A study of Italian pediatric celiac disease patients confirms that the primary HLA association is to the DQ(alpha 1*0501, beta 1*0201) heterodimer.

Celiac disease (CD) has been recently reported to be primarily associated with the DQ(alpha 1*0501, beta 1*0201) heterodimer encoded in cis on DR3 haplotype and in trans in DR5,7 heterozygous individuals. The high incidence of DR5,7 heterozygotes, reflecting the high frequency of the DR5 allele in Italy, makes the analysis of the Italian CD patients critical. Polymerase chain reaction-amplified DNA from 50 CD patients and 50 controls, serologically typed for DR and DQw antigens, was hybridized with five DQA1-specific oligonucleotide probes detecting DQA1*0101 + 0102 + 0103, DQA1*0201, DQA1*0301 + 0302, DQA1*0401 + 0501 + 0601, and DQA1*0501 and a DQB1-sequence-specific oligonucleotide probe recognizing DQB1*0201 allele. As expected by the DR-DQ disequilibria, DQA1*0201 [62% in patients versus 26% in controls, relative risk (RR) = 5] and DQA1*0501 (96% versus 56%, RR = 19) show positive association with the disease. Of CD patients, 92% (50% DR3 and 42% DR5,7) compared to 18% of the controls carry both DQA1*0501 and DQB1*0201 alleles, so that the combination confers an RR of 52, higher than both the risks of the single alleles (DQA1*0501 RR = 19, DQB1*0201 RR = 30), confirming the primary role of the dimer in determining genetic predisposition to CD both in DR3 and in DR5,7 subjects.

Different binding of NF-Y transcriptional factor to DQA1 promoter variants.

Polymorphism in the HLA-DQA1 promoter (QAP) sequences could influence the gene expression through a differential binding of transcriptional factors. Considering the main role played by the Y-box in the transcription, we focused on the QAP4 variants differing for a G vs A transition from the QAP Y-box consensus sequence. Electrophoretic Mobility Shift Assay using the two Y-box sequences was performed to determine whether this mutation could be reflected in an allele-specific binding of transcriptional factors. Indeed, the NF-Y specific band, recognised by supershift experiments, was clearly observed using the Y-box consensus probe but it was barely detectable with the QAP4 one. On the contrary, two other complexes were found to more strongly interact with QAP4 Y-box in comparison to the consensus sequence. The analysis of a selected panel of HLA homozygous lymphoblastoid cell lines by competitive RT-PCR and by Northern blotting revealed that the DQA1 *0401, *0501,*0601 alleles regulated by the QAP4 promoters were less expressed at the mRNA level than the DQA1* 0201 allele regulated by the QAP2.1 variant. In conclusion, these results show an evident reduction of NF-Y binding to the mutated QAP4 Y-box and a decreased mRNA accumulation of the DQA1 alleles regulated by these variants.

Absence of in vivo DNA-protein interactions in the DQA2 and DQB2 promoter regions.

The DQ subregion of the human major histocompatibility complex (HLA) contains two pairs of loci: the DQA1/B1 genes (hereafter called DQ1), coding for the DQ molecules, and the DQA2/B2 pseudogenes (hereafter called DQ2). These pseudogenes are highly homologous to the functional DQ1 genes and they have no apparent abnormal features in their sequences that could prevent their activity. Only recently a low expression of the DQA2 gene has been observed whereas the DQB2 transcript was never found. The comparison between the DQ1 and DQ2 regulatory sequences revealed several differences in their W, X, and Y cis-acting elements. To examine the DNA/protein interactions in the DQ promoter regions, we performed in vivo footprinting experiments. Whereas the functional DQ1 loci showed a series of DNA-protein contact points in the X and Y boxes, the promoters of the DQ2 pseudogenes displayed an unoccupied phenotype. These findings suggest that the very low level of DQA2 expression and the apparent lack of DQB2 activity are caused by the reduced binding affinity of specific transcription factors.

Different dose effect of HLA-DQ alpha beta heterodimers in insulin-dependent diabetes mellitus and celiac disease susceptibility.

To compare the quantitative effect of the DQ alpha beta heterodimers DQ alpha 52 Arg+, beta 57 Asp- and DQ alpha 1*0501, beta 1*0201 on susceptibility to IDDM and CD, we characterized, at the genomic level, the DQ alpha 52 and DQ beta 57 residues of 50 IDDM Italian patients observed in Rome. The results were compared with those of a previous study concerning the oligotyping of DQ dimers in a group of CD children belonging to the same population. Our data confirm that both diseases are primarily associated with HLA-DQ alpha beta heterodimers, but the distributions of the respective susceptible DQA1 and DQB1 alleles in the two diseases were different. In fact, the highest risk of IDDM is for subjects alpha SS, beta SS that could express, by either cis- or trans-association, four susceptible heterodimers and decreases in proportion to the number of these; in regard to CD, the highest risk was found for individuals who carried only one predisposing heterodimer.

Distinctive features of the alpha 1-domain alpha helix of HLA-C heavy chains free of beta 2-microglobulin.

Only a few monoclonal antibodies are available with a restricted specificity to HLA-C products. In the present report, we demonstrate that antibody L31, previously shown to react with beta 2m-less (free) class I MHC heavy chains, binds to an epitope (residues 66-68 of the alpha 1 domain alpha helix) present on all the HLA-C alleles corresponding to the accepted (CW1 through CW8) serologic specificities, and on a few HLA-B heavy chains sharing with HLA-C an aromatic residue at position 67. Extensive IEF blot testing of HLA homozygous, EBV-transformed B-lymphoid cells indicates that HLA-C molecules are present at significantly lower levels than HLA-B polypeptides not only at cell surface, as previously demonstrated, but also in total cellular extracts. Testing of metabolically labeled HLA-CW1, -CW5, and -CW6 transfectants and HLA homozygous lymphoid cells, particularly HLA-CW1-expressing cells, demonstrates that the L31 epitope is present on a subpopulation of naturally occurring HLA-C molecules distinct from that identified by antibody W6/32 to beta 2m-associated heavy chains. Pulse-chase experiments demonstrate that this epitope is transiently made available to antibody binding at early biosynthetic stages, but becomes hidden upon assembly with beta 2m. Thus, free HLA-C and other Y/F67+ heavy chains are characterized by distinctive antibody binding features in a region (residues 66-68) included in a previously identified HLA-C restricted motif, which has been suggested to be the primary cause of distinctive features of the antigen-binding groove, low affinity for endogenous peptide antigens and beta 2m, and preferential uptake of exogenous peptides, possibly of viral origin. We also show that HLA-CW1 heavy chains, both free and beta 2m associated, acquire sialilation. Free HLA-CW1 heavy chains are expressed at the cell surface even when unsialilated, albeit at low levels.

A mouse monoclonal antibody against a polymorphic determinant in a defined subset of DR molecules.

The hybridoma technique was used to produce a mouse monoclonal antibody, designated as XI 21.4, which belongs to the IgG2a class. It is active in complement-dependent cytotoxicity and detects a B-cell antigenic determinant associated with DR1, DR4, DRw10, and, possibly, DRw9. Microfingerprinting of the immunoprecipitate from a homozygous DR4 cell line shows a typical alpha DR pattern and a beta pattern coinciding with that of DR4 molecules.

A study of HLA class II antigens in an Italian paediatric population with coeliac disease.

One hundred and twenty-one Italian children with coeliac disease (CD) have been compared with a control population from the same geographical area for the distribution of HLA-DR and DQ antigens. The pattern of an increase in DR3, DR7, and of heterozygotes DR5/7 was associated with an excess of heterozygotes DQw2/DQw3 in the CD population. These findings suggest that epitopes determined by specific combinations of DQ alpha and beta chains (combinatorial determinants) predispose to the disease.

HLA antigens and adult rheumatoid arthritis: a study with a monoclonal antibody.

Adult rheumatoid arthritis (RA) is a very heterogeneous disease that is associated with HLA-antigens, although no absolute association has been found with any particular HLA type. Forty-one seropositive RA patients have been studied with a local monoclonal antibody named X1 21.4 (9w940), strongly associated with HLA-DRI, DR4, Drw10 antigens, to verify a possible correlation with the disease. The results obtained have also been compared with the data reported on MC1, a serologically defined determinant correlated with RA. X1 21.4 monoclonal antibody appears to be associated with the disease and it could identify one epitope involved in the susceptibility to RA.

A nonneutralizing human IgM monoclonal antibody inhibiting hemagglutination of H3N2 influenza A strains.

A mouse-human hybridoma has been produced by fusing human splenocytes from a Cooley's anemia patient with the murine myeloma P3-NS1/1-Ag 4-1. The hybridoma is stable after 18 months and secretes human IgM. The antibody reacts with some H3N2 influenza A strains and detects an epitope that is part of the hemagglutinin antigen, but does not affect virus infectivity.

Polymorphism in the upstream regulatory region of DQA1 gene in the Italian population.

Polymorphism in the 5'-upstream regulatory region of the DQA1 gene has been recently described. Using PCR-SSO method and SSCP analysis we have investigated this polymorphism in a group of 111 Italian blood donors which had been oligotyped for DRB1, DQA1 and DQB1 genes. Eight allelic variants were detected. Looking at the relationships among QAP sequences and DQA1 and DRB1 genes, three alternative situations were found: 1. a one-to-one relation between QAP and DQA1 alleles, independently of the other class II genes; 2. the same QAP allele in association with different DQA1-DRB1 haplotypes; 3. the same DQA1 allele with different QAP sequences according to the DRB1 specificity. No unexpected associations with DQB1 gene were found. These results must be interpreted considering that DQA1 and DRB1 genes are transcribed in opposite directions so that the promoter region of DQA1 gene lies between DQA1 and DRB1, close to the former but several hundreds kb away from the latter.

A mouse monoclonal antibody detecting the allospecificity HLA-A3.

Balb/c mice were immunized with a human B-lymphoblastoid cell line typed HLA-A3, B7. The splenocytes of the immunized mice were fused with a murine myeloma. Supernatants of the cultures were screened against the immunizing cell line in fluorochromasia. Positive cultures were expanded and cloned. One of the clones, X 15.4, was expanded and brought to ascites in Balb/c mice. Monoclonality of the antibody X 15.4, which belongs to the class IgM and immunoprecipitates a molecule of 44000 daltons, was demonstrated by isoelectric focusing. By complement dependent cytotoxicity the ascites only reacted with the lymphocytes of all HLA-A3 individuals from a panel of 146 donors, showing no crossreactions. X 15.4 appears to be one of the very rare xenomonoclonal antibodies suitable for HLA typing.

Oligotyping of Italian celiac patients with the 11th International Histocompatibility Workshop reagents.

Oligotyping for HLA-DRB1, DQA1 and DQB1 specificities has been performed on PCR-amplified DNA from 55 Italian celiac children, living in Rome, and 50 blood donors. 52.6% of CD patients were DR3;DQA1*0501;DQB1*0201-positive versus 14% of controls (RR = 6.85) and 34.5% were DR5,7;DQA1*DQB1*0201-positive versus 2% of controls (RR = 25.86). 7 patients (12.7%) were negative for the DQA1*0501/B1*0201 dimer: 3 of them were DR4 (5.4%) and the others typed as DR1,5; 1,7; 5,7 and w6,7. No patient was negative for both DQA1*0501 and DQB1*0201 alleles.

Single-strand conformation polymorphism (SSCP) analysis of HLA-DRB1*1101-06.

Single-strand conformation polymorphism (SSCP) has been developed as a method for detecting the presence of mutations in a segment of DNA. We applied it to the subtyping of the DR11 group of alleles. The SSCP patterns of DRB1-DR52 group-specific products were defined in cell lines representing the DRB1*1101-06 alleles, using non-denaturing acrylamide gel electrophoresis and silver staining. Only one set of gel electrophoresis conditions was able to discriminate the DR11 alleles tested. The protocol was validated in an analysis of 105 DR11-positive individuals previously typed by oligonucleotides probing. The study demonstrates the suitability of the SSCP technique to define the DRB1*1101-06 alleles, the technique being particularly valuable in confirming and extending the oligotyping of DRB1-DR52 heterozygous samples.

HLA and chronic active hepatitis.

42 patients (33 males and 9 females) with chromic active hepatitis (CAH) mostly HBsAg+, were typed for 24 alleles of the A and B loci. Diagnosis was performed according to the criteria outlined by the European Association for the Study of the Liver. The increased frequency of HLA-A3 (47.6% instead of 19.1% in 266 healthy controls) is significant after correction. The relative risk is 3.83. The phenotypic association A3/Bw35 is also increased (28.5% instead of 6.0 in the control group). The risk of the A3/Bw35 association is 6.25. The risk of A3 calculated in patients lacking Bw35 is 1.6. A family study in 5 patients over 5 showed an haplotype A3/Bw35.

Oligotyping of celiac multiplex families with the 11th International Histocompatibility Workshop reagents.

Using PCR and SSO probes from the 11th International Histocompatibility Workshop, we oligotyped for HLA-DRB1 gene and DQA1*0501, DQB1*0201 alleles 10 celiac families each with 2 affected children. All families belong to the Italian population except for one, whose mother is originally from Cape Verde island. 8/10 sibling pairs share the DQA1*0501/B1*0201 heterodimer, inherited in cis or in trans arrangement. All the dimer-negative patients were DR4-positive.

Search for coeliac disease susceptibility loci on 7q11.23 candidate region: absence of association with the ELN17 microsatellite marker.

The involvement of HLA genes in the susceptibility to coeliac disease (CD) has been well documented and represents the only consistently observed genetic feature of this multifactorial disease. In the present study, the search for new susceptibility genes has been devoted to a candidate region suggested by the association of CD with Williams syndrome (WS). This genetic disorder is due to a deletion in the 7q11.23 region that includes the elastin (ELN) gene. An increased prevalence of CD in WS patients has been previously reported and a case of CD-WS is also described in the present study. We used the ELN17 microsatellite marker mapped within the ELN gene to look for a possible contribution of this region to the susceptibility to CD. The analysis of 74 Italian CD families provided no evidence of association with the ELN17 marker.

mAb KUL/05 identifies a denaturation-resistant determinant shared by class II MHC products DR, DQ and DP.

mAb KUL/05, a novel murine monoclonal antibody, reacts with molecules displaying the typical tissue distribution and molecular profile of class II MHC antigens. An extensive scrutiny employing serological and immunochemical assays on DR homozygous and DR alpha- mutant cell lines has shown that this reagent displays some additional, interesting features, namely mAb KUL/05 (a) binds in a broadly monomorphic fashion to cells of DR1 through seven specificities, (b) recognizes a determinant shared by a large proportion of DR, DQ and DP beta chains from most haplotypes, in both their monomeric and alpha chain-associated forms, and (c) reacts with frozen, acetone-fixed, as well as conventional, formalin-fixed, paraffin embedded tissues. Thus, mAb KUL/05 is likely to represent a useful adjunct for the study of the expression of class II MHC products in normal and pathological tissue specimens.

HLA-ABC and DR antigens in celiac disease. A study in a pediatric Italian population.

HLA phenotypes of 64 Italian pediatric patients with celiac disease (CD) were compared with those of a group of healthy controls. DR3 and DR7 are significantly increased as reported in other populations. In addition an increase of heterozygotes DR5/DR7 was observed in our patients. The Hardy-Weinberg distribution in the patients group shows a disequilibrium due to the genotype DR5/DR7. Our data confirm that more than one HLA gene product is associated with CD: one with DR3 and the other with DR7.

Delineation of syndromes due to partial 6q imbalances. Trisomy 6q21 leads to qter and monosomy 6q221 leads to qter in two unrelated patients.

Two unrelated patients carrying imbalances involving the long arm of chromosome 6 are described. In the first trisomy 6q21 leads to qter had segregated from a maternal translocation t(6;16)(q15;q24). The clinical data of the proposita are compared with those of three other published cases. A partial 6q trisomy syndrome is postulated characterized by: growth deficiency of prenatal onset, psychomotor retardation, craniofacial abnormalities (microcephalia, hypertelorism, downward slanting palpebral fissures, flattened nasal bridge, long philtrum, hypoplastic perioral features, large jaw resulting in a round appearance of the face, receding chin, malformed ears) and dysmorphic extremities (contractures of limbs due to short flexor tendons, hypoplastic fingers, toes and nails). In the second case, monosomy 6q221 leads to qter resulted from a de novo rearrangement and was responsible for mental retardation and facial dysmorphism (reduced biparietal diameter, hypotelorism, absent eyebrows, prominent nose, ptosis, receding chin, dysmorphic ears). Studies of HLA and PGM3 segregation showed normal inheritance patterns and ruled out the location of these genes in bands 6q221 leads to qter.