Krzysztof Wanic

Genome-Wide Association Scan for Diabetic Nephropathy Susceptibility Genes in Type 1 Diabetes

OBJECTIVE Despite extensive evidence for genetic susceptibility to diabetic nephropathy, the identification of susceptibility genes and their variants has had limited success. To search for genes that contribute to diabetic nephropathy, a genome-wide association scan was implemented on the Genetics of Kidneys in Diabetes collection. RESEARCH DESIGN AND METHODS We genotyped ∼360,000 single nucleotide polymorphisms (SNPs) in 820 case subjects (284 with proteinuria and 536 with end-stage renal disease) and 885 control subjects with type 1 diabetes. Confirmation of implicated SNPs was sought in 1,304 participants of the Diabetes Control and Complications Trial (DCCT)/Epidemiology of Diabetes Interventions and Complications (EDIC) study, a long-term, prospective investigation of the development of diabetes-associated complications. RESULTS A total of 13 SNPs located in four genomic loci were associated with diabetic nephropathy with P < 1 × 10−5. The strongest association was at the FRMD3 (4.1 protein ezrin, radixin, moesin [FERM] domain containing 3) locus (odds ratio [OR] = 1.45, P = 5.0 × 10−7). A strong association was also identified at the CARS (cysteinyl-tRNA synthetase) locus (OR = 1.36, P = 3.1 × 10−6). Associations between both loci and time to onset of diabetic nephropathy were supported in the DCCT/EDIC study (hazard ratio [HR] = 1.33, P = 0.02, and HR = 1.32, P = 0.01, respectively). We demonstratedexpression of both FRMD3 and CARS in human kidney. CONCLUSIONS We identified genetic associations for susceptibility to diabetic nephropathy at two novel candidate loci near the FRMD3 and CARS genes. Their identification implicates previously unsuspected pathways in the pathogenesis of this important late complication of type 1 diabetes.

The pseudokinase tribbles homolog 3 interacts with ATF4 to negatively regulate insulin exocytosis in human and mouse β cells

Insufficient insulin secretion and reduced pancreatic beta cell mass are hallmarks of type 2 diabetes (T2DM). Here, we confirm that a previously identified polymorphism (rs2295490/Q84R) in exon 2 of the pseudokinase-encoding gene tribbles 3 (TRB3) is associated with an increased risk for T2DM in 2 populations of people of mixed European descent. Carriers of the 84R allele had substantially reduced plasma levels of C-peptide, the product of proinsulin processing to insulin, suggesting a role for TRB3 in beta cell function. Overexpression of TRB3 84R in mouse beta cells, human islet cells, and the murine beta cell line MIN6 revealed reduced insulin exocytosis, associated with a marked reduction in docked insulin granules visualized by electron microscopy. Conversely, knockdown of TRB3 in MIN6 cells restored insulin secretion and expression of exocytosis genes. Further analysis in MIN6 cells demonstrated that TRB3 interacted with the transcription factor ATF4 and that this complex acted as a competitive inhibitor of cAMP response element-binding (CREB) transcription factor in the regulation of key exocytosis genes. In addition, the 84R TRB3 variant exhibited greater protein stability than wild-type TRB3 and increased binding affinity to Akt. Mice overexpressing TRB3 84R in beta cells displayed decreased beta cell mass, associated with reduced proliferation and enhanced apoptosis rates. These data link a missense polymorphism in human TRB3 to impaired insulin exocytosis and thus increased risk for T2DM.

New Polymorphism of ENPP1 (PC-1) Is Associated With Increased Risk of Type 2 Diabetes Among Obese Individuals

The K121Q polymorphism in ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) is associated with type 2 diabetes and obesity. The possibility of other ENPP1 polymorphisms influencing these phenotypes has received little attention. Our aim was to examine the associations of tagging single nucleotide polymorphisms (SNPs) and haplotypes of the linkage disequilibrium (LD) block containing K121Q polymorphism with type 2 diabetes in a Polish population, controlling for any effect of obesity. We genotyped 426 type 2 diabetic case and 370 control subjects for seven SNPs in ENPP1. In the total group, neither type 2 diabetes nor obesity was significantly associated with any SNP. However, in obese subjects, two SNPs were significantly associated with type 2 diabetes: the Q allele of K121Q (odds ratio 1.6 [95% CI 1.003–2.6]) and T allele of rs997509 (4.7 [1.6–13.9]). In the LD block, four SNPs plus the K121Q polymorphism distinguished six haplotypes, three of which carried the Q allele. Interestingly, the T allele of rs997509 sufficed to distinguish a 121Q-carrying haplotype that was significantly more associated with type 2 diabetes than the other two (4.2 [1.3–13.5]). These other two 121Q-carrying haplotypes were not associated with type 2 diabetes. In conclusion, we found a new SNP, rs997509, in intron 1 that is strongly associated with risk of type 2 diabetes in obese individuals. The molecular mechanisms underlying this association are unknown.

Exclusion of polymorphisms in carnosinase genes (CNDP1 and CNDP2) as a cause of diabetic nephropathy in type 1 diabetes: results of large case-control and follow-up studies.

OBJECTIVES— Recently, an association was found between diabetic nephropathy and the D18S880 microsatellite, located in the carnosinase gene (CNDP1) on chromosome 18q. Alleles of this microsatellite encode for a variable number of leucine residues (from four to seven) in the leader peptide of the carnosinase precursor. The frequency of subjects homozygous for the five leucines was higher in control subjects than in case subjects in studies focusing on type 2 diabetic patients. To test whether this finding can be extended to type 1 diabetic patients, we carried out a comprehensive study on association between diabetic nephropathy and the D18S880 microsatellite and 21 additional SNPs that tagged the genomic region containing CNDP1 and CNDP2. RESEARCH DESIGN AND METHODS— Overall, 1,269 Caucasian patients with type 1 diabetes were included in the study, including 613 patients with normoalbuminuria and a long duration of diabetes, 445 patients with persistent proteinuria, and 211 patients with end-stage renal disease (ESRD). All patients were genotyped for selected polymorphisms, the associations with diabetic nephropathy were tested by a χ2 test, and odds ratios were calculated. RESULTS— We did not find any significant association between diabetic nephropathy and any examined genetic markers. The negative findings of the case-control study were supported further by negative findings obtained from the 6-year follow-up study of 445 patients with persistent proteinuria, during which 135 patients developed ESRD. CONCLUSIONS— Our large, comprehensive study did not find an association between the D18S880 microsatellite or any other polymorphisms in the CNDP2–CNDP1 genomic region and susceptibility for diabetic nephropathy in type 1 diabetes.

High-density single nucleotide polymorphism genome-wide linkage scan for susceptibility genes for diabetic nephropathy in type 1 diabetes : discordant sibpair approach

OBJECTIVE— Epidemiological and family studies have demonstrated that susceptibility genes play an important role in the etiology of diabetic nephropathy, defined as persistent proteinuria or end-stage renal disease (ESRD) in type 1 diabetes. RESEARCH DESIGN AND METHODS— To efficiently search for genomic regions harboring diabetic nephropathy genes, we conducted a scan using 5,382 informative single nucleotide polymorphisms on 100 sibpairs concordant for type 1 diabetes but discordant for diabetic nephropathy. In addition to being powerful for detecting linkage to diabetic nephropathy, this design allows linkage analysis on type 1 diabetes via traditional affected sibpair (ASP) analysis. In weighing the evidence for linkage, we considered maximum logarithm of odds score (maximum likelihood score [MLS]) values and corresponding allelic sharing patterns, calculated and viewed graphically using the software package SPLAT. RESULTS— Our primary finding for diabetic nephropathy, broadly defined, is on chromosome 19q (MLS = 3.1), and a secondary peak exists on chromosome 2q (MLS = 2.1). Stratification of discordant sibpairs based on whether disease had progressed to ESRD suggested four tertiary peaks on chromosome 1q (ESRD only), chromosome 20p (proteinuria only), and chromosome 3q (two loci 58 cm apart, one for ESRD only and another for proteinuria only). Additionally, analysis of 130 ASPs for type 1 diabetes confirmed the linkage to the HLA region on chromosome 6p (MLS = 9.2) and IDDM15 on chromosome 6q (MLS = 3.1). CONCLUSIONS— This study identified several novel loci as candidates for diabetic nephropathy, none of which appear to be the sole genetic determinant of diabetic nephropathy in type 1 diabetes. In addition, this study confirms two previously reported type 1 diabetes loci.

Evaluation of Apolipoprotein M Serum Concentration as a Biomarker of HNF-1alpha MODY

Apolipoprotein M (apoM) is a 26-kDa protein expressed mainly in the liver and kidneys. It is present predominantly in high-density lipoproteins (HDL). ApoM expression is influenced by the hepatocyte nuclear factor-1alpha (HNF-1alpha), which is a transcription factor associated with the pathogenesis of MODY. Some earlier data suggested that apoM levels were lower in the serum of HNF-1alpha MODY subjects, than in that of other diabetics and healthy controls. The aim of this study was to evaluate apoM as a biomarker for HNF-1alpha MODY. We included in this study 48 HNF-1alpha mutation carriers (40 diabetic patients and 8 subjects with normal glucose levels in the fasted state) from the Polish Nationwide Registry of MODY. In addition, we examined 55 T2DM patients and 55 apparently healthy volunteers who had normal fasting glucose levels. ApoM was measured by the sandwich dot-blot technique with recombinant apoM (Abnova) as a protein standard, mouse anti-human apoM monoclonal primary antibody and rat anti-mouse HRP-conjugated secondary antibody (BD Biosciences). Mean apoM level in the MODY group was 13.6 mug/ml, SD 1.9 (13.5 mug/ml, SD 1.7 in diabetic subjects and 13.9 mug/ml, SD 2.0 in non-diabetic mutation carriers respectively). In the T2DM group, mean apoM level was 13.7 mug/ml, SD 2.1, while it reached 13.8 mug/ml, SD 2.0 in healthy controls. There was no difference between apoM serum concentrations in all the study groups. In summary, our study showed no association between HNF-1alpha mutations resulting in MODY phenotype and apoM levels. Thus, we cannot confirm the clinical usefulness of apoM as a biomarker of HNF-1alpha MODY.

Transfer to Sulphonylurea Therapy in Adult Subjects With Permanent Neonatal Diabetes Due to KCNJ11-Activating Mutations Evidence for improvement in insulin sensitivity

A ctivating mutations in the KCJN11 gene encoding in the ATP-sensitive K channel (KATP channel) subunit Kir6.2 were reported (1) as the most common cause of permanent neonatal diabetes (PND). Recently, it has been shown that most subjects with Kir6.2 mutations could be switched from insulin to sulfonylurea and that such treatment is both safe and highly effective, at least in the short term (2,3). Notably, the majority of reported successfully transferred patients were children. Data on adults are very scarce, and there are few mutation carriers transferred off insulin (2,4). Moreover, some adult subjects are unable to switch from insulin to sulfonylurea (2). We have recently identified four adult carriers of a Kir6.2 mutation and provided evidence that they, before the sulfonylurea exposure, were characterized by decreased insulin sensitivity (5). Here, we report their successful transfer to sulfonylurea.

High-density SNP genome wide linkage scan for susceptibility genes for diabetic nephropathy in type 1 diabetes: Discordant sib-pair approach.

OBJECTIVE— Epidemiological and family studies have demonstrated that susceptibility genes play an important role in the etiology of diabetic nephropathy, defined as persistent proteinuria or end-stage renal disease (ESRD) in type 1 diabetes. RESEARCH DESIGN AND METHODS— To efficiently search for genomic regions harboring diabetic nephropathy genes, we conducted a scan using 5,382 informative single nucleotide polymorphisms on 100 sibpairs concordant for type 1 diabetes but discordant for diabetic nephropathy. In addition to being powerful for detecting linkage to diabetic nephropathy, this design allows linkage analysis on type 1 diabetes via traditional affected sibpair (ASP) analysis. In weighing the evidence for linkage, we considered maximum logarithm of odds score (maximum likelihood score [MLS]) values and corresponding allelic sharing patterns, calculated and viewed graphically using the software package SPLAT. RESULTS— Our primary finding for diabetic nephropathy, broadly defined, is on chromosome 19q (MLS = 3.1), and a secondary peak exists on chromosome 2q (MLS = 2.1). Stratification of discordant sibpairs based on whether disease had progressed to ESRD suggested four tertiary peaks on chromosome 1q (ESRD only), chromosome 20p (proteinuria only), and chromosome 3q (two loci 58 cm apart, one for ESRD only and another for proteinuria only). Additionally, analysis of 130 ASPs for type 1 diabetes confirmed the linkage to the HLA region on chromosome 6p (MLS = 9.2) and IDDM15 on chromosome 6q (MLS = 3.1). CONCLUSIONS— This study identified several novel loci as candidates for diabetic nephropathy, none of which appear to be the sole genetic determinant of diabetic nephropathy in type 1 diabetes. In addition, this study confirms two previously reported type 1 diabetes loci.

Clinical Application of 1,5-Anhydroglucitol Measurements in Patients with Hepatocyte Nuclear Factor-1α Maturity-Onset Diabetes of the Young

OBJECTIVE—1,5-anhydroglucitol (1,5-AG) is a short-term marker of metabolic control in diabetes. Its renal loss is stimulated in hyperglycemic conditions by glycosuria, which results in a lowered plasma concentration. As a low renal threshold for glucose has been described in hepatocyte nuclear factor-1α (HNF-1α) maturity-onset diabetes of the young (MODY), the 1,5-AG level may be altered in these patients. The purpose of this study was to assess the 1,5-AG levels in patients with HNF-1α MODY and in type 2 diabetic subjects with a similar degree of metabolic control. In addition, we aimed to evaluate this particle as a biomarker for HNF-1α MODY. RESEARCH DESIGN AND METHODS—We included 33 diabetic patients from the Polish Nationwide Registry of MODY. In addition, we examined 43 type 2 diabetic patients and 47 nondiabetic control subjects. The 1,5-AG concentration was measured with an enzymatic assay (GlycoMark). Receiver operating characteristic (ROC) curve analysis was used to evaluate 1,5-AG as a screening marker for HNF-1α MODY. RESULTS—The mean 1,5-AG plasma concentration in diabetic HNF-1α mutation carriers was 5.9 μg/ml, and it was lower than that in type 2 diabetic patients (11.0 μg/ml, P = 0.003) and in nondiabetic control subjects (23.9 μg/ml, P < 0.00005). The ROC curve analysis revealed 85.7% sensitivity and 80.0% specificity of 1,5-AG in screening for HNF-1α MODY at the criterion of <6.5 μg/ml in patients with an A1C level between 6.5 and 9.0%. CONCLUSIONS—1,5-AG may be a useful biomarker for differential diagnosis of patients with HNF-1α MODY with a specific range of A1C, although this requires further investigation. However, the clinical use of this particle in diabetic HNF-1α mutation carriers for metabolic control has substantial limitations.

Exclusion of Polymorphisms in Carnosinase Genes (CNDP1 and CNDP2) as a Cause of Diabetic Nephropathy in Type 1 Diabetes

OBJECTIVES— Recently, an association was found between diabetic nephropathy and the D18S880 microsatellite, located in the carnosinase gene (CNDP1) on chromosome 18q. Alleles of this microsatellite encode for a variable number of leucine residues (from four to seven) in the leader peptide of the carnosinase precursor. The frequency of subjects homozygous for the five leucines was higher in control subjects than in case subjects in studies focusing on type 2 diabetic patients. To test whether this finding can be extended to type 1 diabetic patients, we carried out a comprehensive study on association between diabetic nephropathy and the D18S880 microsatellite and 21 additional SNPs that tagged the genomic region containing CNDP1 and CNDP2. RESEARCH DESIGN AND METHODS— Overall, 1,269 Caucasian patients with type 1 diabetes were included in the study, including 613 patients with normoalbuminuria and a long duration of diabetes, 445 patients with persistent proteinuria, and 211 patients with end-stage renal disease (ESRD). All patients were genotyped for selected polymorphisms, the associations with diabetic nephropathy were tested by a χ2 test, and odds ratios were calculated. RESULTS— We did not find any significant association between diabetic nephropathy and any examined genetic markers. The negative findings of the case-control study were supported further by negative findings obtained from the 6-year follow-up study of 445 patients with persistent proteinuria, during which 135 patients developed ESRD. CONCLUSIONS— Our large, comprehensive study did not find an association between the D18S880 microsatellite or any other polymorphisms in the CNDP2–CNDP1 genomic region and susceptibility for diabetic nephropathy in type 1 diabetes.