Molecular Diagnosis of Phenylketonuria in 157 Chinese Families and the Results of Prenatal Diagnosis in These Families
Phenylketonuria (PKU) is an autosomal recessive genetic disorder caused by pathogenic variants in the phenylalanine hydroxylase (PAH) gene, which encodes the enzyme phenylalanine hydroxylase. This enzyme is crucial for the metabolism of phenylalanine. Early intervention with a low-phenylalanine diet can mitigate most neuropsychological disorders associated with PKU, but maintaining this diet long-term is challenging. To date, 1184 variants in the PAH gene have been identified, including missense, splicing, nonsense, insertion, and deletion variants. The distribution of these variants varies significantly across different ethnic groups. Genetic testing and prenatal diagnosis are effective strategies for preventing PKU families from transmitting pathogenic PAH alleles to their offspring. However, there are few reports on the prenatal diagnosis of PKU in northern China. This study summarizes the results of variant detection in 157 probands and their parents, as well as prenatal diagnosis in 103 fetuses from 95 PKU families.
The study was conducted at Peking University First Hospital and was approved by the hospital’s Research Ethics Committee. Informed consent was obtained from the probands or their guardians and family members. The study included 157 probands and their parents, with the probands’ ages ranging from 1 month to 17 years and a male-to-female ratio of 1:0.92. Most participants were from northern China. All probands had elevated plasma phenylalanine levels (>2 mg/dL), and tetrahydrobiopterin (BH4) deficiency was excluded via a BH4-loading test.
Genomic DNA was extracted from peripheral lymphocytes of the probands and their parents using the QuickGene DNA Whole Blood Kit. The 13 exons and their flanking sequences of the PAH gene were amplified by polymerase chain reaction (PCR). The PCR products were purified and sequenced using an ABI 3130XL DNA Analyzer. Sequencing results were compared with the PAH gene transcript (NM_000277) and its genomic sequence (GRCh38/hg38). Detected variants were further searched in the PAHvdb, ClinVar, and HGMD databases. Novel variants were evaluated using predictive tools such as SIFT, PROVEAN, and PolyPhen2 to assess their pathogenic effects. Variants found in the probands were also examined in their parents. For probands without pathogenic variants or only one pathogenic variant, DNA samples were subjected to multiplex ligation-dependent probe amplification (MLPA) to detect large insertions, deletions, or duplications in the PAH gene.
In the 157 families, prenatal diagnosis was performed for 95 pregnant mothers, with eight mothers undergoing prenatal diagnosis twice. DNA samples were extracted from chorionic villi, amniotic fluid, or abortion tissues and subjected to the same PCR-direct sequencing and/or MLPA procedures. Additionally, six short tandem repeat (STR) markers near the PAH gene were amplified by PCR and separated on an ABI 3130XL Genetic Analyzer. Genotypes of the six STR markers were compared between the mother and the fetus to exclude false results due to maternal blood contamination.
Among the 157 probands, 145 had two pathogenic alleles, including one proband with two pathogenic variants in one allele and one pathogenic variant in another allele. Ten probands had only one pathogenic allele, and no pathogenic alleles were found in two probands. A total of 80 kinds of pathogenic variants were identified, including 71 point nucleotide substitutions, seven small insertions/deletions, and two large deletions. These variants resulted in 52 missense variants in 159 alleles, 13 splicing variants in 82 alleles, seven premature terminations in 46 alleles, five frame shifts in six alleles, two large deletions in four alleles, and one amino acid deletion in four alleles. The most prevalent variants were R243Q, the splicing variant due to c.611A>G, and the splicing variant due to c.1197A>T, accounting for 17.9%, 9.0%, and 8.3% of the variant alleles, respectively. The highest frequency of pathogenic variants (excluding large deletions/duplications) was found in exon 7, followed by exon 11, exon 6, exon 12, and exon 3.
MLPA was performed in 13 probands, revealing one large deletion of exon 1 and its upstream region in two probands and one large deletion of exon 4/exon 5 in the other two probands.
Prenatal diagnosis of PKU was performed in 103 fetuses from 95 families. Thirty fetuses (29.1%) were identified as having PKU (carrying two pathogenic variants), and all families chose abortion, with the pathogenic variants confirmed by testing the abortion tissues. Fifty-two fetuses (50.5%) were identified as PKU carriers (carrying one pathogenic variant), and 21 fetuses (20.4%) were identified as normal (no pathogenic variant found). Most carrier and normal fetuses were born, and their PAH gene genotypes were confirmed by testing peripheral blood after birth.
The study highlighted the importance of molecular diagnosis and prenatal diagnosis for PKU in a family. After molecular diagnosis of PKU in the proband and her parents, the proband’s aunt and uncle were also at risk of having a PKU baby. Molecular diagnosis revealed that both the aunt and uncle were carriers of PKU, and genetic counseling was provided to prevent the birth of another PKU case.
The PAH gene is located on human chromosome 12q23.2 and consists of 13 exons encoding a polypeptide of 452 amino acid residues. Mutant phenylalanine hydroxylase blocks the metabolism of phenylalanine to tyrosine, leading to the accumulation of phenylalanine. This accumulation alters cerebral myelination and protein synthesis and reduces levels of serotonin, dopamine, and noradrenaline in the brain, resulting in severe mental retardation and neurobehavioral abnormalities. Neonatal screening for PKU is useful for early treatment, but it does not prevent the birth of affected infants. From 2014 to 2017, 176,340 newborns in the Haidian District of Beijing were screened for PKU, with 33 confirmed cases, indicating an incidence of 1/5344.
In this cohort of PKU probands, the most prevalent variants were R243Q, c.611A>G, and c.1197A>T, accounting for 35.2% of the variants, similar to reports from other regions in China and Korea. In contrast, the most prevalent variant in Japan is R413P. The R243Q variant results in a mutant phenylalanine hydroxylase with less than 10% normal activity in a eukaryotic cell expression system. The novel variant 163_164insATAT causes a frameshift and premature termination of the polypeptide, making it a definite pathogenic variant.
Two large deletions, exon 1 and its upstream region in two cases and exon 4/exon 5 in two other cases, were identified by MLPA in 13 probands where two variant alleles were not found by PCR-Sanger sequencing. Previous studies have reported similar large deletions in Chinese PKU patients, indicating that these deletions may be relatively common in this population.
No variant hotspot in the PAH gene was found in this cohort. Variants were distributed across all 13 exons, with the highest frequency in exon 7, followed by exon 11, exon 6, exon 12, and exon 3. Previous studies have also found that most variants concentrate in exon 7, followed by exons 6, 11, and 3, suggesting that these exons should be prioritized for variant screening.
Next-generation sequencing technology has become a powerful tool for diagnosing genetic diseases. However, in this cohort, regular PCR-Sanger sequencing and MLPA detected 95.6% of pathogenic alleles in the PAH gene, indicating that classic methods remain effective for the genetic diagnosis of PKU.
In families where both parents carry a pathogenic variant in the PAH gene, there is a 25% chance of having a PKU baby. Prenatal diagnosis is the only way for these families to prevent the birth of an affected infant. Genotyping of several STR markers is essential to prevent misdiagnosis due to maternal blood contamination in fetal samples. The six highly polymorphic STR markers used in this study were located around the PAH gene, ensuring accurate results even in cases of maternal blood contamination.
Prenatal diagnosis using chorionic villi is typically performed at 11 to 13 weeks of gestation, allowing for earlier molecular diagnosis and less physical and psychological damage to the pregnant woman if the fetus is affected and the pregnancy is terminated. However, chorionic villi sampling carries a relatively high risk of abortion, and placental chimerism may affect the accuracy of the results. In contrast, amniocentesis is usually performed at 16 to 23 weeks of gestation and is relatively safe, but the later the molecular diagnosis, the higher the risk of abortion if the fetus is affected.
In conclusion, this study presents the spectrum of variants in the PAH gene in PKU patients in northern China. No variant hotspot was found, with variants frequently detected in exon 7. Prenatal diagnosis is the only way to prevent the birth of an infant with PKU in families where both parents are carriers of pathogenic variants.
doi.org/10.1097/CM9.0000000000001469
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