C-Reactive Protein and Diabetes: proving a negative, for a change?

Eric Brunner PhD FFPHReader in Epidemiology and Public Health

MRC Centre for Causal Analyses in Translational Epidemiology2 March 2009Bristol

Objective

To conduct a definitive study of the relation between CRP and type 2 diabetes with focus on the residual confounding explanation for the adjusted epidemiological effect

Background

Raised serum hs-CRP is a non-specific marker of low-grade inflammation and a risk factor for type 2 diabetes (T2D)Papers in Diabetes, Diabetes Care, Arch Int Med, JAMA suggest CRP may be causally related to insulin resistance

Prospective studiesAge, sex, BMI adjusted risk ratios

Dehghan et al Diabetes 2007

Correlated risk factors measured imprecisely:residual confounding writ large

HDL-C and TG measured with differing degrees of error Observed correlation r= -0.44

Estimated β for CHDTG effect HDL effectuniv adj for HDL univ adj for TG

Observed 0.26 0.12 -0.39 -0.32Repeat r 0.6/0.9 0.46 0.35 -0.44 -0.15Repeat r 0.7/0.9 0.38 0.17 -0.43 -0.30

Phillips and Davey Smith JCE 1991

So how are we to interpret the evidence in the case of

C-reactive protein type 2 diabetes

?

Is the relation between serum CRP and diabetes causal?

Brunner et al PLoS Med 2008

Ridker’s Medical News

Model (279 cases, total N=4291 ) HR 95% CI P

Age, sex, CRP>10 mg/L 1.40 (1.29-1.51) <0.0001

+ occupational status 1.39 (1.28-1.50) <0.0001

+ prevalent CHD, infectious symptoms 1.39 (1.28-1.50) <0.0001

+ BMI categories, waist circumference 1.22 (1.11-1.33) <0.0001

+ systolic BP, diastolic BP, BP treatment 1.20 (1.10-1.32) <0.0001

+ serum HDL-cholesterol, TG 1.17 (1.07-1.28) 0.001

Hazard ratio (95%CI) for diabetes per doubling of serum CRP at age 49 with sequential adjustments. 13.1 year follow-up

Whitehall II study

Alternative explanations for the observed risk factor-disease relation

Adapted from Hingorani and Humphries 2005

Confounding Reverse causation True causation

T2DM T2DM T2DMβ cell death

Intepretation?

CRP-T2D effect attenuated by 53% on adjustment

Is the cup half full or half empty?

What are the potential explanations?

Mendelian randomization

Taking advantage of natural genetic randomisation during sexual reproduction

MR technique compares the effect of phenotype and genotype

Two concepts

1. Random allocation of parental alleles to offspring leads to lifetime exposure to differing levels of the risk factor, in the present case CRP haplotype and heritable CRP level.

2. The genetically-influenced component of variation in the risk factor will generally be unaffected by confounding (Mendel’s 2nd law) and reverse causation, in contrast to the variation associated with environmental influences.

Study design: Whitehall II cohort

Diabetes: phenotype defined by glucose tolerance tests and self report at baseline and follow-up (13 years)

Glycaemia: HbA1c measured at follow-up (2003-4)Insulin resistance: HOMA-IR at follow-up (fasting GLU*INS)/22.5)

Phenotype: serum hs-CRP by immunonephelometry at baseline & follow-up

CRP haplotype: 3 tSNPs genotyped in the CRP gene +1444T>C, +2303G>A, +4899T>GABI Prism sequence detection system; genotyping error rate <1% (n=553)+1444 not in HWE (P=0.003) (n=678 repeats)

Sample: 5274 white Europeans with CRP haplotype plus HbA1c or HOMA-IR

Statistical methods

Conventional linear, logistic and Cox regression

Instrumental variables analysisCRP haplotypes used as instrumental variables for the unconfounded and

unbiased effect of CRP on HbA1c and HOMA-IRTwo-stage least squares method. 1. model haplotype-serum CRP-outcome association assuming each of a

participant's two haplotypes contributes additively to serum CRP level. Timpson et al Lancet 2005F-statistic used to evaluate strength of the instruments (F>10 indicates sufficient strength to ensure the validity of instrumental variable methods). Staiger and Stock, Econometrica 1997

2. compare results from the instrumental variable estimates of the association of CRP with HbA1c/HOMA-IR to those from ordinary linear regression using the Durbin form of the Durbin-Wu-Hausman statistic.

Analytic framework

CRP gene(haplotype)

serum CRP(phenotype)

Age, socioeconomic status,BMI, BP

insulin resistancehyperglycaemia

T2DM

Instrumented variable

?

Instrumental variables analysisTest of linear regression effect versus instrumented effect

ParticipantsCharacteristic N (%) unless

otherwise statedTotal N

Age, mean (SD), y 60.9 (5.9) 5274

Women 1425 (27.0) 5274

BMI, mean (SD), kg/m2 26.7 (4.3) 5251

Physical inactivity 233 (4.5) 5231

Low occupational status* 432 (8.3) 5223

Current smoking 426 (8.1) 5248

Prevalent CHD† 695 (13.2) 5274

Serum CRP, mean (SD), mg/L 2.58 (5.21) 5274

Previous serum CRP, mean (SD), mg/L†† 1.72 (3.21) 4674

Prevalent diabetes** 348 (7.1) 4883

Haemoglobin A1c, mean (SD), % 5.30 (0.60) 5266

HOMA-IR, mean (SD), (mU/L.mmol/L)/22.5 2.14 (1.89) 4357

†† at mean age 49

Median (IQR) CRP, mg/L

Haplotype of +1444, +2302 and +4899 SNPs

At mean age 61 yearsN=5092

At mean age 49 yearsN=4594

CAT0 (n = 2224/2014) 1.28 (0.70 to 2.51) 0.90 (0.46 to 1.79)1 (n = 2334/2098) 1.14 (0.61 to 2.35) 0.77 (0.39 to 1.53)2 (n = 534/482) 0.90 (0.47 to 1.89) 0.71 (0.37 to 1.29)P for trend† <0.0001 <0.0001

CGG0 (n = 4558/4116) 1.15 (0.61 to 2.33) 0.78 (0.40 to 1.59)1 (n = 516/461) 1.45 (0.80 to 2.81) 0.96 (0.53 to 1.91)2 (n = 18/17) 1.78 (1.13 to 3.73) 1.82 (1.47 to 4.35)P for trend† <0.0001 <0.0001

CGT0 (n = 2457/2193) 1.20 (0.62 to 2.48) 0.84 (0.43 to 1.67)1 (n = 2163/1980) 1.16 (0.62 to 2.30) 0.78 (0.40 to 1.58)2 (n = 472/421) 1.17 (0.67 to 2.45) 0.79 (0.40 to 1.62)P for trend† 0.43 0.01

TGT0 (n = 2403/2169) 1.11 (0.58 to 2.27) 0.77 (0.39 to 1.49)1 (n = 2262/2047) 1.20 (0.65 to 2.43) 0.83 (0.42 to 1.68)2 (n = 427/378) 1.43 (0.75 to 2.73) 0.99 (0.54 to 2.06)P for trend† <0.0001 <0.0001

Association between CRP haplotypes and serum CRP

excludes CRP>10mg/L

CRP haplotype is a determinant of serum CRP phenotype, and therefore a potential instrumental variable

Associations of risk factors for diabetes with serum CRP, HbA1c and HOMA-IR at age 61

log2CRP (mg/L) lnHbA1c (%) lnHOMA-IR

Risk factor Beta* (95% CI) P Beta* (95% CI) P Beta* (95% CI) P

Occupational status

0.086 (0.061-0.112) <0.0001 0.006 (0.004-0.007) <0.0001 0.019 (0.005-0.032) 0.006

BMI 0.126 (0.118-0.135) <0.0001 0.005 (0.005-0.006) <0.0001 0.093 (0.089-0.097) <0.0001

Waist circumference (per 10cm)

0.513 (0.483-0.544) <0.0001 0.022 (0.019-0.024) <0.0001 0.364 (0.350-0.379) <0.0001

Diastolic BP (per 10mmHg)

0.232 (0.196-0.268) <0.0001 0.005 (0.002-0.007) <0.0001 0.184 (0.165-0.202) <0.0001

Physical inactivity†

0.327 (0.142-0.512) 0.001 0.012 (0.001-0.025) 0.07 0.077 (-0.026-0.181) 0.14

linear regression adjusted for age and sex

Strong associations between known risk factors and(a) exposure (serum CRP) (b) outcome (HbA1c, HOMA-IR)

Risk factors confound CRP-T2D relation

Cross-sectional and prospective associations of serum CRP with HbA1c and HOMA-IR

Ratio of geometric means (95% CI) *

Outcome N Adjusted for age, sex and raised CRP

P Age, sex, raised CRP and risk factor

adjusted †

P

HbA1c (%) at mean age 61

Per doubling of CRP concentration at mean age 61

5022 1.011 (1.009-1.013) <0.0001 1.005 (1.003-1.007) <0.0001

Per doubling of CRP concentration at mean age 49

4471 1.010 (1.008-1.011) <0.0001 1.003 (1.001-1.005) 0.003

HOMA-IR at mean age 61

Per doubling of CRP concentration at mean age 61

4184 1.154 (1.138-1.169) <0.0001 1.012 (1.000-1.024) 0.050

Per doubling of CRP concentration at mean age 49

3773 1.120 (1.105-1.134) <0.0001 1.025 (1.012-1.038) <0.0001

† adjusted for age, sex, raised CRP (>10 mg/L), occupational status, prevalent CHD, smoking, physical inactivity, BP, BP medication, BMI categories, waist circumference, serum HDL cholesterol and triglycerides, and additionally at age 61, diabetic medication

CRP phenotype link with HbA1c and HOMA-IR considerably attenuated but not abolished Parallel to the findings with serum CRP and incident T2D

Relation of CRP haplotypes with HbA1c and HOMA-IR at mean age 61

Trend test p-value adjusted for age group and sex

No CRP haplotype effect on HbA1c or HOMA-IR

Comparison of cross-sectional and prospective associations of CRP with HbA1c and HOMA-IR estimated by linear regression and instrumental variables analysis

Linear regression analysis adjusted for age and sex

a Test of equality of linear regression and instrumental variables estimatesDurbin-Wu-Hausman test

Linear regression finds CRP--T2D/HbA1c/HOMA-IR associations

Instrumental variables analysis does not (p > 0.6)

Test of difference between estimates highly or borderline significant

Upper limits of 95%CI for IV analysis compatible with phenotypic effect adjusted for age and sex

CRP haplotype and T2D: POOLED ANALYSIS, WII & NPHSII

Logistic regression, adjusted for study, age (and WII sex)

NPHSII: same haplotypes, 174 T2D cases, 2173 men aged 56

Total 522 T2D cases

3 snps, LD with WII/NPHSII tag SNPs (r2=0.75-1.0)1923 Type 2 diabetes cases, 2932 controls

OR (95% CI)

rs12760041 0.98 (0.92-1.04)rs2592889 1.04 (0.98-1.12)rs11265260 0.91 (0.81-1.03)

REPLICATION ANALYSIS: WTCCC

No association between proxy snps and caseness

Bounds of 95%CI compatible with 20% effect size

DISCUSSION

Significant additional evidence against major causal effects of CRP on T2D and quantitative traits, insulin resistance and hyperglycaemia Study approaches proving the null/negativeLarger studies?Further genetic analysis might involve low frequency intermediate-penetrance (LFIP) variants if identified (Keavney)Ball is in the CRP advocates court