Precision Diagnosis of Maturity-Onset Diabetes of the Young with Next-Generation Sequencing: Findings from the MODY-IST Study in Adult Patients.

Maturity-onset diabetes of the young (MODY) is a highly heterogeneous group of monogenic and nonautoimmune diseases. Misdiagnosis of MODY is a widespread problem and about 5% of patients with type 2 diabetes mellitus and nearly 10% with type 1 diabetes mellitus may actually have MODY. Using next-generation DNA sequencing (NGS) to facilitate accurate diagnosis of MODY, this study investigated mutations in 13 MODY genes (HNF4A, GCK, HNF1A, PDX1, HNF1B, NEUROD1, KLF11, CEL, PAX4, INS, BLK, ABCC8, and KCNJ11). In addition, we comprehensively investigated the clinical phenotypic effects of the genetic variations identified. Fifty-one adult patients with suspected MODY and 64 healthy controls participated in the study. We identified 7 novel and 10 known missense mutations localized in PDX1, HNF1B, KLF11, CEL, BLK, and ABCC8 genes in 29.4% of the patient sample. Importantly, we report several mutations that were classified as "deleterious" as well as those predicted as "benign." Notably, the ABCC8 p.R1103Q, ABCC8 p.V421I, CEL I336T, CEL p.N493H, BLK p.L503P, HNF1B p.S362P, and PDX1 p.E69A mutations were identified for the first time as causative variants for MODY. More aggressive clinical features were observed in three patients with double- and triple-heterozygosity of PDX1-KLF11 (p.E69A/p.S182R), CEL-ABCC8-KCNJ11 (p.I336, p.G157R/p.R1103Q/p.A157A), and HNF1B-KLF11 (p.S362P/p.P261L). Interestingly, the clinical effects of the BLK mutations appear to be exacerbated in the presence of obesity. In conclusion, NGS analyses of the adult patients with suspected MODY appear to be informative in a clinical context. These findings warrant further clinical diagnostic research and development in different world populations suffering from diabetes with genetic underpinnings.

Monogenic Childhood Diabetes: Dissecting Clinical Heterogeneity by Next-Generation Sequencing in Maturity-Onset Diabetes of the Young.

Diabetes is a common disorder with a heterogeneous clinical presentation and an enormous burden on health care worldwide. About 1-6% of patients with diabetes suffer from maturity-onset diabetes of the young (MODY), the most common form of monogenic diabetes with autosomal dominant inheritance. MODY is genetically and clinically heterogeneous and caused by genetic variations in pancreatic ß-cell development and insulin secretion. We report here new findings from targeted next-generation sequencing (NGS) of 13 MODY-related genes. A sample of 22 unrelated pediatric patients with MODY and 13 unrelated healthy controls were recruited from a Turkish population. Targeted NGS was performed with Miseq 4000 (Illumina) to identify genetic variations in 13 MODY-related genes: HNF4A, GCK, HNF1A, PDX1, HNF1B, NEUROD1, KLF11, CEL, PAX4, INS, BLK, ABCC8, and KCNJ11. The NGS data were analyzed adhering to the Genome Analysis ToolKit (GATK) best practices pipeline, and variant filtering and annotation were performed. In the patient sample, we identified 43 MODY-specific genetic variations that were not present in the control group, including 11 missense mutations and 4 synonymous mutations. Importantly, and to the best of our knowledge, the missense mutations NEUROD1 p.D202E, KFL11 p.R461Q, BLK p.G248R, and KCNJ11 p.S385F were first associated with MODY in the present study. These findings contribute to the worldwide knowledge base on MODY and molecular correlates of clinical heterogeneity in monogenic childhood diabetes. Further comparative population genetics and functional genomics studies are called for, with an eye to discovery of novel diagnostics and personalized medicine in MODY. Because MODY is often misdiagnosed as type 1 or type 2 diabetes mellitus, advances in MODY diagnostics with NGS stand to benefit diabetes overall clinical care as well.

The comparison of serum TGF-beta levels and associated polymorphisms in patients with coronary artery ectasia and normal coronary artery.

BACKGROUND: Coronary artery ectasia (CAE) is described as the enlargement of a coronary artery segment by 1.5 times or more, which is generally associated with the atherosclerotic process. Atherosclerotic changes lead to arterial remodeling result in CAE. In our study, we measured serum transforming growth factor (TGF)-ß1 levels, which have a protective role against atherosclerosis. Further, we aimed to assess the TGF-ß1 gene variants rs1800469 (-509C>T, c.-1347C>T) and rs1800470 (c.+29T>C, p.Pro10Leu, rs1982073), which might have an effect on TGF production. Overall, 2877 patients were screened including 56 patients with CAE and 44 patients with normal coronary arteries who were included in the study. Serum TGF-ß1 levels were measured using ELISA and compared between two groups. Additionally, TGF-ß1 rs1800469 and rs1800470 gene variations were determined using TaqMan® SNP Genotyping Assays. RESULTS: Serum TGF-ß1 levels were significantly lower in patients with CAE than in controls (p=0.012). However, there was no difference in terms of the genotype and allele distributions of TGF-ß1 rs1800469 and rs1800470 polymorphisms. Serum TGF-ß1 levels were higher in individuals carrying the TGF-ß1 rs1800470 G allele (GG+AG) than in individuals with normal homozygous AA genotype in the CAE group (p=0.012). CONCLUSION: Our findings suggest that lower serum TGF-ß1 levels are associated with an increased risk for CAE development and that TGF-ß1 polymorphisms exert a protective effect. Furthermore, TGF-ß1 rs1800470 G allele carriers were shown to have higher TGF-ß1 levels in the CAE group. This suggests that having the G allele in the TGF-ß1 rs1800470 polymorphism could prevent CAE development.

Co-existence of BRAF V600E Gene Mutation in Tumor and Non-tumoral Surrounding Tissues in Colorectal Cancer.

BACKGROUND/AIM: The murine sarcoma viral (V-Raf) oncogene homolog B (BRAF) V600E mutation, which increases protein kinase activity in BRAF-mitogen-activated protein kinase kinase (MEK) - extracellular signal-regulated kinases (ERK) (mitogen-activated protein kinase (MAPK)) signaling, is found in 5-40% of all colorectal carcinoma cases. Proteins with this mutation are reported to be 130-fold more active, which results in induced proliferation, differentiation, cellular survival, and angiogenesis. The aim of the present study was to investigate tumor tissues, together with the surrounding non-tumoral tissues, for BRAF mutation presence, which may be an indicator for possible recurrence or prognosis as in the 'field carcinogenesis' model. MATERIALS AND METHODS: The BRAF V600E genotype of 152 colorectal adenocarcinoma paraffin-embedded specimens were determined by mutant-allele-specific amplification-polymerase chain reaction. RESULTS: According to our results, the presence of BRAF mutation increases risk of lymph node invasion by 1.55-fold [χ(2)=3.83, p=0.05, odds ratio (OR)=1.55, 95% confidence interval (CI)=1.00-2.42], histologically medium or high-grade tumor by 1.60-fold (χ(2)=4.34, p=0.030, OR=1.60, 95% CI=1.03-2.48), vascular invasion by 1.55-fold (χ(2)=3.55, p=0.05, OR=1.55, 95% CI=0.99-2.42), perineural invasion by 1.50-fold (χ(2)=3.16, p=0.07, OR=1.5, 95% CI=0.96-2.33) and the combination of these poor prognostic features by 1.54-fold (χ(2)=2.47, p=0.11, OR=1.54, 95% CI=0.93-2.53). We also found that females are more prone to having the mutation and that being female increases the risk of having this mutation by 1.54-fold (χ(2)=3.58, p=0.05, OR=1.54, 95% CI=0.97-2.44). CONCLUSION: BRAF V600E mutation in non-tumoral surrounding tissue in patients with colorectal cancer may be used as a valuable marker to foresee clinical outcome or a possible recurrence. To our knowledge, this was the first study to take into consideration the non-tumoral surrounding tissues in addition to the tumor tissue.

Investigation of the common paraoxonase 1 variants with paraoxonase activity on bone fragility in Turkish patients.

There is increasing evidence of a biochemical link between oxidative stress and bone metabolism. Oxidative stress has been shown to be involved in bone resorption as it causes loss of bone mineral density (BMD). Paraoxonase 1 (PON1), can prevent these effects of the oxidative stress on bone formation. It has been suggested that the PON1 gene as possibly implicated in reduced BMD in bone fragility cases. It has been hypothesized that PON1 gene polymorphisms may influence both the risk of osteoporosis and osteopenia occurrence and prognosis. The aim of our study is to evaluate the relationship between PON1 polymorphisms and bone fragility development. Seventy-four osteoporotic, 121 osteopenic and 79 nonosteoporotic postmenopausal women were recruited. For detection of the polymorphisms, polymerase chain reaction-restriction fragment length polymorphism techniques have been used. BMD was measured at the lumbar spine and hip by dual-energy X-ray absorptiometry. Distributions of PON1 (PON 192 and PON 55) polymorphisms in study groups were not significantly different. But, there was medium strength connection between in the osteopenic with control groups regarding PON1 55-PON1 192 haplotypes and we found a power strength connection between in the osteoporosis with control groups regarding PON1 55-PON1 192 haplotypes. Furthermore, subjects with PON1 192RR and PON1 55LL genotypes had lower PON activity values of osteoporotic subject compared to healthy control and this difference was statistically significant (p < 0.05). This result suggest that PON1 genotypes could be higher risk for osteoporosis, as determined by reduced BMD.

MCP-1 and CCR2 gene variants and the risk for osteoporosis and osteopenia.

AIM: In this study, we investigated whether monocyte chemotactic protein 1 (MCP-1) and CC chemokine receptor 2 (CCR2) gene polymorphisms account for an increased risk of osteoporosis or osteopenia. METHODS: Three hundred three postmenopausal women, 80 osteoporotic, 123 osteopenic, and 100 unrelated age-matched healthy controls, were included in the study. Genotyping of MCP-1 A2518G and CCR2 V64I gene polymorphisms were detected by PCR-RFLP. RESULTS: We, for the first time, demonstrated the positive association of MCP-1 GG, CCR2 Val/Ile, and CCR2 Val+ genotype with osteoporosis risk. However, CCR2 Ile/Ile genotype frequencies were high in the control group compared with those of the patients with osteoporosis and osteopenia. Haplotype analysis confirmed the association of MCP-1/CCR2 gene variants with osteopenia and revealed that the frequency of MCP-1 A:CCR2 Val haplotype was significantly higher in patients when compared with controls. CONCLUSIONS: In conclusion, our findings have suggested that MCP-1 and CCR2 gene variants were risk factors for osteoporosis and osteopenia.

Associations of receptor for advanced glycation end products -374 T/A and Gly82 Ser and peroxisome proliferator-activated receptor gamma Pro12Ala polymorphisms in Turkish coronary artery disease patients.

AIM: The aim of the present study was to investigate the individual and combined effects of receptor for advanced glycation end products (RAGE) -374T/A, RAGE Gly82Ser, and peroxisome proliferator-activated receptor gamma (PPAR-γ) Pro12Ala polymorphisms on the development of coronary artery disease (CAD). MATERIALS AND METHODS: This study was carried out in 87 patients with CAD and 52 CAD-free healthy controls. Polymerase chain reaction, restriction fragment length polymorphism, and agarose gel electrophoresis techniques were used to determine RAGE -374T/A, RAGE Gly82 Ser, and PPAR-γ Pro12 Ala. RESULTS: Individual allele and genotype frequencies of RAGE -374T/A, RAGE Gly82Ser, and PPAR-γ Pro12Ala polymorphisms were not significantly different between study groups. However, compared with the control group, wild-type T allele frequency was found to be higher in patients with diabetes (p=0.009). To investigate the combined effects of RAGE and PPAR polymorphisms, haplotype analysis was elevated and there was no statistical difference between the haplotypes of RAGE Gly82Ser with RAGE-374T/A or PPAR Pro12Ala. However, the frequency of RAGE-374T/PPAR12Ala haplotype was found to be higher in both the patient group (p=0.024) and in patients without diabetes (p=0.037). CONCLUSION: The results of the present study demonstrated that possessing the A allele of RAGE -374T/A polymorphism by diabetic CAD patients and possessing the-374T/Ala12 haplotype of RAGE -374T/A and PPAR-γ Pro12 Ala polymorphisms by the patients group were the most important risk factors for CAD.

Association of monocyte chemotactic protein-1 and CC chemokine receptor 2 gene variants with preeclampsia.

Preeclampsia complicates 10% of pregnancies in developing countries. It is one of the leading causes of maternal and fetal/neonatal mortality and morbidity worldwide. It has been suggested that maladaptation of the maternal immune response during pregnancy might be a causal factor for preeclampsia. According to immune maladaptation hypothesis, preeclampsia is due to an inappropriate regulation of normally Th2-deviated maternal immune responses, leading to a shift toward harmful Th1 immunity. Several studies indicate that monocyte chemotactic protein-1 (MCP-1) and CC chemokine receptor 2 (CCR2) are involved in Th1 and Th2 immunity. In this study, we investigated the association between MCP-1 A-2518G and CCR2-V64I polymorphisms and preeclampsia. One hundred eighty preeclamptic pregnant women and 145 healthy controls were included in the study. We observed that in preeclamptic women, MCP-1 G: CCR2 Val haplotype was significantly higher when compared with other haplotypes. In conclusion, we stated that MCP-1 and CCR2 gene variants might be associated with preeclampsia.

Associations of -374T/A polymorphism of receptor for advanced glycation end products (RAGE) gene in Turkish diabetic and non-diabetic patients with coronary artery disease.

BACKGROUND: In this study we aimed to determine the possible risks for the development of coronary artery disease (CAD) in diabetic (DM(+)) and non-diabetic (DM(-)) patients according to the -374T/A polymorphism of the receptor for advanced glycation end products (RAGE) gene which affects the function of RAGE itself. MATERIALS AND METHODS: This study was carried out in 52 non-diabetic and 62 diabetic patients with CAD, and 55 CAD-free, healthy volunteers as controls. The A-T transversion polymorphism at position -374 in the promotor region of the RAGE gene was analyzed by polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) techniques. RESULTS: The -374T/A AA genotype frequency was statistically higher in the whole patient group when compared with the control group (p=0.034), and statistically higher in the DM(+) group when compared with the control group (p=0.003). Homozygosity for the -374A allele was found to be higher, but not statistically meaningful, in DM(-) patients (17.3%) when compared with the control group (13.2%). In this study, in contrast with other studies, we found possesion of the A allele to be an independent risk factor in CAD in patients with diabetes mellitus. CONCLUSION: Possesion of the -374A allele may contribute to the CAD in diabetic patients with triggering macrophages by increased levels of AGEs.

Is LOX-1 K167N polymorphism protective for coronary artery disease?

BACKGROUND: Human lectin-like oxidized low-density lipoprotein receptor 1 (LOX-1, OLR1) has been identified as a cell surface endocytosis receptor for oxidized low-density lipoprotein (oxLDL) on vascular endothelial cells. OxLDLs are avidly ingested by macrophages, resulting in foam cell formation. OxLDLs are also involved in inducing smooth muscle cell migration, proliferation and transformation. A single nucleotide polymorphism K167N (G501C) of the LOX-1 gene results in an amino acid dimorphism (Lys/Asn) at residue 167. Replacement of this Lys residue causes reduced binding and internalization of oxLDL. The purpose of this study was to investigate the effect of the LOX-1 K167N gene polymorphism in Turkish patients with coronary artery disease (CAD). MATERIALS AND METHODS: K167N polymorphism were studied in 91 patients with CAD and 72 healthy controls by the PCR-RFLP method. RESULTS: The frequencies of the KK genotype and the K allele were higher in the CAD group than the controls (p<0.05), while the frequency of the NN genotype was higher in the control group than in the CAD group (p<0.05). It was observed that the decreased CAD risk in patients who had the N allele was reversed by male sex (OR: 0.400 -->0.481) and smoking (OR: 0.400 -->0.949). Although male sex and smoking were lower than other cardiovascular risk factors in patients with the N allele they were higher than other cardiovascular risk factors in patients with the K allele. CONCLUSION: Male sex and smoking decrease the protective effects of the N allele. The adverse effects of the K allele on the CAD risk resulting from the K167N polymorphism appear to be independent of other cardiovascular risk factors.

Associations of lipoprotein lipase S447X and apolipoprotein E genotypes with low-density lipoprotein subfractions in Turkish patients with coronary artery disease.

BACKGROUND: This study investigated associations of specific lipoprotein lipase (LPL) S447X and apolipoprotein (Apo)E allelic patterns with low-density lipoprotein (LDL) size and subfraction profiles in patients with coronary artery disease (CAD) and healthy individuals. PATIENTS AND METHODS: Forty-one cases with CAD and 23 controls were compared regarding the occurrence of the Ser-->Stop codon of the LPL and ApoE polymorphism. Polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) techniques were utilized to perform genotyping, and LDL size and subfractions were assessed by a high-resolution, nongradient polyacrylamide gel electrophoresis technique. RESULTS: The lowest small dense (sd) LDL level was observed for the homozygous LPLX447 genotype (6.00 +/- 4.00 mg/dl) while the highest sdLDL level was observed for LPLX447(+)/ApoE4(+) carriers (14.33 +/- 20.55 mg/dl) in the patient group. No protective effect of LPLX447 allele on the atherogenic LDL profile was observed when it was together with the ApoE4 allele. Furthermore, the detrimental effect of LPLS447 on the atherogenic LDL profile increased when it was present together with the ApoE4 allele. CONCLUSION: The X447 allele of the LPL gene may protect from atherogenic LDL subfraction, although this effect is small. We suggest that the S447X polymorphism of the LPL gene may modify the risk of atherogenic sdLDL fraction in an ApoE-dependent fashion.