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Biomedical subjects

N S Thomas

Publications and source records attributed to N S Thomas.

At least 55 records · Page 3Linked to original sources
PubMed1999

Characterization of 34 novel and six known MTM1 gene mutations in 47 unrelated X-linked myotubular myopathy patients.

X-linked myotubular myopathy (XLMTM) is a congenital muscle disorder mainly affecting newborn males. Neonatal muscle weakness and hypotonia usually leads to a rapid demise. The responsible gene, MTM1, was isolated in 1996, and mutational data derived from 90 patients have been published. We report on our findings in a further 53 patients, using genomic DNA and mRNA screening protocols. Thirty-four novel mutations were identified in 37 cases, and six known mutations found in 10 other patients. The 34 new mutations include five large deletions, eight nonsense, six frameshift, five missense, and eight splice-site mutations, whereas two intronic variants causing partial exon skipping represent the first report on such a mechanism in MTM1. Two deletions, one involving exon 1, and the second exon 15, are the first defects to be identified in these exons. The heterogeneity of the mutations, their mutational origins, and the varied ethnic backgrounds of the patients, indicate that the majority of XLMTM families are affected by unique MTM1 mutations.

Adult↗
PubMed1999

Hardy-Weinberg quality control.

An efficient test of deviation from Hardy-Weinberg frequencies with one degree of freedom was applied to 44 marker loci in a genome scan, and 7 loci had a significant excess of apparent homozygotes (chi2 (1) > 6) suggestive of typing error. In this example evidence for linkage did not increase when outliers were censored. Statistical quality control is an essential part of genotyping, and the effect of mistyping and map error should be considered in evaluating any genome scan.

Asthma↗
PubMed1999

Cord blood progenitor cells have greater transendothelial migratory activity and increased responses to SDF-1 and MIP-3beta compared with mobilized adult progenitor cells.

When cord blood is used as a source of haemopoietic stem cells for transplantation, fewer cells are required per kg of recipient. This greater engraftment efficiency of cord blood cells may relate to an increased ability to traverse sinusoidal endothelium, a crucial step in the homing of stem cells. We report that freshly isolated cord blood progenitors migrated more efficiently than mobilized adult cells. Cord blood progenitors responded rapidly to growth factor stimulation with an increase in migratory ability within 24 h whereas mobilized adult cells responded only after 72 h (P < 0.01). Cord blood cells also exited G0/G1 rapidly; after 24 h of growth factor exposure, 20.2 +/- 1.2% of cord blood CD34+ cells were in S + G2/M compared to 6.9 +/- 1.2% of adult CD34+ cells (P < 0.01). Proliferating CFC migrated more efficiently (13.3 +/- 3.4% for GM-CFC) than non-proliferating CFC (1.4 +/- 0.5%, P < 0.01) as determined using a 3H-thymidine suicide assay. Cord blood progenitor cells also demonstrated a greater transmigratory response to chemokine stimulation compared with adult cells; this was manifested as a differential response of freshly isolated cells to SDF-1, and of growth factor activated cells to MIP-3beta. Finally, cord blood CD34+ cells express higher levels of the chemokine receptor for SDF-1, CXCR4, when compared with mobilized adult CD34+ cells (P < 0. 05).

Adult↗
PubMed1998

Linkage of asthma to markers on chromosome 12 in a sample of 240 families using quantitative phenotype scores.

We present evidence of linkage between markers on chromosome 12 and asthma using the BETA program for nonparametric single- and multipoint linkage analysis. We have used quantitative scores as our phenotypic variables, combining data into indices for asthma and atopy and maximizing heritability. The largest single-locus LODs were achieved for asthma: D12S342 and asthma score (LOD 2.255), D12S324 and asthma affection (LOD 2.214), and D12S366 and wheeze (LOD 3.307). The region of interest identified using multipoint analysis, with a maximum LOD of 2.29, centers around D12S97 at location 173.5 cM with a standard error of 6.5 for the asthma score and close agreement for asthma affection and wheeze. Such evidence merits further investigation of this area in an attempt to define the region with greater precision with a view to identifying candidate genes. We hope that the methods presented will encourage researchers to use phenotypic information in a way that encourages meta-analysis.

Adolescent↗
PubMed1998

p130, p107, and pRb are differentially regulated in proliferating cells and during cell cycle arrest by alpha-interferon.

We have determined how the phosphorylation of the retinoblastoma family (pRb, p107, and p130) is governed in individual cell cycle phases of Daudi B-cells during cell cycle exit triggered by alpha-interferon (alpha-IFN). alpha-IFN causes dephosphorylation of pRb and loss of p130 phosphorylated Form 3. However, the change in p130 phosphorylation in response to alpha-IFN occurs before dephosphorylation of pRb is complete because loss of p130 Form 3 occurs throughout the cell cycle prior to complete arrest in G1, whereas pRb is dephosphorylated only in G1. In contrast, p107 is dephosphorylated and is then depleted from cells as they exit the cell cycle. p130, predominantly in Form 1, and hypophosphorylated pRb bind an E2F DNA binding site; p130 complexes E2F-4, whereas pRb binds both E2F-4 and E2F-1. The phosphorylated forms of E2F-4 that bind to the E2F DNA site are different from hyperphosphorylated E2F-4, which predominates in primary hemopoietic cells in G0. We conclude that although cell cycle arrest induced by alpha-IFN may be mediated in part by formation of a complex containing p130 and E2F-4, alpha-IFN does not induce hyperphosphorylation of E2F-4, which characterizes primary hemopoietic cells in G0.

B-Lymphocytes↗
PubMed1998

The A-Myb transcription factor is a marker of centroblasts in vivo.

The A-Myb transcription factor is structurally related to the c-myb proto-oncogene and is involved in the control of proliferation and/or differentiation of mature B lymphocytes. We have shown previously by PCR analysis that A-myb is preferentially expressed in CD38+ CD39- sIgM- mature B cells. We demonstrate here, using in situ hybridization, that A-myb expression is restricted to the dark zone of human tonsils and lymph nodes. Furthermore, we show that A-Myb expression is cell cycle regulated both in tonsillar B cells and in Burkitt's lymphoma cell lines, being detectable only in the S and G2/M phases of the cell cycle and not in G0/G1 phase. Strong proliferation of resting human B cells induced in vitro by a variety of physiologic signals, including anti-mu, CD40 ligand, IL-2, IL-4, IL-6, IL-13, IFN-gamma, TNF-alpha, anti-CD19, and anti-CD20, failed to induce A-myb expression, suggesting that proliferation alone is not sufficient for A-myb expression in the absence of induction of a true centroblast phenotype. Finally, we show that differentiation of germinal center B cells in vitro toward either memory or plasma cells is accompanied by rapid down-regulation of A-myb expression. We conclude that A-myb is a marker of centroblasts generated in vivo.

B-Lymphocytes↗
PubMed1998

A study of females with deletions of the short arm of the X chromosome.

We have undertaken a clinical and molecular study of 25 females with deletions of the short arm of the X chromosome. We have determined the deletion breakpoints, the parental origin and the activation status of the deleted X chromosomes. Genotype-phenotype correlations suggest that the presence of a single copy of the DFFRX gene, previously postulated as a gene involved in the ovarian failure seen in Turner syndrome, may be compatible with normal ovarian function, and that there may be a gene for Turner-like features located in distal Xp22.3.

Adolescent↗
PubMed1998

Pure populations of transduced primary human cells can be produced using GFP expressing herpes virus vectors and flow cytometry.

Herpes simplex virus (HSV) has often been suggested as a vector for gene delivery to the nervous system although it is also capable of infecting many other cell types. HSV also has the ability to package large genetic insertions so the expression of multiple genes from a single virus is possible. Here we show that a green fluorescent protein (GFP) expressing HSV1 vector can transduce two primary human cell types--quiescent human CD34+ hematopoietic progenitor cells and dendritic cells--which are both hard to transduce by other means. We also show that GFP is an effective marker when expressed from an HSV vector in vivo in the mouse brain. When GFP is expressed together with a second gene (in this case lacZ) from a single virus, transduced GFP-positive CD34+ hematopoietic progenitor cells or dendritic cells can both be generated at an effective efficiency of 100% for the second gene. Here transduction with the vector is combined with flow cytometry allowing GFP-positive cells to be sorted from the untransduced population. Such completely transduced populations of quiescent CD34+ hematopoietic progenitor and dendritic cells cannot easily be achieved by other means, and might thus allow experimental or therapeutic protocols to be carried out requiring high-level transduction which would not otherwise be possible. Such an approach using HSV vectors might also be applicable to other cell types for which transduction is as yet unreliable or of low efficiency.

Animals↗
PubMed1998

Scarcity of mutations detected in families with X linked hypohidrotic ectodermal dysplasia: diagnostic implications.

Indirect molecular diagnosis of X linked hypohidrotic ectodermal dysplasia (XLHED), a congenital disorder of hair, teeth, and eccrine sweat glands, has been possible by linkage analysis. Direct mutation detection would enable carrier detection in female relatives of sporadic cases, as well as help distinguish XLHED from the rarer, clinically indistinguishable, autosomal recessive disorder ARHED. Recently, a candidate gene for XLHED has been identified. Genomic DNA from 162 affected males and 21 females, who were either obligate carriers or had manifestations of the disorder, were screened by SSCP analysis. A subset of the patients had been previously screened for large genomic deletions and had limited screening of a single exon by SSCP analysis. The two known exons were amplified using flanking primers. Approximately 7% of patients, all males, had putative mutations identified within exon 1, but no variants were found within exon 2. Ten different putative mutations and four probable polymorphisms were identified. Both of the known exons were sequenced in 10 patients who had no detectable SSCP changes, but no additional mutations were found. No correlation between phenotype and genotype was evident between either affected subjects or subjects with or without detectable mutations. The results of the study indicate that only a small minority of affected males can be diagnosed by direct mutation analysis, and that the remainder of the patients are likely to have mutations in as yet unidentified exons of the EDA gene. Linkage analysis, in informative situations, therefore remains the only practical diagnostic option available.

Cohort Studies↗
PubMed1997

Characterization of cell cycle status and E2F complexes in mobilized CD34+ cells before and after cytokine stimulation.

Mobilized peripheral blood progenitors (CD34+ cells) have been shown to be either in the G0 or G1 phase of the cell cycle. In this study, it is shown that they are small cells with low protein content suggestive of G0. Support for this is provided by showing that the principal E2F complex consists of hypophosphorylated p130, E2F-4, and DP-1. The E2F-4 is more highly phosphorylated than in quiescent T cells. In response to cytokines in vitro, the CD34+ cells start to enter G1 within 8 hours and enter S-phase at about 48 hours. As cells enter G1, E2F-4 is dephosphorylated to several hypophosphorylated forms and three new DNA-binding complexes appear, including one containing E2F-4, DP-1, and p107. We suggest that mobilized CD34+ cells may be maintained in G0 by p130, E2F-4, and DP-1 and the coordinate dephosphorylation of E2F-4 and hyperphosphorylation of p130 may be central to the initiation of proliferation.

Antigens, CD34↗
PubMed1997

Acylated C-21 steroidal bisdesmosidic glycosides from Caraluma umbellata.

From the whole plant of Caraluma umbellata, three new C-21 steroidal glycosides, named as carumbellosides III-V, were isolated and their structures elucidated by extensive spectroscopic experiments, devoid of any derivatisation, as caralumagenin 3-O-beta-D-glucopyranosyl(1-->4)-beta-D-digitalopyranoside-20-O-be ta- D-glucopyranoside, caralumagenin 3-O-beta-D-glucopyranosy(1-->4)- beta-D-digitalopyranoside-20-O-(2-O- benzoyl)-beta-D-glucopyranoside and caralumagenin 3-O-[6-O-benzoyl-beta-D-glucopyranosyl(1-->4)]-beta-D- digitalopyranoside-20-O-(2-O-benzoyl)-beta-D-glucopyranoside. The determination of the absolute configuration of the aglycone as (20 R), the conformations of the sugars and the unambiguous assignments of their NMR spectroscopic signals were achieved by a combination of 2D-NMR techniques. The isolates were devoid of significant cytotoxity in the UIC human cancer cell panel.

Carbohydrate Conformation↗
PubMed1997

The glutamine 27 beta2-adrenoceptor polymorphism is associated with elevated IgE levels in asthmatic families.

BACKGROUND: The beta2-adrenoceptor polymorphisms occurring at amino acid positions 16 (arginine to glycine) and 27 (glutamine to glutamate) are known to be functionally relevant and also disease-modifying in subjects with asthma. However, the contribution of these polymorphisms to the development of the asthmatic phenotype or other markers for allergic disease remains to be established. OBJECTIVE: This large family study examines the contributions of these polymorphisms in determining the heritable component of markers for allergic disease in asthmatic families. METHODS: Three hundred twenty-four individuals from 60 families multiplex for asthma selected by means of an asthmatic proband were characterized for the following markers of allergic disease: asthma, atopy, and serum IgE. The polymerase chain reaction was used to generate a 234 base pair fragment spanning the region of interest, and the beta2-adrenoceptor polymorphism was then defined by allele-specific oligonucleotide hybridization. Segregation analysis was then performed. RESULTS: We found a significant association (p = 0.009) between the glutamine 27 beta2-adrenoceptor polymorphism and elevated levels of IgE, which was supported by the observation of linkage between IgE and beta2-adrenoceptor polymorphisms at locus 27 (p = 0.037). However, there was no association between either the arginine-glycine 16 or the glutamine-glutamate 27 beta2-adrenoceptor polymorphism and an increased risk of asthma or atopy per se. CONCLUSION: The glutamine 27 beta2-adrenoceptor polymorphism appears to contribute to IgE variability in families with asthma. However, it seems that although both amino acid 16 and 27 beta2-adrenoceptor polymorphisms are disease-modifying in subjects with asthma, they do not contribute markedly to the development of the asthmatic phenotype.

Adult↗
PubMed1997

The predominant E2F complex in human primary haemopoietic cells and in AML blasts contains E2F-4, DP-1 and p130.

The E2F family of transcription factors are thought to play an important role in the control of cell cycle progression. There is now also increasing evidence that some family members may act as oncogenes or tumour suppressor genes. The characterization of these proteins in human primary haemopoietic cells and acute myeloid leukaemia (AML) blasts may thus give an insight to the molecular mechanisms governing proliferation and leukaemogenesis in these cells. Therefore we analysed the expression of E2F-DNA binding activity and the constituent proteins found in the complexes in human primary haemopoietic cells of various lineages. We also studied blasts from 18 patients with acute myeloid leukaemia (AML). On electromobility shift assays (EMSA) a single E2F-DNA binding complex was detected in T cells, B cells and monocytes which was shown to contain E2F-4, DP-1 and p130, indicating that all quiescent haemopoietic cells have the same complex. Examination of 18 AML samples by EMSA revealed the presence of E2F binding and no gross abnormalities were detected. An E2F-4/p130 complex was detected in representative samples of all FAB types analysed. Thus abnormalities of E2F function are unlikely to play a primary pathogenic role in AML.

Blotting, Western↗
PubMed1997

Plasmid diversity in Chlamydia.

Chlamydiae exhibit low interspecies DNA homology and plasmids from different chlamydial species can be readily distinguished by Southern blot analysis and restriction enzyme profiling. In contrast, available plasmid sequence data from within the species Chlamydia trachomatis indicate that plasmids from human isolates are highly conserved. To evaluate the nature and extent of plasmid variation, the complete nucleotide sequences were determined for novel plasmids from three diverse non-human chlamydial isolates: pCpA1 from avian Chlamydia psittaci (N352); pCpnE1 from equine Chlamydia pneumoniae (N16); and pMoPn from C. trachomatis mouse pneumonitis. Comparison of the sequence data did not identify an overall biological function for the plasmid but did reveal considerable sequence conservation (> 60%) and a remarkably consistent genomic arrangement comprising eight major ORFs and four 22 bp tandem repeats. The plasmid sequences were close to 7500 nucleotides in length (pCpA1, 7553 bp; pMoPn, 7502 bp) however the equine C. pneumoniae plasmid was smaller (7362 bp) than all other chlamydial plasmids. The reduced size of this plasmid was due to a single large deletion occurring within ORF 1; this potentially generates two smaller ORFs. The disruption of ORF 1 is the only significant variation identified amongst the chlamydial plasmids and could prove important for future vector development studies.

Amino Acid Sequence↗
PubMed1997

The candidate region approach to the genetics of asthma and allergy.

To date, the strongest linkage claims for genes underlying asthma and atopy have been for chromosomes 5 and 11. Chromosome 5q contains the cytokine cluster and the beta2 adrenoceptor, and the beta chain of the high-affinity IgE receptor (FCepsilonRI-beta) is encoded on chromosome 11q13. We have attempted to replicate these findings in two distinct sample populations from the United Kingdom. Allelic associations were identified in both regions, but there was no significant evidence for linkage. Although we could not substantiate the existence of the nucleotide changes reported within exon 6 of the FCepsilonRI-beta gene, an amino acid substitution in exon 7 was strongly linked to asthma and atopy. We have also identified positive linkage and allelic associations to several markers on chromosome 12q in both our UK populations. Independent evidence from another study also supports linkage to 12q, so although our data could not confirm linkage to chromosomes 5 or 11, we have identified an additional region of the genome that could be important for the genetic predisposition to asthma and atopy.

Asthma↗
PubMed1997

Candidate locus approach for studying the genetics of asthma and atopy.

Insights into the complex genetic factors underlying asthma and atopy have arisen from investigating genes whose functions are likely to be involved in the pathogenesis of these disorders. Candidate genes have now been studied on 13 human autosomes, and genetic linkage analysis and association studies have identified potentially important genes on chromosomes 5, 6, 11 and 14. However, these four regions do not account for all the heritability of these disorders, and additional regions are now being identified by systematic searches of the whole genome.

Asthma↗