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1.
Epigenetics ; 8(12): 1261-7, 2013 Dec.
Article in English | MEDLINE | ID: mdl-24135723

ABSTRACT

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/microbiology
2.
Pediatrics ; 130(4): e1034-9, 2012 Oct.
Article in English | MEDLINE | ID: mdl-22966035

ABSTRACT

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 Adult
3.
Clin Chem ; 57(9): 1286-94, 2011 Sep.
Article in English | MEDLINE | ID: mdl-21771947

ABSTRACT

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 Specificity
4.
Eur Arch Otorhinolaryngol ; 268(11): 1639-46, 2011 Nov.
Article in English | MEDLINE | ID: mdl-21331782

ABSTRACT

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 → 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/pathology
5.
Wien Klin Wochenschr ; 122(21-22): 607-13, 2010 Nov.
Article in English | MEDLINE | ID: mdl-20938748

ABSTRACT

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 Factors
6.
Mol Genet Metab ; 100(1): 42-5, 2010 May.
Article in English | MEDLINE | ID: mdl-20083419

ABSTRACT

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 Specificity
7.
Clin Chim Acta ; 411(5-6): 345-50, 2010 Mar.
Article in English | MEDLINE | ID: mdl-19954743

ABSTRACT

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/economics
8.
J Surg Oncol ; 101(2): 127-30, 2010 Feb 01.
Article in English | MEDLINE | ID: mdl-19950209

ABSTRACT

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 Studies
9.
J Craniomaxillofac Surg ; 33(5): 301-6, 2005 Oct.
Article in English | MEDLINE | ID: mdl-16122939

ABSTRACT

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 Factors
10.
Mol Genet Metab ; 77(4): 326-31, 2002 Dec.
Article in English | MEDLINE | ID: mdl-12468279

ABSTRACT

Arginine:glycine amidinotransferase (AGAT, EC 2.1.4.1) deficiency is a recently recognized autosomal recessive inborn error of creatine biosynthesis, characterized by mental retardation and severe language impairment. We extensively investigated a third 5-year-old patient with AGAT deficiency, discovered in the pedigree of the same Italian family as the two index cases. At the age of 2 years he presented with psychomotor and language delay, and autistic-like behavior. Brain MRI was normal, but brain 1H-MRS disclosed brain creatine depletion, which almost completely normalized following creatine monohydrate supplementation. A remarkable clinical improvement paralleled the restoration of brain creatine concentration. AGAT and GAMT (guanidinoacetate:methyltransferase) genes were analyzed in the proband and in 26 relatives, including the two cousins with AGAT deficiency. Sequencing of the proband's AGAT gene disclosed the same homozygous mutation at nt position 9093 converting a tryptophan (TGG) to a stop codon (TAG) at residue 149 (W149X), as already described in the two previously reported cases. The proband's parents and 10 additional subjects of the pedigree were carriers for this mutation. AGAT deficiency was further confirmed by undetectable AGAT activity in the patient's lymphoblasts. Mutation analysis of the GAMT gene revealed a sequence variation in exon 6 (T209M), not in the proband, but in 15 additional subjects from the pedigree. The silent nature of this sequence variation is supported by its homozygosity in one AGAT deficient cousin and in one asymptomatic adult, both with normal GAMT activity.


Subject(s)
Amidinotransferases/deficiency , Creatine/metabolism , Glycine/analogs & derivatives , Psychomotor Disorders/genetics , Amidinotransferases/genetics , Amidinotransferases/metabolism , Child, Preschool , Creatine/deficiency , Female , Glycine/urine , Guanidinoacetate N-Methyltransferase , Humans , Male , Methyltransferases/genetics , Methyltransferases/metabolism , Pedigree , Psychomotor Disorders/metabolism
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