Genetic testing and prenatal diagnosis of two families with phenylketonuria caused by PAH gene deep intronic heterozygous mutation
Objective To perform whole exome sequencing in two phenylketonuria(PKU)families carrying a single heterozygous mutation,to explore the PAH gene mutation,and to make prenatal diagnosis.Methods In March 2023,two PKU families underwent genetic testing and prenatal diagnosis in the First Affiliated Hospital of Zhengzhou University.Family 1:the proband,male,7 years old,had developmental delay after birth and severe jaundice in the neonatal period;brain CT showed demyelination of white matter,and the serum phenylalanine was elevated;the genetic test in the foreign hospital before admission showed that the proband carried the c.1068C＞A(p.Y356X)of PAH gene.Family 2:the proband,female,8 years old,was diagnosed with PKU in neonatal screening after birth,and the genetic test in the foreign hospital found that the proband carried the c.611A＞G(p.Y204C)of PAH gene.The peripheral venous blood was collected from two probands and their parents,the pregnant woman underwent amniocentesis to extract amniotic fluid at 18 weeks of gestation,and DNA was extracted for whole exome sequencing.The related literature was searched in PubMed database,and newly discovered pathogenic mutation loci were included in the target region for probe design and capture sequencing.The imbrication multilayer probe design scheme was adopted.The whole exome sequencing results were annotated and analyzed,and the candidate pathogenic mutation loci were screened to refer to the human genome reference sequence GRCh37.The candidate loci mutation database inclusion and pathogenicity were searched in the dbSNP database,the population frequency of the mutation was queried in the gnomAD database.According to the classification standards and guidelines of genetic variation of the American Society of Medical Genetics and Genomics,the pathogenicity of the variation was graded and determined.The carrier status of candidate mutations in the parents of the probands were verified by PCR amplification and Sanger sequencing,and the prenatal diagnosis of candidate loci was performed on the fetus.Results(1)The proband in family 1 was found compound heterozygous mutations of PAH gene c.1068C＞A(p.Y356X)and c.1065+241C＞A,and the proband in family 2 was found compound heterozygous mutations of PAH gene c.611A＞G(p.Y204C)and c.1065+241C＞A.(2)The c.611A＞G(p.Y204C)and c.1068C＞A(p.Y356X)of PAH gene were the pathogenic hotspot mutation of PKU.The PAH gene c.1065+241C＞A was not found in the dbSNP database and did not belong to the polymorphism locus,its frequency in the population was extremely low,and the gnomAD database did not query the frequency of this mutation in the population.According to the American Society of Medical Genetics and Genomics guidelines,the PAH gene c.1065+241C＞A was determined to be pathogenic(PM3_Very Strong,PP4_Moderate,PM2_Supporting).(3)The results of Sanger verification of the probands were consistent with the whole exome sequencing results,and their parents were the carriers of the related pathogenic mutation genes.The fetus in family 1 carried the c.1065+241C＞A of PAH gene heterozygous mutation,and the fetus in family 2 carried the c.611A＞G(p.Y204C)of PAH gene heterozygous mutation.Conclusion To design multi-layer probe for the target region and to update the target region of whole exome sequencing timely according to the literature contribute to the detection of pathogenic mutation in deep intron region of PAH gene by whole exome sequencing.
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