应用二代测序技术对四个低血磷性佝偻病家系的基因突变检测

Mutation analysis of four pedigrees affected with hypophosphatemic rickets through targeted next-generation sequencing

目的对4个家系6例低血磷性佝偻病（hypophosphatemie rickets，HR）患者进行PHEX基因的突变分析，并对家系中高危胎儿进行产前诊断。方法应用二代测序对4个HR家系的先证者进行PHEX、FGF23、DMP1、ENPP1、CLCN5、SLC34A3基因的全外显子检测，在家系成员和200名健康个体采用Sanger双向测序对点突变进行验证分析，并在家系成员和20名健康个体应用多重连接探针扩增技术对基因的缺失突变进行验证分析。在确定致病突变后，对其中1个家系中的高危胎儿进行孕早期产前诊断。结果在4个家系中均检出PHEX基因突变，分别为c．850-3C〉G、第11外显子缺失突变、第13外显子缺失突变、c．1753G〉A（p．G585R），其中第11外显子缺失突变、第13外显子缺失突变、c．1753G〉A（p．G585R）尚未见报道，在家系正常成员和健康对照中均未发现这3个突变。对家系3的高危胎儿行产前基因诊断，未携带PHEX基因突变，选择继续妊娠，随访至出生1年后新生儿表型均无异常。结论PHEX基因突变是4个HR家系的致病病因，二代测序结合Sanger测序方法和多重连接探针扩增技术检测可以快速且准确地进行该病的基因诊断和产前诊断。

Objective To detect potential mutations of PHEX gene in four pedigrees affected with hypophosphatemic rickets （HR） and provide prenatal diagnosis for a fetus at 13th gestational week. Methods The coding regions and exon/intron boundaries of PHEX, FGF23, DMP1, ENPP1, CLCN5 and SLC34A3 genes of the probands were analyzed by targeted next-generation sequencing （NGS）. Suspected mutations were verified by Sanger sequencing among unaffected relatives and 200 unrelated healthy individuals. Deletions were confirmed by multiplex ligation-dependent probe amplification （MLPA） detection of probands, unaffected relatives and 20 unrelated healthy individuals. Prenatal diagnosis for a fetus with high risk was carried out through MLPA analysis. Results Four PHEX mutations were respectively detected in the pedigrees, which included c. 850-3C〉G, exon 11 deletion, exon la deletion and c. 1753G〉A （p. G585R）. Among these, exon 11 deletion, exon 13 deletion and c. 1753G〉A （p. G585R） were novel mutations and not found among unaffected relatives and healthy controls. In pedigree 3, the same mutation was not found in the fetus. Conclusion Mutations of the PHEX gene probably underlies the disease among the four pedigrees. NGS combined with Sanger sequencing and/or MLPA detection can ensure accurate diagnosis for this disease.