CAJ | 학술논문

目的探讨基于目的基因捕获的高通量测序技术对串联质谱联合气相色谱-质谱技术在遗传代谢病(IMD)诊断中的应用价值.方法收集中山大学附属第六医院遗传代谢病实验室通过串联质谱联合气相色谱-质谱技术检测的疑似阳性10例患儿及其父母外周血样本,通过包含有153个常见的IMD致病基因捕获探针的高通量测序技术对患儿进行基因检测,采用Sanger测序技术对患儿阳性突变位点进行验证,并对其父母相应的位点进行检测.结果 10例疑似IMD患儿,通过高通量测序技术确诊8例,包括全羧化酶合成酶缺陷症3例,甲基丙二酸血症mut型、甲基丙二酸血症cblB型、枫糖尿症Ia型、鸟氨酸氨甲酰转移酶缺乏症、Citrin缺陷症各1例.其中有1例经串联质谱联合气相色谱-质谱诊断为异戊酸血症的患儿,其基因检测发现1个错义突变(c.158G ＞C,p.R53P)和1个同义突变(c.732C＞T,p.D244D);另外1例疑诊为Citrin缺陷症的患儿,其基因检测发现1个错义突变(c.1156G ＞A,p.G386S)和1个同义突变(c.1194A＞G,p.L398L),这2例基因检测结果在理论上尚不能确诊.10例患儿中鸟氨酸氨甲酰转移酶缺乏症患儿的致病突变属于自身新发生的基因突变,而其他患儿的致病突变均遗传于各自的父母.此外,高通量测序所检测的结果均经Sanger测序验证,二者结果全部一致.结论基于目的基因捕获的高通量测序技术在疑似IMD的检测中结果精确,不仅可为临床医师提供可靠的分子诊断依据,还能精准地进行疾病分型,有效地为后续临床治疗和遗传咨询提供依据.
목적 탐토기우목적기인포획적고통량측서기술대천련질보연합기상색보-질보기술재유전대사병(IMD)진단중적응용개치.방법 수집중산대학부속제륙의원유전대사병실험실통과천련질보연합기상색보-질보기술검측적의사양성10례환인급기부모외주혈양본,통과포함유153개상견적IMD치병기인포획탐침적고통량측서기술대환인진행기인검측,채용Sanger측서기술대환인양성돌변위점진행험증,병대기부모상응적위점진행검측.결과 10례의사IMD환인,통과고통량측서기술학진8례,포괄전최화매합성매결함증3례,갑기병이산혈증mut형、갑기병이산혈증cblB형、풍당뇨증Ia형、조안산안갑선전이매결핍증、Citrin결함증각1례.기중유1례경천련질보연합기상색보-질보진단위이무산혈증적환인,기기인검측발현1개착의돌변(c.158G ＞C,p.R53P)화1개동의돌변(c.732C＞T,p.D244D);령외1례의진위Citrin결함증적환인,기기인검측발현1개착의돌변(c.1156G ＞A,p.G386S)화1개동의돌변(c.1194A＞G,p.L398L),저2례기인검측결과재이론상상불능학진.10례환인중조안산안갑선전이매결핍증환인적치병돌변속우자신신발생적기인돌변,이기타환인적치병돌변균유전우각자적부모.차외,고통량측서소검측적결과균경Sanger측서험증,이자결과전부일치.결론 기우목적기인포획적고통량측서기술재의사IMD적검측중결과정학,불부가위림상의사제공가고적분자진단의거,환능정준지진행질병분형,유효지위후속림상치료화유전자순제공의거.
Objective To evaluate the application value of high throughput targeted exome sequencing in inherited metabolic disorders(IMD) diagnosed by tandem mass spectrometry combined with gas chromatography-mass spectrometry.Methods Ten cases of peripheral blood were collected from the parents and children with suspected inborn errors of metabolism detected by tandem mass spectrometry combined with gas chromatography mass spectrometry in the testing center of the Sixth Affiliated Hospital of Sun Yat-Sen University.One hundred and fifty-three genes related to genetics metabolic disorders were analyzed to obtain the positive mutations utlizing the high throughput targeted exome sequencing technology.Then all the mutations were validated by Sanger sequencing, as well as their parents' corresponding sites.Results Eight of the 10 patients with suspected IMD were confirmed by high throughput sequencing targeted exome sequencing.Among the 8 patients, 1 patient with methylmalonic academia-mut type, 1 patient with methylmalonic academia-cblB type, 1 patient with maple syrup urine disease-I a type, 1 patient with ornithine carbamoyltransferase deficiency, 1 patient with citrin deficiency,and 3 patients with holocarboxylase synthetase deficiency.However,there were still 2 cases which could not be verified, 1 patient with the suspected isovaleric acidaemia diagnosed by gas chromatography-mass spectrome spectrometry who had a missense mutation c.158G ＞ C(p.R53P) and a same sense mutation c.732C ＞ T(p.D244D) ,the other one with citrin suspected deficiency had a missense mutation c.1156G ＞ A(p.G386S) and a same sense mutation c.1194A ＞ G(p.L398L).Moreover, the positive gene sites of the patient with ornithine carbamoyltransferase deficiency (X-linked recessive inheritance) mutated spontaneously which differed from the other 9 cases inherited from their own parents.Besides, all the data of high-throughput sequencing were confirmed by Sanger sequencing and in accordance with each other.Conclusions The use of high throughput targeted exome sequencing can make a precise diagnosis for patients with high risk of IMD.It can not only provide reliable molecular diagnosis, but also proceed accurate disease classification affording the basis for clinical and genetic conseling.