RESUMO
Clinical exome sequencing (CES) is increasingly being used as an effective diagnostic tool in the field of pediatric genetics. We sought to evaluate the parental experience, understanding and psychological impact of CES by conducting a survey study of English-speaking parents of children who had diagnostic CES. Parents of 192 unique patients participated. The parent's interpretation of the child's result agreed with the clinician's interpretation in 79% of cases, with more frequent discordance when the clinician's interpretation was uncertain. The majority (79%) reported no regret with the decision to have CES. Most (65%) reported complete satisfaction with the genetic counseling experience, and satisfaction was positively associated with years of genetic counselor (GC) experience. The psychological impact of CES was greatest for parents of children with positive results and for parents with anxiety or depression. The results of this study are important for helping clinicians to prepare families for the possible results and variable psychological impact of CES. The frequency of parental misinterpretation of test results indicates the need for additional clarity in the communication of results. Finally, while the majority of patients were satisfied with their genetic counseling, satisfaction was lower for new GCs, suggesting a need for targeted GC training for genomic testing.
Assuntos
Deficiências do Desenvolvimento/genética , Sequenciamento do Exoma/métodos , Exoma/genética , Aconselhamento Genético , Adulto , Criança , Deficiências do Desenvolvimento/fisiopatologia , Revelação , Feminino , Testes Genéticos , Humanos , Masculino , Pais , Inquéritos e QuestionáriosRESUMO
UNLABELLED: Blood ammonia and glutamine levels are used as biomarkers of control in patients with urea cycle disorders (UCDs). This study was undertaken to evaluate glutamine variability and utility as a predictor of hyperammonemic crises (HACs) in UCD patients. METHODS: The relationships between glutamine and ammonia levels and the incidence and timing of HACs were evaluated in over 100 adult and pediatric UCD patients who participated in clinical trials of glycerol phenylbutyrate. RESULTS: The median (range) intra-subject 24-hour coefficient of variation for glutamine was 15% (8-29%) as compared with 56% (28%-154%) for ammonia, and the correlation coefficient between glutamine and concurrent ammonia levels varied from 0.17 to 0.29. Patients with baseline (fasting) glutamine values >900 µmol/L had higher baseline ammonia levels (mean [SD]: 39.6 [26.2]µmol/L) than patients with baseline glutamine ≤ 900 µmol/L (26.6 [18.0]µmol/L). Glutamine values >900 µmol/L during the study were associated with an approximately 2-fold higher HAC risk (odds ratio [OR]=1.98; p=0.173). However, glutamine lost predictive significance (OR=1.47; p=0.439) when concomitant ammonia was taken into account, whereas the predictive value of baseline ammonia ≥ 1.0 upper limit of normal (ULN) was highly statistically significant (OR=4.96; p=0.013). There was no significant effect of glutamine >900 µmol/L on time to first HAC crisis (hazard ratio [HR]=1.14; p=0.813), but there was a significant effect of baseline ammonia ≥ 1.0 ULN (HR=4.62; p=0.0011). CONCLUSIONS: The findings in this UCD population suggest that glutamine is a weaker predictor of HACs than ammonia and that the utility of the predictive value of glutamine will need to take into account concurrent ammonia levels.
Assuntos
Amônia/sangue , Glutamina/sangue , Hiperamonemia/sangue , Distúrbios Congênitos do Ciclo da Ureia/sangue , Adolescente , Adulto , Biomarcadores/sangue , Criança , Pré-Escolar , Jejum , Feminino , Glicerol/análogos & derivados , Glicerol/uso terapêutico , Humanos , Hiperamonemia/etiologia , Masculino , Fenilbutiratos/uso terapêutico , Valor Preditivo dos Testes , Distúrbios Congênitos do Ciclo da Ureia/tratamento farmacológico , Adulto JovemRESUMO
BACKGROUND: Phenylacetic acid (PAA) is the active moiety in sodium phenylbutyrate (NaPBA) and glycerol phenylbutyrate (GPB, HPN-100). Both are approved for treatment of urea cycle disorders (UCDs) - rare genetic disorders characterized by hyperammonemia. PAA is conjugated with glutamine in the liver to form phenylacetyleglutamine (PAGN), which is excreted in urine. PAA plasma levels ≥ 500 µg/dL have been reported to be associated with reversible neurological adverse events (AEs) in cancer patients receiving PAA intravenously. Therefore, we have investigated the relationship between PAA levels and neurological AEs in patients treated with these PAA pro-drugs as well as approaches to identifying patients most likely to experience high PAA levels. METHODS: The relationship between nervous system AEs, PAA levels and the ratio of plasma PAA to PAGN were examined in 4683 blood samples taken serially from: [1] healthy adults [2], UCD patients of ≥ 2 months of age, and [3] patients with cirrhosis and hepatic encephalopathy (HE). The plasma ratio of PAA to PAGN was analyzed with respect to its utility in identifying patients at risk of high PAA values. RESULTS: Only 0.2% (11) of 4683 samples exceeded 500 µg/ml. There was no relationship between neurological AEs and PAA levels in UCD or HE patients, but transient AEs including headache and nausea that correlated with PAA levels were observed in healthy adults. Irrespective of population, a curvilinear relationship was observed between PAA levels and the plasma PAA:PAGN ratio, and a ratio>2.5 (both in µg/mL) in a random blood draw identified patients at risk for PAA levels>500 µg/ml. CONCLUSIONS: The presence of a relationship between PAA levels and reversible AEs in healthy adults but not in UCD or HE patients may reflect intrinsic differences among the populations and/or metabolic adaptation with continued dosing. The plasma PAA:PAGN ratio is a functional measure of the rate of PAA metabolism and represents a useful dosing biomarker.
Assuntos
Glutamina/análogos & derivados , Encefalopatia Hepática/sangue , Fenilacetatos/sangue , Distúrbios Congênitos do Ciclo da Ureia/sangue , Biomarcadores/sangue , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos/sangue , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos/etiologia , Glutamina/administração & dosagem , Glutamina/sangue , Glicerol/administração & dosagem , Glicerol/análogos & derivados , Encefalopatia Hepática/etiologia , Encefalopatia Hepática/patologia , Humanos , Fígado/efeitos dos fármacos , Fígado/metabolismo , Neoplasias/complicações , Neoplasias/tratamento farmacológico , Fenilacetatos/administração & dosagem , Fenilbutiratos/administração & dosagem , Ensaios Clínicos Controlados Aleatórios como Assunto , Distúrbios Congênitos do Ciclo da Ureia/epidemiologia , Distúrbios Congênitos do Ciclo da Ureia/etiologia , Distúrbios Congênitos do Ciclo da Ureia/patologiaRESUMO
UNLABELLED: We have analyzed pharmacokinetic data for glycerol phenylbutyrate (also GT4P or HPN-100) and sodium phenylbutyrate with respect to possible dosing biomarkers in patients with urea cycle disorders (UCD). STUDY DESIGN: These analyses are based on over 3000 urine and plasma data points from 54 adult and 11 pediatric UCD patients (ages 6-17) who participated in three clinical studies comparing ammonia control and pharmacokinetics during steady state treatment with glycerol phenylbutyrate or sodium phenylbutyrate. All patients received phenylbutyric acid equivalent doses of glycerol phenylbutyrate or sodium phenylbutyrate in a cross over fashion and underwent 24-hour blood samples and urine sampling for phenylbutyric acid, phenylacetic acid and phenylacetylglutamine. RESULTS: Patients received phenylbutyric acid equivalent doses of glycerol phenylbutyrate ranging from 1.5 to 31.8 g/day and of sodium phenylbutyrate ranging from 1.3 to 31.7 g/day. Plasma metabolite levels varied widely, with average fluctuation indices ranging from 1979% to 5690% for phenylbutyric acid, 843% to 3931% for phenylacetic acid, and 881% to 1434% for phenylacetylglutamine. Mean percent recovery of phenylbutyric acid as urinary phenylacetylglutamine was 66.4 and 69.0 for pediatric patients and 68.7 and 71.4 for adult patients on glycerol phenylbutyrate and sodium phenylbutyrate, respectively. The correlation with dose was strongest for urinary phenylacetylglutamine excretion, either as morning spot urine (r = 0.730, p < 0.001) or as total 24-hour excretion (r = 0.791 p<0.001), followed by plasma phenylacetylglutamine AUC(24-hour), plasma phenylacetic acid AUC(24-hour) and phenylbutyric acid AUC(24-hour). Plasma phenylacetic acid levels in adult and pediatric patients did not show a consistent relationship with either urinary phenylacetylglutamine or ammonia control. CONCLUSION: The findings are collectively consistent with substantial yet variable pre-systemic (1st pass) conversion of phenylbutyric acid to phenylacetic acid and/or phenylacetylglutamine. The variability of blood metabolite levels during the day, their weaker correlation with dose, the need for multiple blood samples to capture trough and peak, and the inconsistency between phenylacetic acid and urinary phenylacetylglutamine as a marker of waste nitrogen scavenging limit the utility of plasma levels for therapeutic monitoring. By contrast, 24-hour urinary phenylacetylglutamine and morning spot urine phenylacetylglutamine correlate strongly with dose and appear to be clinically useful non-invasive biomarkers for compliance and therapeutic monitoring.
Assuntos
Amônia/urina , Glutamina/análogos & derivados , Glicerol/análogos & derivados , Fenilacetatos/urina , Fenilbutiratos/urina , Distúrbios Congênitos do Ciclo da Ureia/tratamento farmacológico , Distúrbios Congênitos do Ciclo da Ureia/urina , Adolescente , Adulto , Amônia/sangue , Biomarcadores Farmacológicos/sangue , Biomarcadores Farmacológicos/urina , Criança , Estudos Cross-Over , Esquema de Medicação , Feminino , Glutamina/sangue , Glutamina/urina , Glicerol/sangue , Glicerol/farmacocinética , Glicerol/urina , Humanos , Masculino , Fenilacetatos/sangue , Fenilbutiratos/sangue , Fenilbutiratos/farmacocinética , Distúrbios Congênitos do Ciclo da Ureia/sangueRESUMO
Acute hyperammonaemia (HA) causes cerebral oedema and severe brain damage in patients with urea cycle disorders (UCDs) or acute liver failure (ALF). Chronic HA is associated with developmental delay and intellectual disability in patients with UCDs and with neuropsychiatric symptoms in patients with chronic liver failure. Treatment often cannot prevent severe brain injury and neurological sequelae. The causes of the brain oedema in hyperammonaemic encephalopathy (HAE) have been subject of intense controversy among physicians and scientists working in this field. Currently favoured hypotheses are astrocyte swelling due to increased intracellular glutamine content and neuronal cell death due to excitotoxicity caused by elevated extracellular glutamate levels. While many researchers focus on these mechanisms of cytotoxicity, others emphasize vascular causes of brain oedema. New data gleaned from expression profiling of astrocytes acutely isolated from hyperammonaemic mouse brains point to disturbed water and potassium homeostasis as regulated by astrocytes at the brain microvasculature and in the perisynaptic space as a potential mechanism of brain oedema development in hyperammonaemia.
Assuntos
Astrócitos/metabolismo , Astrócitos/patologia , Encefalopatias/etiologia , Encefalopatias/genética , Perfilação da Expressão Gênica , Hiperamonemia/genética , Doença Aguda , Animais , Encefalopatias/metabolismo , Encefalopatias/patologia , Células Cultivadas , Modelos Animais de Doenças , Humanos , Hiperamonemia/complicações , Hiperamonemia/metabolismo , Hiperamonemia/patologia , Análise de Sequência com Séries de OligonucleotídeosRESUMO
Urinary phenylacetylglutamine (U-PAGN) concentrations in spot urine samples were analyzed as a dosing biomarker during glycerol phenylbutyrate (GPB) dosing in 68 healthy adults and 66 adult and pediatric patients with urea cycle disorders who participated in GPB clinical trials. Age- and body surface area (BSA)-specific 25th percentile cutoff points for spot U-PAGN concentrations (<~9000 µg/mL for < 2 years old patients, < 7000 µg/mL for > 2 years with BSA ≤ 1.3 m2, and <~5000 µg/mL for > 2 years of age with BSA > 1.3 m2) were determined as an approach to identify patients for whom increased dosing and/or adherence to prescribed dosing should be assessed.
RESUMO
Genotyping of the phenylalanine hydroxylating system offers a new way of characterizing patients with phenylalanine hydroxylase (PAH) deficiency. This paper investigates the power of genotyping as a parameter for differential diagnosis and as a measure of the risk factor of brain damage in well-treated patients with phenylketonuria (PKU). Thirty-three PKU patients were followed up over 9 years and the quality of dietary treatment, plasma phenylalanine (phe) in the newborn period before treatment and intellectual outcome at the age of 9 years were measured and correlated with the predicted residual activity (PRA) of the phe hydroxylase system as estimated from mutation analysis of the PAH gene. Patients were grouped in group Ia (PRA = 0%), group Ib (PRA = 5-15%) and group II (PRA > or = 25% of the normal activity). Mean plasma phe levels in the newborn in group Ia were 37.9 +/- 6.5 (2296 +/- 394), in group Ib 40.8 +/- 15.9 (2472 +/- 963) and in group II 16.2 +/- 4.2 (981 +/- 254) mg/dl (mumol/l). Difference in mean plasma values of groups Ia and Ib on the one hand and group II on the other were highly significant (P < 0.0001). No difference could be seen between groups Ia and Ib. There was a higher mean IQ at the age of 9 years in group II (97.4 +/- 5.4) in comparison with groups Ia (92.7 +/- 12.8) and Ib (85.0 +/- 14.4). The difference between group Ib and group II was significant (P < 0.040).(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Fenilcetonúrias/genética , Criança , Pré-Escolar , DNA/análise , Genótipo , Humanos , Lactente , Recém-Nascido , Testes de Inteligência , Fenótipo , Fenilalanina/sangue , Fenilalanina Hidroxilase/deficiência , Fenilalanina Hidroxilase/genética , Fenilcetonúrias/dietoterapia , Fenilcetonúrias/psicologia , Estudos Prospectivos , Fatores de RiscoRESUMO
Sodium phenylbutyrate and glycerol phenylbutyrate mediate waste nitrogen excretion in the form of urinary phenylacetylglutamine (PAGN) in patients with urea cycle disorders (UCDs); rare genetic disorders characterized by impaired urea synthesis and hyperammonemia. Sodium phenylbutyrate is approved for UCD treatment; the development of glycerol phenylbutyrate afforded the opportunity to characterize the pharmacokinetics (PK) of both compounds. A population PK model was developed using data from four Phase II/III trials that collectively enrolled patients ages 2 months to 72 years. Dose simulations were performed with particular attention to phenylacetic acid (PAA), which has been associated with adverse events in non-UCD populations. The final model described metabolite levels in plasma and urine for both drugs and was characterized by (a) partial presystemic metabolism of phenylbutyric acid (PBA) to PAA and/or PAGN, (b) slower PBA absorption and greater presystemic conversion with glycerol phenylbutyrate, (c) similar systemic disposition with saturable conversion of PAA to PAGN for both drugs, and (d) body surface area (BSA) as a significant covariate accounting for age-related PK differences. Dose simulations demonstrated similar PAA exposure following mole-equivalent PBA dosing of both drugs and greater PAA exposure in younger patients based on BSA.
Assuntos
Glicerol/análogos & derivados , Modelos Biológicos , Fenilbutiratos/administração & dosagem , Fenilbutiratos/farmacocinética , Distúrbios Congênitos do Ciclo da Ureia/tratamento farmacológico , Distúrbios Congênitos do Ciclo da Ureia/metabolismo , Adulto , Criança , Pré-Escolar , Ensaios Clínicos Fase II como Assunto , Ensaios Clínicos Fase III como Assunto , Simulação por Computador , Feminino , Glutamina/análogos & derivados , Glutamina/urina , Glicerol/administração & dosagem , Glicerol/farmacocinética , Humanos , Masculino , Nitrogênio/urina , Ensaios Clínicos Controlados Aleatórios como Assunto , Doenças Raras/tratamento farmacológico , Doenças Raras/metabolismo , Distúrbios Congênitos do Ciclo da Ureia/urinaRESUMO
Lowe syndrome is a rare X-linked disease characterized by congenital cataracts, defects in renal tubule cell function, and mental retardation. Mutations in the OCRL1 gene, which encodes ocrl1, a phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P(2)) 5-phosphatase, are the cause of Lowe syndrome. PtdIns(4,5)P(2), a substrate of ocrl1, is an important signaling molecule within the cell. OCRL1 is ubiquitously expressed and co-localizes with the trans-Golgi network (TGN) and endosomal proteins. The ocrl1 protein contains two recognizable domains, one a conserved Ptd(4,5)P(2) 5-phosphatase domain and the other with homology to Rho GTPase activating proteins (RhoGAPs). The objective of our study was to further characterize the ocrl1 RhoGAP-homology domain by analyzing the effect of two missense mutations in this domain, I751N and A780P, which were previously reported in Lowe syndrome patients. Both mutant proteins were expressed at levels similar to wild-type but their enzyme activity was reduced by 85-90%, indicating that the RhoGAP-homology domain is important for the enzymatic function of ocrl1. Study of a C-terminal region of wild-type ocrl1 containing this domain detected no GAP activity, eliminating the possibility of an effect by mutations in this domain on GTPase activation. Because members of the Arf family of small G-proteins are directly involved in (Ptd(4,5)P(2)) signaling and localize to the TGN like ocrl1, we analyzed by immunoprecipitation the interaction of ocrl1 with Arf1 and Arf6 via its RhoGAP-homology domain. Wild-type ocrl1, but not the I751N mutant protein, co-immunoprecipitated with these two Arf proteins. These results indicate that wild-type ocrl1 and Arf proteins can interact and that this interaction is disrupted by the mutation. It remains unknown whether a disrupted interaction between Arf and ocrl1 plays a role in the Lowe syndrome phenotype.
Assuntos
Fatores de Ribosilação do ADP/metabolismo , Síndrome Oculocerebrorrenal/genética , Monoéster Fosfórico Hidrolases/genética , Monoéster Fosfórico Hidrolases/metabolismo , Ativação Enzimática , Fibroblastos/enzimologia , GTP Fosfo-Hidrolases/análise , Proteínas Ativadoras de GTPase/metabolismo , Humanos , Imunoprecipitação , Mutação de Sentido Incorreto , Síndrome Oculocerebrorrenal/enzimologia , Monoéster Fosfórico Hidrolases/análise , Estrutura Terciária de ProteínaRESUMO
The hyperphenylalaninemic disorders of classic phenylketonuria (PKU), mild phenylketonuria, and hyperphenylalaninemia (HPA), result from a deficiency of the hepatic enzyme phenylalanine hydroxylase (PAH) or its cofactor (tetrahydrobiopterin). Use of the complementary DNA of this enzyme has allowed the establishment of a restriction fragment length polymorphism (RFLP) haplotype-analysis system. This haplotype analysis system provides the means for determination of mutant PAH alleles in most affected families and is the basis for mutational analysis of the PKU locus. This review is focused on two major areas of current PKU research: (1) the use of DNA haplotype analysis in the study of the population genetics of PAH deficiency, and (2) the study of genotypes, and their various combinations, as a means of explaining and predicting the phenotypic variability observed for the disorders of PAH deficiency.
Assuntos
Alelos , Genética Populacional , Mutação , Fenilalanina Hidroxilase/genética , Fenilcetonúrias/genética , Mapeamento Cromossômico , Haplótipos , Humanos , Fenilalanina Hidroxilase/deficiência , Polimorfismo de Fragmento de RestriçãoRESUMO
Five sequence polymorphisms at the phenylalanine hydroxylase (PAH) gene locus were observed to be in tight association with specific alleles of this locus. Since these polymorphisms can be detected using polymerase chain reaction (PCR) methodology, application of a combination of these polymorphisms reduces the effort involved in PAH DNA haplotype analysis, which is needed for population genetic analysis or diagnosis of the disease status. In addition our results indicate the evolution of haplotype 3, 4 and 7 PAH alleles from a common ancestor, whereas PAH haplotypes 5, 6, and 11 arose from another common ancestor allele. These data reveal that two of the polymorphisms investigated originated before the separation of races.
Assuntos
Alelos , Fenilalanina Hidroxilase/genética , Polimorfismo Genético , Sequência de Bases , DNA/análise , Éxons , Haplótipos , Humanos , Íntrons , Reação em Cadeia da PolimeraseRESUMO
Phenylalanine hydroxylase (PAH) is the key enzyme in phenylalanine metabolism. PAH deficiency results in hyperphenylalaninemia, leading to severe mental retardation in the classical form of the disease, phenylketonuria (PKU). Previously the expression of PAH could only unambiguously be demonstrated in human liver, whereas in rodents PAH expression has been established in kidney and liver. Reports concerning PAH activity in other human or rodent tissues were severely questioned by subsequent investigations such that they did not gain general recognition. Conducting Northern blot analyses, we detected the PAH transcript in RNA isolated from human liver, kidney, pancreas, and brain. PAH gene expression in human kidney was subsequently investigated by RNase protection assay analyses, RNA in situ hybridization, immunohistochemistry, enzyme assay, and cDNA isolation. These experiments allowed the conclusive verification of a functional PAH enzyme in human kidney. The primary structure of the kidney transcript corresponded to the structure of the liver transcript. Human kidney PAH may play a significant role in phenylalanine homeostasis of the organism, as impaired phenylalanine hydroxylation has been observed in renal failure and differences in the regulation of the kidney versus the liver enzyme have been indicated. These results provide new aspects to research into the basis for the heterogeneity of hyperphenylalaninemia phenotypes and establish that the expression of the human PAH gene is not limited to the liver.
Assuntos
Rim/enzimologia , Fígado/enzimologia , Fenilalanina Hidroxilase/genética , Northern Blotting , Encéfalo/enzimologia , Feminino , Regulação Enzimológica da Expressão Gênica , Humanos , Imuno-Histoquímica , Hibridização In Situ , Pâncreas/enzimologia , Fenilalanina Hidroxilase/metabolismo , Placenta/enzimologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Distribuição TecidualRESUMO
The isolation and sequencing of 15 independent human lysosome-associated membrane protein-2 (h-lamp-2) recombinants from a primary human liver cDNA library has resulted in the determination of a transcript sequence significantly longer than previously reported and reveals the utilization of each of the four potential polyadenylation signals (AATAAA) present in the 3' untranslated region. The most 5' extending cDNA clone initiates upstream of the proposed transcription initiation site. A number of differences with published sequences for the h-lamp-2 transcript were observed, some of which result in amino acid changes in the predicted primary structure of the h-lamp-2 protein, and two of which give rise to restriction fragment length polymorphisms. The knowledge of these sequence alterations and polymorphisms is an important consideration for the further analysis of the h-lamp-2 locus with regard to the delineation of function and association with human inherited disorders.
Assuntos
Antígenos CD , Lisossomos , Glicoproteínas de Membrana/genética , Proteínas de Membrana/genética , Sequência de Aminoácidos , Sequência de Bases , DNA Complementar , Humanos , Fígado/metabolismo , Proteínas de Membrana Lisossomal , Dados de Sequência MolecularRESUMO
Human phenylalanine hydroxylase (PAH) is expressed in a liver-specific manner and catalyzes the enzymatic conversion of phenylalanine to tyrosine. Genetic deficiency of PAH results in the autosomal-recessive disorder phenylketonuria (PKU). Through the application of genomic and cDNA cloning, primer extension studies, SI mapping experiments, and PCR methodologies, the transcription initiation (CAP) site has been identified and the 5'-flanking region determined. The most upstream CAP site for the human hepatic PAH gene transcript is located 154 nucleotides upstream of the first translation codon. The genomic and cDNA sequences analyzed demonstrated that the previously reported cDNA sequence, phPAH247 [Kwok et al. (1985) Biochemistry 24, 556-561], contained a 164-nucleotide cloning artifact at its 5'-end. The 319 base pair region immediately upstream of the CAP site is characterized by the lack of a proximal TATA box and the presence of sequences similar to GC boxes, CACCC boxes, CCAAT boxes, activator protein 2 (Ap-2) sites, partial glucocorticoid response elements (GREs), and partial cyclic AMP response elements (CREs). This suggests that the human PAH gene has a TATA-less promoter regulated by multiple transcription factors.
Assuntos
Fenilalanina Hidroxilase/genética , Sequência de Bases , Carcinoma Hepatocelular , Linhagem Celular , Clonagem Molecular , Códon/genética , Cosmídeos , DNA/genética , DNA/isolamento & purificação , Éxons , Biblioteca Gênica , Humanos , Fígado/enzimologia , Neoplasias Hepáticas , Dados de Sequência Molecular , Oligodesoxirribonucleotídeos , Fenilalanina Hidroxilase/isolamento & purificação , Reação em Cadeia da Polimerase , Biossíntese de Proteínas , Proteínas Recombinantes/isolamento & purificação , Mapeamento por Restrição , Transcrição GênicaRESUMO
Autosomal dominant renal Fanconi syndrome is a genetic model for the study of proximal renal tubular transport pathology. We were able to map the locus for this disease to human chromosome 15q15.3 by genotyping a central Wisconsin pedigree with 10 affected individuals. After a whole-genome scan with highly polymorphic simple sequence repeat markers, a maximum LOD score of 3.01 was calculated for marker D15S659 on chromosome 15q15.3. Linkage and haplotype analysis for an additional 24 markers flanking D15S659 narrowed the interval to approximately 3 cM, with the two highest single-point LOD scores observed being 4.44 and 4.68 (for D15S182 and D15S537, respectively). Subsequently, a complete bacterial artificial chromosome contig was constructed, from the High Throughput Genomic Sequence Database, for the region bounded by D15S182 and D15S143. The identification of the gene and gene product altered in autosomal dominant renal Fanconi syndrome will allow the study of the physiology of proximal renal tubular transport.
Assuntos
Mapeamento Cromossômico , Cromossomos Humanos Par 15/genética , Síndrome de Fanconi/genética , Genes Dominantes/genética , Cromossomos Artificiais Bacterianos/genética , Mapeamento de Sequências Contíguas , Síndrome de Fanconi/fisiopatologia , Feminino , Marcadores Genéticos/genética , Haplótipos/genética , Humanos , Escore Lod , Masculino , Linhagem , WisconsinRESUMO
Thirteen children with acute lymphoblastic leukemia (ALL) were investigated before and during cytotoxic therapy. EEG findings were correlated with the clinical course and the therapy protocol and compared with normal data obtained from 295 healthy children. Frequency analysis of the background activity of the EEG revealed an initial slowing of the background activity prior to therapy and further slowing each time a combination of vincristine (VCR), daunorubicin (DAU) or adriblastine (ADR), prednisone (PRED), and L-asparaginase (L-ASP) was administered. The slowing of the background activity correlated only with the administration of these drugs. DAU, ADR, and PRED are not known to influence the EEG; therefore, VCR and L-ASP remain the primary candidates responsible for the central nervous system alteration.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Encefalopatias/induzido quimicamente , Eletroencefalografia/efeitos dos fármacos , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Adolescente , Asparaginase/administração & dosagem , Asparaginase/farmacologia , Encefalopatias/fisiopatologia , Criança , Pré-Escolar , Terapia Combinada , Irradiação Craniana , Daunorrubicina/administração & dosagem , Daunorrubicina/farmacologia , Doxorrubicina/administração & dosagem , Doxorrubicina/farmacologia , Feminino , Humanos , Masculino , Leucemia-Linfoma Linfoblástico de Células Precursoras/complicações , Leucemia-Linfoma Linfoblástico de Células Precursoras/radioterapia , Prednisona/administração & dosagem , Prednisona/farmacologia , Vincristina/administração & dosagem , Vincristina/farmacologiaRESUMO
Forty-six individuals having phenylketonuria (PKU) alleles at the phenylalanine hydroxylase (PAH) locus were tested for the haplotype 2 PKU mutation by allele-specific hybridization following in vitro DNA amplification. Patients and carriers previously shown to have a mutant haplotype 2 PAH allele demonstrated conservation of this mutation. In vitro DNA amplification greatly facilitated this analysis and provides the possibility of population screening for 37% of the mutant German PAH alleles.
Assuntos
Amplificação de Genes , Mutação , Fenilcetonúrias/genética , Alelos , Southern Blotting , DNA Polimerase Dirigida por DNA/análise , Triagem de Portadores Genéticos , Humanos , Hibridização de Ácido Nucleico , Fenilalanina Hidroxilase/genéticaRESUMO
DNA sequence analysis of the 13 exons and intron/exon boundaries of the phenylalanine hydroxylase (PAH) gene has detected two base transitions, resulting in mis-sense mutations, in the genomic DNA of a Turkish patient (E1) with phenylketonuria (PKU). The Leu48----Ser amino acid substitution was associated with the mutant haplotype 3 allele and the Glu221----Gly amino acid substitution with the mutant haplotype 4 allele of this family. Allele-specific oligonucleotide (ASO) dot-blot analysis subsequently detected the Leu48----Ser mutation in the haplotype 4 PKU alleles of nine (18.8%) of the 48 unrelated Caucasian PKU families investigated. This mutation results in mild PKU in the homozygous state. The Glu221----Gly mutation has only been detected within patient E1 and his father.
Assuntos
Mutação , Fenilalanina Hidroxilase/genética , Fenilcetonúrias/genética , Alelos , Sequência de Bases , Mapeamento Cromossômico , DNA/genética , Análise Mutacional de DNA , Glutamatos/genética , Ácido Glutâmico , Glicina/genética , Homozigoto , Humanos , Dados de Sequência Molecular , Hibridização de Ácido Nucleico , Fenilalanina Hidroxilase/deficiência , Reação em Cadeia da Polimerase , Polimorfismo de Fragmento de Restrição , TurquiaRESUMO
We report the isolation of an alternatively spliced human lysosome-associated membrane protein-2 (h-lamp-2) transcript which is overexpressed in human muscle. The cloning of this transcript is an indication for the tissue-specific expression of lysosomal membrane proteins and implicates the possibility of multiple functions for the protein products of the h-lamp-2 gene, as well as other lysosome-associated membrane proteins. The new transcript, designated h-lamp-2b, results from the alternative splicing of the last exon, exon 9, the alternative form of which is approximately 2800 bp in length. The resulting protein is identical in length to the previously reported h-lamp-2 protein, 410 amino acids including the leader peptide. This final exon, which encodes the last eleven amino acids of the luminal domain, the 24 amino acid transmembrane spanning region, and an eleven amino acid cytoplasmic tail, shows complete conservation of the Gly.Tyr.X.X lysosomal targeting signal with regard to its position relative to the transmembrane spanning region and the carboxy terminus of the protein. Immune electron microscopy studies verified localization of this alternative gene product to the lysosomal membrane.
Assuntos
Processamento Alternativo , Antígenos CD/biossíntese , Expressão Gênica , Glicoproteínas de Membrana/biossíntese , Sequência de Aminoácidos , Animais , Antígenos CD/isolamento & purificação , Sequência de Bases , Northern Blotting , Clonagem Molecular , Primers do DNA , DNA Complementar , Éxons , Humanos , Proteínas de Membrana Lisossomal , Glicoproteínas de Membrana/isolamento & purificação , Camundongos , Microscopia Imunoeletrônica , Dados de Sequência Molecular , Músculo Esquelético/metabolismo , Músculo Esquelético/ultraestrutura , Especificidade de Órgãos , Organelas/metabolismo , Organelas/ultraestrutura , Reação em Cadeia da Polimerase , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/isolamento & purificação , Homologia de Sequência de Aminoácidos , Transcrição GênicaRESUMO
In order to elucidate the molecular basis of phenylketonuria (PKU) in Portugal, a detailed study of the Portuguese mutant phenylalanine hydroxylase (PAH) genes was performed. A total of 222 mutant alleles from 111 PKU families were analysed for 26 mutations and restriction fragment length polymorphismlvariable number tandem repeat (RFLP/VNTR) haplotypes. It was possible to characterise 55% of the mutant alleles, in which 14 different mutations (R261Q, V388M, IVS10nt-11, I65T, P281L, R252W, R158Q, L348V, Y414C, L311P, Y198fsdel22bp, R408W, R270K, and R261X) and three polymorphisms (Q232Q, V245V, and L385L) were identified. A total of 14 different haplotypes were observed, with a high prevalence of haplotype 1 among mutant and normal alleles. The results reported in this study show considerable genetic heterogeneity in the Portuguese PKU population, as has also been described for other southern European populations.