Diagnostic Delay of Celiac Disease in Childhood.

Importance: The extent and factors associated with risk of diagnostic delay in pediatric celiac disease (CD) are poorly understood. Objectives: To investigate the diagnostic delay of CD in childhood, and to assess factors associated with this delay. Design, Setting, and Participants: Multicenter, retrospective, cross-sectional study (2010-2019) of pediatric (aged 0-18 years) patients with CD from 13 pediatric tertiary referral centers in Italy. Data were analyzed from January to June 2023. Main Outcomes and Measures: The overall diagnostic delay (ie, the time lapse occurring from the first symptoms or clinical data indicative of CD and the definitive diagnosis), further split into preconsultation and postconsultation diagnostic delay, were described. Univariable and multivariable linear regression models for factors associated with diagnostic delay were fitted. Factors associated with extreme diagnostic delay (ie, 1.5 × 75th percentile) and misdiagnosis were assessed. Results: A total of 3171 patients with CD were included. The mean (SD) age was 6.2 (3.9) years; 2010 patients (63.4%) were female; and 10 patients (0.3%) were Asian, 41 (1.3%) were Northern African, and 3115 (98.3%) were White. The median (IQR) overall diagnostic delay was 5 (2-11) months, and preconsultation and postconsultation diagnostic delay were 2 (0-6) months and 1 (0-3) month, respectively. The median (IQR) extreme overall diagnostic delay (586 cases [18.5%]) was 11 (5-131) months, and the preconsultation and postconsultation delays were 6 (2-120) and 3 (1-131) months, respectively. Patients who had a first diagnosis when aged less than 3 years (650 patients [20.5%]) showed a shorter diagnostic delay, both overall (median [IQR], 4 [1-7] months for patients aged less than 3 years vs 5 [2-12] months for others) and postconsultation (median [IQR], 1 [0-2] month for patients aged less than 3 years vs 2 [0-4] months for others). A shorter delay was registered in male patients, both overall (median [IQR], 4 [1-10] months for male patients vs 5 [2-12] months for female patients) and preconsultation (median [IQR], 1 [0-6] month for male patients vs 2 [0-6] months for female patients). Family history of CD was associated with lower preconsultation delay (odds ratio [OR], 0.59; 95% CI, 0.47-0.74) and lower overall extreme diagnostic delay (OR, 0.75; 95% CI, 0.56-0.99). Neurological symptoms (78 patients [21.5%]; OR, 1.35; 95% CI, 1.03-1.78), gastroesophageal reflux (9 patients [28.1%]; OR, 1.87; 95% CI, 1.02-3.42), and failure to thrive (215 patients [22.6%]; OR, 1.62; 95% CI, 1.31-2.00) showed a more frequent extreme diagnostic delay. A previous misdiagnosis (124 patients [4.0%]) was more frequently associated with gastroesophageal reflux disease, diarrhea, bloating, abdominal pain, constipation, fatigue, osteopenia, and villous atrophy (Marsh 3 classification). Conclusions and Relevance: In this cross-sectional study of pediatric CD, the diagnostic delay was rather short. Some factors associated with risk for longer diagnostic delay and misdiagnosis emerged, and these should be addressed in future studies.

Peripheral blood mononuclear cells reactivity in recent-onset type I diabetes patients is directed against the leader peptide of preproinsulin, GAD65271-285 and GAD65431-450.

Introduction: T cell reactivity against pancreatic autoantigens is considered one of the main contributors to the destruction of insulin-producing cells in type 1 diabetes (T1D). Over the years, peptide epitopes derived from these autoantigens have been described in NOD mice and in both HLA class II transgenic mice and humans. However, which ones are involved in the early onset or in the progressive phases of the disease is still unclear. Methods: In this work we have investigated, in early-onset T1D pediatric patients and HLA-matched controls from Sardinia, the potential of preproinsulin (PPI) and glutamate decarboxylase 65 (GAD65)-derived peptides to induce spontaneous T cell proliferation responses of peripheral blood mononuclear cells (PBMCs). Results: Significant T cell responses against PPI1-18, PPI7-19 and PPI31-49, the first two belonging to the leader sequence of PPI, and GAD65271-285 and GAD65431-450, were found in HLA-DR4, -DQ8 and -DR3, -DQ2 T1D children. Conclusions: These data show that cryptic epitopes from the leader sequence of the PPI and GAD65271-285 and GAD65431-450 peptides might be among the critical antigenic epitopes eliciting the primary autoreactive responses in the early phases of the disease. These results may have implications in the design of immunogenic PPI and GAD65 peptides for peptide-based immunotherapy.

DNA methylation of the TPMT gene and azathioprine pharmacokinetics in children with very early onset inflammatory bowel disease.

BACKGROUND: Thiopurine methyltransferase (TPMT) is a crucial enzyme for azathioprine biotransformation and its activity is higher in very early onset inflammatory bowel disease (VEO-IBD) patients than in adolescents with IBD (aIBD). AIMS: The aims of this pharmacoepigenetic study were to evaluate differences in peripheral blood DNA methylation of the TPMT gene and in azathioprine pharmacokinetics in patients with VEO-IBD compared to aIBD. METHODS: The association of age with whole genome DNA methylation profile was evaluated in a pilot group of patients and confirmed by a meta-analysis on 3 cohorts of patients available on the public functional genomics data repository. Effects of candidate CpG sites in the TPMT gene were validated in a larger cohort using pyrosequencing. TPMT activity and azathioprine metabolites (TGN) were measured in patients' erythrocytes by HPLC and associated with patients' age group and TPMT DNA methylation. RESULTS: Whole genome DNA methylation pilot analysis, combined with the meta-analysis revealed cg22736354, located on TPMT downstream neighboring region, as the only statistically significant CpG whose methylation increases with age, resulting lower in VEO-IBD patients compared to aIBD (median 9.6% vs 12%, p = 0.029). Pyrosequencing confirmed lower cg22736354 methylation in VEO-IBD patients (median 4.0% vs 6.0%, p = 4.6 ×10-5). No differences in TPMT promoter methylation were found. Reduced cg22736354 methylation was associated with lower TGN concentrations (rho = 0.31, p = 0.01) in patients with VEO-IBD and aIBD. CONCLUSION: Methylation of cg22736354 in TPMT gene neighborhood is lower in patients with VEO-IBD and is associated with reduced azathioprine inactivation and increased TGN concentrations.

Toward the renal vesicle: Ultrastructural investigation of the cap mesenchyme splitting process in the developing kidney.

Background: A complex sequence of morphogenetic events leads to the development of the adult mouse kidney. In the present study, we investigated the morphological events that characterize the early stages of the mesenchymal-to-epithelial transition of cap mesenchymal cells, analyzing in depth the relationship between cap mesenchymal induction and ureteric bud (UB) branching. Design and methods: Normal kidneys of newborn non-obese diabetic (NOD) mice were excised and prepared for light and electron microscopic examination. Results: Nephrogenesis was evident in the outer portion of the renal cortex of all examined samples. This process was mainly due to the interaction of two primordial derivatives, the ureteric bud and the metanephric mesenchyme. Early renal developmental stages were initially characterized by the formation of a continuous layer of condensed mesenchymal cells around the tips of the ureteric buds. These caps of mesenchymal cells affected the epithelial cells of the underlying ureteric bud, possibly inducing their growth and branching. Conclusions: The present study provides morphological evidence of the reciprocal induction between the ureteric bud and the metanephric mesenchyme showing that the ureteric buds convert mesenchyme to epithelium that in turn stimulates the growth and the branching of the ureteric bud.

Low-Risk Human Leukocyte Antigen Genes and Mild Villous Atrophy Typify Celiac Disease With Immunoglobulin A Deficiency.

OBJECTIVES: We aimed to establish if in celiac disease (CD) with immunoglobulin A deficiency (IgAD) duodenal histopathology is influenced by human leukocyte antigen (HLA)-DQB1∗02 alleles dosage. Clinical differences between patients with CD and patients with CD and IgAD (CD-IgAD) were also evaluated. METHODS: Five hundred and sixteen CD and 16 patients with CD-IgAD, enrolled over the time of 8 years, took part in this study. The severity of duodenal histopathology and frequency of CD at-risk HLA class II genes were compared in patients with CD versus patients with CD-IgAD. HLA class II genotypes were subdivided into two categories of genetic risk: high: HLA-DR3/DR7, -DR3/DR3, -DR4/DR4 -DR3/DR4 and low: HLA-DR5/DR7, -DR3/X, -DR4/X and X/X, where X means neither -DR3 nor -DR4. Then, they were compared with two types of duodenal histopathology: 0, 1, 2 and 3a of mild villous atrophy (MVA) and 3b and 3c of severe villous atrophy (SVA) according to the Marsh-Oberhuber classification. Clinical data concerning gender, number of esophagogastroduodenoscopies (EGDs) and association with other autoimmune diseases were obtained from medical records. RESULTS: In comparison with CD, CD-IgAD showed an increased frequency of MVA (P < 0.0001). Furthermore, CD-IgAD with MVA showed an increase of HLA low-risk genotypes (P = 0.036) and half HLA-DQ2 heterodimers (P = 0.0443). Interestingly, CD-IgAD demanded an increased number of EGDs to reach the diagnosis of CD (P = 0.0104) and autoimmune liver diseases were more frequent compared to CD (P = 0.0049). CONCLUSIONS: CD-IgAD is associated with MVA, low-risk HLA class II genes, an increased number of EGDs and autoimmune liver diseases.

Phenotype and Natural History of Children With Coexistent Inflammatory Bowel Disease and Celiac Disease.

BACKGROUND: Adult patients with both inflammatory bowel disease (IBD) and celiac disease (CeD) have peculiar phenotypic features. This study aimed at describing the characteristics and natural history of children with both IBD and CeD. METHODS: This was a case-control study based on a national registry. Cases included children diagnosed with both IBD and CeD. Two matched IBD controls without CeD, and 2 matched CeD controls were selected for each case. Inflammatory bowel disease phenotype and natural history, comprising growth and pubertal development, were compared between groups. RESULTS: Forty-nine (1.75%) patients with IBD and CeD were identified out of 2800 patients with IBD. Compared with patients with IBD alone, patients with IBD and CeD presented more frequently with autoimmune diseases (odds ratio, 2.81; 95% CI, 0.97-8.37; P = 0.04). Ileocolonic localization (46.1% vs 73.1%), treatment with azathioprine (46.2% vs 71.2%), and anti-TNF biologics (46.2% vs 69.2%) were less common in patients with Crohn's disease and CeD than in patients with Crohn's disease alone. Patients with ulcerative colitis and CeD had an increased risk of colectomy despite similar medical treatments compared with patients with ulcerative colitis alone (13.0% vs 0%). Pubertal delay was more common in patients with IBD and CeD compared with patients with IBD alone (14.9% vs 3.2%; odds artio, 5.24; 95% CI, 1.13-33.0; P = 0.02) and CeD alone (14.9% vs 1.1%; P = 0.002). CONCLUSIONS: Children with IBD and CeD may have peculiar features with a higher risk for autoimmune diseases, colectomy, and pubertal delay compared with IBD alone.

Prudence is necessary in the application of the new ESPGHAN criteria for celiac disease omitting duodenal biopsy: a case report.

New guidelines for celiac disease (CD) diagnosis from the European Society for Paediatric Gastroenterology, Hepatology, and Nutrition (ESPGHAN) propose the option to omit the duodenal biopsy in the diagnosis of CD. For this option, all four of the following criteria have to apply in children and adolescents: signs and symptoms suggestive of CD, anti-transglutaminase type 2 antibody (anti-TG2) levels more than 10 times the upper limit of normal, positive confirmation tests of anti-endomysium-IgA antibodies (EMA), and at-risk HLA-DQ2 or HLA-DQ8. Here, we report the case of a female patient, 2 years old, with chronic diarrhea that started after an acute viral gastroenteritis. The patient had anti-TG2 levels of more than 10 times the upper limit of normal, positivity for EMA, antigliadin IgA, and IgG (AGA-IgA, AGA-IgG, respectively), and the at-risk HLA-DRB1*0301, DQB1*0201/DRB1*10, DQB1*0501 genotype, thus fulfilling all criteria for the diagnosis of CD. Although the diarrhea disappeared after about 5 weeks, anti-TG2, EMA, and AGA-IgG remained positive. Therefore, a duodenal biopsy was performed and evidenced a normal mucosa (Marsh 0). After about 18 months, the antibody titer for AGA-IgG, anti-TG2, and EMA became negative. The patient was all the time on a normal, gluten-containing diet. This clinical case represents an exception to the new ESPGHAN guidelines for CD diagnosis. During 5 weeks, the new ESPGHAN criteria were all fulfilled, allowing to propose for this patient the diagnosis of CD without performing a duodenal biopsy. Therefore, a prudent approach is suggested when the pediatric gastroenterologist makes a diagnosis of CD without duodenal biopsy.

Anti-actin IgA antibodies identify celiac disease patients with a Marsh 3 intestinal damage among subjects with moderate anti-TG2 levels.

A new diagnostic tool (algorithm-1) for coeliac disease (CD) permitting the diagnosis without performing the duodenal biopsy has been recently proposed by the European Society for Paediatric Gastroenterology, Hepatology, and Nutrition (ESPGHAN). It combines symptoms associated with CD, high anti-transglutaminase type 2 antibody (anti-TG2) levels, anti-endomysium-IgA antibodies (EMA), and at-risk HLA. Our aims were (i) to evaluate retrospectively in 227 individuals (149 CD patients and 78 controls) the algorithm-1, (ii) to reduce the number of duodenal biopsies among CD patients for whom algorithm-1 is not applicable through the addition of antiactin IgA antibodies (AAA-IgA), and (iii) to evaluate prospectively algorithm-1 and AAA-IgA in 50 patients with suspected CD. Algorithm-1 identified 70 out of 149 CD patients with Marsh 3 lesions. Adding AAA-IgA to the remaining patients with anti-TG2 levels comprised between 4 and 10 times upper limit of normal (ULN) allowed the detection of further 20 patients with a Marsh 3 damage. In our prospective study, algorithm-1 identified 23 out of 50 patients, whilst further 7 were recognized adding AAA-IgA. We confirm that algorithm-1 may avoid the duodenal biopsy in many CD patients and underscores the usefulness of AAA-IgA in reducing the number of duodenal biopsies in patients with moderate anti-TG2 levels.

Genetic variants regulating immune cell levels in health and disease.

The complex network of specialized cells and molecules in the immune system has evolved to defend against pathogens, but inadvertent immune system attacks on "self" result in autoimmune disease. Both genetic regulation of immune cell levels and their relationships with autoimmunity are largely undetermined. Here, we report genetic contributions to quantitative levels of 95 cell types encompassing 272 immune traits, in a cohort of 1,629 individuals from four clustered Sardinian villages. We first estimated trait heritability, showing that it can be substantial, accounting for up to 87% of the variance (mean 41%). Next, by assessing ∼8.2 million variants that we identified and confirmed in an extended set of 2,870 individuals, 23 independent variants at 13 loci associated with at least one trait. Notably, variants at three loci (HLA, IL2RA, and SH2B3/ATXN2) overlap with known autoimmune disease associations. These results connect specific cellular phenotypes to specific genetic variants, helping to explicate their involvement in disease.

High meat consumption is associated with type 1 diabetes mellitus in a Sardinian case-control study.

The large worldwide variation in type 1 diabetes incidence and increasing incidence over time points toward important environmental risk factors. Among them, nutrition plays an important role. The objective was to investigate the relationship between type 1 diabetes and nutritional factors in pregnancy and early in life. We carried out, using semi-quantitative food frequency questionnaires, a retrospective case-control study in 298 children of 0-15 years old, 145 of which were affected by type 1 diabetes. The diet of all children and of their mothers during pregnancy and lactation was assessed. In children, a statistically significant dose-response association between type 1 diabetes and the amount of meat consumption was found while no other nutritional factors were associated with the disease. High meat consumption seems to be an important early in life cofactor for type 1 diabetes development, although these findings need to be confirmed in wider prospective follow-up studies.

Autoimmune polyendocrine syndrome type 1: an extensive longitudinal study in Sardinian patients.

CONTEXT: Autoimmune polyendocrine syndrome type 1 (APS1) is a childhood-onset monogenic disorder caused by mutations in the autoimmune regulator (AIRE) gene, including the distinctive R139X in Sardinia. Its rarity and great variability in manifestations/onset ages make early diagnosis difficult. To date, very few longitudinal studies of APS1 patients have been reported. OBJECTIVE: The aim of this study was to describe the features and clinical course of APS1 and correlate them with AIRE and HLA class II genotypes in a large homogeneous cohort of Sardinian patients followed for up to 25 yr. PATIENTS: Twenty-two pediatric APS1 patients were studied prospectively. RESULTS: This Sardinian series (female/male ratio, 1.44; median current age, 30.7 yr; range, 1.8-46 yr) showed early disease onset (age range, 0.3-10 yr; median, 3.5 yr) and severe phenotype (on average, seven manifestations per patient). Besides the classic triad of chronic mucocutaneous candidiasis, hypoparathyroidism, and Addison's disease, autoimmune hepatitis was a serious and surprisingly common/early/presenting feature (27%; two deaths), with a 5:1 female bias (median age, 6 yr; range, 2.5-11 yr). By contrast, type 1 diabetes was rare (one patient), and hypothyroidism was not seen. Additional disease components (several of them potentially life-threatening) appeared in adulthood. The major nonsense mutation, R139X, was found in 93% of the mutant AIRE alleles. High-titer interferon (IFN)-ω and IFN-α autoantibodies were detected in all patients tested, even preclinically at 4 months of age in one sibling. HLA alleles appear to influence the exact phenotype-the most interesting apparent association being between HLA-DRB1*0301-DQB1*0201, liver-kidney microsome autoantibodies (anti-CYP1A2), and autoimmune hepatitis. CONCLUSION: APS1 in Sardinia is characterized by severe phenotype, marked clinical heterogeneity, and relative genetic homogeneity. The single AIRE mutation, R139X, and the anti-IFN-ω and IFN-α autoantibodies are helpful for earlier diagnosis, especially when APS1 presents unusually. HLA genotypes can modify the phenotype.

High frequency of low-risk human leukocyte antigen class II genotypes in latent celiac disease.

Human leukocyte antigen (HLA) class II genotypes in latent celiac disease, a clinical variant of celiac disease (CD) have been scarcely studied. The aim of this work was to investigate whether latent CD and CD share similar frequencies of HLA class II genotypes. HLA class II genotypes of CD patients compared with controls were subdivided in the following at-risk groups: DQB1*02/DQB1*02 (43.0%, odds ratio [OR] 8.02, p < 0.0001), DQB1*0302/DQB1*02 (12.2%, OR 2.77, p = 0.0002), DQB1*02/DQB1*X (39.2%, OR 1.23, p = 0.1903), DQB1*0302/DQB1*X (3.4%, OR 0.35, p = 0.0064) and DQB1*X/DQB1*X (0.8%, OR 0.01, p = 0.0001) where X is neither DQB1*0302 nor DQB1*02. Next, HLA class II genotypes of 21 latent CD patients were compared with the above at-risk groups. Only one latent CD patient (4.8%) was found in the high risk DQB1*02/DQB1*02 group, three (14.3%) were DQB1*0302/DQB1*02, one (4.8%) was DQB1*0302/DQB1*X and the remaining 16 (76.2%) showed the DQB1*02/DQB1*X genotype. Noteworthy, the only 1 patient with the DQB1*02/DQB1*02 high risk genotype did not carry the DR3-DQB1*02/DR3-DQB1*02 or the DR3-DQB1*02/DR7-DQB1*02 but the uncommon DR3-DQB1*02/DR4-DQB1*02 genotype. These data suggest that latent CD is prevalently associated with low-risk HLA class II genotypes.

Prevalence of autoimmune thyroiditis in children with celiac disease and effect of gluten withdrawal.

OBJECTIVE: To study the prevalence of autoimmune thyroiditis (AT) in Sardinian children with celiac disease (CD) and the effects of a gluten-free diet (GFD) on thyroid function. STUDY DESIGN: Children with biopsy-proven CD (n = 324; female:male 2:1; mean age, 6.6 years) followed from 1 to 15 years, were retrospectively evaluated for AT at onset of CD and during GFD. Serum thyroid peroxidase and thyroglobulin antibodies (AbTPO, AbTG), thyroid-stimulating hormone (TSH), free thyroxine (FT4), free triiodothyronine (FT3), and thyroid ultrasonography were considered. Age-matched Sardinian schoolchildren (n = 8040), previously evaluated for antithyroid antibodies and thyroid function, were used as controls. RESULTS: Thirty-four patients with CD (10.5%) developed AT (female:male 4,5:1; mean age, 10.5 years), 11 at onset of CD and 23 during GFD, with a higher prevalence than controls (P = 2.9(-13)). Twenty-eight patients were euthyroid and 6 hypothyroid. AbTPO and/or AbTG persisted elevated for 2 to 9 years despite the GFD in 9 of 11 patients with AT at onset of CD. CONCLUSIONS: AT is strongly associated with CD in Sardinian children, has an age of onset of 10.5 years, and appears to be gluten-independent. In children with CD with AT, the female:male bias reported in adult AT is present before puberty.

Further evidence of a primary, causal association of the PTPN22 620W variant with type 1 diabetes.

OBJECTIVE: The minor allele of the nonsynonymous single nucleotide polymorphism (SNP) +1858C>T within the PTPN22 gene is positively associated with type 1 diabetes and other autoimmune diseases. Genetic and functional data underline its causal effect, but some studies suggest that this polymorphism does not entirely explain disease association of the PTPN22 region. The aim of this study was to evaluate type 1 diabetes association within this gene in the Sardinian population. RESEARCH DESIGN AND METHODS: We resequenced the exons and potentially relevant portions of PTPN22 and detected 24 polymorphisms (23 SNPs and 1 deletion insertion polymorphism [DIP]), 8 of which were novel. A representative set of 14 SNPs and the DIP were sequentially genotyped and assessed for disease association in 794 families, 490 sporadic patients, and 721 matched control subjects. RESULTS: The +1858C>T variant, albeit rare in the general Sardinian population (allele frequency 0.014), was positively associated with type 1 diabetes (P(one tail) = 3.7 x 10(-3)). In contrast, the background haplotype in which this mutation occurred was common (haplotype frequency 0.117) and neutrally associated with disease. We did not confirm disease associations reported in other populations for non +1858C>T variants (rs2488457, rs1310182, and rs3811021), although they were present in appreciable frequencies in Sardinia. Additional weak disease associations with rare variants were detected in the Sardinian families but not confirmed in independent case-control sample sets and are most likely spurious. CONCLUSIONS: We provide further evidence that the +1858C>T polymorphism is primarily associated with type 1 diabetes and exclude major contributions from other purportedly relevant variants within this gene.

Decline of lactase activity and c/t-13910 variant in Sardinian childhood.

OBJECTIVES: Our study aims to determine the age of onset of adult-type hypolactasia in Sardinians, and to establish the age at which genotyping of the C/T-13910 variant can be used reliably in the diagnosis of lactose malabsorption. PATIENTS AND METHODS: A lactose breath hydrogen test was given to 383 randomly selected patients, from 3 to 19 years old. RESULTS: The C/C-13910 genotype was found in 90% of patients; the frequency of the positive lactose breath hydrogen test increased with age and reached a prevalence of 85% at 9 years. CONCLUSIONS: In Sardinians, adult-type hypolactasia becomes phenotypically evident in all individuals older than 9 years, suggesting that this should be considered the minimum age at which the genetic test for lactase nonpersistence should be applied.

Genotyping of the lactase-phlorizin hydrolase c/t-13910 polymorphism by means of a new rapid denaturing high-performance liquid chromatography-based assay in healthy subjects and colorectal cancer patients.

Adult-type hypolactasia results from the progressive decline of lactase-phlorizin hydrolase activity in enterocytes after weaning. Lactase nonpersistence may determine a primary lactose intolerance with reduced diary product consumption, which is possibly related to an increased risk of colon cancer. Recently, a genetic variant C/T(-13910) upstream of the lactase-phlorizin hydrolase (LCT) gene has been strongly correlated with the lactase persistence/nonpersistence trait in both family and case-control studies. The authors validate a denaturing high-performance liquid chromatography (dHPLC)-based assay versus conventional genotype sequencing in detecting the C/T(-13910) polymorphism of LCT and evaluate its prevalence in 2 different Italian geographical areas and in colorectal cancer patients. DNA samples of 157 healthy subjects and 124 colon cancer patients from Apulia and of 97 healthy subjects from Sardinia were evaluated for the C/T(-13910) polymorphism by dHPLC, sequencing, and restriction fragment length polymorphism (RFLP). Under optimized conditions, dHPLC was as sensitive as DNA sequencing and detected a new genetic variant (T/C(-13913)) in 2 individuals that was not identified by RFLP assay. Frequency of lactase nonpersistence genotype (C/C(-13910)) was similar in healthy subjects from 2 different Italian geographical areas and not increased in patients with colorectal cancer. The results indicate that the dHPLC method may be used as a rapid, noninvasive, and labor-saving screening tool for genotyping C/T(-13910) polymorphism, with high success, low cost, and reproducibility.

HLA-DQB1*0201 homozygosis predisposes to severe intestinal damage in celiac disease.

OBJECTIVE: Celiac disease (CD) is a T-lymphocyte-mediated small intestinal enteropathy triggered and maintained by dietary gluten, with a strong genetic component mapping to the HLA genes encoding for the class II DQ(alpha1*0501, beta1*02) molecule. Damage of the small intestine may cause a variety of clinical signs ranging from isolated long-standing iron-deficiency anemia refractory to iron supplementation to forms of severe malnutrition that may become life threatening. However, patients carrying the typical intestinal lesions of CD and presenting no symptoms at all (silent CD) are also a common clinical observation. Since it is commonly assumed that clinical signs and symptoms tend to correlate with the severity of the intestinal damage, the purpose of this study was to investigate whether particular HLA class II genotypes might also influence the extent of intestinal damage and consequently the clinical presentation of the disease. MATERIAL AND METHODS: We retrospectively compared histological grading of celiac disease intestinal biopsies with HLA haplotype, age at onset of disease and clinical signs and symptoms. RESULTS: Our findings showed that homozygosis for the DQB1*0201 allele is associated with a higher severity of the histological score (p<0.008). Of note for the clinician, this work also suggests that the same type 3c of intestinal damage causes a different clinical syndrome, depending on the patient's age. CONCLUSIONS: The genetic predisposition at the HLA-DQB1 locus influences the severity of the mucosal damage in a dose-dependent manner, but not the clinical presentation, of celiac disease.

DRB1*0401-restricted human T cell clone specific for the major proinsulin73-90 epitope expresses a down-regulatory T helper 2 phenotype.

Recently, we have identified proinsulin (P-Ins)(73-90) as an immunodominant T cell epitope of HLA-DRB1*0401 (DR4) subjects with beta-islet cell autoimmunity and of HLA-DR4/CD4 double-transgenic mice immunized with human P-Ins. We have compared the fine specificities of one human CD4 T cell clone and two mouse T cell hybridoma clones recognizing this epitope, and, although these three clones all recognized the same core region (LALEGSLQK), there were major differences in how they interacted with the peptide (p)/HLA complex, reflecting the fact that human P-Ins is a foreign antigen in the mouse and an autoantigen in the type 1 diabetes patient. The human T cell clone was forkhead transcription factor 3 (Foxp3)-positive, a marker for regulatory T cell lineages, and secreted predominantly IL-5, IL-10, and low levels of IFNgamma in response to P-Ins(73-90). This finding is compatible with the previously detected regulatory cytokine pattern in subjects with beta-cell autoimmunity. However, added N- or C-terminal amino acids drastically changed HLA and tetramer binding capacity as well as T cell reactivity and the cytokine phenotype of the P-Ins(73-90)-specific human CD4 T cell clone, suggesting a potential for this P-Ins epitope as a target for therapeutic intervention in HLA-DR4-positive humans with beta-islet cell autoimmunity or recent-onset type 1 diabetes.

Autoimmune-associated lymphoid tyrosine phosphatase is a gain-of-function variant.

A SNP in the gene PTPN22 is associated with type 1 diabetes, rheumatoid arthritis, lupus, Graves thyroiditis, Addison disease and other autoimmune disorders. T cells from carriers of the predisposing allele produce less interleukin-2 upon TCR stimulation, and the encoded phosphatase has higher catalytic activity and is a more potent negative regulator of T lymphocyte activation. We conclude that the autoimmune-predisposing allele is a gain-of-function mutant.

Heterogeneity in the magnitude of the insulin gene effect on HLA risk in type 1 diabetes.

There is still uncertainty concerning the joint action of the two established type 1 diabetes susceptibility loci, the HLA class II DQB1 and DRB1 genes (IDDM1) and the insulin gene (INS) promoter (IDDM2). Some previous studies reported independence, whereas others suggested heterogeneity in the relative effects of the genotypes at these disease loci. In this study, we have assessed the combined effects of the HLA-DQB1/DRB1 and INS genotypes in 944 type 1 diabetic patients and 1,023 control subjects, all from Sardinia. Genotype variation at INS significantly influenced disease susceptibility in all HLA genotype risk categories. However, there was a significant heterogeneity (P = 2.4 x 10(-4)) in the distribution of the INS genotypes in patients with different HLA genotypes. The INS predisposing genotype was less frequent (74.9%) in high-risk HLA genotype-positive patients than in those with HLA intermediate-risk (86.1%) and low-risk (84.8%) categories. Gene-gene interaction modeling led to rejection of the additive model, whereas a multiplicative model showed a better, albeit still partial, fit to the observed data. These genetic results are consistent with an interaction between the protein products of the HLA and INS alleles, in which both the affinity of the various HLA class II molecules for a preproinsulin-derived peptide and the levels of this peptide in the thymus act jointly as key regulators of type 1 diabetes autoimmunity.