Methods
A two-sample MR study was conducted to investigate the association of
genetically proxied inhibition of IL-6 signalling with different
measures of renal function: estimated glomerular filtration rate (eGFR),
CKD and blood urea nitrogen (BUN). Two-sample refers to the fact that
the instrument-exposure and instrument-outcome estimate are obtained
from two different genome wide association study (GWAS), increasing the
statistical power of the MR study.
Genetic variants for downregulated IL-6 signalling were selected as
independent (linkage disequilibrium r2< 0.1) single-nucleotide polymorphism (SNPs) within 300 kB of
the IL-6 receptor gene (IL6R ) that strongly associated with
circulating CRP (p < 5×10-8 with independent
associations as described by Georgakis et al8), a
downstream target of IL-6 signalling. The association between the IL6R
genetic variants and CRP level were taken from the summary data of the
Cohorts for Heart and Aging Research in Genomic Epidemiology (CHARGE)
Inflammation Working Group GWAS of 204,402 individuals of European
ancestry.13 In this GWAS, association estimates are
expressed as change in natural log transformed CRP (mg/L). The variance
in CRP levels explained by the genetic variants,R2 , was calculated using the formula:
R2 = [2 × MAF × (1–MAF) × β2]/var(log(CRP)),
where MAF is the minor allele frequency and β is the effect estimate of
the SNP on CRP levels. F-statistics, a measure of instrument strength in
MR, were calculated using the formula: F =
𝑅2×(𝑁−2)/(1−𝑅2) whereR 2 is the variance of CRP explained by the
specific variant and N the number of individuals in the GWAS
analysis.
Summary GWAS data from the Chronic Kidney Disease Genetics (CKDGen)
Consortium meta-analyses were used for the primary outcome associates of
log eGFR (61 studies, 765,348 participants), BUN (33 studies, 416,178
participants), CKD (30 studies, 64,164 cases and 561,055
controls).14 Log eGFR was calculated using the Chronic
Kidney Disease Epidemiology Collaboration (CKD-EPI) equation in adults
and using the Schwartz formula for participants who were 18 years or
younger. CKD was constructed as a binary outcome based on an eGFR
< 60 ml min−1 per 1.73 m2.
BUN was calculated as 2.8 x blood urea (mg/dl).
Data for the exposure and outcome were harmonised according to the
effect allele and no exclusions were made for palindromic variants.
Random-effects inverse-variance weighted method was used as the primary
MR analysis. To assess potential pleiotropy, we conducted sensitivity
analyses using the simple median, weighted median and MR-Egger methods.
The median methods are robust even with up to 50% of the contribution
to the MR estimates coming from invalid instrumental
variables.15 MR-Egger provides robust estimates even
when all instrumental variables are invalid, as long as the INstrument
Strength Independent of Direct Effect (INSIDE) assumption holds - that
any pleiotropic effect of the variants on the outcome are independent of
the strength of their association with the exposure.15The estimated MR-Egger intercept is indicative of the average
pleiotropic effect of the variants used.15 We tested
for such pleiotropy by assessing whether our intercept was significantly
different from zero.15 Results are presented as effect
estimates and corresponding 95% confidence intervals in forest plots.
For eGFR and BUN respectively, estimates represent the change in log
eGFR or blood urea nitrogen per genetically predicted decrease in
natural log of CRP. For CKD the results are expressed as odds ratio of
CKD, with 95% confidence intervals. All data analyses were performed by
the “TwoSampleMR” package version 4.26 in R software.