[Analysis of pathogenicity and genotype-phenotype correlation of the c.158G>A variant of phenylalanine hydroxylase gene].

OBJECTIVE: To explore the pathogenicity and genotype-phenotype correlation of the c.158G>A variant of phenylalanine hydroxylase (PAH) gene among patients with PAH deficiency. METHODS: Thirty seven children diagnosed with PAH deficiency at the Obstetrics and Gynecology Hospital Affiliated to Nanjing Medical University between July 2016 and June 2021 were selected as the study subjects. Clinical data and results of genetic testing were retrospectively analyzed. RESULTS: Among the 37 patients, mild hyperphenylalaninemia (HPA) was observed in 34 cases, two PAH variants (including c.158G>A), which formed a compound heterozygous mutation genotype, were detected in 33 patients, and the remainder one was found to harbor three PAH variants, including homozygous c.158G>A variants and a heterozygous c.842+2T>A variant. Classical phenylketonuria (PKU) was observed in 3 patients, and three PAH variants were detected in each of them, including two with c.[158G>A,842+2T>A]/c.728G>A and c.[158G>A,842+2T>A]/c.611A>G, respectively, and one with c.[158G>A, c.722G>A]/c.728G>A. The c.158G>A variant has a minimal influence on the PAH activity and is associated with a mild HPA phenotype. The variant should thereby be classified as likely benign. CONCLUSION: When the c.158G>A variant and other pathogenic variants are arranged in cis position, the ultimate phenotype will be determined by the pathogenicity of other variants.

[Genetic analysis of eighteen patients from Gansu Province with Tetrahydrobiopterin deficiency].

OBJECTIVE: To explore the genetic basis of eighteen patients with Tetrahydrobiopterin deficiency (BH4D) from Gansu Province. METHODS: Eighteen patients diagnosed with BH4D at Gansu Provincial Maternal and Child Health Care Hospital from January 2018 to December 2021 were selected as the study subjects. Whole exome sequencing was carried out, and candidate variants were verified by Sanger sequencing. RESULTS: All of the thirty-six alleles of the eighteen patients were successfully determined by molecular genetic testing. Sixteen patients were found to harbor variants of the PTS gene, and two had harbored variants of the QDPR gene. Ten variants were detected in the PTS gene, with the most common ones being c.259C>T (34.38%) and c.286G>A (15.63%). Based on the guidelines from the American College of Medical Genetics and Genomics (ACMG), the c.259C>T was classified as a pathogenic variant, whilst the c.286G>A, c.166G>A, c.200C>T, c.272A>G, c.402A>C, c.421G>T, c.84_291A>G and c.317C>T were classified as likely pathogenic variants. A novel c.289_290insCTT variant was classified as likely pathogenic (PM1+PM2_Supporting+PM3+PP3+PP4). The two variants (c.478C>T and c.665C>T) detected in the QDPR gene were both classified as variants of uncertain significance (PM1+PM2_Supporting+PP3+PP4). CONCLUSION: Genetic testing has clarified the pathogenic variants in these BH4D patients, which has enabled timely and accurate clinical intervention and treatment, and provided a reference for genetic counseling and reproductive guidance for their families.

An unusual case of trisomy 8 mosaicism complicated by coexistence of phenylketonuria.

Trisomy 8 syndrome, also known as " Warkany syndrome type 2 ", was first reported in 1971. Complete trisomy 8 are mostly aborted spontaneouslyinthe first trimester. Trisomy 8 mosaicism (T8M), predominated in the current cases reported. Itisahighlyheterogeneous Chromosome disorder. We know little about its effects on fertility. In this case, a patient with T8M combined with phenylketonuria was diagnosed. She's mentally retarded. After evaluating the anatomy and function of the reproductive system, the patient conceived through preimplantationgenetictesting-intracytoplasmicsperminjection-embryotransfer (PGT-ICSI-ET) and obtained a healthy fetus, which is the first report. The study focuses on the maintenance of fertility in patients with T8M, the effects of phenylketonuria and genetic counseling.

A base editing strategy using mRNA-LNPs for in vivo correction of the most frequent phenylketonuria variant.

The c.1222C>T (p.Arg408Trp) phenylalanine hydroxylase (PAH) variant is the most frequent cause of phenylketonuria (PKU), an autosomal recessive disorder characterized by accumulation of blood phenylalanine (Phe) to neurotoxic levels. Here we devised a therapeutic base editing strategy to correct the variant, using prime-edited hepatocyte cell lines engineered with the c.1222C>T variant to screen a variety of adenine base editors and guide RNAs in vitro, followed by assessment in c.1222C>T humanized mice in vivo. We found that upon delivery of a selected adenine base editor mRNA/guide RNA combination into mice via lipid nanoparticles (LNPs), there was sufficient PAH editing in the liver to fully normalize blood Phe levels within 48 h. This work establishes the viability of a base editing strategy to correct the most common pathogenic variant found in individuals with the most common inborn error of metabolism, albeit with potential limitations compared with other genome editing approaches.

State-of-the-art 2023 on gene therapy for phenylketonuria.

Phenylketonuria (PKU) or hyperphenylalaninemia is considered a paradigm for an inherited (metabolic) liver defect and is, based on murine models that replicate all human pathology, an exemplar model for experimental studies on liver gene therapy. Variants in the PAH gene that lead to hyperphenylalaninemia are never fatal (although devastating if untreated), newborn screening has been available for two generations, and dietary treatment has been considered for a long time as therapeutic and satisfactory. However, significant shortcomings of contemporary dietary treatment of PKU remain. A long list of various gene therapeutic experimental approaches using the classical model for human PKU, the homozygous enu2/2 mouse, witnesses the value of this model to develop treatment for a genetic liver defect. The list of experiments for proof of principle includes recombinant viral (AdV, AAV, and LV) and non-viral (naked DNA or LNP-mRNA) vector delivery methods, combined with gene addition, genome, gene or base editing, and gene insertion or replacement. In addition, a list of current and planned clinical trials for PKU gene therapy is included. This review summarizes, compares, and evaluates the various approaches for the sake of scientific understanding and efficacy testing that may eventually pave the way for safe and efficient human application.

Genotypic variants of the tetrahydrobiopterin (BH4) biosynthesis genes in patients with hyperphenylalaninemia from different regions of Iran.

BACKGROUND: Hyperphenylalaninemia (HPA) is a metabolic disorder classified into phenylalanine-4-hydroxylase (PAH) and non-PAH deficiency. The latter is produced by mutations in genes involved in the tetrahydrobiopterin (BH4) biosynthesis pathway and DNAJC12 pathogenetic variants. The BH4 metabolism, including de novo biosynthesis involved genes (i.e., guanosine 5'-triphosphate cyclohydrolase I (GTPCH/GCH1), sepiapterin reductase (SR/SPR), 6-pyruvoyl-tetrahydropterin synthase (PTPS/PTS)), and two genes that play roles in cofactor regeneration pathway (i.e., dihydropteridine reductase (DHPR/QDPR) and pterin-4α-carbinolamine dehydratase (PCD/PCBD1)). The subsequent systemic hyperphenylalaninemia and monoamine neurotransmitter deficiency lead to neurological consequences. The high rate of consanguineous marriages in Iran substantially increases the incidence of BH4 deficiency. METHODS: We utilized the Sanger sequencing technique in this study to investigate 14 Iranian patients with non-PAH deficiency. All affected subjects in this study had HPA and no mutation was detected in their PAH gene. RESULTS: We successfully identified six mutant alleles in BH4-deficiency-associated genes, including three novel mutations: one in QDPR, one in PTS, and one in the PCBD1 gene, thus giving a definite diagnosis to these patients. CONCLUSION: In this light, appropriate patient management may follow. The clinical effect of reported variants is essential for genetic counseling and prenatal diagnosis in the patients' families and significant for the improvement of precision medicine.

DNAJC12 in Monoamine Metabolism, Neurodevelopment, and Neurodegeneration.

Recent studies show that pathogenic variants in DNAJC12, a co-chaperone for monoamine synthesis, may cause mild hyperphenylalaninemia with infantile dystonia, young-onset parkinsonism, developmental delay and cognitive deficits. DNAJC12 has been included in newborn screening, most revealingly in Spain, and those results highlight the importance of genetic diagnosis and early intervention in combating human disease. However, practitioners may be unaware of these advances and it is probable that many patients, especially adults, have yet to receive molecular testing for DNAJC12. Hence, this review summarizes genotype-phenotype relationships and treatment paradigms for patients with pathogenic variants in DNAJC12. It provides an overview of the structure of DNAJC12 protein, known genetic variants, domains, and binding partners, and elaborates on its role in monoamine synthesis, disease etiology, and pathogenesis. © 2023 International Parkinson and Movement Disorder Society.

Systems Biology and Inborn Error of Metabolism: Analytical Strategy in Investigating Different Biochemical/Genetic Parameters.

Inborn errors of metabolism (IEM) are a group of about 500 rare genetic diseases with large diversity and complexity due to number of metabolic pathways involved in. Establishing a correct diagnosis and identifying the specific clinical phenotype is consequently a difficult task. However, an inclusive diagnosis able in capturing the different clinical phenotypes is mandatory for successful treatment. However, in contrast with Garrod's basic assumption "one-gene one-disease," no "simple" correlation between genotype-phenotype can be vindicated in IEMs. An illustrative example of IEM is Phenylketonuria (PKU), an autosomal recessive inborn error of L-phenylalanine (Phe) metabolism, ascribed to variants of the phenylalanine hydroxylase (PAH) gene encoding for the enzyme complex phenylalanine-hydroxylase. Blood values of Phe allow classifying PKU into different clinical phenotypes, albeit the participation of other genetic/biochemical pathways in the pathogenetic mechanisms remains elusive. Indeed, it has been shown that the most serious complications, such as cognitive impairment, are not only related to the gene dysfunction but also to the patient's background and the participation of several nongenetic factors.Therefore, a Systems Biology-based strategy is required in addressing IEM complexity, and in identifying the interplay between different pathways in shaping the clinical phenotype. Such an approach should entail the concerted investigation of genomic, transcriptomics, proteomics, metabolomics profiles altogether with phenylalanine and amino acids metabolism. Noticeably, this "omic" perspective could be instrumental in planning personalized treatment, tailored accordingly to the disease profile and prognosis.

Results of neonatal screening for congenital hypothyroidism and hyperphenylalaninemia in Zhejiang province from 1999 to 2022. / 浙江省1200ä¸‡ä¾‹æ–°ç”Ÿå„¿å ˆå¤©æ€§ç”²çŠ¶è ºåŠŸèƒ½å‡é€€ç—‡ã€é«˜è‹¯ä¸™æ°¨é ¸è¡€ç—‡ç­›æŸ¥ç»“æžœåˆ†æž.

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.

Analysis of gene variation and long-term follow-up in children with phenylalanine hydroxylase deficiency diagnosed by newborn screening. / æ–°ç”Ÿå„¿ç­›æŸ¥ç¡®è¯Šè‹¯ä¸™æ°¨é ¸ç¾ŸåŒ–é ¶ç¼ºä¹ç—‡æ‚£è€ åŸºå› å˜å¼‚åŠé•¿æœŸéšè®¿åˆ†æž.

OBJECTIVES: To retrospectively analyze the variation and characteristics of phenylalanine hydroxylase (PAH) gene, and to observe the long-term treatment effect and follow-up of newborns with PAH deficiency. METHODS: Clinical data, treatment and follow-up results of 198 patients with PAH deficiency diagnosed by newborn screening in Jinan from 1996 to 2021 were collected. The genetic analysis of 55 patients with PAH deficiency diagnosed by newborn screening in Jinan and 213 patients referred from the surrounding areas of Jinan were summarized. Gene variations were checked by a customized Panel gene detection method. Blood phenylalanine-concentration and physical development indicators including height and weight were regularly monitored. Intellectual development was assessed using a neuropsychological development scale for patients aged 0-6 years and academic performance, and brain injury in patients was assessed using brain magnetic resonance imaging. RESULTS: c.728G>A, c.158G>A, c.721C>T, c.1068C>A, c.611A>G variations were common in PAH gene. The genotype of c.158G>A variation is compound heterozygous variation, with mainly a mild hyperpheny-lalaninemia. 168 patients with PAH deficiency who were followed-up regularly had normal physical development without dwarfism or malnutrition. Among the 33 preschool patients who underwent mental development assessment, 2 were mentally retarded and the initial treatment age was older than 6 months. Nine patients with an average age of (17.13±2.42) years completed brain magnetic resonance imaging, one case was normal, and 8 cases were abnormal. There were patchy or patchy hyperintense foci near the bilateral lateral ventricles on T2WI, and the intellectual development was normal. Compared with the other eight patients, the blood phenylalanine concentration of the normal child was better and stably controlled within the ideal range. CONCLUSIONS: c.728G>A, c.158G>A, c.721C>T, c.1068C>A, c.611A>G variations were common in PAH gene. After standardized treatment, most patients with PAH deficiency diagnosed by screening can obtain normal growth and intellectual development in adolescence, but there are different degrees of organic lesions in the cerebral white matter.

Preliminary analysis of oral and gut microbiome of an elderly patient with late-diagnosed phenylketonuria.

Phenylketonuria (PKU) is a metabolic and genetic disorder caused by a phenylalanine hydroxylase (PAH) gene deficiency that raises Phe levels in organs. Dietary therapy involves an elimination diet and Phe-free items, which may alter microbiota. The study examined the oral and intestinal microbiomes of a 63-year-old PKU patient and a control man, living in rural areas. iSeq100 (Illumina) sequenced the stool and oral 16S rRNA gene V3-V4 region. PKU guts had more Firmicutes and fewer Bacteroidetes than control. Clostridia predominated in PKU, while Bacteroidia dominated in control. Oral Bacteroidetes. Firmicutes, Proteobacteria, and Fusobacteria phyla were similar in both men. The microbiome may differ from those fed a Phe-free diet from birth due to late diagnosis and treatment of PKU. Due to the age of the 63-year-old patient's and late therapy, the results differ from earlier studies. No study has compared an older PKU patient's gut and oral microbiomes.

Efficient in vivo prime editing corrects the most frequent phenylketonuria variant, associated with high unmet medical need.

The c.1222C>T (p.Arg408Trp) variant in the phenylalanine hydroxylase gene (PAH) is the most frequent cause of phenylketonuria (PKU), the most common inborn error of metabolism. This autosomal-recessive disorder is characterized by accumulation of blood phenylalanine (Phe) to neurotoxic levels. Using real-world data, we observed that despite dietary and medical interventions, most PKU individuals harboring at least one c.1222C>T variant experience chronic, severe Phe elevations and do not comply with Phe monitoring guidelines. Motivated by these findings, we generated an edited c.1222C>T hepatocyte cell line and humanized c.1222C>T mouse models, with which we demonstrated efficient in vitro and in vivo correction of the variant with prime editing. Delivery via adeno-associated viral (AAV) vectors reproducibly achieved complete normalization of blood Phe levels in PKU mice, with up to 52% whole-liver corrective PAH editing. These studies validate a strategy involving prime editing as a potential treatment for a large proportion of individuals with PKU.

[Development of analytics in newborn screening-from the Guthrie card to genetics]. / Entwicklung der Analytik im Neugeborenen-Screening ­ Von der Guthrie-Karte zur Genetik.

For more than five decades, all newborns in Germany have been offered a screening examination for the early detection of congenital treatable diseases. Since its inception, about 35 million children have been screened in this way.Originally, screening exams only included early detection of phenylketonuria, which, without timely treatment, would lead to mental retardation that could no longer be corrected. The bacteriological Guthrie test allowed the detection of elevated concentrations of phenylalanine. The methods used today are the result of decades of development. They have been expanded to include tests to determine enzyme activities, immunoassays for the early detection of important hormonal disorders such as congenital hypothyroidism, and high-pressure liquid chromatography for the diagnosis of pathologic hemoglobins. The very sophisticated tandem mass spectrometry enables the simultaneous detection of amino acid and fatty acid compounds. Steroids can also be identified. The specificity can be further increased by combining tandem mass spectrometry with chromatographic pre-separation. In recent years, chemical-analytical analyses have been supplemented by genetic diagnostic methods such as quantitative or qualitative polymerase chain reaction (PCR).The current state of laboratory technology is by no means final. Both classical analytics and especially genetic methods are facing further rapid development. Although the expansion of screening is also a consequence of technical development, the inclusion of further congenital diseases is fundamentally dependent on the given therapy. But it is precisely here that many innovations are currently being investigated. Gene therapy is at the forefront of interest.

Predictors of eventual requirement of phenylalanine-restricted diet in young infants with phenylalanine hydroxylase deficiency initially managed with sapropterin monotherapy.

BACKGROUND: Phenylalanine (Phe)-restricted diet is associated with lower quality of life for patients with phenylketonuria (PKU), and a concern for caregivers of recently-diagnosed infants. Sapropterin is an oral drug used as an alternative or adjunct to dietary treatment. We have observed that some of the young infants initially managed successfully with sapropterin monotherapy have required dietary treatment in long-term follow-up. We aimed to determine the baseline factors associated with future initiation of dietary treatment in these patients. METHODS: Data were obtained retrospectively from the medical records of 80 PKU patients started on sapropterin monotherapy before 3 months of age between 2011 and 2021. RESULTS: The patients were followed for a median of 3.9 years (Q1-Q3: 2.5-5.75 years). Sapropterin was tapered down and discontinued in 5 patients (6.3%) as their Phe levels remained below 360 µmol/L without treatment. Sapropterin monotherapy was sufficient in 62 patients (77.5%), while 13 (16.2%) required dietary treatment. Phe and tyrosine (Tyr) levels, and Phe:Tyr ratios differed significantly among the patients maintained on sapropterin monotherapy and those started on dietary treatment, but the Phe:Tyr ratio at diagnosis was the most important independent baseline variable (OR: 1.61, 95% CI: 1.15-2.27, p = 0.006), with Phe:Tyr ratio at diagnosis >5.25 associated with dietary treatment (sensitivity: 90.0%, specificity: 81.8%). Genotypic phenotype value (GPV), unavailable at baseline, was also associated with dietary treatment (median GPV 9.2 vs. 3.8, p = 0.006), but some genotypes were not specific to the final treatment modality. DISCUSSION: We propose that the Phe:Tyr ratio at diagnosis is an important indicator to predict dietary requirement in young infants initially managed with sapropterin monotherapy.

Carriers of autosomal recessive conditions: are they really 'unaffected?'

Mendel's Law of Dominance suggests that recessive disease expression requires the inheritance of two mutated alleles as the dominant, wildtype allele suppresses disease presentation leading to the expression of physiological normal phenotypes. However, there is existing evidence that challenges this school of thought. Here, we summarise existing literature evaluating metabolic and health impacts among carriers of autosomal recessive conditions, focusing on phenylketonuria (PKU), classical homocystinuria, galactosemia and Usher syndrome as examples. Our findings suggest that carriers, often described as 'unaffected', may actually display attenuated symptoms for the recessive disease they are carrying. For instance, PKU is an inborn error of metabolism characterised by the build-up of plasma phenylalanine attributed to the deficiency of the phenylalanine hydroxylase (PAH) enzyme. While less severe, PKU carriers also exhibit this impaired enzymatic activity, leading to elevated plasma phenylalanine levels, especially after phenylalanine consumption. Related to these metabolic alterations in the PAH pathway, there is early evidence to suggest that PKU carriers may have compromised cognitive and mental health outcomes. Overall, research on the health and metabolic impacts of PKU carriers is sparse, with most studies conducted several decades ago. However, early evidence suggests that intermediate phenotypes among carriers of autosomal recessive conditions are plausible. The illustrated possible intermediate phenotypes observed among carriers necessitates future research to determine possible clinical implications among this population.

The treatment of biochemical genetic diseases: From substrate reduction to nucleic acid therapies.

Newborn screening (NBS) began a revolution in the management of biochemical genetic diseases, greatly increasing the number of patients for whom dietary therapy would be beneficial in preventing complications in phenylketonuria as well as in a few similar disorders. The advent of next generation sequencing and expansion of NBS have markedly increased the number of biochemical genetic diseases as well as the number of patients identified each year. With the avalanche of new and proposed therapies, a second wave of options for the treatment of biochemical genetic disorders has emerged. These therapies range from simple substrate reduction to enzyme replacement, and now ex vivo gene therapy with autologous cell transplantation. In some instances, it may be optimal to introduce nucleic acid therapy during the prenatal period to avoid fetopathy. However, as with any new therapy, complications may occur. It is important for physicians and other caregivers, along with ethicists, to determine what new therapies might be beneficial to the patient, and which therapies have to be avoided for those individuals who have less severe problems and for which standard treatments are available. The purpose of this review is to discuss the "Standard" treatment plans that have been in place for many years and to identify the newest and upcoming therapies, to assist the physician and other healthcare workers in making the right decisions regarding the initiation of both the "Standard" and new therapies. We have utilized several diseases to illustrate the applications of these different modalities and discussed for which disorders they may be suitable. The future is bright, but optimal care of the patient, including and especially the newborn infant, requires a deep knowledge of the disease process and careful consideration of the necessary treatment plan, not just based on the different genetic defects but also with regards to different variants within a gene itself.

Mutational landscape of phenylketonuria in Iran.

To date more than 1000 different variants in the PAH gene have been identified in patients with phenylketonuria (PKU). In Iran, several studies have been performed to investigate the genetics bases of the PKU in different parts of the country. In this study, we have analysed and present an update of the mutational landscape of the PAH gene as well as the population genetics and frequencies of detected variants for each cohort. Published articles on PKU mutations in Iran were identified through a comprehensive PubMed, Google Scholar, Web of Science (ISI), SCOPUS, Elsevier, Wiley Online Library and SID literature search using the terms: "phenylketonuria", "hyperphenylalaninemia", and "PKU" in combination with "Iran", "Iranian population", "mutation analysis", and "Molecular genetics". Among the literature-related to genetics of PKU, 18 studies were on the PKU mutations. According to these studies, in different populations of Iran 1497 patients were included for mutation detection that resulted in detection of 129 different mutations. Results of genetic analysis of the different cohorts of Iranian PKU patients show that the most prevalent mutation in Iran is the pathogenic splice variant c.1066-11G > A, occurring in 19.54% of alleles in the cohort. Four other common mutations were p.Arg261Gln, p.Pro281Leu, c.168 + 5G > C and p.Arg243Ter (8.18%, 6.45%, 5.88% and 3.7%, respectively). One notable feature of the studied populations is its high rate of consanguineous marriages. Considering this feature, determining the prevalent PKU mutations could be advantageous for designing screening and diagnostic panels in Iran.

[Analysis of screening results for genetic metabolic diseases among 352 449 newborns from Changsha].

OBJECTIVE: To retrospectively analyze the screening results for genetic metabolic diseases among newborns from Changsha in order to determine the prevalence of single diseases and their mutational spectrum. METHODS: 352 449 neonates born from January 2016 to December 2021 in Changsha were subjected to tandem mass spectrometry. Suspected cases were further analyzed by biochemical and genetic testing. RESULTS: Among the 352 449 newborns, 6 170 were positive for the screening, which yielded a positive rate of 1.75%. 5 437 cases were recalled, and 92 were confirmed, with the overall prevalence being 1∶3 831 and positive predictive value of 1.69%. Eighteen genetic metabolic diseases were detected among the 92 children, including 33 amino acid metabolic disorders, among which 20 were phenylalanine hydroxylase deficiency (60.60%). 17 cases had organic acid metabolic disorders, among which 4 were 2-methyl-dehydrogenase deficiency (23.50%). 42 had fatty acid metabolic disorders, among which 27 (64.30%) were primary carnitine deficiency and 12 were short-chain acyl-CoA dehydrogenase deficiency (28.60%). In total 90 genetic variants were identified, with the most common ones including c.51C>G, c.1400C>G, c.760C>T, c.1031A>G and c.1165A>G. CONCLUSION: The common neonatal genetic metabolic diseases in Changsha include primary carnitine deficiency, phenylalanine hydroxylase deficiency and short-chain acyl-CoA dehydrogenase deficiency. The preliminary delineation of mutational spectrum for genetic metabolic diseases in Changsha can facilitate early diagnosis and intervention, so as to improve the quality of newborn population.

Mutation Characteristics of Phenylalanine Hydroxylase Gene in Children with Phenylketonuria in Yinchuan City.

BACKGROUND: Phenylalanine hydroxylase deficiency (PAHD) is an autosomal recessive disorder affecting phenylalanine (Phe) metabolism caused by mutations in the phenylalanine hydroxylase (PAH) gene. It has a complex phenotype with many variants and genotypes in various populations. This study sets out to analyze the screening results of children with phenylketonuria (PKU) in Yinchuan City and characterize the mutation variants of the PAH gene. METHODS: Phenylketonuria screening results were retrospectively analyzed in 398,605 neonates (207,361 males and 191,244 females) born in different maternity hospitals in Yinchuan City between January 2017 and December 2021. Screening for genetic metabolic diseases was performed with parental consent at their own expense. A comprehensive diagnosis was performed by integrating tandem mass spectrometry (MS/MS) findings with clinical presentations. High-throughput sequencing (HTS) was used to detect genetic and metabolic disease-associated genes in children with PKU who were clinically diagnosed and voluntarily tested. The identified loci were validated through Sanger sequencing and parental verification. RESULTS: Among the screened newborns, 45 (11.3/100,000) PKU cases were diagnosed. In the 38 cases that underwent self-financed PAH sequencing, 56 mutations were detected in 76 chromosomes, with an overall detection rate of 73.7%. All patients harbored mutant genes, and the 56 mutations detected identified represented 14 variants, including 8 missense mutations, 2 splicing mutations, 2 nonsense mutations, and 2 silent mutations. The mutations were primarily distributed in exons 2, 3, 6, 7, 9, 11, and intron 4, with the highest frequency observed in exon 7 (25 [44.7%]), followed by exon 11 (15 [26.7%]). The most prevalent mutations were exon 7-p.R252W (10 [17.9%]) and exon 7-p.R261Q (8 [14.3%]). CONCLUSIONS: The PAH gene mutations in children with PKU in Yinchuan City are predominantly concentrated in exons 6, 7, and 11, with the highest detection rates observed for p.R252W and p.R261Q mutations.

Phenylketonuria (PKU) Urinary Metabolomic Phenotype Is Defined by Genotype and Metabolite Imbalance: Results in 51 Early Treated Patients Using Ex Vivo 1H-NMR Analysis.

Phenylketonuria (PKU) is a rare metabolic disorder caused by mutations in the phenylalanine hydroxylase gene. Depending on the severity of the genetic mutation, medical treatment, and patient dietary management, elevated phenylalanine (Phe) may occur in blood and brain tissues. Research has recently shown that high Phe not only impacts the central nervous system, but also other organ systems (e.g., heart and microbiome). This study used ex vivo proton nuclear magnetic resonance (1H-NMR) analysis of urine samples from PKU patients (mean 14.9 ± 9.2 years, n = 51) to identify the impact of elevated blood Phe and PKU treatment on metabolic profiles. Our results found that 24 out of 98 urinary metabolites showed a significant difference (p < 0.05) for PKU patients compared to age-matched healthy controls (n = 51) based on an analysis of urinary metabolome. These altered urinary metabolites were related to Phe metabolism, dysbiosis, creatine synthesis or intake, the tricarboxylic acid (TCA) cycle, end products of nicotinamide-adenine dinucleotide degradation, and metabolites associated with a low Phe diet. There was an excellent correlation between the metabolome and genotype of PKU patients and healthy controls of 96.7% in a confusion matrix model. Metabolomic investigations may contribute to a better understanding of PKU pathophysiology.