RESUMEN
Phenylketonuria (PKU) is a genetic disorder caused by variants in the gene encoding phenylalanine hydroxylase (PAH), resulting in accumulation of phenylalanine to neurotoxic levels. Here, we analyzed the cellular stability, localization, and interaction with wild-type PAH of 20 selected PKU-linked PAH protein missense variants. Several were present at reduced levels in human cells, and the levels increased in the presence of a proteasome inhibitor, indicating that proteins are proteasome targets. We found that all the tested PAH variants retained their ability to associate with wild-type PAH, and none formed aggregates, suggesting that they are only mildly destabilized in structure. In all cases, PAH variants were stabilized by the cofactor tetrahydrobiopterin (BH4 ), a molecule known to alleviate symptoms in certain PKU patients. Biophysical calculations on all possible single-site missense variants using the full-length structure of PAH revealed a strong correlation between the predicted protein stability and the observed stability in cells. This observation rationalizes previously observed correlations between predicted loss of protein destabilization and disease severity, a correlation that we also observed using new calculations. We thus propose that many disease-linked PAH variants are structurally destabilized, which in turn leads to proteasomal degradation and insufficient amounts of cellular PAH protein.
Asunto(s)
Estudios de Asociación Genética , Predisposición Genética a la Enfermedad , Genotipo , Fenilalanina Hidroxilasa/genética , Fenilcetonurias/diagnóstico , Fenilcetonurias/genética , Alelos , Línea Celular , Activación Enzimática , Estudios de Asociación Genética/métodos , Humanos , Modelos Moleculares , Mutación , Fenilalanina Hidroxilasa/sangre , Fenilalanina Hidroxilasa/química , Complejo de la Endopetidasa Proteasomal/metabolismo , Conformación Proteica , Estabilidad Proteica , Relación Estructura-ActividadRESUMEN
BACKGROUND: Phenylketonuria (PKU) is due to the deficit of the enzyme phenylalanine hydroxylase, the first step of dopamine synthesis. If not early treated the disease results in severe neurological impairment. Minor neurological signs have been reported in early treated PKU (ETPKU) subjects. Prolactin level is affected by (and reflects) brain dopamine availability. Object of the study was to assess the occurrence, age at onset, distribution, associated neurological signs, and possible pathogenetic biomarkers of tremor in ETPKU. METHODS: Fifty-nine ETPKU and 43 control subjects (age range 7-54) underwent individual and familiar tremor history, clinical assessment of tremor by means of the Fahn-Tolosa-Marin Tremor Rating Scale, and IQ evaluation. Historical and concomitant biochemical data (blood levels of Phe) and serum prolactin were included in the analysis. RESULTS: Thirty-two percent of ETPKU patients were affected by postural and kinetic tremor. We found a significant correlation between severity of tremor and: prolactin level at the day of examination (part A: rsâ¯=â¯0.320; pâ¯=â¯.014; part C: rsâ¯=â¯0.319; pâ¯=â¯.014), Phe fluctuations from 12â¯years onwards (part B: rsâ¯=â¯0.300; pâ¯=â¯.036). We also found a significant correlation between prolactin (18.2⯱â¯9.6â¯ng/ml) and Phe levels (852⯱â¯472⯵mol/l) on the day of assessment (rsâ¯=â¯0.470; pâ¯<â¯.001). CONCLUSIONS: The main clinical features of tremor in ETPKU evoke those of essential tremor, although with a higher prevalence and an earlier onset than in general population. The severity of tremor was related to concomitant prolactin rather than Phe levels. This pattern suggests that metabolic alterations associated with PKU may result in an anticipation of the tremor onset in subjects who are possibly prone to this disorder.
Asunto(s)
Fenilcetonurias/complicaciones , Fenilcetonurias/fisiopatología , Temblor/etiología , Adolescente , Adulto , Biomarcadores , Niño , Estudios Transversales , Femenino , Humanos , Estudios Longitudinales , Masculino , Persona de Mediana Edad , Fenilalanina Hidroxilasa/sangre , Fenilcetonurias/diagnóstico , Prolactina/sangre , Adulto JovenRESUMEN
Phenylketonuria (PKU) is an autosomal recessive inborn error of metabolism caused by pathogenic variants in the phenylalanine hydroxylase gene (PAH). The correlation between genotype and phenotype can be complex and sometimes variable but often very useful for categorizing and predicting dietary tolerance and potential outcome. We reviewed medical records for 367 patients diagnosed with PKU or persistent mild hyperphenylalaninemia (MHP) between 1950 and 2015 who had PAH genotyping. In 351 we had the full PAH genotype as well as phenotypic characteristics such as phenylalanine (Phe) concentrations (at newborn screening, confirmation, and highest known), and dietary Phe tolerance. On 716 mutant chromosomes, including 14 in genotypes with only one identified variant, we identified 114 different pathogenic variants. The most frequent, p.R408W, was present in 15.4% of the alleles; other frequent variants were c.1315â¯+â¯1Gâ¯>â¯A (6.1%), p.I65T (5.7%), and p.R261Q (5.7%). Three variants, c.142â¯Tâ¯>â¯G (p.L48â¯V), c.615Gâ¯>â¯C (p.E205D), and c.1342_1345delCTCC, were novel. We used the phenotypic parameters of variants paired with null alleles (functional hemizygotes) to assign the variants as classic PKU, moderate PKU, mild PKU, MHP-gray zone, or MHP. We also included the phenotype association(s) for all of the full genotypes. In 103 patients, we also could assign sapropterin dihydrochloride responsiveness, which is a synthetic form of the tetrahydrobiopterin (BH4) PAH cofactor. This compilation from a single metabolic center provides further information on PAH variants in the United States and the correlations between genotype and phenotype.
Asunto(s)
Estudios de Asociación Genética , Genotipo , Mutación , Fenotipo , Fenilalanina Hidroxilasa/genética , Fenilcetonurias/diagnóstico , Fenilcetonurias/genética , Alelos , Sustitución de Aminoácidos , Biopterinas/análogos & derivados , Biopterinas/uso terapéutico , Femenino , Humanos , Recién Nacido , Masculino , Tamizaje Neonatal , Fenilalanina Hidroxilasa/sangre , Fenilcetonurias/tratamiento farmacológico , Fenilcetonurias/epidemiología , Resultado del Tratamiento , Estados Unidos/epidemiologíaRESUMEN
The aim of this study is to investigate the ability to prenatally diagnose phenylketonuria (PKU) by using phenylalanine hydroxylase (PAH) gene mutation analysis combined with short tandem repeat (STR) linkage analysis in 118 fetuses from 112 Chinese families. Genomic DNA was extracted from the peripheral blood from members of 112 families and the exons and exon-intron boundaries of the PAH gene were amplified by PCR. PCR products were analyzed by bi-directional Sanger sequencing and multiplex ligation-dependent probe amplification (MLPA). The three variable number of tandem repeat (VNTR) markers PAH-1, PAH-26, PAH-32 were used in the prenatal diagnosis for the PKU families. We identified a spectrum of 63 different mutations, including 61 point mutations and indels, two large exon deletion mutations, and five novel mutations. A substantial proportion of mutant alleles were accounted for by p.R243Q (15.62%), EX6-96AG (9.82%), p.V399V (7.59%), p.Y356X (6.70%), and p.R413P (5.36%). The same mutations were identified in 31 prenatally genotyped fetuses. We identified 58 fetuses that carried only one mutant allele and 29 fetuses that carried no mutations of PAH and were presumed normal. PAH gene mutation analysis combined with STR linkage analysis can provide rapid and accurate prenatal diagnosis for PKU families.
Asunto(s)
Fenilalanina Hidroxilasa/genética , Fenilcetonurias/genética , Diagnóstico Prenatal , Alelos , Pueblo Asiatico , Exones , Femenino , Ligamiento Genético , Genotipo , Humanos , Intrones/genética , Repeticiones de Microsatélite/genética , Fenilalanina Hidroxilasa/sangre , Fenilcetonurias/sangre , Mutación Puntual , Embarazo , Eliminación de Secuencia/genéticaRESUMEN
OBJECTIVE: To investigate the feature of phenylalanine hydroxylase (PAH) gene mutations and provide guidance for genetic and prenatal diagnosis of patients with phenylketonuria from Shaanxi. METHODS: For 55 patients whose blood Phe concentration was over 2.0 mg/dL, potential mutations in 13 exons and flanking sequences of the PAH gene were detected by PCR and DNA sequencing. RESULTS: A total of 98 mutations were detected in 110 PAH alleles, with the detection rate being 89.10%. Nine mutations have been identified in exon 7, which accounted for 33.67% of all. Exon 12 (14.29%) and exon 3 (12.24%) have followed. Thirty eight mutations, locating in exon2-exon12 and the flanking sequence, were detected in the 55 PKU patients. p.R243Q (24.49%) was the commonest mutation, whilstp.A47E, p.I65S and p.A259T were first discovered in China. After querying international databases including PAHdb and HGMD, the p.C334X was verified as the novel PAH gene mutation. CONCLUSION: The mutation spectrum of the PAH gene in Shaanxi has been identified. And a novel mutation has been identified. This may facilitate the diagnosis of PKU in the future.
Asunto(s)
Mutación , Fenilalanina Hidroxilasa/genética , Fenilcetonurias/enzimología , Fenilcetonurias/genética , Alelos , Secuencia de Bases , Niño , Preescolar , China , Femenino , Humanos , Lactante , Recién Nacido , Masculino , Fenilalanina Hidroxilasa/sangreAsunto(s)
Biomarcadores/sangre , Biopterinas/análogos & derivados , Fenilcetonurias/dietoterapia , Adolescente , Adulto , Biopterinas/administración & dosificación , Biopterinas/efectos adversos , Biopterinas/genética , Biopterinas/metabolismo , Índice de Masa Corporal , Niño , Preescolar , Femenino , Humanos , Lactante , Masculino , Fenilalanina Hidroxilasa/sangre , Fenilcetonurias/sangre , Fenilcetonurias/patología , Adulto JovenRESUMEN
UNLABELLED: Sapropterin dihydrochloride, a synthetic tetrahydrobiopterin (BH4), works as a chaperone of phenylalanine hydroxylase (PAH) in phenylketonuria (PKU) to facilitate and stabilize folding of PAH into its most active conformation. No standard pharmacogenetic tests exist to identify responsive genotypes. Previous studies have failed to identify genotypes that consistently predict response; they are weakened by varied: 1) doses; 2) response definitions; 3) duration; 4) phenylalanine (PHE) test times during different protein catabolic states; 5) control of dietary PHE. START (sapropterin therapy actual response test) protocol is a double blind, placebo-controlled, 4-week clinical test that obviates the confounders aforementioned. START results were evaluated for response-genotype correlates and trends in molecular characteristics. RESULTS: Seventy-four patients completed START. Thirty-six patients (48.6%) responded, 55 patients' genotypes are known, 38 unique genotypes are present. Alleles consistently associated with response include Y414C (8/8 patients, 6 genotypes) and I65T (9/9 patients, 6 genotypes). The p.R408W mutation, in which substitution of straight chain arginine with bulky aromatic amine, tryptophan, at the crux of a strategic hinge site activating folding of PAH, amino acid sequence 408, was strongly associated with non-response (21/29 patients non-responsive, 12/17 genotypes non-responsive). Genotypes containing at least one allele with ≥25% residual activity compared to wild type, were strongly associated with response. CONCLUSIONS: The START protocol provides a rigorous pharmacogenetic test to identify sapropterin responsiveness and genotypes associated with responsiveness and non-responsiveness. Some genotypes were found to be predictive of responsiveness or non-responsiveness, and responsiveness was associated with specific alleles. The START protocol provides a reliable test for sapropterin responsiveness and will continue to improve understanding of how PKU mutations impact PAH protein-folding dynamics and enhance understanding of PKU disease and its management.
Asunto(s)
Biopterinas/análogos & derivados , Fenilalanina Hidroxilasa/metabolismo , Fenilalanina/metabolismo , Fenilcetonurias/tratamiento farmacológico , Adulto , Alelos , Biopterinas/farmacocinética , Biopterinas/farmacología , Biopterinas/uso terapéutico , Preescolar , Relación Dosis-Respuesta a Droga , Método Doble Ciego , Activación Enzimática/efectos de los fármacos , Femenino , Genotipo , Humanos , Masculino , Mutación , Fenilalanina/genética , Fenilalanina Hidroxilasa/sangre , Fenilalanina Hidroxilasa/genética , Fenilcetonurias/metabolismoRESUMEN
Owing to our special genetic heritage, phenylketonuria is very rare in Finland, but the situation will change as the number of immigrants from populations with a larger incidence increases. In persons with this disorder, the enzyme phenylalanine hydroxylase, which metabolizes phenylalanine to tyrosine, is not functioning normally, leading to the accumulation phenylalanine within the body. High levels of phenylalanine are toxic to the central nervous system. A newborn affected with phenylketonuria is asymptomatic but will rapidly become disabled without therapy. The leading principle of the therapy is careful limitation of phenylalanine intake.
Asunto(s)
Tamizaje Neonatal , Fenilcetonurias/dietoterapia , Fenilcetonurias/diagnóstico , Emigrantes e Inmigrantes , Finlandia , Humanos , Recién Nacido , Fenilalanina Hidroxilasa/sangre , Fenilcetonurias/enzimologíaRESUMEN
Here we developed a haplotype-based noninvasive prenatal diagnosis method for hyperphenylalaninemia (HPA) and demonstrated its accuracy and feasibility during early pregnancy. Capture sequencing was performed on genomic DNA from parents and probands using customized hybridization probes targeting highly heterozygous single-nucleotide polymorphisms located within the 1 M region flanking phenylalanine hydroxylase (PAH) and 6-pyruvoyltetrahydropterin (PTS) and its coding region to determine the parental haplotypes and linkage to pathogenic mutations. Maternal plasma DNA obtained at 12-20 weeks of gestation was also subjected to targeted sequencing to deduce the fetal haplotypes based on the parental haplotypes. The fetal genotypes were further validated by invasive prenatal diagnosis. Haplotype-based noninvasive prenatal testing was successfully performed in 13 families. Five fetuses were identified to harbor bi-allelic pathogenic variants of PAH, four fetuses were carriers of one heterozygous PAH variant, three fetuses were normal, and the fetus of the 6-pyruvoyl tetrahydrobiopterin synthase family was identified as normal. The fetal genotypes at two gestational weeks from the same PAH family were identical. All results were consistent with the prenatal diagnosis based on amniotic fluid. Haplotype-based noninvasive prenatal testing for HPA through targeted sequencing is accurate and feasible during early gestation.
Asunto(s)
Haplotipos , Fenilalanina Hidroxilasa/sangre , Fenilalanina Hidroxilasa/genética , Fenilcetonurias/sangre , Fenilcetonurias/genética , Alelos , Femenino , Genotipo , Edad Gestacional , Humanos , Mutación , Fenilcetonurias/diagnóstico , Polimorfismo de Nucleótido Simple , Embarazo , Diagnóstico Prenatal , Reproducibilidad de los Resultados , Análisis de Secuencia de ADNRESUMEN
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.
Asunto(s)
Trastorno Autístico/genética , Fenilcetonurias/genética , Adulto , Trastorno Autístico/diagnóstico , Niño , Consanguinidad , Manual Diagnóstico y Estadístico de los Trastornos Mentales , Femenino , Genotipo , Homocigoto , Humanos , Masculino , Mutación , Fenilalanina Hidroxilasa/sangre , Fenilcetonurias/diagnóstico , Fenilcetonurias/dietoterapiaRESUMEN
OBJECTIVES: Although high phenylalanine (phe) exposure has been shown to influence the DNA methylation status of leukocytes in hyperphenylalaninemia (HPA), the potential of DNA methylation changes as a biomarker of pretreatment high phe exposure in diet free newborns with HPA has not been explored. We therefore investigated the DNA methylation pattern of the phenylalanine hydroxylase (PAH) gene promoter at different phe levels, and the possibility of DNA methylation pattern changes being a biomarker of high phe exposure in diet free newborns with HPA. DESIGN AND METHODS: With a combination of methylated PCR, high resolution melting, and sequencing, the cytosine phosphodiester bond guanine (CpG) dinucleotides in the 5' untranslated region of the PAH gene were analysed 2-15days after birth using leukocyte DNA from diet free 16 newborns with HPA and 16 healthy controls. RESULTS: In 2-3days blood cards, GTGTG and GTGC/TG alleles were both detected at similar low mean phe levels in healthy controls (59.39±14.62 and 55.33±13.43µmol/L) and non-phenylketonuria (PKU) HPA (265.00 and 244.25±73.73µmol/L). In HPA with PKU, the GTGTG and GTGC/TG alleles were both detected at dissimilar elevated mean phe levels (380.80±64.62 and 589.00±191.96µmol/L). In ≥7day blood cards, GTGTG and GTGC/TG alleles were both detected at similar excess mean phe levels in HPA with PKU (2297±374.38 and 1562.66±718.23µmol/L). CONCLUSION: The demethylated GTGTG and partial methylated GTGC/TG alleles are not pathogenic alleles. Our results suggest a specific remodeling of the DNA methylated alleles of the PAH promoter at elevated, but not excess phe levels in diet free newborns with PKU.
Asunto(s)
Alelos , Metilación de ADN , Fenilalanina Hidroxilasa/genética , Fenilalanina/sangre , Fenilcetonurias/genética , Regiones Promotoras Genéticas , Regiones no Traducidas 5' , Estudios de Casos y Controles , Islas de CpG , Femenino , Humanos , Recién Nacido , Masculino , Desnaturalización de Ácido Nucleico , Fenilalanina Hidroxilasa/sangre , Fenilcetonurias/sangre , Fenilcetonurias/diagnóstico , Fenilcetonurias/patología , Reacción en Cadena de la Polimerasa , Análisis de Secuencia de ADNRESUMEN
Since 2007, synthetic tetrahydrobiopterin (BH4) has been approved as a therapeutic option in BH4-responsive phenylketonuria (PKU) and since 2015 extended to infants younger than 4 years in Europe. The current definition of BH4 responsiveness relies on the observation of a 20% to 30% blood phenylalanine (Phe) decrease after BH4 administration, under nonstandardized conditions. By this definition, however, patients with the same genotype or even the same patients were alternatively reported as responsive or nonresponsive to the cofactor. These inconsistencies are troubling, as frustrating patient expectations and impairing cost-effectiveness of BH4-therapy. Here we tried a quantitative procedure through the comparison of the outcome of a simple Phe and a combined Phe plus BH4 loading in a series of infants with PKU, most of them harboring genotypes already reported as BH4 responsive. Under these ideal conditions, blood Phe clearance did not significantly differ after the 2 types of loading, and a 20% to 30% decrease of blood Phe occurred irrespective of BH4 administration in milder forms of PKU. Such early screening for BH4 responsiveness, based on a quantitative assay, is essential for warranting an evidence-based and cost-effective therapy in those patients with PKU eventually but definitely diagnosed as responsive to the cofactor.
Asunto(s)
Biopterinas/análogos & derivados , Diagnóstico Precoz , Tamizaje Masivo , Fenilalanina/sangre , Fenilcetonurias/sangre , Fenilcetonurias/tratamiento farmacológico , Biopterinas/sangre , Biopterinas/uso terapéutico , Análisis Mutacional de ADN , Humanos , Lactante , Fenilalanina Hidroxilasa/sangre , Fenilalanina Hidroxilasa/genética , Fenilcetonurias/diagnóstico , Fenilcetonurias/genética , Resultado del TratamientoRESUMEN
Phenylketonuria (PKU) is a genetic disorder caused by a partial or complete mutation of the enzyme phenylalanine hydroxylase (PHA), fact that produces high levels of phenylalanine in blood resulting in mental retardation if not diagnosed during the neonatal period. Treatment consists of a phenylalanine (Phe) restricted diet. Several studies have shown that due to restriction of animal protein, this diet is deficient in fatty acids such as alfalinolenic acid (ALA) and provides high levels of linoleic acid (LA). The objective of this study was to determine the lipid composition of the diet consumed by children with early-diagnosed PKU. Lipid composition of the Phenylalanine restricted diet consumed by 29 children with PKU and in follow-up at INTA, University of Chile, were analyzed. Children were paired by sex and age with a control group. A twenty-four hour dietary recall was performed for 3 consecutive days and total fatty acid intake, including saturated, monounsaturated, polyunsaturated, LA and ALA, were calculated. In the restricted diet of children with PKU, 31.8% of total calories are from fat, 13% of which are LA and 0.2% ALA, showing significant differences as compared to the control group. The ratio of saturated:monounsaturated:polyunsaturated fatty acids was 1:1.7:3.9 and the ratio of LA:ALA was ten-fold higher than the recommended ratio of 115:1. It is concluded that the Phenyalanine restricted diet of Chilean children with PKU is high in LA and low in ALA.
Asunto(s)
Grasas de la Dieta/sangre , Fenilcetonurias/sangre , Niño , Preescolar , Estudios Transversales , Grasas de la Dieta/administración & dosificación , Femenino , Humanos , Lactante , Ácidos Linoleicos/administración & dosificación , Ácidos Linoleicos/sangre , Masculino , Estado Nutricional , Fenilalanina Hidroxilasa/sangre , Fenilcetonurias/dietoterapia , Encuestas y Cuestionarios , Ácido alfa-Linolénico/administración & dosificación , Ácido alfa-Linolénico/sangreRESUMEN
The majority of mutations in the human phenylalanine hydroxylase (PAH) gene that lead to the recessive disease phenylketonuria (PKU) are believed to affect the activity or stability of the PAH enzyme. In this study we have performed in vivo analyses of lymphocyte PAH mRNA from PKU patients homozygous for the PKU missense mutations P281L and R408Q as well as the nonsense mutations G272X and Y356X. The mutations G272X, P281L and R408Q, which are located outside the consensus splice site sequence, result in transcripts with one or more exons skipped in addition to full-length transcripts. The mutation Y356X results in transcripts with one or more exons skipped, but no full-length transcripts. Our findings question the value of functional and structural predictions of mutations at the protein level without analyses of the corresponding transcript.
Asunto(s)
Linfocitos/enzimología , Mutación , Fenilalanina Hidroxilasa/genética , Fenilcetonurias/genética , Células Cultivadas , Homocigoto , Humanos , Mutación Missense , Fenilalanina Hidroxilasa/sangre , Empalme del ARN , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transcripción GenéticaRESUMEN
The effect of serum from patients with renal failure on phenylalanine hydroxylase activity has been measured in normal individuals, patients with steady-state chronic renal failure and patients undergoing haemodialysis. Significant inhibition of enzyme activity was induced by serum from patients with steady-state chronic renal failure but not from patients undergoing haemodialysis. Inhibitor(s) was readily diffusible in vitro and appeared to have an approximate molecular mass of 800. The results suggest that the low-plasma tyrosine levels observed in patients with chronic renal failure are due, at least in part, to the inhibition of phenylalanine hydroxylase.
Asunto(s)
Fallo Renal Crónico/enzimología , Fenilalanina Hidroxilasa/sangre , Diálisis Renal , Adulto , Anciano , Cromatografía en Gel , Femenino , Humanos , Fallo Renal Crónico/terapia , Masculino , Persona de Mediana Edad , Fenilalanina Hidroxilasa/antagonistas & inhibidores , Tirosina/sangreRESUMEN
Phenylalanine hydroxylase (PAH) deficiency is caused by mutations in the PAH gene (12q22-q24) resulting in a primary deficiency of the PAH enzyme activity, intolerance to the dietary intake of phenylalanine (Phe) and production of the phenylketonuria (PKU) disease. To date there have been no reports on the molecular analysis of PKU in Iranian population. In this study, the states of the PKU disease in terms of prevalence and mutation spectrum among patients reside in the institutions for mentally retarded in Isfahan was investigated. In the first step, 611 out of 1541 patients with PKU phenotype or severe mental retardation were screened for the PKU disease using the Guthrie bacterial inhibition assay (GBIA) followed by HPLC. Among the patients screened 34 (5.56%) were found positive with abnormal serum Phe of above 7mg/dl. In the next step, the presence of 18 common mutations of the PAH gene in 26 of the patients with classical PKU (serum Phe above 20mg/dl) was investigated, using the polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP). Of the 52 independent mutant alleles that were analyzed, 34 (65.38%) were genotyped showing 8 mutations as follows: R252W (15.38%), Q232Q (13.46%), R261Q (7.69%), delL364 (7.69%), IVS10-11g>a (5.77%), L333F (5.77%), V245V (5.77%) and S67P (3.85%). The results from this study may serve as a reference to analyze the PKU mutations in other part of Iran, and to establish diagnostic tests for carrier detection and prenatal diagnosis of the PKU disease in Iranian population.
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Discapacidad Intelectual , Mutación/genética , Fenilcetonurias/etnología , Fenilcetonurias/genética , Cromatografía Líquida de Alta Presión , Frecuencia de los Genes , Humanos , Institucionalización , Irán/epidemiología , Leucocitos/metabolismo , Tamizaje Masivo , Pruebas de Sensibilidad Microbiana , Fenilalanina/sangre , Fenilalanina Hidroxilasa/sangre , Fenilalanina Hidroxilasa/genética , Fenilcetonurias/enzimología , Reacción en Cadena de la Polimerasa , Polimorfismo de Longitud del Fragmento de Restricción , Mapeo RestrictivoRESUMEN
OBJECTIVE: To evaluate the results of systematizing preventive and health promotion actions among phenylketonuria (PKU) patients. METHODS: Results of phenylketonuria patients attended in the Prenatal PKU Screening Program in the State of Paraná, Brazil, from 1996 to 2001, were evaluated. Socioeconomic data were investigated and the gross motor function measure was applied to determine the motor score among 32 PKU infants with early diagnosis and treatment. Pearson's correlation coefficient was adopted to investigate the relationship between the target variable (motor score) and other quantitative variables (mean post-treatment serum phenylalanine level, parents' educational level, infant's age at the start of treatment, and family income). RESULTS: Among all the children evaluated, 93.7% showed development within normal limits as reported in the literature. Treatment was initiated in the first month of life in 71.9% of the PKU cases. Socioeconomic data showed 39.5% of parents having completed the fourth grade of primary school or less. There was a significant correlation between infant's motor score and parents' educational level (N=32), as well as between motor score and early treatment (N=27). CONCLUSIONS: The results highlighted the program's effectiveness. The correlation between parents' low educational level and lower motor score emphasizes the importance of support for parents in their use of diet therapy. The association between motor score and early initiation of treatment confirms the need for immediate admission into the program. The paucity of other evaluation studies in the literature makes generalization of the results difficult.
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Promoción de la Salud , Fenilcetonurias/prevención & control , Brasil , Niño , Desarrollo Infantil/fisiología , Preescolar , Humanos , Lactante , Recién Nacido , Destreza Motora/fisiología , Tamizaje Neonatal , Fenilalanina Hidroxilasa/sangre , Fenilcetonurias/diagnóstico , Factores Socioeconómicos , Resultado del TratamientoRESUMEN
INTRODUCTION: PKU is an autosomal recessive disorder. There is a broad spectrum phenotype which depends mainly on residual enzymatic activity and also on other factors such as modifying genes and non-genetic factors. This fact makes us consider that a multidisciplinary study of these patients is necessary to improve knowledge of the condition. OBJECTIVE: To establish phenotype-genotype correlation and classify nine new mutations according to severity. PATIENTS AND METHODS: We evaluated the clinical data obtained from a multidisciplinary trial of 11 patients with PKU/HPA who presented with nine new mutations (P275S, P279fsdelC, V388delTG, N61/I62/T63fsdel5bp, P281S, P362T, H1OOR, I164V and Y168H) identified during a molecular study of the PAH gene done in Catalonia (Spain). RESULTS AND CONCLUSION: In our patients the genotype is correlated with the biochemical phenotype whereas the cognitive phenotype depends on determining factors such as early diagnosis and diet. Therefore, although PKU may be considered to be a complex characteristic, the mutations in the PAH gene are the main determining factor of the metabolic phenotype of PKU. A multidisciplinary study is the best way to understand and control these patients.
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Mutación , Fenilalanina Hidroxilasa/genética , Fenilcetonurias/genética , Niño , Preescolar , Trastornos del Conocimiento/diagnóstico , Femenino , Expresión Génica , Genotipo , Humanos , Lactante , Recién Nacido , Masculino , Pruebas Neuropsicológicas , Fenotipo , Fenilalanina Hidroxilasa/sangre , Fenilcetonurias/sangre , Fenilcetonurias/diagnóstico , Índice de Severidad de la EnfermedadRESUMEN
INTRODUCTION: The hyperphenylalaninemias (HFA) form a diverse group of recessive autosomic disorders. They are caused by defects in the hepatic system for hydroxylation of the amino acid phenylalamine to tyrosine. The estimated incidence is approximately 10 cases per 100,000 live births. Children with this metabolic disorder may present with varied neurological symptoms. Control of plasma levels, so that they are more normal as soon as possible after birth, significantly prevents mental retardation and other neuropsychological dysfunction. For this reason HFA has been included in neonatal screening. However, some patients are not detected on screening. When they are adults, these patients pose problems of diagnosis for neurologists who attend adults. CLINICAL CASE: We describe an adolescent with mental and linguistic retardation, in whom neonatal screening to rule out metabolic defects was normal. At the age of 15, the phenylalanine in blood and urine were found to be raised. On cerebral magnetic resonance changes typical of pheynylketonuria (PKU) were seen. CONCLUSIONS: The HFA should be considered as causes of cerebral dysfunction in adults, since in spite of neonatal screening, false negatives may occur. We describe a clinical case and consider different forms of hyperphenylaleninemias. Their diagnosis and treatment.
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Discapacidad Intelectual/etiología , Fenilcetonurias/complicaciones , Fenilcetonurias/diagnóstico , Adolescente , Encéfalo/patología , Humanos , Discapacidad Intelectual/diagnóstico , Imagen por Resonancia Magnética , Masculino , Fenilalanina Hidroxilasa/sangre , Fenilcetonurias/dietoterapia , Factores de Tiempo , Tomografía Computarizada por Rayos XRESUMEN
Incubation of nitrocellulose filters containing proteins in solutions of organic alcohols (ethanol, methanol, isopropanol) enabled to increase the immunoblotting sensitivity after denaturating electrophoresis in presence of SDS. Maximal elevation of the label sensitivity (4-6-fold) was observed after incubation of these filters in 30% isopropanol within 2 hrs. The effect of sensitivity elevation appears to be caused by the antigen renaturation due to SDS washing off. The modified procedure of immunoblotting allowed to detect phenylalanine hydroxylase antigen in human thrombocytes. The antigen had electrophoretic mobility similar to that of phenylalanine hydroxylase from liver tissue; its concentration constituted less than 0.1 microgram per 1 mg of protein in thrombocytes.