Molecular diagnosis of Phenylketonuria in 157 Families and Prenatal Diagnosis of Phenylketonuria

Background Phenylketonuria (PKU) is a genetic metabolic disease with a relatively higher incidence, but only a few studies about the prenatal diagnosis of PKU have been reported so far in China. The aim of this study was to characterize the spectrum of mutations in PAH gene in PKU probands and the prenatal diagnosis of PKU in north China. Methods A total of 157 families in which PKU patients had been diagnosed were included in the study. The 13 exons and their flanking sequences of PAH gene were amplified by PCR and sequenced in the probands. If none or only one mutant allele was found in the probands, the sample was subjected to MLPA for large deletions/duplication detection in PAH gene. Prenatal diagnosis was performed for pregnant women in these families. Results Pathogenic mutation in PAH was found in 2 alleles in 148 probands and in one allele in 7 probands, and the mutation was not detected in 2 probands. There were 289 point mutants, 10 frame-shift mutations and 4 large deletions with a total of 80 kinds of mutations. The most prevalent mutations were R243Q (17.2%), EX6-96A>G (8.6%) and V399V (8.0%). We also found a novel mutation of 163_164insATAT. Prenatal diagnosis of 95 families found 21 healthy fetus (20.4%), 52 carriers (50.5%) and 30 patients (29.1%), and the accuracy of prenatal diagnosis was confirmed after birth of the fetuses. Conclusion We present here a spectrum of mutations in PAH gene in PKU patients in north China. Prenatal diagnosis for PKU is useful for PKU families to prevent birth of another PKU case. amniotic fluid cells or chorionic villi cells the only way to get accurate results. Here we present a spectrum of mutations in PAH gene in PKU patients in north China. No hotspot mutation was found. The mutations were frequently detected in exon 7. Prenatal diagnosis is an essential way to prevent the birth of PKU cases, but related information is rare in China. We show the results of prenatal diagnosis for PKU in north China.


Abstract
Background Phenylketonuria (PKU) is a genetic metabolic disease with a relatively higher incidence, but only a few studies about the prenatal diagnosis of PKU have been reported so far in China. The aim of this study was to characterize the spectrum of mutations in PAH gene in PKU probands and the prenatal diagnosis of PKU in north China.
Methods A total of 157 families in which PKU patients had been diagnosed were included in the study. The 13 exons and their flanking sequences of PAH gene were amplified by PCR and sequenced in the probands. If none or only one mutant allele was found in the probands, the sample was subjected to MLPA for large deletions/duplication detection in PAH gene. Prenatal diagnosis was performed for pregnant women in these families.
Results Pathogenic mutation in PAH was found in 2 alleles in 148 probands and in one allele in 7 probands, and the mutation was not detected in 2 probands. There were 289 point mutants, 10 frame-shift mutations and 4 large deletions with a total of 80 kinds of mutations. The most prevalent mutations were R243Q (17.2%), EX6-96A>G (8.6%) and V399V (8.0%). We also found a novel mutation of 163_164insATAT. Prenatal diagnosis of 95 families found 21 healthy fetus (20.4%), 52 carriers (50.5%) and 30 patients (29.1%), and the accuracy of prenatal diagnosis was confirmed after birth of the fetuses.
Conclusion We present here a spectrum of mutations in PAH gene in PKU patients in north China. Prenatal diagnosis for PKU is useful for PKU families to prevent birth of another PKU case.
Background PKU is an autosomal recessive genetic disease caused by mutations in PAH gene coding phenylalanine hydroxylase, a key enzyme in the metabolism of phenylalanine. Early dietary therapy improves most of the neuropsychological disorders, but is hard to be maintained for a long period of time [1]. To date more than 700 mutations in PAH gene, including missense, splicing, nonsense, insertion and deletion mutations, have been identified. The distribution of the mutations are heterogeneic in ethic groups. Genetic testing and prenatal diagnosis can prevent PAH families from transmitting PKU to their progeny. However, only a few reports about the prenatal diagnosis of PKU from north of subjected to the same PCR-direct sequencing and/or MLPA procedures as described before.
In addition, PCR amplification of the 5 STR markers nearby PAH and separation of the PCR products by ABI 3130XL Genetic Analyzer were also included to exclude false results due to maternal blood contamination.

Spectrum of mutations in PAH gene
Among the 157 probands, pathogenic mutation in PAH was found in 2 alleles in 148 probands and in one allele in 7 probands, and the mutation was not detected in 2 probands. There are totally 303 mutations including 289 point mutants, 10 frame shift mutations and 4 large deletions. Table 1 lists the spectrum of the 303 mutations we detected, in which the mutation of 163_164insATAT is a novel mutation not stored in the 4 databases. The most prevalent mutations are R243Q, EX6-96A>G and V399V accounting for 17.2%, 8.6% and 8.0% of the mutant alleles, respectively. The highest prevalence of exon and its flanking sequences where mutations locate in PAH gene is exon 7, followed by exon 11, exon 12, exon 6, exon 3, exon 5 and exon 2 ( Figure   1). 3 carriers' spouses accepted prenatal counseling and PAH sequencing. One was found a pathogenic mutant (c.1068C>A, p.Y356X), which is shown in Figure 2. Advice of PKU prenatal diagnosis was given to them. Neonatal screening for PKU are useful for the early treatment of PKU. During the period from 1999 to 2009 in Beijing, a total of 1,166,218 newborns were screened for PKU, and a total of 145 newborns were confirmed to have PKU with the incidence of 1/8,064 [2] In this cohort of PKU probands, the 3 prevalent mutations of R243Q, EX6-96A>G and V399V accounted for 33.8% of the mutations, similar to the reports from other regions in China [3][4][5] and from Korea [6]. In contrast in Japan, the most prevalent mutation is R413P [7]. The mutations of R243Q and EX6-96A>G have been reported in Chinese PKU cases; the R243Q mutation derived an abnormal PAH that only has <10% of normal PAH activity in a eukaryotic cell expression system [8]. The novel mutation of 163_164insATAT we found causes frame shift and premature termination of the polypeptide chain and is definitely a pathogenic mutation.
Three pathogenic mutations in a proband were detected in two of our probands. One carried c.59A>C and c.60G>C from mother and c.721C>T from father, and the other carried c.194T>C and c.510T>A from mother and c.739G>C from father. The c.59A>C and c.60G>C mutations in one allele have been reported previously [9,10]. The genotype of 3 pathogenic mutations in a patient is not uncommon, for an example, a total 35 PKU patients with this genotype were detected from 796 PKU patients by next-generation sequencing [3].
Thirteen probands not carrying two mutations in two alleles were subjected to MLPA, and two large deletions, exon 1 and its upstream region in 2 probands and exon 4/exon 5 in other 2 probands were identified in these probands. Chen et al. reported that 3 large deletion alleles (exon 1 and its upstream region, exon 4/exon 5, and exon 5) were disclosed in 17 PKU families without having two pathogenic mutations [11]. Yan et al.
identified 24 (51.1%) large deletion/duplication alleles by MLPA in 22 of the 43 PKU patients with none or only one mutant allele, in which Ex1del3758 was detected in 10 cases and Ex4_5del in 4 cases; the authors considered that Ex1del3758 large deletion is a hotspot in Chinese PKU patients, similar to our findings [12].   Ratio of the mutants in this cohort of PKU patients