Newborn screening and genetic features of patients with hyperphenylalaninemia in a southern Chinese population.

BACKGROUND AND AIMS: Hyperphenylalaninemia (HPA) is the most common congenital amino acid metabolism-related defect, but its incidence differs substantially between northern and southern China. We aimed to elucidate the incidence, proportion, and genetic features of HPA in a southern Chinese population. MATERIALS AND METHODS: We analyzed the HPA screening results for 580,460 newborns from 2014 to 2021. RESULTS: Of the 296 newborns who tested HPA positive, 56 were diagnosed with HPA, including 47 with phenylalanine hydroxylase deficiency and nine with tetrahydrobiopterin deficiency (BH4D). HPA incidence was estimated to be 1:10,365 newborns. All patients had elevated Phe and Phe/Tyr levels. Thirty-three PAH variants and five PTS variants were detected in HPA patients; 80.6 % PAH variants and 100 % PTS variants were classified as pathogenic or likely pathogenic. In silico tools predicted the remaining variants to be damaging. PAH variants clustered in exons 3, 5, 7, 11, and 12 and PTS variants clustered in exons 2 and 5. The most common PAH variants were c.158G > A (p.R53H, 22.3 %) and c.721C > T (p.R241C, 14.9 %). The most common PTS variants were c.155A > G (p.N52S, 50.0 %) and c.259C > T (p.P87S, 33.3 %). CONCLUSION: Newborn screening is an effective method for early detection of HPA, but differential diagnosis of BH4D is necessary.

The pathogenicity classification of PAH gene variants in the Iranian population.

Till now not many studies have been conducted to classify PAH gene variants according to American College of Medical Genetics and Genomics (ACMG-AMP) guidelines. The aim of this study was to collect all PAH gene variants reported among Iranian population and investigate their pathogenicity based on ACMG-AMP guidelines. Systematic collection of PAH gene variants, verification of variants, in silico analysis, and application of ACMG-AMP guidelines were the main steps in performing the present study. A total of 267 unique variants were identified; according to ACMG-AMP guidelines, 90, 40, 71, 14, and 52 variants were classified as pathogenic (P), likely pathogenic (LP), variant of uncertain significance (VUS), likely benign (LB), and benign (B), respectively. The need to pay more attention to synonymous and missense variants with low or no impact on protein function as well as intronic variants, whether they are deep or are close to intron/exon boundaries, was a highlight of this study. Due to the fact that few functional studies are performed on these variants, it is suggested that they be analyzed first using bioinformatics tools, and if positive results are obtained, then functional studies can be designed.

The effect of casein glycomacropeptide versus free synthetic amino acids for early treatment of phenylketonuria in a mice model.

INTRODUCTION: Management of phenylketonuria (PKU) is mainly achieved through dietary control with limited intake of phenylalanine (Phe) from food, supplemented with low protein (LP) food and a mixture of free synthetic (FS) amino acids (AA) (FSAA). Casein glycomacropeptide (CGMP) is a natural peptide released in whey during cheese making by the action of the enzyme chymosin. Because CGMP in its pure form does not contain Phe, it is nutritionally suitable as a supplement in the diet for PKU when enriched with specific AAs. Lacprodan® CGMP-20 (= CGMP) used in this study contained only trace amounts of Phe due to minor presence of other proteins/peptides. OBJECTIVE: The aims were to address the following questions in a classical PKU mouse model: Study 1, off diet: Can pure CGMP or CGMP supplemented with Large Neutral Amino Acids (LNAA) as a supplement to normal diet significantly lower the content of Phe in the brain compared to a control group on normal diet, and does supplementation of selected LNAA results in significant lower brain Phe level?. Study 2, on diet: Does a combination of CGMP, essential (non-Phe) EAAs and LP diet, provide similar plasma and brain Phe levels, growth and behavioral skills as a formula which alone consist of FSAA, with a similar composition?. MATERIAL AND METHODS: 45 female mice homozygous for the Pahenu2 mutation were treated for 12 weeks in five different groups; G1(N-CGMP), fed on Normal (N) casein diet (75%) in combination with CGMP (25%); G2 (N-CGMP-LNAA), fed on Normal (N) casein diet (75%) in combination with CGMP (19,7%) and selected LNAA (5,3% Leu, Tyr and Trp); G3 (N), fed on normal casein diet (100%); G4 (CGMP-EAA-LP), fed on CGMP (70,4%) in combination with essential AA (19,6%) and LP diet; G5 (FSAA-LP), fed on FSAA (100%) and LP diet. The following parameters were measured during the treatment period: Plasma AA profiles including Phe and Tyr, growth, food and water intake and number of teeth cut. At the end of the treatment period, a body scan (fat and lean body mass) and a behavioral test (Barnes Maze) were performed. Finally, the brains were examined for content of Phe, Tyr, Trp, dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC), serotonin (5-HT) and 5-hydroxyindole-acetic acid (5-HIAA), and the bone density and bone mineral content were determined by dual-energy x-ray absorptiometry. RESULTS: Study 1: Mice off diet supplemented with CGMP (G1 (N-CGMP)) or supplemented with CGMP in combination with LNAA (G2 (N-CGMP-LNAA)) had significantly lower Phe in plasma and in the brain compared to mice fed only casein (G3 (N)). Extra LNAA (Tyr, Trp and Leu) to CGMP did not have any significant impact on Phe levels in the plasma and brain, but an increase in serotonin was measured in the brain of G2 mice compared to G1. Study 2: PKU mice fed with mixture of CGMP and EAA as supplement to LP diet (G4 (CGMP-EAA-LP)) demonstrated lower plasma-Phe levels but similar brain- Phe levels and growth as mice fed on an almost identical combination of FSAA (G5 (FSAA-LP)). CONCLUSION: CGMP can be a relevant supplement for the treatment of PKU.

A noncoding RNA modulator potentiates phenylalanine metabolism in mice.

The functional role of long noncoding RNAs (lncRNAs) in inherited metabolic disorders, including phenylketonuria (PKU), is unknown. Here, we demonstrate that the mouse lncRNA Pair and human HULC associate with phenylalanine hydroxylase (PAH). Pair-knockout mice exhibited excessive blood phenylalanine (Phe), musty odor, hypopigmentation, growth retardation, and progressive neurological symptoms including seizures, which faithfully models human PKU. HULC depletion led to reduced PAH enzymatic activities in human induced pluripotent stem cell-differentiated hepatocytes. Mechanistically, HULC modulated the enzymatic activities of PAH by facilitating PAH-substrate and PAH-cofactor interactions. To develop a therapeutic strategy for restoring liver lncRNAs, we designed GalNAc-tagged lncRNA mimics that exhibit liver enrichment. Treatment with GalNAc-HULC mimics reduced excessive Phe in Pair -/- and Pah R408W/R408W mice and improved the Phe tolerance of these mice.

A New View of Bone Loss in Phenylketonuria.

Osteopenia is common in phenylalanine hydroxylase deficient phenylketonuria (PKU). PKU is managed by limiting dietary phenylalanine. Osteopenia in PKU might reflect a therapeutic diet, with reduced bone forming materials. However, osteopenia occurs in patients who never received dietary therapy or following short-term therapy. Humans and animal studies find no correlation between bone loss, plasma hyperphenylalaninemia, bone formation, and resorption markers. Work in the Pahenu2 mouse recently showed a mesenchymal stem cell (MSC) developmental defect in the osteoblast pathway. Specifically, Pahenu2 MSCs are affected by energy dysregulation and oxidative stress. In PKU, MSCs oximetry and respirometry show mitochondrial respiratory-chain complex 1 deficit and over-representation of superoxide, producing reactive oxygen species affecting mitochondrial function. Similar mechanisms are involved in aging bone and other rare defects including alkaptonuria and homocysteinemia. Novel interventions to support energy and reduce oxidative stress may restore bone formation PKU patients, and in metabolic diseases with related mechanisms.

BH4-deficient hyperphenylalaninemia in Russia.

A timely detection of patients with tetrahydrobiopterin (BH4) -deficient types of hyperphenylalaninemia (HPABH4) is important for assignment of correct therapy, allowing to avoid complications. Often HPABH4 patients receive the same therapy as phenylalanine hydroxylase (PAH) -deficiency (phenylketonuria) patients-dietary treatment-and do not receive substitutive BH4 therapy until the diagnosis is confirmed by molecular genetic means. In this study, we present a cohort of 30 Russian patients with HPABH4 with detected variants in genes causing different types of HPA. Family diagnostics and biochemical urinary pterin spectrum analyses were carried out. HPABH4A is shown to be the prevalent type, 83.3% of all HPABH4 cases. The mutation spectrum for the PTS gene was defined, the most common variants in Russia were p.Thr106Met-32%, p.Asn72Lys-20%, p.Arg9His-8%, p.Ser32Gly-6%. We also detected 7 novel PTS variants and 3 novel QDPR variants. HPABH4 prevalence was estimated to be 0.5-0.9% of all HPA cases in Russia, which is significantly lower than in European countries on average, China, and Saudi Arabia. The results of this research show the necessity of introducing differential diagnostics for HPABH4 into neonatal screening practice.

Variants of the phenylalanine hydroxylase gene in neonates with phenylketonuria in Hainan, China.

OBJECTIVE: To investigate the incidence of phenylalanine hydroxylase (PAH) deficiency and PAH genotypes in neonates in Hainan, China. Methods: We performed heal stick to collect blood and obtain dry blood spot specimens from newborns in Hainan from January 2007 to December 2016. Phenylalanine (Phe) concentration in these dry blood spots was measured by the fluorescence method to screen phenylketonuria (PKU). For suspicious samples, the genotypes of the PAH gene were amplified by biotin labeled oligonucleotide primers. Polymerase chain reaction (PCR) products were then analyzed by flow-through hybridization to detect genotypes. At the same time, peripheral blood samples of children suspicious of PKU and their parents were used to perform gene sequencing. Results: Of the 914,520 newborns screened, 29 of them had PAH deficiency. The incidence of PAH deficiency in Hainan was 3.17/100,000. A total of 58 mutant alleles belonging to 15 different types were identified in the 29 patients. In terms of genotypes frequency, the top 4 were: c.611A > G 20.7% (12/58) , c.728G > A 17.2%, c.158G > A 15.2% (9/58) and c.721C > T 13.8% (8/58). The frequencies of other genotypes were all below 10%. Conclusion: The incidence of PAH deficiency in Hainan is relatively high among all provinces in southern China. With a total frequency of 67.2%, c.611A > G, c.728G > A, c.158G > A and c.721C > T, and are the most common PAH gene genotypes.

Maternal PKU: Defining phenylalanine tolerance and its variation during pregnancy, according to genetic background.

BACKGROUND AND AIMS: Phenylketonuria (PKU)-affected women may become pregnant, and dietary phenylalanine (Phe) intake must be adjusted according to Phe tolerance. We report our experience with maternal PKU in relation to genotype PKU heterogeneity. METHODS AND RESULTS: A total of 10 pregnancies in 7 PKU women (7 different genotypes) were followed up as part of personalized care. Phe tolerance during preconception and pregnancy was assessed by strict dietary control and weekly Phe measurement (blood spots) in relation to genotype. Most women had stopped PKU diet during childhood or adolescence and six pregnancies were unplanned; a phenylalanine-restricted diet was reinstituted soon after conception. Women were classified according to their Phe levels at birth screening and genotype. Phe tolerance increased systematically in the course of pregnancy in all cases, but the increase was different in subjects with classic PKU (cPKU) when compared with cases with mild hyperphenylalaninemia (mHPA), both on average (+297 mg/day in cPKU vs. 597 in mHPA; P = 0.017) and as percentage (+107% in cPKU vs. +17% in mHPA). Notably, Phe tolerance also varied in the same women in the course of different pregnancies, when body weight gain was also different. Two newborns from the same cPKU mother (unplanned pregnancies on free diet) were affected by congenital alterations. CONCLUSIONS: Several factors influence metabolic phenotype in maternal PKU, to an unpredictable extent even in the same woman. The number of maternal PKU cases is growing in dedicated Nutrition Units, and the burden associated with careful management of this condition for the health care system should be adequately considered.

Tyrosine metabolism in health and disease: slow-release amino acids therapy improves tyrosine homeostasis in phenylketonuria.

OBJECTIVES: Phenylalanine (Phe) hydroxylase (PAH) deficiency leads to hyperphenylalaninemia (HPA) and tyrosine (Tyr) depletion. We investigated Tyr homeostasis in patients with PAH deficiency and the effect of a slow-release amino acids therapy in phenylketonuria (PKU). METHODS: We performed four complementary investigations: (1) Tyr concentrations were monitored in 114 patients (10.6 ± 11.9 years) with PKU on dietary treatment supplemented with traditional amino acid formulations (n=52, 1175 samples) or non-PKU HPA on a free diet (n=62, 430 samples); (2) Tyr metabolism in PKU was quantitatively evaluated in three patients by a simple Tyr oral loading test (100 mg/kg); (3) diurnal and (4) long-term Tyr concentrations were evaluated in 5 and 13 patients with PKU, respectively, who switched from traditional to slow-release amino acids therapy. RESULTS: 1) Tyr concentrations in the PKU population were subnormal and significantly lower than in non-PKU HPA (p<0.01); (2) the response to a Tyr loading test in PKU was normal, with basal Tyr concentrations reached within 12 h; (3) the diurnal metabolic profile in patients on slow-release amino acids therapy revealed higher morning fasting and nocturnal Tyr concentrations with respect to traditional therapy (p<0.01); (4) this picture was confirmed at follow-up, with normalization of morning fasting Tyr concentrations in patients on slow-release amino acids therapy (p<0.01) and unchanged Phe control (p=0.19). CONCLUSIONS: Slow-release amino acids therapy can improve Tyr homeostasis in PKU. If associated to optimized Phe control, such a metabolic goal may allow long-term clinical benefits in patients with PKU.

Metabolomics analysis reveals perturbations of cerebrocortical metabolic pathways in the Pahenu2 mouse model of phenylketonuria.

AIMS: Phenylketonuria (PKU), which is caused by mutations in the phenylalanine hydroxylase (PAH) gene, is one of the most common inherited diseases of amino acid metabolism. Phenylketonuria is characterized by an abnormal accumulation of phenylalanine and its metabolites in body fluids and brain tissues, subsequently leading to severe brain dysfunction. Various pathophysiological and molecular mechanisms underlying brain dysfunction in PKU have been described. However, the metabolic changes and their impacts on the function of cerebral cortices of patients with PKU remain largely unknown. METHODS: We measured the levels of small molecule metabolites in the cerebrocortical tissues of PKU mice and wild-type control mice using liquid chromatography-mass spectrometry (LC-MS)-based metabolome analysis. Differential metabolites were further subjected to metabolic pathway and enrichment analysis. RESULTS: Metabolome analysis revealed 35 compounds among 143 detected metabolites were significantly changed in PKU mice as compared to those in their wild-type littermates. Metabolic pathway and enrichment analysis of these differential metabolites showed that multiple metabolic pathways, including phenylalanine, tyrosine, and tryptophan biosynthesis; valine, leucine, and isoleucine biosynthesis; alanine, aspartate, and glutamate metabolism; purine metabolism; arginine and proline metabolism and methionine metabolism, were impacted in the cerebral cortices of PKU mice. CONCLUSIONS: The data revealed that multiple metabolic pathways in cerebral cortices of PKU mice were disturbed, suggesting that the disturbances of the metabolic pathways might contribute to neurological or neurodevelopmental dysfunction in PKU, which could thus provide new insights into brain pathogenic mechanisms in PKU as well as mechanistic insights for better understanding the complexity of the metabolic mechanisms of the brain dysfunction in PKU.

Examining the blood amino acid status in pretherapeutic patients with hyperphenylalaninemia.

BACKGROUND: Hyperphenylalaninemia is the most common genetic metabolic disease. Early treatment prevents brain injury effectively. The present study aimed to detect the exact amino acid status of patients with hyperphenylalaninemia before treatment. METHODS: Data of 116 newborn patients from our Newborn Screening Center and 161 older patients from our clinic before treatment were collected. The content of 17 amino acids in their blood was determined by tandem mass spectrometry and compared with normal controls. Relationship between phenylalanine and other amino acids in patients was analyzed using the smoothing curve fitting and threshold effect analysis. RESULTS: Most amino acids in the blood of patients were within the normal range; however, they were different significantly from those of the normal children. Newborn patients showed higher phenylalanine (346.30 vs 45.90 µmol/L), valine (121.50 vs 110.30 µmol/L), citrulline, ornithine and lower tyrosine (52.97 vs 66.12 µmol/L), threonine (68.68 vs 78.21 µmol/L), glutamine levels than observed in normal newborns. Older patients showed significantly higher phenylalanine (844.00 vs 51.82 µmol/L), valine (117.60 vs 110.90 µmol/L), histidine, serine and lower tyrosine (55.97 vs 67.31 µmol/L), threonine (35.94 vs 51.89 µmol/L), alanine, asparagine, glutamic acid, methionine, arginine, glycine, ornithine, glutamine content than found in matched normal children. Tyrosine, valine, ornithine, and threonine in newborn patients and tyrosine, glycine, glutamine, and threonine in older patients had a nonlinear correlation with phenylalanine levels with obvious threshold effect and clear inflection points. CONCLUSION: Significant difference was observed in the amino acid status between pretherapeutic hyperphenylalaninemia patients and normal children. Some amino acids showed notable threshold effect with phenylalanine level in a nonlinear pattern.

5-year retrospective analysis of patients with phenylketonuria (PKU) and hyperphenylalaninemia treated at two specialized clinics.

BACKGROUND: Phenylketonuria (PKU) is an autosomal recessive disease caused by mutations in the PAH gene, resulting in deficiency of phenylalanine hydroxylase (PAH), an enzyme that converts phenylalanine (Phe) to tyrosine (Tyr). The purpose of this study was to capture real-world data associated with managing PKU under current standard of care and to characterize a representative population for a planned gene therapy trial. METHODS: A retrospective chart review was conducted at two U.S. clinics for individuals 10-40 years old diagnosed with PKU-related hyperphenylalaninemia (HPA). Demographics, medical history, treatments and blood Phe data were collected from electronic medical records spanning a five-year period ending in November 2017. RESULTS: 152 patients were enrolled (65.8% had classical PKU). Although >95% of patients were prescribed a Phe-restricted diet, blood Phe concentrations remained substantially elevated, particularly in patients diagnosed with classical PKU. As the Phe threshold was lowered (Phe < 600, 360, 120 or 30 µmol/L), the number of patients with consecutive lab values below the threshold decreased, suggesting that many patients' Phe levels are inadequately controlled. 62.5% of patients were reported as having a history of at least one neuropsychiatric comorbidity, and adults were more likely than adolescents (69.5% vs. 54.3%). 92 of 98 PAH genotypes collected were distinct mutations; the 6 null-null genotypes were associated with classical PKU. Overall the demographics and clinical data were consistent across both sites. CONCLUSION: Despite dietary restrictions, mean Phe concentrations were > 360 µmol/L (a level considered well-controlled based on current U.S. treatment guidelines) for mild, moderate, and classical PKU patients. There remains an unmet need for therapies to control Phe concentrations.

State-of-the-Art 2019 on Gene Therapy for Phenylketonuria.

Phenylketonuria (PKU) is considered to be a paradigm for a monogenic metabolic disorder but was never thought to be a primary application for human gene therapy due to established alternative treatment. However, somewhat unanticipated improvement in neuropsychiatric outcome upon long-term treatment of adults with PKU with enzyme substitution therapy might slowly change this assumption. In parallel, PKU was for a long time considered to be an excellent test system for experimental gene therapy of a Mendelian autosomal recessive defect of the liver due to an outstanding mouse model and the easy to analyze and well-defined therapeutic end point, that is, blood l-phenylalanine concentration. Lifelong treatment by targeting the mouse liver (or skeletal muscle) was achieved using different approaches, including (1) recombinant adeno-associated viral (rAAV) or nonviral naked DNA vector-based gene addition, (2) genome editing using base editors delivered by rAAV vectors, and (3) by delivering rAAVs for promoter-less insertion of the PAH-cDNA into the Pah locus. In this article we summarize the gene therapeutic attempts of correcting a mouse model for PKU and discuss the future implications for human gene therapy.

Retrospective Study of Patients with Hyperphenylalaninemia- Experience from a Tertiary Care Center in Pakistan.

OBJECTIVE: To assess the clinical and biochemical features as well as outcome of hyperphenylalaninemia patients. Methods: The descriptive retrospective study was conducted at the Aga Khan University Hospital, Karachi, and comprised data from January 2013 to February 2017 of plasma amino acid analysed at the Biochemical Genetic Laboratory of patients with phenylalanine levels >120 umol/L. Medical charts of patients registered with the Metabolic Clinics were reviewed, while outside referrals were contacted by telephone to collect data on a pre-structured questionnaire. Data was analysed using SPSS 21. RESULTS: Of the 18 patients, 13(72%) were males. Overall median age was 606 days (interquartile range: 761) and median phenylalanine levels were 1280 (interquartile range: 935) umol/L. Phenylalanine hydroxylase deficiency was present in 5(28%) patients while 3(16.6%) had defects in the metabolism or regeneration of tetrahydrobiopterin. The most common clinical features was intellectual deficit and seizures 14(78%) each, followed by lighter hair colour 10(55.5%) and hypotonia 11(61%). High treatment cost was the leading reason for cessation of therapy in 7(39%) followed by refusal by patient's family 5(28%). CONCLUSIONS: Most hyperphenylalaninemia cases were diagnosed late when intellectual disability had already developed.

Long-term dietary intervention with low Phe and/or a specific nutrient combination improve certain aspects of brain functioning in phenylketonuria (PKU).

INTRODUCTION: In phenylketonuria (PKU), a gene mutation in the phenylalanine metabolic pathway causes accumulation of phenylalanine (Phe) in blood and brain. Although early introduction of a Phe-restricted diet can prevent severe symptoms from developing, patients who are diagnosed and treated early still experience deficits in cognitive functioning indicating shortcomings of current treatment. In the search for new and/or additional treatment strategies, a specific nutrient combination (SNC) was postulated to improve brain function in PKU. In this study, a long-term dietary intervention with a low-Phe diet, a specific combination of nutrients designed to improve brain function, or both concepts together was investigated in male and female BTBR PKU and WT mice. MATERIAL & METHODS: 48 homozygous wild-types (WT, +/+) and 96 PKU BTBRPah2 (-/-) male and female mice received dietary interventions from postnatal day 31 till 10 months of age and were distributed in the following six groups: high Phe diet (WT C-HP, PKU C-HP), high Phe plus specific nutrient combination (WT SNC-HP, PKU SNC-HP), PKU low-Phe diet (PKU C-LP), and PKU low-Phe diet plus specific nutrient combination (PKU SNC- LP). Memory and motor function were tested at time points 3, 6, and 9 months after treatment initiation in the open field (OF), novel object recognition test (NOR), spatial object recognition test (SOR), and the balance beam (BB). At the end of the experiments, brain neurotransmitter concentrations were determined. RESULTS: In the NOR, we found that PKU mice, despite being subjected to high Phe conditions, could master the task on all three time points when supplemented with SNC. Under low Phe conditions, PKU mice on control diet could master the NOR at all three time points, while PKU mice on the SNC supplemented diet could master the task at time points 6 and 9 months. SNC supplementation did not consistently influence the performance in the OF, SOR or BB in PKU mice. The low Phe diet was able to normalize concentrations of norepinephrine and serotonin; however, these neurotransmitters were not influenced by SNC supplementation. CONCLUSION: This study demonstrates that both a long-lasting low Phe diet, the diet enriched with SNC, as well as the combined diet was able to ameliorate some, but not all of these PKU-induced abnormalities. Specifically, this study is the first long-term intervention study in BTBR PKU mice that shows that SNC supplementation can specifically improve novel object recognition.

Prevalence of comorbid conditions among adult patients diagnosed with phenylketonuria.

BACKGROUND: Phenylalanine hydroxylase (PAH) deficiency, otherwise known as phenylketonuria (PKU), is an inborn error of metabolism that requires treatment to be initiated in the newborn period and continued throughout life. Due to the challenges of treatment adherence and the resulting cumulative effects of high and labile blood phenylalanine, PKU exerts a significant burden of disease. Retrospective studies using large databases allow for unique perspectives on comorbidities associated with rare diseases. An evaluation of comorbidities across various organ systems is warranted to understand the disease burden in adult patients. OBJECTIVES: The aim of this insurance claim-based observational study was to assess the prevalence of comorbid conditions across various organ systems (e.g. dermatological, renal, respiratory, gastrointestinal, hematological, and others) among adult PKU patients compared with matched controls from the general population. METHODS: This retrospective, case-controlled study selected patients from United States insurance claims databases from 1998 to 2014 using International Classification of Diseases, Ninth Revision (ICD-9) codes for diagnosis of PKU. The date of first diagnosis during the study period was index date and this was not necessarily the first time the patient was diagnosed with PKU. Cases were matched with a 1:5 ratio with general population (non-PKU controls) on age, sex, race, geographic location, duration of time in the database and insurance type. Prevalence and prevalence ratio (PR) calculations for comorbidities across various organ systems among adults (≥20 years old) with PKU were compared with the general population (non-PKU controls). The conditions were selected based on complications associated with PKU and feedback from clinicians treating PKU patients. RESULTS: A total of 3691 PKU patients and 18,455 matched, non-PKU controls were selected, with an average age of 35 years. The mean healthcare costs incurred by the PKU patients during baseline, were approximately 4 times that of the controls ($4141 vs $1283; p < .0001). The prevalence rates of comorbidities across various organ systems during the follow-up period were significantly higher for those with PKU than in the control group. After adjusting for baseline characteristics, the adjusted prevalence ratios (PR) of 15 conditions studied (asthma, alopecia, urticaria, gallbladder disease, rhinitis, esophageal disorders, anemia, overweight, GERD, eczema, renal insufficiency, osteoporosis, gastritis/esophagitis and kidney calculus) were all above PR = 1.24 and significantly higher for the PKU cohort (p ≤ .001). The highest adjusted PR were for renal insufficiency with hypertension (PR [95% CI]: 2.20 [1.60-3.00]; p < .0001) and overweight (PR [95%CI]: 2.06 [1.85-2.30]; p < .0001). CONCLUSIONS: The prevalence of selected comorbidities across several organ systems is significantly higher among PKU patients than for general population controls. Regular screening for common co-morbidities may be warranted as part of PKU management.

Mutational spectrum of the phenylalanine hydroxylase gene in patients with phenylketonuria in the central region of China.

Phenylketonuria (PKU, OMIM 261600) caused by phenylalanine hydroxylase (PAH) deficiency is an autosomal recessive disease that is characterized by abnormalities of phenylalanine metabolism. In this study, a total of 77 patients, originating from the central region of China and who were diagnosed with PAH deficiency at the third affiliated hospital of Zhengzhou University, were enrolled in this study. The 13 exons and 12 flanking introns of the PAH gene were analyzed by Sanger sequencing and next generation sequencing. The sequencing data were aligned to the hg19, PAHvdb and HGMD databases to characterize the genotypes of PKU patients, and genotype-phenotype correlations and BH4 responsiveness predictions were performed using BIOPKUdb. In total, 149 alleles were characterized among the 154 PKU alleles. These mutations were located in exons 2-13, and intron 12 of the PAH gene, with a relative frequency of ≥5%, for EX6-96A>G, p.R241C, p.R243Q, p.V399V and p.R53H. Additionally, a novel variant, p.D84G, was identified. The genotype correlated with clinical symptoms in 33.3-100% of the cases, depending on the disease severity, and BH4 responsiveness predictions show that only five patients with MHP-PKU and one patient with Mild-PKU were predicted to be BH4 responsive. In conclusion, we have characterized the mutational spectrum of PAH in the central region of China and have identified a novel mutation. The hotspot mutation information might be useful for screening, diagnosis and treatment of PKU.

Reassessing the significance of the PAH c.158G>A (p.Arg53His) variant in patients with hyperphenylalaninemia.

BACKGROUND: The accurate interpretation of sequence variation is critical for successful molecular diagnoses. It is also fundamental to the accurate diagnosis and treatment of phenylketonuria (PKU). This study aims to evaluate the significance of the c.158G>A (p.Arg53His) variant in the PAH gene, which was previously reported to be a pathogenic mutation that results in decreased phenylalanine hydroxylase enzyme activity in hyperphenylalaninemia (HPA) patients. METHODS: Seven unrelated Korean patients with HPA genotyped with the c.158G>A variant were included in this study. The variant c.158G>A was classified by the standards and guidelines for the interpretation of sequence variants by the American College of Medical Genetics and Genomics and the Association for Molecular Pathology. RESULTS: By both directly collecting genetic data and comprehensively reviewing the existing literature, we found that this variant is more appropriately classified as "Likely benign" rather than pathogenic. The allele's frequency is 2.57% in the general Korean population, which was greater than expected for phenylketonuria. This variant was observed to be homozygous in healthy subjects and was also observed in cis with other pathogenic variants. It is common in East Asian populations (especially in Koreans) compared to Western populations. There is a possibility that it causes decreased enzyme activity without leading to the full pathology of phenylketonuria. CONCLUSIONS: This study expands our understanding of the consequences of variation in PAH and its relationship to HPA.