Identification of Variants Underlying Phenylalanine Hydroxylase Deficiency in Saudi Arabia.

Background: Deleterious mutations in the human gene phenylalanine hydroxylase (PAH) encoding the phenylalanine hydroxylase enzyme give rise to classic phenylketonuria and hyperphenylalaninemia. Our study was designed to characterize the spectrum of variants in the PAH gene in Saudi patients. Materials and Methods: We screened a cohort of 72 Saudi patients with clinical and biochemical diagnoses of hyperphenylalaninemia at the largest tertiary care center in Saudi Arabia; the King Faisal Specialist Hospital and Research Center (KFSH&RC), Riyadh. All patient's charts were reviewed under an approved study by Institutional Review Board. Results: Twenty-one different PAH variants were identified among the 144 PAH alleles assessed by targeted gene sequencing. Within the studied cohort, 60 of 72 patients had homozygous mutations with the the remaining 12 being compound heterozygotes. The most prevalent of the disease alleles identified in this study was the p.(Arg252Trp) mutation, which accounted for 38 of 144 alleles (26.4%). With the high incidence of genetic disorders in the population, religiously permissible preventive reproductive measures are a priority in our practice. Prenatal diagnoses carried out on four fetuses revealed two that were homozygous for PAH pathogenic variants. In addition, pre-implantation genetic diagnoses were initiated for 19 families. Eight of these families completed more than one full cycle of treatment, from which one healthy newborn was delivered. Conclusions: This study describes the spectrum of PAH variants in the Saudi population and highlights the molecular heterogeneity underlying phenylketonuria and hyperphenylalaninemia. These results add to the existing knowledge about PAH variants in Middle Eastern Countries. These results can be further translated to provide: informed counseling; cascade carrier testing in extended family members; and pre-marital screening.

Hyperphenylalaninemias genotyping: Results of over 60 years of history in Lombardy, Italy.

BACKGROUND: Hyperphenylalaninemias (HPA) are due to several gene mutations, of which the PAH gene is the most frequently involved. Prevalence and incidence of disease vary between populations, with genotype/phenotype correlations not always capable to correctly predict disease severity. The aim of this study was to give an overview of PAH mutations among one of the largest cohort of patients among Europe, born in Lombardy (Italy) starting from late 1970 s and including over a 60 years of activity; furthermore, to evaluate and discuss identified genotype/phenotype correlations and related reliability. PATIENTS/METHODS: Eight hundred and twenty-six HPA patients in current follow-up at the San Paolo Hospital in Milan (Italy) were retrospectively reviewed, including molecular results and allelic phenotype and genotype values (attributed on the basis of the APV/GPV system) to verify genotype-phenotype correlations. RESULTS: A total of 166 different PAH variants were reviewed; of those, seven variants were identified as not previously described in literature. Most frequently reported variant was p.Ala403Val, followed by p.Arg261Gln, p.Val245Ala, IVS10-11 g>a, p.Tyr414Cys and p.Leu48Ser. Phenotype prediction, based on APV/GPV, matched the actual phenotype in most cases, but not always. CONCLUSION/DISCUSSION: The cohort of patients included in this study constitute a representative sample of the HPA population worldwide. Studies on this sample may allow to improve clinical and genetic evaluation performances for affected patients, consequently to develop personalized medicine interventions and provide more precise indications on the correct treatment approach based on the accumulated evidence, also in light of a prognostically reliable but not always conclusive APV/GPV system.

Mild hyperphenylalaninemia (hpa) presenting as orthostatic tremor: a case report.

BACKGROUND: Orthostatic tremor (OT) is a type of postural tremor of the lower extremities that has not been described in either phenylketonuria (PKU) or hyperphenylalaninemia (HPA). Because little is known about the clinical features and therapeutic responses of OT in mild HPA, we describe a mild HPA patient who presented with OT as an initial symptom. CASE PRESENTATION: A 22-year-old male was admitted for bilateral leg tremor while standing, with symptom onset eight months prior. One month before admission, the tremor disappeared in the left leg but persisted in the right leg. Electromyography recorded from the right gastrocnemius revealed a 6-8 Hz tremor, which appeared when the patient was standing and disappeared when he was resting or walking. Blood screening showed a phenylalanine/tyrosine ratio of 2.06 and a phenylalanine level of 140 µmol/L. Urine metabolic screening was negative. Whole-exome sequencing confirmed the presence of a compound heterozygous mutation, c.158G > A and c.728G > A, in phenylalanine hydroxylase (PAH) gene. After three months of levodopa/benserazide tablets (250 mg, tid) and a low-phenylalanine diet treatment, the tremor disappeared. CONCLUSIONS: Young-onset mild HPA is a relatively rare autosomal recessive metabolic disease, and slow OT is a rare clinical feature. Metabolic screening and genetic testing are the keys to early diagnosis and treatment. For adolescents and young adults, appropriate medication and long-term dietary therapy remain important treatments. This case expanded the disease spectrum of slow OT.

Genetic etiology and clinical challenges of phenylketonuria.

This review discusses the epidemiology, pathophysiology, genetic etiology, and management of phenylketonuria (PKU). PKU, an autosomal recessive disease, is an inborn error of phenylalanine (Phe) metabolism caused by pathogenic variants in the phenylalanine hydroxylase (PAH) gene. The prevalence of PKU varies widely among ethnicities and geographic regions, affecting approximately 1 in 24,000 individuals worldwide. Deficiency in the PAH enzyme or, in rare cases, the cofactor tetrahydrobiopterin results in high blood Phe concentrations, causing brain dysfunction. Untreated PKU, also known as PAH deficiency, results in severe and irreversible intellectual disability, epilepsy, behavioral disorders, and clinical features such as acquired microcephaly, seizures, psychological signs, and generalized hypopigmentation of skin (including hair and eyes). Severe phenotypes are classic PKU, and less severe forms of PAH deficiency are moderate PKU, mild PKU, mild hyperphenylalaninaemia (HPA), or benign HPA. Early diagnosis and intervention must start shortly after birth to prevent major cognitive and neurological effects. Dietary treatment, including natural protein restriction and Phe-free supplements, must be used to maintain blood Phe concentrations of 120-360 µmol/L throughout the life span. Additional treatments include the casein glycomacropeptide (GMP), which contains very limited aromatic amino acids and may improve immunological function, and large neutral amino acid (LNAA) supplementation to prevent plasma Phe transport into the brain. The synthetic BH4 analog, sapropterin hydrochloride (i.e., Kuvan®, BioMarin), is another potential treatment that activates residual PAH, thus decreasing Phe concentrations in the blood of PKU patients. Moreover, daily subcutaneous injection of pegylated Phe ammonia-lyase (i.e., pegvaliase; PALYNZIQ®, BioMarin) has promised gene therapy in recent clinical trials, and mRNA approaches are also being studied.

Next-generation probiotics as a therapeutic strategy for the treatment of phenylketonuria: a review.

Phenylketonuria (PKU) is a rare genetic disease that causes brain toxicity due to the inability of the body to convert dietary phenylalanine to tyrosine by the action of phenylalanine hydroxylase. The only treatment for PKU so far is lifelong dietary intervention to ensure normal human growth and neurodevelopment. However, in adults, low long-term adherence to this type of dietary intervention has been observed. Given the important role of the intestinal microbiota in the process of digestion and disease prevention, probiotics could be a therapeutic strategy to help degrade dietary phenylalanine, reducing its levels before ingestion. Genetically modified probiotics designed as live biotherapeutic agents for the treatment of specific diseases are sophisticated alternative therapeutic strategies. In this review, the focus is on demonstrating what has been elucidated so far about the use of next-generation probiotics as a therapeutic strategy in the treatment of individuals with PKU. The results described in the literature are encouraging and use genetically modified engineered probiotics showing efficacy both in vitro and in vivo. These probiotics appear to be suitable for meeting the unmet need for new drugs for PKU.

Treatment of a metabolic liver disease by in vivo genome base editing in adult mice.

CRISPR-Cas-based genome editing holds great promise for targeting genetic disorders, including inborn errors of hepatocyte metabolism. Precise correction of disease-causing mutations in adult tissues in vivo, however, is challenging. It requires repair of Cas9-induced double-stranded DNA (dsDNA) breaks by homology-directed mechanisms, which are highly inefficient in nondividing cells. Here we corrected the disease phenotype of adult phenylalanine hydroxylase (Pah)enu2 mice, a model for the human autosomal recessive liver disease phenylketonuria (PKU)1, using recently developed CRISPR-Cas-associated base editors2-4. These systems enable conversion of C∙G to T∙A base pairs and vice versa, independent of dsDNA break formation and homology-directed repair (HDR). We engineered and validated an intein-split base editor, which allows splitting of the fusion protein into two parts, thereby circumventing the limited cargo capacity of adeno-associated virus (AAV) vectors. Intravenous injection of AAV-base editor systems resulted in Pahenu2 gene correction rates that restored physiological blood phenylalanine (L-Phe) levels below 120 µmol/l [5]. We observed mRNA correction rates up to 63%, restoration of phenylalanine hydroxylase (PAH) enzyme activity, and reversion of the light fur phenotype in Pahenu2 mice. Our findings suggest that targeting genetic diseases in vivo using AAV-mediated delivery of base-editing agents is feasible, demonstrating potential for therapeutic application.

Informe de evaluación científica en la evidencia disponible: fenilcetonuria / Scientific evaluation report on the available evidence: phenylketonuria

INTRODUCCIÓN: La fenilcetonuria es una enfermedad congénita del metabolismo, causada en la mayoría de los casos por el déficit de la enzima hepática fenilalanina hidroxilasa responsable de transformar la fenilalanina a tirosina. Esto da como resultado concentraciones elevadas de fenilalanina y sus metabolitos, fenilacetato y fenilactato en la sangre. Además, al ser interferida la vía de transformación de fenilalanina en tirosina producto de que la fenilalanina hidroxilasa no se habrá formado bien, se traduciría en un retardo mental. La incidencia de fenilcetonuria es de 13.500 a 19.000 nacimientos en EE.UU. Este informe evalúa sapropterina para pacientes con fenilcetonuria que hayan mostrado responder a este tratamiento. Este tratamiento no cuenta con cobertura financiera en la actualidad. Sin embargo, los pacientes con fenilcetonuria cuentan con cobertura de alimentos y con un programa de screening neonatal. Se considerarán para su evaluación aquellas solicitudes realizadas conforme al Reglamento que establece el proceso destinado a determinar los diagnósticos y tratamientos de alto costo con sistema de protección financiera, según lo establecido en los artículos 7° y 8° de la Ley N° 20.850. Estas solicitudes no son vinculantes para el Ministerio de Salud, debiendo, sin embargo, tomar especialmente en cuenta aquellas solicitudes y opiniones que hayan sido realizadas por sus comisiones técnicas asesoras y por las asociaciones de pacientes incluidas en el Registro de Asociaciones de Pacientes que crea la Ley 20.850. De igual forma, para ser incorporadas en el proceso de evaluación científica de la evidencia, cada intervención debe cumplir con los criterios establecidos en el Artículo 6o del Reglamento mencionado, según lo indicado en el Numeral 9 del presente informe. TECNOLOGÍAS SANITARIA DE INTERÉS: Sapropterina es una versión sintética de la 6R-BH4 natural, que es un cofactor de las hidrolasas de fenilalanina, tirosina y triptófano. La justificación de la administración de sapropterina en pacientes con fenilcetonuria que responde a BH4 (tetrahidrobiopterina) es aumentar la actividad de la fenilalanina hidroxilasa defectuosa y así aumentar o restituir el metabolismo oxidativo de la fenilalanina lo suficiente para reducir o mantener los niveles plasmáticos de fenilalanina, evitar o reducir aún más la acumulación de fenilalanina y aumentar la tolerancia a la ingesta de fenilalanina en la dieta. EFICACIA DE LOS TRATAMIENTOS: Se encontraron tres revisiones sistemáticas, las cuales incluyen dos estudios controlados aleatorizados. Además, se encontraron otros cinco estudios con diferente diseño. De acuerdo a esta evidencia, sapropterina podría reducir la concentración de fenilalanina en la sangre y además podría aumentar la tolerancia a la fenilalanina, ambos resultados con una certeza de evidencia baja. ALTERNATIVAS DISPONIBLES: Sustituto proteico y alimentos hipoproteicos, los cuales están actualmente cubiertos por el programa de alimentación complementaria, tanto en niños como en adultos. RESULTADOS DE LA BÚSQUEDA DE EVIDENCIA: Se encontraron 3 revisiones sistemáticas (15­17) relevantes para la pregunta que se está intentando responder, dentro de las cuales se reportaron 2 ensayos controlados aleatorizados que abordaban la pregunta de interés. CONCLUSIÓN: Para dar cumplimiento al artículo 28° del Reglamento que establece el proceso destinado a determinar los diagnósticos y tratamientos de alto costo con Sistema de Protección Financiera, según lo establecido en los artículos 7°y 8° de la ley N°20.850, aprobado por el decreto N°13 del Ministerio de Salud, se concluye que el presente informe de evaluación se considera favorable, de acuerdo a lo establecido en el Título III. de las Evaluaciones Favorables de la Norma Técnica N° 0192 de este mismo Ministerio.

Informe de evaluación científica en la evidencia disponible: fenilcetonuria / Relatório de avaliação científica das evidências disponíveis: fenilcetonúria

INTRODUCCIÓN: La fenilcetonuria es una enfermedad congénita del metabolismo, causada en la mayoría de los casos por el déficit de la enzima hepática fenilalanina hidroxilasa (1,2), responsable de transformar la fenilalanina a tirosina, esto da como resultado concentraciones elevadas de fenilalanina y sus metabolitos, fenilacetato y fenilactato en sangre. Además la fenilalanina tendrá interferida su vía de transformación en tirosina, ya que la fenilalanina hidroxilasa no se habrá formado bien, lo que se traduce en un retardo mental. La incidencia de fenilcetonuria es de 13.500 a 19.000 nacimientos en EEUU. Las manifestaciones clínicas de la fenilcetonuria en los recién nacidos son raras, en general son asintomáticos y no puede causar síntomas hasta la infancia temprana. En los pacientes no tratados la enfermedad se manifiesta con la incapacidad intelectual, un área de cognición que es particularmente afectada es la función ejecutiva. Otros hallazgos incluyen anormalidades de la marcha y postura. TECNOLOGÍAS SANITARIA DE INTERÉS: Sapropterina: es una versión sintética de la 6R-BH4 natural, que es un cofactor de las hidrolasas de fenilalanina, tirosina y triptófano. La justificación de la administración de sapropterina en pacientes con fenilcetonuria que responde a BH4 (tetrahidrobiopterina) es aumentar la actividad de la fenilalanina hidroxilasa defectuosa y así aumentar o restituir el metabolismo oxidativo de la fenilalanina lo suficiente para reducir o mantener los niveles plasmáticos de fenilalanina, evitar o reducir aún más la acumulación de fenilalanina y aumentar la tolerancia a la ingesta de fenilalanina en la dieta. La justificación de la administración de la administración de sapropterina en pacientes con deficiencia de BH4 es restituir los niveles deficitarios, restableciendo la actividad de la fenilalanina hidroxilasa (EMA). EFICACIA DE LOS TRATAMIENTOS: La información sobre los efectos de la Sapropterina está basada en dos estudios. El resumen de los resultados es el siguiente. Los resultados arrojan que existe un beneficio del tratamiento de la Fenilcetonuria con Sapropterina para pacientes que responden a la misma. El tratamiento resultó eficaz en disminuir considerablemente la concentración de Fenililanina en sangre (3,4) y una tolerancia mayor a la misma. A pesar de lo anterior, cabe destacar que es necesario estudios que consideren la efectividad de la Sapropterina en el largo plazo, que estudie desenlaces finales y no solo intermedios. A pesar de que existen estudios que consideran un período relativamente largo de estudio (5), dando indicios de buenos resultados del tratamiento con Sapropterina, se necesitan estudios que aborden la investigación con mayor control sobre la población (ensayos aleatorios controlados) para evaluar la efectividad en el largo plazo con mayor certeza. Es también importante mayor investigación que genere evidencia sobre los efectos del tratamiento según sea la severidad de la enfermedad, ya que los estudios encontrados no permiten tales aseveraciones. ALTERNATIVAS DISPONIBLES: Dieta: Sustituto proteico y Alimentos hipoprotéicos. RESULTADOS DE LA BÚSQUEDA DE EVIDENCIA: La información sobre los efectos de la Sapropterina está basada en dos estudios. El resumen de los resultados es el siguiente. Los resultados arrojan que existe un beneficio del tratamiento de la Fenilcetonuria con Sapropterina para pacientes que responden a la misma. El tratamiento resultó eficaz en disminuir considerablemente la concentración de Fenilalanina en sangre y una tolerancia mayor a la misma. A pesar de lo anterior, cabe destacar que es necesario estudios que consideren la efectividad de la Sapropterina en el largo plazo, que estudie desenlaces finales y no solo intermedios. A pesar de que existen estudios que consideran un período relativamente largo de estudio, dando indicios de buenos resultados del tratamiento con Sapropterina, se necesitan estudios que aborden la investigación con mayor control sobre la población (ensayos aleatorios controlados) para evaluar la efectividad en el largo plazo con mayor certeza. Es también importante mayor investigación que genere evidencia sobre los efectos del tratamiento según sea la severidad de la enfermedad, ya que los estudios encontrados no permiten tales aseveraciones. CONCLUSIÓN: Para dar cumplimiento al artículo 28° del Reglamento que establece el proceso destinado a determinar los diagnósticos y tratamientos de alto costo con Sistema de Protección Financiera, según lo establecido en los artículos 7°y 8° de la ley N°20.850, aprobado por el decreto N°13 del Ministerio de Salud, se concluye que el presente informe de evaluación se considera no favorable, dado que su impacto presupuestario supera el 80% del fondo disponible, de acuerdo a lo establecido en el Título III. de las Evaluaciones Favorables de la Norma Técnica N° 0192 de este mismo Ministerio.

A novel approach for enzyme replacement therapy. The use of phenylalanine hydroxylase-based fusion proteins for the treatment of phenylketonuria.

Metabolic diseases arise from mutations in key enzymes of major metabolic pathways. One promising approach for the treatment of such diseases is based on the administration of a wild-type enzyme to substitute the activity of the impaired enzyme by the use of enzyme replacement therapy, yet it is important to deliver this enzyme to the specific deficient tissue. We suggest a new concept for the treatment of metabolic diseases using fusion proteins. We examined the feasibility of this concept in the well characterized metabolic disease, phenylketonuria (PKU), which results from a mutation in the liver enzyme phenylalanine hydroxylase (PAH). PAH is a key enzyme in the metabolic pathway of phenylalanine. Deficiency in PAH leads to high and persistent levels of this amino acid in the plasma of PKU patients, causing permanent neurological damage. Currently a low protein diet is still considered the only effective treatment for most PKU patients. To restore PAH activity in the liver of PKU patients, we constructed PAH-based fusion proteins with delivery moieties based on the HIV-transactivator of transcription peptide, and fragments of human hepatocyte growth factor aiming to specifically target PAH to the liver. We show that these new fusion proteins can be delivered into a variety of human liver cell lines and retain PAH activity after being internalized. We also show that plasma phenylalanine levels were dramatically lowered in mice treated with PAH-based fusion proteins after intravenous administration. We therefore suggest an alternative concept for the treatment of PKU using targeted fusion proteins, which may also be applied to the treatment of other metabolic diseases.

Trends in enzyme therapy for phenylketonuria.

Phenylketonuria (PKU) is an inborn error of amino acid metabolism caused by phenylalanine hydroxylase (PAH) deficiency. Dietary treatment has been the cornerstone for controlling systemic phenylalanine (Phe) levels in PKU for the past 4 decades. Over the years, it has become clear that blood Phe concentration needs to be controlled for the life of the patient, a difficult task taking into consideration that the diet becomes very difficult to maintain. Therefore alternative models of therapy are being pursued. This review describes the progress made in enzyme replacement therapy for PKU. Two modalities are discussed, PAH and phenylalanine ammonia-lyase PAH. Developing stable and functional forms of both enzymes has proven difficult, but recent success in producing polyethylene glycol-modified forms of active and stable PAH shows promise.

Toward PKU enzyme replacement therapy: PEGylation with activity retention for three forms of recombinant phenylalanine hydroxylase.

Phenylketonuria (PKU) is a disease in which phenylalanine and phenylalanine-derived metabolites build up to neurotoxic levels due to mutations in the phenylalanine hydroxylase gene (PAH). Enzyme replacement therapy is a viable option to supply active PAH. However, the inherent protease sensitivity and potential immunogenicity of PAH have precluded adoption of this approach. In this report, we have used polyethylene glycol derivatization (PEGylation) to produce protected forms of PAH for potential therapeutic use. Three recombinantly produced PAH enzymes were reacted with activated PEG species, with the aim of developing a stable and active PKU enzyme replacement. Tetrameric full-length human PAH, dimeric double-truncated (DeltaN102-DeltaC428) human PAH, and monomeric Chromobacterium violaceum PAH were PEGylated with succinimidyl succinate polyethylene glycol of molecular weight 5000 or 20,000 Da. Characterization of the PEGylated species was accomplished with MALDI-TOF mass spectrometry, SDS-PAGE, and specific activity measurements using ESI mass spectrometry. All PEG-derivatized PAH species retained catalytic activity, and, at low numbers of PEG molecules attached, these PEGylated PAH proteins were found to be more active and more stable than their non-derivatized PAH counterparts.

Expression of phenylalanine hydroxylase (PAH) in erythrogenic bone marrow does not correct hyperphenylalaninemia in Pah(enu2) mice.

BACKGROUND: Treatment of many inherited liver enzyme deficiencies requires the removal of toxic intermediate metabolites from the blood of affected individuals. We propose that circulating toxins can be adequately cleared and disease phenotype influenced by enzyme expressed in tissues other than the liver, such as bone marrow. Our specific hypothesis was that phenylalanine hydroxylase (PAH) expressed in bone marrow would lower blood phenylalanine levels in hyperphenylalaninemic Pah(enu2) mice, a model of human phenylketonuria (PKU). METHODS: Germline-modified marrow PAH-expressing mice were developed using a transgene that contained the mouse liver PAH cDNA under the transcriptional control of a human beta-globin promoter. Marrow PAH-expressing mice were bred to Pah(enu2) mice to generate progeny that lacked liver PAH activity but expressed PAH in bone marrow. RESULTS: Marrow PAH expression did not affect the health, function, or reproductive capacity of transgenic animals. Hyperphenylalaninemia persisted in transgenic Pah(enu2) homozygous mice despite PAH activity in marrow lysates, and was not altered following supplementation with tetrahydrobiopterin (BH(4)), a required cofactor for PAH. PAH activity measured in intact marrow cells was significantly lower than in marrow lysates; no such difference was measured in isolated hepatocytes vs. liver homogenate. CONCLUSIONS: Marrow PAH expression did not correct hyperphenylalaninemia in Pah(enu2) mice. Phenylalanine clearance may have been limited by the natural perfusion rate of the marrow compartment, by insufficient PAH expression in marrow, or by other cellular factors affecting phenylalanine metabolism in intact marrow cells. Differences in PAH activity measured in intact marrow cells vs. cell lysates suggest that hepatocytes and PAH-expressing marrow cells are fundamentally different in their ability to metabolize phenylalanine. The efficacy of bone-marrow-directed gene therapy as a metabolic sink in the treatment of phenylketonuria may be limited, although further experiments with greater marrow PAH expression levels will be necessary to definitively prove this conclusion.