Age-dependent association of idiopathic achalasia with vasoactive intestinal peptide receptor 1 gene.

Idiopathic achalasia is a rare disorder of the oesophagus of unknown aetio-pathogenesis characterized by a myenteric inflammation, aperistalsis and insufficient lower oesophageal sphincter relaxation. Vasoactive intestinal peptide (VIP), present in the myenteric plexus, is involved in smooth muscle relaxation and acts as an anti-inflammatory cytokine. The human VIP receptor 1 gene (VIPR1) is highly polymorphic and may play a role in idiopathic achalasia. One hundred and four consecutive patients and 300 random controls from the same geographic area were typed for five SNPs mapping in the VIPR1 gene. Patients with idiopathic achalasia show a significant difference in allele, genotype and phenotype distribution of SNP rs437876 mapping in intron 4. This association, however, was almost entirely due to the group of patients with late disease onset (P = 0.0005). These results strongly suggest that idiopathic achalasia is a heterogeneous disease with a different aetiology in cases with early or late disease onset.

A functional polymorphism of the vasoactive intestinal peptide receptor 1 gene correlates with the presence of HLA-B*2705 in Sardinia.

The association of HLA-B27 with ankylosing spondylitis (AS) is the strongest among all inflammatory diseases. However, the exact role of these molecules in disease pathogenesis is still unknown. The existence of HLA-B27 variants rarely found in patients introduces a further level of complexity. It is now accepted that other genes of minor impact contribute to modify disease susceptibility and these genes might be diverse in different populations depending on the genetic background. We report here a study performed in Sardinia, an outlier population in which two major HLA-B27 subtypes are present, B (*)2705 strongly associated with AS and B (*)2709 which is not, and show the co-occurrence of the B (*)2705 allele with a single nucleotide polymorphism (SNP) mapping at 3'-UTR of the receptor 1 (VIPR1) for the vasoactive intestinal peptide (VIP), a neuropeptide with anti-inflammatory properties. This same SNP is associated with a different kinetics of down-modulation of the VIPR1 mRNA in monocytes after exposure to lipopolysaccharide (P=0.004). This particular setting, HLA-B (*)2705 and a functional polymorphism in VIPR1 gene, might be due to a founder effect or might be the result of a selective pressure. Irrespectively, the consequent downregulation of this receptor in the presence of a 'danger' signal might influence susceptibility to AS.

Association of DRB1*04-DQB1*0301 haplotype and lack of association of two polymorphic sites at CTLA-4 gene with Hashimoto's thyroiditis in an Italian population.

Hashimoto's thyroiditis (HT) is an autoimmune disease resulting from a complex interaction between genetic and environmental factors. The genetic loci conferring susceptibility need to be still defined. The aim of the present study was to determine whether Cytotoxic T-Lymphocyte-Associated Antigen-4 (CTLA-4), HLA DRB1, and DQB1 genes were associated to HT in an Italian population. We evaluated the allele distribution of the following loci: CTLA-4 exon 1 A49G dimorphism, which resulted in an amino acidic exchange (Thr/Ala) in the leader peptide, CTLA-4 3' microsatellite, HLA DRB1 and DQB1 in 126 patients with HT and in 301 control subjects from an Italian population (Lazio region). CTLA-4 exon 1 A49G dimorphism was typed by Polymerase Chain Reaction and Restriction Fragment Length Polymorphism (PCR-RFLP); CTLA-4 3' microsatellite alleles were defined using a fluorescence-based method. HLA DRB1 and DQB1 alleles were typed using a SSO reverse line blot method and a probeless procedure based on allele group-specific amplification followed by DNA heteroduplex analysis, respectively. Data were initially analyzed by chi2 test or Fisher's exact test. Multiple logistic regression analysis was then applied on factors with significant crude odds ratios and on CTLA-4 exon 1 A49G dimorphism to investigate their independent effects. The two polymorphic sites at CTLA-4 gene did not increase the risk for HT. The distribution of HLA DRB1 and DQB1 alleles did not show any significant difference between patients and controls, however, the DRB1*04-DQB1*0301 haplotype was significantly increased in patients. Other factors that increase the risk of disease were gender and age. Females showed approximately 18 times more risk than males; subjects older than 50 years had an odds ratio of 6.6. These data suggest that these two polymorphic sites at CTLA-4 do not play a major role in the susceptibility of the disease in an Italian population while female gender, age over 50 years, HLA DRB1*04-DQB1*0301 haplotype increase the risk of developing HT.

The distribution of HLA class II susceptible/protective haplotypes could partially explain the low incidence of type 1 diabetes in continental Italy (Lazio region).

HLA class II is the primary susceptibility gene to type 1 diabetes and the analysis of HLA class II association could help to clarify the relative weight of genetic contribution to the incidence of the disease. Here we present an extensive typing for HLA class II alleles and their haplotypes in a homogenous population of type 1 diabetic patients (n=134) and controls (n=128) and in simplex (n=100) and multiplex families (n=50) from continental Italy (Lazio region). Among the various haplotypes tested, the DRB1*0301-DQA1*0501-DQB1*0201 was the most frequent found in type 1 diabetic patients and was transmitted in 82% of affected siblings, whereas DRB1*0402-DQA1*0301-DQB1*0302 appeared to have the highest odds ratio (10.4), this haplotype was transmitted in 96.3% of affected siblings, followed by DRB1*0405-DQA1*0301-DQB1*0302, DRB1*0405-DQA1*0301-DQB1*0201, DRB1*0401-DQA1*0301-DQB1*0302 and DRB1*0404-DQA1*0301-DQB1*0302. The following haplotypes showed a significant decreased transmission to diabetic siblings: DRB1*0701-DQA1*0201-DQB1*0303, DR2-DQA1*01-DQB1*0602, DR5-DQA1*0501-DQB1*0301. We suggest that the HLA DR/DQ haplotype/genotype frequencies observed could in part explain the low incidence of type 1 diabetes registered in Lazio region (8.1/100.000/year), for a number of reasons: i) the low frequency, in the general control population, of the most susceptible haplotypes and genotype for type 1 diabetes DRB1*0301-DQA1*0501-DQB1*0201 (14%), and DR4-DQA1*0301-DQB1*0302 (9%) and DRB1*0301-DQA1*0501-DQB1*0201/DR4-DQA1*0301-DQB1*0302 (0.8%) compared to other countries characterised by high incidence rate of the disease, Sardinia and Finland, respectively; ii) a significant lower ratio, in the control population, between the susceptible DRB1*0301-DQA1*0501-DQB1*0201 and the neutral DRB1*0701-DQA1*0501-DQB1*0201 haplotypes compared to the Sardinian population; iii) the high frequency of protection haplotypes/genotypes as the DR5-DQA1*0501-DQB1*0301, and DR5-DQA1*0501-DQB1*0301/DR5-DQA1*0501-DQB1*0301 very common in the control population of Lazio region and the DRB1*1401-DQA1*0101-DQB1*0503 haplotype.

An N-terminal domain shared by Fas/Apo-1 (CD95) soluble variants prevents cell death in vitro.

Fas/Apo-1 molecule, also designated as CD95, is a member of the TNF receptor family. Fas cross-linking by its natural ligand or by agonistic mAbs results in rapid induction of apoptosis in susceptible cells. in addition to the Fas full-length mRNA, human activated PBMC and tumor cell lines express several mRNA Fas variants that derive from alternative splicing of the primary transcript. All five variants identified, two of which are newly described here, code for soluble proteins that, with the exception of FasTMDel, are truncated in the extracytoplasmic region and possess short C-terminal amino acid sequences corresponding to a different reading frame. We have identified Abs that recognize all splicing variants and established a sandwich ELISA by which the soluble Fas molecules could be detected in culture supernatants of transfected cell lines and in PBMC following T cell activation. Next, we have studied in detail the functional role of these variants by apoptosis inhibition studies. We found that all soluble proteins block the apoptosis induced by either an agonistic Ab or, more importantly, by the natural Fas ligand in Fas-positive sensitive cell lines. interestingly, this functional property can be assigned to the first 49 amino acids of the mature protein that is the only region shared by the five soluble Fas molecules.

Fas/Apo-1 (CD95) receptor lacking the intracytoplasmic signaling domain protects tumor cells from Fas-mediated apoptosis.

FAS/Apo-1 (CD95) is an apoptosis-signaling cell surface receptor belonging to the TNF receptor family. Tumor cells resistant to Fas-mediated apoptosis have been described, but to date, the mechanisms responsible for this resistance are not well understood. We found that a series of apoptosis-resistant clones from human HUT78 lymphoma cells express a splicing variant coding for a truncated Fas molecule that lacks the intracellular death-signaling domain. The mutation responsible for the FasExo8Del expression was identified as a deletion-insertion in the intron 7/exon 8 region of the Fas gene. Moreover this mutation affects the phenotype in a dominant negative fashion, i.e., even in the presence of the normal receptor.

Soluble Fas/Apo-1 splicing variants and apoptosis.

In addition to the full length mRNA activated human peripheral blood mononuclear cells (PBMC) and T cell tumor lines express several alternatively spliced Fas variants. At least five of these code for soluble Fas (CD95) molecules. In vitro studies suggest that these soluble Fas isoforms inhibit apoptosis induced by agonistic antibodies and, more importantly, by the natural Fas ligand in Fas-bearing sensitive cells. Interestingly, this functional property can be assigned to the first 49 aminoacids of the mature protein, the only region shared by the soluble Fas molecules.

Three functional soluble forms of the human apoptosis-inducing Fas molecule are produced by alternative splicing.

Fas/Apo-1 molecule is an apoptosis-signaling cell surface Ag belonging to the TNFR family. To investigate the possibility that soluble forms of the Fas receptor are expressed in human cells, we analyzed Fas mRNA transcripts obtained from activated peripheral mononuclear cells of healthy donors and from human tumor cell lines. We identified and characterized three human mRNA Fas variants: FasTMDel, FasDel2, and FasDel3. To determine whether the three transcripts were derived by alternative splicing, the Fas genomic intron/exon organization of the regions surrounding the deleted sequences was analyzed in Fas clones isolated from a human genomic library. Expression of the transcripts was studied in COS cells transiently transfected with the FasTMDel, FasDel2, and FasDel3 cDNAs. Immunocytochemical and in vitro apoptosis inhibition studies suggest that the transcripts are expressed as soluble Fas proteins that may play a functional role in the regulation of apoptosis.

HLA supratypes in an Italian population.

A supratype analysis of a North Italian population was performed, using 16 polymorphisms in the HLA region spanning the HLA-A-DP segment. Fourteen supratypes were identified, mostly corresponding to those found in other Caucasoid populations. The degree of their conservation both within the B-DR/DQ region and in the regions telomeric and centromeric from HLA-A and DP was evaluated and linkage disequilibria among several DR and DP alleles were identified. Notably, the degree of association with DP increased when the DR marker was part of a conserved B-DR/DQ supratype. These data are relevant to the definition of the genetic structure of the population and to the prediction of probabilities of histocompatibility matching between unrelated individuals.

Gametic association of HSP70-1 promoter region alleles and their inclusion in extended HLA haplotypes.

Gametic associations of a three-allele polymorphism of the HSP70-1 promoter region were analyzed in a random North Italian population, in 69 HLA homozygous cell lines and in 29 families in Boston, all typed for HLA class I, class II and complement alleles. Significant phenotypic associations were detected in the random population between HSP70-1 alleles and several HLA markers carried by extended haplotypes. The inclusion of HSP70-1 alleles in extended haplotypes, suggested by population analysis, was confirmed in genotyped cells, including 10W HLA homozygous cell lines and families, selected for the presence of the whole set of alleles reported for conserved extended haplotypes. Every tested extended haplotype was exclusively associated with a given HSP70-1 allele, except those carrying DR1. HSP70-1 C was included in both [HLA-B8, SC01, DR3] and [HLA-B18, F1C30, DR3] extended haplotypes, accounting for the previously observed strong association with DR3. In addition the same allele was found on the [HLA-B13, SC31, DR7], on the [HLA-B62, SB42, DR4] and on the [HLA-B60, SC02, DR13] extended haplotypes. The HSP70-1 A allele was carried by all DR4+ extended haplotypes except the one above cited. HSP70-1 B correlated with DR10, DQB1*0501 and BF*F. Thus the HSP70-1 promoter alleles provide new precisely located markers of extended haplotypes.

Strong genetic association between HLA-DR3 and a polymorphic variation in the regulatory region of the HSP70-1 gene.

A three-allele polymorphic system is detectable by direct electrophoretic analysis of the amplified 5' untranslated und 5' flanking regions of the HLA-linked HSP70-1 gene. Single nucleotide differences at two sites, i.e., -110 and +120, are responsible for changes in the bending pattern and, consequently, in the electrophoretic mobility of the three alleles. The presence of the sequence AAACCCC around position -110 is strongly associated with DR3.

Subgrouping of DR4 alleles by DNA heteroduplex analysis.

Amplified DNA molecules from six DR4 alleles at the DRB1 locus were denatured and cross-hybridized pairwise. Several of the DNA heteroduplexes thus generated were found to possess distinct mobilities in polyacrylamide gel electrophoresis. The degree of retardation as compared to homoduplexes depends strongly on the position of mismatched nucleotide pairs. In critical positions, the type of mispairing also influences the number of heteroduplex bands since a transversion-type substitution yields two reciprocal pairings (purine-purine or pyrimidine-pyrimidine) whereas a transition-type substitution generates symmetrical (purine-pyrimidine) pairings. Based on their heteroduplex pattern the DR4 alleles can be subdivided in four groups: group I = DRB*0401, group II = 02 and 06, group III = 03 and 04, and group IV = 05. Each group can be recognized by the heteroduplex bands generated by cross-hybridizing with group II reference DNA (either the 02 or 06). This subgrouping is obtained with a single electrophoretic run and without the use of probes. However, the alleles within groups II and III, and notably the alleles 03 and 04, which are both present in the Caucasoid population, can be distinguished only by oligonucleotide hybridization (dot blot) analysis. With this limitation, the method can be recommended either in conjunction with dot blot typing or independently, thus avoiding completely the use of probes in the cases where it is not essential to discriminate between 03 and 04. The data also show that distinguishable heteroduplexes may be generated by a single mismatch. This opens the possibility of applying the same technique to genetic systems of lower degree of polymorphism.

Probe-less genomic typing of Arg52 (type 1 diabetes-associated) and non-Arg52 (non-type 1 diabetes-associated) HLA-DQA1 alleles.

According to recent evidence, the presence of an Arg residue at position 52 in the HLA-DQ alpha chain may confer susceptibility to Type 1 diabetes and thus be possibly used to define quantitatively the genetic risk of this disease. Arg52 and non-Arg52 DQA1 alleles cannot be typed by the conventional cytotoxicity test and they must be distinguished at the genomic level. We describe a simple procedure which discriminates the DQA1 alleles based on the differential electrophoretic migration of the DNA heteroduplexes they form with a reference DNA fragment. A major advantage of this procedure is the fact that no hybridization probe is required. Practically, this typing procedure consists of an electrophoretic run of the products of a selective PCR in polyacrylamide gel.

Alloreactive T-cell recognition of two DQ beta allelic specificities associated with DQw1.

Two sets of alloreactive T-cell clones from different original mixed lymphocyte reactions (MLR) discriminate between two DQ beta allelic forms when these are expressed in association with the same DQ alpha form. From these results we concluded that it is possible to guide a DQ specific response of alloreactive T-cell clones by setting up an appropriate responder-stimulator combination in the original MLR; DQw1 is associated with two allelic forms of the DQ molecule recognized as lymphocyte activating determinants by alloreactive T-cell clones; alloreactive T cells recognize specific alpha-beta chain combination of the DQ molecule; the allelic forms that differentiate the alpha or the beta chains of the DQ molecule from each other can also be recognized at the molecular level by DQ alpha and DQ beta RFLP analysis.

Allelic forms of the alpha- and beta-chain genes encoding DQw1-positive heterodimers.

On chromosome 6, in the HLA region, the DQ subregion is located immediately centromeric to the DR subregion. Even though only three serological specificities to date have been officially recognized (DQw1, DQw2, and DQw3), it seems likely that the phenotypical polymorphism expressed by DQ molecules is much more complex. There are reasons to believe that fixed alpha-beta combinations exist, each of them associated with a different DR allele. DQw1 is a determinant present on DQ molecules that are found associated with DR1-, DR2-, and DRw6-positive haplotypes. By restriction fragment length polymorphism analysis, we recognized three allelic DQ-alpha and three allelic DQ-beta patterns associated with DQw1. In addition, one of these alpha/beta pairs associated with DR1, two with DR2, and a fourth with DRw6. We have obtained evidence using nucleotide sequencing that there are as many allelic forms of DQ-alpha and DQ-beta genes as there are different molecular DQ-alpha and DQ-beta patterns. The DQ-alpha and DQ-beta chains of DQw1-positive molecules each are encoded by at least three distinctly different allelic genes, and particular alpha/beta gene combinations are associated with the same DR alleles as their corresponding molecular alpha/beta pairs.