Cell-free DNA barcode-enabled single-molecule test for non-invasive prenatal diagnosis of phenylketonuria: an analysis of four cases / 中华围产医学杂志

ABSTRACT

Objective:To analyze the applicability and feasibility of a cell-free DNA barcode-enabled single-molecule test (cfBEST) in non-invasive prenatal diagnosis of phenylketonuria.Methods:This study recruited four pregnant women who were prenatally diagnosed as heterozygous carriers of hot spot mutations in the PAH gene from pedigrees with phenylketonuria at the First Affiliated Hospital of Zhengzhou University from July to September 2019. The frequency of mutations in maternal plasma cell-free DNA and the fetuses' genotypes were analyzed by cfBEST. Nested polymerase chain reaction primers were designed to amplify the mutation sites in each pedigree. The results of cfBEST were compared with those of invasive prenatal diagnosis. Descriptive analysis was used for data analysis. Results:In pedigree 1, the frequency of c.603T>G and c.842+2T>A mutations in maternal plasma cell-free DNA were 48.40% (291/601) and 9.70% (61/628), which was detected by cfBEST. The fetus was diagnosed with phenylketonuria with two heterozygous mutations. In pedigree 2, the frequency of c.1238G>C and c.842+2T>A mutations in maternal plasma cell-free DNA was 43.70% (786/1 798) and 0% (0/1 550), respectively. Both mutations were wild-type, and the fetus was neither phenylketonuria nor a carrier. In pedigree 3, the frequency of c.1045T>G and c.728G>A mutations in maternal plasma cell-free DNA was 44.00% (930/2 112) and 0% (0/705), respectively, suggesting that both mutations in the fetus were wild-type, and the fetus was neither phenylketonuria nor a carrier. In pedigree 4, the frequency of c.755G>A and c.728G>A mutations were 45.40% (743/1 637) and 4.50% (28/849), respectively, which indicated that the former was wild-type, and the latter was heterozygous; namely the fetus was a carrier of phenylketonuria. The results of cfBEST were consistent with those of invasive prenatal diagnosis. Three pedigrees (Pedigree 2, 3 and 4) continued the pregnancy to full-term, and the phenylalanine levels in the neonates were all below 120 μmol/L. No abnormalities were reported in those three infants during follow-ups at one, three, and six months after birth.Conclusions:The cfBEST could be used for non-invasive prenatal diagnosis of phenylketonuria caused by PAH gene mutation, but further studies with a larger sample size are needed.