Cleide Boccazzi

Molecular characterization of G2m(n+) and G2m(n−) allotypes

Immunoglobulin (Ig) allotype typing is usually performed with serological methods based on hemagglutination inhibition. The recent development of molecular techniques has allowed the molecular typing of several Ig markers. The hinge, CH2, and CH3 domains of the G2 gene from six unrelated individuals (three G2m(n+) and three G2m(n−)) were amplified and cloned to establish the molecular basis of the G2mn+ and G2mn−. Comparison of the allele sequences revealed three changes: two (codons 308 and 437) are silent exonic substitutions, one is a G to A transition corresponding to an amino acid difference in position 282: Val (GTG) in G2mn−, Met (ATG) in G2mn+. These substitutions were identified via two approaches: 282 polymorphism, after digestion of a specific polymerase chain reaction product with Nla III followed by acrylamide electrophoresis; 308 and 437, by a dot-blot technique using allele-specific oligonucleotides. These molecular typing results correspond exactly to those obtained serologically; moreover, the three substitutions defining the G2mn+ and G2mn− alleles are always associated in a strict linkage disequilibrium.

Variability of the immunoglobulin heavy chain constant region locus: a population study

The Immunoglobulin Heavy chain Constant region (IGHC) locus is a multigene family composed of highly homologous segments often involved in unequal crossings over that lead to deleted and duplicated haplotypes. The frequencies of these haplotypes in 558 individuals from Lombardy, Veneto, Puglia and Sardinia were determined by Pulsed Field Gel Electrophoresis (PFGE), followed by Southern blotting with four IGHC probes, and compared with those observed in 110 subjects from Piedmont. Twenty deletions and 60 duplications were characterized, all in heterozygous individuals except for 2 homozygous deletions. The differences in frequency between the five populations were not significant. The deletions/duplications involved one or more genes: GP-A2, A1-E and G4 duplications, and A1-E and GP-A2 deletions were the most common. Four new duplications are described: three, involving the genes from GP to A2, from G2 to G4, and G4, are counterparts of known deletions. The fourth duplication spans from GP to G2. A G1 deleted heterozygous individual never previously described in Italy is reported. All the rearranged haplotypes seem to be the result of unequal crossing over. The difference between the number of duplications and deletions was significant in Sardinia, Lombardy, Puglia and in the total of 668 subjects (P < 0.001). This may be due to selection or genetic drift.

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.

The G4 gene is duplicated in 44% of human immunoglobulin heavy chain constant region haplotypes

Abstract The structure of the human immunoglobulin heavy chain constant region (IGHC), on chromosome 14q32, comprises nine CH genes and two pseudogenes, all originating from multiple duplication events. Continuing evolution of the region is demonstrated by the finding of various types of duplicated and deleted haplotypes, which together add up to 6%. Here we provide molecular and genetic evidence that the G4 gene is duplicated in 44% of IGHC haplotypes in the Italian population. The duplication spans about 20 kb of genomic DNA and probably originated through unequal crossing over. Refined characterisation of the genomic region downstream from the G4 gene improves our knowledge of the evolutionary history of CH genes.

A simplified procedure for haptoglobin subtyping.

SummaryA simplified procedure is described for haptoglobin subtyping employing minute amount of serum. It consists of a partial purification of haptoglobin by cellulose-acetate electrophoresis, followed by acidic urea polyacrylamide gel electrophoresis of reduced and alkylated samples.

Serum Immunoglobulin Levels in Heterozygous Subjects With Immunoglobulin Heavy-chain Constant-region Gene Deletions

SummaryThe immunoglobulin heavy chain constant region locus is a multigene family composed of nine genes and two pseudogenes, whose high homology is often responsible for meiotic mispairings leading to deleted and duplicated haplotypes. These rearrangements have a population frequency of about 1.5% and 4.5% respectively, with a significant difference between deletions and duplications (P<0.001). Both positive selection of duplications or negative selection against deletions can account for this imbalance. Serum levels of IgG and IgA subclasses, of IgE, of isohemagglutinins and of IgG antibodies to tetanus toxoid and pneumococcal antigens were evaluated in 11 heterozygous carriers of constant region deletions. There was no gross abnormality in serum IgG and IgA subclass levels, with the possible exception of G1-deleted individuals; furthermore, isohemagglutinins and anti-tetanus toxoid and pneumococcal IgG antibodies are in the normal range, suggesting that the humoral immune response is normal in these carriers. The influence of single and multiple immunoglobulin heavy chain constant region gene deletions on the humoral response is discussed.

Immunochemical and structural characterization of an IgG1 heavy chain disease.

SummaryThe immunochemical and structural analysis of an IgG1 heavy chain disease is reported. The abnormal component is characterized by extensive deletion including the CH1 domain and most or all of the VH region.

A rapid alpha-fetoprotein radioimmunoassay.

SummaryA simple, reliable and inexpensiveα-fetoprotein radioimmunoassay is described, in which polyethylene-glycol (PEG) is used as a precipitating agent of antibody-bound AFP. The concentrations of AFP found in amniotic fluid, newborn and maternal sera are in agreement with those reported by others. This assay fulfills the requirements of a screening test for fetal abnormalities and other clinical applications.