Diego Bailetti

Loss-of-Function Mutations in APPL1 in Familial Diabetes Mellitus

Diabetes mellitus is a highly heterogeneous disorder encompassing several distinct forms with different clinical manifestations including a wide spectrum of age at onset. Despite many advances, the causal genetic defect remains unknown for many subtypes of the disease, including some of those forms with an apparent Mendelian mode of inheritance. Here we report two loss-of-function mutations (c.1655T>A [p.Leu552(∗)] and c.280G>A [p.Asp94Asn]) in the gene for the Adaptor Protein, Phosphotyrosine Interaction, PH domain, and leucine zipper containing 1 (APPL1) that were identified by means of whole-exome sequencing in two large families with a high prevalence of diabetes not due to mutations in known genes involved in maturity onset diabetes of the young (MODY). APPL1 binds to AKT2, a key molecule in the insulin signaling pathway, thereby enhancing insulin-induced AKT2 activation and downstream signaling leading to insulin action and secretion. Both mutations cause APPL1 loss of function. The p.Leu552(∗) alteration totally abolishes APPL1 protein expression in HepG2 transfected cells and the p.Asp94Asn alteration causes significant reduction in the enhancement of the insulin-stimulated AKT2 and GSK3β phosphorylation that is observed after wild-type APPL1 transfection. These findings-linking APPL1 mutations to familial forms of diabetes-reaffirm the critical role of APPL1 in glucose homeostasis.

Joint Effect of Insulin Signaling Genes on Insulin Secretion and Glucose Homeostasis

CONTEXT Reduced insulin signaling in insulin secreting β-cells causes defective insulin secretion and hyperglycemia in mice. OBJECTIVE We investigated whether functional polymorphisms affecting insulin signaling (ie, ENPP1 K121Q, rs1044498; IRS1 G972R, rs1801278; and TRIB3 Q84R, rs2295490) exert a joint effect on insulin secretion and abnormal glucose homeostasis (AGH). DESIGN Insulin secretion was evaluated by 1) the disposition index (DI) from an oral glucose tolerance test (OGTT) in 829 individuals; 2) insulin secretion stimulation index (SI) in islets from nondiabetic donors after glucose (n = 92) or glibenclamide (n = 89) stimulation. AGH (including impaired fasting glucose and/or impaired glucose tolerance or type 2 diabetes; T2D) was evaluated in case-control studies from the GENetics of Type 2 Diabetes in Italy and the United States (GENIUS T2D) Consortium (n = 6607). RESULTS Genotype risk score, obtained by totaling individual weighted risk allele effects, was associated with the following: 1) DI (P = .005); 2) glucose and glibenclamide SI (P = .046 and P = .009); or 3) AGH (odds ratio 1.08, 95% confidence interval 1.03-1.13; P = .001). We observed an inverse relationship between genetic effect and age at AGH onset, as indicated by a linear correlation between AGH-genotype risk score odds ratios and age-at-diagnosis cutoffs (R(2) = 0.80, P < .001). CONCLUSIONS Functional polymorphisms affecting insulin signaling exert a joint effect on both in vivo and in vitro insulin secretion as well as on early-onset AGH. Our data provide further evidence that abnormal insulin signaling reduces β-cell function and impairs glucose homeostasis.

Genetic variant at the GLUL locus predicts all-cause mortality in patients with type 2 diabetes

Single nucleotide polymorphism (SNP) rs10911021 at the glutamate-ammonia ligase (GLUL) locus has been associated with an increased risk of coronary heart disease in individuals with type 2 diabetes. The effect of this SNP on mortality was investigated among 1,242 white subjects with type 2 diabetes from the Joslin Kidney Study (JKS) (n = 416) and the Gargano Mortality Study (GMS) (n = 826). During a mean follow-up of 12.8 ± 5.8 and 7.5 ± 2.2 years, respectively, a total of 215 and 164 deaths were observed in the two studies. In both cohorts, the all-cause mortality rate significantly increased with the number of rs10911021 risk alleles, with allelic hazard ratios (HRs) of 1.32 (95% CI 1.07–1.64, P = 0.01), 1.30 (1.10–1.69, P = 0.04), and 1.32 (1.12–1.55, P = 0.0011), respectively, in the JKS, the GMS, and the two studies combined. These associations were not affected by adjustment for possible confounders. In the JKS, for which data on causes of death were available, the HR for cardiovascular mortality was 1.51 (1.12–2.04, P = 0.0077) as opposed to 1.15 (0.84–1.55, P = 0.39) for mortality from noncardiovascular causes. These findings point to SNP rs10911021 as an independent modulator of mortality in patients with type 2 diabetes and, together with the previous observation, suggest that this results from an effect of this variant on cardiovascular risk.

IRS1 G972R Missense Polymorphism Is Associated With Failure to Oral Antidiabetes Drugs in White Patients With Type 2 Diabetes From Italy

This study tried to replicate in a large sample of white patients with type 2 diabetes (T2D) from Italy a previously reported association of the IRS1 G972R polymorphism with failure to oral antidiabetes drugs (OAD). A total of 2,409 patients from four independent studies were investigated. Case subjects (n = 1,193) were patients in whom, because of uncontrolled diabetes (i.e., HbA1c >8%), insulin therapy had been added either on, or instead of, maximal or near-maximal doses of OAD, mostly metformin and sulfonylureas; control subjects (n = 1,216) were patients with HbA1c <8% in the absence of insulin therapy. The IRS1 G972R polymorphism was typed by TaqMan allele discrimination. In all samples, individuals carrying the IRS1 R972 risk variant tended to be more frequent among case than control subjects, though reaching statistical significance only in one case. As no IRS1 G972R-by-study sample interaction was observed, data from the four samples were analyzed together; a significant association was observed (allelic odds ratio [OR] 1.30, 95% CI 1.03–1.63). When our present data were meta-analyzed with those obtained in a previous study, an overall R972 allelic OR of 1.37 (1.12–1.69) was observed. This study confirms in a large and ethnically homogeneous sample that IRS1 G972R polymorphism is associated with failure to OAD among patients with T2D.

Joint effect of insulin signaling genes on all-cause mortality

OBJECTIVE We have previously reported the combined effect of SNPs perturbing insulin signaling (ENPP1 K121Q, rs1044498; IRS1 G972R, rs1801278; TRIB3 Q84R, rs2295490) on insulin resistance (IR), type 2 diabetes (T2D) and cardiovascular events. We here investigated whether such a combined effect affects also all-cause mortality in a sample of 1851 Whites of European ancestry. METHODS We investigated a first sample of 721 patients, 232 deaths, 3389 person-years (py). Replication was assessed in two samples of patients with T2D: the Gargano Mortality Study (GMS) of 714 patients, 127 deaths, 5426 py and the Joslin Kidney Study (JKS) comprising 416 patients, 214 deaths, 5325 py. RESULTS In the first sample, individuals carrying 1 or ≥ 2 risk alleles had 33% (p = 0.06) and 51% (p = 0.02) increased risk of mortality, as compared with individuals with no risk alleles. A similar, though not significant, trend was obtained in the two replication samples only for subject carrying ≥ 2 risk alleles. In a pooled analysis, individuals carrying ≥ 2 risk alleles had higher mortality rate as compared to those carrying 0 risk alleles (HR = 1.34, 95%CI = 1.08-1.67; p = 0.008), and as compared to those carrying only one risk allele (HR = 1.41, 95%CI = 1.13-1.75; p = 0.002). This association was independent from several possible confounders including sex, age, BMI, hypertension and diabetes status. CONCLUSION Our data suggest that variants affecting insulin signaling exert a joint effect on all-cause mortality and is consistent with a role of abnormal insulin signaling on mortality risk.

The SH2B1 obesity locus and abnormal glucose homeostasis: Lack of evidence for association from a meta-analysis in individuals of European ancestry

BACKGROUND/AIMS The development of type 2 diabetes (T2D) is influenced both by environmental and by genetic determinants. Obesity is an important risk factor for T2D, mostly mediated by obesity-related insulin resistance. Obesity and insulin resistance are also modulated by the genetic milieu; thus, genes affecting risk of obesity and insulin resistance might also modulate risk of T2D. Recently, 32 loci have been associated with body mass index (BMI) by genome-wide studies, including one locus on chromosome 16p11 containing the SH2B1 gene. Animal studies have suggested that SH2B1 is a physiological enhancer of the insulin receptor and humans with rare deletions or mutations at SH2B1 are obese with a disproportionately high insulin resistance. Thus, the role of SH2B1 in both obesity and insulin resistance makes it a strong candidate for T2D. However, published data on the role of SH2B1 variability on the risk for T2D are conflicting, ranging from no effect at all to a robust association. METHODS The SH2B1 tag SNP rs4788102 (SNP, single nucleotide polymorphism) was genotyped in 6978 individuals from six studies for abnormal glucose homeostasis (AGH), including impaired fasting glucose, impaired glucose tolerance or T2D, from the GENetics of Type 2 Diabetes in Italy and the United States (GENIUS T2D) consortium. Data from these studies were then meta-analyzed, in a Bayesian fashion, with those from DIAGRAM+ (n = 47,117) and four other published studies (n = 39,448). RESULTS Variability at the SH2B1 obesity locus was not associated with AGH either in the GENIUS consortium (overall odds ratio (OR) = 0.96; 0.89-1.04) or in the meta-analysis (OR = 1.01; 0.98-1.05). CONCLUSION Our data exclude a role for the SH2B1 obesity locus in the modulation of AGH.

The 9p21 coronary artery disease locus and kidney dysfunction in patients with Type 2 diabetes mellitus

BACKGROUND We investigated whether the coronary artery disease (CAD) locus on chromosome 9p21 (as represented by single nucleotide polymorphism rs2383206) is associated with low estimated glomerular filtration rate (eGFR) or increased urinary albumin excretion in patients with Type 2 diabetes mellitus (T2DM). METHODS Four samples, including a total of 3167 patients, were studied. The presence of low eGFR (<60 mL/min/1.73m(2)) was estimated from serum creatinine by means of the Modification of Diet in Renal Disease Study equation. Increased urinary albumin excretion was defined as an albumin-creatinine ratio (ACR) ≥2.5 mg/mmol in men and ≥3.5 mg/mmol in women. RESULTS No association was found between rs2383206 and low eGFR or increased ACR in each sample as well as in a pooled analysis (overall odds ratio = 1.07, 95% confidence interval 0.94-1.22, P = 0.31 and overall odds ratio = 1.00, 95% confidence interval 0.90-1.12, P = 0.95, respectively). No interaction was observed between rs2383206 and poor glycemic control [HbA1c was above the median in the pooled sample (7.7%) in modulating eGFR or ACR (P for interaction = 0.42 and 0.90, respectively)]. CONCLUSION Variability at the 9p21 CAD locus is unlikely to play a role in modulating susceptibility to kidney dysfunction in patients with T2DM.

The vitamin D receptor functional variant rs2228570 (C>T) does not associate with type 2 diabetes mellitus

ABSTRACT Aim: Vitamin D acts through the binding to the vitamin D receptor (VDR). Several polymorphisms in VDR gene have been studied. Among these, the rs2228570 C>T (FokI) variant has been demonstrated to be functional, leading to a protein with a different size and activity. So far, genetic studies on the association between VDR gene rs2228570 single nucleotide polymorphism (SNP) and type 2 diabetes mellitus (T2DM) showed contradictory results. Thus, we performed an association study in a large cohort of adult Italian subjects with T2DM and in nondiabetic controls. Materials and methods: For this study, 1713 subjects, 883 T2DM patients and 830 controls, were genotyped for the polymorphism. All participants without a diagnosis of diabetes underwent oral glucose tolerance test (OGTT), with measurement of glucose and insulin levels. Indices of insulin resistance (Homeostatic model assessment of insulin resistance, insulin sensitivity index), secretion (homeostatic model assessment for beta-cell, corrected insulin response at 30 minutes) and disposition index were calculated. Results: Genotype distributions and allele frequencies did not show difference between T2DM subjects and controls. We did not find significant differences among the three genotypes regarding gender, age, BMI, waist, hip, waist-to-hip ratio, and blood pressure. There were also no significant differences in lipid parameters, aspartate aminotransferase, and alanine aminotransferase levels. We tested for association with OGTT-derived data and surrogate indices of insulin resistance and secretion. We did not find significant differences among the genotypes in any of above-mentioned parameters. Furthermore, vitamin D levels were measured in a subgroup of subjects. We did not find significant differences among the genotypes. Conclusions: Our study does not provide evidence for the association of the rs2228570 polymorphism with T2DM in a Caucasian population.

The IRS1 G972R polymorphism and glomerular filtration rate in patients with type 2 diabetes of European ancestry

BACKGROUND In Mexican Americans, the IRS1 G972R polymorphism (rs1801278) has been associated to such a marked reduction in glomerular filtration rate (GFR) (i.e. β = -8.3 mL/min/1.73 m(2)) to be considered a major determinant of kidney function. METHODS This was a cross-sectional study to investigate whether a similarly strong effect can also be observed among individuals of European ancestry. We investigated a total of 3973 White patients with type 2 diabetes. Standardized serum creatinine was measured by the modified kinetic Jaffè reaction and estimated GFR (eGFR) calculated by the modification diet renal disease (MDRD) formula; rs1801278 was genotyped by TaqMan assay. RESULTS No significant association was observed, with R972 carriers showing only a modestly, not significant, lower eGFR level as compared with other subjects (β = -1.82 mL/min/1.73 m(2), P = 0.086). CONCLUSIONS Our data indicate that IRS1 G972R is not a strong determinant of GFR in diabetic patients of European ancestry as in Mexican Americans. Since we had 100% power to detect the previously reported association, the risk our finding is a false negative one is minimal.

Evaluation of Polygenic Determinants of Non-Alcoholic Fatty Liver Disease (NAFLD) By a Candidate Genes Resequencing Strategy

NAFLD is a polygenic condition but the individual and cumulative contribution of identified genes remains to be established. To get additional insight into the genetic architecture of NAFLD, GWAS-identified GCKR, PPP1R3B, NCAN, LYPLAL1 and TM6SF2 genes were resequenced by next generation sequencing in a cohort of 218 NAFLD subjects and 227 controls, where PNPLA3 rs738409 and MBOAT7 rs641738 genotypes were also obtained. A total of 168 sequence variants were detected and 47 were annotated as functional. When all functional variants within each gene were considered, only those in TM6SF2 accumulate in NAFLD subjects compared to controls (P = 0.04). Among individual variants, rs1260326 in GCKR and rs641738 in MBOAT7 (recessive), rs58542926 in TM6SF2 and rs738409 in PNPLA3 (dominant) emerged as associated to NAFLD, with PNPLA3 rs738409 being the strongest predictor (OR 3.12, 95% CI, 1.8-5.5, P < 0.001). A 4-SNPs weighted genetic risk score value >0.28 was associated with a 3-fold increased risk of NAFLD. Interestingly, rs61756425 in PPP1R3B and rs641738 in MBOAT7 genes were predictors of NAFLD severity. Overall, TM6SF2, GCKR, PNPLA3 and MBOAT7 were confirmed to be associated with NAFLD and a score based on these genes was highly predictive of this condition. In addition, PPP1R3B and MBOAT7 might influence NAFLD severity.