1. Prospective Evaluation of B-type Natriuretic Peptide Concentrations and the Risk of Type 2 Diabetes in Women B.M. Everett, N. Cook, D.I. Chasman, M.C. Magnone, M. Bobadilla, N. Rifai, P.M. Ridker, and A.D. Pradhan March 2013 www.clinchem.org/content/59/3/557.full © Copyright 2013 by the American Association for Clinical Chemistry

2. © Copyright 2009 by the American Association for Clinical Chemistry Introduction  Natriuretic Peptides in Heart Failure  Promote vasodilation, natriuresis and diuresis in normal physiology  Assays for the active form of the B-type natriuretic peptide (BNP) and the inactive amino-terminal fragment of the peptide (NT-proBNP) are commercially available  Commonly used to diagnosis of heart failure in acute- care settings

3. © Copyright 2009 by the American Association for Clinical Chemistry  Natriuretic Peptides in Metabolism  Natriuretic peptides play an important role in regulating lipolysis in both humans and animals  Several cross-sectional studies in humans have reported inverse associations between natriuretic peptide levels and features of the metabolic syndrome, including  Body mass index  Glucose concentrations  Measures of insulin resistance  Blood pressure  However, prospective studies of natriuretic peptides and incident type 2 diabetes (T2DM) are scarce and results have been inconsistent Introduction

4. © Copyright 2009 by the American Association for Clinical Chemistry  To determine the correlations between plasma concentrations of natriuretic peptides and other circulating markers of metabolism and inflammation  To determine whether baseline concentrations of B-type natriuretic peptides associate with incident T2DM in a cohort of women followed prospectively for T2DM  To determine whether common genetic variants in the natriuretic peptide promoter region (NPPA-NPPB), which are known to affect B-type natriuretic peptide concentrations, also associate with incident T2DM Aims

5. © Copyright 2009 by the American Association for Clinical Chemistry  Describe the strengths and weaknesses of evaluating the relationship between B-type natriuretic peptide concentrations and T2DM in a prospective as compared to a cross-sectional study.  Are these strengths and weaknesses more or less important when studying diabetes? Question

6. © Copyright 2009 by the American Association for Clinical Chemistry  Laboratory Analysis Plasma NT-proBNP was measured using an electrochemiluminescent immunassay from Roche Diagnostics Plasma concentrations of a variety of metabolic, lipid, and inflammatory biomarkers were also measured DNA was genotyped using Illumina Infinium II platform. Included samples were successfully genotyped for 98% of assayed single nucleotide polymorphisms (SNPs) and did not deviate from Hardy-Weinberg 4 SNPs in natriuretic peptide A and B (NPPA-NPPB) region previously associated with natriuretic peptide levels, type 2 diabetes, and/or metabolic syndrome were identified from the literature: rs198358, rs5068, rs632793, rs198389 rs198358 was directly genotyped, and estimated allele dose for remaining SNPs calculated using MACH (http://www.sph.umich.edu/csg/abecasis/mach/index.html) Materials and Methods: Laboratory Studies

7. © Copyright 2009 by the American Association for Clinical Chemistry  Prospective association of NT-proBNP and T2DM  491 women with incident T2DM  561 women selected as the reference subcohort  Correlations between NT-proBNP and other biomarkers were analyzed in the reference subcohort of 561 women  Cross-sectional association of genotype and NT-proBNP levels  Subset (n=458) of the 561 women selected as the reference subcohort who had genotyping information available  Correlations between NT-proBNP and other biomarkers were analyzed in the reference subcohort of 561 women  Prospective association of genotype and incident T2DM  1372 cases of incident T2DM in 22,607 women at risk Materials and Methods: Study Participants

8. © Copyright 2009 by the American Association for Clinical Chemistry  How do you determine which covariates to include in a statistical model testing the relationship between an exposure (e.g., NT-proBNP concentrations) and an outcome (e.g., T2DM)?  The minor allele frequency at each of the tested SNPs varies from approximately 6% to 40%.  At which SNP are you likely to have the most power to detect an association with either NT-proBNP concentrations or incident T2DM?  How should a null result at the lower frequency SNPs be interpreted? Questions

9. © Copyright 2009 by the American Association for Clinical Chemistry Correlations between NT-proBNP and other metabolic biomarkers

10. © Copyright 2009 by the American Association for Clinical Chemistry Risk of incident T2DM by NT-proBNP concentration at baseline

11. © Copyright 2009 by the American Association for Clinical Chemistry Genetic variants in NPPA-NPPB and NT- proBNP concentrations

12. © Copyright 2009 by the American Association for Clinical Chemistry Association between variants in NPPA- NPPB and incident T2DM Table 4. Each of the four SNPs previously noted to be associated with either natriuretic peptide levels, metabolic syndrome traits, and/or type 2 diabetes were tested for association with incident type 2 diabetes in the Women’s Genome Health Study. For this analysis, 22,607 women of European ancestry without type 2 diabetes at baseline who had available genotyping information were followed for the development of incident type 2 diabetes. In total, 1372 women developed type 2 diabetes during follow up. SNP Per-Allele Relative Risk (95% CI) P-value rs1983590.94 (0.86-1.03)0.18 rs50680.98 (0.83-1.16)0.84 rs6327930.91 (0.84-0.989)0.026 rs1983890.92 (0.85-0.995)0.036

13. © Copyright 2009 by the American Association for Clinical Chemistry  What aspects of this study support a causal link between natriuretic peptide concentrations and type 2 diabetes?  What aspects of this study do not support such a link? Questions

14. © Copyright 2009 by the American Association for Clinical Chemistry  NT-proBNP concentrations that are high, but still within the reference interval, associate with incident T2DM in a prospective study of initially healthy women  Authors observed a nearly 50% reduction in the risk of incident type 2 diabetes among women with NT-proBNP concentrations >117 ng/L, a concentration that is both within the reference interval and routinely observed in clinical practice  NT-proBNP concentrations correlate with fasting insulin levels in healthy women, even after adjusting for age and body mass index Conclusions (1)

15. © Copyright 2009 by the American Association for Clinical Chemistry  Common variants in the NPPA-NPPB promoter region associate with a lower risk of T2DM  These same variants associate with increased plasma concentrations of NT-proBNP  The direction of their effect on the risk of T2DM is consistent with their direction of effect on NT-proBNP concentrations, and with the observation that increased NT-proBNP concentrations associate with lower T2DM risk  These prospective data, in combination with other studies in both humans and animals, support a causal role for the natriuretic peptide system in the development of type 2 diabetes Conclusions (2)

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