Autosomal-Recessive Mutations in SLC34A1 Encoding Sodium-Phosphate Cotransporter 2A Cause Idiopathic Infantile Hypercalcemia

CLINICAL RESEARCH www.jasn.org

† ‡ † Karl P. Schlingmann,* Justyna Ruminska, Martin Kaufmann, Ismail Dursun,*§ Monica Patti, | | Birgitta Kranz,* Ewa Pronicka, Elzbieta Ciara, Teoman Akcay,¶ Derya Bulus,** †† ‡‡ || Elisabeth A.M. Cornelissen, Aneta Gawlik, Przemysław Sikora,§§ Ludwig Patzer, ††† ‡‡‡ Matthias Galiano,¶¶ Veselin Boyadzhiev,*** Miroslav Dumic, Asaf Vivante, ‡‡‡ ||| Robert Kleta,§§§ Benjamin Dekel, Elena Levtchenko, René J. Bindels,¶¶¶ Stephan Rust,* † † ‡ † Ian C. Forster, Nati Hernando, Glenville Jones, **** Carsten A. Wagner, and Martin Konrad*

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

ABSTRACT Idiopathic infantile hypercalcemia (IIH) is characterized by severe hypercalcemia with failure to thrive, vomiting, dehydration, and nephrocalcinosis. Recently, mutations in the vitamin D catabolizing enzyme 25-hydroxyvitamin

D3-24-hydroxylase (CYP24A1) were described that lead to increased sensitivity to vitamin D due to accumulation of theactivemetabolite 1,25-(OH)2D3. In a subgroup of patients who presented in early infancy with renal phosphate wasting and symptomatic hypercalcemia, mutations in CYP24A1 were excluded. Four patients from families with parental consanguinity were subjected to homozygosity mapping that identified a second IIH gene locus on chromosome 5q35 with a maximum logarithm of odds (LOD) score of 6.79. The sequence analysis of the most promising candidate gene, SLC34A1 encoding renal sodium-phosphate cotransporter 2A (NaPi-IIa), revealed autosomal-recessive mutations in the four index cases and in 12 patients with sporadic IIH. Functional studies of mutant NaPi-IIa in Xenopus oocytes and opossum kidney (OK) cells demonstrated disturbed trafficking to the plasma membrane and loss of phosphate transport activity. Analysis of calcium and phosphate metabolism in Slc34a1-knockout mice highlighted the effect of phosphate depletion and fibroblast growth factor-23 suppression on the development of the IIH phenotype. The human and mice data together demonstrate that primary renal phosphate wasting caused by defective NaPi-IIa function induces inappropriate production of 1,25-(OH)2D3 with subsequent symptomatic hypercalcemia. Clinical and laboratory findings persist despite cessation of vitamin D prophylaxis but rapidly respond to phosphate supplementation. Therefore, early differentiation between SLC34A1 (NaPi-IIa) and CYP24A1 (24-hydroxylase) defects appears critical for targeted therapy in patients with IIH.

J Am Soc Nephrol 27: 604–614, 2016. doi: 10.1681/ASN.2014101025

Serum calcium levels are primarily maintained by (FGF23) that primarily regulates phosphate metab- vitamin D and parathyroid hormone (PTH). The olism limits the action of vitamin D by inhibiting its conversion of vitamin D to its biologically active form 1,25-(OH)2D3, as well as its inactivation, in- volve sequential hydroxylation steps which are Received October 22, 2014. Accepted April 3, 2015. tightly regulated (Figure 1). The biologic activity Published online ahead of print. Publication date available at of both key activating and deactivating enzymes www.jasn.org. a 25-OH-D3-1 -hydroxylase (CYP27B1) and 25- Correspondence: Martin Konrad, Department of General Pediatrics, ’ OH-D3-24-hydroxylase (CYP24A1) is mainly con- University Children s Hospital Münster, Waldeyerstrasse 22, D-48149 Münster, Germany. Email: [email protected] trolled by 1,25-(OH)2D3 itself, calcium, phosphate, and PTH. In addition, ﬁbroblast growth factor 23 Copyright © 2016 by the American Society of Nephrology

604 ISSN : 1046-6673/2702-604 J Am Soc Nephrol 27: 604–614, 2016 www.jasn.org CLINICAL RESEARCH activation via 1a-hydroxylase and promoting its degradation knockout mice that normalize calcium metabolism following via 24-hydroxylase. Defects in vitamin D activation and action phosphate supplementation but not after omission of vitamin D cause different forms of vitamin D-dependent rickets whereas supplementation alone. impaired degradation of 1,25-(OH)2D3 underlies idiopathic infantile hypercalcemia (IIH).1,2 IIH (OMIM #143880) was first described in the 1950s after RESULTS an epidemic occurrence of unexplained hypercalcemia in infants receiving increased amounts of vitamin D via fortified Genome-Wide Linkage and Mutational Analysis milk products for the prevention of rickets.3,4 Although a link We identified four patients from three consanguineous families to exogenous vitamin D was recognized early, the pathophys- (F1–F3) with typical IIH without mutations in CYP24A1 iology remained unknown until the recent identification of (Supplemental Figure 1). Homozygosity mapping revealed a inactivating mutations in CYP24A1.2 Meanwhile, CYP24A1 single region on chromosome 5q35 with a maximum multi- mutations have also been described in adults who primarily point logarithm of odds score of 6.79 (Supplemental Figure 2). presented with nephrolithiasis while remaining asymptomatic The critical interval of approximately 1.66 Mb contains 30 during infancy.5 known RefSeq genes as well as 15 putative transcripts (Sup- Here, we demonstrate genetic heterogeneity of IIH by the plemental Table 1) including SLC34A1 as the most promising identification of autosomal-recessive loss-of-function mutations positional candidate. Direct sequencing of SLC34A1 yielded in SLC34A1 encoding the renal sodium-phosphate cotrans- homozygous mutations in all four patients subjected to ho- porter NaPi-IIa. Affected patients, in addition to hypercalcemia mozygosity mapping. Next, the SLC34A1 gene was screened and suppressed PTH levels, also exhibit hypophosphatemia due in a larger cohort of sporadic IIH patients without CYP24A1 to renal phosphate wasting. The critical role of phosphate de- mutations (n=126). Biallelic mutations were identified in 11 ficiency for the development of IIH is replicated in Slc34a1 patients (Supplemental Figures 1 and 3). Solely in patient F5.1, only one heterozygous mutation could be identified. Cosegregation analysis was com- patible with autosomal-recessive inheritance (Supplemental Figure 1). In total, 16 different mutations were identified (Figure 2A/B). All mutations were excluded in at least 204 healthy Caucasian control alleles. Further- more, we identified an in-frame deletion of seven amino acids (91del7) that has been described previously6 and is listed in the human exome variant server with an allele frequency of approximately 2.6% in the European American population (http:// evs.gs.washington.edu). For this variant, a larger sample was tested, yielding an allele frequency of approximately 1.6% (8 out of 512 alleles).

Clinical Findings All 15 patients with proven SLC34A1 mutations (F1.1–F14.1) were clinically re- evaluated, as well as one patient with early-onset nephrocalcinosis (F15.1) (Table 1, Supplemental Figure 1). Clinical details of patient F4.1 have been reported previously (patient 3 in Lameris et al.7). All patients received vitamin D supplements from birth Figure 1. Integrated scheme of calcium and phosphate metabolism. The activation of according to their home country’s recom- vitamn D to its biologically active form 1,25-(OH) D by 1a-hydroxylase (CYP27B1) as well 2 3 mendations. The age at onset varied between as its degradation by 24-hydroxylase (CYP24A1) are tightly controlled by 1,25-(OH)2D3 itself, serum calcium, and PTH (lower part). In addition, vitamin D metabolism is critically 20 days and 10 months with failure to thrive inﬂuenced by phosphate homeostasis via the action of the primary phosphaturic hor- and polyuria being the most frequent clinical mone FGF23 that limits the action of active 1,25-(OH)2D3 by inhibiting 1a-hydroxylase symptoms. Renal ultrasound demonstrated (CYP27B1) and activating 24-hydroxylase (CYP24A1) (upper part). medullary nephrocalcinosis in all infants. A

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retrospective analysis of laboratory data at the time of initial manifestation revealed hypercalcemia and suppressed intact parathyroid hormone (iPTH) levels. During hypercalcemia, active 1,25-(OH)2D3 was determined in 11/15 patients and found to be elevated in 6/11 patients (Table 1, Supplemental Table 2, median=82 pg/ml). In patient F15.1, nephrocalcinosis was discovered accidentally at 18 months of age. In this patient, only polyuria had been no- ticed before. The laboratory evaluation revealed a high–normal serum calcium and an iPTH level at the lower normal limit (Table 1). A thorough re-evaluation of phosphate metabolism revealed hypophosphatemia (S-PO4 median=1.1 mmol/l (25.0SDS, adjusted for age)). Impaired renal phosphate conservation could be demonstrated in four out of seven patients with available data (tubular maximum phosphate reabsorption per glomerular filtration rate, TmP/GFR#22SDS). During acute treat- ment of hypercalcemia vitamin D supplements were stopped in all patients, additional therapeutic measures included intravenous rehydration, furosemide, corticosteroids, and ketoconazole (Supplemental Table 2). A low calcium diet was initiated in four patients. Thereafter, serum calcium levels decreased but tended to be continuously elevated during follow-up in six patients. Six out of 16 patients were treated with oral phosphate (sodium phosphate or sodium glycerophosphate). The determination of TmP/GFR (13/16 patients with data) during follow-uprevealedlowvalues(TmP/ GFR#22SDS) in four patients, seven patients exhibited TmP/GFR in the lower part of the normal range. In the majority of patients (11/16), iPTH levels normalized during follow-up (median follow-up Figure 2. Genetic and functional analyses of SLC34A1/NaPi-IIa. (A) Identified mutations of 3.5 years). FGF23 levels were determined in the SLC34A1 gene. In total, 16 different mutations were identified including six mis- in 8/16 patients and found to be within the sense mutations, two frame-shift mutations, one in-frame deletion, two stop mutations, normal range during follow-up in the pres- and five splice-site mutations. (B) Secondary topology of the human NaPi-IIa protein ence of normophosphatemia and normal- (adapted from Fenollar-Ferrer et al.36) with mutations indicated. (C) Phosphate transport ized calcium metabolism. activity of wild-type and mutant hNaPi-IIa. Uptakes were performed in Xenopus oocytes 3 In the context of the efficacy of the days after injection of cRNA encoding hNaPi-IIa. n=2, each 8–10 oocytes; NI, non- mentioned therapeutic measures, patient injected; wt, wild-type. (D) Expression of human NaPi-IIa cotransporters in OK cells. Cells F9.1 is of special importance because were transfected with pEGFP plasmids containing either wild-type or mutant hNaPi-IIa, as SLC34A1 mutations were detected during well as with the empty pEGFP plasmid. Confluent cultures were processed for confocal acute disease manifestation while still being microscopy. (a) Focal planes of lateral projections. (b) Focal planes of apical projections. (c) Cross-sections. NaPi-IIa signal is shown in green and the actin staining in red. hypercalcemic and exhibiting phosphate deficiency. Prior to manipulation of dietary

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Table 1. Clinical and biochemical characteristics of the patient cohort

27: Sporadic IIH cases F15.1 Patient F1.1 F1.2 F2.1 F3.1

604 F4.1–F14.1 (n=11) nephrocalcinosis –

1,2016 614, Origin Turkey Turkey Turkey Turkey Poland Age at presentation 20 days 1 month 2 months 2 months 1–10 months 18 months Vitamin D prophylaxis 400IE 400IE 500IE 400IE 200–2000IE (n=11) 800IE Weight loss/failure to thrive Yes Yes No Yes 8/11 No Polyuria/dehydration Yes Yes No Yes 8/10 Yes Muscular hypotonia Yes No No No 3/10 No Nephrocalcinosis Yes Yes Yes Yes 11/11 Yes Hypercalciuria Yes Yes Yes Yes 8/11 No Initial serum calcium (mM) (2.1–2.6) 3.5 2.9 3.1 3.2 3.1 (2.6–3.8) 2.6 Initial serum phosphate (mmol/l) 1.0 (25.8SDS) 0.5 (28.6SDS) 1.5 (22.8SDS) 0.7 (27.2SDS) 1.2 (0.6–1.9) (24.1SDS) 1.7 (+0.4SDS) TmP/GFR (mmol/l GF) 0.9 (23.3SDS) n.d. 1.4 (20.3SDS) n.d. 0.9 (0.5–1.6) (22.0SDS) n.d. Initial iPTH (pg/ml)(14–72) 1.0 15 5.5 31 ,3(,1–4.9) 13 Initial 25-OH-D3 (ng/ml) (10–65) 28 n.d. 45 46 33.7 (15.9–56.3) 21 Initial 1,25-(OH)2D3 (pg/ml) (17–74) 139 n.d. 146 26 72.3 (50.8–271) 32 Therapy (acute phase) Ketoconazole Rehydration No Rehydration steroids Rehydration (10/10) No phosphate furosemide Steroids (3/10) Bisphosphonates (1/10) Therapy (long term) Oral phosphate Oral phosphate No Low calcium diet Low calcium diet (3/11) Potassium citrate hydrochlorothiazide hydrochlorothiazide Hydrochlorothiazide (1/11) Potassium citrate (2/11) Oral phosphate (3/11) Follow-up Age at last visit 1.5 years 7 years 6 years 1.5 years 5 years (0.5–17) 3.5 years Last serum calcium (mM) (2.1–2.6) 2.7 2.5 2.4 2.8 2.6 (2.5–2.7) (11/11) 2.7 2 2 –

aiIaadIfnieHypercalcemia Infantile and NaPi-IIa Last serum phosphate (mM) 0.9 ( 4.1SDS) 1.1 ( 1.7SDS) 1.5 (+0.5SDS) 1.8 (+0.9SDS) 1.5 (0.9 1.8) (+0.1SDS) 1.7 (+1.3SDS) 2 2 2 2

TmP/GFR (mmol/l GF) 0.8 ( 3.0SDS) 1.0 ( 2.0SDS) 1.3 ( 0.5SDS) n.d. 1.3 ( 0.5SDS) 1.6 (+1.0SDS) www.jasn.org Last PTH (pg/ml)(14–72) 21 23 20 42 16 (7.1–33) 22 FGF23 (kRU/l)(26–110) 136 n.d. n.d. 77 51 (29–114) n.d. Last 25-OH-D3 (ng/ml) (10–65) 8 12 26 36 26 (15–63) 38 Last 1,25-(OH)2D3 (pg/ml) (17–74) 57 75 58 n.d. 59 (35–146) 54 SLC34A1 mutations nucleotide level c.644(+1)g.a homoz. c.644(+1)g.a homoz. c.458G.T homoz. c.1006(+1)g.a homoz. See Supplemental Material c.271_91del homoz. RESEARCH CLINICAL SLC34A1 mutations protein level IVS6(+1)g.a homoz. IVS6(+1)g.a homoz. p.G153V homoz. IVS9(+1)g.a homoz. See Supplemental Material p.91del7 homoz. The individual data are provided for the patients from consanguinous families F1–F3 used for homozygosity mapping as well as for patient F15.1 who presented with polyuria and nephrocalcinosis. The data of sporadic cases of IIH (F4.1–F14.1) are summarized. For clinical symptoms the proportion of affected patients is provided, the values indicated for laboratory parameters correspond to median and range. For serum phosphate and TmP/GFR, age-dependent SDS are provided (for sporadic cases median SDS). GF, glomerular filtrate; n.d., no data available. 607 CLINICAL RESEARCH www.jasn.org phosphate intake, oral rehydration as well as a diet containing low calcium and de- void of vitamin D supplements had been unable to correct the hypercalcemia. Therefore, a supplementation with oral phosphate in a dose of 0.5–1 mmol/kg/day was initiated leading to a rapid correction of hypophosphatemia, a rapid normalization of calcium metabolism, and a significant clinical improvement reflected by a rapid weight gain (Figure 3). Clinicaland/or biochemicaldataofparents was available for 12 families (Supplemental Tables 3 and 4). Whereas nephrocalcinosis was not identified in any relative, both parents of patient F5.1 had suffered from kidney stone disease, the mother of patient F9.1 underwent nephrectomy in adolescence after recurrent pyelonephritis and a stag- horn calculus. The remaining carriers of heterozygous SLC34A1 mutations with available data were free of renal pathology. Bio- chemical analyses indicated normocalcemia, normophosphatemia and iPTH levels within the normal range; only the father of patient F13.1 with heterozygous SLC34A1 mutation p.V408E displayed hypophosphatemia (0.6 mmol/l) and a suppressed iPTH (,3 pg/ml), while being normocalcemic and free of clinical symptoms. However, in 4/6 carriers of heterozygous SLC34A1 mutations, TmP/GFR was in the lower normal range (0.8 mmol/l).

NaPi-IIa-Mediated Phosphate Uptake in Xenopus Oocytes Human wild-type and mutant NaPi-IIa constructs were functionally expressed in Xenopus oocytes and transport of labeled 32 phosphate ( Pi) was measured. Overex- pression of wild-type NaPi-IIa induced a 32 Figure 3. Clinical course of patient F9.1 during acute disease manifestation. Whereas significant Pi uptake as described previously.8 In contrast, injection of mutant rehydration and omitting of vitamin D prophylaxis did not lead to correction of hypercalcemia and clinical improvement, phosphate supplementation implemented after NaPi-IIa complementary RNA (cRNA) 32 fi genetic diagnosis of SLC34A1 mutations resulted in normophosphatemia, a normali- did not produce Pi uptakes signi cantly zation of calcium metabolism, a reduction in calcium excretion, as well as a rapid different from non-injected controls, com- clinical recovery and weight gain. The inset shows severe medullary nephrocalcinosis patible with a loss of function and/or de- on renal ultrasonography in this infant. fective trafficking to the membrane of identified mutations. Only the 91del7 var- 32 iant yielded Pi uptakes comparable to localizationwas studied by confocal microscopy (Figure 2D). For the wild-type construct (Figure 2C). wild-type NaPi-IIa, a regular localization at the plasma membrane was observed (visible as patchy apical accumulations on Subcellular Localization of Mutant NaPi-IIa in OK Cells focal as well as cross-sectional planes) in colocalization with EGFP-tagged human NaPi-IIa constructs were transiently actin. Mutant NaPi-IIa constructs displayed a complete intracel- transfected into opossum kidney (OK) cells and the subcellular lular retention and no detectable actin colocalization. The

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91del7 variant was found to be expressed both in intracellular compartments as well as at the plasma membrane, indicating a partial retention of this variant inside the cell (Figure 2D).

Animal Data To study the mechanisms behind the immediate clinical improvement upon phosphate supplementation in patient F9.1, the influence of phosphate and vitamin D was studied in Slc34a1 knockout mice. After weaning, mice were fed diets with low or high phosphate (lowP/highP) as well as low or high vitamin D content (lowD/highD). As expected, lowP diets led to severe hypophosphatemia in knockout mice whereas wild- type animals remained normophosphatemic (Figure 4). The highD diets resulted in a vitamin D overload in knockout and wild-type animals reflected by high 25-OH-D3 levels. In re- sponse, wild-type mice were able to adequately down-regulate vitamin D activation. They exhibited low CYP27B1 mRNA as well as high CYP24A1 mRNA levels and showed very low serum levels of active 1,25-(OH)2D3. In contrast, knockout animals showed significantly higher levels of CYP27B1 mRNA (0.110 in knockout versus 0.005 in wild-type, P=0.001) while CYP24A1 expression was low (0.34 in knockout versus 7.78 in wild-type, P,0.001). As a consequence, levels of 1,25-(OH)2D3 stayed significantly higher (16.5 ng/ml in knockout versus 7.4 ng/ml in wild-type, P=0.01), contributing to an exacerbation of hypercalcemia and hypercalciuria. The impaired inhibition of vitamin D activation in hypophosphatemic knockout mice likely occurred in consequence of suppressed FGF23 levels (14 pg/ml in knockout versus 571 pg/ml in wild-type, P,0.05). Equally low FGF23 levels were observed in knockout animals on a lowP/ lowD diet. Despite low vitamin D supply, these mice showed an augmented vitamin D activation with higher levels of active 1,25-(OH)2D3 resulting in hypercalcemia and hypercalciuria. In contrast, the highP diets normalized serum phosphate levels and FGF23. Consecutively, levels of 1,25-(OH)2D3 as well as Figure 4. Biochemical data of Slc34a1 knockout mice in com- serum calcium returned to their physiologic ranges. For a sum- parison to wild-type animals. Both mice were fed diets with low mary of results see Figure 4 (the full data set is provided in or high phosphate content (lowP/highP) and vitamin D (lowD/ Supplemental Table 5). highD), respectively. HighD diets resulted in a vitamin D overload in both knockout and wild-type mice. Wild-type mice adequately limited vitamin D activation by downregulating Cyp27b1 and activating Cyp24a1 expression. In contrast, phosphate-depleted DISCUSSION Slc34a1 knockout mice exhibited low FGF23 levels, provoking a reverse regulation of Cyp27b1 (1a-hydroxylase) and Cyp24a1 Using a positional candidate gene approach, we identified loss- (24-hydroxylase) expression. Consequently, these mice were not of-function mutations in SLC34A1 encoding renal proximal able to limit vitamin D activation, leading to an aggravation tubular sodium-phosphate cotransporter NaPi-IIa in a cohort of hypercalcemia. Dysregulated calcium homeostasis in these of infants with IIH without mutations in CYP24A1. Cosegre- knockout mice was only slightly improved by limiting vitamin D gation analysis indicates autosomal-recessive inheritance. supply with persistence of hypercalcemia and hypercalciuria. In NaPi-IIa (SLC34A1), together with its close homolog NaPi-IIc contrast, high phosphate supplementation restored serum levels (SLC34A3), mediates the conservation of filtered phosphate of phosphate and FGF23 enabling a normalization of 1,25-(OH)2D3 and serum calcium levels. Significant differences between knockout from primary urine.9 The importance of NaPi-IIa and NaPi- and wild-type mice under lowP/highD diet are indicated by bold IIc for renal phosphate conservation could be deduced from letters, significance levels are: *P,0.05; **P,0.01; ***P,0.001; # = animal studies as well as hereditary human disease. NaPi-IIa normalized Cyp27b1 and Cyp24a1 expression levels (for details see knockout mice exhibit urinary phosphate wasting with consec- Supplemental Material). 10 utive hypophosphatemia. They also show high 1,25-(OH)2D3 levels, resulting in hypercalcemia and hypercalciuria. In contrast,

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NaPi-IIc knockout mice do not exhibit overt renal phosphate in our patients at initial manifestation are mostly lacking. FGF23 wasting, but only display hypercalciuria and elevated 1,25- was solely measured in patient F9.1 during hypophosphatemia 11 (OH)2D3 levels. These findings for NaPi-IIc in mice differed and hypercalcemia (Figure 3) and in the low–normal range from initial observations in humans, where inactivating NaPi-IIc (33 kRU/ml) (for details concerning the employed assay please mutations were shown to lead to hereditary hypophosphatemic see Supplemental Material). During follow-up, in face of 12,13 rickets with hypercalciuria (OMIM #241530). Meanwhile, normophosphatemia (median S-PO4=1.5 mmol/l), FGF23 lev- patients with NaPi-IIc defects presenting with isolated hypercal- els were normal (8/16 patients with available data). While this ciuria and nephrolithiasis have been described.14,15 human data incompletely traces all proposed pathophysiologic The identification of autosomal-recessive SLC34A1 muta- changes, these have been delineated accurately in Slc34a1 knock- tions in infants with IIH now demonstrates a crucial role of out mice.10,16,18 NaPi-IIa for calcium metabolism as well as phosphate balance To examine the concomitant disturbances in calcium and in humans, which are tightly linked because they share major phosphate metabolism in more detail and their dependence on control mechanisms comprising vitamin D, iPTH, and FGF23. phosphate and vitamin D supply, we re-examined Slc34a1 FGF23 exerts two major effects in the proximal tubule. In the knockout mice fed diets with variable phosphate and vitamin first place, FGF23 inhibits phosphate reabsorption via NaPi-IIa D content. First, the lowP/highD diet was used to simulate the and NaPi-IIc. Secondly, it inhibits 1a-hydroxylase (CYP27B1) situation of human infants who receive breast milk with low and activates 24-hydroxylase (CYP24A1), decreasing circulating phosphate content (~0.1%–0.15%) together with the recom- levels of active 1,25-(OH)2D3 (Figure 1). Excess levels of FGF23, mended vitamin D prophylaxis (500 IU/day orally) for the as present in different forms of hereditary hypophosphatemic prevention of rickets. Under this diet, Slc34a1 knockout rickets, therefore lead to secondary renal phosphate wasting to- mice developed hypophosphatemia, hypercalcemia, and hy- gether with low levels of active vitamin D. percalciuria (Figure 4, Supplemental Table 5). Although some In contrast, IIH patients with NaPi-IIa mutations exhibit a changes in calcium metabolism caused by excess vitamin D primary defect in proximal tubular phosphate reabsorption. were also observed in wild-type mice under identical diet, the Subsequent hypophosphatemia as present in our patients at analysis of hormonal factors and vitamin D metabolizing en- initial manifestation (Table 1, Supplemental Table 2) induces a zymes revealed the critical changes in the regulation of vitamin decrease in circulating FGF23 levels.16,17 Both hypophosphatemia D (Figure 4). Wild-type mice are able to limit vitamin D ac- and low FGF23 levels are known to increase CYP27B1 expres- tivation by increased action of FGF23, while phosphate-deficient sion and 1a-hydroxylase activity as well as inhibiting CYP24A1 Slc34a1 knockout mice display an unlimited vitamin D activa- expression and 24-hydroxylase activity.16 These effects to- tion caused by lack of the regulatory counterpart FGF23. These gether promote an increase of 1,25-(OH)2D3 with subsequent abnormalities are only slightly mitigated by limiting vitamin D hypercalcemia (Figure 5). supply with persistence of hypercalcemia and hypercalciuria As laboratory parameters during acute hypercalcemia were compiled retrospectively, the available data set is incomplete. At initial manifestation, 1,25-(OH)2D3 was elevated in 6/11 IIH patients with available data (Table 1, Supplemental Table 2). Comparable 1,25-(OH)2D3 levels have been identified previously in hypercalcemic patients with CYP24A1 defects.2 In contrast, in non-vitamin D-mediated hypercalcemic conditions, levels of 1,25-(OH)2D3 are expected to be low. In IIH patients with NaPi-IIa defects, the disturbances in calcium homeostasis clearly outmatch the primary defect in phosphate metabolism. Although hypophosphatemia and renal phosphate wasting are detected at Figure 5. Pathophysiology of disturbed NaPi-IIa function in the proximal renal tubule. initial presentation and to a lesser extent (A) Under physiologic conditions, proximal tubular phosphate reabsorption via NaPi-IIa (and NaPi-IIc, not shown) ensures the maintenance of phosphate homeostasis. Phos- during follow-up, a clinical correlate, e.g., phate reabsorption via NaPi-IIa is limited by the concerted action of PTH (not shown) signs of rickets, is lacking. Unfortunately, and FGF23. Besides its phosphaturic effect, FGF23 negatively regulates the action of an accurate determination of FGF23 levels 1,25-(OH)2D3 by inhibiting the expression of 1a-hydroxylase (CYP27B1) and activating poses a challenge in the clinical setting due 24-hydroxylase (CYP24A1). (B) As a consequence of a NaPi-IIa defect, phosphate to the hormone’s instability (see Supplemen- depletion leads to a decrease of FGF23 levels. In turn, an unrestricted activation of tal Material). Therefore data on FGF23 levels 1,25-(OH)2D3 results in hypercalcemia, hypercalciuria, and nephrocalcinosis.

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(Figure 4). In contrast, Slc34a1 knockout mice respond to a high second non-functional allele (Supplemental Table 2). These phosphate diet with normalization of serum phosphate and patients display a clinical phenotype that is indistinguishable FGF23 levels enabling a return of 1,25-(OH)2D3 into the phys- from that of IIH patients with two non-functional SLC34A1 iologic range, normocalcemia, and normocalciuria. Similar ob- alleles. Our functional analyses confirmed the impaired traf- servations were made by Tenenhouse and colleagues who ficking of NaPi-IIa-91del7 in HEK293 cells while phosphate describe a normalization of calcium metabolism in Slc34a1 uptake in the Xenopus oocyte system was largely preserved. knockout mice by either additional ablation of 1a-hydroxylase Thepathophysiologicrelevanceofthe91del7variantin (CYP27B1) or by high phosphate supplementation.19 Impor- compound-heterozygous state is also supported by a report tantly, these data are in line with the clinical observation made on an infant with Sotos syndrome and clinical IIH.26 The patient in patient P9.1 who remained hypercalcemic after rehydration carried a genomic deletion including SLC34A1 that is typical and cessation of vitamin D supplements (Figure 3), but showed a for Sotos syndrome on one allele and the 91del7 variant on the rapid improvement of his clinical condition and a complete re- remaining SLC34A1 allele.26 Finally, we identified this mutant versal of biochemical abnormalities, including an increase in in homozygous state in a girl (F15.1) who presented with in- FGF23, after phosphate supplementation. cidental nephrocalcinosis and polyuria. Despite no obvious The SLC34A1 mutations observed in IIH patients clearly disturbance in phosphate metabolism, she had borderline hy- indicate autosomal-recessive inheritance. The spectrum of percalcemia and hypercalciuria, a clinical phenotype that mutations comprises truncating mutations as well as missense could be considered a mild IIH variant. mutations. For the latter, functional analyses demonstrated a In summary, the discovery of autosomal-recessive loss-of- loss-of-function character which may be mostly due to im- function mutations in SLC34A1 (NaPi-IIa) highlights a novel paired trafficking of the mutant transporter to the membrane pathophysiologic pathway in IIH. In affected patients, primary as indicated by the OK cell experiments (Figure 2D). Mutations renal phosphate wasting and suppression of FGF23 leads to an in SLC34A1 have been described before, either in homozygous/ inappropriate activation of 1,25-(OH)2D3 with subsequent compound-heterozygous or heterozygous state. A recessive loss- hypercalcemia. As the clinical phenotype strongly resembles of-function mutation in SLC34A1 was described in two siblings that of patients with CYP24A1 defects, all infants clinically with renal Fanconi’s syndrome, hypophosphatemic rickets, hy- presenting with IIH require a careful evaluation of phosphate 20,21 percalciuria, and elevated 1,25-(OH)2D3 levels. Unfortu- metabolism. Beyond omitting vitamin D prophylaxis and cal- nately, no data regarding the clinical course during infancy cium restriction, infants with defective NaPi-IIa require phos- were reported. A recent report describes two siblings with a ho- phate supplementation in order to restore serum phosphate mozygous missense mutation who presented with hypophos- levels and normalize vitamin D and calcium metabolism. Fu- phatemia and nephrocalcinosis.22 The laboratory evaluation of ture studies will have to address the definite impact of this the asymptomatic younger sister performed in early infancy re- combined disturbance of phosphate and calcium metabolism vealed hypercalcemia, suppressed iPTH, and elevated levels of for the development of hypercalciuria, nephrocalcinosis, and 1,25-(OH)2D3 as present in our IIH patients. Thus, the bio- nephrolithiasis in later life as already described for defects in chemical profile resembles IIH, noteworthy without clinical CYP24A1.5 symptoms related to hypercalcemia. Compound-heterozygous mutations were described in a patient who presented during infancy with early-onset nephrocalcinosis and hyperoxaluria.23 CONCISE METHODS Furthermore, heterozygous missense mutations in SLC34A1 were identified in adult patients with nephrolithiasis, Patients bone demineralization, and renal phosphate leak.6,24 Func- Weidentifiedfourpatientsfromthreeconsanguineousfamilies(F1–F3) tional studies demonstrated a loss of phosphate transport as well as 12 unrelated patients with typical IIH who did not exhibit activity for the reported mutations, but failed to identify a mutations in CYP24A1. Clinical and laboratory data of affected patients dominant-negative effect that had initially been postu- were collected retrospectively from medical charts except for patient lated.24,25 We observed nephrolithiasis in 3/26 heterozygous F9.1 who was recruited during the acute phase of hypercalcemia. Dur- first-degree relatives of our patients (Supplemental Table 4). ing follow-up, all patients were clinically re-evaluated and current bio- Therefore, a heterozygous carrier status appears to represent a chemical data obtained. In addition, clinical, radiologic, and laboratory predisposition for the development of kidney stone disease, data of available parents were obtained and analyzed using standardized presumably requiring additional genetic or lifestyle factors as questionnaires (summarized in Supplemental Tables 3 and 4). Detailed 24 already suggested by Prié and colleagues. descriptions of the analyses of serum iPTH, FGF23, 25-OH-D3, and Previously identified sequence variants included the 91del7 1,25-(OH)2D3 are provided in the Supplemental Material. Renal phos- 6 variant, for which the authors demonstrated a reduced ex- phate handling was assessed as TmP/GFR=SPO4–(UPO43SCr/UCr). For 27,28 pression level in HEK293 cells as well as a reduction in SPO4 and TmP/GFR, age-dependent reference values were used. All phosphate-induced currents in Xenopus oocytes. We here describe investigations, including genetic studies, were approved by the Ethics three patients (F6.1, F8.1, and F13.1) who carry the 91del7 Committee of the Westfälische Wilhelms University, Münster. Patients mutant in compound-heterozygous state together with a or their parents provided written informed consent.

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Genetic Studies Animal Studies Genomic DNA of affected individuals and available family members was Experiments were performed on 4–12 week old C57BL/6 and homo- extractedfromperipheralbloodusingstandardmethods.Agenomescan zygous Slc34a1 knockout mice obtained from heterozygous crossings for shared homozygous regions was performed in patients F1.1–F3.1 (littermates). Generation, breeding, and genotyping of the Slc34a1 using Illumina Human660W-Quad and Human OmniExpress Bead- knockout mice have been described before.10,33 All experiments were Chips. Multi-point parametric linkage analysis was performed using performed according to Swiss animal welfare laws and approved by Merlin 1.1.2 as described (see Supplemental Material).29 Alistofcan- the local veterinary authority. Animals had free access to water, and didate genes within the critical interval was generated on the basis of received for 8 weeks a standard rodent diet (ssniff, Soest, Germany) Ensembl Genome assembly GRCh37 (www.ensembl.org). The coding supplemented with a high (1.2%) or low (0.1%) Pi content, the latter region and splice sites of SLC34A1 were conventionally Sanger se- with high (10 IU/g chow) or low (0.3–0.5 IU/g chow) Vitamin D3. quenced. Newly identified SLC34A1 sequence variants were tested for Spontaneous urine samples were collected on the last day and frozen their frequency in at least 204 ethnically matched control alleles by until further analysis. Blood samples were collected before sacrificing Sanger sequencing. The presence of the previously reported sequence the mice by puncture of the vena cava. Serum electrolytes and creat- variation 91del7 was analyzed in 512 control alleles. inine were analyzed using commercial kits (Sigma-Aldrich, St. Louis, MO and Wako Chemicals, Neuss, Germany). Plasma concentrations Preparation of Plasmid Constructs of iPTH and intact FGF23 were determined by ELISA (Immunotopics, NaPi-IIa mutations 91del7, G153A, G153V, L155P, C336G, V408E, San Clemente, CA and Kainos, Tokyo, Japan, respectively). L475fs, and W488R were introduced into human SLC34A1 cDNA 25-OH-D3 and 1,25-(OH)2D3 were assayed simultaneously by liquid subcloned into pEGFP-C1 and KSM vectors as described.25,30 Details chromatography-tandem mass spectrometry following derivatiza- 34 concerning mutagenesis are provided in the Supplemental Material. tion as described. The detection of 1,25-(OH)2D3 required an For oocyte expression, capped cRNA was synthesized in vitro using immunopurification step involving commercially available antibodies Megascript T3 kit (Ambion) in the presence of cap analog (New to 1,25-(OH)2D3. England Biolabs). Renal CYP27B1 and CYP24A1 expression was quantified by real-time PCR. After purification of mRNA from kidney (RNeasy 32 Mini Kit, Qiagen), cDNA was synthesized using reverse transcription Pi Uptake and Two Electrode Voltage Clamp Experiments upon Expression of NaPi-IIa in Xenopus (TaqMan Reverse Transcription Kit, Applied Biosystems) and used laevis Oocytes as a template for real-time PCR. The expression of both enzymes was X. laevis oocytes were obtained, selected, and maintained as described normalized to the expression of hypoxanthine-guanine-phosphoribosyl- previously (fordetails see Supplemental Material). All animal procedures transferase (HPRT) (see Supplemental Material). were approved by the Swiss Cantonal Authority and in accordance with the Swiss Animal Protection Law. Oocytes were injected with 10 ng of wild-type and mutant NaPi-IIa cRNA. Experiments were performed ACKNOWLEDGMENTS 3 days post-injection. Oocytes were incubated in 100 Na solution 31 containing 1 mM cold Pi and Pi (specificactivity10mCi/mmolPi; We thank the following clinicians and researchers for their con- PerkinElmer) for 10 minutes. Scintillation counting (Tri-Carb 29000TR; tributions to this work: Lea Haisch (Department of General Pediatrics, Packard) was performed after washing and lysis in 2% SDS. University Children’s Hospital, Münster, Germany), Carla Bettoni Voltage clamp experiments were performed as reported25 (for details (Institute of Physiology and Zurich Center for Integrative Human see Supplemental Material). Steady-state currents were obtained using a Physiology [ZIHP], University of Zurich, Zurich, Switzerland), Tülay protocol in which membrane voltage steps were made from the holding Güran (Department of Pediatrics, Division of Pediatric Endocri- potential (Vh)=260 mV,to test voltages in the range 2180 to +80 mV in nology, Marmara University, Istanbul, Turkey), Leo A. Monnens

20 mV increments. The steady-state Pi-dependent current (IPi)wasobtained (Pediatric Nephrology, Radboud University Nijmegen Medical by subtracting control traces (in 100Na solution) from the corresponding Center, Nijmegen, The Netherlands), Anke L. Lameris (Department traces in the presence of Pi. of Physiology, Radboud University Medical Center, Nijmegen, The Netherlands), Wolfgang Rascher (Department of Pediatrics, Friedrich- Cell Culture and Transient Transfections Alexander-University, Erlangen, Germany), Einat Azaria (Sheba Med- OK cells were cultured as previously reported31 and transfected with ical Center, Tel Aviv, Israel), Yair Anikster (Sheba Medical Center, Tel either wild-type or mutant NaPi-IIa fused to EGFP (enhanced green Aviv, Israel), Joost G. Hoenderop (Department of Physiology, Radboud ﬂuorescent protein). Two independent experiments were performed, University Medical Center, Nijmegen, The Netherlands), Francesco each in duplicates or triplicates. Upon expression of clear patches of Emma (Division of Nephrology and Dialysis, Children’s Hospital wild-type cotransporter, cells were ﬁxed and permeabilized with sa- Bambino Gesù, IRCCS, Rome, Italy), and Monika Stoll (Department of ponin as described previously.32 Actin was stained by incubation with Human Genetics, University Hospital, Münster, Germany). Texas Red-X phalloidin (Invitrogen). After washing with saponin/ This work was supported by the ERA-Net E-Rare Research Pro- PBS, coverslips were mounted on microscope slides. The subcellular gramme for Rare Diseases (IIH-ECC) (to K.P.S., G.J., and C.A.W.), the localization of transfected constructs was analyzed by Confocal Innovative Medizinische Forschung of the Westfalian Wilhelms- Laser Scanning Microscopy (Leica SP2). University (to K.P.S. and M.K.), the B’nai B’Brith Leo Baeck (London)

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Lodge Trust Fund (to A.V., and R.K.) and the European Union, FP7 14. Yu Y, Sanderson SR, Reyes M, Sharma A, Dunbar N, Srivastava T, (grant agreement 2013-305608) (to R.K.), and the Swiss National Jüppner H, Bergwitz C: Novel NaPi-IIc mutations causing HHRH and Science Foundation supported National Center for Excellence in idiopathic hypercalciuria in several unrelated families: long-term follow-up in one kindred. Bone 50: 1100–1106, 2012 Research NCCR Kidney.CH (to C.A.W.). 15. Dasgupta D, Wee MJ, Reyes M, Li Y, Simm PJ, Sharma A, Schlingmann KP, Janner M, Biggin A, Lazier J, Gessner M, Chrysis D, Tuchman S, Baluarte HJ, Levine MA, Tiosano D, Insogna K, Hanley DA, Carpenter TO, Ichikawa S, Hoppe B, Konrad M, Sävendahl L, Munns CF, Lee H, DISCLOSURES Jüppner H, Bergwitz C: Mutations in SLC34A3/NPT2c are associated None. with kidney stones and nephrocalcinosis. JAmSocNephrol25: 2366– 2375, 2014 16. Perwad F, Azam N, Zhang MY, Yamashita T, Tenenhouse HS, Portale AA: Dietary and serum phosphorus regulate fibroblast growth factor 23 REFERENCES expression and 1,25-dihydroxyvitamin D metabolism in mice. Endo- crinology 146: 5358–5364, 2005 1. Kato S, Yoshizazawa T, Kitanaka S, Murayama A, Takeyama K: Molec- 17. Antoniucci DM, Yamashita T, Portale AA: Dietary phosphorus regulates ular genetics of vitamin D-dependent hereditary rickets. Horm Res 57: serum fibroblast growth factor-23 concentrations in healthy men. J Clin 73–78, 2002 Endocrinol Metab 91: 3144–3149, 2006 2. Schlingmann KP, Kaufmann M, Weber S, Irwin A, Goos C, John U, 18. Tenenhouse HS: Regulation of phosphorus homeostasis by the type iia Misselwitz J, Klaus G, Kuwertz-Bröking E, Fehrenbach H, Wingen AM, na/phosphate cotransporter. Annu Rev Nutr 25: 197–214, 2005 Güran T, Hoenderop JG, Bindels RJ, Prosser DE, Jones G, Konrad M: 19. 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32. Pﬁster MF, Lederer E, Forgo J, Ziegler U, Lötscher M, Quabius ES, quantitation of 25-hydroxyvitamin D and 24,25-dihydroxyvitamin D by Biber J, Murer H: Parathyroid hormone-dependent degradation of LC-MS/MS involving derivatization with DMEQ-TAD. J Clin Endocrinol type II Na+/Pi cotransporters. J Biol Chem 272: 20125–20130, Metab 99: 2567–2574, 2014 1997 35. Fenollar-Ferrer C, Patti M, Knöpfel T, Werner A, Forster IC, Forrest LR: 33. Nowik M, Picard N, Stange G, Capuano P, Tenenhouse HS, Biber J, Structural fold and binding sites of the human Na⁺-phosphate co- Murer H, Wagner CA: Renal phosphaturia during metabolic acidosis transporter NaPi-II. Biophys J 106: 1268–1279, 2014 revisited: molecular mechanisms for decreased renal phosphate reabsorption. Pﬂugers Arch 457: 539–549, 2008 34. Kaufmann M, Gallagher JC, Peacock M, Schlingmann KP, Konrad M, DeLuca HF, Sigueiro R, Lopez B, Mourino A, Maestro M, St-Arnaud R, This article contains supplemental material online at http://jasn.asnjournals. Finkelstein JS, Cooper DP, Jones G: Clinical utility of simultaneous org/lookup/suppl/doi:10.1681/ASN.2014101025/-/DCSupplemental.

AFFILIATIONS

† *Department of General Pediatrics, University Children’sHospital,Münster,Germany; Institute of Physiology and Zurich Center for Integrative ‡ Human Physiology (ZIHP), University of Zurich, Zurich, Switzerland; Department of Biomedical and Molecular Sciences, Queen’s University, | Kingston, Ontario, Canada; §Department of Pediatrics, Kayseri University, Kayseri, Turkey; Department of Medical Genetics, The Children’s Memorial Health Institute, Warsaw, Poland; ¶Department of Pediatrics, Division of Pediatric Endocrinology, Marmara University, Istanbul, Turkey; †† **Department of Pediatric Endocrinology, Keçiören Research and Educational Hospital, Ankara, Turkey; Pediatric Nephrology, Radboud ‡‡ University Nijmegen Medical Center, Nijmegen, The Netherlands; Department of Pediatrics, Medical University of Silesia, Katowice, Poland; || §§Department of Pediatric Nephrology, Medical University of Lublin, Lublin, Poland; Children’s Hospital St. Elisabeth and St. Barbara, Halle/ Saale, Germany; ¶¶Department of Pediatrics, Friedrich-Alexander-University, Erlangen, Germany; ***Department of Pediatrics, University Hospital ††† ‡‡‡ St. Marina, Varna Medical University, Varna, Bulgaria; Department of Pediatrics, University Hospital Center, Zagreb, Croatia; Sheba Medical ||| Center, Tel Aviv, Israel; §§§University College London, London, United Kingdom; Department of Pediatric Nephrology, University Hospitals Leuven, Leuven, Belgium; ¶¶¶Department of Physiology, Radboud University Medical Center, Nijmegen, The Netherlands; and ****Department of Medicine, Queen’s University, Kingston, Ontario, Canada

614 Journal of the American Society of Nephrology J Am Soc Nephrol 27: 604–614, 2016 Supplemental Material

Methods

Laboratory Analyses

We measured levels of serum and urine electrolytes and creatinine in samples obtained from all patients using routine methods. Intact parathyroid hormone (iPTH) levels were measured by chemiluminescent immunoassay (CLIA Immulite 2000XP, Siemens, Germany), FGF-23 plasma concentrations were determined by ELISA (Human FGF23 C-terminal Elisa kit, Immutopics International, San Clemente, CA, USA) (reference range 26-110 kRU/mL)(for details see below), 25-OH-D3 was measured by a direct competitive chemiluminescent immunoassay (CLIA, Liaison Analyzer, Diasorin S.p.A.), and 1,25-(OH)2D3 was measured after purification by radioimmunoassay (IDS kit, Immunodiagnostic Systems, Germany).

Tubular phosphate reabsorption

Renal phosphate handling was assessed according to Stark et al. as TmP/GFR = SPO4 – (UPO4 x 1 1,2 SCr / UCr) . For SPO4 and TmP/GFR, age dependent reference values were used . Mean and SD for TmP/GFR were converted to mmol/L GFR resulting in the following reference ranges (mean ±2SD): Newborn=1.4-3.0mmol/l; 1month to 2years = 0.9-2.1mmol/l, 2-12years = 1.0- 1.8mmol/l, 12-16years = 0.9-1.7mmol/l, >16years = 0.8-1.2mmol/l.

Genome wide linkage / LOD score calculation

DNA of four affected individuals shown in the pedigrees F1, F2, F3 (Figure S1) was used to map the disease locus. Individuals F1.1, F1.2 and F2.1 were analyzed using Illumina Human660W-Quad BeadChips, individual F3.1 was run on a Human OmniExpress BeadChip. For combined analysis only those SNPs present on both chip types were used where a genotype was obtained for all four individuals (about 239.000 SNPs). Due to the very dense SNP map, some series of SNPs may create a small homozygous region just by chance or since they represent a major haplotype in the population. Those sites will show high LOD score spikes, despite not representing true candidate regions. This phenomenon occurs since the multipoint calculation by the software has an effect like creating a very low haplotype frequency within that homozygous SNP series by multiplication of the individual allele frequencies. To avoid resulting inflationally high LOD scores in small series of homozygous SNPs, the number of SNPs used for calculating LOD scores was reduced. To this end, first only those SNPs were included where the minor allele was present in at least one individual (181.351 SNPs). The genome was divided into 1/3 cM (centi-Morgan) slices and from the SNPs within each slice, that one SNP was selected to represent that slice that showed the lowest minor allele frequency of all the SNPs within the slice. Thus, the final set of SNPs consisted of 10299 SNPs. Multipoint LOD scores were obtained using Merlin 1.1.2 and the heterogeneity LOD score (HLOD) was calculated and plotted for all autosomes 3.

Preparation of Plasmid Constructs/Mutagenesis

Full-length hSLC34A1 was subcloned into a KSM oocyte expression vector. The G153A, G153V, L155P, C336G, V408E, L475fs, and W488R mutations were then introduced by site- directed mutagenesis using the Quick-Change kit (Stratagene). Mutagenic primers were designed using the Stratagene web based QuickChange® Primer Design software Program. The 91del7 variant was generated in two steps. First, the in-frame 21bp deletion was generated by PCR amplification of the two NaPi-IIa fragments excluding the deletion region. PCR primers contained Xho I/Pvu II (N-terminal fragment) and Nae I/Hind III (C-terminal fragment) respectively. Pvu II and Nae I were introduced at the deletion site. Following amplification, the two NaPi IIa PCR fragments were double digested with Xho I/Pvu II and Nae I/Hind III, respectively. After purification, all fragments were ligated and the resulting construct was amplified. This step generated a t>a point mutation at position 269 of NaPi-IIa, which was then re-converted to the original sequence by a new round of Site-Directed Mutagenesis. XL1-Blue ultracompetent cells were transformed with either the PCR products from the Site-Directed Mutagenesis (point mutations) or the ligation product from the first step of the 91del7 generation. The plasmids containing the generated mutations were isolated using QIAprep Spin Miniprep (Qiagen, 27106) and all mutations were confirmed by sequencing (Microsynth). One clone from each construct was used for the experiments.

For oocyte expression, capped cRNA was synthesized in vitro using Megascript T3 kit (Ambion) in the presence of cap analog (New England Biolabs). To study the expression of wildtype and mutant NaPi-IIa transporter (SLC34A1) in mammalian cells, both wildtype and mutant NaPi-IIa were fused C-terminally to GFP by subcloning the cDNAs into the pEGFP-C1 vector (Clontech).

Cell culture and transient transfections

Opossum kidney cells (clone 3B/2) were cultured in DMEM / Ham's F-12 medium (1:1) supplemented with 10 % fetal calf serum, 2 mM glutamine, 20 mM HEPES and 50 IU/ml penicillin/streptomycin as previously reported 4. Cells were plated on coverslips in 12 multiwell plates (TPP), and cultures were transfected with either wild-type hNaPi-IIa fused to pEGFP-C1 vector or with the mutants described above. Cultures were transfected at about 70 % confluency by an overnight incubation in 500 l OPTIMEM (GIBCO, 31985-047) containing 1 μg of DNA and 3 μl of Lipofectamine™ 2000 Reagent (Invitrogen, 11668-019). We performed two independent experiments, each in duplicates or triplicates.

Actin staining and confocal microscopy

Upon expression of clear patches of wt NaPi-IIa signal (two to three days after transfection), cells were fixed and permeabilised with saponin as described previously 5. Thereafter, actin was stained by incubation with Texas Red®-X phalloidin (Invitrogen) diluted 1:500. After incubation for 30 min in the dark, cells were washed three times with PBS/saponin and once with PBS. The coverslips were then mounted on microscope slides using Dako Glycergel® Mounting Medium. The subcellular final locations of the transfected cotransporters were analysed by Confocal Laser Scanning Microscopy (Leica SP2) using a 63x oil immersion objective at the Center for Microscopy and Image Analysis at the University of Zurich.

Expression of hNaPi-IIa and mutants in Xenopus laevis oocytes

Female X. laevis frogs were purchased from Xenopus Express (France) or African Xenopus Facility (R. South Africa). Portions of ovaries were surgically removed from frogs anesthetized in MS222 (tricaine methansulphonate) and cut in small pieces. Oocytes were treated for 45 min with collagenase (crude type 1A) 1 mg/mL in 100Na solution (without Ca2+) in presence of 0.1 mg/mL trypsin inhibitor type III-O. Healthy stage V-VI oocytes were selected, maintained in modified Barth’s solution at 16°C. All animal procedures were conducted in accordance with the Swiss Cantonal and Federal legislation relating to animal experimentation.

Solutions and reagents

The standard extracellular solution for oocyte experiments (100 Na) contained (in mM): 100

NaCl, 2 KCl, 1.8 CaCl2, 10 HEPES, pH 7.4 adjusted with Tris. Pi was added from a 1M K2HPO4/

KH2PO4 stock premixed to give pH 7.4. Modified Barth’s solution for storing oocytes contained (in mM): 88 NaCl, 1 KCl, 0.41 CaCl2, 0.82 MgSO4, 2.5 NaHCO3, 2 Ca(NO3)2, 7.5 HEPES, pH 7.5 adjusted with Tris and supplemented with 5 mg/L doxycyclin and 5 mg/L gentamicin. All standard reagents were obtained from either Sigma-Aldrich or Fluka (Buchs, Switzerland).

32 Pi uptake

Oocytes were injected with 50 nl of cRNA (200 ηg/μL) for wild-type (wt) or mutant hNaPi-IIa constructs. Experiments were performed 3 days after injection. Non-injected control oocytes (NI), oocytes expressing hNaPi-IIa wt and mutants (6-10 oocytes/group) were first allowed to equilibrate in 100 Na solution without tracer. After aspiration of this solution, oocytes were 32 incubated in 100 Na solution containing 1 mM cold Pi and Pi (specific activity 10 mCi/mmol

Pi, Perkin Elmer). Uptake proceeded for 10 min, then oocytes were washed 4 times with ice- cold 0 Na solution containing 2 mM Pi and lysed individually in 2% SDS for 10 min before addition of the scintillation cocktail. The amount of radioactivity in each oocyte was measured by scintillation counting (Tri-Carb 29000TR, Packard).

Two-electrode voltage clamp experiments

All voltage clamp experiments were performed using a two-electrode voltage clamp (TEC- 10CX, NPI, Tamm, Germany). Oocytes were impaled with microelectrodes filled with 3 M KCl, with a typical resistance of <1 MΩ. The temperature of the recording chamber was monitored using a thermistor probe (TS-2, NPI, Tamm, Germany) placed close to the oocyte. The temperature of the recording chamber and incoming superfusate were regulated using Peltier cooling elements driven by a continuous feedback controller (TC-10, NPI Tamm, Germany). Data acquisition was performed using a 1440 Digidata (Molecular Devices Corp, USA). For recordings at constant holding potential, currents were acquired at >20 samples/s and filtered at 10 Hz. Faster sampling rates (up to 20 k samples/s) were used for voltage step recordings with filtering (digital and analog) adjusted accordingly.

Steady-state currents were obtained using a protocol in which membrane voltage steps were made from the holding potential (V h ) = −60 mV, to test voltages in the range −180 to

+80 mV in 20 mV increments. The steady-state Pi -dependent current (IPi) was obtained by subtracting control traces (in 100 Na solution) from the corresponding traces in the presence of Pi. The current was measured in a region where all presteady-state relaxations were completed. Data was rejected if contaminated by endogenous Cl-currents. Steady state Pi activation was determined by varying the Pi concentration in presence of 100 Na and subtracting the respective currents in 100 Na from those in 100 Na + Pi .

Animal studies

Plasma concentrations of intact parathyroid hormone (iPTH) and intact FGF-23 were determined by ELISA (Immutopics, San Clemente, CA, USA and Kainos, Tokyo, Japan, respectively). The kit to measure PTH contains streptavidin-coated wells together with biotinylated and horseradish peroxidase (HRP)-conjugated antibodies against PTH. For details concerning FGF-23 determination please see below. Renal mRNAs of Cyp27b1 and Cyp24a1 were quantified by real time PCR. For that, total RNA purified from kidneys (RNeasy Mini Kit, Qiagen) was incubated with reverse transcriptase (TaqMan Reverse Transcription Kit, Applied Biosystems) to produce cDNA that was then used as template for real time PCR. The expression of both hydroxylases was normalized to the expression of hypoxanthine guanine phosphoribosyl transferase (HPRT) according to the formula R = 2[Ct(HPRT)-Ct(test gene)], where R is the relative ratio and Ct indicates the cycle number at the threshold of 0.2. All primers and probes were obtained from Taqman Gene Expression Assays.

Determination of human and mouse FGF-23

For the determination of FGF-23 in patients the Immutopics Human FGF-23 (C-terminal) ELISA kit was used (Immutopics International, San Clemente, CA, USA). FGF-23 in mice was measured using the Kainos intact FGF-23 ELISA kit (TECOmedical AG, Sissach, Switzerland). Both assays are two-step enzyme-linked immunosorbent assays. The Kainos assay only detects full-length or intact FGF-23 (iFGF-23) by using antibodies directed against C- and N- terminal regions of FGF-23. In contrast, the C-terminal antibody used in the Immutopics assay detects both intact FGF-23 as well as processed C-terminal fragments of FGF-23 (cFGF- 23) 6. Whereas Immutopics recommends to measure cFGF-23 in EDTA plasma, the Kainos iFGF-23 kit can be used with either serum or plasma. However, using serum for measurement of intact FGF-23 potentially yields falsely low concentrations caused by preanalytical instability 7.

In general, obtaining reliable FGF-23 values is challenging due to the instability of the FGF-23 hormone. Within hours after blood-taking, levels of intact FGF-23 decrease as a result of protease cleavage, whereas FGF-23 levels measured by C-terminal assay which also detects inactive fragments remains relatively stable over time 8. Therefore, immediate separation of plasma or serum and subsequent testing is recommended especially for the determination of iFGF-23. Alternatively, samples may be frozen at -20 °C. As the C-terminal assay detects both iFGF-23 as well as processed C-terminal fragments (cFGF-23), it might be considered less susceptible to suboptimal probe processing (see above). Obtaining falsely lower measured values for FGF-23 would be particularly problematic in patients with a suspected suppression of FGF-23 including patients with NaPi-IIa (SLC34A1) or NaPi-IIc (SLC34A3) defects. For this reason, the cFGF-23, the parameter considered more stable, was measured in human patients, whereas iFGF-23 was determined in mice where experimental conditions allow for a uniform and rapid sample processing. Previously, FGF-23 levels have been reported in a single family with SLC34A1 mutations by Magen and colleagues 9. They measured iFGF-23 as well as cFGF-23 in their two patients with a homozygous SLC34A1 mutation and report values at the lower end of the reference ranges for both assays.

Intra- and inter-assay variances for the Immutopics Human FGF-23 (C-terminal) ELISA kit (provided in the assay`s manual, copyright Immutopics, Inc., San Clemente, CA, USA):

To assess intra-assay precision the mean and coefficient of variation were calculated from 20 duplicate determinations of two samples each performed in a single assay. Mean Value (RU/mL) Coefficient of Variation 33.7 2.4% 302 1.4%

To assess inter-assay precision the mean and coefficient of variation were calculated from duplicate determinations of two samples performed in 10 assays. Mean Value (RU/mL) Coefficient of Variation 33.6 4.7% 293 2.4%

Intra- and inter-assay variances for the Kainos intact FGF-23 (C-terminal) ELISA kit are provided on the manufacturer`s homepage (www.kainos.co.jp)

(http://www.kainos.co.jp/eng/products/fgf23_e/fgf23_e_4.html)

Intra-assay precision: Mean Value (pg/mL) Coefficient of Variation 14.2 3.0% 28.7 2.8% 33.6 2.0%

Inter-assay precision: Mean Value (pg/mL) Coefficient of Variation 19.5 3.8% 42.4 2.1% 119 2.4%

Results

Patients

Figure S2:

Figure S2: Pedigrees of IIH families F1 – F15. Families F1 to F3 with parental consanguinity were used for homozygosity mapping. F4 to F14 represent families with sporadic cases. The patient from family F15 did not present clinically with IIH, but exhibited nephrocalcinosis as an incidental finding. Affected family members are indicated with solid circles (girls) and squares (boys), double horizontal lines indicate parental consanguinity. Mutated alleles are denoted by (+), wild-type alleles by (-).

Genome wide linkage / LOD score calculation

Figure S1:

Figure S1. Linkage analysis. (A) Multipoint LOD scores were obtained using Merlin 1.1.2 and the heterogeneity LOD score (HLOD) was plotted for all autosomes. The maximum HLOD was 6,791 in a homozygosity region of about 2 Mb close to chromosome 5-qter. (B) The candidate region of panel A zoomed in. At top the HLOD is shown as a blue background area. The GenBank accession numbers of SNPs are given in the bottom line. The genotypes are denoted as "1=1" for homozygous major allele, "2=2" for homozygous minor allele and "1/2" for heterozygous state. The position of the candidate gene SLC34A1 is indicated. The SNP rs7708314 (C/T, base position 176.809.618 on chromosome 5 [GRCh37], minor allele frequency 0,11) is located close to the SLC34A1 gene (NM_003052.4: c.- 1918C>T). The length of the homozygosity region between the next heterozygous markers used for LOD score calculations is 1,9 Mb. Detailed review including also the markers that were not used for the Merlin-calculations demonstrated that the true homozygosity region is 1,66 Mb (between heterozygous markers rs3762974 and rs185493, not shown).

Table S1. List of candidate gene within the critical interval

SNP rs3762974; Chr5, 176.901.335bp (GRCh38)

RefSeq genes Description RefSeq Acc-No UIMC1 ubiquitin interaction motif containing 1 NM_016290 ZNF346 zinc finger protein 346 NM_012279 FGFR4 fibroblast growth factor receptor 4 NM_002011 NSD1 nuclear receptor binding SET domain protein 1 NM_022455 RAB24 RAB24, member RAS oncogene family NM_130781 PRELID1 PRELI domain containing 1 NM_013237 MXD3 MAX dimerization protein 3 NM_031300 LMAN2 lectin, mannose-binding 2 NM_006816 RGS14 regulator of G-protein signaling 14 NM_006480 SLC34A1 solute carrier family 34 (NaPi), member 1 NM_003052 PFN3 profilin 3 NM_001029886 F12 coagulation factor XII (Hageman factor) NM_000505 GRK6 G protein-coupled receptor kinase 6 NM_002082 PRR7 proline rich 7 (synaptic) NM_030567 DBN1 drebrin 1 NM_080881 PDLIM7 PDZ and LIM domain 7 (enigma) NM_005451 DOK3 docking protein 3 NM_024872 DDX41 DEAD (Asp-Glu-Ala-Asp) box polypeptide 41 NM_016222 FAM193B family with sequence similarity 193, member B NM_001190946 TMED9 transmembrane emp24 protein transport domain containing 9 NM_017510 B4GALT7 xylosylprotein beta 1,4-galactosyltransferase, polypeptide 7 NM_007255 FAM153A family with sequence similarity 153, member A NM_173663 PROP1 PROP paired-like homeobox 1 NM_006261 FAM153C family with sequence similarity 153, member C NM_001079527 N4BP3 NEDD4 binding protein 3 NM_015111 RMND5B required for meiotic nuclear division 5 homolog B NM_022762 NHP2 NHP2 ribonucleoprotein NM_017838 HNRNPAB heterogeneous nuclear ribonucleoprotein A/B NM_031266 PHYKPL 5-phosphohydroxy-L-lysine phospho-lyase NM_153373 COL23A1 collagen, type XXIII, alpha 1 NM_173465

Processed transcripts ZNF346-IT1 ZNF346 intronic transcript 1 (non-protein coding) HGNC:41423 CTD-2301A4.3 ribosomal protein L21 (RPL21) pseudogene PRMT1P1 protein arginine methyltransferase 1 pseudogene 1 HGNC:49611 CTD-2301A4.1 ribosomal protein S20 (RPS20) pseudogene PRR7-AS1 PRR7 antisense RNA 1 HGNC:27961 RP11-1334A24.5 to be experimentally confirmed RP11-1334A24.6 putative novel antisense transcript RP11-1277A3.1 novel antisense transcript RP11-1101H11.1 putative novel antisense transcript RP11-1026M7.2 novel transcript antisense to FAM153A RP11-423H2.3 novel transcript, antisense to PROP1 RP11-423H2.4 to be experimentally confirmed RP11-423H2.5 novel antisense transcript CTB-26E19.1 novel sense intronic transcript RP11-1259L22.1 putative novel antisense transcript

SNP rs185493; Chr5, 178.564.257bp

Mutational Analysis

Figure S3:

Figure S3. Electropherograms of identified SLC34A1 mutations. Sequencing was performed in affected patients and for cosegregation analysis in available family members. SLC34A1 mutations appear in homozygous or heterozygous state, respectively. If a mutation was discovered in more than a single patient, a representative electropherogram of one patient is shown. On top, the consequence on protein level is indicated above the corresponding change on nucleotide level. Clinical data

Table S2

Patient F1.2 F1.2 F2.1 F3.1 F4.1 F5.1 F6.1 F7.1 F8.1 F9.1 F10.1 F11.1 F12.1 F13.1 F14.1 F15.1

Origin Turkey Turkey Turkey Turkey Netherlands Poland Poland Poland Germany Germany Turkey Bulgaria Israel Belgium Italy Poland Age at Presentation 20 days 1 month 2 months 2 months 2 months 4 months 9 months 10 months 1month 3 months 3 months 4 months 7 months 6 months 3 months 18 months Vitamin D 400IE 400IE 500IE 400IE 500IU 2000IU 2000IU 400IU 500IU 500IU 200IU 1334IU 200IU 500IU 500IU 800IE Prophylaxis

Weight loss/ Yes Yes No Yes Yes Yes Yes Yes Yes No Yes Yes No No Yes No Failure to thrive Polyuria/Dehydratio Yes Yes No Yes No Yes Yes Yes Yes No Yes Yes Yes No Yes Yes n Muscular Hypotonia Yes No No No No Yes Yes No No Yes No No No No No No Nephrocalcinosis Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes

Hypercalciuria Yes Yes Yes Yes Yes Yes Yes Yes Yes No Yes No Yes No Yes No initial serum calcium 3.5 2.9 3.1 3.2 3.8 3.1 3.0 3.4 3.4 3.4 2.8 3.6 3.1 2.6 2.8 2.6 (mM)(2.1-2.6) initial serum 1.0 0.5 1.5 0.7 1.1 1.2 1.3 1.8 1.7 1.0 0.6 1.0 1.0 1.6 1.8 1.7 phosphate (mM) (-5.8SDS) (-8.6SDS) (-2.8SDS) (-7.2SDS) (-5.0SDS) (-4.1SDS) (-2.8SDS) (+0.1SDS) (-1.9SDS) (-5.4SDS) (-7.6SDS) (-5.2SDS) (-4.7SDS) (-1.5SDS) (+0.1SDS) (+0.4SDS) TmP/GFR 0.9 n.d. 1.4 n.d. 0.9 1.2 n.d. n.d. 1.6 0.9 0.5 n.d. n.d. n.d. n.d. n.d. (mmol/L GF) (-3.3SDS) (-0.3SDS) (-2.0SDS) (-1.0SDS) (+0.3SDS) (-2.0SDS) (-3.3SDS) initial PTH (pg/mL) 1.0 15 5.5 31 2.8 * * <3 4,9 <3 <3 <3 <1 1.9 <3 13 (14-72) initial 25-OH-D3 28 n.d. 44.6 46.2 20.4 55.7 23.4 33.7 n.d. 56.3 15.9 70 28.1 48.3 n.d. 21 (ng/mL)(10-65) initial 1,25-(OH)2D3 139 n.d. 146.3 25.8 50.8 62.5 82.1 271 n.d. 63.2 n.d. 135 53 131.7 n.d. 32 (pg/mL)(17-74)

Therapy Keto- Rehydration no Rehydration Rehydration Rehydration Rehydration Rehydration Rehydration Rehydration Rehydration Rehydration Rehydration Rehydration Rehydration no (acute phase) conazole Steroids Steroids Steroids Steroids Phosphate Furosemide Bisphos- phonates Therapy oral oral no low calcium no oral oral low calcium hydrochlo- oral oral no no no potassium potassium (long term) phosphate phosphate diet phosphate phosphate diet rothiazide phosphate phosphate citrate citrate hydrochlo- hydrochlo- low calcium low calcium potassium rothiazide rothiazide diet diet citrate

Follow-up Age at last visit 1.5 years 7 years 6 years 1.5 years 14 years 10 years 17 years 3 years 3 years 0.9years 1 year 0.9years 11 years 6 years 1 year 3.5 years last serum calcium 2.7 2.5 2.4 2.8 2.7 2.7 2.6 2.6 2.5 2.6 2.6 2.6 2.5 2.5 2.7 2.7 (mM)(2.1-2.6) last serum 0.9 1.1 1.5 1.8 1.0 1.5 0.9 1.5 1.7 1.5 1.8 1.5 1.2 1.6 1.8 1.7 phosphate (mM) (-4.1SDS) (-1.7SDS) (+0.5SDS) (+0.9SDS) (-2.3SDS) (+0.5SDS) (-2.8SDS) (+0.1SDS) (+1.2SDS) (-1.6SDS) (+0.4SDS) (-1.6SDS) (-1.2SDS) (+1.0SDS) (+0.4SDS) (+1.3SDS) TP/GFR 0.8 1.0 1.3 n.d. 0.7 1.3 0.7 1.3 1.4 1.4 1.3 1.3 1.1 n.d. n.d. 1.6 (mmol/L GF) (-3.0SDS) (-2.0SDS) (-0.5SDS) (-3.0SDS) (-0.5SDS) (-3.0SDS) (-0.5SDS) (±0.0SDS) (-0.5SDS) (-1.0SDS) (--1.0SDS) (-1.5SDS) (+1.0SDS) last PTH (pg/mL) 21 23 20 42 33 21 8.2 6.9 19 12 36 16 7.1 19 10 22 (14-72) FGF-23 (kRU/L) 136 n.d. n.d. 77 82 29 54 47 114 33 n.d. n.d. n.d. n.d. n.d. n.d. (26-110) last 25-OH-D3 8 12 26 36 53 22 15 26 16 38 n.d. 63 25 53 n.d. 38 (ng/mL) (10-65) last 1,25-(OH)2D3 57 75 58 n.d. 58 49 53 103 35 59 n.d. 146 n.d. 79 n.d. 54 (pg/mL)(17-74) Mutation SLC34A1 c.644(+1) c.644(+1) C458 c.1006(+1) c.458 c.1425_26 c.271_91 c.458 c.271_91 c.458 c.458 c.1006 c.644 c.271_91 c.555_556 c.271_91 (nucleotide level) g>a g>a G>T g>a G>C del del G>C del G>C G>T (+3_6)del G>A del del del homoz. homoz. homoz. homoz. + + + + + + + + homoz. + + homoz. c.1006 ? c.464T>C c.1416(+5) c.914 c.1223 c.1462 c.1614 c.1223 c.1416(+5) T>G g>a G>A T>A T>C G>A T>A g>a Mutation NaPi-IIa IVS6(+1)g> IVS6(+1)g> p.G153V IVS9(+1)g>a p.G153A p.L475fs p.91del7 p.G153A p.91del7 p.G153A p.G153V IVS9(+3_6) p.R215W/ p.91del7 p.I185fs p.91del7 (protein level) a a homoz. homoz. + + + + + + + del + + homoz. homoz. homoz. p.C336G ? p.L155P IVS12(+5) p.W305* p.V408E p.W488R + homoz. p.V408E IVS12(+5) g>a p.W538* (splice site) g>a * = instead of intact PTH, whole PTH was measured and within the lower normal range

Table S2. Full set of clinical, biochemical and genetic data of all 15 patients including sporadic IIH cases. For serum phosphate as well as TmP/GFR instead of a reference range age-specific SDS are provided according to 1, for age >16 years adult values were used (= 3.2 (±0.3) identical to the nomogram by Walton/Bijvoet )10. The c.644G.A mutation in patient F12.1 leads to an exchange of arginine 215 to tryptophane but primarily affects the donor splice site of exon 6.

Table S3. Clinical, nephrocalcinosis/ S-Ca S-PO4 PTH 25-OH-D 1,25-OH -D TmP-GFR Urine Ca/Crea NaPi-IIa 3 2 3 Family 1 nephrolithiasis (mmol/L) (mmol/L) (pg/mL) (ng/mL) (pg/mL) (mmol/L GF) (mg/mg) biochemical, and genetic data mutation (2,1-2,6) (0,9-1,5) (14-72) (10-65) (17-74) (0,8-1,4) (<0.25) of parents of families F1 and father F1.1 IVS6(+1)g>a het. no 2,5 1,0 42,7 5 56 0.9 0,20 F9. mother F1.1 IVS6(+1)g>a het. no 2,3 1,0 23,1 11 67 0,8 0,17 father F1.2 IVS6(+1)g>a het. no 2,4 0,9 23,5 6 54 0,8 0,49 mother F1.2 IVS6(+1)g>a het. no 2,5 1,1 109 4 60 1,1 0,16 father F9.1 p.G153A het. no 2,4 0,9 110 34 77 0,8 0,06 mother F9.1 p.V408E het. no 2,5 0,9 14 20 61 0,8 0,18 Table S4: Available clinical and biochemical data of parents of families F1 to F15.

S-Ca S-PO4 PTH Obligate Heterozygotes Genetic Data Nephrolithiasis (mmol/L) (mmol/L) (pg/mL) F1.3 father F1.1 IVS6(+1)g>a het. no 2,5 1,0 42,7 F1.4 mother F1.1 IVS6(+1)g>a het. no 2,3 1,0 23,1 F1.5 father F1.2 IVS6(+1)g>a het. no 2,4 0,9 23,5 F1.6 mother F1.2 IVS6(+1)g>a het. no 2,5 1,1 109 F3.2 father IVS9(+1)g>a het. no 2,6 1,5 45,5 F3.2 mother IVS9(+1)g>a het. no 2,5 1,4 38,6 F4.2 father - no - - - F4.3 mother - no - - - F5.2 father - yes 2,3 - 18 F5.3 mother - yes 2,4 - - F7.2 father p.G153A het. no 2,2 0,9 23,4 F7.3 mother IVS12(+5)g>a het. no 2,3 1,2 55,1 F8.2 father p.W305* het. no - - - F8.3 mother p.91del7 het. no - - - F9.2 father p.G153A het. no 2,4 0,9 110 F9.3 mother p.V408E het. yes 2,5 1,0 14 F10.2 father - no - - - F10.3 mother - no - - - F11.2 father - no - - - F11.3 mother - no - - - F13.2 father - no 2,2 0,6 3 F13.3 mother - no 2,4 1,2 - F14.2 father - no - - - F14.3 mother - no - - - F15.2 father p.91del7 het. no - - - F15.3 mother p.91del7 het. no - - - 2.4 (2.2-2.6) 1.0 (0.6-1.5) 32 (3-110) 3/26 (n=14) (n=12) (n=12)

Animal data

Table S4:

Serum/Plasma and Urine parameters:

Slc34a1-/- Slc34a1+/+

LowP, highD LowP, lowD HighP, lowD LowP, highD LowP, lowD HighP, lowD Serum/Plasma

Pi (mmol/L) 0.90 ± 0.03** 0.98 ± 0.12ns 2.08 ± 0.20°° 1.88 ± 0.25 1.64 ± 0.22 2.93 ± 0.30

Ca2+ (mmol/L) 3.23 ± 0.23* 2.97 ± 0.12ns 2.05 ± 0.08°°°° 2.84 ± 0. 12 2.97 ± 0.24 1.86 ± 0.25

PTH (pg/ml) 36.9 ± 0.3ns 37.6 ± 0.6ns 392.1 ± 175.0ns 40.3 ± 1.4 48.1 ± 13.1 91.3 ± 15.6

FGF23 (pg/ml) 14.2 ± 1.6* 11.8 ± 1.9ns 51.8 ± 13.3° 570.6 ± 222.2 18.8 ± 0.9 108.2 ± 48.1

25-OH- D ns ns ns 3 200.8 ± 24.7 12.8 ± 0.7 16.1 ± 1.3 165.2 ± 14.6 11.5 ± 0.8 16.7 ± 1.4 (ng/ml)

1,25-(OH) D 2 3 16.5 ± 2.5* 64.4 ± 8.4ns 30.3 ± 3.6ns 7.4 ± 1.0 44.9 ± 6.0 35.2 ± 3.7 (pg/ml)

Cyp27b1 mRNA * 0.110 ± 0.021** 0.411 ± 0.077°° 0.040 ± 0.004° 0.005 ± 0.001 0.290 ± 0.024 0.060 ± 0.007 Cyp24a1 mRNA * 0.38 ± 0.19*** 0.10 ± 0.03ns 0.38 ± 0.15ns 7.78 ± 0.88 0.10 ± 0.03 1.57 ± 0.41

Urine Ca/Crea ns ns 13.12 ± 0.90 11.88 ± 1.02 0.45 ± 0.11°°° 10.07 ± 2.60 8.71 ± 1.13 0.11 ± 0.11 (mol/mol)

Table S4. Full set of biochemical data under high and low vitamin D and phosphate diets. Low vitamin D content (“lowD”) = 0.3-0.5IU/g chow, high vitamin D content (“ highD”) = 10IU/g chow, low phosphate content (“lowP”) = 0.1% P, high phosphate content (“highP”) = 1.2% P (for standard diet compare 11 = 4.5IU/g chow vitamin D and 0.6% phosphate). “*” signs indicate significant differences between knockout and wild-type mice under lowP/highD diet. “°” signs indicate statistically significant differences between knockout mice on lowP/lowD or highP/lowD diets, respectively, in comparison to knockout mice on lowP/highD. The levels of significance in the respective columns are indicated as follows: */° = p<0.05, **/°° = p<0.01, ***/°°° = p<0.001, ns = non significant).

Supplemental references

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3. Abecasis GR, Cherny SS, Cookson WO, Cardon LR. Merlin--rapid analysis of dense genetic maps using sparse gene flow trees. Nat Genet 2002;30:97-101.

4. Reshkin SJ, Forgo J, Murer H. Functional asymmetry of phosphate transport and its regulation in opossum kidney cells: phosphate transport. Pflugers Arch 1990;416:554-60.

5. Pfister MF, Lederer E, Forgo J, et al. Parathyroid hormone-dependent degradation of type II Na+/Pi cotransporters. J Biol Chem 1997;272:20125-30.

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7. Fassbender WJ, Brandenburg V, Schmitz S, et al. Evaluation of human fibroblast growth factor 23 (FGF-23) C-terminal and intact enzyme-linked immunosorbent-assays in end-stage renal disease patients. Clin Lab 2009;55:144-52.

8. Smith ER, Ford ML, Tomlinson LA, et al. Instability of fibroblast growth factor-23 (FGF-23): implications for clinical studies. Clin Chim Acta. Netherlands: 2011 Elsevier B.V; 2011:1008-11.

9. Magen D, Berger L, Coady MJ, et al. A loss-of-function mutation in NaPi-IIa and renal Fanconi's syndrome. N Engl J Med 2010;362:1102-9.

10. Walton RJ, Bijvoet OL. Nomogram for derivation of renal threshold phosphate concentration. Lancet 1975;2:309-10.

11. Beck L, Karaplis AC, Amizuka N, Hewson AS, Ozawa H, Tenenhouse HS. Targeted inactivation of Npt2 in mice leads to severe renal phosphate wasting, hypercalciuria, and skeletal abnormalities. Proc Natl Acad Sci U S A 1998;95:5372-7.