翼序列進行Sanger測序，隨后對陰性樣本行74個耳聾基因的靶向富集測序，并利用測序數(shù)據(jù)進行了CNV分析(包括基因)。隊列總體診斷率為48%，其中基因占24%。2018年Guan等[48]報道了一種耳聾基因梯級檢測策略，第一階段對編碼區(qū)和兩個線粒體基因進行Sanger測序，并利用SNP陣列分析和大片段缺失，第二階段對陰性樣本行耳聾基因外顯子靶向富集測序分析。結(jié)果33例NSHL患者中有11例可以明確診斷(33%)，有8例可能診斷(24.2%)，第一階段診斷率為30.3%(10/33)，最高診斷率為57.2%。2021年Wang等[51]提出了一種由多重PCR加上WES組成的雙層策略，首先多重PCR應(yīng)用于檢測、和，然后進行WES分析，共有64%(59/92)的患者被診斷，其中44例為多重PCR診斷(47.8%)。

第二種檢測策略的第一步直接對多個耳聾基因的已知突變或者基因panel進行檢測；第二步進行耳聾基因靶向富集測序或者WES。2016年Sakuma等[53]對52名日本聽力損失患者進行了包含兩個階段的梯級檢測，首先檢測13個耳聾基因的46個已知突變，并對檢測出或基因單雜合突變的患者進行基因編碼區(qū)的Sanger測序；第二階段對63個已知耳聾基因進行外顯子靶向富集測序。該研究第一階段診斷率為17%(9/52)，總體診斷率為40%(21/52)。2020年Budde等[50]通過對兩個大小不同的耳聾基因panel進行靶向富集測序，揭示了61個埃及非綜合征型聽力損失近親家系的遺傳病因?？傮w診斷率為78.7%(48/61)。另外，在2019年，Li等[52]在一個耳聾家系中進行了熱點變異篩選和隨后的WES檢測，通過逐步遺傳分析首次發(fā)現(xiàn)基因與人類NSHL有關(guān)。現(xiàn)階段，根據(jù)不同的研究隊列及梯級策略的制定差異，以上兩種策略第一步檢測所得診斷率大概在17%～47.8%，當進行了第二階段的檢測后，總體診斷率有一定的提高，這體現(xiàn)了利用梯級策略進行分子診斷的優(yōu)勢。

根據(jù)不同人群和種族，制定診斷策略也會各有差異。Guan等[48]和Baux等[49]提出的診斷策略針對的是歐美人群，突變致聾占比較少，未將基因列入第一步檢測，而在中國，是第二大耳聾致病基因，因此，他們的策略不適合中國聽力損失患者。在東亞聽力損失患者中有很多基因的研究報道，Yokota等[34]發(fā)現(xiàn)在日本輕中度聽力損失患者中基因純合缺失的貢獻率為4.3%(17/398)；Kim等[35]發(fā)現(xiàn)基因CNVs解釋了1/3(27/83，32.5%)的韓國輕中度非綜合征型聽力損失病例。雖然中國對基因?qū)е侣犃p失的研究較少，但這些研究強調(diào)基因是東亞人群輕中度聽力損失的主要原因，然而在Budde等[50]、Wang等[51]和Sakuma等[53]的檢測策略中并沒有包含對CNVs的分析。若患者隊列存在輕中度耳聾患者，那么基因的MLPA檢測在梯級策略中也至關(guān)重要。另外，上述研究除Baux等[49]的檢測策略外都沒有對基因外顯子1進行檢測，然而根據(jù)Yuan等[20]和Yu等[89]報道，c.–23+1G>A在中國耳聾患者中占0.2%～1.89%，因此對中國耳聾患者而言，這些方法可能低估了基因的診斷率。外顯子1的Sanger測序應(yīng)包括在單等位致病變異患者的常規(guī)檢測中。

綜上所述，在多個聽力損失基因診斷研究中，梯級檢測策略體現(xiàn)出了優(yōu)勢和一些不足。因此，本研究團隊提出一種適用于中國聽力損失患者分子診斷的梯級檢測策略(圖2)：首先對聽力損失患者進行臨床表型及家族史分析，對于非綜合征型聽力損失患者，第一步，利用多重PCR結(jié)合高通量測序檢測常見耳聾基因和；第二步，對攜帶單雜合突變的陰性樣本進行第1外顯子的Sanger測序；第三步，對輕中度聽力損失患者使用MLPA進行基因檢測；第四步，將上述步驟檢測為陰性的樣本進行外顯子組測序，并分析CNVs；對于綜合征型耳聾或表型具有高度基因特異性的患者(如前庭導(dǎo)水管擴大、內(nèi)耳畸形IP-III表型、鰓-耳-腎綜合征)，可以考慮直接進行單基因測序或WES等檢測；經(jīng)以上檢測仍然為陰性的樣本，可考慮進行WGS、三代測序或RNA-seq以探索潛在遺傳病因?；驒z測技術(shù)和數(shù)據(jù)的生物信息學(xué)分析等不同層面的技術(shù)一直在快速發(fā)展，該策略需要在實踐中進行檢驗，并根據(jù)技術(shù)的發(fā)展及回顧分析做適時的調(diào)整。

圖2 聽力損失患者分子診斷的梯級檢測策略圖

橙色框表示患者類型，紫色框表示基因檢測方法，藍色框表示診斷結(jié)果。對于非綜合征型聽力損失患者，經(jīng)基因芯片或多重PCR聯(lián)合高通量測序檢測的患者可歸類為常見耳聾基因、或診斷，對單雜合突變患者進行外顯子1的一代測序主要是檢測c.–23+1G>A的突變情況，需要對輕中度聽力損失患者進行MLPA檢測以確定基因的CNV情況，應(yīng)用以上方法未診斷患者可進行WES確定遺傳病因。對于綜合征型耳聾或表型具有高度基因特異性的患者，可直接進行單基因測序、多基因panel檢測、WES或染色體微陣列分析等檢測。經(jīng)以上檢測仍然為陰性的樣本，可利用WGS、RNA-seq或三代測序等手段探索潛在遺傳病因。

3 結(jié)語與展望

高通量檢測技術(shù)在遺傳性耳聾研究領(lǐng)域已取得令人矚目的成績。在WGS費用昂貴、數(shù)據(jù)分析負擔大、非編碼區(qū)域與疾病的關(guān)系尚不明確的情況下，一代測序、耳聾基因靶向測序、MLPA和WES等多種檢測方法聯(lián)合使用，形成適合研究隊列的梯級檢測策略，將成為未來數(shù)年內(nèi)進行遺傳性耳聾分子診斷最重要的手段。梯級檢測策略可以提高診斷率并節(jié)約成本，為遺傳咨詢及耳聾出生缺陷防控提供堅實的基礎(chǔ)。相信隨著遺傳性耳聾致病基因的發(fā)現(xiàn)、高通量檢測技術(shù)與生物信息學(xué)的不斷發(fā)展以及梯級檢測策略的不斷完善，遺傳性耳聾的分子診斷必將實現(xiàn)重大進展。

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Molecular diagnosis of hereditary deafness and application of stepwise testing strategy

Beiping Zeng1, Hongen Xu2, Lu Mao2, Wenxue Tang3

Hereditary deafness is one of the most common sensory disorders in humans, and exhibits high genetic heterogeneity. At present, the commonly used molecular diagnostic methods include gene chip, Sanger sequencing, targeted enrichment sequencing, and whole-exome sequencing, with diagnosis rates reaching 33.5%–56.67%. However, there are still a considerable number of patients who can not get a timely and definitive molecular diagnosis. Furthermore, considering the economic burden on patients’ families and the relatively high cost of whole-exomeor whole-genome sequencing, it is vital to provide stepwise strategies combining multiple detection methods according to the phenotypes of patients. In this review, we evaluate and discuss the utility of molecular diagnosis and the application of stepwise testing strategies in hereditary deafness to provide reference for the selection of diagnostic strategies.

hereditary deafness; genetic heterogeneity; molecular diagnosis; high-throughput screening; stepwise testing strategy

2022-09-22;

2022-10-29;

2022-11-04

鄭州市協(xié)同創(chuàng)新項目(編號：18XTZX12004)，河南省醫(yī)學(xué)科技攻關(guān)計劃聯(lián)合共建項目(編號：LHGJ20190317)[Supported by the Collaborative Innovation Project of Zhengzhou (No. 18XTZX12004), and the Joint Project of Medical Science and Technology Research in Henan Province(No. LHGJ20190317)]

曾焙枰，碩士研究生，專業(yè)方向：遺傳性耳聾分子診斷。E-mail: beipingzeng1120@163.com

許紅恩，博士，講師，研究方向：遺傳性耳聾與腎病。E-mail: hongen_xu@zzu.edu.cn

曾焙枰和許紅恩并列第一作者。

湯文學(xué)，教授，研究方向：遺傳性耳聾分子診斷及突變功能研究。E-mail: twx@zzu.edu.cn

10.16288/j.yczz.22-206

(責(zé)任編委: 方向東)