This study aimed to determine the incidence and disease spectrum of inherited metabolic diseases (IMDs) among newborns in Huai'an City, China. Expanded newborn screening for IMDs using tandem mass spectrometry (MS/MS) enables the simultaneous analysis of more than 40 metabolites and the identification of approximately 50 types of IMDs. Next-generation sequencing (NGS), targeting hundreds of IMD-associated genes, was subsequently performed for genetic analysis of patients identified through screening. Between June 2018 and December 2024, in total, 161,966 newborns in Huai'an were screened using MS/MS. Ultimately, 57 patients were diagnosed with IMDs based on plasma amino acid and acylcarnitine profiling, urinary organic acid analysis, and molecular genetic testing, performed via NGS. Data were analyzed using descriptive statistics. Fifty-seven cases of IMDs were diagnosed, corresponding to an overall incidence rate of 1 in 2,842. Among these, 28 cases involved amino acid metabolism disorders (1 in 5,785), 17 cases of organic acid metabolism disorders (1 in 9,527), and 12 cases of fatty acid oxidation disorders (1 in 13,497). The three most common IMDs were phenylalanine hydroxylase deficiency (1 in 8,098), primary carnitine deficiency (1 in 23,138), and methylmalonic acidemia (1 in 32,393). Genetic testing revealed variants in all 57 patients, with 75 variants identified across 17 IMD-associated genes. Recurrent variants were observed in five IMDs, including PAH gene variants c.728G>A, c.611A>G, and c.721C>T for phenylketonuria, PAH c.158G>A, c.721C>T, and c.728G>A for mild hyperphenylalaninemia, SLC22A5 c.1400C>G for primary carnitine deficiency, MMACHC c.609G>A, c.567dup and c.482G>A for methylmalonic acidemia, ACADS c.1055C>T, and c.1130C>T for short-chain acyl-CoA dehydrogenase deficiency, and ACADSB c.923G>A for 2-methylbutyrylglycinuria. All these recurrent variants were reported as pathogenic or likely pathogenic, except PAH c.158G>A, which was classified as a variant of uncertain significance. The majority of IMD patients in Huai'an carried pathogenic or potentially pathogenic variants identified through expanded newborn screening. Although MS/MS newborn screening followed by NGS confirmation cannot prevent the occurrence of IMDs, timely diagnosis combined with appropriate treatment and management can effectively prevent morbidity, reduce mortality, and support long-term symptom control. Overall, this study demonstrates that MS/MS-based newborn screening combined with molecular diagnosis was highly effective for the study early detection and management of IMDs in the Huai'an population.

To investigate the molecular epidemiology of children with phenylketonuria (PKU) in Gansu, China, providing foundational data for intervention strategies. A retrospective analysis was conducted on 1 159 PKU families who attended Gansu Provincial Maternity and Child Care Hospital from January 2012 to December 2024. Sanger sequencing, multiplex ligation-dependent probe amplification, whole exome sequencing, and deep intronic variant analysis were used to analyze the PAH gene. For the 1 159 children with PKU, 2 295 variants were identified in 2 318 alleles, resulting in a detection rate of 99.01%. The detection rates were 100% (914/914) in 457 classic PKU families, 99.45% (907/912) in 456 mild PKU families, and 96.34% (474/492) in 246 mild hyperphenylalaninemia families. The 2 295 variants detected comprised 208 distinct mutation types, among which c.728G>A (14.95%, 343/2 295) had the highest frequency, followed by c.611A>G (4.88%, 112/2 295) and c.721C>T (4.79%, 110/2 295). The cumulative frequency of the top 23 hotspot variants reached 70.28% (1 613/2 295), and most variant alleles were detected in exon 7 (29.19%, 670/2 295). Deep intronic variant analysis of the PAH gene can improve the genetic diagnostic rate of PKU. The development of targeted detection kits for PAH hotspot variants may enable precision screening programs and enhance preventive strategies for PKU. 目的: 对甘肃地区苯丙酮尿症（phenylketonuria, PKU）患儿进行分子流行病学调查，以期为PKU的干预策略提供基础性数据。方法: 回顾性收集2012年1月—2024年12月甘肃省妇幼保健院就诊的1 159个PKU患儿家系，通过Sanger测序、多重连接依赖探针扩增技术、全外显子组测序以及深部内含子变异分析对PAH基因进行分析。结果: 1 159例PKU患儿中，患儿的2 318个等位基因中检测到2 295个变异，检出率为99.01%。其中457个经典型PKU患儿家系检出率为100%（914/914），456个轻度PKU患儿家系检出率为99.45%（907/912），246个轻度高苯丙氨酸血症患儿家系检出率为96.34%（474/492）。检测到的2 295个等位基因变异分属于208种变异，其中出现频率最高的是c.728G>A（14.95%，343/2 295），其次为c.611A>G（4.88%，112/2 295），随后为c.721C>T（4.79%，110/2 295）。前23位热点变异的累计频率达70.28%（1 613/2 295）。外显子7上检测到的变异等位最多（29.19%，670/2 295）。结论: 对PAH基因进行深部内含子变异分析，可以进一步提高PKU基因诊断率。针对PAH基因热点变异位点与区域设计研发相应检测试剂盒可能有助于PKU的精准筛查与防控。.

Phenylketonuria (PKU) is an autosomal recessive disorder of phenylalanine metabolism, in which especially high phenylalanine concentrations cause brain dysfunction. If untreated, this brain dysfunction results in severe intellectual disability, epilepsy, and behavioural problems. We aimed to investigate demographic, clinical, biochemical, and molecular genetic data in patients with phenylalanine metabolism disorder. This study included 99 predominantly Turkish patients diagnosed with phenylalanine metabolism disorder, primarily referred through newborn screening programs. These patients were evaluated at a single center over a 9-year period, from 2013 to 2021. Demographic, clinical, molecular and laboratory data were collected retrospectively. Among the 99 patients, 93 (93.9%) had hyperphenylalaninemia-phenylketonuria, 2 (2.0%) had tetrahydrobiopterin metabolism disorders [one due to 6-pyruvoyl-tetrahydropterin synthase (PTPS) deficiency and the other due to dihydropteridine reductase (DHPR) deficiency], 3 (3.0%) had maternal PKU syndrome (one of whom also had mild phenylketonuria), and 1 (1.0%) had transient hyperphenylalaninemia. The majority of patients belonged to the mild hyperphenylalaninemia-not requiring treatment group. A total of 33 different alleles and 40 genotypes (59.6% compound heterozygous) were identified in the PAH gene, with missense variants accounting for the largest proportion (72.7%). The most frequent PAH gene variants were c.898G > T p.(Ala300Ser) (14.9%), c.1066-11G > A (8.5%), and c.1208C > T p.(Ala403Val) (8.5%), while the most common genotypes were c.898G > T p.(Ala300Ser)/c.898G > T p.(Ala300Ser) (6.4%) and c.898G > T p.(Ala300Ser) /c.1066-11G > A (6.4%), respectively. Among patients with mild hyperphenylalaninemia-not requiring treatment, the predominant genotypes were c.898G > T p.(Ala300Ser)/c.898G > T p.(Ala300Ser) (11.1%), c.898G > T p.(Ala300Ser)/c.1066-11G > A (11.1%), and c.1208C > T p.(Ala403Val)/c.1208C > T p.(Ala403Val) (7.4%), whereas c.842C > T p.(Pro281Leu)/c.842C > T p.(Pro281Leu) (33.3%) was frequently observed in classic PKU patients. The national newborn screening program has significantly improved the prognosis and quality of life for patients through early diagnosis and timely treatment. While the prevalence of hyperphenylalaninemia-phenylketonuria remains high in Turkey, the higher frequency of the hyperphenylalaninemia-not requiring treatment group, compared to European and Asian countries, is considered a favorable outcome. Additionally, the PAH genotype is identified as the primary determinant of the PKU phenotype.

Achieving and maintaining metabolic control is critical for children with phenylalanine hydroxylase (PAH) deficiency. This retrospective longitudinal cohort study investigated metabolic control and monitoring frequency of children with PAH deficiency (≤ 12 years) treated at one of 12 pediatric metabolic centres across Canada. We abstracted data from medical charts and analyzed outcomes by age and diagnostic classification, using mixed effects regression. Of 215 children included in the study, 43% had a chart diagnosis of classic phenylketonuria (PKU); the remainder had a diagnosis of mild PKU or mild hyperphenylalaninemia (grouped as "less severe PAH deficiency"). During the first month of life, blood phenylalanine levels of children with classic PKU reached the target therapeutic range of 120-360 μmol/L at a median age of 15 days, but 74.3% and 32.9% had ≥ 1 and ≥ 3 values below 120 μmol/L, respectively. From age > 1 month to 12 years, mean blood phenylalanine values were 260.6 and 236.7 μmol/L for children with classic PKU and less severe PAH deficiency, respectively, with a trend of increased blood phenylalanine levels with increasing age (p < 0.001). Fewer children with classic PKU (37.2%) versus less severe PAH deficiency (77.9%) had > 60% of values in the therapeutic range, indicating less optimal metabolic control. Frequency of blood phenylalanine testing and communication with metabolic centres decreased with age. Our findings suggest a need to better understand the reasons for blood phenylalanine variability across child age and disease severity in order to inform supports for children with PAH deficiency and their caregivers to maintain metabolic control.