Common Variants in CLDN14 Are Associated with Differential Excretion

Common Variants in CLDN14 are Associated with Differential Excretion of Magnesium over Calcium in Urine

Tanguy Corre1,2,3,*, Eric Olinger4,*, Sarah E. Harris5,6, Michela Traglia7, Sheila Ulivi8, Stefania Lenarduzzi8, Hendrica Belge4, Sonia Youhanna4, Natsuko Tokonami4, Olivier Bonny9, Pascal Houillier10, Ozren Polasek11,12, Ian J. Deary5,13,, John M. Starr5,14, Daniela Toniolo7, Paolo Gasparini15,16, Peter Vollenweider9, Caroline Hayward17,#, Murielle Bochud1,#, Olivier Devuyst4,#

SUPPLEMENTARY MATERIAL

Suppl. Fig. S1: Forest plots showing the heterogeneity for each associated locus. Suppl. Fig. S2: Linkage disequilibrium structure of SNP’s downstream of CLDN14. Suppl. Fig. S3. Effect of Mg2+ diets on urinary Mg2+ and Ca2+ excretion and expression of the Ca2+-sensing receptor. Suppl. Table S1: Study genotyping characteristics. Suppl. Table S2: Effect of Mg2+ diets on mouse urinary parameters. Suppl. Table S3: Effect of Mg2+ diets on mouse body weight and plasma parameters. Suppl. Table S4: Primers used in real-time PCR analyses.

1 Suppl. Fig. S1. Forest plots showing the heterogeneity for each associated locus. The X axis represents the effect size. Each cohort is represented by a square, of size proportional to 1/SE, with whiskers proportional to SE. The bottom line of each panel represents the combined effect, centered at combined effect size value, and of width spanning the combined SE.

2 3 Suppl. Fig. S2. Linkage disequilibrium structure (from Hapmap CEU v22) of SNP’s downstream of CLDN14. rs172639 is depicted in a green box and the SNP’s previously associated with kidney stones [40] are boxed in blue (LD expressed in D’). The bottom left and right insets show the actual D’ and r2 values of LD, respectively, between the five SNP’s of interest.

4 5 Suppl. Fig. S3. Effect of Mg2+ diets on urinary Mg2+ and Ca2+ excretion and expression of the Ca2+-sensing receptor.

(A & B) C57BL/6J mice were assigned to a low Mg2+ diet (0.005% wt/wt Mg2+), a high Mg2+ diet (0.48% wt/wt Mg2+) or a control diet (0.19% wt/wt Mg2+) for 10 days. Urinary Mg2+ (A) and Ca2+ (B) excretion rates (ng/min) are depicted before (baseline) and after 5 and 10 days on the respective diets. Bars indicate means ± SEM; N see Suppl Table S2; *** p<0.001 compared to control diet. (C) Western blot for the calcium-sensing receptor (CaSR) in total murine kidney lysates after 10 days of low Mg2+ diet (0.005% wt/wt Mg2+), high Mg2+ diet (0.48% wt/wt Mg2+) or control diet (0.19% wt/wt Mg2+). B-actin is shown as a loading control. Densitometry analysis of CaSR/β-actin reveals no significant change in renal CaSR expression. Bars indicate mean ± SEM.

6 Suppl. Table S1. Study genotyping characteristics.

Study CoLaus INGI-Val Borbera INGI- CROATIA-Vis CROATIA CROATIA-Split LBC1936 Carlantino -Korcula

Array type Affymetrix Illumina 370k Illumina Illumina Illumina Illumina Illumina 610 500K 370k HumanHap300v1 HumanHap HumanHap370CNV 370CNV

Genotype BRLMM BeadStudio analysis BeadStudio Genome Studio Genome Genome Studio Genome studio calling software analysis Studio software

QC filters for pHWE<1e-7; call rate >= 90%; MAF >= call rate >= SNP Call rate<=0.98, SNP Call SNP Call rate<=0.98, pHWE

0.0001 90%; MAF MAF<0..01, pHWE<1e- rate<=0.98, MAF<0..01, pHWE<1e- 3 individual call SNPs used rate <90%; >= 1%; 6, Individual Call MAF<0..01 6, Individual Call rate <95%; SNP for SNP call call rate<98%; pHWE p > rate<0.95 , rate<0.95 imputation rate<70%; MAF<0.01 MAF<0.01 0.0001 pHWE<1e- 6, Individual Call rate<0.95

No of SNPs 390'631 332'887 309’430 289'827 307'625 321'456 ~500,000 used for imputation

Imputation IMPUTE v0.2 MACH MACH MACH 1.0.16 MACH MACH 1.0.16 MACH 1.0.16

Imputation HapMap HapMap release 22 (build HapMap HapMap release 22 (build HapMap HapMap release 22 (build HapMap II (build Backbone for release 21 36) release 22 36) release 22 36) 36) phased CEU (build 35) (build 36) (build 36) haplotypes (NCBI build)

Filtering of none Rsq>0.3 Rsq>0.3 None None None none imputed

7 genotypes

Data QUICKTEST R, GenABEL, ProbABEL R, R, GenABEL, ProbABEL R, R, GenABEL, ProbABEL mach2qtl management (mmscore function was GenABEL, (mmscore function was GenABEL, (mmscore function was and statistical used to account for ProbABEL used to account for ProbABEL used to account for analysis relatedness) (mmscore relatedness) (mmscore relatedness) function function was used to was used to account for account for relatedness) relatedness)

Population We included We included the first 3 We We included the first 3 We We included the first 3 We included the stratification the first 3 principal components as included the principal components as included principal components as first 4 principal or Principal principal covariates first 3 covariates the first 3 covariates components as Components components as covariates principal principal covariates components components as as covariates covariates

8 Suppl. Table S2. Effect of Mg2+ diets on mouse urinary parameters.

Day 5 Control diet Low Mg2+ diet High Mg2+ diet N Diuresis (µl/min.gBW) 0.050 ± 0.005 0.055 ± 0.004 0.044 ± 0.003 20 / 22 / 20 U Na+ (µg/min) 3.19 ± 0.27 3.07 ± 0.32 2.52 ± 0.27 20 / 22 / 20 U Cl- (µg/min) 6.86 ± 0.62 7.26 ± 0.60 5.65 ± 0.62 20 / 22 / 20 U K+ (µg/min) 13.1 ± 1.14 13.2 ± 1.07 11.7 ± 1.20 20 / 22 / 20 U Mg²+ (ng/min) 428 ± 33 22 ± 3.9*** 1018 ± 114*** 20 / 22 / 20 U Ca2+ (ng/min) 34.3 ± 3.8 40.9 ± 5.1 37.7 ± 5.6 13 / 12 / 17

Day 10 Control diet Low Mg2+ diet High Mg2+ diet N Diuresis (µl/min.gBW) 0.053 ± 0.005 0.055 ± 0.003 0.051 ± 0.003 20 / 22 / 20 U Na+ (µg/min) 3.32 ± 0.34 3.31 ± 0.32 3.19 ± 0.29 20 / 22 / 20 U Cl- (µg/min) 7.89 ± 0.79 8.26 ± 0.69 6.95 ± 0.67 20 / 22 / 20 U K+ (µg/min) 15.4 ± 1.67 16.0 ± 1.15 15.1 ± 1.35 20 / 22 / 20 U Mg²+ (ng/min) 527 ± 59 9.6 ± 1.4*** 1311 ± 115*** 20 / 22 / 20 U Ca2+ (ng/min) 38.4 ± 3.8 34.8 ± 6.1 41.8 ± 4.4 16 / 7 / 15

*** p < 0.001 versus control diet (two-tailed unpaired t-test) N: number of mice/parameters in each group (Control/Low Mg2+/High Mg2+); diets for 5 or 10 days. U, urine

9 Suppl. Table S3. Effect of Mg2+ diets on mouse body weight and plasma parameters.

Day 10 Control diet Low Mg2+ diet High Mg2+ diet N Body weight (g) 21.6 ± 0.6 21.7 ± 0.7 22.8 ± 0.8 20 / 22 / 20 P urea (mg/dl) 51.9 ± 1.9 66.9 ± 2.4*** 50.6 ± 1.7 20 / 22 / 20 P creatinine (mg/dl) 0.070 ± 0.004 0.077 ± 0.005 0.077 ± 0.004 20 / 22 / 20 P Na+ (mmol/l) 153 ± 0.5 156 ± 0.5*** 152 ± 0.4 20 / 22 / 20 P Cl- (mmol/l) 111 ± 0.6 111 ± 0.8 109 ± 0.6 20 / 22 / 20 P K+ (mmol/l) 4.3 ± 0.1 4.4 ± 0.1 4.6 ± 0.1 20 / 22 / 20 P Mg2+ (mg/dl) 2.97 ± 0.06 1.22 ± 0.09*** 3.22 ± 0.09* 20 / 22 / 20 P Ca2+ (mmol/l) 2.27 ± 0.02 2.13 ± 0.03*** 2.33 ± 0.02 20 / 22 / 20

* p < 0.05 and *** p < 0.001 versus control diet (two-tailed unpaired t-test) N: number of parameters in each group (Control/Low Mg2+/High Mg2+) P, plasma

10 Suppl. Table S4. Primers used in real-time PCR analyses.

Gene PCR Forward primer (5’-3’) Reverse primer (5’-3’) Efficiency product Product (bp)

Gapdh TGCACCACCAACTGCTTAGC GGATGCAGGGATGGGGGAGA 176 1.04 ± 0.03

Cldn14 AAGACCACCTTTGCAGTGCT TGGCCGATTTCAAACTTCAT 146 1.00 ± 0.03

Cldn16 TTTGATGGGATTCGAACCTG CAGTCCAGACCAAGGAGCA 143 0.99 ± 0.02

Cldn19 ACTGCTGTCTCCTGGTACGC GTGCAGCAGAGAAAGGAACC 152 1.02 ± 0.03

Kcnj1 GGCATGACATCAGCCTTTCT ATGGCACCACACATGAAAGA 155 0.98 ± 0.03

Trpm6 TCTTCCTTCGAGAGCCATCA TCCACCAGGATTGGAGTCAC 151 0.97 ± 0.03

Casr CTCTGCTGCTTCTCCAGCT GGCCTCAAATACCAGGAGGA 150 0.99 ± 0.02

Trpv5 ACACCACCAGGAGCAGAATC GGACAAAGGAGGTGTTTGGA 165 0.98 ± 0.03

Slc12a1 CCGTGGCCTACATAGGTGTT GGCTCGTGTTGACATCTTGA 154 1.01 ± 0.03