Significance of utilizing in silico structural analysis and phenotypic data to characterize phenylalanine hydroxylase variants: A PAH landscape.

Phenylketonuria (PKU) is a genetic disorder caused by variations in the phenylalanine hydroxylase (PAH) gene. Among the 3369 reported PAH variants, 33.7% are missense alterations. Unfortunately, 30% of these missense variants are classified as variants of unknown significance (VUS), posing challenges for genetic risk assessment. In our study, we focused on analyzing 836 missense PAH variants following the American College of Medical Genetics and Genomics/Association for Molecular Pathology (ACMG/AMP) guidelines specified by ClinGen PAH Variant Curation Expert Panel (VCEP) criteria. We utilized and compared variant annotator tools like Franklin and Varsome, conducted 3D structural analysis of PAH, and examined active and regulatory site hotspots. In addition, we assessed potential splicing effect of apparent missense variants. By evaluating phenotype data from 22962 PKU patients, our aim was to reassess the pathogenicity of missense variants. Our comprehensive approach successfully reclassified 309 VUSs out of 836 missense variants as likely pathogenic or pathogenic (37%), upgraded 370 likely pathogenic variants to pathogenic, and reclassified one previously considered likely benign variant as likely pathogenic. Phenotypic information was available for 636 missense variants, with 441 undergoing 3D structural analysis and active site hotspot identification for 180 variants. After our analysis, only 6% of missense variants were classified as VUSs, and three of them (c.23A>C/p.Asn8Thr, c.59_60delinsCC/p.Gln20Pro, and c.278A >T/p.Asn93Ile) may be influenced by abnormal splicing. Moreover, a pathogenic variant (c.168G>T/p.Glu56Asp) was identified to have a risk exceeding 98% for modifications of the consensus splice site, with high scores indicating a donor loss of 0.94. The integration of ACMG/AMP guidelines with in silico structural analysis and phenotypic data significantly reduced the number of missense VUSs, providing a strong basis for genetic counseling and emphasizing the importance of metabolic phenotype information in variant curation. This study also sheds light on the current landscape of PAH variants.

Clinical and biochemical footprints of congenital disorders of glycosylation: Proposed nosology.

We have identified 200 congenital disorders of glycosylation (CDG) caused by 189 different gene defects and have proposed a classification system for CDG based on the mode of action. This classification includes 8 categories: 1. Disorders of monosaccharide synthesis and interconversion, 2. Disorders of nucleotide sugar synthesis and transport, 3. Disorders of N-linked protein glycosylation, 4. Disorders of O-linked protein glycosylation, 5. Disorders of lipid glycosylation, 6. Disorders of vesicular trafficking, 7. Disorders of multiple glycosylation pathways and 8. Disorders of glycoprotein/glycan degradation. Additionally, using information from IEMbase, we have described the clinical involvement of 19 organs and systems, as well as essential laboratory investigations for each type of CDG. Neurological, dysmorphic, skeletal, and ocular manifestations were the most prevalent, occurring in 81%, 56%, 53%, and 46% of CDG, respectively. This was followed by digestive, cardiovascular, dermatological, endocrine, and hematological symptoms (17-34%). Immunological, genitourinary, respiratory, psychiatric, and renal symptoms were less frequently reported (8-12%), with hair and dental abnormalities present in only 4-7% of CDG. The information provided in this study, including our proposed classification system for CDG, may be beneficial for healthcare providers caring for individuals with metabolic conditions associated with CDG.

Consensus guidelines for the diagnosis and management of succinic semialdehyde dehydrogenase deficiency.

Succinic semialdehyde dehydrogenase deficiency (SSADHD) (OMIM #271980) is a rare autosomal recessive metabolic disorder caused by pathogenic variants of ALDH5A1. Deficiency of SSADH results in accumulation of γ-aminobutyric acid (GABA) and other GABA-related metabolites. The clinical phenotype of SSADHD includes a broad spectrum of non-pathognomonic symptoms such as cognitive disabilities, communication and language deficits, movement disorders, epilepsy, sleep disturbances, attention problems, anxiety, and obsessive-compulsive traits. Current treatment options for SSADHD remain supportive, but there are ongoing attempts to develop targeted genetic therapies. This study aimed to create consensus guidelines for the diagnosis and management of SSADHD. Thirty relevant statements were initially addressed by a systematic literature review, resulting in different evidence levels of strength according to the Grading of Recommendations Assessment, Development, and Evaluation (GRADE) criteria. The highest level of evidence (level A), based on randomized controlled trials, was unavailable for any of the statements. Based on cohort studies, Level B evidence was available for 12 (40%) of the statements. Thereupon, through a process following the Delphi Method and directed by the Appraisal of Guidelines for Research and Evaluation (AGREE II) criteria, expert opinion was sought, and members of an SSADHD Consensus Group evaluated all the statements. The group consisted of neurologists, epileptologists, neuropsychologists, neurophysiologists, metabolic disease specialists, clinical and biochemical geneticists, and laboratory scientists affiliated with 19 institutions from 11 countries who have clinical experience with SSADHD patients and have studied the disorder. Representatives from parent groups were also included in the Consensus Group. An analysis of the survey's results yielded 25 (83%) strong and 5 (17%) weak agreement strengths. These first-of-their-kind consensus guidelines intend to consolidate and unify the optimal care that can be provided to individuals with SSADHD.

Does hyperphenylalaninemia induce brain glucose hypometabolism? Cerebral spinal fluid findings in treated adult phenylketonuric patients.

Despite numerous studies in human patients and animal models for phenylketonuria (PKU; OMIM#261600), the pathophysiology of PKU and the underlying causes of brain dysfunction and cognitive problems in PKU patients are not well understood. In this study, lumbar cerebral spinal fluid (CSF) was obtained immediately after blood sampling from early-treated adult PKU patients who had fasted overnight. Metabolite and amino acid concentrations in the CSF of PKU patients were compared with those of non-PKU controls. The CSF concentrations and CSF/plasma ratios for glucose and lactate were found to be below normal, similar to what has been reported for glucose transporter1 (GLUT1) deficiency patients who exhibit many of the same clinical symptoms as untreated PKU patients. CSF glucose and lactate levels were negatively correlated with CSF phenylalanine (Phe), while CSF glutamine and glutamate levels were positively correlated with CSF Phe levels. Plasma glucose levels were negatively correlated with plasma Phe concentrations in PKU subjects, which partly explains the reduced CSF glucose concentrations. Although brain glucose concentrations are unlikely to be low enough to impair brain glucose utilization, it is possible that the metabolism of Phe in the brain to produce phenyllactate, which can be transported across the blood-brain barrier to the blood, may consume glucose and/or lactate to generate the carbon backbone for glutamate. This glutamate is then converted to glutamine and carries the Phe-derived ammonia from the brain to the blood. While this mechanism remains to be tested, it may explain the correlations of CSF glutamine, glucose, and lactate concentrations with CSF Phe.

CYP2U1: An emerging treatable neurometabolic disease with cerebral folate deficiency in 2 Chinese brothers.

With the rapid advancement of medical technologies in genomic and molecular medicine, the number of treatable neurometabolic diseases is quickly expanding. Spastic paraplegia 56 (SPG56), one of the severe autosomal recessive forms of neurodegenerative disorders caused by pathogenic variants in the CYP2U1 gene, has no reported specific targeted treatment yet. Here we report 2 Chinese brothers with CYP2U1 bi-allelic pathogenic variants with cerebral folate deficiency who were treated for over a decade with folinic acid supplement. Patients have remained stable under therapy.

Clinical and biochemical footprints of inherited metabolic disease. XVI. Hematological abnormalities.

Many classical inherited metabolic diseases (IMDs) are associated with significant hematological complications such as anemia or thrombosis. While these may not be the prominent presenting feature of these conditions, management of these issues is important for optimal outcomes in people with IMDs. Some disorders that are included in the nosology of inherited metabolic disorders, such as inherited disorders of red cell energy metabolism, have purely hematological features, and have typically been cared for by a hematologist. In the 16th issue of the Footprints series, we identified 265 IMDs associated with hematological abnormalities. We review the major hematological manifestations of IMDs, suggest further investigation of hematological findings, and discuss treatment options available for specific hematological complications of IMDs.

Clinical and biochemical footprints of inherited metabolic diseases. XV. Epilepsies.

We provide a comprehensive overview of inherited metabolic disorders (IMDs) in which epilepsy is a prominent manifestation. Our unique database search has identified 256 IMDs associated with various types of epilepsies, which we classified according to the classic pathophysiology-based classification of IMDs, and according to selected seizure-related factors (neonatal seizures, infantile spasms, myoclonic seizures, and characteristic EEG patterns) and treatability for the underlying metabolic defect. Our findings indicate that inherited metabolic epilepsies are more likely to present in the neonatal period, with infantile spasms or myoclonic seizures. Additionally, the ∼20% of treatable inherited metabolic epilepsies found by our search were mainly associated with the IMD groups of "cofactor and mineral metabolism" and "Intermediary nutrient metabolism." The information provided by this study, including a comprehensive list of IMDs with epilepsy stratified according to age of onset, and seizure type and characteristics, along with an overview of the key clinical features and proposed diagnostic and therapeutic approaches, may benefit any epileptologist and healthcare provider caring for individuals with metabolic conditions.

Clinical and biochemical footprints of inherited metabolic diseases. XIV. Metabolic kidney diseases.

Kidney disease is a global health burden with high morbidity and mortality. Causes of kidney disease are numerous, extending from common disease groups like diabetes and arterial hypertension to rare conditions including inherited metabolic diseases (IMDs). Given its unique anatomy and function, the kidney is a target organ in about 10% of known IMDs, emphasizing the relevant contribution of IMDs to kidney disease. The pattern of injury affects all segments of the nephron including glomerular disease, proximal and distal tubular damage, kidney cyst formation, built-up of nephrocalcinosis and stones as well as severe malformations. We revised and updated the list of known metabolic etiologies associated with kidney involvement and found 190 relevant IMDs. This represents the 14th of a series of educational articles providing a comprehensive and revised list of metabolic differential diagnoses according to system involvement.

Clinical and biochemical footprints of inherited metabolic diseases. XIII. Respiratory manifestations.

At any age, respiratory manifestations are a major cause of increased morbidity and mortality of inherited metabolic diseases (IMDs). Type and severity are extremely variable, this depending on the type of the underlying disorder. Symptoms and signs originating from upper or lower airways and/or thoracic wall and/or respiratory muscles involvement can occur either at presentation or in the late clinical course. Acute respiratory symptoms can trigger metabolic decompensation which, in turn, makes airway symptoms worse, creating a vicious circle. We have identified 181 IMDs associated with various types of respiratory symptoms which were classified into seven groups according to the type of clinical manifestations affecting the respiratory system: (i) respiratory failure, (ii) restrictive lung disease, (iii) interstitial lung disease, (iv) lower airway disease, (v) upper airway obstruction, (vi) apnea, and (vii) other. We also provided a list of investigations to be performed based on the respiratory phenotypes and indicated the therapeutic strategies currently available for IMD-associated airway disease. This represents the thirteenth issue in a series of educational summaries providing a comprehensive and updated list of metabolic differential diagnoses according to system involvement.

Prevalence of DDC genotypes in patients with aromatic L-amino acid decarboxylase (AADC) deficiency and in silico prediction of structural protein changes.

Aromatic L-amino acid decarboxylase (AADC) deficiency is a rare autosomal recessive genetic disorder affecting the biosynthesis of dopamine, a precursor of both norepinephrine and epinephrine, and serotonin. Diagnosis is based on the analysis of CSF or plasma metabolites, AADC activity in plasma and genetic testing for variants in the DDC gene. The exact prevalence of AADC deficiency, the number of patients, and the variant and genotype prevalence are not known. Here, we present the DDC variant (n = 143) and genotype (n = 151) prevalence of 348 patients with AADC deficiency, 121 of whom were previously not reported. In addition, we report 26 new DDC variants, classify them according to the ACMG/AMP/ACGS recommendations for pathogenicity and score them based on the predicted structural effect. The splice variant c.714+4A>T, with a founder effect in Taiwan and China, was the most common variant (allele frequency = 32.4%), and c.[714+4A>T];[714+4A>T] was the most common genotype (genotype frequency = 21.3%). Approximately 90% of genotypes had variants classified as pathogenic or likely pathogenic, while 7% had one VUS allele and 3% had two VUS alleles. Only one benign variant was reported. Homozygous and compound heterozygous genotypes were interpreted in terms of AADC protein and categorized as: i) devoid of full-length AADC, ii) bearing one type of AADC homodimeric variant or iii) producing an AADC protein population composed of two homodimeric and one heterodimeric variant. Based on structural features, a score was attributed for all homodimers, and a tentative prediction was advanced for the heterodimer. Almost all AADC protein variants were pathogenic or likely pathogenic.

Clinical and biochemical footprints of inherited metabolic diseases. XII. Immunological defects.

Immunological problems are increasingly acknowledged manifestations in many inherited metabolic diseases (IMDs), ranging from exaggerated inflammation, autoimmunity and abnormal cell counts to recurrent microbial infections. A subgroup of IMDs, the congenital disorders of glycosylation (CDG), includes CDG types that are even classified as primary immunodeficiencies. Here, we reviewed the list of metabolic disorders reported to be associated with various immunological defects and identified 171 IMDs accompanied by immunological manifestations. Most IMDs are accompanied by immune dysfunctions of which immunodeficiency and infections, innate immune defects, and autoimmunity are the most common abnormalities reported in 144/171 (84%), 44/171 (26%) and 33/171 (19%) of IMDs with immune system involvement, respectively, followed by autoinflammation 17/171 (10%). This article belongs to a series aiming at creating and maintaining a comprehensive list of clinical and metabolic differential diagnoses according to organ system involvement.

Clinical and biochemical footprints of inherited metabolic disorders. XI. Gastrointestinal symptoms.

Inherited metabolic disorders presenting with gastrointestinal (GI) symptoms are characterized by the dysfunction of the esophagus, stomach, small and large intestines, and pancreas. We have summarized associations of signs and symptoms in 339 inherited metabolic diseases presenting with GI symptoms. Feeding difficulties represent the most common abnormality reported for IMDs with GI involvement (37%) followed by intestinal problems (30%), vomiting (22%), stomach and pancreas involvement (8% each), and esophagus involvement (4%). This represents the eleventh of a series of articles attempting to create and maintain a comprehensive list of clinical and metabolic differential diagnoses according to system involvement.

Spectrum of DDC variants causing aromatic l-amino acid decarboxylase (AADC) deficiency and pathogenicity interpretation using ACMG-AMP/ACGS recommendations.

Pathogenic variants in dopa decarboxylase (DDC), the gene encoding the aromatic l-amino acid decarboxylase (AADC) enzyme, lead to a severe deficiency of neurotransmitters, resulting in neurological, neuromuscular, and behavioral manifestations clinically characterized by developmental delays, oculogyric crises, dystonia, and severe neurologic dysfunction in infancy. Historically, therapy has been aimed at compensating for neurotransmitter abnormalities, but response to pharmacologic therapy varies, and in most cases, the therapy shows little or no benefit. A novel human DDC gene therapy was recently approved in the European Union that targets the underlying genetic cause of the disorder, providing a new treatment option for patients with AADC deficiency. However, the applicability of human DDC gene therapy depends on the ability of laboratories and clinicians to interpret the results of genetic testing accurately enough to diagnose the patient. An accurate interpretation of genetic variants depends in turn on expert-guided curation of locus-specific databases. The purpose of this research was to identify previously uncharacterized DDC variants that are of pathologic significance in AADC deficiency as well as characterize and curate variants of unknown significance (VUSs) to further advance the diagnostic accuracy of genetic testing for this condition. DDC variants were identified using existing databases and the literature. The pathogenicity of the variants was classified using modified American College of Medical Genetics and Genomics/Association for Molecular Pathology/Association for Clinical Genomic Science (ACMG-AMP/ACGS) criteria. To improve the current variant interpretation recommendations, in silico variant interpretation tools were combined with structural 3D modeling of protein variants and applied comparative analysis to predict the impact of the variant on protein function. A total of 422 variants were identified (http://biopku.org/home/pnddb.asp). Variants were identified on nearly all introns and exons of the DDC gene, as well as the 3' and 5' untranslated regions. The largest percentage of the identified variants (48%) were classified as missense variants. The molecular effects of these missense variants were then predicted, and the pathogenicity of each was classified using a number of variant effect predictors. Using ACMG-AMP/ACGS criteria, 7% of variants were classified as pathogenic, 32% as likely pathogenic, 58% as VUSs of varying subclassifications, 1% as likely benign, and 1% as benign. For 101 out of 108 reported genotypes, at least one allele was classified as pathogenic or likely pathogenic. In silico variant pathogenicity interpretation tools, combined with structural 3D modeling of variant proteins and applied comparative analysis, have improved the current DDC variant interpretation recommendations, particularly of VUSs.

Clinical and biochemical footprints of inherited metabolic diseases. IX. Metabolic ear disease.

Damages to the ear are very diverse and can depend on the type of inherited metabolic diseases (IMD). Indeed, IMDs can affect all parts of the auditory system, from the outer ear to the central auditory process. We have identified 219 IMDs associated with various types of ear involvement which we classified into five groups according to the lesion site of the auditory system: congenital external ear abnormalities, acquired external ear abnormalities, middle ear involvement, inner ear or retrocochlear involvement, and unspecified hearing loss. This represents the ninth issue in a series of educational summaries providing a comprehensive and updated list of metabolic differential diagnoses according to system involvement.

Clinical and biochemical footprints of inherited metabolic disorders: X. Metabolic myopathies.

Metabolic myopathies are characterized by the deficiency or dysfunction of essential metabolites or fuels to generate energy for muscle contraction; they most commonly manifest with neuromuscular symptoms due to impaired muscle development or functioning. We have summarized associations of signs and symptoms in 358 inherited metabolic diseases presenting with myopathies. This represents the tenth of a series of articles attempting to create and maintain a comprehensive list of clinical and metabolic differential diagnoses according to system involvement.

Clinical and biochemical footprints of inherited metabolic diseases. VIII. Neoplasias.

Cancer, caused by multiple cumulative pathogenic variants in tumor suppressor genes and proto-oncogenes, is a leading cause of mortality worldwide. The uncontrolled and rapid cell growth of the tumors requires a reprogramming of the complex cellular metabolic network to favor anabolism. Adequate management and treatment of certain inherited metabolic diseases might prevent the development of certain neoplasias, such as hepatocellular carcinoma in tyrosinemia type 1 or hepatocellular adenomas in glycogen storage disorder type 1a. We reviewed and updated the list of known metabolic etiologies associated with various types of benign and malignant neoplasias, finding 64 relevant inborn errors of metabolism. This is the eighth article of the series attempting to create a comprehensive list of clinical and metabolic differential diagnosis by system involvement.

Clinical and biochemical footprints of inherited metabolic disorders. VII. Ocular phenotypes.

Ocular manifestations are observed in approximately one third of all inherited metabolic disorders (IMDs). Although ocular involvement is not life-threatening, it can result in severe vision loss, thereby leading to an additional burden for the patient. Retinal degeneration with or without optic atrophy is the most frequent phenotype, followed by oculomotor problems, involvement of the cornea and lens, and refractive errors. These phenotypes can provide valuable clues that contribute to its diagnosis. In this issue we found 577 relevant IMDs leading to ophthalmologic manifestations. This article is the seventh of a series attempting to create and maintain a comprehensive list of clinical and metabolic differential diagnoses according to system involvement.

Importance of the long non-coding RNA (lncRNA) transcript HULC for the regulation of phenylalanine hydroxylase and treatment of phenylketonuria.

More than 1280 variants in the phenylalanine hydroxylase (PAH) gene are responsible for a broad spectrum of phenylketonuria (PKU) phenotypes. While the genotype-phenotype correlation is reaching 88%, for some inconsistent phenotypes with the same genotype additional factors like tetrahydrobiopterin (BH4), the PAH co-chaperone DNAJC12, phosphorylation of the PAH residues or epigenetic factors may play an important role. Very recently an additional player, the long non-coding RNA (lncRNA) transcript HULC, was described to regulate PAH activity and enhance residual enzyme activity of some PAH variants (e.g., the most common p.R408W) by using HULC mimics. In this review we present an overview of the lncRNA function and in particular the interplay of the HUCL transcript with the PAH and discuss potential applications for the future treatment of some PKU patients.