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D M Layton

Publications and source records attributed to D M Layton.

At least 19 recordsLinked to original sources
PubMed2005

Identification and characterization of the novel FAD-binding lobe G75S mutation in cytochrome b(5) reductase: an aid to determine recessive congenital methemoglobinemia status in an infant.

NADH-cytochrome b(5) reductase deficiency results clinically in either type I or type II recessive congenital methemoglobinemia. The more severe type II form is associated with a global deficiency of cytochrome b(5) reductase and is characterized by cyanosis with neurological dysfunction. In contrast, the only symptom for type I is cyanosis. We have identified a novel G to A mutation at position 15,635 in the DIAI gene of a 4-month-old baby that results in a glycine to serine substitution at codon 75 in the cytochrome b(5) reductase protein. The G75S mutation, located in the FAD-binding lobe of cytochrome b(5) reductase, was found in association with the previously described V252M variant. The V252M mutation is present in the NADH-binding domain and associated with both types I and II recessive congenital methemoglobinemia. Since the G75S and V252M mutations represent radical changes in differing regions of cytochrome b(5) reductase, generating and characterizing these variants singly and in combination using a rat heterologous expression system would provide insight into the differences between types I and II disease at the molecular level. Although all three variants were found to retain stoichiometric levels of FAD with spectroscopic and thermodynamic properties comparable to those of native cytochrome b(5) reductase, all exhibited decreased catalytic efficiency and reduced protein stability reflecting the position of the mutations in the primary structure. The G75S variant retained only 11% of the catalytic efficiency of the wild-type enzyme. Thus, cytochrome b(5) reductase deficient patients who are heterozygous for either FAD- or NADH-binding lobe mutations can exhibit the clinically less severe type I phenotype.

Amino Acid Sequence↗
PubMed2005

A statistical analysis of RNA folding algorithms through thermodynamic parameter perturbation.

Computational RNA secondary structure prediction is rather well established. However, such prediction algorithms always depend on a large number of experimentally measured parameters. Here, we study how sensitive structure prediction algorithms are to changes in these parameters. We found already that for changes corresponding to the actual experimental error to which these parameters have been determined, 30% of the structure are falsely predicted whereas the ground state structure is preserved under parameter perturbation in only 5% of all the cases. We establish that base-pairing probabilities calculated in a thermal ensemble are viable although not a perfect measure for the reliability of the prediction of individual structure elements. Here, a new measure of stability using parameter perturbation is proposed, and its limitations are discussed.

Algorithms↗
PubMed2004

Dominant influence of gamma-globin promoter polymorphisms on fetal haemoglobin expression in sickle cell disease.

Polymorphisms of multiple cis-acting elements in the beta-globin locus are associated with variable fetal haemoglobin (HbF) level in sickle cell disease. We developed a multiplex assay permitting simultaneous analysis of three polymorphic cis elements spanning 53 kb of the beta-globin locus. We identified concordance between polymorphic alleles in gamma- and beta-globin promoters however a significant number of betaS-chromosomes were identified with polymorphisms in hypersensitive site 2 (HS2) of the beta-globin locus control region juxtaposed to atypical cis alleles in the gamma-promoter. Analysis of an unusually large number of such hybrid haplotype chromosomes provided unique insight into HbF level associated with specific cis alleles. Associations between cis alleles and HbF level in patients were verified by in vitro functional analysis. Our findings indicate that compared to HS2, polymorphism in the gamma-promoter exerts a dominant influence on HbF level in sickle cell disease.

Alleles↗
PubMed2001

Mass spectral analysis of asymmetric hemoglobin hybrids: demonstration of Hb FS (alpha2gammabetaS) in sickle cell disease.

Formation of the asymmetric hemoglobin hybrid FS (alpha2gammabetaS) inhibits hemoglobin S (Hb S) polymerization in vitro and underlies the protective effect of fetal hemoglobin (Hb F) in homozygous sickle cell disease. Conventional methods for separating Hb reveal only symmetric Hb tetramers because of the rapid dissociation of tetramers to dimers relative to the separation time for electrophoresis and chromatography. To gain insight into the quantitative distribution of asymmetric Hb FS and other tetrameric species in sickle cell disease, the noncovalent association of Hb subunits in hemolysates was studied by a novel application of electrospray ionization mass spectrometry (ESI-MS). Mass spectra of both patient and fetal blood revealed predominance of tetrameric species with dimer and monomer subunits in lower abundance. ESI-MS analysis revealed the hybrid Hb AF (alpha2gammabetaA) in hemolysates shown by conventional high-performance liquid chromatography to contain only the symmetric species Hb A (alpha2betaA2) and Hb F (alpha2gamma2). A unique tetramer of average mass 64,558 Da was identified in hemolysates from patients with sickle cell disease in accordance with the calculated mass of the asymmetric Hb hybrid FS. Hybrid Hb species were stable under the ESI-MS conditions employed allowing concurrent determination of the proportions of Hb FS and the symmetrical Hb S (alpha2betaS2). The ratios of Hb FS to Hb S correlated closely (r2 = 0.96) with those predicted under physiological conditions.

Adolescent↗
PubMed2000

Molecular analysis of the genotype-phenotype relationship in factor X deficiency.

Factor X deficiency is a rare haemorrhagic condition, normally inherited as an autosomal recessive trait, in which a variable clinical presentation correlates poorly with laboratory phenotype. The factor X (F10) genes of 14 unrelated individuals with factor X deficiency (12 familial and two sporadic cases) were sequenced yielding a total of 13 novel mutations. Family studies were performed in order to distinguish the contributions of individual mutant F10 alleles to the clinical and laboratory phenotypes. Missense mutations were studied by means of molecular modelling, whereas single basepair substitutions in splice sites and the 5' flanking region were examined by in vitro splicing assay and luciferase reporter gene assay respectively. The deletion allele of a novel hexanucleotide insertion/deletion polymorphism in the F10 gene promoter region was shown by reporter gene assay, to reduce promoter activity by approximately 20%. One family manifesting an autosomal dominant pattern of inheritance possessed three clinically affected members who were heterozygous for a splice-site mutation that was predicted to lead to the production of a truncated protein product. A model which accounts for the dominant negative effect of this lesion is presented. Variation in the antigen level of heterozygous relatives of probands was found to be significantly higher between families than within families, consistent with the view that the nature of the F10 lesion(s) segregating in a given family is a prime determinant of the laboratory phenotype. By contrast, no such relationship could be discerned between laboratory phenotype and polymorphism genotype.

Base Sequence↗
PubMed1999

Reversal of metabolic block in glycolysis by enzyme replacement in triosephosphate isomerase-deficient cells.

Inherited deficiency of the housekeeping enzyme triosephosphate isomerase (TPI) is the most severe clinical disorder of glycolysis. Homozygotes manifest congenital hemolytic anemia and progressive neuromuscular impairment, which in most cases pursues an inexorable course with fatal outcome in early childhood. No effective therapy is available. Hitherto specific enzyme replacement has not been attempted in disorders of glycolysis. Primary skeletal muscle myoblasts and Epstein-Barr virus (EBV)-transformed lymphoblastoid cell lines generated from homozygous TPI-deficient patients were cultured in the presence of exogenous enzyme or cocultured with human K562 erythroleukemia cells as an exogenous source of TPI. Uptake of active enzyme by TPI-deficient cells resulted in reversal of intracellular substrate accumulation, with a reduction in dihydroxyacetone phosphate (DHAP) concentration to levels seen in TPI-competent cells. Evidence of successful metabolic correction of TPI deficiency in vitro establishes the feasibility of enzyme replacement therapy, and has important implications for the potential role of allogeneic bone marrow transplantation and gene therapy as a means of sustained delivery of functional enzyme in vivo.

Adolescent↗
PubMed1999

Localisation of cis regulatory elements at the beta-globin locus: analysis of hybrid haplotype chromosomes.

Several cis elements at the beta-globin gene cluster and the upstream locus control region (LCR) have been implicated in modulation of fetal haemoglobin (Hb F) level in beta-globin disorders. To determine the role of elements at the LCR and the beta-globin gene cluster on HbF level among sickle cell anaemia (SCA) patients, hybrid haplotype betaS chromosomes exhibiting variation in the association of alleles of LCR hypersensitive site 2 (HS2) and the beta-globin gene cluster restriction fragment length polymorphosim (RFLP) haplotypes were identified in an unselected population of 100 patients. On 15 chromosomes the polymorphic HS2 short tandem repeat(TA)xN10-12(TA)y containing a Hox2 binding motif differed from that typically associated with the corresponding beta-globin gene cluster RFLP haplotype. Among patients homozygous for the Benin RFLP haplotype, in whom one chromosome carried the (TA)9N10(TA)10 allele, no effect on HbF level was observed. Polymorphism of the pre-Ggamma framework, an enhancer located 25 kb downstream of HS2 localised the breakpoint for each of these 'hybrid' haplotype chromosomes upstream of this element. Previously described hybrid haplotype chromosomes with the (TA)9N10(TA)10 HS2 allele associated with raised HbF by contrast arise by recombination 1 kb downstream of the pre-Ggamma framework. This study suggests that variability in HbF level associated with polymorphisn of the HS2 enhancer depend on downstream determinant (s) in tight linkage disequilibrium with HS2. The pre-Ggamma framework is the only known polymorphic cis-active determinant in this region.

Adolescent↗
PubMed1999

Ancestral origin of variation in the triosephosphate isomerase gene promoter.

A high frequency of nucleotide substitutions -5A/G, -8G/A, -24T/G in the triosephosphate isomerase (TPI) gene promoter has been demonstrated in African-Americans. The biological significance of these promoter variants, two of which, -8G/A and -24T/G, occur within regulatory elements essential for transcription, is controversial. The geographical distribution and frequency of allelic variation in the TPI promoter was determined in 378 unrelated normal subjects from sub-Saharan African (n = 103), Caribbean (n = 26), Northern European (n = 57), Mediterranean (n = 55), Middle Eastern (n = 42), Asian Indian (n = 48) and Oriental (n = 47) populations. Five haplotypes were identified: the common haplotype, -5A-8G-24T, -5G, -8A, -5G-8A, and -5G-8A-24G. All, with the exception of the -8A haplotype, were present in geographically dispersed populations. The -5G allele, which was found at varying frequency in all groups, has attained high frequency in the African, Caribbean and Oriental populations. Phylogenetic comparison suggests this may represent the ancestral promoter haplotype. Homozygosity for the -5G-8A haplotype identified in four subjects confirms that these variants are not responsible for a null allele as formerly postulated. Linkage disequilibrium between related TPI promoter haplotypes, -5G, -5G-8A and -5G-8A-24G, and a single nucleotide polymorphism at nt2262 of the TPI gene supports a single ancestral origin for these mutations which precedes the separation of African and non-African populations.

Africa↗
PubMed1999

Localization of a gene for familial hemophagocytic lymphohistiocytosis at chromosome 9q21.3-22 by homozygosity mapping.

Familial hemophagocytic lymphohistiocytosis (FHL), also known as familial erythrophagocytic lymphohistiocytosis and familial histiocytic reticulosis, is a rare autosomal recessive disorder of early childhood characterized by excessive immune activation. Linkage of the disease gene to an approximately 7.8-cM region between markers D9S1867 and D9S1790 at 9q21.3-22 was identified by homozygosity mapping in four inbred FHL families of Pakistani descent with a combined maximum multipoint LOD score of 6.05. This is the first genetic locus to be described in FHL. However, homozygosity by descent across this interval could not be demonstrated in an additional affected kindred of Arab origin, whose maximum multipoint LOD score was -0.12. The combined sample revealed significant evidence for linkage to 9q markers (LOD score with heterogeneity, 5.00). Identification of the gene(s) involved in the pathogenesis of FHL will contribute to an understanding of the control of T-lymphocyte and macrophage activation, which is central to homeostasis in the immune system.

Adolescent↗
PubMed1999

Regulation of triosephosphate isomerase (TPI) gene expression in TPI deficient lymphoblastoid cells.

The metabolic defect of triosephosphate isomerase (TPI) deficiency is reversible in deficient lymphoblastoid cells when cultured in the presence of human K562 erythroleukemia cells or plasma as exogenous source of functional enzyme. However, plasma contains a variety of undefined biological response modifiers whose effects on TPI gene expression are unknown. In the present study, TPI deficient lymphoblastoid cells were cultured in serum-free medium for 24 h (controls) and stimulated with fresh frozen plasma (FFP) at final concentrations of 20, 40, and 60% for 9 h. Changes in TPI mRNA expression were monitored by slot and Northern blot hybridisations using a specific human TPI cDNA probe. For equivalent loading of total RNA, TPI mRNA expression in FFP-treated lymphoblastoid cells exceeded that for controls by on average 20-fold. Additional studies with the transcription inhibitor, actinomycin D, revealed a rapid degradation of TPI mRNA in controls compared to FFP-treated cells, indicating that the stability of the TPI transcript was affected by plasma. These data suggest that functional or regulatory elements within the TPI gene promoter can be modulated by biological response modifiers. An understanding of the transcriptional control of TPI may provide useful insights into the development of gene therapy strategies for TPI deficiency.

Cell Line↗
PubMed1998

Quantification of Ggamma- and Agamma-globins by electrospray ionisation mass spectrometry.

Elucidation of the molecular basis for persistent fetal haemoglobin (Hb F) production in adult life has important implications for the pathophysiology and treatment of human beta haemoglobinopathies. Electrospray ionisation mass spectrometry (ESMS) was applied to analyse the pattern of gamma-globin expression in patients with hereditary persistence of fetal haemoglobin (HPFH) and sickle cell anaemia (SCA). Ggamma and Agamma-globin chains were identified by their measured molecular masses and distinguished by mass difference (14 Da) following deconvolution of ESMS spectra using maximum entropy based software. Prediction of HPFH type by ESMS was confirmed by molecular analysis. Direct determination of Ggamma:Agamma globin chain ratio from whole blood by the novel application of ESMS provides a rapid and sensitive approach to characterisation of gamma-globins and facilitates correlation of gamma-globin level and polymorphism of cis-active elements at the beta-globin locus.

Adult↗
PubMed1997

Evidence for founder effect of the Glu104Asp substitution and identification of new mutations in triosephosphate isomerase deficiency.

Triosephosphate isomerase (TPI) deficiency is an autosomal recessive disorder of glycolysis characterized by multisystem disease and lethality in early childhood. Among seven unrelated Northern European kindreds with clinical TPI deficiency studied, a single missense mutation at codon 104 (GAG;Glu-->GAC;Asp) predominated, accounting for 11/14 (79%) mutant alleles. In three families molecular analysis revealed compound heterozygosity for Glu104Asp and novel missense mutations. In two cases the second mutation was a Cys to Tyr substitution at codon 41 (TGT-->TAT) and in one an Ile to Val substitution at codon 170(ATT-->GTT). The origin of the Glu104Asp mutation was defined by haplotype analysis using a novel G/A polymorphism at nucleotide 2898 of the TPI gene. Cosegregation of the low frequency 2898A allele with the G-->C base change at nucleotide 315 supports a single origin for the Glu104Asp mutation in a common ancestor.

Anemia, Hemolytic, Congenital Nonspherocytic↗
PubMed1997

Predominance of the HLA-H Cys282Tyr mutation in Austrian patients with genetic haemochromatosis.

BACKGROUND/AIMS: Genetic haemochromatosis is the most common autosomal recessive disorder in Northern European populations. A major histocompatibility complex class I-like gene, HLA-H, has been proposed to be responsible for genetic haemochromatosis. The prevalence of HLA-H gene mutations 282(TGC; Cys/TAC; Tyr) and 63(CAT; His/GAT; Asp) was determined in patients of Austrian origin. METHODS: DNA extracted from the blood of 40 Austrian patients and 271 controls was used to amplify HLA-H gene fragments by the polymerase chain reaction method. The base changes responsible for mutations Cys282Tyr and His63Asp alter recognition sites for restriction enzymes SnaB I and Bcl I, respectively. Digestion products were separated by agarose gel electrophoresis and visualised by ethidium bromide staining. RESULTS: Thirty-one (77.5%) genetic haemochromatosis patients were homozygous for mutation Cys282Tyr and three compound heterozygous for mutations Cys282Tyr and His63Asp. One patient was homozygous for mutation His63Asp but normal for mutation Cys282Tyr. Four patients were normal at both genetic loci and one patient was heterozygous for mutation His63Asp. One control subject homozygous for mutation Cys282Tyr was found on investigation to fulfill diagnostic criteria for haemochromatosis. Eight control subjects homozygous for mutation His63Asp showed no biochemical or clinical evidence of haemochromatosis indicating that this variant is not directly responsible for haemochromatosis. Absence of the Cys282Tyr mutation in six genetic haemochromatosis patients with distinct haplotypes indicates mutations within the HLA-H gene or at alternative genetic loci are the cause of genetic haemochromatosis in these patients. CONCLUSIONS: The HLA-H Cys282Tyr defect is likely to play a key role in the pathogenesis of haemochromatosis in most patients. Predominance of a single HLA-H gene mutation in haemochromatosis allows presymptomatic screening by genotypic analysis.

Adult↗
PubMed1996

Two novel mutations in the reduced nicotinamide adenine dinucleotide (NADH)-cytochrome b5 reductase gene of a patient with generalized type, hereditary methemoglobinemia.

Hereditary methemoglobinemia due to reduced nicotinamide adenine dinucleotide (NADH) cytochrome b5 reductase (b5R) deficiency is classified into two types, an erythrocyte (type I) and a generalized (type II). We investigated the b5R gene of a patient with type II from a white United Kingdom (UK) family and found that the patient was a compound heterozygote for two novel mutations. The first mutation was a C-to-A transversion changing codon 42 (TAC: Tyr) to a stop codon in the one allele. From this mutant allele, the product without the catalytic portion of the enzyme is generated. The second one was a missense mutation at codon 95 (CCC-->CAC) in the other allele with the result that Pro changed to His within the flavin adenine dinucleotide (FAD)-binding domain of the enzyme. To characterize effects of this missense mutation on the enzyme function, we compared glutathione S-transferase (GST)-fused b5R with the GST-fused mutant enzyme with the codon 95 missense mutation (P95H) expressed in Escherichia coll. The mutant enzyme showed less catalytic activity, less thermostability, and a greater susceptibility to trypsin than did the normal counterpart. The absorption spectrum of the mutant enzyme in the visual region differed from that of the wild-type. These results suggest that this amino acid substitution influences both secondary structure and catalytic activity of the enzyme. The compound heterozygosity for the nonsense and the missense mutations apparently caused hereditary methemoglobinemia type II in this patient.

Alleles↗
PubMed1996

Prenatal diagnosis of triosephosphate isomerase deficiency.

First-trimester prenatal diagnosis was undertaken by chorionic villus DNA analysis in two unrelated families with the inherited glycolytic disorder triosephosphate isomerase (TPI) deficiency. The propositus in each family was shown to be homozygous for a missense mutation (GAG --> GAC) at codon 104 of the TPI gene. In the first case the fetus was heterozygous for the codon 104 mutation and therefore clinically unaffected. Prenatal diagnosis in the second case showed the fetus to be homozygous for the codon 104 mutation and thus affected by TPI deficiency. This represents the first molecular diagnosis during early pregnancy of a human glycolytic enzyme disorder.

Base Sequence↗
PubMed1995

Hereditary red cell enzymopathies.

The hereditary red cell enzymopathies are an uncommon but important cause of chronic haemolytic anaemia. Their clinical diversity is mirrored by increasingly evident heterogeneity at the molecular level. The structure, function, and expression of the genes encoding red cell enzymes and the nature of the gene defects in the deficient state are examined.

Amino Acid Sequence↗