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Biomedical subjects

M E Conley

Publications and source records attributed to M E Conley.

At least 37 records · Page 2Linked to original sources
PubMed1999

Correction of X-linked immunodeficient mice by competitive reconstitution with limiting numbers of normal bone marrow cells.

Gene therapy for inherited disorders is more likely to succeed if gene-corrected cells have a proliferative or survival advantage compared with mutant cells. We used a competitive reconstitution model to evaluate the strength of the selective advantage that Btk normal cells have in Btk-deficient xid mice. Whereas 2,500 normal bone marrow cells when mixed with 497,500 xid cells restored serum IgM and IgG3 levels to near normal concentrations in 3 of 5 lethally irradiated mice, 25,000 normal cells mixed with 475,000 xid cells reliably restored serum IgM and IgG3 concentrations and the thymus-independent antibody response in all transplanted mice. Reconstitution was not dependent on lethal irradiation, because sublethally irradiated mice all had elevated serum IgM and IgG3 by 30 weeks postreconstitution when receiving 25,000 normal cells. Furthermore, the xid defect was corrected with as few as 10% of the splenic B cells expressing a normal Btk. When normal donor cells were sorted into B220(+)/CD19(+) committed B cells and B220(-)/CD19(-) cell populations, only the B220(-)/CD19(-) cells provided long-term B-cell reconstitution in sublethally irradiated mice. These findings suggest that even inefficient gene therapy may provide clinical benefit for patients with XLA.

Adoptive Transfer↗
PubMed1999

Novel mechanisms control the folding and assembly of lambda5/14.1 and VpreB to produce an intact surrogate light chain.

Surrogate light chain, which escorts the mu heavy chain to the cell surface, is a critical component of the pre-B cell receptor complex. The two proteins that comprise the surrogate light chain, VpreB and lambda5/14.1, contain both unique regions and Ig-like domains. The unique regions have been postulated to function in the assembly of the surrogate light chain. However, by using transient transfection of COS7 cells, we show that deletion of the unique regions of both proteins did not inhibit the assembly of surrogate light chain. Instead, in vivo folding studies showed that the unique region of lambda5/14.1 acts as an intramolecular chaperone by preventing the folding of this protein when it is expressed in the absence of its partner, VpreB. The Ig domains of both lambda5/14.1 and VpreB are atypical. The one in VpreB lacks one of the canonical beta strands whereas the one in lambda5/14.1 has an extra beta strand. Deletion of the extra beta strand in lambda5/14.1 completely abrogated the formation of the surrogate light chain, demonstrating that complementation of the incomplete Ig domain in VpreB by the extra beta strand in lambda5/14.1 was necessary and sufficient for the folding and assembly of these proteins. Our studies reveal two novel mechanisms for regulating surrogate light chain formation: (i) the presence of an intramolecular chaperone that prevents folding of the unassembled subunit but that remains part of the mature assembled protein, and (ii) splitting an Ig domain between two proteins to control their folding and assembly.

Animals↗
PubMed1999

Mutations of the human BTK gene coding for bruton tyrosine kinase in X-linked agammaglobulinemia.

X-linked agammaglobulinemia (XLA) is an immunodeficiency caused by mutations in the gene coding for Bruton agammaglobulinemia tyrosine kinase (BTK). A database (BTKbase) of BTK mutations lists 544 mutation entries from 471 unrelated families showing 341 unique molecular events. In addition to mutations, a number of variants or polymorphisms have been found. Mutations in all the five domains of BTK cause the disease, the single most common event being missense mutations. Most mutations lead to truncation of the enzyme. The mutations appear almost uniformly throughout the molecule. About one-third of point mutations affect CpG sites, which usually code for arginine residues. The putative structural implications of all the missense mutations are provided in the database. BTKbase is available at http://www.uta.fi/imt/bioinfo.

Agammaglobulinaemia Tyrosine Kinase↗
PubMed1999

Unusual mutations in Btk: an insertion, a duplication, an inversion, and four large deletions.

Mutations in Bruton's tyrosine kinase (Btk) result in the immunodeficiency X-linked agammaglobulinemia (XLA). In a previous study of 101 patients with presumed XLA, we identified seven patients with large genomic alterations in Btk. The recent completion of 100 kb of contiguous DNA sequence at the Btk locus has allowed us to characterize these mutations in detail and to identify four different types of alterations. These alterations included a 253-bp retroposon insertion at position +5 within intron 9, an inversion of greater than 48 kb that disrupted Btk between exons 4 and 5, a 12.9-kb duplication including Btk exons 2 to 5, and four deletions ranging from 2.8 to 38 kb in size. The duplication and three of the deletions resulted from unequal crossovers of Alu repeats. Further, three of the deletions terminated within a repeat-rich cluster spanning 30 kb of sequence 3' of Btk exon 19, suggesting that this region was more susceptible to unequal crossovers than the rest of the Btk gene. These studies describe the first reports of an insertion, an inversion, and a duplication in Btk and demonstrate the utility of large-scale sequencing in the elucidation of disease-causing mutations.

Agammaglobulinaemia Tyrosine Kinase↗
PubMed1999

Gene conversion events contribute to the polymorphic variation of the surrogate light chain gene lambda 5/14.1.

Normally occurring and experimentally induced models of immunodeficiency indicate that B cell development and antibody production are influenced by genetic factors. It is highly likely that polymorphic variants in genes that encode receptors for growth and differentiation factors, signal transduction molecules, and components of the B cell and pre-B-cell receptor complex contribute to this genetic control. We have identified a surprisingly large number of polymorphic variants in lambda5/14.1. Together with VpreB, lambda5/14.1 forms the surrogate light chain in the pre-B-cell receptor complex. Thirteen variant alleles of lambda5/14.1 were found in 134 unrelated individuals. Nine of these variants result in changes in the amino acid sequence of this small protein. The majority of the single base pair substitutions in lambda5/14.1 could be attributed to gene conversion events in which donor sequences from the lambda5 pseudogenes, 16.1, 16.2, and Glambda1, replace the wild-type sequence in the lambda5/14.1 functional gene. These findings indicate that gene conversion events play a major role in generating diversity that could affect stability or expression of the pre-B-cell receptor complex.

Alleles↗
PubMed1999

Recent progress in the diagnosis and treatment of patients with defects in early B-cell development.

Mutation detection for X-linked agammaglobulinemia (XLA) has revealed the heterogeneity of the clinical phenotype of patients with defects in Bruton's tyrosine kinase (Btk), the gene that is abnormal in XLA. Over 50% of patients with mutations in Btk have no family history of the disease because their cases are the first manifestation of a new mutation in their family. In 10% to 20% of patients, the serum immunoglobulins are higher than expected or the onset of disease is delayed; however, a marked reduction in B-cell numbers is consistent in all patients. Mutation detection has also shown that not all patients with presumed XLA have mutations in Btk. Mutations in mu heavy chain, and other components of the pre-B cell receptor complex, including lambda 5/14.1, cause a disorder that is clinically identical to XLA. Although new strategies for therapy are not yet available, the groundwork is being laid for cell or gene therapy.

Agammaglobulinemia↗
PubMed1999

Mutations in Igalpha (CD79a) result in a complete block in B-cell development.

Mutations in Btk, mu heavy chain, or the surrogate light chain account for 85-90% of patients with early onset hypogammaglobulinemia and absent B cells. The nature of the defect in the remaining patients is unknown. We screened 25 such patients for mutations in genes encoding components of the pre-B-cell receptor (pre-BCR) complex. A 2-year-old girl was found to have a homozygous splice defect in Igalpha, a transmembrane protein that forms part of the Igalpha/Igbeta signal-transduction module of the pre-BCR. Studies in mice suggest that the Igbeta component of the pre-BCR influences V-DJ rearrangement before cell-surface expression of mu heavy chain. To determine whether Igalpha plays a similar role, we compared B-cell development in an Igalpha-deficient patient with that seen in a mu heavy chain-deficient patient. By immunofluorescence, both patients had a complete block in B-cell development at the pro-B to pre-B transition; both patients also had an equivalent number and diversity of rearranged V-DJ sequences. These results indicate that mutations in Igalpha can be a cause of agammaglobulinemia. Furthermore, they suggest that Igalpha does not play a critical role in B-cell development until it is expressed, along with mu heavy chain, as part of the pre-BCR.

Agammaglobulinemia↗
PubMed1998

Expression and activation of the nonreceptor tyrosine kinase Tec in human B cells.

The tyrosine kinase Tec belongs to a new group of structurally related nonreceptor tyrosine kinases that also includes Btk and Itk. Previous studies have suggested that these kinases have lineage-specific roles, with Tec being involved mainly in the regulation of cytokine-mediated myeloid cell growth and differentiation. In this study, we investigated expression and activation of Tec in human B-lymphoid cell lines representing different stages of B-cell maturation, including pro-B (RS4;11, 380, REH), pre-B (NALM6), and mature B (Ramos, and one Epstein-Barr virus [EBV]-transformed lymphoblastoid line) cells. Like Btk, Tec protein was expressed in all B-cell lines tested. Tec was also highly expressed in two EBV-transformed lymphoblastoid lines derived from patients with X-linked agammaglobulinemia (XLA) lacking Btk expression, as well as in tonsillar lymphoid cells. In surface immunoglobulin-positive B cells (Ramos), ligation of the B-cell antigen receptor (BCR) with anti-IgM antibodies caused marked tyrosine phosphorylation of Tec and increased Tec tyrosine kinase activity. Likewise, cross-linking of CD19 with a monoclonal antibody in BCR-negative pro-B (RS4;11, 380) and pre-B (NALM6) cells induced Tec tyrosine phosphorylation and increased Tec autophosphorylation, as well as Btk activation. Tyrosine phosphorylation of Tec, but not of Btk, was detectable in RS4;11 cells after CD38 ligation, suggesting that these kinases are regulated differently. We conclude that Tec is expressed and can be stimulated throughout human B-cell differentiation, implying that this tyrosine kinase plays a role in B-cell development and activation.

ADP-ribosyl Cyclase↗
PubMed1998

Mutations in the human lambda5/14.1 gene result in B cell deficiency and agammaglobulinemia.

B cell precursors transiently express a pre-B cell receptor complex consisting of a rearranged mu heavy chain, a surrogate light chain composed of lambda5/14.1 and VpreB, and the immunoglobulin (Ig)-associated signal transducing chains, Igalpha and Igbeta. Mutations in the mu heavy chain are associated with a complete failure of B cell development in both humans and mice, whereas mutations in murine lambda5 result in a leaky phenotype with detectable humoral responses. In evaluating patients with agammaglobulinemia and markedly reduced numbers of B cells, we identified a boy with mutations on both alleles of the gene for lambda5/14.1. The maternal allele carried a premature stop codon in the first exon of lambda5/14.1 and the paternal allele demonstrated three basepair substitutions in a 33-basepair sequence in exon 3. The three substitutions correspond to the sequence in the lambda5/14. 1 pseudogene 16.1 and result in an amino acid substitution at an invariant proline. When expressed in COS cells, the allele carrying the pseudogene sequence resulted in defective folding and secretion of mutant lambda5/14.1. These findings indicate that expression of the functional lambda5/14.1 is critical for B cell development in the human.

Agammaglobulinemia↗
PubMed1998

Transcriptional regulatory elements within the first intron of Bruton's tyrosine kinase.

Defects in the gene for Bruton's tyrosine kinase (Btk) result in the disorder X-linked agammaglobulinemia (XLA). Whereas XLA is characterized by a profound defect in B-cell development, Btk is expressed in both the B lymphocyte and myeloid cell lineages. We evaluated a patient with XLA who had reduced amounts of Btk transcript but no abnormalities in his coding sequence. A single base-pair substitution in the first intron of Btk was identified in this patient, suggesting that this region may contain regulatory elements. Using reporter constructs we identified two transcriptional control elements in the first 500 bp of intron 1. A strong positive regulator, active in both pre-B cells and B cells, was identified within the first 43 bp of the intron. Gel-shift assays identified two Sp1 binding sites within this element. The patient's mutation results in an altered binding specificity of the proximal Sp1 binding site. A negative regulator, active in pre-B cells only, was located between base pairs 281 and 491 of the intron. These findings indicate that regulation of Btk transcription is complex and may involve several transcriptional regulatory factors at the different stages of B-cell differentiation.

Agammaglobulinaemia Tyrosine Kinase↗
PubMed1998

Two novel mutations in exon 11 of the PAH gene (V1163del TG and P362T) associated with classic phenylketonuira and mild phenylketonuria. Mutations in brief no. 143. Online.

PKU is one of the commonest genetic disease in man, affecting 1/10,000 individuals. It presents a wide phenotypical spectrum, from classic PKU to moderate Hyperpheylalaninemia depending on the residual enzymatic activity. Two novel mutations 1163/1164delTG and P362T in exon 11 have been detected during the mutational screening of the PAH gene in 84 families. 1163/1164delTC can be confused with V388M if the mutational screening is performed with BsaAI restriction enzyme, this mutation in heterozigosis presents a moderate phenotype. P362T mutation in heterozigosis with V388M shows a classical PKU phenotype. We report here two new mutations in exon 11 of the PAH gene (GenBank U49897), V1163delTG and P362T (using cDNA sequence), detected during the analysis of 57 PKU and 36 HPA patients belonging to 84 unrelated families detected under a neonatal screening program performed in Catalonia.

DNA Mutational Analysis↗
PubMed1998

Genetic basis of abnormal B cell development.

A susceptibility gene in the MHC class III region may underlie the defective B-cell differentiation in familial IgA deficiency and common variable immunodeficiency. Mutations in Bruton's tyrosine kinase, immunoglobulin heavy chain and lambda 5/14.1 surrogate light chain loci disrupt B-cell development to cause profound antibody deficiency. Mutational, biochemical and transgenic studies offer insight into the function of these and other 'antibody deficiency genes'.

Agammaglobulinaemia Tyrosine Kinase↗
PubMed1998

Mutations in btk in patients with presumed X-linked agammaglobulinemia.

In 1993, two groups showed that X-linked agammaglobulinemia (XLA) was due to mutations in a tyrosine kinase now called Btk. Most laboratories have been able to detect mutations in Btk in 80%-90% of males with presumed XLA. The remaining patients may have mutations in Btk that are difficult to identify, or they may have defects that are phenotypically similar to XLA but genotypically different. We analyzed 101 families in which affected males were diagnosed as having XLA. Mutations in Btk were identified in 38 of 40 families with more than one affected family member and in 56 of 61 families with sporadic disease. Excluding the patients in whom the marked decrease in B cell numbers characteristic of XLA could not be confirmed by immunofluorescence studies, mutations in Btk were identified in 43 of 46 patients with presumed sporadic XLA. Two of the three remaining patients had defects in other genes required for normal B cell development, and the third patient was unlikely to have XLA, on the basis of results of extensive Btk analysis. Our techniques were unable to identify a mutation in Btk in one male with both a family history and laboratory findings suggestive of XLA. DNA samples from 41 of 49 of the mothers of males with sporadic disease and proven mutations in Btk were positive for the mutation found in their son. In the other 8 families, the mutation appeared to arise in the maternal germ line. In 20 families, haplotype analysis showed that the new mutation originated in the maternal grandfather or great-grandfather. These studies indicate that 90%-95% of males with presumed XLA have mutations in Btk. The other patients are likely to have defects in other genes.

Agammaglobulinaemia Tyrosine Kinase↗
PubMed1997

Mutation analysis of IL2RG in human X-linked severe combined immunodeficiency.

Severe combined immunodeficiency (SCID) is a syndrome of profoundly impaired cellular and humoral immunity. In humans, SCID is most commonly caused by mutations in the X-linked gene IL2RG, which encodes the common gamma chain, gamma c, of the leukocyte receptors for interleukin-2 and multiple other cytokines. To investigate the frequency and variety of IL2RG mutations that cause SCID, we analyzed DNA, RNA, and B-cell lines from a total of 103 unrelated SCID-affected males and their relatives using a combination of molecular and immunologic techniques. Sixty-two different mutations spanning all eight IL2RG exons were found in 87 cases, making possible correlations between mutation type and functional consequences. Although skewed maternal X chromosome inactivation, single-strand conformation polymorphism, mRNA expression, and cell surface staining with anti-gamma c antibodies were all helpful in establishing IL2RG defects as the cause of SCID, only dideoxy fingerprinting and DNA sequence determination each detected 100% of the IL2RG mutations in our series. Abnormal gamma c chains may be expressed in the lymphocytes of as many as two thirds of patients with X-linked SCID. Specific mutation diagnosis thus remains technically challenging, but it is important for genetic counseling and perhaps for helping to select appropriate subjects for retroviral gene therapy trials, This is a US government work. There are no restrictions on its use.

DNA Fingerprinting↗