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.

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.

Strong linkage disequilibrium of TAP1*0301 and TAP2D alleles with the HLA A1-B35-DRB1*1104-DQA1*0103-DQB1*0603 extended haplotype in Ashkenazi Jews.

On the basis of data collected during the 12th International Histocompatibility Workshop, we postulated a possible linkage disequilibrium between the TAP1C allele and the DRB1*1104-DQA1*0103-DQB1*0603 haplotype characteristic of Ashkenazi Jews. In order to confirm and extend this preliminary observation, a group of 34 subjects carrying this haplotype was analysed for TAP1 and TAP2 polymorphisms and compared with two control groups sharing either the DRB1 or the DQA1 and DQB1 alleles with the test group. The TAP1*0301 and TAP2D alleles were found to be strongly associated with the entire haplotype, but not with the DRB1*1104 or the DQA1*0103-DQB1*0603 alleles when carried separately. These data show a strong linkage disequilibrium of TAP1*0301 and TAP2D alleles with the DRB1*1104-DQA1*0103-DQB1*0603 haplotype of Ashkenazi Jews, extended on the centromeric and telomeric side to the DPB1*0201 and A1-B35 alleles, respectively.

Molecular prenatal diagnosis of ataxia telangiectasia heterozygosity by direct mutational assays.

Ataxia telangiectasia (AT) is a severe autosomal recessive disease, rare but not infrequent in Italy. Owing to the seriousness of the disease, prenatal diagnosis has been attempted in the past by means of cytogenetic, biochemical, radio-biological and indirect molecular analyses. We performed the first direct molecular prenatal diagnosis of AT on a chorionic villi sample from a 37-year-old woman at the 10th week of pregnancy. She had two previous children suffering AT and two induced abortions. At molecular analysis her affected children were compound heterozygotes for mutations 7792C-->T in exon 55 (from the mother) and 8283delTC in exon 59 (from the father). The prenatal diagnosis was performed by two different operators in double-blind form. Mutation 7792C-->T was studied by restriction enzyme analysis using TaqI. Mutation 8283delTC was screened by heteroduplex analysis. The fetus was heterozygous for the mutation 7792C-->T (confirmed by sequencing). In order to verify the possible contamination by maternal DNA, polymorphic loci HLA-DRB1 and HLA-DQA1, together with microsatellite markers D6S259, D11S2000, D11S29, D11S1778 and D11S2179, were examined. All these loci were informative, showing that the fetus received only one allele from each parent. The heterozygosity for ATM mutation 7792C-->T was confirmed by molecular studies after the birth of a healthy male baby.

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.

Genetic contribution of the HLA region to the familial clustering of coeliac disease.

In order to assess the effect of the HLA region on familiality of coeliac disease (CD), we carried out a study on 121 CD index cases and 325 first degree relatives. The transmission disequilibrium test confirmed the importance of the HLA-DR3 haplotype in CD susceptibility. However, the different distortion found in affected children inheriting maternal or paternal DR3 alleles suggested that the sex of the parent might influence the risk conferred by this haplotype. The increase in risk to siblings of affected individuals relative to the risk in the general population (lambda s) and the contribution of the HLA genes to this clustering (lambda sHLA) have also been estimated. Non-overlapping data from the literature have been collected and combined with our sample to extend such analysis. Then, the percentage contribution of the HLA region to the development of CD among siblings was 36.2%. This result confirms that the HLA genotypes are an important genetic background to CD development but shows that additional susceptibility factors remain to be identified.

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.

Gluten-sensitive disease with mild enteropathy.

BACKGROUND & AIMS: Celiac disease is a permanent gluten intolerance strongly associated with HLA class II antigens and possibly showing milder changes of mucosal architecture. Ten patients with symptoms suggesting celiac disease and serum antiendomysium antibodies with normal mucosal architecture were studied. METHODS: Immunohistochemical detection of mucosal immune activation and HLA typings were performed. RESULTS: Mucosal immune activation, with normal mucosal architecture and normal gamma/delta+ intraepithelial lymphocytes counts, was found on a gluten-containing diet. In 3 of 6 patients, multiple biopsy specimens showed one sample with severe villous atrophy. Clinical and immunomorphologic features were strictly gluten dependent. The mucosal immune activation was elicited in vitro by gliadin. Only 4 patients had the typical HLA typing of celiac disease. CONCLUSIONS: Gluten-sensitive celiac-like symptoms may occur in patients with serum antiendomysium antibodies, apparently normal intestinal mucosa, and HLA typing not commonly associated with celiac disease. These patients should undergo multiple biopsies, and signs of immunologic activation should be sought accurately; in the presence of mucosal immune activation, a trial with a gluten-free diet should be encouraged to detect gluten dependency. In vitro immunologic response of small intestinal mucosa to gliadin may support the diagnosis of gluten-sensitive enteropathy.

Frequency and clinical pattern of celiac disease among siblings of celiac children.

To investigate the prevalence and clinical and genetic patterns of celiac disease (CD) among siblings of CD patients, 103 siblings and one twin of 80 celiac children were evaluated by means of their clinical history, physical examination, blood indices of nutritional status, and antigliadin antibodies (AGA). Antiendomysium antibody (AEA) levels were determined in 70 patients and 85 subjects were human leucocyte antigen (HLA) typed. On the basis of clinical or laboratory data or both, 21 siblings (20.2%) were submitted to intestinal biopsy, whereas intestinal biopsy in six siblings with positive serologic screening (AGA IgA or AEA or both) was not performed because of parental refusal. In a high percentage of cases (18%), all on a gluten-containing diet, the intestinal mucosa was atrophic, and CD was subsequently diagnosed. Because we could not submit all the siblings to intestinal biopsy, this figure could underestimate the real prevalence of the disease in our series; consequently, it was not possible to calculate accurately the sensitivity and specificity of AGA and AEA. Nevertheless, AEA (positive in all the nine siblings with mucosal atrophy), followed by AGA IgA, proved to be the best screening for CD. Eighteen of 19 CD siblings showed HLA-predisposing antigens. Among the 19 CD siblings, one showed a typical form with gastrointestinal symptoms, two had short stature, one suffered from recurrent vomiting, and in 15, the disease was clinically silent. On the contrary, among siblings who were first diagnosed (index cases), the majority (73.7%) had a typical form of CD, and no clinically silent cases were observed. We did not find any difference between index cases and CD siblings in food habits and distribution of HLA antigens. In 15 of 18 cases, the sibling diagnosed subsequently was the older one. Finally, the typical form of CD was significantly more frequent among the younger brother than the older. In conclusion, the high prevalence of the silent form of CD in our cases indicates that siblings of CD subjects should always be screened for CD. The combination of AGA IgA and AEA represent a good screening method to use in selecting children for the intestinal biopsy.

Definition of the initial immunologic modifications upon in vitro gliadin challenge in the small intestine of celiac patients.

BACKGROUND & AIMS: Mucosal cell-mediated immune response is considered the central event in the pathogenesis of celiac disease. In cultured intestinal explants from celiacs in remission, we have characterized the early stages of gliadin-induced immune activation. METHODS: Intestinal biopsy specimens (15 treated celiacs and 13 controls) were cultured with gliadin or maize prolamine digests for 24 hours as well as for 1, 2, 4, 6, 8, and 12 hours in some subjects. The expression of immunologic markers was detected by immunocytochemistry. RESULTS: Gliadin challenge may initiate two parallel pathways, one of which leads to T-cell activation and another that precedes it. Epithelial cells overexpress DR molecules after 1 hour, and in a second stage T lymphocytes become fully activated. Moreover, T lymphocytes migrate in the upper mucosal layers. T lymphocytes that migrate in the higher lamina propria compartments are mainly CD4+ and show markers of activation; migrating intraepithelial lymphocytes are CD8+ and do not express these markers. CONCLUSIONS: In vitro gliadin challenge is a suitable model to reproduce various immunologic features of celiac lesions; these may be caused by different pathways. The comprehension of these phenomena is essential to clarify the distinctive pathogenic mechanisms leading to disease and may help in defining novel therapeutic approaches.

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.

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.

Coeliac disease, enamel defects and HLA typing.

The presence of dental enamel defects in coeliac disease and their relation to hypocalcaemia or a particular HLA class in 82 Italian children with coeliac disease was studied. Demarcated opacities or hypoplasia were detected in 23 subjects (group 1) while minimal or no dental lesions were found in the remaining 59 patients (group 2); in 189 normal controls, enamel lesions were significantly less frequent than in patients with coeliac disease (14.8% versus 28.0%; p < 0.005). No statistically significant differences were found for age at diagnosis and calcium concentrations between groups 1 and 2. Regression analysis showed a correlation between age at diagnosis and number of teeth with enamel defects. In our patients, the presence of HLA DR3 antigen significantly increased the risk of dental lesions, while genotype DR5,7 seemed to protect against enamel defects. A logistic regression analysis of the variables age, serum calcium concentrations, number of affected teeth, type of enamel defect and DR antigens showed that only DR antigens discriminated coeliac disease patients with from those without enamel defects.

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.

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.

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.

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.