Mauro Congia

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.

Deferoxamine-induced growth retardation in patients with thalassemia major

In the retrospective study reported here, we compared the longitudinal growth in three groups of children with thalassemia major who received a similar transfusion program but different schedules of chelation treatment. In those patients who initiated deferoxamine (DF) administration by daily subcutaneous infusion (50 to 80 mg/kg/day) simultaneously with the beginning of transfusion (at 8 +/- 6 months), mean height at 2 to 6 years of age was significantly reduced in comparison (1) with those patients who initiated DF subcutaneous treatment after 3 years at similar doses and (2) with those who were treated intramuscularly with small doses. In the patients treated at an early stage, those with more marked stunted growth had a clinical and radiologic ricketslike syndrome associated with joint stiffness. Mineral metabolism studies in these patients showed a reduction of hair and leukocyte zinc levels and leukocyte alkaline phosphatase activity. Our findings indicate that DF administration at high doses by continuous infusion before iron overload has been established adversely affects longitudinal growth. By contrast, after 3 years of age, even large doses (in the order of 100/mg/kg/day) did not result in growth retardation. The growth retardation observed may be related to chelation of other trace elements, including zinc, in the presence of low iron burden, to the direct toxic effect of unchelated DF by interference with critical iron-dependent enzymes, or both. These results indicate that in patients with thalassemia major, DF administration should be initiated only after iron accumulation is established, namely, around 3 years of age, after 20 to 30 transfusions, which are usually associated with ferritin levels in the range of 800 to 1000 ng/ml. At this age, deferoxamine doses should be established on the basis of iron balance studies and dose response curves. Doses higher than 50 to 60 mg/kg do not adversely affect growth but produce toxic side effects on acoustic and visual pathways and therefore should not be used. Longitudinal growth monitoring of DF-treated patients is warranted.

Effect of iron overload on the response to recombinant interferon-alfa treatment in transfusion-dependent patients with thalassemia major and chronic hepatitis C

The purpose of this study was to determine whether interferon-alfa (IFN-alpha) therapy benefits patients with transfusion-dependent thalassemia and chronic active hepatitis C, and whether their iron burden modifies the response to this therapy. We conducted a controlled trial of recombinant IFN-alpha (3 million units per square meter of body surface area, three times a week for 15 months) in 65 patients with thalassaemia major and chronic active hepatitis C; 14 of them were untreated control subjects. In 21 of the 51 treated patients, alanine aminotransferase values returned to normal within 6 months, and hepatitis C virus ribonucleic acid was no longer detected in serum; no changes were detected among control subjects. The response to IFN-alpha therapy was inversely related (p < 0.002) to the liver iron burden as assessed by atomic absorption, the histologic semiquantitative method, or both methods. During 3 years of follow-up, two responder patients had relapses. We conclude that IFN-alpha represents a useful therapeutic option for children with transfusion-dependent thalassemia and chronic active hepatitis C with a mild to moderate iron burden.

Combinations of specific DRB1, DQA1, DQB1 haplotypes are associated with insulin-dependent diabetes mellitus in sardinia

The Sardinian population has an extremely high incidence of IDDM (30.2 of 100.000 in the age group of 0-14 years). This study reports the molecular characterization of HLA class II genes in 120 IDDM sporadic patients and 89 healthy subjects of Sardinian origin. Compared with other Caucasians, both Sardinian patients and controls had an unusual distribution of haplotypes and genotypes. In particular, there was a high gene frequency of the DRB1*0301, DQA1*0501, DQB1*0201 susceptibility haplotype both in patients (0.58) and controls (0.23) while a reduction of the DRB1*1501, DQA1*0102, DQB1*0602 protective haplotype (0.03) was observed in the healthy population. This distribution may partially explain the high incidence of IDDM reported in Sardinia. The analysis of the DQ beta 57 and DQ alpha 52 residues showed that the absence of Asp 57 and the presence of Arg 52 were associated with IDDM in a dose-response manner. On the other hand, we found that (a) a very similar distribution of these residues was found when comparing Sardinians with another healthy Caucasian population from the same latitude but with a lower rate of IDDM incidence; (b) several genotypes encoding the identical DQ alpha 52/DQ beta 57 phenotype carried very different relative risks; and (c) the DRB1*0403, DQA1*0301, DQB1*0304 haplotype (DQ beta 57 Asp-neg and DQ alpha 52 Arg-pos) was found in 40% of the DR4-positive controls but not in patients (p = 0.00034), while the DRB1*0405, DQA1*0301, and DQB1*0302 haplotype carrying the same residues at the same positions was found in 70% of the DR4-positive patients and in only one control (p = 0.00003). These findings suggest that IDDM susceptibility cannot be completely explained by the model in which only DQ alpha 52 and DQ beta 57 residues are taken into account.

A gene dosage effect of the DQA1*0501/DQB1*0201 allelic combination influences the clinical heterogeneity of Celiac disease

This study reports the HLA-DR and DQ molecular characterization of 62 CD patients of Sardinian descent. Patients were divided in two groups (36 in group I and 26 in group II) according to the clinical features at the disease onset. Among the patients of group I, having the fully expressed form of CD and a mean age of 3 years at disease onset, a significant increase of DRB1*0301, DQA1*0501, DQB1*0201 homozygotes, encoding in cis two DQ (alpha 1*0501, beta 1*0201) susceptibility heterodimers, was observed when compared either with the patients of group II (pIII < 0.012) or with healthy individuals (pI < 10(-6)). On the other hand, in the patients of group II, presenting oligosymptomatic forms and a mean age of 5.7 years at the disease onset, the haplotype combinations encoding in cis or in trans only one DQ (alpha 1*0501, beta 1*0201) heterodimer were significantly increased in comparison either with the patients of group I (pIII < 0.026) or with controls (pII < 10(-6)). These findings suggest that a double dose of DQA1*0501, DQB1*0201 genes may predispose a person to an earlier onset and to more severe disease manifestations. Genotype analysis showed that only three patients (all in group I) failed to form in trans or in cis the DQ (alpha 1*0501, beta 1*0201) heterodimer and carried the DQA1*0101,DQB1*0501 haplotype, suggesting its possible role in CD susceptibility. In addition, a significant increment of DQB1*0501 gene (pc < 0.0065) was found comparing the frequency of DQB1 alleles in CD patients and healthy controls, after exclusion of DQB1*0201 chromosomes.(ABSTRACT TRUNCATED AT 250 WORDS)

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 Addisons 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.

Enterocyte actin autoantibody detection: a new diagnostic tool in celiac disease diagnosis: results of a multicenter study.

OBJECTIVES:This study describes a new method to detect autoantibodies against actin filaments (AAA) as a serological marker of intestinal villous atrophy (IVA) in celiac disease (CD), and reports the results of an Italian double-blind multicenter study.METHODS:IgA-AAA were analyzed by immunofluorescence using a newly developed method based on intestinal epithelial cells cultured in presence of colchicine. IgA-AAA were blindly evaluated prospectively in 223 antiendomysial antibody (AEA) and/or antitransglutaminase antibody (TGA) positive subjects and in 78 AEA and TGA negative subjects. IgA-AAA positive patients underwent an intestinal biopsy to confirm the diagnosis. Moreover, IgA-AAA were retrospectively investigated in 84 biopsy-proven CD patients and in 2,000 new consecutively collected serum samples from AEA and TGA negative nonbiopsied subjects.RESULTS:IgA-AAA were positive in 98.2% of the CD patients with flat mucosa, in 89% with subtotal villous atrophy, and in 30% with partial villous atrophy. IgA-AAA were present in none of the AEA and TGA negative nonbiopsied controls. In AEA and/or TGA positive CD patients IgA-AAA positivity significantly correlated with IVA (p < 0.000 in the prospective study, p= 0.005 in the retrospective study). In the prospective study, the values of sensitivity, specificity, the positive predictive value, the negative predictive value, and the efficiency of the IgA-AAA test to identify patients with IVA were, respectively, 83.9%, 95.1%, 97.8%, 69.2%, and 87.0%. Furthermore, a significant correlation (p < 0.0001) was found between the IgA-AAA serum titre and the degree of IVA (rs 0.56).CONCLUSIONS:The results of this multicenter study show that the new method for IgA-AAA detection could represent a practical diagnostic tool in AEA and/or TGA positive subjects, which would be especially useful when IVA shows a patchy distribution, when the histological picture is difficult to interpret, or when a biopsy could represent a life-threatening risk.

Overexpression of the Cytokine BAFF and Autoimmunity Risk

BACKGROUND Genomewide association studies of autoimmune diseases have mapped hundreds of susceptibility regions in the genome. However, only for a few association signals has the causal gene been identified, and for even fewer have the causal variant and underlying mechanism been defined. Coincident associations of DNA variants affecting both the risk of autoimmune disease and quantitative immune variables provide an informative route to explore disease mechanisms and drug‐targetable pathways. METHODS Using case–control samples from Sardinia, Italy, we performed a genomewide association study in multiple sclerosis followed by TNFSF13B locus–specific association testing in systemic lupus erythematosus (SLE). Extensive phenotyping of quantitative immune variables, sequence‐based fine mapping, cross‐population and cross‐phenotype analyses, and gene‐expression studies were used to identify the causal variant and elucidate its mechanism of action. Signatures of positive selection were also investigated. RESULTS A variant in TNFSF13B, encoding the cytokine and drug target B‐cell activating factor (BAFF), was associated with multiple sclerosis as well as SLE. The disease‐risk allele was also associated with up‐regulated humoral immunity through increased levels of soluble BAFF, B lymphocytes, and immunoglobulins. The causal variant was identified: an insertion–deletion variant, GCTGT→A (in which A is the risk allele), yielded a shorter transcript that escaped microRNA inhibition and increased production of soluble BAFF, which in turn up‐regulated humoral immunity. Population genetic signatures indicated that this autoimmunity variant has been evolutionarily advantageous, most likely by augmenting resistance to malaria. CONCLUSIONS A TNFSF13B variant was associated with multiple sclerosis and SLE, and its effects were clarified at the population, cellular, and molecular levels. (Funded by the Italian Foundation for Multiple Sclerosis and others.)

Serum lipoproteins and cancer

BACKGROUND AND AIMS In haematological and solid tumours the blood lipoprotein profile has been reported to be altered; while decreased levels of total cholesterol and increased values of triglycerides have been observed. The mechanism and meaning of these changes are, however, not fully understood. The aim of the present study was to determine relationships between cancer progression and serum lipoproteins. METHODS AND RESULTS We performed a case-control study. We included cancer patients admitted to the 1st Division of Medical Oncology, Businco Hospital of Cagliari, Italy, between 1984 and 1998; 519 patients with any type of solid tumours and 928 healthy controls. We considered total cholesterol (C), high-density lipoprotein (HDL)-C, low-density lipoprotein (LDL)-C, triglycerides and apolipoprotein A-1; other parameters examined were glycaemia, insulinaemia, body mass index (BMI), homeostasis model assessment-estimated insulin resistance (HOMA-IR), C reactive protein (CRP) and tumour necrosis factor-alpha (TNF-alpha). In the cancer group HDL-C and apolipoprotein A-1 were lower (p<0.05) and triglycerides were higher (p<0.05) than in controls; HDL-C (mg/dl) females: 48 vs. 64; males, 40 vs. 52; Apo-A-1 (mg/dl) females: 125 vs. 173; males, 120 vs. 152; triglycerides (mg/dl) females: 133 vs. 96; males, 152 vs. 117. Glucose (mg/dl) was lower in the cancer group (p<0.05); females, 72.3 vs. 80.0; males, 75.7 vs. 78.4. CONCLUSION Using multivariate analysis we were able to rule out cardiovascular and inflammatory diseases as causes of low HDL-C, and also demonstrate that these alterations can be shown as a specific consequence of the presence of a malignant tumour with a diagnostic and prognostic significance.

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.