苯丙酮尿症43个家系的 PAH基因变异分析及产前诊断

Analysis of PAH gene variants and prenatal diagnosis for 43 Chinese pedigrees affected with Phenylketonuria

目的:探讨43个苯丙酮尿症(PKU)家系苯丙氨酸羟化酶(phenylalanine hydroxylase,PAH)基因的变异特点及其产前诊断方法。方法:选择2019年至2021年郑州大学第一附属医院确诊的43个PKU家系作为研究对象,通过高通量测序对其先证者进行PAH基因的变异筛查,并用Sanger测序法对候选变异进行家系验证。对未明确变异的先证者用多重连接探针扩增(MLPA)技术检测PAH基因的大片段缺失或重复。对风险妊娠联合使用Sanger测序、MLPA和多态性连锁分析进行产前诊断。结果:43例先证者的86个等位基因经检测共发现了78个变异和3个大片段缺失,检出率分别为90.70%和3.49%。共检出21种错义变异、5种剪接变异、4种无义变异、2种微小缺失、1种插入和2种大片段缺失。常见的变异包括p.Arg243Gln(23.26%)、p.Arg111Ter(8.14%)、EX6-96A>G(6.98%)、p.Val399Val(5.81%)和p.Arg413Pro(4.65%)。变异热点包括第7、11、3、6和12外显子。产前诊断在43个家系中共确诊PKU受累胎儿9例(20.45%)、杂合变异携带者20例(45.45%)、未携带相同变异者15例(34.09%)。胎儿出生后随访至6个月,产前诊断的准确率为100%。结论:联合高通量测序、Sanger测序、MLPA和多态性连锁性分析可以提高PKU患者的诊断率,并准确进行产前诊断。

Objective To explore the characteristics of phenylalanine hydroxylase(PAH)gene variants and prenatal diagnosis for 43 Chinese pedigrees affected with Phenylketonuria(PKU).Methods Forty three PKU pedigrees diagnosed at the First Affiliated Hospital of Zhengzhou University between 2019 and 2021 were selected as the study subjects.Variants of the PAH gene of the probands were screened by high-throughput sequencing,and candidate variants were verified by Sanger sequencing.Negative cases were further analyzed by multiplex ligation-dependent probe amplification(MLPA)to detect large fragment deletions and duplications of the PAH gene.For 43 women undergoing subsequent pregnancy,Sanger sequencing,MLPA,combined with short tandem repeats(STR)sequence-based linkage analysis,were carried out for prenatal diagnosis.Results Among the 86 alleles carried by the 43 probands,78 nucleotide variants(90.70%)and 3 large deletions(3.49%)were found based on high-throughput sequencing and MLPA.The 81 mutant alleles had included 21 missense variants,5 splice site variants,4 nonsense variants,2 microdeletions,1 insertional variant and 2 large fragment deletions.Relatively common variants have included p.Arg243Gln(23.26%),p.Arg111Ter(8.14%),EX6-96A>G(6.98%),p.Val399Val(5.81%)and p.Arg413Pro(4.65%).Most of the variants were located in exons 7,11,3,6 and 12.For the 43 families undergoing prenatal diagnosis,9 fetuses(20.45%)were diagnosed with PKU,20(45.45%)were heterozygous carriers,and 15(34.09%)did not carry the same pathogenic allele as the proband.All neonates were followed up till 6 months old,and the accuracy of prenatal diagnosis was 100%.Conclusion The combination of high-throughput sequencing,Sanger sequencing,MLPA and linkage analysis can increase the diagnostic rate of PKU and attain accurate prenatal diagnosis.