J. Ramón Bilbao

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.

Association of Insulin-dependent Diabetes Mellitus and Celiac Disease: A Study Based on Serologic Markers

BACKGROUND The association of celiac disease and insulin-dependent diabetes mellitus has been known for some time. In an attempt to clarify this association, the prevalence of celiac disease among diabetic children was determined, and the risk of insulin-dependent diabetes mellitus was defined in pediatric patients with celiac disease. METHODS Ninety-three children with diabetes were analyzed for the presence of celiac disease-related markers (antigliadin and antiendomysial antibodies) and characteristic alterations in the intestinal mucosa. In another group, 93 children with celiac disease were screened for pancreatic autoantibodies and pancreatic beta-cell function. RESULTS Among children with insulin-dependent diabetes mellitus, a 6.45% prevalence of celiac disease was observed, a value significantly higher than that found among healthy controls. In contrast, only three celiac disease patients showed potential autoimmunity toward the pancreatic beta cell, a proportion not significantly different from that in the general population. Additionally, no alteration of glucose metabolism was observed in the antibody-positive patients. CONCLUSION The increased risk of celiac disease among patients with diabetes requires a long follow-up to determine the presence of celiac disease markers among patients with diabetes, to avoid potential malignant disease derived from untreated celiac disease. In contrast, there is no evidence to support an increased risk of insulin-dependent diabetes mellitus among children with celiac disease. In accordance with the accepted influence of diet in the development of autoimmune diabetes, a hypothetical mechanism of protection against insulin-dependent diabetes mellitus that is mediated by environmental factors related to restricted diet is suggested in this population.

HLA-DRB1 and MICA in Autoimmunity

Abstract: Autoimmune disorders such as type 1 diabetes (T1DM), celiac disease (CD), and Addisons 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.

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, αβ and γδ 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.

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.

A novel postzygotic nonsense mutation in SRY in familial XY gonadal dysgenesis

The Y chromosome gene SRY plays an important role in normal male sexual development and is thought to be the testis-determining factor. We describe a familial nonsense mutation in SRY, shared by two XY sisters with complete gonadal dysgenesis and, in a mosaic manner, by their father. This mutation, consisting of a C to T transition in position 1 of codon 97 of SRY, results in a truncated peptide with an incomplete DNA-binding domain. The mutation is also present in the father of the two cases, but a portion of wild-type SRY also remains. Our data suggest that the father suffered a postzygotic mutation early in development, but that he retained a remnant of functional SRY protein that accounts for his normal development.

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

Abstract: 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 Addisons 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 (Pc= 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.

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 γ δ T cells containing the TCR variable region Vδ1, 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.

5′-Insulin Gene VNTR Polymorphism Is Specific for Type 1 Diabetes

Abstract: 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 Addisons 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.

Anti-Insulin Activity in Normal Newborn Cord-Blood Serum: Absence of IgG-Mediated Insulin Binding

Insulin autoantibodies (IAAs) are present in ∼60% of type I diabetes patients at onset and are used as predictors for the disease. Although the prevalence of IAAs in the general population has been reported to be <1%, preliminary data have pointed out a higher proportion of IAA positivity in newborn cord-blood serum, and some authors have suggested that they are immunoglobulin G antibodies, resulting from a hypothetical gestational insulitis. To characterize this insulin-binding activity, we analyzed cord-blood sera from 100 healthy newborns, as well as serum from 21 of their mothers at delivery, 179 new-onset type I diabetic patients, and 200 healthy control subjects. IAAs were present in 0.5% of the control subjects and 54% of newonset type I diabetic patients. On the other hand, 96% of the newborn cord-blood sera showed anti-insulin activity, while it was detected in only 14% of their mothers. No significant differences were observed between cord sera and the general population for isletcell or anti-GAD autoantibodies. Anti-insulin activity in cord serum was not bound by protein A or protein G, in contrast with type I diabetes–related IAA activity. We conclude that this insulin-binding activity, present in most newborn cord sera and specific to the child, is not IgG mediated. These data, together with the absence of other pancreatic autoimmunity markers in this population, suggest that it is an isolated phenomenon not related to type I diabetes or other pancreatic autoimmune processes and is due to the presence of a cross-reacting molecule in cord blood that has yet to be identified.