应用二代测序技术对两个低磷酸酯酶症家系的基因突变检测

Mutation analysis for two hypophosphatasia families with targeted next-generation sequencing

目的对2个中国汉族的围产期型低磷酸酯酶症（HPP）家系进行碱性磷酸酯酶基因（ALPL）突变分析，以探讨该病的致病机制。方法应用二代测序（NGS）技术对2015年7月至2016年3月就诊于郑州大学第一附属医院遗传与产前诊断中心的2个家系的HPP患儿生育史的母亲进行骨病检测包基因检测，发现可疑致病位点后，利用聚合酶链式反应（PCR）和Sanger测序直接对患儿的父亲进行突变筛查，初步确定该家系的可疑致病位点，并对父母双亲、患病胎儿和200名健康个体的进行突变验证分析。结果家系1中临床表现正常的患病胎儿母亲、父亲为杂合子，分别携带ALPL基因c．333delC（P．Gly112 AlafsX10）及c．568_570delAAC（P．190delAsn）碱基缺失突变，患病胎儿为复合杂合突变，同时携带c．333delC（P．Gly112AlafsX10）和c．568_570delAAC（P．190delAsn）碱基缺失突变。家系2中患病胎儿的母亲和父亲分别携带ALPL基因已知致病性突变c．1250A〉G（P．Asn417Ser）和c．1166C〉A（P．Thr389Asn），患病胎儿为c．1166C〉A（P．Thr389Asn）和c．1250A〉G（P．Asn417Ser）复合杂合突变个体。2家系均符合常染色体隐性遗传，ALPL基因c．333delC（P．Gly112AlafsX10）为首次报道的致病性突变，200名无关健康个体验证未发现该突变存在。结论ALPL基因突变是2个HPP家系的致病原因，NGS结合Sanger测序方法可以高效准确地对该病进行基因诊断。

Objective To detect the mutations in alkaline phosphatase （ALPL） gene of two Chinese families with perinatal hypophosphatasia （HPP）, in order to explore the mechanism of this condition. Methods Next-generation sequencing （NGS） of ostcology system panel was carried out for exome sequencing in the mothers of 2 HPP fetuses, who visited Prenatal Diagnosis Center of the First Affiliated Hospital of Zhengzhou University. Further polymerase chain reaction （PCR） and Sanger sequencing v＇,didation was performed in the parents, affected fetuses and 200 unrelated healthy individuals to verify the mutation sites. Results The mother and father of No. 1 family carried ALPL gene c. 333delC （ p. Gly112AlafsX10） and c. 568_570delAAC （ p. 190delAsn） base deletions, respectively. The affected fetus carried compound heterozygotes of the two mutations. Two mutations in ALPL gent known to be associated with hypophosphatasia were found in No. 2 family, c. 1250A 〉 G （p. Asn417Ser） in the mother and e. 1166C 〉 A （p. Thr389Asn） in the father, while the fetus was a compound heterozygote carrying both of the two mutations. Both families met the pattern of autosomal recessive inheritance. ALPL gene c. 333delc （p. Gly112AlafsX10） was a novel mutation,and it was not found in the 200 unrelated healthy individuals. Conclusions The mutations in ALPL gene may be the cause of HPP in the 2 families. NGS technology combined with Sangcr sequencing could be an efficient and accurate diagnostic method.