Polygenic risk scores predict diabetes complications and their response to intensive blood pressure and glucose control.

AIMS/HYPOTHESIS: Type 2 diabetes increases the risk of cardiovascular and renal complications, but early risk prediction could lead to timely intervention and better outcomes. Genetic information can be used to enable early detection of risk. METHODS: We developed a multi-polygenic risk score (multiPRS) that combines ten weighted PRSs (10 wPRS) composed of 598 SNPs associated with main risk factors and outcomes of type 2 diabetes, derived from summary statistics data of genome-wide association studies. The 10 wPRS, first principal component of ethnicity, sex, age at onset and diabetes duration were included into one logistic regression model to predict micro- and macrovascular outcomes in 4098 participants in the ADVANCE study and 17,604 individuals with type 2 diabetes in the UK Biobank study. RESULTS: The model showed a similar predictive performance for cardiovascular and renal complications in different cohorts. It identified the top 30% of ADVANCE participants with a mean of 3.1-fold increased risk of major micro- and macrovascular events (p = 6.3 × 10-21 and p = 9.6 × 10-31, respectively) and a 4.4-fold (p = 6.8 × 10-33) higher risk of cardiovascular death. While in ADVANCE overall, combined intensive blood pressure and glucose control decreased cardiovascular death by 24%, the model identified a high-risk group in whom it decreased the mortality rate by 47%, and a low-risk group in whom it had no discernible effect. High-risk individuals had the greatest absolute risk reduction with a number needed to treat of 12 to prevent one cardiovascular death over 5 years. CONCLUSIONS/INTERPRETATION: This novel multiPRS model stratified individuals with type 2 diabetes according to risk of complications and helped to target earlier those who would receive greater benefit from intensive therapy.

A Variant in the Nicotinic Acetylcholine Receptor Alpha 3 Subunit Gene Is Associated With Hypertension Risks in Hypogonadic Patients.

Ephb6 gene knockout causes hypertension in castrated mice. EPHB6 controls catecholamine secretion by adrenal gland chromaffin cells (AGCCs) in a testosterone-dependent way. Nicotinic acetylcholine receptor (nAChR) is a ligand-gated Ca2+/Na+ channel, and its opening is the first signaling event leading to catecholamine secretion by AGCCs. There is a possibility that nAChR might be involved in EPHB6 signaling, and thus sequence variants of its subunit genes are associated with hypertension risks. CHRNA3 is the major subunit of nAChR used in human and mouse AGCCs. We conducted a human genetic study to assess the association of CHRNA3 variants with hypertension risks in hypogonadic males. The study cohort included 1,500 hypogonadic Chinese males with (750 patients) or without (750 patients) hypertension. The result revealed that SNV rs3743076 in the fourth intron of CHRNA3 was significantly associated with hypertension risks in the hypogonadic males. We further showed that EPHB6 physically interacted with CHRNA3 in AGCCs, providing a molecular basis for nAChR being in the EPHB6 signaling pathway.

EPHA4 regulates vascular smooth muscle cell contractility and is a sex-specific hypertension risk gene in individuals with type 2 diabetes.

OBJECTIVE: We investigated the association of genetic variants of EPHA4, a receptor tyrosine kinase, with hypertension, and its role in vascular smooth muscle cell (VSMC) contractility. METHODS: Data from two human genetic studies, ADVANCE and HCHS/SOL, were analyzed for association of EPHA4 single nucleotide variants (SNVs) with hypertension risks. The effect of EPHA4 signalling on mouse VSMC contractility was assessed. RESULTS: We identified a SNV (rs75843691 hg19 chr2:g.222395371 C>G), located in the third intron of EPHA4 gene, being significantly associated with hypertension in human female patients (P value = 8.3 × 10, below the Bonferroni-corrected critical P value) but not male patients with type 2 diabetes from the ADVANCE clinical trial. We found that EPHA4 was expressed in VSMCs and its stimulation by anti-EPHA4 antibody led to reduced VSMC contractility. Estrogen enhanced the contractility-lowering effect of EPHA4 stimulation. Conversely, siRNA knockdown of Epha4 expression in VSMCs resulted in increased contractility of VSMCs from female mice but not from male mice. CONCLUSION: EPHA4 appears to be a sex-specific hypertension risk gene in type 2 diabetic patients. Forward EPHA4 signalling reduces VSMC contractility, and estrogen is a modifier of this effect. The effect of EPHA4 on VSMCs contractility explains the association of EPHA4 gene with hypertension risks in female patients.

Uromodulin in a Pathway Between Decreased Renal Urate Excretion and Albuminuria.

BACKGROUND: The mechanism explaining the inverse association between renal urate and albumin excretion remains unclear. First, we evaluated the impact of candidate variants in the main urate transporter genes (i.e., SLC2A9, SLC22A12, ABCG2) on the association between fractional excretion of uric acid (FEUA) and urinary albumin/creatinine ratio (uACR). Second, we examined uromodulin and sodium excretion as mediators of the association between FEUA and uACR. METHODS: We performed cross-sectional analysis of 737 French Canadians from the CARTaGENE cohort, a random sample of the Quebec population aged 40-69 years (a total of 20,004 individuals). Individuals with available genotyping and urinary data were obtained from a sub-study including gender-matched pairs with high and low Framingham Risk Score and vascular rigidity index. We further excluded individuals with an estimated glomerular filtration rate <60 ml/min/1.73 m2, glycosuria, and use of confounding medication. A spot urine sample was analyzed. Genotyping was performed using the Illumina Omni2.5-8 BeadChips. Genetic variants were analyzed using an additive model. RESULTS: Final analyses included 593 individuals (45.5% of men; mean age 54.3 ± 8.6). We observed an antagonistic interaction between rs13129697 variant of the SLC2A9 gene and FEUA tertiles on uACR (P = 0.002). Using the mediation analysis, uromodulin explained 32%, fractional excretion of sodium (FENa) 44%, and uromodulin together with FENa explained 70% of the inverse relationship between FEUA and uACR. Bootstrapping process confirmed the role of both mediators. CONCLUSIONS: Our data suggest that the association of albuminuria with decreased renal urate excretion may be modified by the transporter SLC2A9, and mediated by uromodulin and sodium handling.

Analysis of the association of EPHB6, EFNB1 and EFNB3 variants with hypertension risks in males with hypogonadism.

Several members of the EPH kinase family and their ligands are involved in blood pressure regulation, and such regulation is often sex- or sex hormone-dependent, based on animal and human genetic studies. EPHB6 gene knockout (KO) in mice leads to hypertension in castrated males but not in un-manipulated KO males or females. To assess whether this finding in mice is relevant to human hypertension, we conducted a human genetic study for the association of EPHB6 and its two ligands, EFNB1 and EFNB3, with hypertension in hypogonadic patients. Seven hundred and fifty hypertensive and 750 normotensive Han Chinese patients, all of whom were hypogonadic, were genotyped for single nucleotide polymorphisms (SNPs) within the regions of the genes, plus an additional 50 kb 5' of the genes for EPHB6, EFNB1 and EFNB3. An imputed insertion/deletion polymorphism, rs35530071, was found to be associated with hypertension at p-values below the Bonferroni-corrected significance level of 0.0024. This marker is located 5' upstream of the EFNB3 gene start site. Previous animal studies showed that while male EFNB3 gene knockout mice were normotensive, castration of these mice resulted in hypertension, corroborating the results of the human genetic study. Considering the significant associations of EFNB3 SNPs with hypertension in hypogonadic males and supporting evidence from castrated EFNB3 KO mice, we conclude that loss-of-function variants of molecules in the EPHB6 signaling pathway in the presence of testosterone are protective against hypertension in humans.

Evidence from single nucleotide polymorphism analyses of ADVANCE study demonstrates EFNB3 as a hypertension risk gene.

EPH kinases and their ligands, ephrins (EFNs), have vital and diverse biological functions. We recently reported that Efnb3 gene deletion results in hypertension in female but not male mice. These data suggest that EFNB3 regulates blood pressure in a sex- and sex hormone-dependent way. In the present study, we conducted a human genetic study to assess the association of EFNB3 single nucleotide polymorphisms with human hypertension risks, using 3,448 patients with type 2 diabetes from the ADVANCE study (Action in Diabetes and Vascular Disease: Peterax and Diamicron MR Controlled Evaluation). We have observed significant association between 2 SNPs in the 3' untranslated region or within the adjacent region just 3' of the EFNB3 gene with hypertension, corroborating our findings from the mouse model. Thus, our investigation has shown that EFNB3 is a hypertension risk gene in certain individuals.

PROX1 gene CC genotype as a major determinant of early onset of type 2 diabetes in slavic study participants from Action in Diabetes and Vascular Disease: Preterax and Diamicron MR Controlled Evaluation study.

BACKGROUND: The prevalence of diabetic nephropathy varies according to ethnicity. Environmental as well as genetic factors contribute to the heterogeneity in the presentation of diabetic nephropathy. Our objective was to evaluate this heterogeneity within the Caucasian population. METHODS: The geo-ethnic origin of the 3409 genotyped Caucasian type 2 diabetes (T2D) patients of Action in Diabetes and Vascular Disease: Preterax and Diamicron MR Controlled Evaluation was determined using principal component analysis. Genome-wide association studies analyses of age of onset of T2D were performed for geo-ethnic groups separately and combined. RESULTS: The first principal component separated the Caucasian study participants into Slavic and Celtic ethnic origins. Age of onset of diabetes was significantly lower in Slavic patients (P = 7.3 × 10), whereas the prevalence of hypertension (P = 4.9 × 10) and albuminuria (5.1 × 10) were significantly higher. Age of onset of T2D and albuminuria appear to have an important genetic component as the values of these traits were also different between Slavic and Celtic individuals living in the same countries. Common and geo-ethnic-specific loci were found to be associated to age of onset of diabetes. Among the latter, the PROX1/PROX1-AS1 genes (rs340841) had the highest impact. Single-nucleotide polymorphism rs340841 CC genotype was associated with a 4.4 year earlier onset of T2D in Slavic patients living or not in countries with predominant Slavic populations. CONCLUSION: These results reveal the presence of distinct genetic architectures between Caucasian ethnic groups that likely have clinical relevance, among them PROX1 gene is a strong candidate of early onset of diabetes with variations depending on ethnicity.

Reduced blood pressure after smooth muscle EFNB2 deletion and the potential association of EFNB2 mutation with human hypertension risk.

Ephrin B2 (EFNB2) is a ligand for erythropoietin-producing hepatocellular kinases (EPH), the largest family of receptor tyrosine kinases. It has critical functions in many biological systems, but is not known to regulate blood pressure. We generated mice with a smooth muscle cell (SMC)-specific deletion of EFNB2 and investigated its roles in blood pressure regulation and vascular SMC (VSMC) contractility. Male Efnb2 knockout (KO) mice presented reduced blood pressure, whereas female KO mice had no such reduction. Both forward signaling from EFNB2 to EPHs and reverse signaling from EPHs to EFNB2 were involved in regulating VSMC contractility, with EPHB4 serving as a critical molecule for forward signaling, based on crosslinking studies. We also found that a region from aa 313 to aa 331 in the intracellular tail of EFNB2 was essential for reverse signaling regulating VSMC contractility, based on deletion mutation studies. In a human genetic study, we identified five SNPs in the 3' region of the EFNB2 gene, which were in linkage disequilibrium and were significantly associated with hypertension for male but not female subjects, consistent with our findings in mice. The coding (minor) alleles of these five SNPs were protective in males. We have thus discovered a previously unknown blood pressure-lowering mechanism mediated by EFNB2 and identified EFNB2 as a gene associated with hypertension risk in humans.

Transcriptional divergence plays a role in the rewiring of protein interaction networks after gene duplication.

Gene duplication plays a key role in the evolution of protein-protein interaction (PPI) networks. After a gene duplication event, paralogous proteins may diverge through the gain and loss of PPIs. This divergence can be explained by two non-exclusive mechanisms. First, mutations may accumulate in the coding sequences of the paralogs and affect their protein sequences, which can modify, for instance, their binding interfaces and thus their interaction specificity. Second, mutations may accumulate in the non-coding region of the genes and affect their regulatory sequences. The resulting changes in expression profiles can lead to paralogous proteins being differentially expressed and occurring in the cell with different sets of potential interaction partners. These changes could also alter splicing regulation and lead to the inclusion or exclusion of alternative exons. The evolutionary role of these regulatory mechanisms remains largely unexplored. We use bioinformatics analyses of existing PPI data and proteome-wide PPI screening to show that the divergence of transcriptional regulation between paralogs plays a significant role in determining their PPI specificity. Because many gene duplication events are followed by rapid changes in transcriptional regulation, our results suggest that PPI networks may be rewired by gene duplication, without the need for protein to diverge in their binding specificities. This article is part of a Special Issue entitled: From protein structures to clinical applications.