High resolution definition of HLA-DRB haplotypes by a simplified microsatellite typing technique.

In humans, the region configurations DR1, DR8, DR51, DR52 and DR53 are known to display copy number as well as allelic variation, rendering high resolution typing of HLA-DRB haplotypes cumbersome. Advantage was taken of microsatellite D6S2878, present in all DRB genes/pseudogenes with an intact exon 2-intron 2 segment. This DRB-STR is highly polymorphic in composition and length. Recently, it was proven that all exon 2 sequences could be linked to a certain DRB-STR that segregates with the respective DRB allele. Because haplotypes show differential copy numbers and compositions of exon 2-positive DRB genes/pseudogenes, unique DRB-STR patterns could be described that appear to be specific for a particular DRB haplotype. The aim of this workshop project was to approve and to qualify this simple typing protocol in a larger panel covering different European populations. All participants succeeded in correctly defining the DRB-STR amplicons varying from 135 to 222 base pair (bp) lengths. The panel of 101 samples covered 50 DRB alleles distributed over 37 different haplotypes as defined by exon 2 sequence-based typing. These haplotypes could be refined into 105 haplotypes by DRB-STR typing. Thus, discrimination of exon 2-identical DRB alleles was feasible, as well as the exact description of three different crossing-over events that resulted in the generation of hybrid DR region configurations. This typing procedure appears to be a quick and highly robust technique that can easily be performed by different laboratories, even without experience in microsatellite typing; thus, it is suitable for a variety of researchers in diverse research areas.

Monoclonal antibodies against different domains of human IgA: specificities determined by immunoblotting and haemagglutination-inhibition.

The specificity of 14 monoclonal antibodies has been determined by immunoblotting (IB) and haemagglutination-inhibition (HAI) analysis using IgA1 and IgA2 myeloma proteins and eight different IgA1 fragments. Two antibodies probably recognized epitopes on the CH1 domain of IgA. They reacted with all Fab-containing fragments irrespective of whether these originated from the same or different IgA proteins. Seven antibodies were directed against epitopes on the CH2 domain. These antibodies were reactive with F(abc)2 fragments. They failed to react with Fab, Fab' and F(ab')2 fragments. Two out of these seven antibodies did not react with two-chain IgA half-molecules and Fabc fragments containing a single heavy and a single light chain. This suggests that these two antibodies recognized an epitope whose structure is dependent on disulfide linked heavy chains. Five other antibodies showed specificity for the CH3 domain. They were reactive with all CH3-containing molecules, irrespective of whether they comprised one or two alpha chains. Our study demonstrates that IB is an appropriate technique to determine domain specificity of monoclonal anti-immunoglobulin reagents. Although the IB tests were performed on denatured proteins the results agreed surprisingly well with those of the HAI analyses. Moreover, the IB technique could be used on fragments which could not be purified well enough for HAI analyses.

Uniparental maternal disomy 6 in a renal transplant patient.

HLA analysis of the family of a renal transplant patient revealed an extremely rare condition. On repeated typings the only demonstrable HLA antigens shown in the propositus were from the maternal haplotype, HLA-A11,-B46,-CW1,-DR14,-DQ1. No paternal antigens could be demonstrated either by serologic or by DNA-typing methods. A paternity investigation was carried out to exclude the possibility of the legal father not being the biological father. The results of this investigation showed a paternity index I = > 20000 and a fatherhood probability W = > 99.995%. Karyotyping of the patient showed two normal chromosomes 6 and no other chromosomal abnormalities. Maternal isodisomy was demonstrated from the analysis of polymorphic DNA markers, involving the short as well as the long arm of chromosome 6. These data are consistent with this patient having the first uniparental maternal disomy 6 reported (inheritance of two identical chromosome 6 haplotypes from the mother and none from the father).

Genes on chromosome 14q and their role in the pathogenesis of HLA-B27 associated diseases.

Genetic factors other than HLA-B27 may play a role in the pathogenesis of ankylosing spondylitis (AS), acute anterior uveitis (AAU) and Reiter's syndrome (RS). Studies by Brewerton et al. and Kijlstra et al. showed associations between the MZ phenotype of alpha 1-antitrypsin and the Gm phenotype zafngb of IgG in patients with AAU, who developed AS. The loci for alpha 1-antitrypsin (PI) and Gm allotypes (IGH) are situated on the tip of the long arm of chromosome 14. In the present study we tried to clarify and extend the above studies. In 41 B27+ AAU patients with AS the alpha 1-antitrypsin and Gm phenotype and allotype frequencies were not statistically different from those in B27+ AS patients developing AAU and in B27+ AAU patients without AS, in B27+ AS patients without AAU, B27+ patients with Reiter's syndrome, B27+ patients with low back pain, B27- AAU patients and normal controls. It is therefore unlikely that genes on the tip of chromosome 14 play a role in the pathogenesis of B27 associated diseases. A hypothesis was formed suggesting that a bacterial-derived modifying factor may replace the position of beta 2 microglobulin in the HLA-B27 molecule resulting in an impaired cytotoxic T cell reactivity.

Size calculation of restriction enzyme HaeIII-generated fragments detected by probe YNH24 by comparison of data from two laboratories: the generation of fragment-size frequencies.

Restriction fragment-length polymorphism of locus D2S44 detected by the highly polymorphic probe YNH24 and restriction endonuclease HaeIII can be used to improve parentage testing when representative fragment-size frequencies can be obtained. By joining the results of different laboratories, it is possible to set up a meaningful databank. Therefore, the same randomly chosen samples were tested for the HaeIII RFLP detected by probe YNH24 in Düsseldorf (DUS) and Amsterdam (AMS). The results of the different fragment-size calculations obtained by using internal markers and a computerized system (DUS-cad and AMS-cad), and by using external markers and manual calculations (DUS-man), were analyzed. Comparing these results, no statistically significant differences were seen. The results obtained with probe YNH24 and enzyme HaeIII in Düsseldorf and Amsterdam can be used to attain a sufficient number of samples to generate relevant fragment-size frequencies.

Attempt to calculate the allele frequency distribution of a TaqI RFLP detected by the highly polymorphic probe YNH24.

To improve the analysis of parentage testing with the additional technique of DNA polymorphisms, the usefulness of probe YNH24 was studied. The allele frequency distribution of restriction fragments detected by probe YNH24 on TaqI-digested genomic DNA from 100 unrelated individuals was determined. For this purpose, the size of the fragments was calculated by making use of HindIII-digested lambda DNA as an internal marker and of a digitizing tablet coupled to a computer. The size of the fragments ranged from 2.53 kb to 5.89 kb. The mean standard deviation was 0.05 kb. The differences between the fragment sizes appeared to be smaller than the standard deviation. For this reason, it was not possible to calculate the allele frequency distribution of this highly polymorphic genetic system.

Exclusion of paternity by DNA analysis in addition to blood group testing in a family case.

Three men, all related to the mother were involved in a paternity case. Only one of them, an uncle of the mother, could be excluded by conventional blood testing of 22 polymorphic systems including HLA and Gm. Because it was not possible to exclude the other men (the brother and the father of the mother) by these systems restriction fragment length polymorphisms as detected by the probes YNH24 and hMF1 were analyzed. The results with probe hMF1 did not reveal additional information. Both Taq I and Pst I digests, probed with YNH24 excluded the father of the mother from paternity of the child under investigation.

Association between leprosy and immunoglobulin allotypes: Gm-A2m and Km frequencies in Vietnamese.

The relationship between immunoglobulin allotypes and leprosy was studied in 91 unrelated patients and 100 healthy controls from Vietnam. Twenty Vietnamese patients with tuberculosis were also typed for the Gm, A2m and Km allotypes. The results were compared with those from the healthy controls. No significant association was found for the allotypes G1m(z,a,x,f) G2m(n), G3m(g,b), A2m(1,2) and Km(1,3) between the two groups of patients and the controls. Heterogeneity in the distribution of G2m(n), G3m(b), A2m(2) and Km(3) was found when 60 polar-lepromatous (LL) patients and 27 borderline-tuberculoid (BT) patients were separated out of the 91 leprosy patients. In the LL patients there appeared to be a significantly higher frequency of G2m(n), G3m(b) and A2m(2) in comparison with the BT patients (P less than 0.05). A significantly lower frequency of Km(3) was found in the LL patients in comparison with the healthy control group (P less than 0.05). The frequencies of the Gm-A2m haplotypes and of the occurrence of the Km(1) and Km(3) in the Vietnamese population were calculated on the basis of the results in the 100 samples of healthy controls. The main haplotype is Gmaf;n;b (frequency 0.676), occurring with A2m1 (0.200), as well as with A2m2 (0.476).

Gm haplotype sharing by siblings with rheumatoid arthritis.

Gm haplotype sharing by 9 sibling pairs with rheumatoid arthritis was random. This suggests that the model of a single, uncommon, susceptibility gene linked to Gm which predisposes to RA is unlikely.

Distribution of antigenic determinants on human IgA1.

The distribution of antigenic determinants on human IgA was studied with fragments and mutants of IgA1. F(abc)2 and Fabc, lacking the CH3 domain, F(ab')2 and Fab', lacking the CH2 and CH3 domain, Fab that further lacks most of the hinge region, and Fc fragments were included in our investigations. Antibodies specific for the CH3 domain of IgA1 were found in antisera raised against an alpha 1-HCD protein and in anti-Fc alpha antisera. The antisera detected different antigenic determinants on CH3 as was shown by inhibition with sera from nonhuman primates. An anti-Fc5 mu antiserum detected a determinant on Fc common to IgA and IgM. The serum of an IgA-deficient individual reacted with a determinant on CH2 of four-chain molecules only. A subclass-specific anti-Fabc antiserum detected a determinant which needed interaction of CH2 and CH1 or the hinge region. Anti-Fab antisera reacted with class or subclass specific determinants on CH1. The isoallotype nA2m(2) is probably located on CH1. Its expression requires two alpha-chains stabilized in a conformation attributed to by CH2.

Immunoglobulin allotypes in a Chinese population: comparison of haplotype frequencies with other Asian groups.

Two groups of Chinese individuals of the city of Wuhan in the People's Republic of China, consisting of 98 blood bank donors and 278 workers in a steel factory, respectively, have been typed for G1m, G2m, G3m, A2m and Km allotypes. The Gm-A2m haplotype frequencies as well as the Km gene frequencies appeared to be quite similar in these two groups of people, the main haplotypes being Gmaf;n;b A2m2 and Gmza;..;g A2m1. After joining the data of the two groups, the frequencies of the main Gm haplotypes and the A2m and Km gene frequencies have been compared with those of six other Asian populations. In Asia, going from the south to the north, an increasing frequency of Gmza;..;g and a decreasing frequency of Gmaf;n;b were observed. The frequencies found in the investigated Chinese population lie between the frequencies found in the Chinese from Taiwan and the Japanese.

Monoclonal antibodies against IgG allotypes G1m(z), G1m(a), G1m(f), G3m(b1/u) and G3m(g1): their usefulness in HAI and capture ELISA.

Monoclonal antibodies (McAbs) were produced against the IgG allotypes G1m(z), G1m(a), G1m(f), G3m(b1/u) and G3m(g1). Four out of the six McAbs described in this paper showed in the haemagglutination assay cross-reactivity with some or all IgG-coated cell samples. In the haemagglutination inhibition assay, all six McAbs are useful as typing reagent for the above allotypes. In this assay, two of the McAbs show two different specificities, which depend on the Ig-coated cell sample used. Five McAbs are useful for allotyping in a capture ELISA. The results with four of these are promising for the development of a quantitative determination of Gm allotypes.

Human IgG allotypes co-occurring in more than one IgG subclass.

Inheritance of an excess of immunoglobulin allotypes in one haplotype was encountered which could not be explained by the assumption of a duplicated locus. The surplus of allotypes was related to markers on the CH3 domain of gamma 3 chains. Two such cases were investigated extensively. The IgG3 molecules were isolated by gel filtration and by absorption on protein A. Only the usual combination of allotypes appeared to be present on the IgG3 molecules. The supernumerary markers were found in one case on IgG2 molecules and in the other case on IgG1 molecules. This followed from investigations of eluates after separation of the subclasses by immune absorptions. A hypothesis was proposed to explain these events by mutation of a particular position of an otherwise homologous stretch of gamma-subclass DNA.

Altered antibody isotype in cystic fibrosis: impaired natural antibody response to polysaccharide antigens.

Patients with cystic fibrosis (CF) have impaired natural (preinfection) IgG2 antibody responses to Pseudomonas aeruginosa lipopolysaccharide. To investigate the basis for this defect, we measured natural IgG and IgG1-4 antibody levels to Haemophilus influenzae type b polyribophosphate (PRP) and tetanus toxoid by enzyme-linked immunosorbent assay in 24 adult CF patients and 20 normal controls. Immunoglobulin heavy- and light-chain allotypes were determined on 146 Caucasian CF patients and 96 controls. The tetanus toxoid-specific IgG response was predominantly IgG1. CF and control subjects had similar IgG and IgG1 antibody levels. The PRP-specific IgG response was predominantly IgG2. In contrast to tetanus toxoid results, CF patients had lower geometric mean level of PRP-specific IgG compared to normal controls (p = 0.0036). ELISA results were confirmed by liquid-phase 3H-PRP-binding assay: CF patients had a geometric mean serum antibody level of 395 versus 922 ng/ml in controls (p = 0.0044). PRP-specific IgG2 levels were also depressed in CF patients (p = 0.03). CF patients had a lower prevalence of the A2m(2) allotype than the local racially matched control sample (p less than 0.025). Other allotype prevalences including G2m(n) and Km(1) were similar. Impaired IgG2 antibody responses to microbial polysaccharide surface antigens in CF patients might predispose them to persistent endobronchial infection and lead to production of nonopsonizing isotype responses. The potential role of A2m(2), coded for in the H chain locus on chromosome 14, is unknown, but could be related to mucosal IgA2 antibody responses.