Genetic loci linked to type 1 diabetes and multiple sclerosis families in Sardinia.

BACKGROUND: The Mediterranean island of Sardinia has a strikingly high incidence of the autoimmune disorders Type 1 Diabetes (T1D) and Multiple Sclerosis (MS). Furthermore, the two diseases tend to be co-inherited in the same individuals and in the same families. These observations suggest that some unknown autoimmunity variant with relevant effect size could be fairly common in this founder population and could be detected using linkage analysis. METHODS: To search for T1D and MS loci as well as any that predispose to both diseases, we performed a whole genome linkage scan, sequentially genotyping 593 microsatellite marker loci in 954 individuals distributed in 175 Sardinian families. In total, 413 patients were studied; 285 with T1D, 116 with MS and 12 with both disorders. Model-free linkage analysis was performed on the genotyped samples using the Kong and Cox logarithm of odds (LOD) score statistic. RESULTS: In T1D, aside from the HLA locus, we found four regions showing a lod-score > or =1; 1p31.1, 6q26, 10q21.2 and 22q11.22. In MS we found three regions showing a lod-score > or =1; 1q42.2, 18p11.21 and 20p12.3. In the combined T1D-MS scan for shared autoimmunity loci, four regions showed a LOD >1, including 6q26, 10q21.2, 20p12.3 and 22q11.22. When we typed more markers in these intervals we obtained suggestive evidence of linkage in the T1D scan at 10q21.2 (LOD = 2.1), in the MS scan at 1q42.2 (LOD = 2.5) and at 18p11.22 (LOD = 2.6). When all T1D and MS families were analysed jointly we obtained suggestive evidence in two regions: at 10q21.1 (LOD score = 2.3) and at 20p12.3 (LOD score = 2.5). CONCLUSION: This suggestive evidence of linkage with T1D, MS and both diseases indicates critical chromosome intervals to be followed up in downstream association studies.

Sourdough-leavened bread improves postprandial glucose and insulin plasma levels in subjects with impaired glucose tolerance.

Sourdough bread has been reported to improve glucose metabolism in healthy subjects. In this study postprandial glycaemic and insulinaemic responses were evaluated in subjects with impaired glucose tolerance (IGT) who had a meal containing sourdough bread leavened with lactobacilli, in comparison to a reference meal containing bread leavened with baker yeast. Sixteen IGT subjects (age range 52-75, average BMI 29.9 +/- 4.2 kg/ m2) were randomly given a meal containing sourdough bread (A) and a meal containing the reference bread (B) in two separate occasions at the beginning of the study and after 7 days. Sourdough bread was leavened for 8 h using a starter containing autochthonous Saccharomyces cerevisiae and several bacilli able to produce a significant amount of D-and L-lactic acid, whereas the reference bread was leavened for 2 h with commercial baker yeast containing Saccharomyces cerevisiae. Plasma glucose and insulin levels were measured at time 0, 30, 60, 120, and 180 min. In IGT subjects sourdough bread induced a significantly lower plasma glucose response at 30 minutes (p = 0.048) and a smaller incremental area under curve (AUC) delta 0-30 and delta 0-60 min (p = 0.020 and 0.018 respectively) in comparison to the bread leavened with baker yeast. Plasma insulin response to this type of bread showed lower values at 30 min (p = 0.045) and a smaller AUC delta 0-30 min (p = 0.018). This study shows that in subjects with IGT glycaemic and insulinaemic responses after the consumption of sourdough bread are lower than after the bread leavened with baker yeast. This effect is likely due to the lactic acid produced during dough leavening as well as the reduced availability of simple carbohydrates. Thus, sour-dough bread may potentially be of benefit in subjects with impaired glucose metabolism.

No association between variation of the FOXP3 gene and common type 1 diabetes in the Sardinian population.

Mutations of the forkhead/winged helix transcription factor FOXP3 gene on chromosome Xp11.23 cause a rare recessive monogenic disorder called IPEX (immune dysregulation, polyendocrinopathy, including type 1 diabetes, enteropathy, and X-linked syndrome). FOXP3 is necessary for the differentiation of a key immune suppressive subset of T-cells, the CD4+CD25+ regulatory T-cells. Previously, we reported a significant male-female bias in the common, multifactorial form of type 1 diabetes in Sardinia and evidence of linkage of chromosome Xp11 to the disease. These findings indicate that FOXP3 is a prime functional and positional candidate locus for the common form of type 1 diabetes. In the present study, we initially scanned 82 kb of the FOXP3 region for common polymorphisms, including sequencing all of the coding and functionally relevant portions of the gene in 64 Sardinian individuals. Then the most informative polymorphisms in 418 type 1 diabetic families and in 268 male case and 326 male control subjects were sequentially genotyped and tested for disease association. There is no evidence that variants in the FOXP3 regions analyzed are associated with type 1 diabetes and account for the male-female bias observed in Sardinia. Our data indicate that allelic variation in or near the coding regions of the FOXP3 gene does not have a major role in the inherited susceptibility to the common form of type 1 diabetes.

Sex-related bias and exclusion mapping of the nonrecombinant portion of chromosome Y in human type 1 diabetes in the isolated founder population of Sardinia.

A male excess in Sardinian type 1 diabetic cases has previously been reported and was largely restricted to those patients carrying the HLA-DR3/nonDR4 genotype. In the present study, we have measured the male- to-female (M:F) ratio in a sample set of 542 newly collected, early-onset type 1 diabetic Sardinian patients. This data not only confirm the excess of male type 1 diabetic patients overall (M:F ratio = 1.3, P = 3.9 x 10(-3)) but also that the bias in male incidence is largely confined to patients with the DR3/nonDR4 genotype (M:F ratio = 1.6, P = 2.0 x 10(-4)). These sex effects could be due to a role for allelic variation of the Y chromosome in the susceptibility to type 1 diabetes, but to date this chromosome has not been evaluated in type 1 diabetes. We, therefore, established the frequencies of the various chromosome Y lineages and haplotypes in 325 Sardinian male patients, which included 180 cases with the DR3/nonDR4 genotype, and 366 Sardinian male control subjects. Our results do not support a significant involvement of the Y chromosome in DR3/nonDR4 type 1 diabetic cases nor in early-onset type 1 diabetes as a whole. Other explanations, such as X chromosome-linked inheritance, are thus required for the male bias in incidence in type 1 diabetes in Sardinia.

Epitope-restricted 65-kilodalton glutamic acid decarboxylase autoantibodies among new-onset Sardinian type 2 diabetes patients define phenotypes of autoimmune diabetes.

The 65-kDa glutamic acid decarboxylase (GAD65) autoantibodies (GAD65Abs), commonly found in type 1 diabetes mellitus (T1DM) patients, are also found at lower frequencies in type 2 diabetes mellitus (T2DM) patients. GAD65Abs in T1DM patients are epitope specific, in contrast to those found in other GAD65Ab-positive individuals, including T2DM patients. Our aim was to assess whether epitope-specific GAD65Abs, or the additional presence of islet antigen 2 (IA-2) autoantibodies, better define T1DM phenotypes among T2DM patients. GAD65 and IA-2 autoantibodies were analyzed in 1436 Sardinian subjects classified with T2DM and in 384 nondiabetic patient controls. Autoantibody binding specificity to the N-terminal, middle (M), and C-terminal (C) portions of the GAD65 molecule was evaluated. Among the T2DM patients, 5.1% had GAD65 (P < 0.001) and 2.4% had IA-2 autoantibodies, compared with 1.3 and 1.6%, respectively, among the controls. GAD65Ab-positive T2DM patients with M+C (epitope-specific) reactivity were found to have the lowest body mass index (P < 0.001), followed by GAD65Ab/IA-2Ab-positive patients (P < 0.01), and non-M+C-reactive (non-epitope-specific) patients (P < 0.02). In GAD65Ab-positive T2DM patients, c-peptide levels were lower in M+C-reactive compared with non-M+C-reactive patients. Sardinian T2DM patients with M+C-predominant GAD65Ab reactivity have clinical features more similar to those of T1DM patients. Thus, GAD65Ab epitope analysis may help to define T1DM phenotypes among newly diagnosed GAD65Ab-positive patients classified with T2DM.

GAD65 and IA-2 autoantibodies are common in a subset of siblings of Sardinian Type 2 diabetes families.

UNLABELLED: Type 1 diabetes in Sardinia is very common in children, and we hypothesized that Latent Autoimmune Diabetes of Adult (LADA) might constitute a significant proportion of diabetes in adult Sardinian subjects. Since Type 2 diabetes is a familial disorder, we tested this hypothesis by investigating the prevalence of GAD65 and IA-2 autoantibodies (Ab) in Type 2 diabetes multiplex families of Sardinian ancestry enrolled in the Study Group for the Genetics of Diabetes in Sardinia. METHODS: A total of 684 individuals were ascertained from 252 Sardinian Type 2 diabetes multiplex families with 2.4 affected siblings per family comprising 190 families with two affected, 37 with three, 15 with four, 7 with five, and 3 with six, in addition to 80 unaffected siblings. Controls were household contacts representing 204 healthy spouses of affected siblings. Diagnosis was at 35-69 years of age and insulin was not given in the first 4 years after diagnosis. GAD65Ab and IA-2Ab were determined in standard radioligand binding assays. RESULTS: Among affected siblings GAD65Ab were positive in 8.8% of insulin-treated (n = 137; P = 0.0006), in 2.5% of non-insulin-treated (n = 467), and in 1.2% of non-diabetic siblings (n = 80) compared with 0.5% of controls (n = 204). IA-2Ab was positive in 6.6% insulin-treated (P = 0.04), 2.1% non-insulin-treated, and 2.5% non-diabetic siblings compared with 1.5% of controls. CONCLUSION: A high frequency of GAD65Ab and IA-2Ab as markers of Type 1 diabetes was found among Type 2 diabetes siblings from Sardinian multiplex families despite excluding those who had been treated with insulin during the first 4 years of disease. Our data support the hypothesis that LADA may be common in Sardinian Type 2 diabetes and stress the importance of investigating markers of Type 1 diabetes in studies of Type 2 diabetes.

Urinary glycosaminoglycan and proteoglycan excretion in normoalbuminuric patients with type 1 diabetes mellitus.

BACKGROUND: Diabetic nephropathy may be related to an abnormal metabolism of glycosaminoglycans (GAG) in the glomerular basement membrane (GBM). The first manifestation of nephropathy is microalbuminuria, whose appearance indicates a loss of GBM selectivity. The present study evaluated whether GAG excretion becomes abnormal in parallel with microalbuminuria, and whether such abnormalities are also present in normoalbuminuric diabetic patients. METHODS: We measured urinary GAG excretion in 60 patients with type 1 (insulin-dependent) diabetes mellitus and in 22 healthy subjects. GAG were isolated from 24-h urine using ion-exchange chromatography on DEAE Sephacel. GAG composition was determined by cellulose acetate electrophoresis and expressed as percentages by densitometric scanning of Alcian Blue stained strips. RESULTS: On subgrouping for albuminuric status and glyco-metabolic control, we found high urinary GAG concentrations in all except the normoalbuminuric patients with good glyco-metabolic control. The urinary GAG electrophoretic pattern showed alterations in chondroitin sulphate (CS) and heparan sulphate (HS) relative contents. A higher frequency of low sulphated chondroitin sulphate-proteoglycan (LSC-PG) was observed in all patients, including those with normoalbuminuria and good glyco-metabolic control. CONCLUSIONS: This urinary pattern may be indicative of an abnormal GBM metabolism. Since GAG play an important role in GBM permeability, these anomalies might consequently represent a first step towards selective changes of GBM in type 1 diabetes mellitus.

T-cell receptor repertoire analysis in monozygotic twins concordant and discordant for type 1 diabetes.

Several data suggest that stochastic rearrangements of the TCR could play a pathogenic role in both disease predisposition and protection in type 1 diabetes (T1D). As twin sets offer an enormous potential in evaluating the role of genetic and environmental factors in susceptibility to disease, the main goal of this study was to assess whether the degree of sharing of the expressed TCR repertoire of twin pairs discordant for T1D differs from that of disease concordant pairs. We performed our analysis in 5 pairs of monozygotic twins, 3 of which were concordant and 2 discordant for T1D, by combining flow cytometry and CDR3 spectratyping on both CD4+ and CD8+ T-cells. Our data show that TCR repertoires show increased level of concordance within each twin pair, especially in CD8+ cells, in terms of mean BV expression levels on flow cytometry as well as of CDR3 patterns and frequencies of skewed or oligoclonal BV subfamilies on spectratyping. It is worth noting that the degree of similarity among twins seems to be independent of concordance or discordance for T1D. Our findings seem to suggest that in monozygotic twins with T1D the TCR repertoire is influenced by genetic factors more than by the presence of the autoimmune disorder itself.

A longitudinal evaluation of urinary glycosaminoglycan excretion in normoalbuminuric type 1 diabetic patients.

BACKGROUND: Previously, we found high urinary glycosaminoglycan (GAG) concentration, together with an altered electrophoretic pattern, in normoalbuminuric type 1 diabetic subjects with hemoglobin A(1c) (HbA(1c)) > or =8.0%. The purpose of this study was long-term evaluation of GAG excretion variations in these patients compared to those with HbA(1c) < 8.0% at baseline who maintained better metabolic control over time. METHODS: We enrolled 26 normotensive, normoalbuminuric type 1 diabetic patients and divided them into two groups according to mean HbA(1c) levels during the follow-up period. GAGs were isolated from 24-h urine samples on two separate occasions, at baseline and after a mean (+/-SD) follow-up of 6.8+/-1.1 years. RESULTS: All patients remained normoalbuminuric at follow-up, and had normal urinary alpha(1)-microglobulin levels. In patients with HbA(1c) <8.0%, total GAG levels and low sulfated chondroitin sulfate-proteoglycan/chondroitin sulfate ratio were almost unchanged during the follow-up period. In contrast, these increased in patients with HbA(1c) > or =8.0% and were significantly related to both HbA(1c) levels and the duration of poor glycemic control. CONCLUSIONS: Our results show a strong influence of hyperglycemic environment on GAG metabolism in diabetes and indicate that the distribution pattern of urinary GAGs, besides their total concentration, may be predictive of altered GAG metabolism in type 1 diabetes.

Plasma lipid composition and LDL oxidation.

Low-density lipoprotein (LDL) oxidation in vivo depends on lipid composition and on plasma antioxidant status. The aim of our study was to investigate the relationship between plasma lipid composition and LDL oxidation and, in particular, to explore whether LDL-cholesterol/triglycerides ratio (LDL-C/TG) and LDL-cholesterol/high-density lipoprotein (HDL)-cholesterol ratio (LDL-C/HDL-C) can be used as predictive parameters of LDL oxidation in vivo. In 87 volunteers over a wide range of age plasma lipids and LDL oxidation were studied. Blood was collected after 12 h overnight fast. LDL oxidation was estimated by the level of conjugated diene (BDC) in the lipid fraction isolated from plasma after gradient ultra-centrifugation. The results were expressed as micromol/l (BDC/l) to evaluate the level of oxidized LDL, and as nmol of BDC for mg of LDL-cholesterol (BDC/LDL-C) for the evaluation of LDL oxidation degree. BDC/l correlated significantly with age, total and LDL-C, apolipoprotein B and TG, while BDC/LDL-C negatively correlated with total cholesterol, apolipoprotein B, LDL/TG and LDL/HDL ratios. Age of subjects significantly correlated with total and LDL-C and apolipoprotein B. TG have a significant inverse correlation with HDL-C. Our results support the hypothesis that among the several factors involved in LDL oxidation the most important determinants are LDL/TG. Plasma triglycerides appear to be very important even when circulating cholesterol levels are within normal limits. Moreover, we found that the LDL/HDL ratio is also very important with regard to the putative protective role of HDL against LDL oxidation in vivo. In conclusion, plasma lipid parameters must be evaluated not only for their absolute values but also for their mutual ratios as expression of plasma lipid homeostasis. Both LDL/TG and LDL/HDL ratios can be used as predictive parameters of in vivo LDL oxidation.

The Trp64Arg beta3-adrenergic receptor amino acid variant confers increased sensitivity to the pressor effects of noradrenaline in Sardinian subjects.

The beta(3)-adrenergic receptor (beta(3)AR) plays a critical role in lipid metabolism, and thus alterations in its function may be involved in the metabolic syndrome. Indeed, we have found previously that the Trp64Arg amino acid variant of the beta(3)AR is associated with hypertension and higher serum triacylglycerol levels in the Sardinian population. The aim of the present study was to evaluate the effect of the Trp64Arg beta(3)AR variant on the regulation of triacylglycerol levels and blood pressure during the exogenous infusion of noradrenaline. We studied groups of non-diabetic normotensive subjects: eight with the wild-type Trp64Trp beta(3)AR and eight with the Trp64Arg variant. The subjects each received, on two different days (randomized, double-blind fashion), a 4 h infusion of either noradrenaline (0.147 nmol.min(-1).kg(-1)) or Emagel (subjects had fasted for at least 12 h). The only available subject with a homozygous mutant Arg64Arg beta(3)AR was also studied. Blood pressure was measured every 10 min using a sphygmomanometer, and blood samples were taken every 30 min from the contralateral vein for biochemical determinations. After a 4 h noradrenaline infusion, significant increases in diastolic blood pressure (from 83+/-2 to 91+/-3 mmHg; P <0.01) and serum triacylglycerol levels (from 1.69+/-0.4 to 1.79+/-0.6 mmol/l; P <0.05) were observed compared with basal values in subjects with the Trp64Arg beta(3)AR variant, whereas subjects with the Trp64Trp beta(3)AR did not show any significant change over the infusion period. Glycaemia had increased significantly only at the end of the first 1 h of infusion in subjects with the Trp64Arg variant (from 5.0+/-0.1 to 5.8+/-0.3 mmol/l; P <0.05), with no significantly different behaviour compared with those subjects with the Trp64Trp beta(3)AR during the remaining infusion period. The effects of noradrenaline infusion were more marked in the subject with the Arg64Arg variant. In conclusion, our data indicate that the Trp64Arg amino acid variant of the beta(3)AR confers increased sensitivity to the pressure effect of noradrenaline. Moreover, this variant also influences blood triacylglycerol levels and, to a degree, glucose metabolism.

Autoantibodies against oxidized low-density lipoprotein (ox-LDL) and LDL oxidation status.

Oxidized low-density lipoproteins (ox-LDLs) and their autoantibodies (OLAB) are involved in the development of atherosclerosis in animal models, but their role in humans is still not clear. For this reason we studied 54 patients with beta-thalassemia major (TM), as a model of chronically low circulating LDLs with a high level of oxidation; 44 patients with primary hypercholesterolemia, as model of chronically high circulating LDLs; 24 type 2 diabetic mellitus patients (T2DM) before and after 3 months of atorvastatin treatment (20 mg/day), as a model of acute changes in circulating LDLs; and 41 normolipidemic subjects as a control group. ox-LDLs were measured by the determination of baseline diene concentration in the plasma LDL lipidic fraction after 12 hours fasting and were expressed as the amount of conjugated dienes/ liter (BDC/I) or BDC/LDL-cholesterol (LDL-C), which indicate respectively LDL oxidation degree and status. OLAB were determined using an enzyme immunoassay and related to LDL oxidation degree (BDC/I). In TM, BDC/I was lower, while BDC/LDL-C was significantly higher, compared to both hypercholesterolemia and normolipidemic subjects. Patients with hypercholesterolemia had higher BDC/I, but lower BDC/LDL-C and OLAB/BDC-I, than normolipidemic subjects. In T2DM patients at diet, BDC/LDL-C and OLAB/BDC-I were lower than in normolipidemic subjects. After 3 months of atorvastatin treatment, BDC/ LDL-C and OLAB/BDC-I ratios increased. When all patients were evaluated together, a significant inverse correlation was evident between OLAB and either LDL or BDC/I. Our findings suggest that a relationship between OLAB titer and oxidation indices (BDC/I and BDC/LDL-C) does exist and we may speculate that an increase in OLAB/BDC-I ratio might be protective against the risk of atherosclerosis.

Insulin sensitivity correlates with glycogen synthesis rate, but not with von Willebrand factor in type 2 diabetes.

BACKGROUND: Whether insulin resistance in type 2 (non-insulin-dependent) diabetes is due to compromised endothelial insulin migration or to impaired intracellular hormone action or both is unclear. Coexistent microalbuminuria reflects possible endothelial pathogenesis in insulin resistance. METHODS: Insulin sensitivity (S(I)) was calculated from an intravenous glucose tolerance test in 23 type 2 albuminuric (AER+), 11 type 2 normoalbuminuric (AER-), and 17 control subjects. Cultured fibroblasts from skin biopsies from these subjects were used to study intracellular insulin action on glycogen synthesis. Endothelial damage in type 2 diabetes was evaluated by plasma concentrations of von Willebrand factor (vWf). Results: S(I) and glycogen synthesis in fibroblasts were lower in AER+ and AER- than in controls. Glycogen synthesis in vitro was related to S(I) in vivo (r=0.55, P<0.001). vWf was 169+/-12% in AER+ and 140+/-5% in AER-, P<0.051. No correlation was observed between vWf and S(I) or plasma insulin clearance. CONCLUSIONS: This study demonstrates that reduced insulin-mediated glucose removal in type 2 diabetes is strictly associated with a decreased glycogen synthesis of cultured skin fibroblasts in vitro, but not with markers of endothelial damage in vivo.

Autosomal recessive hypercholesterolaemia in Sardinia, Italy, and mutations in ARH: a clinical and molecular genetic analysis.

BACKGROUND: Autosomal recessive hypercholesterolaemia (ARH) is caused by mutations in a putative adaptor protein called ARH. This recessive disorder, characterised by severe hypercholesterolaemia, xanthomatosis, and premature coronary artery disease, is rare except on the island of Sardinia, Italy. Our aim was to ascertain why ARH is more common on Sardinia than elsewhere. METHODS: We obtained detailed medical histories, did physical examinations, measured concentrations of lipoproteins, and harvested genomic DNA from 28 Sardinians with ARH from 17 unrelated families. We sequenced the coding regions and consensus splice sites of ARH in probands from these families, and from 40 individuals of non-Sardinian origin who had an autosomal recessive form of hypercholesterolaemia of unknown cause. FINDINGS: Two ARH mutations, a frameshift mutation (c432insA) in exon 4 (ARH1) and a nonsense mutation (c65G-->A) in exon 1 (ARH2), were present in all of the 17 unrelated families with ARH. Three of the ARH alleles contained both mutations, as a result of an ancient recombination between ARH1 and ARH2. No regional clustering of the three mutant alleles within Sardinia was apparent. Furthermore, four Italians from the mainland with autosomal recessive hypercholesterolaemia were homozygous for ARH1. INTERPRETATION: The small number, high frequency, and dispersed distribution of ARH mutations on Sardinia are consistent with these mutations being ancient and maintained in the Sardinian population because of geographic isolation.