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Biomedical subjects

A Bottaro

Publications and source records attributed to A Bottaro.

At least 37 records · Page 2Linked to original sources
PubMed1993

A selective defect in IgG2b switching as a result of targeted mutation of the I gamma 2b promoter and exon.

LPS stimulation of B lymphocytes induces germline transcription of and subsequent switching to the gamma 2b gene. Mature germline transcripts contain an I exon (non-coding) spliced to the C gamma 2b exons. To investigate the role of germline transcription and/or transcripts in heavy chain class switching, we have replaced the germline I gamma 2b promoter and I exon in ES cells with an expressed neomycin resistance gene. The mutated chromosome retains the downstream target sequence for switch recombination (S regions) and all sequences necessary for expression of a switched gamma 2b gene. Wild-type or mutant ES cells were injected into RAG-2 deficient blastocysts to generate somatic chimeras in which all lymphocytes were ES-cell derived. Chimeras derived from injection of heterozygous mutant ES cells had normal levels of serum IgG2b, but their splenic B cells showed a partial decrease in ability to switch to gamma 2b. Strikingly, B lymphocytes from chimeras derived by injection of homozygous mutant ES cells were deficient in IgG2b production both in vivo and in vitro, but normal with respect to production of other Ig heavy chain isotypes. Additional studies demonstrated that lack of ability to produce IgG2b by the mutant B cells correlated with lack of germline transcription and resulted from a specific defect in class-switch recombination to S gamma 2b. Together, these studies demonstrate that the I region is an important regulatory element for control of class-switch recombination.

Animals↗
PubMed1993

Mutations of the intronic IgH enhancer and its flanking sequences differentially affect accessibility of the JH locus.

To investigate the role of intronic immunoglobulin heavy chain (IgH) enhancer (E mu) in generating accessibility of the JH locus for VDJ recombination, we generated ES cells in which E mu or its flanking sequences were mutated by replacement with or insertion of an expressed neor gene. Heterozygous mutant ES cells were used to generate chimeric mice from which pre-B cell lines were derived by transformation of bone marrow cells with Abelson murine leukemia virus (A-MuLV). Comparison of the rearrangement status of the normal and mutated alleles in individual pre-B cell lines allowed us to assay for cis-acting effects of the mutations. Replacement of a 700 bp region immediately downstream from the core E mu [which includes part of the 3' matrix associated region (MAR) and the I mu exon] had no obvious effect on rearrangement of the targeted allele, indicating that insertion of a transcribed neor gene into the JH-C mu intron does not affect JH accessibility. In contrast, replacement of an overlapping 1 kb DNA fragment that contains the E mu resulted in a dramatic cis-acting inhibition of rearrangement, demethylation and germline transcription of the associated JH locus. Surprisingly, insertion of the neor gene into the 5' MAR sequence approximately 100 bp upstream of the core E mu also dramatically decreased recombination of the linked JH locus; but, in many lines, did not prevent demethylation of this locus. We conclude that integrity of the E mu and upstream flanking sequences is required for efficient rearrangement of the JH locus and that demethylation of this locus, per se, does not necessarily make it a good substrate for VDJ recombination.

Animals↗
PubMed1993

Extensive deletion of immunoglobulin heavy chain constant region genes in the absence of recurrent infections: when is IgG subclass deficiency clinically relevant?

This report describes two children with undetectable serum levels of IgA1, IgG2, IgG4, and IgE due to a homozygous deletion encompassing the A1-E genes. The father is a heterozygous carrier of the same deletion and the mother a heterozygous compound carrying the deletion on one chromosome and duplication on the other. In both children, serum IgG, IgG1, and IgG3 were higher than in normal children and IgG antibody response to tetanus toxoid and polysaccharide antigens was normal with increased IgG1 and IgG3 response compared to controls. The latter can be interpreted as a compensatory mechanism for the subclass deficit and may explain the lack of significant infections in both children. The importance of distinguishing IgG subclass deficiency due to gene deletion from that due to immunoregulatory dysfunction is discussed.

Adolescent↗
PubMed1993

Familial clustering of IGHC deletions and duplications: functional and molecular analysis.

The human immunoglobulin heavy chain constant region locus (IGHC) comprises nine genes and two pseudogenes clustered in a 350 kilobase (kb) region on chromosome 14q32. Several IGHC haplotypes with single or multiple gene deletions and duplications have been characterized. The most likely mechanism accounting for these unusual haplotypes is the unequal crossing-over between homologous regions within the locus. Here we report the analysis of an unusual case of familial clustering of deletions/duplications. In the two branches of the BON family, three duplicated and two deleted haplotypes, all probably independent in origin, have been characterized. The structure of the haplotypes, one of which is described here for the first time, supports the hypothesis of homologous unequal crossing-over as the origin of recombinant haplotypes. The analysis of serological markers in a subject carrying one deleted and one duplicated haplotype allowed us the first direct inferences concerning the functions of the duplicated IGHC haplotypes.

Adolescent↗
PubMed1992

The genetics of IgG4 deficiency: role of the immunoglobulin heavy chain constant region and HLA loci.

IgG4 deficiency is very common (1/400 in the Italian population) and provides a good model for analyzing the genetic factors involved in Ig subclass deficiencies. We have previously reported an association between some immunoglobulin heavy chain constant region (IGHC) polymorphisms and the IgG4 deficiency. The associated polymorphisms spanned the region between the GP and the G4 genes. A larger sample composed of 50 healthy blood donors with IgG4 deficiency (less than 0.001 g/l IgG4), not carrying homozygous gene deletions, together with 82 first-degree relatives is now examined. The results confirmed the association of the deficiency with IGHC polymorphisms, and detected a new association with the HLA-D locus with a strong additive effect between the two systems. However, despite these associations and a highly significant risk for IgG4 deficiency within families, close linkage with either IGHC or HLA loci was not apparent by the affected sib pair method. These findings suggest that several concomitant, possibly cooperating, genetic factors may be involved in IgG4 deficiency.

Chromosome Mapping↗
PubMed1992

Immunoglobulin and HLA-DP genes contribute to the susceptibility to juvenile dermatitis herpetiformis.

HLA-DQ genes and gluten diet are the main factors involved in the pathogenesis of Dermatitis Herpetiformis (DH), as well as Coeliac Disease (CD). However other genetic factors are probably relevant, since about 10% of the patients with DH and CD lack the DQA1*0501/B1*0201 heterodimer while the majority of individuals presenting this genotype and also being exposed to gluten diets did not suffer from these diseases. To evaluate the role of other genes, 36 Northern Italian children with DH were analysed for DNA polymorphisms at HLA-DP and immunoglobulin (Ig) heavy chain loci. DPA1*0201 and DPB1*1301 frequencies were higher in patients than in controls (Pc = 0.0357 and Pc = 0.0273). With respect to immunoglobulin heavy chain restriction fragment length polymorphisms (RFLP), the 4.6 kb SacI RFLP at the switch alpha 2 gene was more frequent in patients (0.13) than in controls (0.019; Pc = 0.036). Moreover, rare alleles or duplications in the switch regions occurred more frequently in the patients than in the controls. These results support the hypothesis of a multifactorial inheritance of DH, the HLA and Ig constant heavy chain genes being some of the loci contributing to the susceptibility. In accordance with previous CD studies, these data also confirm that DP subregion is probably involved in the pathogenesis of DH.

Adolescent↗
PubMed1991

Pulsed-field electrophoresis screening for immunoglobulin heavy-chain constant-region (IGHC) multigene deletions and duplications.

Genome regions containing multiple copies of homologous genes, such as the immunoglobulin (Ig) heavy-chain constant-region (IGHC) locus, are often unstable and give rise to duplicated and deleted haplotypes. Analysis of such processes is fundamental to understanding the mechanisms of evolution of multigene families. In the IGHC region, a number of single and multiple gene deletions, derived from either unequal crossing-over or looping-out excision, have been described. To study these haplotypes at the population level, a simple and efficient method for preparing large numbers of DNA samples suitable for pulsed-field gel electrophoresis (PFGE) analysis was set up, and a sample of 110 blood donors was screened. Deletions were found to be frequent, as expected on the basis of previous serological surveys for homozygotes. Furthermore, a number of multigene duplications, never identified before, were detected. The total frequency of individuals bearing rearranged IGHC haplotypes was 10%. The genes involved in these deletions and duplications were assessed by densitometric analysis of standard Southern blots hybridized with several IGHC probes; two types of deletion and two types of duplication could thus be characterized. These data provide further evidence of the instability of the IGHC locus and demonstrate that unequal crossing-over is the most likely origin of rearranged IGHC haplotypes; they also suggest that such recombination events may be relatively frequent. Moreover, the simplicity and effectiveness of the large-scale PFGE screening approach will be of great help in the study of multigene families and of other loci involved in aberrant recombinations.

Chromosome Deletion↗
PubMed1990

Subclass distribution of antigen-specific IgA antibodies in normal donors and individuals with homozygous C alpha 1 or C alpha 2 gene deletions.

To analyze the subclass restriction of Ag-specific IgA, sera and saliva from healthy blood donors and from IgA class or subclass deficient individuals were studied. The latter included donors with or without C alpha 1 or C alpha 2 gene deletions. Monoclonal human IgA1 and a genetically engineered IgA2 antibody, normal human serum and colostrum IgA were used as standards to estimate serum and saliva levels of Ag-specific antibodies. In normal individuals, there was a strong IgA1 preference of naturally acquired antibodies in serum against both polysaccharide Ag (PPS 6A, PPS 23, pneumococcal C-polysaccharide, and LPS from Escherichia coli) and protein Ag (Staphylococcus aureus alpha-toxin and HSV). Specific IgA2 in serum against the tested Ag were frequently not measurable. In contrast, most of the individuals with homozygous C alpha 1 gene deletions displayed substantial amounts of specific IgA2 against protein as well as polysaccharide Ag. The median levels of specific IgA in serum against protein Ag were approximately one-third as compared to normal individuals and one-fifth, or less, against polysaccharide Ag. Normal serum levels of IgA against the tested Ag, restricted to the IgA1 subclass, were noted in two individuals with IgA2 deficiency, one of whom carried a homozygous C alpha 2 gene deletion. Median values of specific IgA, against the tested Ag S. aureus alpha-toxin, HSV, and pneumococcal C-poly-saccharide, from normal healthy donors were approximately four to eight times higher in serum as compared to saliva. Individuals with homozygous C alpha 1 gene deletions displayed increased levels of the various specific IgA2 antibodies in saliva. In conclusion, the individuals with homozygous C alpha 1 gene deletions displayed decreased median levels of specific IgA antibodies in serum despite normal levels of total IgA. Normal levels of both specific IgA and total IgA in saliva were found.

Chromosome Deletion↗
PubMed1990

Multiple levels of analysis of an IGHG4 gene deletion.

Human immunoglobulin heavy chain constant region (IGHC) genes constitute a typical multigene family, usually comprising eleven genes on the telomere of chromosome 14 (14q32). In this region, deleted and duplicated haplotypes have been reported to exist with considerable frequency. Their origin is the result of either unequal crossing-over or looping out excision. In this paper, we report the characterization of a new type of deletion, involving the IGHG4 gene, in a subject who also carries a larger deletion of a previously described type on the second chromosome. Employment of several methods (polymerase chain reaction, standard Southern blot, pulsed field gel electrophoresis, serological techniques) to analyze these deleted haplotypes has resulted in a level of accuracy in their characterization that has not been achieved in previous cases. The site of recombination responsible for the IGHG4 deletion was restricted to a 2.5-kb region 3' of the G4 gene; this rules out any possible involvement of the S regions in the recombination process. The usefulness of the various techniques in the characterization of the deletions is also discussed, together with possible future applications in the field.

Base Sequence↗
PubMed1989

Genetic analysis of new restriction fragment length polymorphisms (RFLP) in the human IgH constant gene locus.

The human immunoglobulin heavy chain constant gene locus (IGHC) is polymorphic at both the protein (Gm and A2m allotypes) and the DNA level [RFLP for the gamma genes (IGHG), the switch mu region (IGHSM) and the switch alpha regions (IGHSA)]. The polymorphisms have been a valuable tool for assessment of the IGHC locus organization and a variety of population genetics and immunological investigations. In this study three new probes, identifying regions related to the IGHG (IGHPG and IGHSG) or IGHA (IGHAT) genes, have been employed to describe 11 different loci, 6 of which were polymorphic. Most of the polymorphisms are probably due to short insertions/deletions, particularly the SG regions, due to their repetitive structure. Ten loci were assigned to the IGHC region on the basis of known restriction maps, deletion mapping and association with mapped RFLP; the 11th, despite a striking sequence similarity with the IGHPG regions, could not be assigned to any known IGHC subregion. Analysis of these and previously known IGHG RFLP in a sample of 65 unrelated subjects plus 15 families allowed us to draw a genetic map, with particularly high resolution in the GP-G2-G4 genes region, revealing a marked discontinuity in the linkage disequilibrium values between pairs of adjacent loci.

Biological Evolution↗
PubMed1989

Human IGHC locus restriction fragment length polymorphisms in IgG4 deficiency: evidence for a structural IGHC defect.

In man, IgG4 is the least abundant of the four IgG subclasses, and its serum levels vary considerably from one subject to another. Its deficiency has been thought to lead to recurrent infections; nevertheless, it is also commonly found in healthy individuals (1/400 in the Italian population). In 39 subjects with IgG4 serum levels less than 1 microgram/ml, we used 4 different probes (described in the accompanying study, Bottaro et al., Eur. J. Immunol. 1989. 19: 2151) to examine 13 loci within the IGHC region and analyzed the RFLP for 7 of them. No aberrant restriction patterns were identified in any of the subjects, showing the absence of major IGHC structural alterations. The allele frequency of some loci, however, was significantly different from that of a control group of 95 random subjects. This variation was shown to depend on a selective increase in the number of homozygotes for the associated alleles, that reached significant levels for the IGHGP, G2, PG2, PG4 and SG4 loci, but not for SG1 and A2T. The highest value was reached for alleles in the PG4 region, just 5' of SG4. These data indicate that a minor structural IGHC defect is probably the cause of a significant fraction of IgG4 deficiencies. Moreover, the different association levels of the PG4 and SG4 regions suggest that this defect is likely to lie in an upstream regulatory region rather than in the structural G4 gene.

Gene Frequency↗
PubMed1989

New types of multiple and single gene deletions in the human IgCH locus.

The locus for human immunoglobulin heavy chain constant region genes (IgCH) is characterized by a significant frequency of deleted or duplicated haplotypes, due to unequal crossing-over events. Four types of deletions and one duplication have been reported so far. We describe here a molecular study of four cases of IgCH deletions. Two of the three types of deletions are reported here for the first time. Analysis of genetic markers associated with the deleted haplotypes pointed to the independent origin of similar deletions and the involvement of intergenic sequences in the mispairing-recombination process. The reduced or absent transcription of the C gamma 4 gene in two C gamma 2-deleted haplotypes offers an insight into the requirements for the isotype switch mechanism.

Blotting, Southern↗
PubMed1989

Pulsed-field gel analysis of human immunoglobulin heavy-chain constant region gene deletions reveals the extent of unmapped regions within the locus.

The human immunoglobulin heavy-chain constant region gene locus is organized in three main gene groups, the physical distances of which are unknown. Different types of gene deletions, originated by unequal crossingover, have been found to encompass one or more genes in the locus. We have analyzed some of these deletions by means of pulsed-field gel electrophoresis, which allows resolution of large DNA fragments. By identifying a fragment containing two of the main gene groups and by observing the size reduction of this fragment in subjects with deletions, we were able to estimate the distance between the two groups and better locate the pseudogene in-between.

Chromosome Deletion↗