Identification of mutations in the pah gene in pku patients in the state of mato grosso / Identificação de mutações no gene da pah em pacientes com fenilcetonúria do estado de mato grosso

ABSTRACT Objective: To identify phenylalanine hydroxylase (PAH) mutations in patients with phenylketonuria (PKU) from the Newborn Screening Service in Mato Grosso, Midwest Brazil. Methods: This is a cross-sectional descriptive study. The sample consisted of 19 PKU patients diagnosed by newborn screening. Molecular analysis: DNA extraction using the "salting-out" method. Detection of IVS10nt-11G>A, V388M, R261Q, R261X, R252W, and R408W mutations by the restriction fragment length polymorphism (RFLP) technique. Results: Two mutant alleles were identified in four patients (21.1%), one allele in five patients (26.2%), and none in the remaining ten patients (52.6%). A total of 13/38 alleles were detected, corresponding to 34.2% of the PAH alleles present. The most prevalent variant was V388M (13.2% of the alleles), followed by R261Q (10.1%) and IVS10nt-11G>A (7.9%). Three variants (R261X, R252W, and R408W) were not found. The most frequent mutation types were: missense mutation in eight alleles (18.4%) and splicing in four alleles (10.5%). The model proposed by Guldberg to determine a genotype/phenotype correlation was applied to four classical PKU patients with two identified mutations. In three of them, the predicted moderate/moderate or moderate PKU phenotype did not coincide with the actual diagnosis. The prediction coincided with the diagnosis of one classic PKU patient. The estimated incidence of PKU for Mato Grosso, Brazil, was 1:33,342 live births from 2003 to 2015. Conclusion: The only mutations found in the analyzed samples were the IVS10nt-11G>A, V388M, and R261Q. The genotype/phenotype correlation only occurred in four (5.3%) patients.

RESUMO Objetivo: Identificar mutações da fenilalanina hidroxilase (PAH) em pacientes com PKU (fenilcetonúria) do Serviço de Triagem Neonatal em Mato Grosso. Métodos: Estudo de corte transversal. Amostra composta de 19 pacientes com PKU através do exame de triagem neonatal biológica. Análise molecular: a) extração de DNA pela metodologia "salting out". B) detecção de mutações IVS10nt-11G>A, V388M, R261Q, R261X, R252W e R408W pela técnica de polimorfismo de comprimento de fragmento de restrição (RFLP). Resultados: Dois alelos foram identificados em quatro pacientes (21,1%), um alelo em cinco pacientes (26,2%) e nenhum nos dez pacientes restantes (52,6%). Um total de 13/38 alelos foram identificados, correspondendo a 34,2% dos alelos PAH presentes. A variante mais prevalente foi a V388M (13,2% dos alelos), seguida de R261Q (10,1%) e IVS10nt-11G>A (7,9%). Três variantes (R261X, R252W e R408W) não foram encontradas. Os tipos de mutações mais frequentes foram: troca de sentido em oito alelos (18,4%) e emenda em quatro alelos (10,5%). O modelo proposto por Guldberg para determinar uma correlação genótipo/fenótipo foi aplicado para quatro pacientes clássicos de PKU, com duas mutações identificadas. Em três, o fenótipo previsto de PKU moderada/moderada ou moderada não coincidiu com o diagnóstico real. A predição coincidiu com o diagnóstico de um paciente PKU clássico. A incidência de PKU estimada para Mato Grosso, Brasil foi de 1:33.342 nascidos vivos para o período de 2003 a 2015. Conclusões: Foram encontradas apenas as mutações IVS10nt-11G>A, V388M, R261Q nas amostras analisadas. A correlação genótipo/fenótipo ocorreu em quatro (5,3%) pacientes.

Fenilcetonúria: aspectos genéticos, diagnóstico e tratamento / Phenylketonuria: genetic aspects, diagnosis and treatment

A fenilcetonúria é uma doença genética e metabólica, com bom prognóstico caso seja detectada e tratada precocemente. É a mais frequente entre os distúrbios metabólicos com significativa implicação clínica. Ela é detectada precocemente pelo Teste do Pezinho na triagem neonatal, e o tratamento padrão consiste em dieta restritiva. Este estudo teve por finalidade informar e atualizar os profissionais da área da saúde sobre base genético-clínica da fenilcetonúria, com destaque para sua etiologia e aconselhamento genético; diagnóstico com enfoque no histórico da triagem neonatal; e tratamento − em especial o dietético. Foi utilizada como fonte a literatura científica especializada, publicada principalmente nos últimos 5 anos.(AU)

Phenylketonuria is a genetic and metabolic disease, with good prognosis if early detected and treated. It is the most frequent among metabolic disorders, with significant clinical implications. It is detected early by Guthrie test in the neonatal screening, and the standard treatment consists of a restrictive diet. This study is aimed at informing and updating healthcare professionals on: 1) genetic-clinical basis of phenylketonuria, highlighting its etiology and genetic counseling, 2) diagnosis focusing on the history of newborn screening, and 3) treatment, in particular the dietetic one. The specialized scientific literature, particularly that published in the last five years, was used as a source.(AU)

¿Qué debe saber el pediatra de las hiperfenilalaninemias? / What should the paediatrician know about hyperphenylalaninaemia?

Las hiperfenilalaninemias se definen por un nivel sanguíneo de fenilalanina sobre 2 mg/dl. La principal causa es una mutación en el gen que codifica la fenilalanina hidroxilasa que cataliza la reacción que transforma la fenilalanina en tirosina. Las hiperfenilalaninemias se clasifican en benignas o leves, y las fenilcetonurias en leves, moderadas y clásicas. Debido a que su detección más allá del periodo neonatal causa retardo mental severo, desde 1992 en Chile su detección, junto con la del hipotirodismo congénito, es parte del Programa Nacional de Pesquisa Neonatal. Este artículo pretende responder las preguntas más comunes que se puede hacer el pediatra cuando enfrenta a un paciente con hiperfenilalaninemias.

Hyperphenylalaninaemias are defined by a blood phenylalanine over 2 mg/dl. The main cause is due to a mutation in the gene that codes the phenylalanine hydroxylase that catalyses the reaction that converts phenylalanine into tyrosine. The hyperphenylalaninaemias are classified into benign or mild hyperphenylalaninaemias, or mild, moderate or classic phenylketonurias. Due to its delayed detection outside the neonatal period it causes severe mental retardation. Its detection along with congenital hypothyroidism has been part of the National Neonatal Screening Program since 1992 in Chile. This article aims to answer the most common questions asked by the paediatrician when faced with a patient with hyperphenylalaninaemias.

The proportion of tetrahydrobiopterin deficiency and PAH gene deficiency variants among cases with hyperphenyalaninemia in Western Iran.

BACKGROUND: Defects either in phenylalanine hydroxylase (PheOH) or in the production and recycling of its cofactor (tetrahydrobiopterin [BH4]) are the causes of primary hyperphenylalaninemia (HPA). The aim of our study was to investigate the current status of different variants of HPA Kurdish patients in Kermanshah province, Iran. MATERIALS AND METHODS: From 33 cases enrolled in our study, 32 were identified as HPA patients. Reassessing of pre‑treatment phenylalanine concentrations and the analysis of urinary pterins was done by high‑performance liquid chromatography method. RESULTS: A total of 30 patients showed PAH deficiency and two patients were diagnosed with BH4 deficiency (BH4/ HPA ratio = 6.25%). Both of these two BH4‑deficient patients were assigned to severe variant of dihydropteridine reductase (DHPR) deficiency. More than 75% of patients with PAH deficiency classified as classic phenylketonuria (PKU) according their levels of pre‑treatment phenylalanine concentrations. CONCLUSION: Based on the performed study, we think that the frequency of milder forms of PKU is higher than those was estimated before and/or our findings here. Furthermore, the frequency of DHPR deficiency seems to be relatively high in our province. Since the clinical symptoms of DHPR deficiency are confusingly similar to that of classic PKU and its prognosis are much worse than classical PKU and cannot be solely treated with the PKU regime, our pilot study support that it is crucial to set up screening for BH4 deficiency, along with PAH deficiency, among all HPA patients diagnosed with HPA.

Responsividade à tetrahidrobiopterina em pacientes com deficiência de fenilalanina hidroxilase / Tetrahydrobiopterin responsiveness of patients with phenylalanine hydroxylase deficiency

OBJETIVO: Identificar indivíduos responsivos à tetrahibrobiopterina (BH4) em uma amostra de pacientes brasileiros com hiperfenilalaninemia por deficiência de fenilalanina-hidroxilase (HPA-PAH). MÉTODOS: Estudo intervencional, amostragem por conveniência. Para serem incluídos no estudo, os pacientes deveriam: possuir diagnóstico bioquímico de HPA-PAH; ter idade > 7 anos; estar em tratamento dietético; e apresentar níveis de fenilalanina (Phe) > 6 mg/dL em todas as medidas realizadas no ano anterior à inclusão no estudo. Os níveis de Phe foram determinados por meio de espectrometria de massas in tandem no dia anterior (dia 1) e nos pontos de hora 0, 4 e 8 h (dia 2) e 24 h (dia 3) após ingestão de BH4. Os critérios utilizados para definir responsividade ao BH4 foram: critério 1-redução > 30 por cento de Phe após 8 h da administração de BH4; e critério 2-redução > 30 por cento de Phe após 24 h da administração. RESULTADOS: Dezoito pacientes foram incluídos no estudo (mediana de idade = 14 anos, sexo masculino = 12). Cinco pacientes foram responsivos ao BH4, sendo três (forma clássica: um; forma leve: dois) de acordo com ambos os critérios, e dois (forma clássica: um; forma não definida: um) de acordo com o critério 2. Os níveis de Phe plasmáticos do dia 1 não demonstraram variação nos pontos de hora (p = 0,523). Entretanto, quando comparamos os níveis de Phe nos pontos de hora dos dias 1 e 2, encontramos uma variação significativa (p = 0,006). A análise da associação genótipo-fenótipo confirmou o caráter multifatorial da responsividade ao BH4. CONCLUSÃO: Os nossos achados estão de acordo com a literatura e indicam que um número relevante de pacientes brasileiros com HPA-PAH é responsivo à BH4.

OBJECTIVE: To identify patients responsive to tetrahydrobiopterin (BH4) in a sample of Brazilians with hyperphenylalaninemia due to phenylalanine hydroxylase deficiency (HPA-PAH). METHODS: Interventional study, convenience sampling. The inclusion criteria were: diagnosis of HPA-PAH; age > 7 years; phenylalanine-restricted diet and phenylalanine (Phe) levels > 6 mg/dL in all blood tests 1 year before inclusion. Blood samples were obtained the day before (day 1) and at 0, 4, 8 (day 2) and 24 h (day 3) after BH4 intake. Phe levels were measured using tandem mass spectrometry. The criteria used to define responsiveness to BH4 were: criterion 1- Phe reduction > 30 percent 8 h after BH4 administration; criterion 2 - Phe reduction > 30 percent 24 h after BH4 administration. RESULTS: Eighteen patients were enrolled (median age, 14 years; 12 boys). Five patients were responsive to BH4, 3 according to both criteria (one classical PKU, two mild PKU); and two according to criterion 2 (one classical PKU; one indefinite PKU type). There were no differences between Phe serum levels on day 1 and at the other time points (p = 0.523). However, Phe levels on days 1 and 2 were significantly different (p = 0.006). The analysis of the phenotype-genotype association confirmed its multifactorial character. CONCLUSION: A relevant number of Brazilian patients with HPA-PAH are responsive to BH4, in agreement with other studies in the literature.

Tissue-specific activation of mitogen-activated protein kinases for expression of transthyretin by phenylalanine and its metabolite, phenylpyruvic acid

Phenylketonuria is an autosomal recessive disorder caused by a deficiency of phenylalanine hydroxylase. Transthyretin has been implicated as an indicator of nutritional status in phenylketonuria patients. In this study, we report that phenylalanine and its metabolite, phenylpyruvic acid, affect MAPK, changing transthyretin expression in a cell- and tissue-specific manner. Treatment of HepG2 cells with phenylalanine or phenylpyruvic acid decreased transcription of the TTR gene and decreased the transcriptional activity of the TTR promoter site, which was partly mediated through HNF4alpha. Decreased levels of p38 MAPK were detected in the liver of phenylketonuria-affected mice compared with wild-type mice. In contrast, treatment with phenylalanine increased transthyretin expression and induced ERK1/2 activation in PC-12 cells; ERK1/2 activation was also elevated in the brainstem of phenylketonuria-affected mice. These findings may explain between-tissue differences in gene expression, including Ttr gene expression, in the phenylketonuria mouse model.

Protective Effect of Recombinant Adeno-Associated Virus 2/8-Mediated Gene Therapy from the Maternal Hyperphenylalaninemia in Offsprings of a Mouse Model of Phenylketonuria

Phenylketonuria (PKU) is an autosomal recessively inherited metabolic disorder caused by a deficiency of phenylalanine hydroxylase (PAH). The accumulation of phenylalanine leads to severe mental and psychomotor retardation, and the fetus of an uncontrolled pregnant female patient presents with maternal PKU syndrome. We have reported previously on the cognitive outcome of biochemical and phenotypic reversal of PKU in a mouse model, Pah(enu2), by the AAV serotype 2-mediated gene delivery of a human PAH transgene. However, the therapeutic efficacy had been limited to only male PKU mice. In this study, we generated a pseudotyped recombinant AAV2/8-hPAH vector and infused it into female PKU mice through the hepatic portal vein or tail vein. Two weeks after injection, complete fur color change to black was observed in female PKU, as in males. The PAH activity in the liver increased to 65-70% of the wild-type activity in female PKU mice and to 90% in male PKU mice. Plasma phenylalanine concentration in female PKU mice decreased to the normal value. In addition, the offsprings of the treated female PKU mice can rescue from the harmful effect of maternal hyperphenylalaninemia. These results indicate that recombinant AAV2/8-mediated gene therapy is a potential therapeutic strategy for PKU.

Genotype and natural history in unrelated individual with phenylketonuria and autistic behavior

We describe three unrelated individuals, two males (ages 35 and 9) and a female (age 8) presenting with late diagnosed phenylketonuria (PKU) and autistic behavior, all showing poor adhesion to the dietary treatment resulting in high plasmatic phenylalanine levels, particularly in the oldest subject. Clinical findings included hair hypopigmentation, microcephaly, severe mental retardation with absent development of verbal language and autistic symptoms in all three patients, whereas variable neurological signs such as seizures, spasticity, ataxia, aggressivity, and hyperactivity were individually found. Homozygosity for the IVS10nt11g/a (IVS10nt546) was found in all. This is the first report of molecular findings in subjects with PKU also presenting with autistic features. The authors discuss if this mutation is particularly involved in the association of autistic symptoms in untreated PKU individuals.

Descrevemos três indivíduos não aparentados, dois do sexo masculino (com idades de 35 e nove anos) e um do sexo feminino (com idade de oito anos) apresentando fenilcetonúria diagnosticada tardiamente e comportamento autístico, todos com adesão limitada ao tratamento dietético resultando em altos níveis plasmáticos de fenilalanina, especialmente no indivíduo mais velho. Os achados clínicos incluem hipopigmentação de cabelos, retardo mental grave com ausência de desenvolvimento da linguagem verbal e sintomas autísticos nos três pacientes, enquanto outros achados neurológicos como convulsões, espasticidade, ataxia, agressividade e hiperatividade são descritos em um indivíduo, cada. Homozigose para a mutação IVS10nt11g/a (IVS10nt546) foi encontrada em todos. Este é o primeiro relato de achados moleculares em indivíduos com fenilcetonúria que desenvolveram características autísticas. Discute-se se essa mutação estaria particularmente envolvida no desenvolvimento de sintomas autísticos em indivíduos com fenilcetonúria não tratada.

The time has come: a new scene for PKU treatment

Phenylketonuria (PKU) is one of the few genetic diseases in which mental retardation can be prevented. Hence, diagnosis and treatment must be established early. PKU treatment consists of a phenylalanine-restricted diet supplemented with a phenylalanine-free mixture of amino acids. However, it is difficult to adhere to this diet. In the last decade, a better comprehension of the biochemistry, genetics and molecular basis of the disease, as well as the need for easier treatment, led to the development of several new therapeutic strategies for PKU. In the present study, we evaluated these new therapeutic options in terms of theoretical basis, methodologies, efficacy, and costs.

Frequencies of phenylalanine hydroxylase mutations I65T, R252W, R261Q, R261X, IVS10nt11, V388M, R408W, Y414C, and IVS12nt1 in Minas Gerais, Brazil

In order to determine the phenylketonuria (PKU) mutation spectrum in the population of Minas Gerais State, Brazil, 78 unrelated PKU patients found by the neonatal screening program from 1993 to 2003 were tested for nine phenylalanine hydroxylase mutations. These mutations were selected due to their high frequencies in other Brazilian populations and in Portugal, where the largest contingent of the Caucasian component of the Brazilian population originated from. The most frequent mutations were V388M (21%), R261Q (16%), IVS10nt11 (13.4%), I65T (5.7%), and R252W (5%). The frequencies of the other four mutations (R261X, R408W, Y414C, and IVS12nt1) did not reach 2%. By testing these nine mutations, we were able to identify 64% of the PKU alleles in our sample. V388M frequency was higher than in any other known population and almost three times larger than that observed in Portugal, probably reflecting genetic drift. The mutation profile, as well as the relative frequency of the different mutations, suggest that the Minas Gerais population more closely resembles that of Portugal than do the other Brazilian populations that have already been tested.

Tamiz neonatal en América Latina: problemas y propuestas derivadas de la práctica clínica / Neonatal screening in Latin America: problems and proposals derived from clinical practice

Las pruebas de tamiz neonatal sirven para detectar a recién nacidos portadores de alguna patología endocrina, infecciosa o errores del metabolismo, antes de que la enfermedad se manifieste y previene, de ser posible, alguna discapacidad física, mental o la muerte. Se considera conveniente establecer un programa nacional de tamiz neonatal e infantil en México y en Latinoamérica, para hipotiroidismo congénito y evaluar la posibilidad de incluir a la hiperplasia adrenal congénita la toxoplasmosis congénita y el neuroblastoma mediante estudios piloto, que consideren un estricto control de calidad, sistemático y automatizado. El costo/beneficio del tamiz neonatal es positivo a la sociedad y evita el daño cerebral permanente, y/o la muerte de los niños

Successful therapy of hyperphenylalaninemia due to defective tetrahydrobiopterin metabolism in two siblings

Alguns pacientes com o diagnóstico inicial de fenilcetonúria (PKU) näo apresentam melhora clínica mesmo apesar de tratados com uma dieta pobre em fenilalanina. Inúmeros trabalhos publicados nos últimos dez anos têm atribuído esses casos a erros no metabolismo da tetra hidrobiopterina (BH4), um cofator essencial para a hidroxilaçäo de fenilalanina, tirosina e triptofânio. Descrevem-se duas irmäs que apresentam um defeito na síntese de BH4, nas quais a administraçäo oral de BH4, L-Dopa, Carbidopa e 5-hidróxi-triptofânio foi seguida de significativa melhora clínica, neurofisiológica e bioquímica. Dados obtidos na nossa regiäo permitem supor que a freqüência de defeitos no metabolismo do BH4, entre os pacientes com hiperfenilalaninemia seja maior que a presentemente estimada. Ao contrário dos indivíduos com PKU clássica, alguns pacientes com deficiência de BH4 parecem responder ao tratamento mesmo quando este é introduzido tardiamente. Enfatiza-se a importância de aplicar o teste para o diagnóstico de deficiência de BH4 em todos os pacientes com hiperfenilalaninemia

Heterozygote detection in two hyperphenylaninemia types: classic phenylketonuria and dihydrobiopterin biosynthesis deficiency

Controles normais (N=41) e heterozigotos comprovados para dois tipos de hiperfenilalaninemia (HP), fenilcetonúria clássica (HP I, N=8) e deficiência de síntese de dihidrobiopterina (HP V, N = 6), foram estudados quanto aos níveis séricos de fenilalanina (P) e tirosina (T), medidos por fluorometria. Foram observados os seguintes valores para P e T, respectivamente: controles, 79,4 + ou - 15,8 e 86,6 + ou - 19,0; HP I, 158,5 + ou - 18,8 e 96,7 + ou - 25,7; HP V, 148,3 + ou - 28,8 e 85,5 + ou - 27,7. A relaçäo p2/T e a funçäo discriminante usando simultaneamente P e T foram os parâmetros que melhor distinguiram controles dos dois tipos de heterozigotos. Näo foi possível separar heterozigotos para HP I daqueles para HP V por nenhum desses parâmetros