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Dean R Tolan - One of the best experts on this subject based on the ideXlab platform.
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mutations in the promoter region of the aldolase b gene that cause Hereditary Fructose Intolerance
Journal of Inherited Metabolic Disease, 2010Co-Authors: Erin M Coffee, Dean R TolanAbstract:Hereditary Fructose Intolerance (HFI) is a potentially fatal inherited metabolic disease caused by a deficiency of aldolase B activity in the liver and kidney. Over 40 disease-causing mutations are known in the protein-coding region of ALDOB. Mutations upstream of the protein-coding portion of ALDOB are reported here for the first time. DNA sequence analysis of 61 HFI patients revealed single base mutations in the promoter, intronic enhancer, and the first exon, which is entirely untranslated. One mutation, g.−132G>A, is located within the promoter at an evolutionarily conserved nucleotide within a transcription factor-binding site. A second mutation, IVS1+1G>C, is at the donor splice site of the first exon. In vitro electrophoretic mobility shift assays show a decrease in nuclear extract-protein binding at the g.−132G>A mutant site. The promoter mutation results in decreased transcription using luciferase reporter plasmids. Analysis of cDNA from cells transfected with plasmids harboring the IVS1+1G>C mutation results in aberrant splicing leading to complete retention of the first intron (~5 kb). The IVS1+1G>C splicing mutation results in loss of luciferase activity from a reporter plasmid. These novel mutations in ALDOB represent 2% of alleles in American HFI patients, with IVS1+1G>C representing a significantly higher allele frequency (6%) among HFI patients of Hispanic and African-American ethnicity.
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Increased prevalence of mutant null alleles that cause Hereditary Fructose Intolerance in the American population
Journal of Inherited Metabolic Disease, 2010Co-Authors: Erin M Coffee, Laura Yerkes, Elizabeth P. Ewen, Tiffany Zee, Dean R TolanAbstract:Mutations in the aldolase B gene ( ALDOB ) impairing enzyme activity toward Fructose-1-phosphate cleavage cause Hereditary Fructose Intolerance (HFI). Diagnosis of the disease is possible by identifying known mutant ALDOB alleles in suspected patients; however, the frequencies of mutant alleles can differ by population. Here, 153 American HFI patients with 268 independent alleles were analyzed to identify the prevalence of seven known HFI-causing alleles (A149P, A174D, N334K, Δ4E4, R59Op, A337V, and L256P) in this population. Allele-specific oligonucleotide hybridization analysis was performed on polymerase chain reaction (PCR)-amplified genomic DNA from these patients. In the American population, the missense mutations A149P and A174D are the two most common alleles, with frequencies of 44% and 9%, respectively. In addition, the nonsense mutations Δ4E4 and R59Op are the next most common alleles, with each having a frequency of 4%. Together, the frequencies of all seven alleles make up 65% of HFI-causing alleles in this population. Worldwide, these same alleles make up 82% of HFI-causing mutations. This difference indicates that screening for common HFI alleles is more difficult in the American population. Nevertheless, a genetic screen for diagnosing HFI in America can be improved by including all seven alleles studied here. Lastly, identification of HFI patients presenting with classic symptoms and who have homozygous null genotypes indicates that aldolase B is not required for proper development or metabolic maintenance.
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structure of the thermolabile mutant aldolase b a149p molecular basis of Hereditary Fructose Intolerance
Journal of Molecular Biology, 2005Co-Authors: Ali D Malay, Karen N Allen, Dean R TolanAbstract:Hereditary Fructose Intolerance (HFI) is a potentially lethal inborn error in metabolism caused by mutations in the aldolase B gene, which is critical for gluconeogenesis and Fructose metabolism. The most common mutation, which accounts for 53% of HFI alleles identified worldwide, results in substitution of Pro for Ala at position 149. Structural and functional investigations of human aldolase B with the A149P substitution (AP-aldolase) have shown that the mutation leads to losses in thermal stability, quaternary structure, and activity. X-ray crystallography is used to reveal the structural basis of these perturbations. Crystals of AP-aldolase are grown at two temperatures (4 °C and 18 °C), and the structure solved to 3.0 A resolution, using the wild-type structure as the phasing model. The structures reveal that the single residue substitution, A149P, causes molecular disorder around the site of mutation (residues 148–159), which is propagated to three adjacent β-strand and loop regions (residues 110–129, 189–199, 235–242). Disorder in the 110–129-loop region, which comprises one subunit–subunit interface, provides an explanation for the disrupted quaternary structure and thermal instability. Greater structural perturbation, particularly at a Glu189-Arg148 salt bridge in the active-site architecture, is observed in the structure determined at 18 °C, which could explain the temperature-dependent loss in activity. The disorder revealed in these structures is far greater than that predicted by homology modeling and underscores the difficulties in predicting perturbations of protein structure and function by homology modeling alone. The AP-aldolase structure reveals the molecular basis of a Hereditary disease and represents one of only a few structures known for mutant proteins at the root of the thousands of other inherited disorders.
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fructo oligosaccharide tolerance in patients with Hereditary Fructose Intolerance a preliminary nonrandomized open challenge short term study
Nutrition Research, 2003Co-Authors: Bruce Barshop, Dean R Tolan, William L Nyhan, Philippe H Steenhout, Wolf Endres, Roger ClemensAbstract:Abstract Hereditary Fructose Intolerance (HFI) caused by Fructose aldolase B deficiency results in hepatic dysfunction unless managed with severely restricted dietary intake of Fructose and sucrose. The potential of FOS to provide a Fructose load in compromised individuals has not been determined. The purpose of this study was to evaluate the safety and tolerance of FOS among subjects with established diagnoses of HFI. Five subjects with HFI (ages 14-52 yrs; 4 male, 1 female) participated in a prospective, non-randomized open challenge with FOS at 6 g/m 2 /d for 2 days. A female infant (5 mos) with resolved neonatal hepatitis was also studied. Diet records were maintained for the 48-hr period and analyzed for dietary Fructose. Tolerance was assessed through evaluation of serum AST, ALT, GGT, glucose, bilirubin, uric acid, phosphorus, and electrolytes, upon initiation and at 12-hr intervals during the challenge. Blood chemistry values were within normal ranges and did not change appreciably during the study period, except for two patients with slight elevations of uric acid. One subject reported gastric discomfort on day 2 of the FOS challenge. These data suggest that FOS providing approximately 4.7 mg Fructose/kg bw/d for 2 days is safe and well tolerated among individuals with diagnosed HFI.
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the temperature dependence of activity and structure for the most prevalent mutant aldolase b associated with Hereditary Fructose Intolerance
Archives of Biochemistry and Biophysics, 2002Co-Authors: Ali D Malay, Sheri L Procious, Dean R TolanAbstract:Abstract Hereditary Fructose Intolerance (HFI) is an autosomal recessive disorder in humans which is caused by mutations in the aldolase B gene. The most common HFI allele encodes an enzyme with an A149P substitution (AP-aldolase). A lysis method suitable for aggregation-prone proteins overexpressed in bacteria was developed. The enzyme’s structure and function is investigated as a function of temperature. Near-UV CD shows a qualitative difference in tertiary structure, whereas far-UV CD shows no difference in overall secondary structure, although both show increased temperature sensitivity for AP-aldolase compared to that seen with wild-type aldolase B. AP-aldolase exists as a dimer at all temperatures tested, unlike the tetrameric wild-type enzyme, thus providing a possible explanation for the loss in thermostability. AP-aldolase has sixfold lower activity than wild type at 10 °C, which decreases substantially at higher temperature. In addition to disruptions at the catalytic center, the kinetic constants toward different substrates suggest that there is a disruption at the C1-phosphate-binding site, which is not sensitive to temperature. The implications of these structural alterations are discussed with regard to the HFI disease.
Francesco Salvatore - One of the best experts on this subject based on the ideXlab platform.
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six novel alleles identified in italian Hereditary Fructose Intolerance patients enlarge the mutation spectrum of the aldolase b gene
Human Mutation, 2004Co-Authors: Gabriella Esposito, Luigi Vitagliano, Antonietta Viola, Luigi Ieno, L Fiori, Rita Santamaria, Adriana Zagari, Giancarlo Parenti, Lucia Zancan, Francesco SalvatoreAbstract:Hereditary Fructose Intolerance (HFI) is a recessively inherited disorder of carbohydrate metabolism caused by impaired functioning of human liver aldolase (B isoform; ALDOB). To-date, 29 enzyme-impairing mutations have been identified in the aldolase B gene. Here we report six novel HFI single nucleotide changes identified by sequence analysis in the aldolase B gene. Three of these are missense mutations (g.6846T>C, g.10236G>T, g.10258T>C), one is a nonsense mutation (g.8187C>T) and two affect splicing sites (g.8180G>C and g.10196A>G). We have expressed in bacterial cells the recombinant proteins corresponding to the g.6846T>C (p.I74T), g.10236G>T (p.V222F), and g.10258T>C (p.L229P) natural mutants to study their effect on aldolase B function and structure. All the new variants were insoluble; molecular graphics data suggest this is due to impaired folding. © 2004 Wiley-Liss, Inc.
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structural and functional analysis of aldolase b mutants related to Hereditary Fructose Intolerance
FEBS Letters, 2002Co-Authors: Gabriella Esposito, Luigi Vitagliano, Antonietta Viola, Rita Santamaria, Adriana Zagari, Francesco SalvatoreAbstract:Hereditary Fructose Intolerance (HFI) is a recessively inherited disorder of carbohydrate metabolism caused by impaired function of human liver aldolase (B isoform). 25 enzyme-impairing mutations have been identified in the aldolase B gene. We have studied the HFI-related mutant recombinant proteins W147R, A149P, A174D, L256P, N334K and Δ6ex6 in relation to aldolase B function and structure using kinetic assays and molecular graphics analysis. We found that these mutations affect aldolase B function by decreasing substrate affinity, maximal velocity and/or enzyme stability. Finally, the functional and structural analyses of the non-natural mutant Q354E provide insight into the catalytic role of Arg303, whose natural mutants are associated to HFI.
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novel six nucleotide deletion in the hepatic Fructose 1 6 bisphosphate aldolase gene in a patient with Hereditary Fructose Intolerance and enzyme structure function implications
European Journal of Human Genetics, 1999Co-Authors: Rita Santamaria, Luigi Vitagliano, Paola Izzo, S Tamasi, Adriana Zagari, Lucia Zancan, Francesco SalvatoreAbstract:Hereditary Fructose Intolerance (HFI) is an autosomal recessive human disease that results from the deficiency of the hepatic aldolase isoenzyme. Affected individuals will succumb to the disease unless it is readily diagnosed and Fructose eliminated from the diet. Simple and non-invasive diagnosis is now possible by direct DNA analysis that scans for known and unknown mutations. Using a combination of several PCR-based methods (restriction enzyme digestion, allele specific oligonucleotide hybridisation, single strand conformation analysis and direct sequencing) we identified a novel six-nucleotide deletion in exon 6 of the aldolase B gene (delta 6ex6) that leads to the elimination of two amino acid residues (Leu182 and Val183) leaving the message inframe. The three-dimensional structural alterations induced in the enzyme by delta 6ex6 have been elucidated by molecular graphics analysis using the crystal structure of the rabbit muscle aldolase as reference model. These studies showed that the elimination of Leu182 and Val183 perturbs the correct orientation of adjacent catalytic residues such as Lys146 and Glu187.
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molecular basis of Hereditary Fructose Intolerance in italy identification of two novel mutations in the aldolase b gene
Journal of Medical Genetics, 1996Co-Authors: Rita Santamaria, Paola Izzo, S Tamasi, Giovanni Piano, C Borrone, Giacomo Faldella, Generoso Andria, Gianfranco Sebastio, Francesco SalvatoreAbstract:We screened the aldolase B gene in 14 unrelated Italian patients with Hereditary Fructose Intolerance (HFI), and found two novel disease related mutations: a single nucleotide deletion in exon 2 (delta A20) that leads to an early stop codon, and a C-->T transition in exon 8 that substitutes an Arg with a Trp residue at codon 303 (R303W).
Rita Santamaria - One of the best experts on this subject based on the ideXlab platform.
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six novel alleles identified in italian Hereditary Fructose Intolerance patients enlarge the mutation spectrum of the aldolase b gene
Human Mutation, 2004Co-Authors: Gabriella Esposito, Luigi Vitagliano, Antonietta Viola, Luigi Ieno, L Fiori, Rita Santamaria, Adriana Zagari, Giancarlo Parenti, Lucia Zancan, Francesco SalvatoreAbstract:Hereditary Fructose Intolerance (HFI) is a recessively inherited disorder of carbohydrate metabolism caused by impaired functioning of human liver aldolase (B isoform; ALDOB). To-date, 29 enzyme-impairing mutations have been identified in the aldolase B gene. Here we report six novel HFI single nucleotide changes identified by sequence analysis in the aldolase B gene. Three of these are missense mutations (g.6846T>C, g.10236G>T, g.10258T>C), one is a nonsense mutation (g.8187C>T) and two affect splicing sites (g.8180G>C and g.10196A>G). We have expressed in bacterial cells the recombinant proteins corresponding to the g.6846T>C (p.I74T), g.10236G>T (p.V222F), and g.10258T>C (p.L229P) natural mutants to study their effect on aldolase B function and structure. All the new variants were insoluble; molecular graphics data suggest this is due to impaired folding. © 2004 Wiley-Liss, Inc.
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structural and functional analysis of aldolase b mutants related to Hereditary Fructose Intolerance
FEBS Letters, 2002Co-Authors: Gabriella Esposito, Luigi Vitagliano, Antonietta Viola, Rita Santamaria, Adriana Zagari, Francesco SalvatoreAbstract:Hereditary Fructose Intolerance (HFI) is a recessively inherited disorder of carbohydrate metabolism caused by impaired function of human liver aldolase (B isoform). 25 enzyme-impairing mutations have been identified in the aldolase B gene. We have studied the HFI-related mutant recombinant proteins W147R, A149P, A174D, L256P, N334K and Δ6ex6 in relation to aldolase B function and structure using kinetic assays and molecular graphics analysis. We found that these mutations affect aldolase B function by decreasing substrate affinity, maximal velocity and/or enzyme stability. Finally, the functional and structural analyses of the non-natural mutant Q354E provide insight into the catalytic role of Arg303, whose natural mutants are associated to HFI.
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novel six nucleotide deletion in the hepatic Fructose 1 6 bisphosphate aldolase gene in a patient with Hereditary Fructose Intolerance and enzyme structure function implications
European Journal of Human Genetics, 1999Co-Authors: Rita Santamaria, Luigi Vitagliano, Paola Izzo, S Tamasi, Adriana Zagari, Lucia Zancan, Francesco SalvatoreAbstract:Hereditary Fructose Intolerance (HFI) is an autosomal recessive human disease that results from the deficiency of the hepatic aldolase isoenzyme. Affected individuals will succumb to the disease unless it is readily diagnosed and Fructose eliminated from the diet. Simple and non-invasive diagnosis is now possible by direct DNA analysis that scans for known and unknown mutations. Using a combination of several PCR-based methods (restriction enzyme digestion, allele specific oligonucleotide hybridisation, single strand conformation analysis and direct sequencing) we identified a novel six-nucleotide deletion in exon 6 of the aldolase B gene (delta 6ex6) that leads to the elimination of two amino acid residues (Leu182 and Val183) leaving the message inframe. The three-dimensional structural alterations induced in the enzyme by delta 6ex6 have been elucidated by molecular graphics analysis using the crystal structure of the rabbit muscle aldolase as reference model. These studies showed that the elimination of Leu182 and Val183 perturbs the correct orientation of adjacent catalytic residues such as Lys146 and Glu187.
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molecular basis of Hereditary Fructose Intolerance in italy identification of two novel mutations in the aldolase b gene
Journal of Medical Genetics, 1996Co-Authors: Rita Santamaria, Paola Izzo, S Tamasi, Giovanni Piano, C Borrone, Giacomo Faldella, Generoso Andria, Gianfranco Sebastio, Francesco SalvatoreAbstract:We screened the aldolase B gene in 14 unrelated Italian patients with Hereditary Fructose Intolerance (HFI), and found two novel disease related mutations: a single nucleotide deletion in exon 2 (delta A20) that leads to an early stop codon, and a C-->T transition in exon 8 that substitutes an Arg with a Trp residue at codon 303 (R303W).
P Rellos - One of the best experts on this subject based on the ideXlab platform.
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expression purification and characterization of natural mutants of human aldolase b role of quaternary structure in catalysis
Journal of Biological Chemistry, 2000Co-Authors: P Rellos, Jurgen SyguschAbstract:Abstract Fructaldolases (EC 4.1.2.13) are ancient enzymes of glycolysis that catalyze the reversible cleavage of phosphoFructose esters into cognate triose (phosphates). Three vertebrate isozymes of Class I aldolase have arisen by gene duplication and display distinct activity profiles with Fructose 1,6-bisphosphate and with Fructose 1-phosphate. We describe the biochemical and biophysical characterization of seven natural human aldolase B variants, identified in patients suffering from Hereditary Fructose Intolerance and expressed as recombinant proteins in E. coli, from which they were purified to homogeneity. The mutant aldolases were all missense variants and could be classified into two principal groups:catalytic mutants, with retained tetrameric structure but altered kinetic properties (W147R, R303W, and A337V), andstructural mutants, in which the homotetramers readily dissociate into subunits with greatly impaired enzymatic activity (A149P, A174D, L256P, and N334K). Investigation of these two classes of mutant enzyme suggests that the integrity of the quaternary structure of aldolase B is critical for maintaining its full catalytic function.
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alteration of substrate specificity by a naturally occurring aldolase b mutation ala337 val in Fructose Intolerance
Biochemical Journal, 1999Co-Authors: P Rellos, Marie Vidailhet, Jurgen SyguschAbstract:A molecular analysis of human aldolase B genes in two newborn infants and a 4-year-old child with Hereditary Fructose Intolerance, the ospring of a consanguineous union, has identified the novel mutation Ala$$( ! Val in homozygous form. This mutation was also detected independently in two other aected individuals who were compound heterozygotes for the prevalent aldolase B allele, Ala"%* ! Pro, indicating that the mutation causes aldolase B deficiency. To test for the eect of the mutation, catalytically active wild-type human aldolase B and the Val$$( variant enzyme were expressed in Escherichia coli. The specific activities of the wild-type recombinant enzyme were 4.8 units}mg and 4.5 units}mg towards Fructose 1,6-bisphosphate (FBP) and Fructose 1-phosphate (F-1-P) as substrates with Michaelis constants of 4 lM and 2.4 mM respectively. The specific activities of purified tetrameric Val$$( aldolase B, which aects an invariant residue in the C-terminal region, were 4.2 units}mg and 2.6 units}mg towards FBP and F-1-P as substrates respectively; the corresponding Michaelis constants were 22 lM and 24 mM. The FBP-to-F-1-P substrate activity ratios were 0.98 and 1.63 for
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neonatal screening for Hereditary Fructose Intolerance frequency of the most common mutant aldolase b allele a149p in the british population
Journal of Medical Genetics, 1996Co-Authors: C L James, P Rellos, A F HeeleyAbstract:Hereditary Fructose Intolerance (HFI) causes severe and sometimes fatal metabolic disturbances in infants and children but responds to dietary treatment. To determine the practicability of screening newborn infants for HFI, we have investigated the frequency of the most common and widespread mutant allele of aldolase B, A149P, in the neonatal population. The polymerase chain reaction was used to amplify aldolase B exon 5 genomic sequences in DNA present in dried blood specimens preserved on Guthrie cards. The A149P mutation was identified by discriminatory hybridisation to allele specific oligonucleotides and confirmed independently by digestion with the restriction endonuclease BsaHI. Twenty-seven A149P heterozygotes were identified by the molecular analysis of aldolase B genes in blood samples obtained from a random cohort of 2050 subjects born in 1994 and 1995, 1.32 +/- 0.49% (95% confidence level). Although no A149P homozygotes were identified, the data allow the frequency of 1 in 23,000 homozygotes for this allele to be predicted. Our findings have implications for establishing an interventional mass screening programme to identify newborn infants with HFI in the UK.
Jennifer A Littlechild - One of the best experts on this subject based on the ideXlab platform.
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the structure of human liver Fructose 1 6 bisphosphate aldolase
Acta Crystallographica Section D-biological Crystallography, 2001Co-Authors: Andrew R Dalby, Dean R Tolan, Jennifer A LittlechildAbstract:The X-ray crystallographic structure of the human liver isozyme of Fructose-1,6-bisphosphate aldolase has been determined by molecular replacement using a tetramer of the human muscle isozyme as a search model. The liver aldolase (B isozyme) crystallized in space group C2, with unit-cell parameters a = 291.1, b = 489.8, c = 103.4 A, α = 90, β = 103.6, γ = 90°. These large unit-cell parameters result from the presence of 18 subunits in the asymmetric unit: four catalytic tetramers and a dimer from a fifth tetramer positioned on the twofold crystallographic axis. This structure provides further insight into the factors affecting isozyme specificity. It reveals small differences in secondary structure that occur in regions previously determined to be isozyme specific. Two of these regions are at the solvent-exposed enzyme surface away from the active site of the enzyme. The most significant changes are in the flexible C-terminal region of the enzyme, where there is an insertion of an extra α-helix. Point mutations of the human liver aldolase are responsible for the disease Hereditary Fructose Intolerance. Sequence information is projected onto the new crystal structure in order to indicate how these mutations bring about reduced enzyme activity and affect structural stability.