Vittorio Tassi

A Functional Variant of the Adipocyte Glycerol Channel Aquaporin 7 Gene Is Associated With Obesity and Related Metabolic Abnormalities

Aquaporin 7 (AQP7), the gateway protein controlling glycerol release, has recently emerged as a modulator of adipocyte metabolism. AQP7 knockout mice develop obesity and hyperglycemia. The contribution of AQP7 to these abnormalities in humans is unknown. We examined whether common single nucleotide polymorphisms (SNPs) in the AQP7 gene modulate the risk of obesity and related abnormalities. Among several SNPs we identified, A-953G in the AQP7 promoter was associated with type 2 diabetes in 977 (530 female/447 male) Caucasians: odds ratio for XG (i.e., AG+GG) versus AA individuals was 1.36 (95% CI 1.01–1.84), P = 0.04. This finding was entirely due to the association among females (1.8 [1.2–2.6], P = 0.004), which was no longer significant when adjusted for BMI. In fact, BMI was higher in XG than in AA females (30.8 ± 6.6 vs. 28.9 ± 5.2, P = 0.002). This association was confirmed in independent case-control study (n = 299 female subjects) for morbid obesity (1.66 [1.01–2.74], P = 0.04). Luciferase and mobility shift assays showed that, compared with −953A, the −953G promoter had reduced transcriptional activity (P = 0.001) and impaired ability to bind CCAAT/enhancer binding protein (C/EBP)β transcription factor (P = 0.01). Finally, AQP7 expression in adipose tissue decreased from AA to AG to GG individuals (P = 0.036). These data strongly suggest that AQP7 downregulation is pathogenic for obesity and/or type 2 diabetes.

Variants of the interleukin-10 promoter gene are associated with obesity and insulin resistance but not type 2 diabetes in caucasian italian subjects

Interleukin (IL)-10 is a major anti-inflammatory cytokine that has been associated with obesity and type 2 diabetes. The three polymorphisms −1082G/A, −819C/T, and −592C/A in the IL10 promoter were reported to influence IL10 transcription. We investigated whether these polymorphisms were associated with type 2 diabetes and related traits in a cohort of Italian Caucasians comprising 551 type 2 diabetic and 1,131 control subjects. The −819C/T and −592C/A polymorphisms were in perfect linkage disequilibrium (r2 = 1.0). The −1082G/A polymorphism was not associated with type 2 diabetes or related traits. Although the −592C/A polymorphism was not associated with type 2 diabetes, nondiabetic homozygous carriers of the A allele showed increased BMI and insulin resistance and lower plasma IL-10 levels compared with the other genotypes. In the nondiabetic group, the ATA haplotype was associated with an increased risk for obesity (odds ratio 1.28 [95% CI 1.02–1.60]; P = 0.02). The ATA/ATA composite genotype was associated with an increased risk for obesity (1.96 [1.16–3.31]; P = 0.01) and insulin resistance (1.99 [1.12–3.53]; P = 0.01). This study suggests that polymorphisms and haplotypes of the IL10 promoter may be associated with obesity and insulin resistance in a large sample of Italian Caucasians.

Interaction Between PPARγ2 Variants and Gender on the Modulation of Body Weight

Conflicting results have been reported regarding the effect of the peroxisome proliferator‐activated receptor‐γ−2 (PPARγ2) Pro12Ala polymorphism, (singly or in combination with the silent C1431T polymorphism) on BMI. Gender‐based dimorphism has been evidenced for genes that affect BMI, but few and conflicting data are available regarding PPARγ2. We sought to investigate whether the Pro12Ala interacts with gender in modulating BMI in 566 nondiabetic unrelated white subjects (men:women = 211:355, age 36.59 ± 11.85; BMI 25.36 ± 4.53). In the whole study population, BMI, fasting glucose and insulin levels, and lipid profile were similar in Ala12 carriers (i.e., XA) and Pro/Pro homozygous subjects. Among the men, but not among the women, X/Ala individuals showed higher BMI (25.9 ± 3.6 vs. 28.2 ± 4.9, P = 0.006) and risk of obesity (odds ratio = 2.85, 95% confidence interval = 1.07–7.62). A significant gene‐gender interaction in modulating BMI was observed (P = 0.039). Among the men, but not among the women, those carrying Ala‐T haplotype (i.e., containing both Ala12 and T1431 variants) showed the highest BMI (haplo‐score = 3.72, P = 0.0014). Our data indicate that in whites from Italy the PPARγ2 Pro12Ala polymorphism interacts with gender in modulating BMI, thereby replicating some, but not all, earlier data obtained in different populations. Whether the PPARγ2‐gender interaction is a general phenomenon across different populations, is still an open question, the answer to which requires additional, specifically designed, studies.

Genetics of Specific Phenotypes of Congenital Hypothyroidism: A Population-Based Approach

Congenital hypothyroidism (CH) may cause severe and irreversible neurologic and developmental abnormalities when not recognized early. Many millions of newborns have now been screened and many thousands of patients with CH have been identified. Approximately 80%-85% have defects of thyroid gland development, while 15%-20% have congenital errors of thyroid hormone biosynthesis. An entire population screened for CH over a long period of time, was studied in the present report, using a population-based approach. In particular, two CH phenotypes, both presenting with in situ thyroid gland (patients with either goiter or with thyroid gland volume ranging from normal to hypoplasic) were analyzed. Mutations were searched in some of the most likely candidate genes: thyroperoxidase (TPO) in patients with CH goiter, Pax8 and thyrotropin receptor (TSHR) in the other group. In the former group (n = 8), four TPO gene mutations were identified in three patients. One patient was a compound heterozygous. In two cases an already described mutation (1277(insGGCC)) was present; in two other cases mutations not previously described (1996(G-->T) and 2295(G-->A)), which induced aminoacid variations with a Glu --> Stop and Val --> Ile changes, respectively, were identified. In all patients mutations were inherited from one of the parents. In the case of the compound heterozygous patient, one mutation was inherited from the mother (1277(insGGCC)) and the other from the father (1996(G-->T), Glu --> Stop). In the latter group (n = 8), a patient with a 16-base pair C(T)(13)CC deletion in TSHR gene intron 8, 42-bp distal to exon/intron 8 splice junction, was identified. No mutation was identified in Pax8 gene.

ACE, PAI-1, Decorin and Werner Helicase Genes are not Associated with the Development of Renal Disease in European Patients with Type 1 Diabetes

Genetic factors are involved in the development of diabetic nephropathy in Type 1 diabetes. We have examined the association of four candidate genes, angiotensin converting enzyme (ACE): insertion/deletion (I/D) polymorphism, plasminogen activator inhibitor‐1 (PAI‐1): 4G/5G polymorphism, decorin: 179/183/185 polymorphism and Werner syndrome helicase: C/R polymorphism, with the presence of diabetic nephropathy in Type 1 diabetic patients.

Cloning and sequence analysis of human thyroid transcription factor 1

The thyroid transcription factor 1 (TTF-1) is a homeodomain-containing transcription factor that activates the transcriptional activity of thyroid-specific gene promoters by binding to them. Hence, TTF-1 is crucial in the maintenance of the thyroid differentiation phenotype. The authors isolated and analysed the human TTF-1 gene, which shows a striking homology with the rat TTF-1 gene.

High insulin levels do not influence PC-1 gene expression and protein content in human muscle tissue and hepatoma cells

To verify whether insulin levels influence PC‐1 tissue content, we studied PC‐1 gene expression and protein content in skeletal muscle of patients with insulinoma, a model of primary hyperinsulinemia. Data were compared with those obtained in matched insulin sensitive or resistant healthy subjects. In addition, the effect of high insulin concentration on PC‐1 protein content was studied in HepG2 cells.

The Decorin Gene 179 Allelic Variant Is Associated with a Slower Progression of Renal Disease in Patients with Type 1 Diabetes

Background genetic factors may influence the variability in the rate of progression of kidney disease in type 1 diabetes. In diabetes, progressive mesangial matrix expansion and glomerular sclerosis are, to a large extent, mediated by TGF-β1. Decorin, a proteoglycan which is a component of the extracellular matrix, regulates TGF-β1 activity and expression. We have examined the relationship between the 179/183/185 polymorphism of the Decorin gene and the progression of diabetic nephropathy. Methods: From a cohort of 175 European patients with diabetic nephropathy, we studied 79 patients who were selected because they had a follow-up of at least 2 years (average 6.5 years; range: 2.5–15 years), and regular measurements of serum creatinine on 5 or more occasions. Creatinine clearance (CrCl) calculated from serum creatinine concentration was used as a measure of derived glomerular filtration rate (dGFR). All patients were on antihypertensive therapy. Results: The rate of dGFR decline in the whole cohort was [median (range)] 4.6 (–3.8 to 18) ml/min/year. No patient with 185 allele was found. Patients with 179/183 and 179/179 genotype (n = 14), who were considered together and named 179 carriers, had a slower rate of GFR decline [2.1 (0.06–11.7) ml/min/year] as compared to patients with Decorin 183/183 genotype (n = 65) [5.6 (–3.8 to 18) ml/min/year; p < 0.001]. In addition, when considering individual data, patients carrying the 179 allele had a 3.0 (95%CI: 1.8–4.2)-fold higher probability to be slow progressors (i.e. GFR decline below the median). This difference could not be accounted for by differences in duration of disease, type and duration of antihypertensive therapy, albumin excretion rate, blood glucose or blood pressure control. In a multivariate logistic analysis albumin excretion rate (p < 0.001), mean arterial pressure (p = 0.07) and Decorin gene polymorphism (p = 0.036), but not HbA1c, were independently correlated with the rate of dGFR fall. Conclusion: The 179 allele variant of the Decorin gene is related to a slower progression of DN in type 1 diabetic patients with albuminuria and receiving antihypertensive therapy.

The protein tyrosine phosphatase receptor type f (PTPRF) locus is associated with coronary artery disease in type 2 diabetes

Dear Sir, Insulin resistance is pathogenic for coronary artery disease particularly amongst patients with type 2 diabetes [1]. Protein Tyrosine Phosphatase Receptor Type F (PTPRF), also know as Leukocyte Antigen-Related (LAR), an inhibitor of insulin signalling, is overexpressed in tissues of insulin resistance subjects [2, 3]. Transgenic mice overexpressing PTPRF are insulin resistant [4]. Furthermore, PTPRF gene variability is associated with insulin resistance [5]. All these data suggest that PTPRF may contribute to the pathogenesis of insulin resistance related conditions. Aim of this study was to verify whether variability at the PTPRF locus is associated with coronary artery disease in patients with type 2 diabetes consecutively recruited at the Scientific Institute CSS-San Giovanni Rotondo, Italy, according to the following criteria: type 2 diabetes diagnosed after age 30 years; insulin treatment not required for at least 2 years after type 2 diabetes diagnosis; absence of clinically evident autoimmune disease, as previously described [6]. Of these, 248 were cases (>50% stenosis at coronary angiography and ⁄or previous myocardial infarction) and 344 controls (asymptomatic patients with no signs of myocardial ischaemia at stress ECG or with stenosis £50% at coronary angiography) for coronary artery disease. Clinical features for cases and controls, respectively, were: male ⁄ female ratio: 155 ⁄73 and 140 ⁄193; age: 64 ± 8 and 60 ± 8 years; BMI: 30 ± 4 and 31 ± 5 kg m; HbA1c: 8.6 ± 2 and 8.5 ± 2%; diabetes duration: 14.7 ± 9 and 11.8 ± 8 years; hypertension: 78% and 74%; dyslipidaemia: 90% and 88%; smokers: 44% and 25%. Four polymorphisms (rs11590627, rs2782641, rs10890257 and rs516790), tagging three linkage disequilibrium (LD) blocks and covering the entire PTPRF gene were genotyped. Coronary artery disease was significantly associated with rs2782641 (+A2518G, minor allele frequency = 0.202 in cases vs. 0.137 in controls, P = 0.035). The association was consistent with a recessive model of inheritance, with GG homozigotes having a 50% increased risk of coronary artery disease than major allele carriers (either AA or AG carriers) after adjusting for age, sex and smoking status (Table 1). The association was still significant after adjusting also for other known risk factors such as BMI, hypertension and dyslipidaemia (OR = 1.96, 95% CI = 1.13–3.28, P = 0.016). As rs2782641 resides within intron 3, its biological relevance is questionable. Looking for possible variants in LD with rs2782641, we genotyped six additional polymorphisms (rs6695915, rs651740, rs2842187, rs2819339, rs2842185, rs11580074) in a range of about 14 kb around it. Of an 8 kb area of high LD (r > 0.8), embedding rs2782641, a 450 bp DNA segment, including exons 4 and 5, were, then, re-sequenced in 100 individuals. No additional variants were found, leaving unanswered the question whether rs2782641 is in LD with other an yet unidentified variants. Unfortunately, we didn’t have the opportunity to replicate our positive association in a second sample of type 2 diabetic patients. However, we took advantage from a recently published genomewide-association case–control study for coronary artery disease in the general population performed by the Wellcome Trust Case Control Consortium (WTCCC) [7] whose results are widely accessible at http://www.wtccc.org.uk/summary_stats.shtml. In this study minor allele at rs2782641 was associated with coronary artery disease in sex-differentiated analyses with P-values of either 0.05 or 0.028 when 2000 coronary artery disease cases were compared with either 3000 or 12 000 controls respectively. Thus, despite data obtained in the general population from the WTCCC study do not represent a formal replication,

Characterization of insulin autoantibodies in a patient with autoimmune hypoglycemia

A 60-year-old man referred because of hypoglycemic bouts was found to have insulin autoantibodies. Total plasma insulin was as high as 1.44 nmol/l. Both plasma free insulin and C-peptide were in the normal range. The indirect immunofluorescence technique showed positivity for antinuclear antibodies. The T-lymphocyte populations in the peripheral blood were normal. When serum binding capacity for pork insulin was measured, antibodies binding pork insulin were not detected. The patient’s serum bound 125l-insulin. The binding protein was identified to be an immunoglobulin G. The kinetics of dissociation, studied by the Scatchard analysis of the autoantibody, showed a curvilinear plot, which was analyzed in two components. Cold human insulin was able to compete with 125l-insulin for the antibody binding site (I.C.50=1.35 nmol/ml). These antibodies were apparently not associated with antibodies directed against the insulin receptor.