ABSTRACT
BACKGROUND: Obesity-associated chronic low-grade inflammation leads to dysregulation of central lipid and glucose metabolism pathways leading to metabolic disorders. MicroRNAs (miRNAs) are known to control regulators of metabolic homeostasis. We aimed to assess the relationship of circulating miRNAs with inflammatory modulators and metabolic disorders in pediatric obesity. METHODS: From a pediatric cohort with severe obesity (n = 109), clinically thoroughly characterized including diverse routine blood parameters, oral glucose tolerance test, and liver MRI, a panel of 16 circulating miRNAs was quantified using qRT-PCR. Additionally, markers of inflammation TNFα, IL1 receptor antagonist, procalcitonin, CRP, and IL-6 were measured. RESULTS: Markers of obesity-associated inflammation, TNFα, IL-1Ra, and procalcitonin, all significantly correlated with concentrations of miRNAs 122 and 192. Concentrations of these miRNAs negatively correlated with serum adiponectin and were among those strongly linked to parameters of dyslipidemia and liver function. Moreover, miRNA122 concentrations correlated with HOMA-IR. Several miRNA levels including miRNAs 34a, 93, 122, and 192 were statistically significantly differing between individuals with prediabetes, impaired glucose tolerance, metabolic syndrome, or nonalcoholic fatty liver disease compared to the respective controls. Additionally, miRNA 192 was significantly elevated in metabolically unhealthy obesity. CONCLUSIONS: A miRNA pattern associated with obesity-associated inflammation and comorbidities may be used to distinguish metabolically healthy from unhealthy pediatric patients with obesity. Moreover, these changes in epigenetic regulation could potentially be involved in the etiology of obesity-linked metabolic disease in children and adolescents.
Subject(s)
Metabolic Syndrome , MicroRNAs/blood , Pediatric Obesity , Adolescent , Child , Female , Humans , Inflammation , Male , Metabolic Syndrome/epidemiology , Metabolic Syndrome/metabolism , Pediatric Obesity/epidemiology , Pediatric Obesity/metabolismABSTRACT
BACKGROUND: Eighty percent of adolescents with severe obesity suffer from non-alcoholic fatty liver disease (NAFLD). Non-invasive prediction models have been tested in adults, however, they performed poorly in paediatric populations. OBJECTIVE: This study aimed to investigate novel biomarkers for NAFLD and to develop a score that predicts liver fat in youth with severe obesity. METHODS: From a population with a BMI >97th percentile aged 9-19 years (n = 68), clinically thoroughly characterized including MRI-derived proton density fat fraction (MRI-PDFF), amino acids and acylcarnitines were measured by HPLC-MS. RESULTS: In children with NAFLD, higher levels of plasma branched-chain amino acids (BCAA) were determined. BCAAs correlated with MRI-PDFF (R = 0.46, p < .01). We identified a linear regression model adjusted for age, sex and pubertal stage consisting of BCAAs, ALT, GGT, ferritin and insulin that predicted MRI-PDFF (R = 0.75, p < .01). ROC analysis of this model revealed AUCs of 0.85, 0.85 and 0.92 for the detection of any, moderate and severe steatosis, respectively, thus markedly outperforming previously published scores. CONCLUSION: BCAAs could be an important link between obesity and other metabolic pathways. A BCAA-based metabolic score can predict steatosis grade in high-risk children and adolescents and may provide a feasible alternative to sophisticated methods like MRI or biopsy in the future.
Subject(s)
Non-alcoholic Fatty Liver Disease , Obesity, Morbid , Adolescent , Amino Acids, Branched-Chain , Child , Female , Humans , Liver , Magnetic Resonance Imaging , Male , Non-alcoholic Fatty Liver Disease/diagnosis , Non-alcoholic Fatty Liver Disease/epidemiology , Obesity, Morbid/diagnosis , Obesity, Morbid/epidemiologyABSTRACT
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.
Subject(s)
Alleles , DNA Methylation , Phenylalanine Hydroxylase/genetics , Phenylalanine/blood , Phenylketonurias/genetics , Promoter Regions, Genetic , 5' Untranslated Regions , Case-Control Studies , CpG Islands , Female , Humans , Infant, Newborn , Male , Nucleic Acid Denaturation , Phenylalanine Hydroxylase/blood , Phenylketonurias/blood , Phenylketonurias/diagnosis , Phenylketonurias/pathology , Polymerase Chain Reaction , Sequence Analysis, DNAABSTRACT
OBJECTIVES: Phenylketonuria (PKU) is characterized by a high phenylalanine (phe) in plasma and oxidative stress. However, the monitoring of oxidative stress in newborns with PKU using the activity levels of antioxidant enzymes is not optimal. We investigated the possibility of monitoring an increased reactive oxygen species (ROS) production using DNA methylation changes of an oxidative stress response element in the promoter region of an enzymatic antioxidant gene. DESIGN AND METHODS: Using DNA extracted from blood leukocytes, the cytosine phosphodiester bond guanine positions of an overlapping CCAAT box/metal response element (CGATTGGCTG) of the glutathione peroxidase 3 promoter activated by oxidative stimuli and expressed in plasma were analysed for methylation changes in 20 newborns with hyperphenylalaninemia and 20 healthy controls. RESULTS: A demethylated allele was detected in a PKU patient at a phe level of 465µmol/L on day 2 after birth, but not in other patients (phe<465µmol/L, ≥day 2 after birth; phe>465µmol/L, ≥day 3 after birth) and healthy controls (phe<465µmol/L, ≥day 2 after birth). CONCLUSIONS: The detection of the demethylated allele could be time and phe concentration dependent. The demethylated allele is suggested as an early epigenetic marker for an extracellular monitoring of an increased ROS production in newborns with PKU.
Subject(s)
Glutathione Peroxidase/genetics , Phenylketonurias/genetics , Promoter Regions, Genetic/genetics , DNA Methylation/genetics , Female , Humans , Infant, Newborn , MaleABSTRACT
Diagnosis of bacterial sepsis in preterm neonates can be difficult when using serum markers that rely on physiological changes because these changes may not necessarily be the result of bacterial infections alone. This retrospective investigation explores the potential use of the DNA methylation pattern of CpG sites in the promoter region of the calcitonin-related polypeptide α (CALCA) gene as an epigenetic biomarker for bacterial sepsis in preterm newborns. Four novel changes in the DNA methylation of eight CpG sites were detected in this gene and are present only in neonates with bacterial sepsis: (1) partial methylation at -769 CpG in gram-negative or gram-positive early onset sepsis (EOS) and late onset sepsis (LOS) episodes; (2) demethylation of 8 CpGs in gram-negative EOS followed by LOS (ELS) and in gram-negative EOS; (3) demethylation of 7 CpGs in gram-positive ELS and gram-positive EOS; (4) -771 C:G>T:A; 5' de novo -778 CpG mutation on both alleles in EOS. These changes were not detected in birth weight and gestational age matched controls or in newborns with isolated infections. Our results indicate that the DNA methylation pattern of the promoter region of the CALCA gene varies in different types of bacterial preterm sepsis, thus suggesting a potential use as an epigenetic biomarker. A prospective confirmation of these results is essential.
Subject(s)
Bacteremia/metabolism , Calcitonin/genetics , DNA Methylation , Epigenesis, Genetic , Infant, Premature, Diseases/metabolism , Protein Precursors/genetics , Sepsis/metabolism , Bacteremia/diagnosis , Bacteremia/microbiology , Biomarkers/blood , Calcitonin/metabolism , Calcitonin Gene-Related Peptide , CpG Islands , Humans , Infant, Newborn , Infant, Premature , Infant, Premature, Diseases/diagnosis , Infant, Premature, Diseases/microbiology , Promoter Regions, Genetic , Protein Precursors/metabolism , Retrospective Studies , Sepsis/diagnosis , Sepsis/microbiologyABSTRACT
Deficiency of ß-1,4 mannosyltransferase (MT-1) congenital disorder of glycosylation (CDG), due to ALG1 gene mutations. Features in 9 patients reported previously consisted of prenatal growth retardation, pregnancy-induced maternal hypertension and fetal hydrops. Four patients died before 5 years of age, and survivors showed a severe psychomotor retardation. We report on 7 patients with psychomotor delay, microcephaly, strabismus and coagulation abnormalities, seizures and abnormal fat distribution. Four children had a stable clinical course, two had visual impairment, and 1 had hearing loss. Thrombotic and vascular events led to deterioration of the clinical outcome in 2 patients. Four novel ALG1 mutations were identified. Pathogenicity was determined in alg1 yeast mutants transformed with hALG1. Functional analyses showed all novel mutations representing hypomorphs associated with residual enzyme activity. We extend the phenotypic spectrum including the first description of deafness in MT1 deficiency, and report on mildly affected patients, surviving to adulthood. The dysmorphic features, including abnormal fat distribution and strabismus highly resemble CDG due to phosphomannomutase-2 deficiency (PMM2-CDG), the most common type of CDG. We suggest testing for ALG1 mutations in unsolved CDG patients with a type 1 transferrin isoelectric focusing pattern, especially with epilepsy, severe visual loss and hemorrhagic/thrombotic events.
Subject(s)
Congenital Disorders of Glycosylation/genetics , Mannosyltransferases/genetics , Child , Child, Preschool , Congenital Disorders of Glycosylation/diagnosis , Fatal Outcome , Female , Genetic Markers , Humans , Infant , Male , Mannosyltransferases/deficiency , Mutation , Phenotype , Young AdultABSTRACT
BACKGROUND: Interest in lysosomal storage disorders, a collection of more than 40 inherited metabolic disorders, has increased because of new therapy options such as enzyme replacement, stem cell transplantation, and substrate reduction therapy. We developed a high-throughput protocol that simplifies analytical challenges such as complex sample preparation and potential interference from excess residual substrate associated with previously reported assays. METHODS: After overnight incubation (16-20 h) of dried blood spots with a cassette of substrates and deuterated internal standards, we used a TLX-2 system to quantify 6 lysosomal enzyme activities for Fabry, Gaucher, Niemann-Pick A/B, Pompe, Krabbe, and mucopolysaccharidosis I disease. This multiplexed, multidimensional ultra-HPLC-tandem mass spectrometry assay included Cyclone P Turbo Flow and Hypersil Gold C8 columns. The method did not require offline sample preparation such as liquid-liquid and solid-phase extraction, or hazardous reagents such as ethyl acetate. RESULTS: Obviating the offline sample preparation steps led to substantial savings in analytical time (approximately 70%) and reagent costs (approximately 50%). In a pilot study, lysosomal enzyme activities of 8586 newborns were measured, including 51 positive controls, and the results demonstrated 100% diagnostic sensitivity and high specificity. The results for Krabbe disease were validated with parallel measurements by the New York State Screening Laboratory. CONCLUSIONS: Turboflow online sample cleanup and the use of an additional analytical column enabled the implementation of lysosomal storage disorder testing in a nationwide screening program while keeping the total analysis time to <2 min per sample.
Subject(s)
Clinical Protocols , Lysosomal Storage Diseases/diagnosis , Neonatal Screening/methods , Chromatography, High Pressure Liquid/methods , Fabry Disease/diagnosis , Gaucher Disease/diagnosis , Glycogen Storage Disease Type II/diagnosis , Humans , Infant, Newborn , Leukodystrophy, Globoid Cell/diagnosis , Mass Spectrometry , Mucopolysaccharidosis I/diagnosis , Niemann-Pick Disease, Type A/diagnosis , Niemann-Pick Disease, Type B/diagnosis , Pilot Projects , Sensitivity and SpecificityABSTRACT
OBJECTIVE: To evaluate the effects of creatine monohydrate (CMH) supplementation on global DNA methylation and disease-specific clinical symptoms in female patients with Rett syndrome (RTT). METHODS: Double-blind, randomized, placebo-controlled crossover trial of female patients with RTT. Participants received 200 mg/kg of either CMH or placebo daily for 6 months and switched following a 4-week washout period. Primary endpoints were change in global DNA methylation and in a RTT-specific symptom score as defined by medical history and clinical evaluation with Rett Syndrome Motor and Behavioral Assessment. Secondary endpoints were changes in biochemical markers of methionine metabolism. RESULTS: Eighteen female patients aged 3 to 25 years with clinically diagnosed typical RTT and MECP2 mutation at clinical Stages III or IV were studied. CMH supplementation resulted in a statistically significant increase of global methylation by 0.11 (95% confidence interval 0.03-0.19, p = .009) compared with placebo. Total and subscores of Rett Syndrome Motor and Behavioral Assessment tended to improve but without statistical significance. CONCLUSION: CMH supplementation increases global DNA methylation statistically significantly. Scores were lower for creatine than for placebo reflecting clinical improvement but not reaching statistical significance. Biochemical variables of methionine-homocysteine remethylation are unaffected. Multicenter studies are urgently warranted to evaluate the long-term effects of CMH supplementation in an optimally homogenous RTT population over a prolonged period.
Subject(s)
Creatine/therapeutic use , Dietary Supplements , Rett Syndrome/drug therapy , Adolescent , Adult , Child , Child, Preschool , Creatine/administration & dosage , Cross-Over Studies , DNA Methylation/drug effects , DNA Methylation/genetics , Double-Blind Method , Female , Humans , Methyl-CpG-Binding Protein 2/genetics , Severity of Illness Index , Tandem Mass Spectrometry , Young AdultABSTRACT
The aim of this retrospective analysis was to evaluate the status of p53 and possible mutations in Merkel cell carcinoma (MCC) cell lines and MCC tissue samples. The p53 mutations are common in different cancer origins but rare in MCCs detected so far. MCCs are highly aggressive neuroendocrine tumors with an enhanced potential to metastasize. Until now, less is known about MCC and new approaches to understand this disease are necessary. RNA and DNA were extracted from two MCC cell lines and 27 archival paraffin-embedded patient samples. After reverse transcription, a real-time PCR and a high-resolution melt analysis were carried out. In both MCC cell lines, we could detect a p53 missense mutation at codon 193 (exon 6) with a change in amino acids (His â Leu). This mutation was equal in both cell lines and was investigated in 27 tissue samples in succession to detect possible accounts for the aggressive behavior of MCCs. Unfortunately, no corresponding p53 mutation could be observed in the investigated tissue samples. A new p53 mutation was detected in MCC cell lines. This mutation could not be determined in patients' samples. Therefore, the aggressiveness of MCC seems to be independent of p53 mutations and other mutations might be responsible for developing MCC.
Subject(s)
Carcinoma, Merkel Cell/genetics , DNA, Neoplasm/genetics , Genes, p53/genetics , Head and Neck Neoplasms/genetics , Mutation , Skin Neoplasms/genetics , Aged , Aged, 80 and over , Blotting, Western , Carcinoma, Merkel Cell/pathology , Cell Line, Tumor , Female , Follow-Up Studies , Head and Neck Neoplasms/pathology , Humans , Immunohistochemistry , Male , Middle Aged , Prognosis , Real-Time Polymerase Chain Reaction , Retrospective Studies , Skin Neoplasms/pathologyABSTRACT
The creatine/phosphocreatine system is essential for cellular phosphate coupled energy storage and production, particularly in tissues subject to high metabolic demands. Male factor infertility is a common condition with unknown etiology in most of the cases. Sperm abnormalities could possibly lead to infertility. As sperm motility depends on intact mitochondrial function and energy levels. Thus reduced intracellular creatine stores may contribute to decreased sperm motility leading to male infertility as creatine /phosphocreatine system plays major role in making and breaking of ATP, thus in energy kinetics. We developed and validated a denaturing high performance liquid chromatograph (DHPLC) method for the molecular analysis of SLC6A8 and GAMT genes involve in creatine biosynthesis and transport as a possible source of human male infertility by analyzing DNA from 64, clinically confirmed, infertile men. No mutation/polymorphism was detected in the exonic regions of both genes in all the patients and in fertile healthy controls indicating that SLC6A8 and GAMT genes may not be directly involved in human male infertility.
Subject(s)
Guanidinoacetate N-Methyltransferase/genetics , Infertility, Male/genetics , Infertility, Male/metabolism , Nerve Tissue Proteins/genetics , Plasma Membrane Neurotransmitter Transport Proteins/genetics , Chromatography, High Pressure Liquid , DNA/genetics , DNA/isolation & purification , DNA Mutational Analysis , DNA Primers , Exons/genetics , Guanidinoacetate N-Methyltransferase/metabolism , Humans , Indicators and Reagents , Infertility, Male/enzymology , Male , Nerve Tissue Proteins/metabolism , Plasma Membrane Neurotransmitter Transport Proteins/metabolism , Protein Denaturation , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
BACKGROUND: the National Austrian Newborn Screening Program for inherited metabolic and endocrinologic disorders was introduced in 1966. The program continuously evolved by expanding the screening panel from phenylketonuria and galactosemia to congenital hypothyroidism, biotinidase deficiency, cystic fibrosis, and congenital adrenal hyperplasia. In 2002, the introduction of tandem mass spectrometry (MS/MS) substantially increased the number of detectable inborn errors of metabolism and now includes disorders of fatty acid oxidation, organic acidurias and various disorders of amino acid metabolism. OBJECTIVE: in this study we report our eight years experience with MS/MS in Austria and give an overview of the incidence of diseases, organization, updates on methods and current development and future aspects. METHODS: a total of 622,489 newborns were screened by MS/MS for more than 20 diseases in Austria between April 2002 and December 2009. Dried blood spot samples were collected and sent to the National Laboratory for Newborn Screening located at the Medical University of Vienna, Vienna, Austria. RESULTS: The resulting overall prevalence of inherited metabolic disorder identified by MS/MS was 1:2855, including 125 newborns with amino acidemias (1:4,980), 46 with organic acidurias (1:13,532), and 47 with fatty acid oxidation disorders (1:13,244). CONCLUSION: the introduction of MS/MS technology in Austria significantly increased the detection of inherited metabolic disorders that were previously not covered. A primary goal is the continuous effort by developing second-tier strategies with the inclusion of more specific markers in order to minimize the risk of false-negatives and to improve the positive predictive value of screening results. Early recognition of these disorders enables diagnosis and treatment before the onset of symptoms.
Subject(s)
Biomarkers/blood , Government Programs/statistics & numerical data , Mass Screening/statistics & numerical data , Mass Spectrometry/statistics & numerical data , Metabolism, Inborn Errors/diagnosis , Metabolism, Inborn Errors/epidemiology , Austria/epidemiology , Female , Humans , Infant, Newborn , Male , Mass Screening/methods , Metabolism, Inborn Errors/blood , Prevalence , Risk Assessment , Risk FactorsABSTRACT
Biotinidase deficiency (BD) is an autosomal recessive disorder of biotin metabolism that causes incomplete recycling of free biotin. The resulting depletion of intracellular biotin leads to impaired activities of biotin-dependent carboxylases. The ensuing clinical phenotype includes progressive neurologic deterioration with epileptic seizures, muscular hypotonia as well as skin eczema. BD may be readily diagnosed by analysing enzyme activity in dried blood spots during newborn screening but typically requires molecular confirmation. More than 100 different mutations in the biotinidase gene have been reported to date. To simplify molecular testing we have developed a rapid and accurate denaturing high pressure liquid chromatography (dHPLC) method of the promoter, 3'UTR, all exons including exon/intron boundaries as a first line screen followed by direct sequencing of the respective PCR products. To validate this method we used DNA from 23 different, newly diagnosed patients with biochemically proven BD from Austria, India, Morocco and Spain. A total of 11 mutations, missense 7, frameshift 3 and 1 nonsense, were screened. Six mutations were novel to this study. All mutations revealed distinct dHPLC pattern thus enabling their accurate detection. This study revealed that dHPLC method is robust, automated, economical and above all highly sensitive for the molecular analysis of biotinidase gene and should be used as a pre-analytical tool followed by sequencing of aberrant heteroduplex forming amplicons.
Subject(s)
Biotinidase/genetics , Chromatography, High Pressure Liquid/methods , Child, Preschool , Humans , Infant , Infant, Newborn , Mutation , Protein Denaturation , Sensitivity and SpecificityABSTRACT
BACKGROUND: Mutations in the alpha-l-iduronidase A (IDUA) gene cause mucopolysaccharidosis type I (MPS I), a progressive multisystem disorder with features ranging over a continuum from mild to severe which is inherited in an autosomal recessive manner. To date over 100 mutations are known, nonetheless genotype-phenotype prediction is complicated and hampered due to attenuating polymorphisms, rare sequence variants, varied genetic backgrounds and environmental effects. METHODS: In this study we report the first development of a denaturating high performance liquid chromatography (dHPLC) protocol for the rapid and accurate detection of recently described mutations in the IDUA gene. Optimal PCR running and dHPLC partial denaturing conditions for mutation detection were established for each PCR amplicon corresponding to 14 IDUA exons and their adjacent intronic/flanking sequences. RESULTS: A total of 12 different mutations, 5 nonsense, 4 missense, 1 deletion, and 2 splice site (intron), in 10 MPS I patients were screened. All mutations revealed a distinct dHPLC pattern thus enabling their accurate detection. CONCLUSIONS: A dHPLC screening method was developed for the detection of mutations and sequence variants in the IDUA gene and the results presented in this study revealed that this promising method proved to be robust, automated, economical and above all, highly sensitive. Costs for the detection of mutations causing MPS I disease should be reduced by using this method as a pre-analytical tool followed by sequencing of aberrant heteroduplex-forming amplicons.
Subject(s)
Iduronidase/analysis , Mucopolysaccharidosis I/genetics , Chromatography, High Pressure Liquid/economics , Cost-Benefit Analysis , DNA Mutational Analysis/economics , Genetic Variation/genetics , Humans , Iduronidase/metabolism , Mutation , Nucleic Acid Denaturation , Polymerase Chain Reaction/economicsABSTRACT
BACKGROUND AND OBJECTIVES: This retrospective study was performed to evaluate the status of p53 in pleomorphic adenomas and carcinomas ex pleomorphic adenoma in the parotid gland. As loss or mutation of p53 can cause malignant transformation, the possible degeneration of pleomorphic adenomas to carcinomas ex pleomorhic adenoma was investigated by mutational analysis. METHODS: Twenty-five Patients including 14 patients with pleomorphic adenomas and 11 patients with carcinoma ex pleomorphic adenoma of the parotid gland were examined for p53 status. DNA was extracted out of paraffin-embedded tissue and PCR was performed for the coding exons 2-11. Denaturing gradient gel electrophoresis (DGGE) was carried out for mutational analysis and DNA sequencing was performed in case of a suspected mutation. RESULTS: Fourteen pleomorphic adenomas and 11 carcinomas ex pleomorphic adenoma were screened for p53 status and potent mutations. Subsequent sequencing of the distinct exons showed no mutation. CONCLUSION: We could not detect mutations of p53 neither in benign nor malignant parotid tumors and we therefore assume that p53 plays no role in the transformation from pleomorphic adenoma to carcinoma ex pleomorphic adenoma.
Subject(s)
Adenoma, Pleomorphic/genetics , Genes, p53/genetics , Parotid Neoplasms/genetics , Adult , Aged , Aged, 80 and over , Female , Humans , Male , Middle Aged , Retrospective StudiesABSTRACT
Mutations in the gene for 4-hydroxyphenylpyruvic acid dioxygenase (HPD) cause either autosomal recessive tyrosinemia type III or autosomal dominant hawkinsinuria. We report a 6-month-old Indian infant who is compound heterozygous for both alleles and who has hawkinsinuria but not tyrosinemia type III based on biochemical investigations. The HPD gene was directly sequenced in the proband and both parents. The mechanistic model of the enzymatic function was built using the known structure of rat HPD. We identified a novel hawkinsinuria mutation, Asn241Ser, and a known tyrosinemia type III mutation, Ile335Met, in trans configuration. The structural analysis of the active site revealed that the IIe335Met mutation is situated in the close vicinity of one of the two highly conserved Phe rings which stack with the phenol ring of the substrate. The Asn241Ser mutation is situated further away from the 4-hydroxyphenylpyruvate binding pocket. Assuming that Asn241Ser causes hawkinsinuria, we propose positioning the dioxygen molecule in the HPD-catalyzed reaction as a novel role for the Asn residue. The IIe335Met allele is equivalent to a null mutation while the Asn241Ser allele results in a partially active enzyme with an uncoupled turnover causing hawkinsinuria.
Subject(s)
4-Hydroxyphenylpyruvate Dioxygenase/genetics , Amino Acids, Sulfur/urine , Cyclohexenes/urine , Tyrosinemias/genetics , Tyrosinemias/urine , 4-Hydroxyphenylpyruvate Dioxygenase/deficiency , DNA/blood , DNA/genetics , DNA/isolation & purification , Genetic Carrier Screening , Humans , Infant , Models, Molecular , Polymerase Chain Reaction , Protein ConformationABSTRACT
This study aimed to estimate the number of infants who died of unrecognized congenital adrenal hyperplasia (CAH) in Austria and the Czech Republic within the past 13 years, before the introduction of adequate neonatal screening. The study was based on retrospective analysis of neonatal screening cards of 242 infants who died suddenly between 7 days and 12 months of age and whose cause of death could not be identified. 17-hydroxyprogesterone (17-OHP) was measured by fluoroimmunoassay and positive samples were subsequently genotyped. Three infants out of 242 may have had unrecognized CAH due to CYP21 (steroid 21-hydroxylase) gene defect. Their newborn 17-OHP levels and CYP21 genotypes were 706 nmol/l and del/conv//del/conv, 53 nmol/l and I2//I2, and 811 nmol/l and I2//Gln318stop, respectively. CAH due to CYP21 defect can lead to sudden unexpected death without prior symptoms typical for the condition. Hence, newborn screening would have prevented these deaths had it been available. In addition, we have shown that the I2 point mutation that is expected to lead to simple virilizing form may lead to a fatal outcome.
Subject(s)
Adrenal Hyperplasia, Congenital/epidemiology , Sudden Infant Death/epidemiology , Austria/epidemiology , Czech Republic/epidemiology , Female , Humans , Infant , Infant, Newborn , Male , Neonatal Screening , PrevalenceABSTRACT
INTRODUCTION: Non-syndromic cleft lip with or without cleft palate (CL/P), is one of the most common birth defects, but its aetiology is largely unknown. The aim of this study was to determine the sequence changes of the Cleft Lip and Palate Transmembrane Protein 1 (CLPTM 1) and Poliovirus Receptor Related 1 (PVRL 1) genes in patients with non-syndromic complete clefts of lip, alveolus and palate and to correlate these findings with clinical features. PATIENTS AND METHODS: 25 patients were analysed (14 male and 11 female, aged 4-10 years) of European descent (9 patients with right, 9 with left and 7 patients with bilateral CLAP) and 25 controls, respectively. Exons 2-14 of the CLPTM1 and exons 1-6 of the PVRL1 gene were analysed by a direct sequencing method using DNA extracted from whole blood. RESULTS: A novel in frame Glu441-Gly442 ins Glu mutation of the PVRL 1 gene in combination with novel exon mutations Gly331Gly, Ala88Ala, Pro309Pro and intron change IVS7-10G/A of the CLPTM 1 gene were found in 9 patients. The Glu441-Gly442 ins Glu mutation and the intron change IVS7-10G/A were not detected in 25 controls. CONCLUSION: These results suggest that a simultaneous occurrence of PVRL1 and CLPTM 1 gene mutations in cleft patients does not correlate with the type of cleft (left, right, bilateral) or the gender of the patients. If a combination of the intron change IVS7-10G/A, exon changes Gly331Gly, Ala88Ala and Pro309Pro of the CLMPT 1 gene and Glu441-Gly442 ins Glu mutation of the PVRL 1 gene could be a genetic factor for non-syndromic clefts of the primary and the secondary palates, it is important to investigate more patients and controls.
Subject(s)
Cell Adhesion Molecules/genetics , Cleft Lip/genetics , Cleft Palate/genetics , Immunoglobulins/genetics , Membrane Proteins/genetics , Mutation/genetics , Receptors, Virus/genetics , Alanine/genetics , Alveolar Process/abnormalities , Child , Child, Preschool , Exons/genetics , Female , Glutamic Acid/genetics , Glycine/genetics , Humans , Introns/genetics , Male , Molecular Sequence Data , Nectins , Proline/genetics , Sequence Analysis, Protein/methods , Sex FactorsABSTRACT
Van der Woude syndrome (VWS) is an autosomal dominant disorder characterized by clefts of the lip and/or palate (CL+/-P), lip pits, bifid uvula and hypodontia. Mutations of the interferon regulatory factor 6 gene (IRF6) have been recently described in patients with VWS. The entire 9 exons of the IRF6 gene in two brothers of Turkish origin clinically diagnosed with Van der Woude syndrome and four healthy family members were screened for mutations using a newly established denaturing gradient gel electrophoresis (DGGE) method. A novel heterozygous mutation in exon 2 (DNA binding region) of the IRF6 gene, p.Arg84Gly, was found in both brothers with VWS and in their clinically asymptomatic mother. Our results suggest a dominant negative effect of the p.Arg84Gly mutation in the VWS of both patients. Non-penetrance of this mutation is suggested in the mother of the patients.