Genome-Wide Meta-Analysis Unravels Interactions Between Magnesium Homeostasis and Metabolic Phenotypes

META-ANALYSIS www.jasn.org

Genome-Wide Meta-Analysis Unravels Interactions between Magnesium Homeostasis and Metabolic Phenotypes

Tanguy Corre,1,2,3 Francisco J. Arjona,4 Caroline Hayward,5 Sonia Youhanna,6 Jeroen H.F. de Baaij,4 Hendrica Belge,6 Nadine Nägele,6 Huguette Debaix,6 Maxime G. Blanchard,4 Michela Traglia,7 Sarah E. Harris,8,9 Sheila Ulivi,10 Rico Rueedi,2,3 David Lamparter,2,3 Aurélien Macé,1,3 Cinzia Sala,7 Stefania Lenarduzzi,10 Belen Ponte,11 Menno Pruijm,12 Daniel Ackermann,13 Georg Ehret,14 Daniela Baptista,14 Ozren Polasek,15 Igor Rudan,16 Toby W. Hurd,5 Nicholas D. Hastie,5 Veronique Vitart,5 Geràrd Waeber,17 Zoltán Kutalik,1,3 Sven Bergmann,2,3,18 Rosa Vargas-Poussou,19,20 Martin Konrad,21 Paolo Gasparini,22,23 Ian J. Deary,9,24 John M. Starr,8,25 Daniela Toniolo,7 Peter Vollenweider,17 Joost G.J. Hoenderop,4 René J.M. Bindels,4 Murielle Bochud,1 and Olivier Devuyst6

Due to the number of contributing authors, the afﬁliations are listed at the end of this article.

ABSTRACT Magnesium (Mg2+) homeostasis is critical for metabolism. However, the genetic determinants of the renal handling of Mg2+, which is crucial for Mg2+ homeostasis, and the potential influence on metabolic traits in the general population are unknown. We obtained plasma and urine parameters from 9099 individuals from seven cohorts, and conducted a genome-wide meta-analysis of Mg2+ homeostasis. We identified two 2 loci associated with urinary magnesium (uMg), rs3824347 (P=4.4310 13)nearTRPM6, which encodes an 2 epithelial Mg2+ channel, and rs35929 (P=2.1310 11), a variant of ARL15, which encodes a GTP-binding protein. Together, these loci account for 2.3% of the variation in 24-hour uMg excretion. In human kidney cells, ARL15 regulated TRPM6-mediated currents. In zebrafish, dietary Mg2+ regulated the expression of the highly conserved ARL15 ortholog arl15b,andarl15b knockdown resulted in renal Mg2+ wasting and metabolic disturbances. Finally, ARL15 rs35929 modified the association of uMg with fasting insulin and fat mass in a general population. In conclusion, this combined observational and experimental approach uncovered a gene–environment interaction linking Mg2+ deficiency to insulin resistance and obesity.

J Am Soc Nephrol 29: 335–348, 2018. doi: https://doi.org/10.1681/ASN.2017030267

Magnesium (Mg2+) is an essential cation for mul- Received March 11, 2017. Accepted July 19, 2017. tiple enzymatic reactions, including those involving T.C., F.J.A., and C.H. contributed equally to this work. DNA and protein synthesis and energy metabo- O.D., M.B., and R.J.M.B. have codirected this study. lism.1 Abnormal Mg2+ levels in serum are associated with common diseases such as diabetes and Published online ahead of print. Publication date available at metabolic disorders.2 Interventional and longitudi- www.jasn.org. nal observational studies in humans show that high Correspondence: Prof. Dr. Olivier Devuyst, Institute of Physiology, dietary Mg2+ protects against the risk of developing Mechanisms of Inherited Kidney Disorders Group, University of Zurich, Winterthurerstrasse 190, 8057 Zürich, Switzerland. Email: 3–6 type 2 diabetes, and improves glycemic control [email protected] or Prof. Murielle Bochud, IUMSP, Route de la in patients with diabetes7 as well as in overweight Corniche 10, 1010 Lausanne, Switzerland. Email: murielle.bochud@ META-ANALYSIS nondiabetic people.8–10 In young adults, high Mg2+ chuv.ch intake is signiﬁcantly associated with lower Copyright © 2018 by the American Society of Nephrology

J Am Soc Nephrol 29: 335–348, 2018 ISSN : 1046-6673/2901-335 335 META-ANALYSIS www.jasn.org incidence of metabolic syndrome.11 Further, there is evidence Significance Statement linking Mg2+ intake to body weight regulation, with low intake potentially impairing lean body mass growth,12,13 whereas Mg2+ Magnesium (Mg2+) is essential for many enzymatic reactions in- 2+ supplementation may increase lean body mass and decrease fat volved in energy metabolism, and abnormal Mg levels are associ- 14 ated with diabetes and metabolic disorders. The genetic factors mass in overweight women. 2+ 2+ regulating Mg homeostasis are poorly characterized. Using ge- Plasma Mg levels are closely regulated and remain con- nome-wide association studies (GWAS) in European populations, we 2+ stant throughout life, despite the fact that dietary Mg intake discovered two loci significantly associated with urinary Mg2+:the and intestinal absorption decrease with age.15 The control of TRPM6 gene coding for a Mg2+ channel and the ARL15 gene, which is Mg2+ balance is ensured by a tightly regulated reabsorption of linked to lipid levels, type 2 diabetes and cardiovascular disease. We fi fl 2+ Mg2+ in the distal tubular segments of the kidney. In particu- nd that ARL15 in uences Mg reabsorption through regulation of fi 2+ TRPM6, suggesting a genetic mechanism for the association of urinary lar, the ltered Mg is reabsorbed in the proximal tubule and Mg2+ excretion with metabolic phenotypes. These findings suggest thick ascending loop of Henle (TAL) via paracellular routes, that gene-diet interactions may contribute to the link between Mg2+ whereas downstream, in the distal convoluted tubule (DCT), homeostasis and metabolic disorders in the general population. Mg2+ is efficiently reabsorbed through transcellular mechanisms involving the transient receptor potential cation channel, subfamily M, member 6 (TRPM6).2 The reabsorption of RESULTS Mg2+ in the DCT determines the final urinary magnesium (uMg) excretion because no reabsorption of Mg2+ occurs in Meta-Analyses of GWAS for Mg2+ Homeostasis posterior segments. People harboring inherited or acquired The initial discovery phase consisted of a GWAS (2.5 M mark- dysfunctions of the renal tubular handling of Mg2+ show in- ers) performed on the population-based CoLaus cohort appropriate urinary loss of Mg2+, causing chronic hypomag- (5150 samples) testing for association with urinary Mg2+-to- nesemia and severe, multisystemic manifestations.2,16,17 creatinine ratio. The analysis revealed a single, genome-wide 2 The genetic component of Mg2+ homeostasis is indicated significant signal at rs3824347 (P=3.6310 8), corresponding by a significant heritability (15%–39%) of serum Mg2+18,19 to the TRPM6 locus on chromosome 9, and six suggestive loci 2 and by rare monogenic disorders disturbing renal tubular with P values ,10 5 (Supplemental Figure 1). These associ- transport of Mg2+.20–23 However, most of the regulatory genes ation results were combined with the genome-wide associa- of renal Mg2+ channels and transporters remain unknown. A tion scans from six additional European cohorts (LBC1936, genome-wide association study (GWAS) on serum Mg2+ in CROATIA-Split, CROATIA-Vis, Carlantino, CROATIA- European ancestry adults identified six loci, one of which in- Korcula, and Val Borbera) into a single meta-analysis (9099 cluded the gene encoding TRPM6.24 Two more recent studies samples). As shown on the Manhattan plot (Figure 1A), two 2 in children25 and in blacks26 did not reveal additional loci. signals showed a P value ,5310 8. The QQ plot showed no 2 However, changes in uMg excretion precede changes in circu- problematic inflation (l=1.014, se=3.16310 5) (Supplemen- lating serum Mg2+ levels, thus being a more sensitive indicator tal Figure 2A). Both the forest plots (Figure 1B) and the low I2 of any disturbance in Mg2+ homeostasis.27 Further, Mg2+ de- values (Table 1) indicated little heterogeneity across cohorts. pletion can be found in individuals with apparently normal No secondary signals were detected in the approximate con- total serum Mg2+ levels,28 which underscores the limitations ditional analysis, using the lead single nucleotide polymor- of total serum Mg2+ as a marker of Mg2+ status. Assessment of phisms (SNPs) as covariates in the regression (Supplemental 2 uMg in GWAS is therefore crucial to elucidate new genes in- Figure 2, B and C). The lowest combined P value (4.4310 13) volved in Mg2+ homeostasis. Further, studying the association was observed for the SNP rs3824347 on chromosome 9, at a between the genetic determinants of uMg and metabolic pheno- locus comprising five genes: TRPM6, C9orf40, C9orf41, types linked to disturbed Mg2+ balance (e.g.,obesityanddiabetes) NMRK1,andOSTF1 (Figure 1C). The second strongest signal 2 may provide novel insights into links between Mg2+ and common maps to chromosome 5, with a P value of 2.1310 11 for the diseases. Indeed, plasma triglycerides and glucose are major de- lead SNP rs35929 (Figure 1D). This SNP and all other genome- terminants of the Mg2+ balance in patients with diabetes.29 wide significant SNPs at this locus lie in the first intron of To shed light on the genetic factors and molecular mecha- the ADP ribosylation factor like GTPase 15 (ARL15) gene. None nisms linking Mg2+ handling and metabolic disorders, we of the two signals showed significant association with urinary performed a meta-analysis of GWAS for the renal handling creatinine. of Mg2+ by combining genetic isolates and population-based Results from the versatile gene-based association study (VE- studies, and investigating the biologic relevance of the identified GAS analysis)30 of urinary Mg2+-to-creatinine ratio identified loci using cellular systems and model organisms. Given the known C9orf40, C9orf41,andTRPM6 as the only genes with a statis- 2 relationships of Mg2+ intake with metabolic disorders, we also ex- tically significant result (P,2.8310 6); however, these signals plored whether the identified loci modified these relationships. are overlapping with the one SNP association in the meta- These studies establish a novel biologic control of renal Mg2+ han- analysis (rs3824347). None of the discovered SNPs tag copy dling, and a novel gene–environment interaction sustaining the link number variations (CNVs). A pathway analysis was per- between Mg2+ homeostasis, insulin resistance, and obesity. formed using the Magenta algorithm, but no enrichment

336 Journal of the American Society of Nephrology J Am Soc Nephrol 29: 335–348, 2018 www.jasn.org META-ANALYSIS

appeared to be significant after multiple testing correction. Sex-stratified analyses did not reveal additional genome-wide significant loci. No significant eQTL is refer- enced for rs35929 nor rs3824347 in the GTEx portal. Ameta-GWASofserumMg2+ levels replicated the previously published locus on chromosome 1 containing, among oth- 2 ers, the gene MUC1 (P=4.45310 14) (Supplemental Figure 3, Supplemental Table 1).24 Our meta-analysis on fractional excretion of magnesium (FEMg) 2 yielded 12 suggestive loci with a P,10 5 (Supplemental Figure 4), but no genome- wide significant association (the sample size was reduced to cohorts having serum andurinefromthesametimepoint, therefore without cohorts LBC1936, Split, and Vis; n=7976). The association of 2 rs35929 (P=2.8310 4) and rs3824347 2 (P=1.4310 5) with FEMg were nominally, but not genome-wide significant.

A Genetic Score Including rs35929 and rs3824347 Associates with uMg Traits Urinary Mg2+-to-creatinine ratio (Figure 2A, CoLaus cohort), FEMg in spot urine (Figure 2B, CoLaus cohort), and uMg excretion in 24-hour urine (Figure 2C, Swiss Kidney Project on Genes in Hypertension [SKIPOGH] cohort), but not serum Mg2+ (Figure 2D, CoLaus) were strongly and linearly associated with an additive unweighted genetic risk score including 2+ Figure 1. A genome-wide meta-analysis for urinary Mg -to-creatinine ratio reveals rs35929-A (ARL15) and rs3824347-G two signals. (A) Manhattan plot showing 2log10(P values) for all SNPs in the genome- 2+ (TRPM6) alleles. The genetic score ex- wide meta-analysis for normalized urinary Mg -to-creatinine ratio in Europeans plained 2.3% of 24-hour uMg excretion (n=9099), ordered by chromosomal position. The values correspond to the association 2+ in the SKIPOGH study and 1.0% of urinary of normalized urinary Mg -to-creatinine ratio, including age and sex as covariates in 2+ the model as well as study-specific covariates if needed. The gene closest to the SNP Mg -to-creatinine ratio in CoLaus. with the lowest P value is listed at each locus. Two loci reached genome-wide sig- 2 nificance (P,5310 8), indicated by the red horizontal line, at combined analysis ARL15 Regulates TRPM6 Channel (TRPM6, rs3824347 and ARL15, rs35929). (B) Forest plot for rs35929 (ARL15)and Activity rs3824347 (TRPM6) showing effect sizes and 95% confidence intervals across studies To validate the association between uMg as well as the summary meta-analysis results. (C) Regional association plot at the excretion and ARL15 and TRPM6 variants, rs3824347 (TRPM6) locus. Regional association plot showing 2log10 (P values) for the we first showed that the highest mRNA association of SNPs at the locus of interest ordered by their chromosomal position expression of Arl15 was in distal nephron 2+ with normalized urinary Mg -to-creatinine ratio. The -log10 (P value) for each SNP is segments including the TAL and DCT, the colored according to the correlation of the corresponding SNP, with the SNP showing DCTalso being strongly positive for Trpm6 the lowest P value (index SNP) within the locus using different colors for selected (Figure 3A). At the protein level, ARL15 is levels of LD (chi-squared). Correlation structures correspond to HapMap 2 CEU. The blue line represents the recombination according to the scale shown on the right-side localized predominantly in the TAL and the y-axis. (D) Regional association plot at the rs35929 (ARL15) locus. DCT segments (Figure 3B), which are enriched in the Tamm–Horsfall protein and 2 the Na+-Cl cotransporter, respectively.

J Am Soc Nephrol 29: 335–348, 2018 Genetic Determinants of Magnesium Homeostasis and Metabolism 337 META-ANALYSIS www.jasn.org

transfected with wild-type (WT) TRPM6 in parallel with a mock trans- fect, WT ARL15, or a dominant negative (T46N) ARL15 mutant. The T46N ARL15 mutant was predicted to be of dominant negative nature by com- parative analysis with T27N ARF6, a dominant negative mutant affecting a protein (ARF6) that belongs to the same protein family as ARL15.32 Patch clamp recordings showed slowly developing outwardly rectifying currents, a signature feature of TRPM6 under our recording conditions (Fig- ure 3C and D). A signiﬁcant increase in TRPM6 channel activity was observed in the presence of WT ARL15, but not the T46N ARL15 mutant (Fig- ure 3E). HEK293 cells express endog- enous TRPM7 channels.33 Here, these currents were small (,50 pA/pF) and were not signiﬁcantly increased by expression of WT ARL15 (Figure 3F).

Physiologic Relevance of ARL15 in Model Organisms To support the important role of ARL15 in Mg2+ homeostasis, we in- vestigated the regulation of TRPM6 and ARL15 gene expression in mice on exposure to variable Mg2+ diets, known to affect the expression of critical genes in the mouse kidney and intestine,34 where Arl15 is expressed (Supplemental Figure 6A). Metabolic profiling (Supplemental Tables 2 and 3) revealed a remarkable adaptation after exposure to high and low Mg2+ diets, reflected by significant changes in the renal mRNA expression of Trpm6 and Pvalb, contrasting Figure 1. Continued. with the stable expression of Arl15 and other segmental markers of the TAL and DCT (Supplemental Figure 6B). Importantly, TRPM6 is also distinctly localized in the DCT A similar effect was observed in the ileum and the cecum, segment.31 The spatial correlation between TRPM6 and also involved in Mg2+ handling2,34 (Supplemental Figure 6, ARL15 expression in the DCT prompted us to postulate a CandD). functional interaction between these two proteins. In addi- We further tested the biologic relevance of ARL15 by using tion, functional protein association network analyses for a zebrafish model, which shows tubular segmentation patterns ARL15 (Supplemental Figure 5) showed that ARL15 interacts that are equivalent to the mammalian kidney.35 Two distinct with key proteins for endocytic trafficking (RAB11 and ARF- orthologs of mammalian ARL15 were identified in zebrafish GEF families), a process that may increase the abundance and (Arl15a and Arl15b), presenting 68% and 83% of amino acid channel activity of TRPM6 in the plasma membrane. identity respectively, when aligned to the human counterpart To assess the potential effect of ARL15 on TRPM6 channel (Supplemental Figure 7A). Given the higher amino acid con- activity, human embryonic kidney 293 (HEK293) cells were servation of Arl15b compared with Arl15a, the former was

338 Journal of the American Society of Nephrology J Am Soc Nephrol 29: 335–348, 2018 www.jasn.org META-ANALYSIS

2 selected to study the function of its mammalian ortholog 7 I 35.7 ARL15. As for mammalian ARL15, arl15b was ubiquitously

value, expressed in all adult zebrafish tissues tested (Supplemental 2 I fi 2+ 2 Figure 7B). When adult zebra sh were fed different Mg diets for 3 weeks, arl15b expression patterns reflected compensa- tory mechanisms to cope with Mg2+ deficiency (kidney and 2+ –

Cohorts gills) or Mg surplus (gut) (Supplemental Figure 7, C E).

of Individual A loss-of-function approach using morpholinos (MOs) 22222 + 2222222

Direction of Effects against arl15b in zebraﬁsh larvae, where arl15b is expressed in the pronephros (Supplemental Figure 8), was used to relate

11 13 2+

2 2 ARL15 function to renal Mg wasting (Supplemental Figures 2+ 10 10 9 and 10). Because intestinal Mg absorption does not take 3 3 Value P

Analysis place during the time frame where the knockdown is applied, a Combined change in zebraﬁsh larvae Mg2+ content reﬂects a change in the renal and/or skin (renal-like tissue36)uptakeofMg2+.To- tal Mg2+ content decreased consistently in arl15b morphants compared with controls, indicating renal Mg2+ wasting be- 0.183) 0.152) 0.12270.1103 2.11 4.38 2 2

2 2 cause the pronephric kidney is the only extrusion route for Combined Effect Size 2+ Analysis (SE) Mg (Figure 4, A and B). In parallel, cardiovascular impairments and morphologic phenotypes characterized by a poorly 7 6 2 2 metabolized yolk in comparison with controls, indicative of 10 10 – 3 3 metabolic disturbances, were also observed (Figure 4, C G). Value

P Coinjection of arl15b-MO with human WT ARL15 cRNA for rs35929 is LBC1936, having an effect close to 0. The last column shows the Replication ”

- induced a rescue of all phenotypes observed, whereas coinjec- “ tion with T46N ARL15 cRNA did not result in any rescue (Figure 4, H–K). These rescue experiments demonstrated the speciﬁcity of the renal Mg2+ wasting and metabolic defects 0.027) ( 0.135 5.95 0.024) ( 0.1125 2.02 2 2 2 associated with dysfunctional ARL15. 2 Effect Size

Replication (SE) Genetic Background Inﬂuences the Link between uMg

6 8 and Metabolism Phenotypes 2 2

or +. The cohort displaying a 2+ 10 10 – The functional relevance of ARL15 for Mg homeostasis, as -to-creatinine ratio 3 3 Value

2+ demonstrated in this study, in combination with the associa- P

Discovery tion between ARL15 and obesity and insulin biology,37 urged us to investigate a possible link between genetic background, renal Mg2+ handling, and metabolic traits in general population cohorts. 2+ 0.02) ( 0.112 5.57 0.109 3.61 0.025) ( Urinary Mg -to-creatinine ratio was positively associated 2 ( 2 2 2 ( , Effect Size with circulating fasting adiponectin (P 0.001) and glucose Discovery (SE) levels (P=0.001), and negatively with body mass index (BMI) (P=0.001), homocysteine (P,0.001), and g-glutamyl transeptidase (GGT) (P,0.001), independently of serum Mg2+, in CoLaus (the largest cohort available). In CoLaus, Mean the positive associations with adiponectin and fasting glucose, Frequency Effect Allele as well as the negative associations with BMI, GGT, and homocysteine, remained signiﬁcant (P,0.05) upon adjustment for all the other metabolic phenotypes. In SKIPOGH, 24-hour Allele Other uMg excretion was negatively associated with fasting serum insulin (P,0.001), even after adjustment for other metabolic covariates (P=0.003). The negative associations of 24-hour Allele Effect uMg excretion with fasting triglycerides or glucose disappeared Summary statistics for the meta-analysis of urinary Mg upon adjustment for fasting insulin; we found no association of 24-hour uMg excretion with BMI nor with fat mass (P.0.40).

SNP In CoLaus, the negative association of the ARL15 rs35929 Table 1. rs35929 aThe effect size sign for each individual cohort in the last but one g column is represented by 0.21 representing the heterogeneity across cohorts. rs3824347 g a 0.41 variant with uMg excretion was stronger in overweight

J Am Soc Nephrol 29: 335–348, 2018 Genetic Determinants of Magnesium Homeostasis and Metabolism 339 META-ANALYSIS www.jasn.org

modify the association between uMG excretion and fasting insulin. The corresponding P values for interaction were 0.14 and 0.14 when replacing fasting insulin with BMI or fat mass, respectively.

DISCUSSION

In this multistep study (Figure 6), we conducted the first genome-wide meta-analysis for uMg levels using data from seven population- based studies of European descent. We identified two genome-wide significant loci associated with urinary Mg2+-to-creatinine ratio: (1) rs3824347 located on chromosome 9, near the TRPM6 gene coding for a Mg2+ channel; and (2) rs35929 located on chromosome 5, an intronic variant of the ARL15 gene known for its association with adiponectin38 and lipid levels,39,40 type 2 diabetes,40 fasting insulin levels,41 and coronary heart disease,38 but without a prior physiologic link to Mg2+ homeostasis (Supplemental Table 4). Next we provide robust evidence that ARL15 influences renal Mg2+ reabsorption through regulation of the Mg2+ channel, TRPM6. An additive genetic score including the rs35929 A (ARL15)and rs3824347 G (TRPM6) alleles is strongly associated with 24-hour uMg excretion, a physiologic test of Mg2+ status, which in- tegrates both intestinal absorption and re- 42 Figure 2. A genetic score including rs35929 and rs3824347 associates with uMg traits. nal wasting. In addition, the association (A–D) Associations of uMg excretion and fractional excretion with an unweighted of ARL15 rs35929 with uMg levels is mod- genetic risk score including rs35929 (ARL15 locus) and rs3824347 (TRPM6 locus). Data ified by the metabolic status, and the asso- are geometric means and whiskers are 95% confidence intervals for Mg2+-related ciation of uMg excretion with metabolic phenotypes in the CoLaus (A, B, D) and SKIPOGH (C) studies. The x-axes represent the phenotypes is modified by ARL15 rs35929 unweighted genetic score generated from rs35929 and rs3824347, using as effect in the general adult population. Altogether, 2+ allele the one associated with lower urinary Mg -to-creatinine ratio, i.e., the A allele these findings suggest a gene–diet interac- for rs35929 and the G allele for rs3824347. P values are from nonparametric trend tion relating Mg2+ homeostasis to metabolic tests across genetic score. The numbers in each genetic score category are listed in all disorders in the general population. If repli- panels. The y-axis represents urinary Mg2+-to-creatinine ratio (milligrams per gram) 2+ cated in independent populations, the ob- (A), FEMg (%) (B), uMg excretion (milligrams per 24 hour) (C), and serum Mg (mil- fi ligrams per deciliter) (D). served genetic effect sizes are suf ciently large to be of clinical and public health relevance. people (P,0.01 for interaction between BMI and rs35929 for In contrast to TRPM6, little is known about the function of their effect on uMg concentration in spot urine), in people the ARL15 gene, except its association with adiponectin and with high fat mass (P interaction =0.02) (Figure 5A), and in lipid levels, type 2 diabetes, and higher fasting insulin levels in people with high fasting insulin levels (P interaction =0.01) humans.38–40,43 We found that ARL15 is highly expressed in (Figure 5B), highlighting an SNP-by-environment interac- the TAL and DCT, where Mg2+ reabsorption is mostly regu- tion. In SKIPOGH, the negative association of daytime lated and the main bulk of Mg2+ is reabsorbed. Particularly, we uMg excretion with fasting insulin was stronger in carriers show that ARL15 is enriched along with the Mg2+ channel of the ARL15 rs35929 A allele (P interaction ,0.04), which is TRPM6 in the DCT, and that ARL15 increases TRPM6-mediated consistent with results in CoLaus in that rs35929 appears to currents in renal epithelial cells. Because TRPM6 is the

340 Journal of the American Society of Nephrology J Am Soc Nephrol 29: 335–348, 2018 www.jasn.org META-ANALYSIS

Figure 3. ARL15 localizes in renal DCT regulating TRPM6 channel activity. (A) Gene expression analyses of Trpm6, Arl15, Podocin, Sglt2, Snat3, Nkcc2, Ncc,andAqp2 in microdissected mouse nephron segments showed coexpression of Arl15 and Trpm6 in DCT. (B) Double immunofluorescence staining of mouse kidney cortex sections for ARL15 (in green) and BCRP (red); TH (red); NCC (red); or AQP2 (red) as markers of the proximal tubule, TAL, DCT, or collecting duct, respectively. For detection, the Alexa Fluor dye was used. (C) Typical current-voltage curves obtained from transfected HEK293 cells 200 seconds after break-in. Outwardly rectifying currents are observed in response to a 500 ms voltage ramp (from 2100 to +100 mV) applied 200 seconds after break-in. (D) The average time development of the current density measured at +80 mV is shown (n$10). The mock plus ARL15 condition is not shown for clarity reasons. (E) WT ARL15 (red, WT, n=47) but not the T46N ARL15 mutant (black, T46N, n=18) increased the whole-cell current density of TRPM6. Asterisk indicates significant difference with respect to the cells transfected with TRPM6 only (blue, “-,” n=44). One-way ANOVA followed by Tukey multiple comparisons post-test; P,0.05. (F) Transfection of HEK293 cells with WT ARL15 did not evoke a significant increase in whole-cell current density when compared with mock-transfected cells (n$10, unpaired t test). gatekeeper in transepithelial Mg2+ transport, its regulation by fed different Mg2+ diets. This discrepancy between the mouse ARL15 supports a key role of the latter in the transepithelial and the zebrafish models might be due to the fact that, in zebra- Mg2+ transport in the distal part of the renal tubule. ARL15 is fish and freshwater fish in general, active transcellular Mg2+ structurally similar to Ras-related GTP-binding proteins, reabsorption is more prominent than in terrestrial vertebrates, which regulate intracellular vesicle trafficking.44 It interacts as freshwater fish are hyperosmotic with respect to the sur- with proteins of the RAB11 and ARFGEF families, which are rounding aquatic medium.46,47 Thus, the dietary Mg2+ challenge involved in endocytic traffic, suggesting that ARL15 regulates performed with mice in this study may not be sufficiently severe the trafficking of vesicular TRPM6 to the plasma membrane, to evoke regulation of Arl15 expression. thereby influencing Mg2+ reabsorption in the kidney. In this The association of the ARL15 rs35929 variant with uMg sense, our in vivo data substantiates ARL15 as a key protein for excretion in the present GWAS predicted a physiologic rele- the maintenance of Mg2+ homeostasis. This conclusion is sup- vance of ARL15 in Mg2+ homeostasis. In agreement with these ported by the high sensitivity of ARL15 expression to dietary findings, knockdown experiments in zebrafish recognized Mg2+, as demonstrated by the regulation of the highly con- ARL15 as a relevant key protein that determines renal Mg2+ served zebrafish ortholog of ARL15, arl15b,inthekidneyand excretion. In agreement with previous GWAS linking ARL15 gills of fish exposed to different Mg2+ diets—as already ob- variants with risk of congestive heart disease,38 cardiovascular served for the zebrafish ortholog of TRPM6.45 Conversely, Arl15 impairments (i.e., deficient tail blood circulation) was recog- expression in intestine and kidney remained unchanged in mice nized in zebrafish arl15b morphants. Further, previous links

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Figure 4. ARL15 dysfunction results in renal Mg2+ wasting and metabolic disturbances. (A and B) Knockdown of arl15b by 0.5–2 (A) and 2–8 (B) ng per embryo of the two arl15b-MOs used (exon skipping 3 and 4 arl15b-MOs) resulted in a dose-dependent decrease of the total Mg2+ content of zebrafish arl15b morphants, reflecting renal Mg2+ wasting. The zero dose represents injection with control-MO (2 [A] and 8 [B] ng MO per embryo). (C) Morphologic phenotypes distinguished in zebrafish larvae (5 days postfertilization) after treatment with arl15b-MO or control-MO (253 magnification). A complete description of each phenotype is detailed in the Supplemental Ma- terial. Metabolic defects (poor metabolization of the yolk) are indicated by arrowheads. (D–G) Distribution of morphologic phenotypes in zebrafish larvae injected with 0.5–2 (D, F) and 2–8 (E, G) ng per embryo of exon skipping 3 (D, E) arl15b-MO, exon skipping 4 (F, G) arl15b-MO, or control-MO (2 [D, F] and 8 [E, G] ng MO per embryo). (H–K) Rescue of renal Mg2+ wasting (H, J) and of metabolism defects (I, K) in morphant zebrafish by coinjection of exon skipping 3 arl15b-MO (0.5 ng MO per embryo [H, I]) or exon skipping 4 arl15b-MO (8 ng MO per embryo [J, K]) with cRNA encoding human WT ARL15 (50 pg cRNA per embryo). Coinjection with cRNA encoding human T46N ARL15 mutant (50 pg cRNA per embryo) did not rescue renal Mg2+ wasting or the defects in metabolism. (D–G, I, K) Numbers on top of the bars indicate the number of animals in each experiment. (A, B, H, J) Data are presented as mean6SEM (n=10, except for [B], where n=6–10). (A, B, H, J) Asterisks indicate significant differences respect to the control condition (one-way ANOVA followed by Tukey multiple comparisons post-test; P,0.05). between ARL15 and lipid metabolism37–39,43 were functionally of the arl15b knockdown (by using doses of 1 and 2 ng exon supported in zebrafish, where arl15b morphants showed a 4–skipping arl15b-MO per embryo), renal Mg2+ wasting was poor mobilization of yolk metabolite reserves. In zebrafish the only phenotype observed in the fish morphants. There- larvae, the yolk serves mainly as a lipid store,48,49 therefore fore, the disturbances in Mg2+ balance observed in arl15b the deficiency in yolk absorption observed in arl15b mor- morphants are a primary effect of ARL15 dysfunction phants revealed poor lipid mobilization. The zebrafish and not a secondary disturbance to the kidney/metabolic/ arl15b morphants also accumulated fluid in the pericardial cardiovascular phenotypes observed. The phenotype rescue cavity and in the pronephros in the form of cysts showing experiments performed with normal or mutant versions of kidney dysfunction. However, when lowering the intensity human ARL15 confirmed that renal Mg2+ wasting and

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uMg excretion) that, unlike circulating Mg2+, was found to be associated with risk of ischemic heart disease.54 In population-based studies, the association between uMg and metabolic phenotypes (i.e., BMI, fat mass, fasting insulin levels) tended to differ by ARL15 rs35929 variants. These results are in line with human studies showing that Mg2+ intake exerts more protective effects on type 2 Figure 5. Genotypes at rs35929 influence the link between uMg and metabolism diabetes in overweight compared with related phenotypes. (A) Effect modification of fat mass on the association of uMg nonoverweight people.3 Such gene–diet concentration with rs35929 (ARL15) genotypes. Data represent adjusted square-root- interaction has already been observed for transformed uMg levels by rs35929 genotypes and fat mass strata in CoLaus. The type 2 diabetes, for which the increased risk model was adjusted for age, sex, height, lean mass, CKD-EPI, urinary creatinine conferred by the rs7903146 TCF7L2 geno- (square root), serum Mg2+, serum and urinary Ca2+ (square root), and menopausal status. P interaction =0.02. Fat mass strata are cut by sex-specific medians (n=4729). type was only observed in the absence of 55 (B) Effect modification of fasting insulin on the association of uMg levels with rs35929 lifestyle intervention. Therefore, the fi (ARL15) genotypes. Data represent adjusted square-root-transformed uMg levels by ARL15 gene may modulate the bene cial 2+ rs35929 genotypes and fasting insulin strata in CoLaus. The model was adjusted for effect of Mg intake on insulin resistance age, sex, height, lean mass, CKD-EPI, urinary creatinine (square root), serum Mg2+, in humans, offering a novel paradigm in serum and urinary Ca2+ (square root), and menopausal status. P interaction =0.01. nutrigenetics. Further, the beneficial effect Fasting insulin strata are cut by sex-specificmedians(n=4729). of high Mg2+ intake on metabolic disorders, such as type 2 diabetes and obesity, may not be uniform across individuals. metabolic defects were directly due to ARL15 knockdown. In conclusion, the translational data described in this study Altogether, the ARL15 functions unraveled in this study illus- offer novel insights for the understanding of Mg2+ homeostasis trate an unprecedented case of shared pathophysiologic genetics and its relation to metabolic phenotypes, and identify ARL15 of renal Mg2+ wasting, cardiovascular disease, and metabolic as a key player for these processes. disorders, and demonstrate the pleiotropic nature of ARL15, inferred from the present and published GWAS. The impor- tance of ARL15 is also supported by recent genetic evidence for CONCISE METHODS its positive selection among adaptive loci identified in cattle.50 Thus far, no mutations in ARL15 have been associated with a Genome-Wide Meta-Analyses monogenic disorder, including unidentified forms of renal Seven European population-based cohorts with Caucasian ethnicity Mg2+ wasting (data not shown). (CoLaus, CROATIA-Vis, CROATIA-Korcula, CROATIA-Split, Lothian Could the metabolic traits linked to ARL15 variants be Birth Cohort 1936, INGI-Val Borbera, and INGI-Carlantino, described connected? By integrating intestinal absorption and renal in the Supplemental Material) participated in the study, with a total of wasting, uMg excretion represents a physiologic test of Mg2+ 9099 individuals. A variety of electrolytes including Mg2+ as well as status more suitable than total serum Mg2+ levels.42 Urinary creatinine, were measured in both urine and serum (methods and mean Mg2+-to-creatinine ratio was associated with multiple values described in the Supplemental Methods and Supplemental metabolic phenotypes, independently of serum Mg2+,inthe Table 5). population-based CoLaus study, providing insights into the All cohorts were whole genome genotyped before imputation us- metabolic consequences of Mg2+ deficiencies. Our results are in ing the HapMap CEU panel as reference (Supplemental Tables 6 and line with longitudinal studies in humans suggesting that self- 7). Serum Mg2+ measurements and FEMg were subjected to quantile- reported dietary Mg2+ intake may protect against type 2 diabetes, quantile normalization in order to reach normality before perform- insulin resistance,51 and metabolic syndrome.52 Growing exper- ing linear regression. uMg measurements expressed in milligrams per imental evidence in favor of a beneficial role of Mg2+ intake on deciliter were first standardized to urinary creatinine, then corrected type 2 diabetes is accumulating in humans. Mg2+ supplements for age-, sex-, and study-specific covariates (such as ancestry princi- were found to significantly improve glucose tolerance and in- pal components, study center) before undergoing a quantile-quantile sulin sensitivity in diabetic and nondiabetic people.53 Whether normalization. Approximately 2.5 M SNPs passing quality control such benefit would persist over years is currently unknown. The checks were subjected to a linear regression of the residuals for public health relevance of dietary Mg2+ intake is further under- each phenotype, and association summary statistics were collected. scored by an association with all-cause mortality and risk of Even though quantile-quantile plots of the individual GWAS cardiovascular disease.51 The novelty of our results is that we showed minimal inflation, ruling out the presence of population benefit from an objective measure of Mg2+ intake (namely substructure, a genomic-control correction on the basis of the

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for association using the summary statistics from genetic association studies. VEGAS assigns SNPs to genes and combines their association P values into a gene-based test statistic. Permuta- tions are used to calculate the null distribution of the test statistic for each gene in order to derive an empirical gene-wise P value. The gene-based approach also reduces the multiple-testing problem of GWAS by only consider- ing statistical tests for 17,787 genes, giving a Bonferroni-corrected threshold of P,2.8E26.

Pathway Analysis We used Magenta58 to check enrichment for pathways in either the BIOCARTA KEGG REACTOME databases, which were contained in the msigdb version 4.0 (http://www.broadinstitute.org/gsea/ msigdb/collections.jsp#C2).

Metabolome Analysis Nuclear magnetic resonance spectroscopy data obtained from urine samples was binned and normalized to produce metabolome features.59 These features were then associated with the SNPs of interest, including relevant covariates among standard covariates (age, sex, genotype principal components) and lifestyle factors (smoking, caffeine use, dietary intake, etc.).

CNV Analysis In-house datasets were used to call CNVs and to check their correlation with the SNPs of interest. The CNVs call was done using pennCNV software.60 An SNP-by-sample matrix with the Figure 6. ARL15 was identified as a key player for metabolism and renal homeostasis. copy number status was created. Then the The figure shows the multistep approach (A) used to investigate the genetic deter- square correlation (Pearson correlation) be- minants of the renal handling of Mg2+ and their influence on metabolic traits in the tween the value of each SNP of interest and general population (B). the SNPs copy number status in a 6100 kb re- gion has been calculated. For the SNPs of interest for which no correspondence has been l factor was applied to the association P values. Subsequently, a meta- found in the datasets, they have been replaced by the closest SNPs in analysis was conducted using the inverse variant weighting method high linkage disequilibrium (LD) and present in the datasets. LD implemented in METAL.56 A new genomic-control correction was between the SNPs of interest and a list of SNPs tagging CNVs from applied to combined statistics. the Genetic Investigation of ANthropometric Traits (GIANT) consortium has also been calculated. The SNPs from the GIANT list are SKIPOGH Cohort in LD .0.8 with their corresponding CNV.60 SKIPOGH is a population-based, family-based, multicenter study focusing on BP regulation and renal function.57 Each participant Laboratory Measurements collected 24-hour urine. The study was approved by the institutional Electrolytes, hematology parameters, and glycaemia were dosed in ethical committees of each participating university hospital and par- the biochemical platform of the University of Zürich using standard ticipants signed written informed consent. clinical laboratory methods. Creatinine was measured using Jaffe kinetic compensated method (Roche Diagnostics, Switzerland; VEGAS Analysis intra-assay variability 0.7%–2.9%). The CKI Epidemiology Col- The SNP association P values from the meta-analysis of uMg were laboration (CKD-EPI) formula was used to calculate the eGFR. analyzed using VEGAS,30 a program for performing gene-based tests In CoLaus, urinary uromodulin, creatinine, electrolytes, and

344 Journal of the American Society of Nephrology J Am Soc Nephrol 29: 335–348, 2018 www.jasn.org META-ANALYSIS osmolality concentrations were measured in morning spot urine comparison post-test. When only two experimental groups were af- samples. fected by the factor of variance, an unpaired t test was used. Statistical All urinary biochemical parameters were measured from samples significance was set at P˂0.05. The datasets and summary statistics are stored at 280°C, using the same biochemical platform UniCel DxC available in the IUMSP Research Data Repository, under the link 800 Synchron Clinical System (Beckman Coulter, Nyon, Switzerland) https://data.iumsp.ch/index.php/catalog/6 (identification number: at the University of Zürich. Appropriate controls and sets of calibra- CHE-GWAS-V01; created on July 12, 2017). tion standards were used before running each sample batch. All cohorts were subjected to the same measurement protocol, in the same Metabolic Factors Associated with uMg Excretion laboratory. We used multiple linear regression to explore the association between square-root-transformed urinary Mg2+-to-creatinine ratio in spot Tissue Distribution and Localization of ARL15 urine and several metabolic markers (i.e., fasting triglycerides, total The tissue distribution of Arl15 and of its highly conserved zebrafish cholesterol, HDL cholesterol, glucose, insulin, homocysteine, adipo- ortholog arl15b was studied in adult mouse, adult zebrafish, and zebra- nectin, GGT, and BMI), taken individually as the dependent variable, fish larvae, as described in the Supplemental Material (for information and also adjusting for serum Mg2+ and other potential confounders on the primer sequences used for the RT-qPCR measurements, see in SKIPOGH and CoLaus cohorts. In SKIPOGH, we used multivar- Supplemental Table 8). Segmental expression of ARL15 in the mouse iate linear mixed effect regression to explore the association of fasting (C57BL/6J) kidney was studied at both protein and mRNA level. Five- insulin with 24-hour uMg excretion, with adjustment for potential micrometer sections of fixed frozen kidney samples were costained for confounders. We used a conservative P value (0.05/9=0.0056) thresh- ARL15 and specific segment markers: breast cancer resistance protein old to consider the associations of uMg with metabolic phenotypes as 2 for the proximal tubule, Tamm–Horsfall protein for the TAL, Na+-Cl statistically significant. cotransporter for the DCT, and aquaporin-2 for the collecting duct. For mRNA expression, kidneys were dissected and minced, before isolation SNP-by-Environment Interaction on uMg Levels of well characterized nephron segments. For more details of the immu- We explored the role of metabolic phenotypes on the association of nohistochemistry, microdissection studies, and mRNA analyses in the ARL15 rs35929 with uMg levels in spot urine among CoLaus partic- nephron segments, see the Supplemental Material. ipants. We conducted multiple linear regression to explore the modifying effect of BMI, fat mass, and fasting insulin levels on the Functional Studies in Cells and Animal Models association of the ARL15 rs35929 with square-root-transformed uMg Tostudy the regulation of TRPM6 channel activity byARL15, HEK293 concentration, including age, sex, height, lean mass, CKD-EPI, uri- cells were transfected with 1 mg of human TRPM6 cDNA33 and 250 ng nary creatinine (square root), serum Mg2+,serumandurineCa2+ of empty vector (mock), human WT ARL15, or a human dominant (square-root), and menopausal status. The P values for interaction negative (T46N) ARL15 mutant as negative control. Whole-cell patch were not corrected for multiple testing because we tested a single clamp recordings were performed to determine the electrophysiolog- global hypothesis (does the association between rs35929 and uMg ical properties of the cells transfected with the constructs mentioned differ by metabolic status?), guided by prior knowledge. above (see details in the Supplemental Material). The regulation of ARL15 gene expression by dietary Mg2+ was studied in C57BL/6J mice and Tupfel long-finzebrafish (details in ACKNOWLEDGMENTS the Supplemental Material. Primer information for RT-qPCR deter- minations is indicated in Supplemental Table 8). These studies were We would like to acknowledge the invaluable contributions of the followed by a loss-of-function approach in zebrafish larvae using two recruitment team in Korcula and Split, the administrative teams in nonoverlapping, splice-site blocking MOs: 59-AAACACTGAAA- Croatia and Edinburgh, and the people of Korcula and Split. The SNP GACGGGACAAAGAC-39 and 59-GTTAAGCGAGTATTAGGT- genotyping for the CROATIA-Korcula cohort was performed in TACCTCT-39 (Gene Tools, Philomath, OR), designated as exon Helmholtz Zentrum München, Neuherberg, Germany. The SNP skipping 3 and 4 arl15b-MO, respectively. These antisense oligonucle- genotyping for the CROATIA-Split cohort was performed by AROS otides were used to knockdown the highly conserved ARL15 ortholog in Applied Biotechnology, Aarhus, Denmark. We thank the LBC1936 the zebrafish, arl15b. Phenotype rescue experiments were performed by (Lothian Birth Cohort 1936) participants, the LBC1936 team for data coinjecting arl15b-MOs with human ARL15 cRNA as previously de- collection and collation, and the staff at the Wellcome Trust Clinical scribed,61 and detailed in the Supplemental Material. The use of two Research Facility for bio-sample collection and genotyping. We thank nonoverlapping arl15b-MOs and the combination with rescue experi- Dr. Sjoerd Verkaart, Anke Hoefnagels and Sami G. Mohammed for ments with human ARL15 cRNA served to rule out potential off-target their excellent technical support during the performance of the effects in our knockdown approach. functional analyses, and acknowledge Dr. Erwin van Wijk and Dr. Gert Flik for providing the facilities for zebrafish experimentation. Statistical Analyses in Experiments Performed in For INGI (Italian Network on Genetic Isolates)-Carlantino, we thank Cellular and Animal Models Anna Morgan and Angela D’Eustacchio for technical support; the All results are depicted as mean6SEM. Statistical analyses were municipal administrators for their collaboration on the project and for conducted by one-way ANOVA followed by the Tukey multiple logistic support; and all participants of the study.

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This work was supported by the Long-Term Fellowship from the 2. de Baaij JHF, Hoenderop JGJ, Bindels RJM: Magnesium in man: Im- European Renal Association European Dialysis and Transplant As- plications for health and disease. Physiol Rev 95: 1–46, 2015 sociation (ERA LTF 175-2014) and the Impulsion Grant from the 3. Dong JY, Xun P, He K, Qin LQ: Magnesium intake and risk of type 2 diabetes: Meta-analysis of prospective cohort studies. Diabetes Care ERA-EDTA Working Group on Inherited Kidney Disorders to F.J.A; 34: 2116–2122, 2011 and by the grant from The Netherlands Organization for Scientific 4. Hruby A, Meigs JB, O’Donnell CJ, Jacques PF, McKeown NM: Higher Research (NWO; VICI 016.130.668) to J.G.J.H. J.H.F.d.B. is supported magnesium intake reduces risk of impaired glucose and insulin me- by a grant from NWO (Rubicon 825.14.021) and R.J.M.B. is supported tabolism and progression from prediabetes to diabetes in middle-aged – by the EURenOmics project from the European Union Seventh americans. Diabetes Care 37: 419 427, 2014 – 5. Lopez-Ridaura R, Willett WC, Rimm EB, Liu S, Stampfer MJ, Manson JE, Framework Programme (FP7/2007 2013, agreement no 305608). Hu FB: Magnesium intake and risk of type 2 diabetes in men and Z.K. received financial support from the Leenaards Foundation, the women. Diabetes Care 27: 134–140, 2004 Swiss National Science Foundation (31003A-143914), and SystemsX. 6. Schulze MB, Schulz M, Heidemann C, Schienkiewitz A, Hoffmann K, Boeing ch (51RTP0_151019). M.B. and T.C. are supported by the Swiss H: Fiber and magnesium intake and incidence of type 2 diabetes: A pro- – National Centre of Competence in Research Kidney Control of Ho- spective study and meta-analysis. Arch Intern Med 167: 956 965, 2007 7. Song Y, He K, Levitan EB, Manson JE, Liu S: Effects of oral magnesium meostasis (NCCR Kidney.C.H.) program. O.D. is supported by the supplementation on glycaemic control in Type 2 diabetes: A meta- European Community’s Seventh Framework Programme (305608 analysis of randomized double-blind controlled trials. Diabet Med 23: EURenOmics), the Swiss National Centre of Competence in Research 1050–1056, 2006 Kidney Control of Homeostasis (NCCR Kidney.C.H.) program, the 8. Chacko SA, Sul J, Song Y, Li X, LeBlanc J, You Y, Butch A, Liu S: Mag- fl Swiss National Science Foundation (31003A_169850), and the Rare nesium supplementation, metabolic and in ammatory markers, and global genomic and proteomic profiling: A randomized, double-blind, Disease Initiative Zurich (radiz), a clinical research priority program controlled, crossover trial in overweight individuals. Am J Clin Nutr 93: of the University of Zurich, Switzerland. The SKIPOGH (Swiss 463–473, 2011 Kidney Project on Genes in Hypertension) study is supported by a 9. Mooren FC, Krüger K, Völker K, Golf SW, Wadepuhl M, Kraus A: Oral grant from the Swiss National Science Foundation (FN 33CM30- magnesium supplementation reduces insulin resistance in non-diabetic 124087). The CoLaus (Cohorte Lausannoise) study is supported by subjects - a double-blind, placebo-controlled, randomized trial. Di- abetes Obes Metab 13: 281–284, 2011 research grants from GlaxoSmithKline, the Faculty of Biology and 10. 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AFFILIATIONS

1Institute of Social and Preventive Medicine, 17Department of Medicine, Internal Medicine, and 12Service of Nephrology, University Hospital of Lausanne, Lausanne, Switzerland; 2Department of Computational Biology, University of Lausanne, Lausanne, Switzerland; 3Swiss Institute of Bioinformatics, Lausanne, Switzerland; 4Department of Physiology, Radboud Institute for Molecular Life Sciences, Radboud University Medical Center, Nijmegen, The Netherlands; 5Medical Research Council Human Genetics Unit, Institute of Genetics and Molecular Medicine, 8Centre for Cognitive Ageing and Cognitive Epidemiology, 16Usher Institute of Population Health Sciences and Informatics, 24Department of Psychology, and 25Alzheimer Scotland Dementia Research Centre, University of Edinburgh, Edinburgh, Scotland, UK; 6Institute of Physiology, University of Zürich, Zurich, Switzerland; 7Division of Genetics and Cell Biology, San Raffaele Scientiﬁc Institute, Milan, Italy; 9Medical Genetics Section, University of Edinburgh Centre for Genomic and Experimental Medicine and Medical Research Council Institute of Genetics and Molecular Medicine, Western General Hospital, Edinburgh, Scotland, UK; 10Department of Medical Genetics, Institute for Maternal and Child Health, Istituto di Ricovero e Cura a Carattere Scientiﬁco “Burlo Garofolo,” Trieste, Italy; 11Service of Nephrology and 14Division of Cardiology, Department of Internal Medicine Specialties, University Hospital of Geneva, Geneva, Switzerland; 13University Clinic for Nephrology and Hypertension, Inselspital, Bern University Hospital, University of Bern, Bern, Switzerland; 15Faculty of Medicine, University of Split, Split, Croatia; 18Department of Integrative Biomedical Sciences, University of Cape Town, Cape Town, South Africa; 19Department of Genetics, Hôpital Européen Georges Pompidou, Assistance Publique Hôpitaux de Paris, Paris, France; 20Centre de Référence des Maladies Rénales Héréditaires de l’Enfant et de l’Adulte, Paris, France; 21Department of General Pediatrics, University Hospital Münster, Munster, Germany; 22Department of Medical, Surgical and Health Sciences, University of Trieste, Trieste, Italy; and 23Department of Experimental Genetics, Sidra, Doha, Qatar

348 Journal of the American Society of Nephrology J Am Soc Nephrol 29: 335–348, 2018