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Objective To explore the gene mutation frequency and characteristics of phenylalanine hydroxylase (PAH) in PAH deficiency children in some regions of Chongqing in order to provide scientific reference for the diagnosis and treatment of the disease. Methods A retrospective analysis was conducted on 45 children with PAH deficiency who were diagnosed in our hospital from January 1, 2014 to October 25, 2020. They was assigned into classic phenylketonuria (PKU), mild PKU and mild hyperphenylalaninemia (HPA). We analyzed the mutations of PAH gene by second-generation high-throughput sequencing and multiplex ligase probe dependent amplification (MLPA) technique, and the results were verified by the detection of corresponding mutation sites of their parents by Sanger sequencing. Results ① There were 2 mutation sites in 43 cases, including 40 cases of compound heterozygous mutation and 3 cases of homozygous mutation. All mutations were detected in the corresponding mutation sites of their parents. The left 2 cases had heterozygous mutations, and had only 1 mutation site. ② There were 34 types of mutations detected in 45 patients with PAH deficiency. Missense mutation was the main mutation type (52.9%). The mutation frequency of c.728G>A was the highest (15.9%, 14/88), followed by c.158G>A (11.4%, 10/ 88), c.1197A>T (9.1%, 8/88), and c.721C > T (9.1%, 8/88). The region of high frequency mutation was in exon 7, which contained 4 mutations and 26 mutations sites (29.5%). ③ There were 11 PAH gene mutations in 13 patients with classic PKU. The mutation frequency of c.728G> A (8/25, 32%) was the highest. There were 9 PAH gene mutations in 8 patients with mild PKU. The mutation frequency of c.728G>A (3/15, 20%) was the highest. There were 24 PAH gene mutations in 24 patients with mild HPA. The mutation frequency of c.158G>A (10/48, 20.8%). Conclusion The mutations in children with PAH deficiency in some regions of Chongqing are mainly compound heterozygosity. Missense mutation is the main type, with obvious hot spot mutations (c.728G>A, c.158G>A, c.721C > T and c.1197A>T).

Objectives: To analyze the results of neonatal screening for congenital hypothyroidism (CH) and hyperphenylalaninemia (HPA) in Zhejiang province from 1999 to 2022., Methods: A total of 11 922 318 newborns were screened from September 1999 and December 2022 in Zhejiang province. The blood thyroid stimulating hormone (TSH) levels were measured by a fluorescence method and blood phenylalanine (Phe) levels were measured by fluorescence method or tandem mass spectrometry. TSH≥9 μIU/mL was considered positive for CH, while Phe>120 μmol/L and/or Phe/Tyr ratio>2.0 were considered positive for HPA. The positive newborns in screening were recalled, and the gene variations were detected by high-throughput sequencing and MassARRAY tests., Results: The overall neonatal screening rate during 1999-2022 was 89.41% (11 922 318/13 333 929) and the screening rate was increased from 6.46% in 1999 to 100.0% in 2022. A total of 8924 cases of CH were diagnosed among screened newborns with an incidence rate of 1/1336. A total of 563 cases of HPA were diagnosed, including 508 cases of classic phenylketonuria (cPKU) and 55 cases of tetrahydrobiopterin deficiency (BH4D), with an incidence rate of 1/21 176. Ninety-seven out of 8924 cases of CH underwent genetic analysis. Gene mutations were detected in 9 CH related genes, the highest frequency mutations were found in DUOX2 gene (69.0%) with c.3329G>A (p.R1110Q) (18.2%) and c.1588A>T (p.K530X) (17.3%) as the hotspot mutations. There were 81 PAH gene variants detected in a total of 250 cases of cPKU, and c728G>A (p.R243Q) (24.4%), c.721C>T (p.R241C) (15.0%) were the hotspot mutations. Meanwhile 7 novel variants in PAH gene were detected: c.107C>A (p.S36*), c.137G>T (p.G46V), c.148A>G(p.K50E), c.285C>T (p.I95I), c.843-10delTTCC, exon4-7del and c.1066-2A>G. There were 12 PTS gene variants detected in 36 cases of BH4D, and c.259C>T (p.P87S) (31.9%) was the hotspot mutation., Conclusions: The incident of CH has increased from 1999 to 2022 in Zhejiang province, and it is higher than that of national and global levels; while the incidence of HPA is similar to the national average. DUOX2 gene variation is the most common in CH patients; c.728G>A (p.R243Q) is the hotspot mutation in cPKU patients, while c.259C>T (p.P87S) is the hotspot mutation in BH4D patients.