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Biomedical subjects

M E Conley

Publications and source records attributed to M E Conley.

At least 55 records · Page 3Linked to original sources
PubMed1996

Mutations in the mu heavy-chain gene in patients with agammaglobulinemia.

BACKGROUND: Most patients with congenital hypogammaglobulinemia and absent B cells are males with X-linked agammaglobulinemia, which is caused by mutations in the gene for Bruton's tyrosine kinase (Btk); however, there are females with a similar disorder who do not have mutations in this gene. We studied two families with autosomal recessive defects in B-cell development and patients with presumed X-linked agammaglobulinemia who did not have mutations in Btk. METHODS: A series of candidate genes that encode proteins involved in B-cell signal-transduction pathways were analyzed by linkage studies and mutation screening. RESULTS: Four different mutations were identified in the mu heavy-chain gene on chromosome 14. In one family, there was a homozygous 75-to-100-kb deletion that included D-region genes, J-region genes, and the mu constant-region gene. In a second family, there was a homozygous base-pair substitution in the alternative splice site of the mu heavy-chain gene. This mutation would inhibit production of the membrane form of the mu chain and produce an amino acid substitution in the secreted form. In addition, a patient previously thought to have X-linked agammaglobulinemia was found to have an amino acid substitution on one chromosome at an invariant cysteine that is required for the intrachain disulfide bond and, on the other chromosome, a large deletion that included the immunoglobulin locus. CONCLUSIONS: Defects in the mu heavy-chain gene are a cause of agammaglobulinemia in humans. This implies that an intact membrane-bound mu chain is essential for B-cell development.

Agammaglobulinemia↗
PubMed1996

A single strand conformation polymorphism study of CD40 ligand. Efficient mutation analysis and carrier detection for X-linked hyper IgM syndrome.

Mutations in the gene for CD40 ligand are responsible for the X-linked form of hyper IgM syndrome. However, no clinical or laboratory findings that reliably distinguish X-linked disease from other forms of hyper IgM syndrome have been reported, nor are there tests available that can be used to confidently provide carrier detection. To identify efficiently mutations in the gene for CD40 ligand, eight pairs of PCR primers that could be used to screen genomic DNA by single strand conformation polymorphism (SSCP) were designed. 11 different mutations were found in DNA from all 13 patients whose activated T cells failed to bind a recombinant CD40 construct. The exact nature of four of these mutations, a deletion and three splice defects, could not be determined by cDNA sequencing. In addition, SSCP analysis permitted rapid carrier detection in two families in whom the source of the mutation was most likely a male with gonadal chimerism who passed the disorder on to some but not all of his daughters. These studies document the utility of SSCP analysis for both mutation detection and carrier detection in X-linked hyper IgM syndrome.

Base Sequence↗
PubMed1996

CD38 signal transduction in human B cell precursors. Rapid induction of tyrosine phosphorylation, activation of syk tyrosine kinase, and phosphorylation of phospholipase C-gamma and phosphatidylinositol 3-kinase.

Ligation of CD38 inhibits proliferation and induces apoptosis of human immature B cells, but the molecular mechanisms underlying this function are unknown. We found that CD38 dimerization with the specific mAbs T16 and IB4 induces rapid and transient tyrosine phosphorylation of several intracellular proteins in the immature B cell lines RS4;11, REH, 380, Nalm6, and OP-1. This effect could be markedly reduced by incubating cells with the tyrosine kinase inhibitors genistein, staurosporine, and herbimycin A. CD38 dimerization induced tyrosine phosphorylation of the protein kinase syk and increased syk kinase activity. CD38 dimerization also induced tyrosine phosphorylation of phospholipase C-gamma and of the p85 subunit of phosphatidylinositol 3-kinase (PI 3-K). The latter was accompanied by a distinct increase in PI 3-kinase activity in the immunoprecipitates obtained with an anti-phosphotyrosine Ab. In contrast to the signaling triggered by surface Ig engagement in B lymphocytes, CD38 ligation did not appear to induce tyrosine phosphorylation of the src-like protein tyrosine kinases lyn, fyn, and btk, or of vav- and ras-GTPase-activating protein, nor did it induce detectable changes in cytosolic CA2+ concentrations. CD38 signaling also differed from cytokine-induced signaling in that it did not cause tyrosine phosphorylation of Jak1 and Jak2. Finally, CD38 ligation did not inhibit IL-3-induced tyrosine phosphorylation of Jak2. These results identify CD38 as a cell surface receptor with signal transduction properties activated by dimerization. Induction of signal transduction by CD38 ligation implies the existence of a yet unidentified natural ligand of CD38.

ADP-ribosyl Cyclase↗
PubMed1996

Neutropenia in X-linked agammaglobulinemia.

X-linked agammaglobulinemia (XLA) is usually considered a disorder of B cell development; however, the gene that is defective in XLA encodes a cytoplasmic tyrosine kinase called Btk, that is expressed throughout myeloid as well as B cell differentiation. A review of medical records of patients in whom mutations in Btk had been identified indicated that 13 of 50 patients (26%) had experienced episodes of profound neutropenia. In 12 of the 13 patients, neutropenia was part of the acute illness that precipitated an evaluation for immunodeficiency. These boys were more likely to be less than a year of age at the time of diagnosis and they were less likely to have a family history of immunodeficiency. Neutropenia was associated with staphylococcal or pseudomonas sepsis in 6 of the patients. The duration of neutropenia was variable but was often more than 1 week. Neutropenia was not seen in any patient with XLA receiving intravenous gammaglobulin. Although neutropenia was not associated with any specific mutation in Btk, most of the alterations in this gene in the patients with XLA and neutropenia resulted in the absence of Btk protein or in amino acid substitutions in sites thought to be critical to Btk function. Btk may not be required for neutrophil production under normal circumstances; however, it may play a role in the response to stress.

Agammaglobulinaemia Tyrosine Kinase↗
PubMed1996

Wiskott-Aldrich syndrome in a family with Fanconi anemia.

Thrombocytopenia may be the presenting finding for both Wiskott-Aldrich syndrome and Fanconi anemia. We examined a sibship of four boys who had features of both of these hematologic disorders. Peripheral blood lymphocytes from three of the boys demonstrated DNA instability when cultured with diepoxybutane, confirming the diagnosis of Fanconi anemia in these patients. However, results of linkage analysis and X chromosome inactivation studies were consistent with the diagnosis of Wiskott-Aldrich syndrome in two of the boys, including one of the boys with Fanconi anemia. These findings could be attributed to the occurrence of two rare genetic disorders in a single family or to an unusual variant of Fanconi anemia. The recent identification of the Wiskott-Aldrich gene permitted us to address this question directly. Epstein-Barr virus-transformed cell lines from the two boys thought to have Wiskott-Aldrich syndrome on the basis of linkage analysis failed to express transcripts for the Wiskott-Aldrich gene. Genomic DNA from these two patients demonstrated a G insertion in the tenth exon of the Wiskott-Aldrich gene, resulting in a frameshift and a premature stop codon. Surprisingly, the patient with Fanconi anemia and a null mutation in the Wiskott-Aldrich gene had typical Fanconi anemia but mild Wiskott-Aldrich syndrome.

Child↗
PubMed1996

Cure of X-linked lymphoproliferative disease (XLP) with allogeneic hematopoietic stem cell transplantation (HSCT): report from the XLP registry.

Seven male patients in the David T Purtilo International X-linked Lymphoproliferative Disease (XLP) Registry have undergone allogeneic hematopoietic stem cell transplantation (HSCT). All patients received HSCT from HLA-identical donors: sibling BM, five; unrelated BM, one; and sibling umbilical cord blood, one. Ages at time of HSCT ranged from 5 to 30 years. Pre-HSCT clinical course varied, but four boys had a significant history of chronic and/or serious infections. Conditioning regimens varied: TBI containing regimens, four, chemotherapy only, three. All patients engrafted. Six developed grade I-II acute GVHD but no chronic GVHD. Four are alive and well with normal immune function greater than 3 years following HSCT. Three died within 100 days: disseminated adenovirus, one; polymicrobial sepsis, one; and multiple organ system failure and bleeding diathesis, one. No EBV-associated post-transplant complications were observed, even though all donors except the umbilical cord blood were EBV-seropositive. Unsuccessful HSCT was associated with age at HSCT (> 15 years), TBI-containing regimen and significant history for pre-HSCT infections. These results provide evidence that HSCT performed during childhood with HLA-identical sibling donors, regardless of EBV serostatus, offers the only curative therapy for XLP.

Adolescent↗
PubMed1995

B-cell-specific demethylation of BTK, the defective gene in X-linked agammaglobulinemia.

BTK, the gene that is defective in X-linked agammaglobulinemia, encodes a cytoplasmic tyrosine kinase that is critical for B-cell proliferation, or survival. To identify regulatory elements that control the expression of BTK we evaluated the methylation pattern of this gene in cell lines and in freshly isolated cells. An Hpa II site that was specifically demethylated in mature B cells but not in pre-B cells, T cells, neutrophils, or nonhematopoietic cells was identified in the tenth intron of BTK. In a 40 kilobase (kb) segment of DNA spanning the entire coding region of BTK plus 3 kb upstream of the first exon there were no other sites that demonstrated lineage-specific demethylation. The B-cell-specific demethylation site in intron 10, which falls within the SH2 domain, 26 kb distal to the first exon, occurs in a region rich in regulatory elements including two E2 boxes, two AP-2 sites, and a cAMP response element. It is likely that this site plays a role in maintaining BTK transcription in mature B cells.

Agammaglobulinaemia Tyrosine Kinase↗
PubMed1995

Effect of intravenous gammaglobulin (IVIG) on the platelet count in patients with Wiskott-Aldrich syndrome.

The ability of IVIG to increase platelet counts in patients with idiopathic thrombocytopenic purpura suggests its potential usefulness in other disease states characterized by low platelet counts. This possibility was evaluated in nine patients with the Wiskott-Aldrich syndrome (WAS) who received IVIG, at a dose of 400 mg/kg every 4 weeks. The mean platelet count prior to institution of IVIG was 32,000/cumm (range 2,400 to 98,000). Following administration of IVIG, the platelet count ranged between 5,000 and 85,000/cumm. There were no immediate increases in platelet counts after IVIG infusion in any patient who had serial platelet counts. During treatment, patients were not given any routine platelet transfusions for low platelet counts. However, while on IVIG, two patients showed a good response to platelet transfusion prior to surgical procedures. In conclusion, chronic IVIG therapy does not appear to affect platelet counts in patients with the Wiskott-Aldrich syndrome.

Child↗
PubMed1994

The genomic structure of human BTK, the defective gene in X-linked agammaglobulinemia.

It has recently been demonstrated that mutations in the gene for Bruton's tyrosine kinase (BTK) are responsible for X-linked agammaglobulinemia. Southern blot analysis and sequencing of cDNA were used to document deletions, insertions, and single base pair substitutions. To facilitate analysis of BTK regulation and to permit the development of assays that could be used to screen genomic DNA for mutations in BTK, we determined the genomic organization of this gene. Subcloning of a cosmid and a yeast artificial chromosome showed that BTK is divided into 19 exons spanning 37 kilobases of genomic DNA. Analysis of the region 5' to the first untranslated exon revealed no consensus TATAA or CAAT boxes; however, three retinoic acid binding sites were identified in this region. Comparison of the structure of BTK with that of other nonreceptor tyrosine kinases, including SRC, FES, and CSK, demonstrated a lack of conservation of exon borders. Information obtained in this study will contribute to our understanding of the evolution of nonreceptor tyrosine kinases. It will also be useful in diagnostic studies, including carrier detection, and in studies directed towards gene therapy or gene replacement.

Agammaglobulinaemia Tyrosine Kinase↗
PubMed1994

X-linked immunodeficiencies.

In the past year, researchers have identified the genes responsible for X-linked severe combined immunodeficiency (encoding a cytokine receptor protein), X-linked agammaglobulinemia (encoding a cytoplasmic tyrosine kinase) and X-linked hyper IgM syndrome (encoding the ligand for CD40). Although these three genes are completely unrelated, it is of interest that all are lineage-specific genes that are involved in the control of lymphocyte proliferation or differentiation.

Agammaglobulinemia↗
PubMed1994

Screening of genomic DNA to identify mutations in the gene for Bruton's tyrosine kinase.

Mutations in the gene for Bruton's tyrosine kinase (Btk) are responsible for X-linked agammaglobulinemia (XLA). Thus far, mutations in this gene have been identified based on alterations in Southern or Northern blot analysis or cDNA sequence. To permit detection of mutations in genomic DNA, we designed PCR primers to flank each of the 19 exons of Btk with splice sites. Two overlapping PCR products were employed for exons longer than 230 base pairs. Single strand conformation polymorphism (SSCP) analysis was used to screen PCR products from 30 unrelated families presumed to carry a Btk mutation. It was possible to amplify DNA in every reaction from every patient, indicating that large deletions in Btk are uncommon. Twenty three different mutations were found in 25 unrelated families, including one family in whom DNA was available from a carrier but not an affected patient. Seven mutations were single base pair substitutions resulting in premature stop codons scattered throughout the gene. Small insertions or deletions causing frameshifts and secondary premature stop codons constituted an additional seven mutations. One patient had a point mutation in the start codon and one patient had a mutation in a splice donor site. Point mutations resulting in amino acid substitutions were seen in nine patients. Northern blot analysis of RNA from three patients with premature stop codons showed an absence of Btk transcript whereas four patients with amino acid substitutions had normal amounts of transcript of normal size. These studies document the considerable variability in the Btk mutations causing XLA and they demonstrate an approach that will be useful for carrier detection as well as mutation identification.

Agammaglobulinaemia Tyrosine Kinase↗
PubMed1994

X-linked agammaglobulinemia: new approaches to old questions based on the identification of the defective gene.

The identification of a cytoplasmic tyrosine kinase, Btk, as the defective protein in human XLA and xid in the mouse, supports the hypothesis that both disorders are due to defects in B-cell activation or differentiation. Phenotypic analysis of B-lineage cells and studies on X-chromosome inactivation patterns in both mice and human patients suggest that mutations in Bth do not affect entry of stem cells into the B-lineage pathway but they do inhibit progression at multiple steps along that pathway. Although the exact function of Btk in signal transduction is not yet known, it is probable that studies which correlate specific mutations in different patients with alterations in Btk function will provide clues about critical sites in the molecule. Diagnosis and genetic counseling for families at risk of carrying the gene for XLA will be improved almost immediately by the identification of the responsible gene. Improvements in therapy may come more slowly. The possibility of curative gene therapy is attractive; however, there are several features of Btk that suggest that this will be a challenging undertaking. Overexpression or expression in inappropriate cell lineages may carry unacceptable risks. Mutant proteins may interfere with the function of wild-type proteins provided by gene therapy. However, it is likely that a better understanding of Btk function and regulation will benefit not only patients with XLA but also other patients with defects in B-cell function.

Agammaglobulinaemia Tyrosine Kinase↗
PubMed1994

Hyper IgM syndrome associated with defective CD40-mediated B cell activation.

Recent studies show that most patients with X-linked hyper IgM syndrome have defects in the gene for CD40 ligand. We evaluated 17 unrelated males suspected of having X-linked hyper IgM syndrome. Activated T cells from 13 of the 17 patients failed to bind a soluble CD40 construct. In these patients, the sequence of CD40 ligand demonstrated mutations. By contrast, T cells from the remaining four patients exhibited normal binding to the CD40 construct. Sequencing of the cDNA for CD40 ligand from these patients did not show mutations. The possibility that hyper IgM syndrome in these four patients was due to abnormalities in the B cell response to CD40-mediated signals was examined. Peripheral blood lymphocytes were stimulated with anti-CD40 alone, IL4 alone or anti-CD40 plus IL4. In comparison with B cells from controls or patients with hyper IgM syndrome and mutant CD40 ligand, B cells from the patients with hyper IgM syndrome and normal CD40 ligand were defective in their ability to secrete IgE (P < 0.02) or express activation markers, CD25 and CD23 (P < 0.02) in response to stimulation with anti-CD40. The failure of these B cells to respond to CD40-mediated activation could not be attributed to a generalized deficiency in B cell activation because IL4 induced normal up-regulation of CD23 and CD25 expression. These findings indicate that hyper IgM syndrome may result from defects in expression of CD40 ligand by activated T cells or defects in CD40-mediated signal transduction in B cells.

Antigens, CD↗
PubMed1994

Successful treatment of neutropenia in the hyper-immunoglobulin M syndrome with granulocyte colony-stimulating factor.

PATIENT: A young boy with hyper-immunoglobulin M (IgM) syndrome had recurrent severe infections, failure to thrive, and chronic neutropenia for 2 years despite treatment with i.v. gammaglobulin (IVIG). METHODS AND RESULTS: With the addition of granulocyte colony-stimulating factor (G-CSF; Filgrastim, Amgen, Inc., Thousand Oaks, CA), increased doses of IVIG, and prophylactic trimethoprim-sulfamethoxazole, his absolute neutrophil count increased from 0.64 x 10(9)/L to 3.36 x 10(9)/L, and he has been free of significant infection for the past 22 months. CONCLUSIONS: The use of G-CSF merits consideration in patients with hyper-IgM syndrome and severe neutropenia.

Child↗
PubMed1993

Correction of Duncan's syndrome by allogeneic bone marrow transplantation.

Duncan's syndrome (X-linked lymphoproliferative disorder) is a fatal immunodeficiency syndrome of unknown aetiology and without cure. We show that allogeneic bone marrow transplantation can correct the lethal immune deficits in a patient with this syndrome. We suggest that the primary abnormality in Duncan's syndrome is in bone-marrow derived cells.

Bone Marrow Transplantation↗