Functional study of a novel single deletion in the TITF1/NKX2.1 homeobox gene that produces congenital hypothyroidism and benign chorea but not pulmonary distress.

CONTEXT: We studied two sisters with congenital hypothyroidism and choreoathetosis but not respiratory distress. OBJECTIVE: The aim of this study was to establish the genetic defect that causes this phenotype and study the molecular mechanisms of the pathology by means of functional analysis. DESIGN: Sequencing of DNA, expression vectors generation, EMSAs, transfections experiments as well as bioinformatics analysis were performed. RESULTS: We found a new single deletion (825delC) in one allele of the TITF1/NKX2.1 gene. The mutation located in the C-terminal domain generates a nonsense thyroid transcription factor 1 (TTF1) protein, with 22 amino less and rich in positive charges. This protein shows diminished binding to DNA, does not interfere with wild-type (wt) TTF1 binding, and fails to activate reporter genes harboring the thyroglobulin (Tg), thyroperoxidase (TPO), or surfactant protein B (SP-B) promoters. In addition, the mutant (mut) protein has a dominant-negative effect on the transcriptional activity of wt TTF1 in a promoter-specific manner, inhibiting the transcription of Tg and TPO but not of SP-B. Using a Gal4 reporter system, we demonstrate that the mut protein is not transcriptionally active and does not likely compete with the wild type for coactivators. Interestingly, the mut protein impairs the wt capacity to synergize with paired box 8 (PAX8). This cooperation is necessary for Tg and TPO transcription but dispensable for SP-B expression. CONCLUSION: These results are concordant with the phenotype of the two sisters studied and demonstrate a differential role for TTF1 in the different tissues in which it is expressed.

MICA response to gliadin in intestinal mucosa from celiac patients.

MHC class I chain-related gene A (MICA), a putative independent susceptibility gene in autoimmune diseases, encodes a surface protein present in epithelial cells that binds to NKG2D, an activating receptor of NK, alphabeta and gammadelta T cells, and could function as a stress-inducible activator of the innate immune response. There is no evidence of a long-term implication of MICA in the celiac autoimmune process. However, it could be that gliadin activation of MICA occurs only during the initial stages of the disease. In order to determine whether MICA is activated in response to gliadin in patients with celiac disease (CD), small intestinal mucosa biopsy samples from ten long-standing celiac patients on a gluten-free diet and from five non-celiac individuals were incubated with and without gliadin for 4 h. Total RNA was purified and MICA, IFNG and NKG2D mRNA were quantified by fluorescent real-time RT-PCR. Expression levels were calculated relative to GAPDH. MICA expression was detected in both patients and controls, but incubation with gliadin induced a strong increase in samples from the treated CD group compared with the non-CD controls (P=0.028), while no differences were observed for IFNG or NKG2D mRNA levels. The gliadin-provoked over-expression of MICA in "normalized" tissues from CD patients suggests a role for this stress-induced activator of the immune response in the early stages of organ-specific autoimmune destruction, probably preceding the onset of inflammation.

Prospective population screening for celiac disease: high prevalence in the first 3 years of life.

BACKGROUND: Celiac disease (CD) is an autoimmune enteropathy that develops in genetically susceptible individuals exposed to gliadin. Early diagnosis of CD may reduce the risk of complications, and several studies have related the duration of gluten exposure to the risk of other autoimmune diseases. It has been proposed that silent CD be diagnosed as soon as possible to avoid potential complications. OBJECTIVES: The purpose of this study was to determine the prevalence of CD among children less than 3 years and to provide treatment to those patients diagnosed with CD. PATIENTS AND METHODS: Parents of 1100 healthy children born between October 1998 and December 1999 were asked at the time of delivery to enroll their children in a program for the early diagnosis of CD. The parents of 830 children agreed to participate. Patients in the study were examined and anti-tissue transglutaminase antibody was first measured at about 1.5 years of age. A second antibody titer was obtained at about 2.5 years of age. Patients with detectable autoantibodies underwent intestinal biopsy for confirmation of CD. RESULTS: Of the 830 children initially enrolled, 613 and 484 returned for the first and second visits, respectively. None had anti-tissue transglutaminase antibodies at the first visit, but 9 had anti-tissue transglutaminase immunoglobulins at the second visit. In 7 of these 9, intestinal biopsy confirmed the diagnosis of CD which suggests a minimum prevalence of CD of 1 per 118 healthy newborns. CONCLUSIONS: The authors observed a very high prevalence of CD, comparable to that observed in other European populations, which might even be higher if all of the children initially examined had returned for their second visit. If general screening for CD were accepted, the authors would recommend age 2-3 years as the best time for measuring tissue transglutaminase antibodies.

No association of INS-VNTR genotype and IAA autoantibodies.

Type 1 diabetes is associated with autoimmune responses against insulin, with insulin autoantibodies (IAA) being a hallmark of the disease. Genetic susceptibility to type 1 diabetes is polygenic and includes the INS VNTR-IDDM2 locus, which has been shown to regulate the expression of insulin in the pancreas and thymus. In order to determine whether insulin autoimmunity could be attributed to a genetic susceptibility conferred by the INS-VNTR (IDDM2) locus, we studied the frequency of INS-VNTR alleles and the presence of IAA in 90 patients with new-onset type 1 diabetes. IAA were detected in 49 of 67 class I/I individuals and 19 of 23 class III/X patients, indicating that there is no association between the INS VNTR-IDDM2-susceptible allele and humoral autoimmunity against insulin. This finding does not support the hypothesis of an allele-specific tolerance induction that could determine susceptibility toward autoimmunity against the insulin protein and subsequently the development of type 1 diabetes.

No evidence of pancreatic autoimmunity among patients with multiple sclerosis.

Although multiple sclerosis (MS) usually appears isolated from other autoimmune disorders, an overlap with type 1 diabetes mellitus (T1DM) has been described in Sardinia, where T1DM-associated haplotype HLA-B18-DR3-DQ2 contributes to MS risk. To determine whether in our population MS patients show signs of pancreatic autoimmunity and share this haplotype, sera from 49 MS patients were tested for GAD, IA2, and CPH autoantibodies, and MICA exon 5 polymorphism was genotyped in 30 patients. Pancreatic autoimmune markers were not present among MS patients, nor was any MICA allele associated with MS. Overall, there is no evidence supporting a T1DM/MS overlap in our population.

5'-Insulin gene VNTR polymorphism is specific for type 1 diabetes: no association with celiac or Addison's disease.

The VNTR region located at the 5'-end of the insulin gene on chromosome 11p15.5 is linked to susceptibility to type 1 diabetes mellitus (T1DM), and class I alleles have been associated with increased risk of disease, whereas class III alleles are considered to be protective. Although a potential effect on the expression level of thymic insulin and a consequent abnormal tolerance have been proposed as an explanation, it is still not clear whether the association is specific for T1DM or whether it is shared by other autoimmune disorders. To investigate the contribution of INS-VNTR to the genetic susceptibility to autoimmune disorders, we analyzed 102 autoantibody-positive T1DM patients, 59 patients with celiac disease (CD), and 57 patients with Addison's disease (ADD), as well as 111 unrelated healthy individuals from the general population. When analyzing the results, class I allele frequencies were 85.8% in the T1DM group, 77% among CD patients, 71% in the ADD group, and 76.1% in the general population. Association with increased risk was seen only in the T1DM group (pc = 0.015). Risk to T1DM was associated with the class I/class I homozygous genotype (RR, 1.92; 95% CI, 1.03-3.6). In conclusion, INS-VNTR does not seem to be involved in the susceptibility to autoimmune diseases other than T1DM and can be considered a diabetes-specific locus.

HLA-DRB1 and MICA in autoimmunity: common associated alleles in autoimmune disorders.

Autoimmune disorders such as type 1 diabetes (T1DM), celiac disease (CD), and Addison's disease (ADD) develop in individuals with genetic susceptibility that are exposed to environmental triggering factors not completely defined. Patients with an autoimmune disease (and their relatives) are at increased risk of developing another disorder, and this might be caused by a common genetic origin of autoimmunity; for example, HLA class II region in 6p21 shows a very strong association with most diseases. The aim of this study was to determine whether shared susceptibility markers extend from the central (DRB1) through the telomeric (MICA) HLA region. We analyzed three independent sets of families with one autoimmune disease, T1DM, CD, or ADD, and genotyped them for HLA-DRB1 and for the exon 5 GCT polymorphism of MICA. For HLA-DRB1, allele DRB1*0301 was the only one associated with risk for all three diseases; in the case of MICA, allele A9 was found to be the common protective allele. Haplotype analysis shows that haplotype A5.1-DRB1*0301 confers risk to autoimmunity. Our results show that there are common risk and protection alleles in both loci, suggesting a core of genetic association with autoimmunity (HLA-DRB1*0301 risk; A9 protection) that could be modulated by other alleles/loci or environmental factors toward one or another disease. Some alleles are part of conserved haplotypes (A5.1-DR3, A5.1-DR2), whereas others seem to have independent effect (A9) and support the idea of two independent loci in this region.

No association of CTLA4 gene with celiac disease in the Basque population.

BACKGROUND: Celiac disease (CD) is an autoimmune disorder caused by intolerance to ingested gluten that develops in genetically susceptible individuals. The contribution of human leukocyte antigen (HLA) genes to the genetic risk to CD has been known for a long time; however, non-HLA genetic factors are likely to be required for the development of the disease. Several studies have associated the CD28/CTLA4 region on chromosome 2q33 with the disease in different populations. The CTLA4 gene encodes a receptor involved in the control of T-cell proliferation and mediates T-cell apoptosis. AIM To determine the contribution of two polymorphisms of the CTLA4 to the disease: the A/G dimorphism at position +49 in exon 1 and the (AT)(n) microsatellite in the 3' untranslated region. PATIENTS: Forty-one celiac families of Basque origin (43 patients with CD and 80 first-degree relatives). METHODS: Restriction enzyme digestion of polymerase chain reaction amplified genomic DNA for the A/G dimorphism and polymerase chain reaction followed by high-resolution electrophoresis for the (AT)(n) microsatellite. For disease association studies, the Affected Family Based Controls approach was used. RESULTS: The frequency of the A allele of 49 A/G polymorphism was 67.47% in the celiac allele group compared with 70.13% in the Affected Family Based Controls group. These differences were not significant. Analysis of the (AT)(n) polymorphism identified 17 different alleles, ranging from 262 to 312 bp in length, but no allele was significantly associated with the disease. CONCLUSIONS: Our results did not show any evidence of association of any of the CTLA4 gene polymorphisms with the disease. This may result from population-specific differences in genetics and environmental susceptibility to CD.

Analysis of the expression of MICA in small intestinal mucosa of patients with celiac disease.

The MHC class I chain-related A gene (MICA) is expressed in gastrointestinal epithelium and functions as an immune activation signal under stress conditions. MICA protein binds to NKG2D, a receptor of gamma delta T cells containing the TCR variable region V(delta)1, which are the most abundant subset of T cells in the intestinal epithelium. Ingested gluten in patients with celiac disease (CD) may function as a stress signal for the epithelial cells, and could enhance MICA expression on their surface. In this study, we have analyzed MICA expression in intestinal biopsy specimens from newly diagnosed and treated CD patients and controls. Quantitative RT-PCR analysis did not show differences in MICA expression among the three groups. With these results, we conclude that overexpression of MICA does not seem to play an important role in the pathogenesis of CD, at least at the time of diagnosis.

Immunoglobulin G autoantibodies against tissue-transglutaminase. A sensitive, cost-effective assay for the screening of celiac disease.

The characterization of target antigens in several autoimmune disorders has made it possible to develop antigen-specific immunoassays that are superior in terms of sensitivity, specificity, reproducibility and ease of standardization, compared to immunohistological methods that are highly subjective, rely on skilled technicians and are not applicable to large-scale studies. In the case of celiac disease (CD), tissue transglutaminase (tTGase) has been identified as a major autoantigen, and antibodies against this molecule are present in most CD patients before gluten is removed from diet. In general, anti-tTGase detection assays detect the presence of IgA class antibodies, but these immunoglobulins are absent among patients with IgA deficiency, a frequent condition in which CD is very prevalent. In this report, we have analyzed 64 patients at diagnosis of CD for the presence of antibodies against tTGase of both IgA (TGA) and IgG (TGG) classes, using anti-IgA antibodies or Protein A, respectively, for the immunoprecipitation of 35S labeled, in vitro transcribed and translated human recombinant tTGase. In our hands, the TGG assay matches TGA in terms of sensitivity (97%) and specificity (100%), and besides, the combination of both assays is able to detect antibodies in all patient samples. The method described uses only 6 microl of serum, can be adapted to automated large-scale analysis and, in combination with other antigens, can be used for the simultaneous screening of other autoimmune diseases, like type 1 diabetes mellitus.

Contribution of MIC-A polymorphism to type 1 diabetes mellitus in Basques.

The maximum genetic susceptibility to type 1 diabetes (T1DM) in Basques is conferred by extended HLA haplotype F1C30-DR3-DQ2-DPB1*0202. Due to the strong linkage disequilibrium within the haplotype, it is difficult to determine which individual allele shows the strongest association with T1DM. Recent studies of the MIC-A gene have shown an HLA-independent association of allele A5 with T1DM and A5.1 with Addison's disease. In order to test for association of the MIC-A exon 5 polymorphism with T1DM and to further characterize risk and protection haplotypes in Basques, we typed 70 Basque families with T1DM for MIC-A exon 5 polymorphism using fluorescent PCR and electrophoresis on an ABI sequencing machine. When analyzed individually, allele A4 was associated with disease [OR = 2.93 (1.58-5.5)], while the presence of A9 conferred protection from T1DM [OR = 0.27 (0.08-0.74)]. In the context of HLA haplotypes, allele A4 was found to be associated to the F1C30-DR3-DQ2-DPB*0202 risk haplotype, both for T1DM and AFBAC alleles (P(c) = 0.0003). Allele A5.1 was strongly associated with protective haplotype SC31-DRB1*1501-DQB1*0602, present only in AFBAC alleles, but also with risk haplotype SC01-DR3-DQ2. In conclusion, polymorphisms in exon 5 of the MIC-A gene are associated with genetic susceptibility/protection to T1DM, but in the context of susceptibility HLA haplotypes. Nevertheless, the protective effect of A9 allele seems independent from HLA, since it does not appear to be associated with any particular extended haplotype.