Mutations in SLC34A3/Npt2c Are Associated with Kidney Stones and Nephrocalcinosis

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Mutations in SLC34A3/NPT2c Are Associated with Kidney Stones and Nephrocalcinosis

†‡ Debayan Dasgupta,* Mark J. Wee,* Monica Reyes,* Yuwen Li,* Peter J. Simm, | † Amita Sharma,§ Karl-Peter Schlingmann, Marco Janner,¶ Andrew Biggin, Joanna Lazier,** †† ‡‡ || Michaela Gessner, Dionisios Chrysis, Shamir Tuchman,§§ H. Jorge Baluarte, ††† ‡‡‡ Michael A. Levine,¶¶ Dov Tiosano,*** Karl Insogna, David A. Hanley, ||| †† | Thomas O. Carpenter,§§§ Shoji Ichikawa, Bernd Hoppe, Martin Konrad, Lars Sävendahl,¶¶¶ †‡ Craig F. Munns, Hang Lee,**** Harald Jüppner,*§ and Clemens Bergwitz*

*Endocrine Unit, and §Pediatric Nephrology Unit, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts; †Institute of Endocrinology and Diabetes, Children’s Hospital at Westmead, Westmead, New South Wales, Australia; ‡Discipline of Pediatrics and Child Health, University of Sydney, Sydney, Australia; |Department of General Pediatrics, University Children’s Hospital, Münster, Germany; ¶Division of Pediatric Endocrinology, Diabetology and Metabolism, University Children’s Hospital, Bern, Germany; **Department of Medical Genetics, University of Calgary, Calgary, Alberta, Canada; ††Division of Pediatric Nephrology, Department of Pediatrics, University Hospital, Köln, Germany; ‡‡Division of Endocrinology, Department of Pediatrics, University of Patras Medical School, Patras, Greece; §§Division of Pediatric Nephrology, Children’s National Medical Center, The George Washington University School of Medicine, Washington, District of Columbia; ||University of Pennsylvania, School of Medicine, Division of Pediatric Nephrology, Children’sHospitalof Philadelphia, Philadelphia, Pennsylvania; ¶¶Division of Endocrinology and Diabetes, Children’s Hospital of Philadelphia, Philadelphia, Pennsylvania; ***Division of Pediatric Endocrinology, Meyer Children’s Hospital, Rambam Health Care Campus, Haifa, Israel; †††Division of Endocrinology, Department of Medicine and §§§Department of Pediatrics (Endocrinology), Yale University School of Medicine, New Haven, Connecticut; ‡‡‡Departments of Medicine, Community Health Sciences, and Oncology, University of Calgary Faculty of Medicine, Calgary, Alberta, Canada; |||Division of Endocrinology, Indiana University School of Medicine, Indianapolis, Indiana; ¶¶¶Pediatric Endocrinology Unit, Department of Women’s and Children´s Health, Karolinska Institutet, Stockholm, Sweden; and ****Biostatistics Center, Massachusetts General Hospital, Boston, Massachusetts

ABSTRACT Compound heterozygous and homozygous (comp/hom) mutations in solute carrier family 34, member 3 (SLC34A3), the gene encoding the sodium (Na+)-dependent phosphate cotransporter 2c (NPT2c), cause hereditary hypophosphatemic rickets with hypercalciuria (HHRH), a disorder characterized by renal phosphate wasting resulting in hypophosphatemia, correspondingly elevated 1,25(OH)2 vitamin D levels, hypercalciuria, and rickets/osteomalacia. Similar, albeit less severe, biochemical changes are observed in heterozygous (het) carriers and indistinguishable from those changes encountered in idiopathic hypercalciuria (IH). Here, we report a review of clinical and laboratory records of 133 individuals from 27 kindreds, including 5 previously unreported HHRH kindreds and two cases with IH, in which known and novel SLC34A3 mutations (c.1357delTTC [p.F453del]; c.G1369A [p.G457S]; c.367delC) were identified. Individuals with mutations affecting both SLC34A3 alleles had a significantly increased risk of kidney stone formation or medullary nephrocalcinosis, namely 46% compared with 6% observed in healthy family members carrying only the wild-type SLC34A3 allele (P=0.005) or 5.64% in the general population (P,0.001). Renal calcifications were also more frequent in het carriers (16%; P=0.003 compared with the general population) and were more likely to occur in comp/hom and het individuals with decreased serum phosphate (odds ratio [OR], 0.75, 95% confidence interval [95% CI], 0.59 to 0.96; P=0.02), decreased tubular reabsorption of phosphate (OR, 0.41; 95% CI, 0.23 to 0.72; P=0.002), and increased serum 1,25(OH)2 vitamin D (OR, 1.22; 95% CI, 1.05 to 1.41; P=0.008). Additional studies are needed to determine whether these biochemical parameters are independent of genotype and can guide therapy to prevent nephrocalcinosis, nephrolithiasis, and potentially, CKD.

J Am Soc Nephrol 25: 2366–2375, 2014. doi: 10.1681/ASN.2013101085

Received October 16, 2013. Accepted February 16, 2014. Blossom Street, Thier Building 1055, Massachusetts General Hospital and Harvard Medical School, Boston, MA 02114. Email: Published online ahead of print. Publication date available at [email protected] www.jasn.org.

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Inactivating mutations on both parental alleles of the solute how therapy should be monitored, whether secondary hyper- carrier family 34, member 3 (SLC34A3), the gene encoding the parathyroidism can develop as observed in FGF23-dependent sodium (Na+)-dependent phosphate cotransporter 2c (NPT2c), hypophosphatemic disorders,15 and whether phosphate re- are the cause of hereditary hypophosphatemic rickets with hy- quirements decrease with age, which has been reported for percalciuria (HHRH; OMIM: 241530)1–3—an autosomal re- ADHR.16 cessive renal phosphate-wasting disorder that was originally In the current study, we investigated five new HHRH described by Tieder et al.4,5 Individuals affected by HHRH kindreds and two new cases with idiopathic hypercalciuria who carry compound heterozygous or homozygous (comp/ (IH), in whomwe discovered known and novel homozygous or hom) SLC34A3/NPT2c mutations show increased urinary compounded heterozygous SLC34A3/NPT2c mutations. Re- phosphate excretion leading to hypophosphatemic rickets, view of the clinical and laboratory findings along with those bowing, and short stature as well as appropriately elevated findings reported for 22 previously published kindreds sug- 1,25(OH)2D levels. Elevated 1,25(OH)2D levels, in turn, result gests that renal calcifications and/or kidney stones may be in hypercalciuria because of enhanced intestinal calcium important, often unrecognized initial findings suggestive of absorption and reduced parathyroid hormone (PTH) - comp/hom SLC34A3/NPT2c mutations. Importantly, hetero- dependent calcium reabsorption in the distal renal tubules. zygous carriers also show an increased frequency of renal cal- Even heterozygous SLC34A3/NPT2c mutations are frequent- cifications and biochemical profiles in plasma and urine that ly associated with hypercalciuria, but none of the carriers are intermediate to those profiles of individuals without of SLC34A3/NPT2c mutations in the originally described SLC34A3/NPT2c mutations and comp/hom changes. Our HHRH patients were reported to have renal calcifications data suggest that serum phosphate, tubular reabsorption of 4,5 and kidney stones. Subsequent investigations, however, re- phosphate (TRP), and serum 1,25(OH)2D levels predict the vealed that these complications affecting the kidneys were development of renal calcifications. However, additional stud- observed in numerous patients with comp/hom SLC34A3/ ies are needed to determine whether these biochemical param- NPT2c mutations.3,6–11 However, the small size of HHRH eters are independent of genotype and can guide therapy to kindreds and the relatively high prevalence of renal calcifica- prevent renal calcifications and potentially, CKD. tions in the general population (5.64%)12,13 have, thus far, pre- vented segregation-based statistical approaches to determine whether SLC34A3/NPT2c mutations do increase the risk of RESULTS developing kidney stones or nephrocalcinosis. Likewise, it is unknown whether loss of NPT2c can lead to additional prox- Novel Mutations in the SLC34A3/NPT2c Gene in imal tubular phenotypes such as Fanconi syndrome, which Kindreds with HHRH and IH has been described in two patients with homozygous Five new kindreds were referred to us for genetic evaluation, SLC34A3/NPT2a mutations14 who developed CKD later in with the index case presenting with classic HHRH during life. childhood. Metabolic bone disease was found to be associated The presence of hypercalciuria, kidney stones, and with hypophosphatemia, and subsequent laboratory studies nephrocalcinosis observed in HHRH kindreds is different were consistent with FGF23-independent renal phosphate from the findings in fibroblast growth factor 23 (FGF23)– wasting. In addition, two unrelated children with IH presented dependent hypophosphatemic disorders, such as X-linked to their pediatrician with renal stones and/or nephrocalcinosis hypophosphatemia (XLH; mutant PHEX),15 autosomal dom- on renal ultrasound and biochemical abnormalities that were inant hypophosphatemic rickets (ADHR; mutant FGF23),16 or consistent with those abnormalities seen in HHRH [i.e.,hy- autosomal recessive hypophosphatemic rickets (ARHR; mu- percalciuria and elevated 1,25(OH)2D levels]. Apparent bone tant DMP1, ENPP1, or FAM20C),17–20 in which affected indi- disease was missing, and mild hypophosphatemia was only viduals show, before treatment with oral phosphate and 1,25 noted on more careful evaluation. Nucleotide sequence anal- (OH)2D, inappropriately normal or suppressed 1,25(OH)2D ysis of SLC34A3/NPT2c revealed known and novel mutations levels despite significant hypophosphatemia and thus, no in- in kindreds A and B and case G (Supplemental Figure 1, A and crease in urinary calcium excretion. Oral phosphate supple- B, Supplemental Tables 1 and 2). A novel homozygous mis- ments combined with active vitamin D analogs are generally sense mutation c.1369G.A(p.G457S) was detected in indi- recommended for treatment of FGF23-dependent hypophos- viduals A/IV-1, A/IV-2, A/IV-3, and A/IV-4, whereas both phatemic disorders.21 In contrast, HHRH is thought to re- parents were heterozygous for this nucleotide change. The index quire phosphate supplements alone,4,5 in part because endog- case B/II-2 inherited the previously described intronic dele- 22 enously elevated 1,25(OH)2D levels are predicted to prevent tion c.560+27_561–38del (g.1440_1469del) from his an increase in PTH secretion triggered by intermittent eleva- mother (B/I-1) and the novel in-frame deletion c.1357delTTC tions in serum phosphate. However, long-term studies are (p. F453del) from his father (B/I-2); c.1369G.A (p.G457S) lacking that determine whether oral phosphate supplementa- and c.1357delTTC (p.F453del) affect highly conserved amino tion alone of HHRH patients is sufficient for prevention of acid residues (Supplemental Table 3). Case G was found to be renal calcifications and bone loss. It is likewise unknown compound heterozygous for a novel deletion c.367delC,

J Am Soc Nephrol 25: 2366–2375, 2014 Frequency of Renal Calcifications in HHRH 2367 CLINICAL RESEARCH www.jasn.org which introduces 14 novel amino acids followed by premature when compared with the prevalence of 5.64% reported in termination of the NPT2c protein after residue p.P48 large cohorts of healthy controls12,13 (P=0.003) (Table 1). (WTLPQLKDPWTLPS-Stop) and the known missense muta- Consistent with an intermediate frequency of renal calcification c.575C.T(p.S192L).2 All three novel mutations are ab- tions, the biochemical findings were, furthermore, intermedi- sent from the 1000 Genome,23 dbSNP,24 and the National ate for heterozygous carriers of SLC34A3/NPT2c mutations. A Heart, Lung, and Blood Institute Gene Ontology Exome Se- combined analysis of the initial biochemical findings in our quencing Project (http://evs.gs.washington.edu/EVS/) data- new HHRH families along with previously published kindreds bases, predicted by Polyphen to reduce protein function,25 (Figures 2 and 3) showed reduced mean serum P, TRP, and and therefore, likely disease-causing. Detailed clinical and intact PTH in carriers of SLC34A3/NPT2c mutations, whereas genotyping information on kindreds A–G is in Supplemental serum Ca, 1,25(OH)2D, and urinary calcium excretion (uCa/ Material, Supplemental Figure 1, A and B, Supplemental Ta- uCrea) ratio were increased. Differences were significant based bles 1 and 2. on one-way ANOVA for these analytes when comparing wild- type, heterozygous, and comp/hom carriers. Despite the small Meta-Analysis of Clinical Information, Including number of healthy siblings, serum P was also significantly re- Previously Reported Kindreds with HHRH duced in heterozygous carriers compared with healthy siblings Seven of eleven com/hom carriers of the identified SLC34A3/ (P,0.03), and heterozygous means for 1,25(OH)2D and TRP NPT2c mutations and one of ten heterozygous carriers pre- were above and below the normal range, respectively. No clear sented initially with renal calcifications or developed these difference in severity was observed for individual mutations changes subsequently. To better understand whether this (Supplemental Figure 2). Renal function was normal in all but high prevalence of renal calcifications in these not previously one heterozygous and two comp/hom carriers of SLC34A3/ reported kindreds can be generalized to all carriers of NPT2c mutations at initial presentation (Figure 2C). SLC34A3/NPT2c mutations and to identify possible predic- Several of the above biochemical parameters have been tors of renal calcifications, we performed a meta-analysis of implicated as predictors of renal calcifications31 and metabolic the clinical information from 13 reports1–3,6–9,11,22,26–29 and bone disease.32 To identify predictors in our HHRH kindreds, two unpublished families (Simm P, Briody J, Reyes M, Gibbons we first performed a univariate logistic regression analysis in P, Alexander S, Rauch F, Jüppner H, Bergwitz C, Munns C, individuals with all genotypes (n=133) (Table 2). As expected, manuscript in preparation). Two reports of heterozygous mu- renal calcifications were more likely to occur in carriers of tations in kindreds with IH and HHRH10,30 were excluded SLC34A3/NPT2c mutations. Furthermore, individuals with from this analysis, because no mutations on the second allele renal calcifications had decreased serum P and TRP but in- had been identified; thus, segregation with the renal phenotype creased serum 1,25(OH)2D. However, only TRP remained sig- in the affected individuals remains uncertain. After including nificant after adjusting for genotype in a multivariate model, our new cases, this analysis comprised 133 individuals from 27 and 43 cases were not sufficient to identify predictors of renal kindreds (Supplemental Table 2). Genotype information is calcifications when limiting the univariate logistic regression available for 117 individuals and four obligate heterozygous analysis to individuals with two mutant SLC34A3/NPT2c alleles. carriers. Genotype was likewise the strongest predictor of metabolic We first evaluated the prevalence of renal calcifications (i.e., bone disease (Table 3). Furthermore, decreased serum P and nephrolithiasis or nephrocalcinosis) in the comp/hom carriers TRP and increased serum 1,25(OH)2Dandserumalkaline of SLC34A3/NPT2c mutations. This was significantly in- phosphatase (ALP) were significantly associated with bone creased (20 of 43 [46%] individuals) compared with the avail- involvement, even after adjusting for genotype in a multivariate able genotyped relatives who carried only the wild-type allele model; 1,25(OH)2D, TRP, and ALP continued to be significant (1 of 16 [6%] individuals; P=0.005) and compared with the when limiting the univariate logistic regression analysis to indi- heterozygote carriers of SLC34A3/NPT2c mutations (10 of 61 viduals with two mutant SLC34A3/NPT2c alleles. [16%] individuals; P=0.002) (Table 1); 15 of 43 (35%) comp/ hom carriers of SLC34A3/NPT2c mutations presented with renal stones (P=0.05), of whom 6 (14%) individuals also DISCUSSION had evidence for nephrocalcinosis. Furthermore, renal ultrasound studies showed medullary nephrocalcinosis in 13 of 43 Prevalence of Renal Calcifications in Carriers of (30%) com/hom individuals (P=0.01) (Table 1). Seven of SLC34A3/NPT2c Mutations Is Increased these individuals (16%) did not have stones. Importantly, 6 Renal calcifications were not previously thought to be part of 61 (10%) heterozygous and 7 of 43 (16%) comp/hom car- of the clinical presentation of HHRH.4,5 More recently, how- riers of SLC34A3/NPT2c mutations presented with renal cal- ever, an increased frequency of medullary nephrocalcinosis cifications, whereas clinical and laboratory findings suggestive and nephrolithiasis has been recognized3,6,7,9,11 in comp/hom of metabolic bone disease were absent (Figure 1). carriers. These observations are further substantiated in the cur- Prevalence of renal calcifications was increased 3-fold in rent report, because the index cases in three of five new kindreds heterozygous carriers of SLC34A3/NPT2c, which is significant showed renal calcifications on initial presentation. Furthermore,

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Table 1. Frequency of renal calciﬁcations is increased in carriers of SLC34A3/NPT2c mutations Genotype Phenotype Normal (n=16) Heterozygous (n=61) Comp/Hom (n=43) NPValue NPValue NPValue Renal Symptomatic 1 1.00 10 0.44/0.003a 20 ,0.01/0.002b/,0.001a Nephrolithiasis 1 1.00 10 0.44/0.003a 15 0.05/0.04b/,0.001a Nephrocalcinosis 0 3 1.00 13 0.01/,0.001b Asymptomatic 15 51 23 Bone Symptomatic 0 8 0.19 33 ,0.001 Rickets/osteomalacia 0 8 0.19 32 ,0.001 Adult osteopenia/osteoporosis 0 0 1 1.00 Asymptomatic 16 53 10 Number (N) of individuals with renal phenotype who presented with nephrolithiasis or nephrocalcinosis and number of individuals (N) with bone phenotype who presented with rickets/osteomalacia or osteopenia/osteoporosis. P values (Fisher exact t test) are shown compared with normal individuals. aAssuming average global prevalence of stones of 5.64% according to the work by Romero et al.12 bP values compared with heterozygous individuals.

it may be underestimated, because most asymptomatic individuals have not had imaging studies. A particularly important strength of our study is the analysis of heterozygous carriers of only those mutations that are clearly disease-causing when combined with another mutation on the second allele or homozygously present. Systematic evaluation of these heterozygous individuals will be an important aspect of future investigations. Several genes that cause rare monogenic Figure 1. Frequency of renal calcifications is increased in carriers of SLC34A3/NPT2c disorders have been associated with hyper- mutations. Shown here is the prevalence of renal calcifications (i.e., nephrolithiasis calciuric nephrolithiasis (i.e., CLCN5, and/or nephrocalcinosis) and bone disease (i.e., rickets/osteomalacia and/or osteo- CASR CLDN16 CLDN19 penia/osteoporosis) among individuals with two normal alleles as well as heterozygous , , , ADCY10, SLC34A1 SLC9A3R1 GLUT2 HSPG2 and comp/hom carriers of SLC34A3/NPT2c mutations. Calc., calcification; dis., dis- , , , , ease. and FN1),31,33 whereas variants of uromo- dulin and fetuin seem to be protective.34 Some of these genes affect tubular handling evaluation of 27 available kindreds indicates that approxi- of calcium and phosphate in a way that is similar to what is mately one half of all comp/hom carriers of SLC34A3/NPT2c observed in our patients with SLC34A3/NPT2c mutations. mutations present with renal calcifications. Renal calcifications, More recently, FAM20A mutations in enamel renal syndrome furthermore, are the only presenting sign in 16%, whereas bone were found to be associated with renal calcifications, but these disease is (apparently) absent. Even heterozygous carriers of individuals do not seem to fit the biochemical profile of IH20; SLC34A3/NPT2c mutations show an approximately 3-fold thus, it is more likely that loss of function of the extracellular higher incidence of renal calcifications, which may be related kinase encoded by FAM20A affects local tissue factors that to their intermediate biochemical profile (Figures 2 and 3). There- contribute to the development of renal calcifications. Fur- fore, all affected individuals and their first-degree relatives thermore, mutations in CYP24A1, the gene encoding the 24- should be examined for renal calcifications. The biochemical hydroxylase, which is the key enzyme leading to inactivation of profile may help identify, in addition to genetic analysis of 1,25(OH)2D, have been reported as a cause of hypercalciuric SLC34A3/NPT2c, those first-degree members in HHRH fam- nephrolithiasis.35 It should be noted that, thus far, genome- ilies who are at risk for developing renal complications as fur- wide association studies for uric acid nephrolithiasis,36 serum ther discussed below. phosphate levels,37 and CKD34 have not supported an associ- Although prevalence of renal calcifications in heterozygous ation of hypercalciuric stone disease with the SLC34A3/NPT2c carriers in this initial survey is only significant compared with locus. However, our meta-analysis clearly suggests that the prevalence reported in large cohorts of healthy controls,12,13 SLC34A3/NPT2c should be added to the above list of

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Serum 1,25(OH)2D, Phosphate, and TRP May Be Predictors of Renal Calcifications in Carriers of SLC34A3/ NPT2c Mutations It is unclear at the moment which genetic and biochemical criteria best predict the risk for renal calcifications in our HHRH kindreds. Based on the current understand- ing of the pathophysiology of HHRH, the defective sodium–phosphate cotransporter causes renal phosphate wasting, which triggers renal 1a-hydroxylase (CYP27B1) enzyme activity; this activity causes an increase in circulating 1,25(OH)2D levels, which in turn, increases intestinal Ca absorption, leading to an increased renal tubular Ca load and hypercalciuria. Thus, hypercalciuria, hyperphosphaturia, and possibly, tissue-specific effects of 1,25 (OH)2D levels may lead to renal calcifications and stones. Hypercalciuria is the most common risk factor of kidney stones, which is present in 40%–50% of adults with recurrent calcium stones and 75%–80% of children with kidney stones. This disease Figure 2. Heterozygous carriers of SLC34A3/NPT2c mutations have intermediate is often referred to as IH because of in- biochemical findings. Summary of biochemical values of the presented and published creased intestinal absorption of calcium, kindreds analyzed with respect to genotype. Individual values with mean695% con- and it can be associated with a mild renal fi dence interval are shown for (A) normalized serum calcium, (B) normalized serum phosphate leak, despite the lack of para- phosphorus, (C) normalized serum creatinine, and (D) serum 1,25(OH)2 vitamin D. het, thyroid hyperactivity.38 Association of in- heterozygous; n/a, not applicable; wt, wild-type. creased 1,25(OH)2D levels with renal calcifications was observed in mouse mod- hypercalciuric stone disease genes. Our finding may have im- els that lack Fgf2339 or Klotho40 or individuals with CYP24A1 portant implications for the general population, because mutations,35 even in the setting of low or normal urinary heterozygous nucleotide changes that alter the amino acid phosphate excretion. Finally, excessive phosphaturia, even sequence or introduce deletions/insertions are relatively fre- with normal or low urinary calcium, can cause nephrocalcinosis quent in NPT2c, but it is currently unknown whether they are in humans as is seen in XLH41 or in phosphate enema–induced of biologic significance. Improved clinical characterization nephrocalcinosis.42 Consistent with the pathophysiology of may provide a rationale for genetic analysis of SLC34A3/ HHRH, we found that increased serum 1,25(OH)2D, low NPT2c in a subpopulation of hypercalciuric stone patients serum P, and decreased TRP may be positive predictors of with clinical and biochemical profiles that resemble the pro- renal calcifications. Only TRP remained associated with files of HHRH. renal calcifications after controlling for genotype; however, To increase sample size, we decided to combine nephro- serum P, 1,25(OH)2D, TRP, and ALP continued to be signif- calcinosis and nephrolithiasis for statistical analysis, because icant predictors of bone involvement. This initial evalua- these clinical findings are both associated with the increased tion suggests that decreased serum P levels and increased urinary calcium excretion observed in HHRH. However, it excretion of phosphate and serum 1,25(OH)2Dmeritaddi- should be noted that 13 of 43 (30%) comp/hom individuals tional evaluation as possible nongenetic predictors of renal showed nephrocalcinosis on ultrasound, which reaches sta- calcifications. tistical significance, compared with normal and heterozygous We wondered whether specific SLC34A3/NPT2c mutations family members (Table 1), and 7 of these individuals did not are associated with an increased serum 1,25(OH)2D, low se- have evidence for renal stones. Future studies may be able to rum P, decreased TRP, and renal calcifications (Supplemental evaluate the possibility that nephrocalcinosis is a unique fea- Figure 2). However, not all individuals with a specific mutation ture of the hypercalciuria and hyperphosphaturia caused by have developed stones, and the numbers are small. It is, there- loss of NPT2c in the proximal tubule, which raises interesting fore, not possible to be certain about a genotype–phenotype pathophysiological questions. effect at the present time.

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Our study is limited by its relatively small sample size, because HHRH is a rare condition affecting, thus far, less than 50 individuals worldwide. Furthermore, clinical and biochemical data are often only available for the initial presentation, and family members who are heterozygous for a SLC34A3/NPT2c mutation or carry no mutation have been studied less well. In addition to a systematic evaluation of these individuals, long-term studies are required that determine whether oral phosphate supplementation alone of HHRH patients is sufficient for the prevention of renal calcifications and bone loss. It is likewise unknown how therapy should be monitored, whether secondary hyperparathyroidism can develop as observed in FGF23-dependent hypophosphatemic disorders,15 and whether phosphate re- quirements decrease with age, which has been reported for ADHR.16 It will also be important to determine whether lack of the NPT2c transporter leads to additional Figure 3. Heterozygous carriers of SLC34A3/NPT2c mutations have intermediate bio- symptoms, such as Fanconi syndrome or chemical findings. Summary of biochemical values of presented kindreds and published CKD, which have been shown for individ- kindreds analyzed with respect to genotype. Individual values with mean695% confi- uals with SLC34A1/NPT2a mutations,14 dence interval are shown for (A) serum PTH levels, (B) normalized ALP, (C) uCa/uCrea, using either animal models of HHRH or and (D) TRP. %UL, percent upper limit of normal; het, heterozygous; wt, wild-type.

Table 2. Univariate and multivariate regression model identifies association of genotype and biochemical variables with renal calcifications Renal Calcifications Unadjusted, All Adjusted, All (for Genotype or Sex/Agea) Unadjusted, Comp/Hom Only Variable Unit 95% CI 95% CI 95% CI N OR P Value N Adjusted OR P Value N OR P Value Lower Upper Lower Upper Lower Upper NPT2c mutation Alleles 122 3.90 1.31 11.63 0.02 89 12.2a 2.04 72.3 ,0.01 Women Yes 133 0.83 0.41 1.69 0.61 43 0.58 0.14 2.36 0.45 Age Year 94 1.00 0.97 1.03 0.86 41 1.02 0.96 1.10 0.51 Serum 25(OH)D 50 nmol/L 71 1.46 0.52 4.12 0.47 71 2.23 0.85 5.87 0.11 33 2.72 0.70 10.59 0.15 Serum calcium 1 SD 79 0.91 0.60 1.37 0.64 77 0.86 0.57 1.31 0.49 33 0.95 0.62 1.46 0.82 Serum phosphate 1 SD 100 0.75 0.59 0.96 0.02 98 0.98 0.71 1.37 0.92 42 0.93 0.58 1.47 0.75 Serum intact PTH 10% UL 78 0.80 0.59 1.07 0.13 76 0.97 0.71 1.32 0.83 32 0.92 0.61 1.40 0.71

Serum 1,25(OH)2D 50 pmol/L 86 1.22 1.05 1.41 ,0.01 85 1.09 0.96 1.25 0.19 39 1.11 0.97 1.26 0.12 uCa/uCrea 50% UL 88 1.39 0.94 2.05 0.10 85 0.92 0.59 1.45 0.73 38 0.87 0.51 1.45 0.58 TRP (%) 10% 68 0.41 0.23 0.72 0.002 66 0.54 0.31 0.92 0.02 27 0.65 0.38 1.10 0.11 Serum ALP 1 SD 73 1.04 0.95 1.15 0.40 72 0.97 0.90 1.04 0.37 29 0.96 0.89 1.04 0.30 Bone disease Yes 133 2.90 1.24 6.77 0.01 122 1.42 0.39 5.22 0.60 43 0.43 0.13 1.40 0.16 A univariate logistic regression model (GENMOD; SAS Institute, Inc.) was applied to estimate the unadjusted odds ratios (ORs) and those ORs were further adjusted for genotype (adjusted OR) as well as sex and age using a multivariate logistic regression model. An OR of one indicates that renal or bone involvement isequally likely to occur in individuals with a given variable. An OR greater than one indicates that renal or bone involvement is more likely to occur in individuals with high measurements of a given variable at the indicated units. An OR less than one indicates that renal or bone involvement is less likely to occur with high measurements of a given variable at the indicated units. 95% CI, 95% conﬁdence interval. aOdds ratios were further adjusted for sex and age by using a multivariate logistic regression model.

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Table 3. Univariate and multivariate regression model identiﬁes association of genotype and biochemical variables with bone disease Bone Disease Unadjusted Adjusted, All (for Genotype or Sex and Agea) Unadjusted, Comp/Hom Only Variable Unit 95% CI 95% CI 95% CI N OR P Value N Adjusted OR P Value N OR P Value Lower Upper Lower Upper Lower Upper NPT2c mutation Alleles 122 20.8 7.84 55.09 ,0.001 89 33.9a 9.52 120.9 ,0.001 Women Yes 133 0.71 0.33 1.55 0.39 43 0.43 0.08 2.41 0.34 Age Year 94 0.96 0.92 0.99 0.02 41 1.02 0.97 1.08 0.45 Serum 25(OH)D 50 nmol/L 71 0.74 0.31 1.79 0.51 71 1.18 0.40 3.49 0.77 33 1.22 0.22 6.79 0.82 Serum calcium 1 SD 79 1.32 0.80 2.18 0.28 77 1.41 0.87 2.28 0.16 33 1.19 0.75 1.88 0.45 Serum phosphate 1 SD 100 0.39 0.24 0.63 ,0.001 98 0.50 0.30 0.83 ,0.01 42 0.45 0.19 1.03 0.06 Serum intact PTH 10% UL 78 0.73 0.62 0.87 ,0.001 76 0.94 0.78 1.14 0.51 32 1.16 0.71 1.88 0.56

Serum 1,25(OH)2D 50 pmol/L 86 1.62 1.20 2.20 0.002 85 1.32 1.04 1.69 0.02 39 1.43 1.01 2.02 0.04 uCa/uCrea 50% UL 88 2.62 1.36 5.05 0.004 85 1.45 0.66 3.21 0.35 38 1.38 0.51 3.72 0.52 TRP(%) 10% 68 0.23 0.13 0.40 ,0.001 66 0.21 0.08 0.56 0.002 27 0.02 0.00 0.94 0.05 Serum ALP 1 SD 73 2.46 1.73 3.50 ,0.001 72 2.00 1.24 3.20 0.004 29 1.45 1.04 2.02 0.03 Renal calcifications Yes 133 2.90 1.24 6.77 0.01 122 1.39 0.36 5.33 0.63 43 0.43 0.13 1.40 0.16 A univariate logistic regression model (GENMOD; SAS Institute, Inc.) was applied to estimate the unadjusted odds ratios (ORs) and those ORs were further adjusted for genotype (adjusted OR) as well as sex and age using a multivariate logistic regression model. An OR of one indicates that renal or bone involvement isequally likely to occur in individuals with a given variable. An OR greater than one indicates that renal or bone involvement is more likely to occur in individuals with high measurements of a given variable at the indicated units. An OR less than one indicates that renal or bone involvement is less likely to occur with high measurements of a given variable at the indicated units. 95% CI, 95% confidence interval. aOdds ratios were further adjusted for sex and age by using a multivariate logistic regression model. large HHRH kindreds, such as those kindreds described by as well as intact FGF23 (Kainos). The renal TRP (in percentage) was Tieder et al.4,5 calculated using the following equation: 1003(12(urine phosphorus3 In summary, we here present five previously unreported serum creatinine)/(serum phosphorus3urine creatinine); when HHRH kindreds and two individuals with IH in whom serum phosphorus is below the reference range for age, TRP should SLC34A3/NPT2c nucleotide sequence analysis identified be above 90. Tubular maximum phosphate reabsorption per known or novel mutations. Review of the clinical presentation GFR (TmP/GFR) was estimated using the Walton and Bijvoet of these kindreds and previously published HHRH kindreds nomogram.43 suggests that renal calcifications and/or renal stones may be an important, often unrecognized initial symptom in carriers of SLC34A3 Genetic Analyses comp/hom SLC34A3/NPT2c mutations. Even heterozygous Mutational and haplotype analysis of SLC34A3 was performed after carriers can be affected by nephrocalcinosis and nephrolithia- informed written consent was obtained using forms approved by the sis, which is consistent with their intermediate biochemical Institutional Review Board of the Massachusetts General Hospital profile. Serum 1,25(OH)2D, phosphate, and TRP may be pre- (MGH). The entire SLC34A3 gene, including approximately 800 bp dictors of renal calcifications, and future studies will help deter- 59 of the transcriptional start site, all intervening sequences, and ap- mine whether these biochemical parameters are independent proximately 200 bp of the 39-untranslated region, was amplified by of genotype and can guide therapy to prevent nephrocalcinosis, PCR from genomic DNA of the index cases followed by nucleotide nephrolithiasis, and potentially CKD. sequence analysis at the MGH DNA Sequencing Core Facility or Genewiz, Inc. as described.1 PCR assays to confirm the findings in index cases and analyze family members and controls were per- CONCISE METHODS formed as described1 using Qiagen reagents or the Expand Long Template PCR System (Roche) at standard PCR cycling conditions Laboratory Assays followed by restriction enzymatic digest or nucleotide sequence anal- With the exception of genetic analyses, all laboratory studies were ysis. Primer sequences are listed in Supplemental Table 4, and con- performed at laboratories used by the different investigators (normal ditions for amplification and detection of the mutations are given in ranges are provided in parentheses after each value). 25-Hydroxy Supplemental Table 1. Searches of the National Center for Biotech- vitamin D levels were measured by liquid chromatography with nology Information–dbSNP24 and the 1000 Genomes Project23 were tandem mass spectroscopy or chemiluminescence immunoassay, and negative for the identified novel mutations, with the exception of Het.

1,25(OH)2D levels were measured by radioimmunoassay or ELISA. c.413C.T(p.S138F) (HGMD: CM060480), which was identiﬁed as a Serum intact PTH levels were determined by electrochemiluminescence rare single nucleotide polymorphism rs141734934, Het: 0.001 based immunoassay, and FGF23 levels were measured by an ELISA that detects on nucleotide sequence analysis of 4352 chromosomes (1000 Ge- the intact hormone and C-terminal fragments thereof (Immutopics) nomes). GenBank accession numbers for SLC34A3 are as follows:

2372 Journal of the American Society of Nephrology J Am Soc Nephrol 25: 2366–2375, 2014 www.jasn.org CLINICAL RESEARCH genomic contig, NT024000.15; cDNA, NM080877.1; protein, REFERENCES NP543153.1. 1. Bergwitz C, Roslin NM, Tieder M, Loredo-Osti JC, Bastepe M, Abu-Zahra Statistical and Data Analyses H, Frappier D, Burkett K, Carpenter TO, Anderson D, Garabedian M, For our meta-analysis, we used clinical and laboratory findings (LFs) Sermet I, Fujiwara TM, Morgan K, Tenenhouse HS, Jüppner H: SLC34A3 mutations in patients with hereditary hypophosphatemic rickets with obtained at presentation before therapy was initiated. For statistical hypercalciuria predict a key role for the sodium-phosphate cotransporter evaluation, we determined the number of individuals affected by (1) NaPi-IIc in maintaining phosphate homeostasis. Am J Hum Genet 78: nephrolithiasis who had history of renal colic or based on imaging 179–192, 2006 or stone analysis, (2) nephrocalcinosis, in whom renal mineral de- 2. Lorenz-Depiereux B, Benet-Pages A, Eckstein G, Tenenbaum-Rakover posits were found on imaging studies (i.e., ultrasound or computed Y, Wagenstaller J, Tiosano D, Gershoni-Baruch R, Albers N, Lichtner P, Schnabel D, Hochberg Z, Strom TM: Hereditary hypophosphatemic tomography), (3) rickets/osteomalacia based on history of bowing, cor- rickets with hypercalciuria is caused by mutations in the sodium- i.e. rective surgery, stress fractures, bone pain, or imaging studies ( , skel- phosphate cotransporter gene SLC34A3. Am J Hum Genet 78: 193– 99m etal radiographic survey and Tc methylene diphosphonate bone 201, 2006 scan), and (4) osteopenia/osteoporosis based on World Health Or- 3. Ichikawa S, Sorenson AH, Imel EA, Friedman NE, Gertner JM, Econs ganization bone densitometry criteria. Asymptomatic individuals MJ: Intronic deletions in the SLC34A3 gene cause hereditary hypophosphatemic rickets with hypercalciuria. JClinEndocrinolMetab91: were negative for the above renal and bone phenotypes either by 4022–4027, 2006 imaging or based on absence of history of symptoms suggesting the 4. Tieder M, Modai D, Samuel R, Arie R, Halabe A, Bab I, Gabizon D, presence of renal calcifications or bone disease. Age-related reference Liberman UA: Hereditary hypophosphatemic rickets with hyper- values (RVs; i.e., 95% confidence intervals) were obtained from http:// calciuria. N Engl J Med 312: 611–617, 1985 www.mayomedicallaboratories.com/, http://cclnprod.cc.nih.gov/ 5. Tieder M, Modai D, Shaked U, Samuel R, Arie R, Halabe A, Maor J, “ dlm/testguide.nsf/Index/1D336E0232533D3285256B9C0059E- Weissgarten J, Averbukh Z, Cohen N, Edelstein S, Liberman UA: Idi- opathic” hypercalciuria and hereditary hypophosphatemic rickets. Two CEA, and respective clinical laboratories; age-related RVs for TmP/ phenotypical expressions of a common genetic defect. NEnglJMed GFR are in refs. 44–46, and age-related RVs for uCa/uCrea are from 316: 125–129, 1987 ref. 47. Assuming normal distribution, LFs can be transformed into 6. Page K, Bergwitz C, Jaureguiberry G, Harinarayan CV, Insogna K: A z scores using means of age-appropriate RVs (RVmean) and SDs patient with hypophosphatemia, a femoral fracture, and recurrent kid- (where SD=RV/4) according to the formula: z score=(LF2RVmean)/ ney stones: Report of a novel mutation in SLC34A3. Endocr Pract 14: 869–874, 2008 48 z SD. Scores were used for all statistical analyses with the fol- 7. Kremke B, Bergwitz C, Ahrens W, Schütt S, Schumacher M, Wagner V, lowing exceptions: absolute values are presented for 25-hydroxy Holterhus PM, Jüppner H, Hiort O: Hypophosphatemic rickets with vitamin D, 1,25(OH)2D, TRP, and TmP/GFR measurements. The hypercalciuria due to mutation in SLC34A3/NaPi-IIc can be masked by data for PTH and age-specific uCa/uCrea are presented as percentage vitamin D deficiency and can be associated with renal calcifications. Exp – of the upper limit of normal RV. These exceptions are important, Clin Endocrinol Diabetes 117: 49 56, 2009 8. Phulwani P, Bergwitz C, Jaureguiberry G, Rasoulpour M, Estrada E: He- because for these assays, upper and lower RVs have not been es- reditary hypophosphatemic rickets with hypercalciuria and nephrolithiasis- fi fi tablished in suf ciently large number of normal age-speci ccon- identification of a novel SLC34A3/NaPi-IIc mutation. Am J Med Genet A trols.49 We used Prism for OSX, version 5.0d (GraphPad Software 155A: 626–633, 2011 Inc.), to perform t tests for unpaired comparisons, one-way ANOVAs 9. Jaureguiberry G, Carpenter TO, Forman S, Jüppner H, Bergwitz C: A novel for multiple comparisons, and linear regression analyses. For uni- missense mutation in SLC34A3 that causes hereditary hypophosphatemic rickets with hypercalciuria in humans identifies threonine 137 as an im- variate and multivariate logisticregressionmodels,weusedSAS portant determinant of sodium-phosphate cotransport in NaPi-IIc. Am J (GENMOD; SAS Institute, Inc.) to estimate the odds ratios. To in- Physiol Renal Physiol 295: F371–F379, 2008 crease sample size, we treated all renal phenotypes (nephrolithiasis and 10. Mejia-Gaviria N, Gil-Peña H, Coto E, Pérez-Menéndez TM, Santos F: nephrocalcinosis) or bone phenotypes (rickets, osteomalacia, and Genetic and clinical peculiarities in a new family with hereditary hy- osteopenia/osteoporosis) collectively. Data are expressed as means695% pophosphatemic rickets with hypercalciuria: A case report. Orphanet J confidence intervals. Rare Dis 5: 1, 2010 11. Tencza AL, Ichikawa S, Dang A, Kenagy D, McCarthy E, Econs MJ, Levine MA: Hypophosphatemic rickets with hypercalciuria due to mu- ACKNOWLEDGMENTS tation in SLC34A3/type IIc sodium-phosphate cotransporter: Pre- sentation as hypercalciuria and nephrolithiasis. J Clin Endocrinol Metab 94: 4433–4438, 2009 This work was supported by National Institutes of Health Grants R01- 12. Romero V, Akpinar H, Assimos DG: Kidney stones: A global picture of DK46718-20 and P01-DK11794, project IV (to H.J.), National In- prevalence, incidence, and associated risk factors. Rev Urol 12: e86– stitutes of Health/National Institute of Diabetes and Digestive and e96, 2010 13. Schissel BL, Johnson BK: Renal stones: Evolving epidemiology and Kidney Diseases Grant 5K08-DK078361 (to C.B.), Young Investigator management. Pediatr Emerg Care 27: 676–681, 2011 Awards by the National Kidney Foundation, and the American As- 14. Magen D, Berger L, Coady MJ, Ilivitzki A, Militianu D, Tieder M, Selig S, sociation for Clinical Investigation (C.B.). Lapointe JY, Zelikovic I, Skorecki K: A loss-of-function mutation in NaPi-IIa and renal Fanconi’s syndrome. N Engl J Med 362: 1102–1109, 2010 DISCLOSURES 15. The HYP Consortium: A gene (PEX) with homologies to endopepti- dases is mutated in patients with X-linked hypophosphatemic rickets. None. Nat Genet 11: 130–136, 1995

J Am Soc Nephrol 25: 2366–2375, 2014 Frequency of Renal Calciﬁcations in HHRH 2373 CLINICAL RESEARCH www.jasn.org

16. ADHR Consortium: Autosomal dominant hypophosphataemic rickets is 29. Ichikawa S, Tuchman S, Padgett LR, Gray AK, Baluarte HJ, Econs MJ: associated with mutations in FGF23. Nat Genet 26: 345–348, 2000 Intronic deletions in the SLC34A3 gene: A cautionary tale for mutation 17. Feng JQ, Ward LM, Liu S, Lu Y, Xie Y, Yuan B, Yu X, Rauch F, Davis SI, analysis of hereditary hypophosphatemic rickets with hypercalciuria. Zhang S, Rios H, Drezner MK, Quarles LD, Bonewald LF, White KE: Loss Bone 59: 53–56, 2014 of DMP1 causes rickets and osteomalacia and identifies a role for os- 30. Yamamoto T, Michigami T, Aranami F, Segawa H, Yoh K, Nakajima S, teocytes in mineral metabolism. Nat Genet 38: 1310–1315, 2006 Miyamoto K, Ozono K: Hereditary hypophosphatemic rickets with hy- 18. Lorenz-Depiereux B, Bastepe M, Benet-Pagès A, Amyere M, percalciuria: A study for the phosphate transporter gene type IIc and Wagenstaller J, Müller-Barth U, Badenhoop K, Kaiser SM, Rittmaster osteoblastic function. JBoneMinerMetab25: 407–413, 2007 RS, Shlossberg AH, Olivares JL, Loris C, Ramos FJ, Glorieux F, Vikkula 31. Stechman MJ, Loh NY, Thakker RV: Genetic causes of hypercalciuric M, Jüppner H, Strom TM: DMP1 mutations in autosomal recessive nephrolithiasis. Pediatr Nephrol 24: 2321–2332, 2009 hypophosphatemia implicate a bone matrix protein in the regulation of 32. Carpenter TO, Imel EA, Holm IA, Jan de Beur SM, Insogna KL: A phosphate homeostasis. Nat Genet 38: 1248–1250, 2006 clinician’s guide to X-linked hypophosphatemia. JBoneMinerRes26: 19. Lorenz-Depiereux B, Schnabel D, Tiosano D, Häusler G, Strom TM: 1381–1388, 2011 Loss-of-function ENPP1 mutations cause both generalized arterial 33. Langman CB: The molecular basis of kidney stones. Curr Opin Pediatr calcification of infancy and autosomal-recessive hypophosphatemic 16: 188–193, 2004 rickets. Am J Hum Genet 86: 267–272, 2010 34. Gudbjartsson DF, Holm H, Indridason OS, Thorleifsson G, Edvardsson 20. Jaureguiberry G, De la Dure-Molla M, Parry D, Quentric M, Himmerkus V, Sulem P, de Vegt F, d’Ancona FC, den Heijer M, Wetzels JF, Franzson N, Koike T, Poulter J, Klootwijk E, Robinette SL, Howie AJ, Patel V, L, Rafnar T, Kristjansson K, Bjornsdottir US, Eyjolfsson GI, Kiemeney LA, Figueres ML, Stanescu HC, Issler N, Nicholson JK, Bockenhauer D, Kong A, Palsson R, Thorsteinsdottir U, Stefansson K: Association of Laing C, Walsh SB, McCredie DA, Povey S, Asselin A, Picard A, variants at UMOD with chronic kidney disease and kidney stones-role of Coulomb A, Medlar AJ, Bailleul-Forestier I, Verloes A, Le Caignec C, age and comorbid diseases. PLoS Genet 6: e1001039, 2010 Roussey G, Guiol J, Isidor B, Logan C, Shore R, Johnson C, Inglehearn 35. Schlingmann KP, Kaufmann M, Weber S, Irwin A, Goos C, John U, C, Al-Bahlani S, Schmittbuhl M, Clauss F, Huckert M, Laugel V, Misselwitz J, Klaus G, Kuwertz-Bröking E, Fehrenbach H, Wingen AM, Ginglinger E, Pajarola S, Spartà G, Bartholdi D, Rauch A, Addor MC, Güran T, Hoenderop JG, Bindels RJ, Prosser DE, Jones G, Konrad M: Yamaguti PM, Safatle HP, Acevedo AC, Martelli-Júnior H, dos Santos Mutations in CYP24A1 and idiopathic infantile hypercalcemia. NEnglJ Netos PE, Coletta RD, Gruessel S, Sandmann C, Ruehmann D, Med 365: 410–421, 2011 Langman CB, Scheinman SJ, Ozdemir-Ozenen D, Hart TC, Hart PS, 36. Tore S, Casula S, Casu G, Concas MP, Pistidda P, Persico I, Sassu A, Neugebauer U, Schlatter E, Houillier P, Gahl WA, Vikkula M, Bloch- Maestrale GB, Mele C, Caruso MR, Bonerba B, Usai P, Deiana I, Thornton Zupan A, Bleich M, Kitagawa H, Unwin RJ, Mighell A, Berdal A, Kleta R: T, Pirastu M, Forabosco P: Application of a new method for GWAS in a Nephrocalcinosis (enamel renal syndrome) caused by autosomal re- related case/control sample with known pedigree structure: Identification cessive FAM20A mutations. Nephron, Physiol 122: 1–6, 2012 of new loci for nephrolithiasis. PLoS Genet 7: e1001281, 2011 21. White AJ, Northcutt MJ, Rohrback SE, Carpenter RO, Niehaus-Sauter 37. Kestenbaum B, Glazer NL, Köttgen A, Felix JF, Hwang SJ, Liu Y, MM, Gao Y, Wheatly MG, Gillen CM: Characterization of sarcoplasmic Lohman K, Kritchevsky SB, Hausman DB, Petersen AK, Gieger C, Ried calcium binding protein (SCP) variants from freshwater crayfish Pro- JS, Meitinger T, Strom TM, Wichmann HE, Campbell H, Hayward C, cambarus clarkii. Comp Biochem Physiol B Biochem Mol Biol 160: 8– Rudan I, de Boer IH, Psaty BM, Rice KM, Chen YD, Li M, Arking DE, 14, 2011 Boerwinkle E, Coresh J, Yang Q, Levy D, van Rooij FJ, Dehghan A, 22. Yu Y, Sanderson SR, Reyes M, Sharma A, Dunbar N, Srivastava T, Rivadeneira F, Uitterlinden AG, Hofman A, van Duijn CM, Shlipak MG, Jüppner H, Bergwitz C: Novel NaPi-IIc mutations causing HHRH and Kao WH, Witteman JC, Siscovick DS, Fox CS: Common genetic variants idiopathic hypercalciuria in several unrelated families: Long-term follow- associate with serum phosphorus concentration. J Am Soc Nephrol 21: up in one kindred. Bone 50: 1100–1106, 2012 1223–1232, 2010 23. Abecasis GR, Altshuler D, Auton A, Brooks LD, Durbin RM, Gibbs RA, 38. Prié D, Ravery V, Boccon-Gibod L, Friedlander G: Frequency of renal Hurles ME, McVean GA; 1000 Genomes Project Consortium: A map of phosphate leak among patients with calcium nephrolithiasis. Kidney Int human genome variation from population-scale sequencing. Nature 60: 272–276, 2001 467: 1061–1073, 2010 39. Hesse M, Fröhlich LF, Zeitz U, Lanske B, Erben RG: Ablation of vitamin D 24. Wheeler DL, Barrett T, Benson DA, Bryant SH, Canese K, Chetvernin V, signaling rescues bone, mineral, and glucose homeostasis in Fgf-23 Church DM, DiCuccio M, Edgar R, Federhen S, Geer LY, Helmberg W, deficient mice. Matrix Biol 26: 75–84, 2007 Kapustin Y, Kenton DL, Khovayko O, Lipman DJ, Madden TL, Maglott 40. Kuro-o M, Matsumura Y, Aizawa H, Kawaguchi H, Suga T, Utsugi T, DR, Ostell J, Pruitt KD, Schuler GD, Schriml LM, Sequeira E, Sherry ST, OhyamaY,KurabayashiM,KanameT,KumeE,IwasakiH,IidaA,Shiraki-Iida Sirotkin K, Souvorov A, Starchenko G, Suzek TO, Tatusov R, Tatusova T, Nishikawa S, Nagai R, Nabeshima YI: Mutation of the mouse klotho TA, Wagner L, Yaschenko E: Database resources of the National Center gene leads to a syndrome resembling ageing. Nature 390: 45–51, 1997 for Biotechnology Information. Nucleic Acids Res 34[Database issue]: 41. Carpenter TO, Imel EA, Holm IA, Jan de Beur SM, Insogna KL: A D173–D180, 2006 clinician’s guide to X-linked hypophosphatemia. JBoneMinerRes26: 25. Adzhubei IA, Schmidt S, Peshkin L, Ramensky VE, Gerasimova A, Bork 1381–1388, 2011 P, Kondrashov AS, Sunyaev SR: A method and server for predicting 42. Gonlusen G, Akgun H, Ertan A, Olivero J, Truong LD: Renal failure and damaging missense mutations. Nat Methods 7: 248–249, 2010 nephrocalcinosis associated with oral sodium phosphate bowel 26. Braithwaite V, Pettifor JM, Prentice A: Novel SLC34A3 mutation caus- cleansing: clinical patterns and renal biopsy findings. Arch Pathol Lab ing hereditary hypophosphataemic rickets with hypercalciuria in a Med 130: 101–106, 2006 Gambian family. Bone 53: 216–220, 2013 43. Walton RJ, Bijvoet OL: A simple slide-rule method for the assessment of 27. Areses-Trapote R, López-García JA, Ubetagoyena-Arrieta M, Eizaguirre renal tubular reaborption of phosphate in man. Clin Chim Acta 81: 273– A, Sáez-Villaverde R: Hereditary hypophosphatemic rickets with hy- 276, 1977 percalciuria: Case report. Nefrologia 32: 529–534, 2012 44. Alon U, Hellerstein S: Assessment and interpretation of the tubular 28. Hasani-Ranjbar S, Amoli MM, Ebrahim-Habibi A, Dehghan E, Soltani A, threshold for phosphate in infants and children. Pediatr Nephrol 8: Amiri P, Larijani B: SLC34A3 intronic deletion in a new kindred with 250–251, 1994 hereditary hypophosphatemic rickets with hypercalciuria. JClinRes 45. Brodehl J, Gellissen K, Weber HP: Postnatal development of tubular Pediatr Endocrinol 4: 89–93, 2012 phosphate reabsorption. Clin Nephrol 17: 163–171, 1982

2374 Journal of the American Society of Nephrology J Am Soc Nephrol 25: 2366–2375, 2014 www.jasn.org CLINICAL RESEARCH

46. Kruse K, Kracht U, Göpfert G: Renal threshold phosphate concentration 49. Lee WT, Jiang J: The resurgence of the importance of vitamin D in bone (TmPO4/GFR). Arch Dis Child 57: 217–223, 1982 health. Asia Pac J Clin Nutr 17[Suppl 1]: 138–142, 2008 47. Mitchell DM, Jüppner H: Regulation of calcium homeostasis and bone metabolism in the fetus and neonate. Curr Opin Endocrinol Diabetes Obes 17: 25–30, 2010 48. Boyd JC, Lacher DA: A multi-stage Gaussian transformation algorithm This article contains supplemental material online at http://jasn.asnjournals. for clinical laboratory data. Clin Chem 28: 1735–1741, 1982 org/lookup/suppl/doi:10.1681/ASN.2013101085/-/DCSupplemental.

J Am Soc Nephrol 25: 2366–2375, 2014 Frequency of Renal Calciﬁcations in HHRH 2375 Supplemental information for Dasgupta et al. “Mutations in SLC34A3/NPT2c are associated with kidney stones and nephrocalcinosis” Supplemental Results: Full case reports of novel HHRH kindreds and IH cases The index case of kindred A (Fig. 1A), A/IV-1 presented at 92/12 years with leg pain when walk- ing and progressive crura vara since age 1, which required surgical correction at age 5. She is the oldest of four children to Afghan parents, who are first-degree cousins. Her physical exam at 91/12 years revealed a weight (W) above 97th percentile, height (H) between the 90th-97th percentile, severe crura vara, flared condyles, large hands and feet, prominent sternum, carious teeth, no enamel defects. Her pubertal development was Tanner breast 3. On presentation, her total calcium (Ca) and ionized Ca (iCa) were normal at 2.4 mmol/l (2.4-2.65) and 1.28 mmol/l (1.1-1.35), respectively, she was hypophosphatemic with a phosphate (P) level of 0.77 mmol/l (1.0-1.8), her alkaline phosphatase (ALP) was elevated to 725 IU/l (<300), parathyroid hormone (iPTH) was suppressed at 1.48 pmol/l (1.6-6.9), and 1,25(OH)2D was at the upper limit of normal at 155 pmol/l (48-160) in the setting of low 25OHD of 55 nmol/l (62-200). Her magnesium (Mg) 0.67 mmol/l (0.73-1), creatinine (Crea) 39 µmol/l (33-68), uCa/Crea ratio 0.86 mol/mol (<0.85) were normal, while tubular reabsorption of phosphate (TRP) of 82% was inappropriately low in the context of hypophosphatemia (>90%). There was no glucosuria, proteinuria, or aminoaciduria. Intact fibroblast growth factor 23 (iFGF23) level was undetectable. On treatment with 20 mg/kg/d of elemental phosphorus, calcitriol 12 ng/kg/d her ALP improved to 585 IU/l, but she developed hypercalciuria; uCa/Crea was 1.28 mol/mol (<0.85) and her renal ultrasound revealed extensive nephrocalcinosis. After discontinuation of calcitriol while continuing treatment with 20 mg/kg/d of elemental phosphorus, her laboratory findings at age 101/12 years improved to ALP 491 U/l, 1,25(OH)2D 136 pmol/l and uCa/Crea 0.5 mol/mol. The proband’s 7-year old brother, A/IV-2, had no clinical symptoms, but nephrocalcinosis was observed on renal ultrasound and ALP was elevated at 342 IU/l (<300). The 24/12 year old sister, A/IV-3, presented with crura vara but had no other rachitic signs and renal ultrasound was normal. Her laboratory results showed an elevated ALP 492 (<300), suppressed iPTH of 1.06 pmol/l (1.6-6.9), and an elevated uCa/Crea ratio of 1.35 mol/mol (<1.00). There was no glucosuria, proteinuria, or aminoaciduria. Intact FGF23 was below the detection limit. The 1 year-old brother, A/IV-4, had a normal physical examination, normal renal ultrasound and his labs were normal with the exception of ALP that was elevated at 320 IU/l (<300). The father, A/III-1, and mother, A/III-2, were healthy and had normal renal ultrasounds. Additional information on kindred A A/IV-1, index case, age 101/12 years: On treatment with sodium-phosphate 20 mg/kg/d, calcitriol 12 ng/kg/d ALP improved to 585 IU/l, and she developed hypercalciuria; uCa/Crea 1.28 mol/mol (<0.85). After discontinuation of calcitriol while continuing treatment with sodium phosphate 20 mg/kg/d, her laboratory findings at age improved further; ALP decreased to 491 U/l. On this therapy, 1,25(OH)2D initially decreased to 136 pmol/l and uCa/Crea improved to 0.5 4/12 mol/mol, but subsequently 1,25(OH)2D again increased 226 pmol/l at 10 years and 284 pmol/l at 107/12 years, presumably due to noncompliance with therapy. On therapy, P ranged from 0.65 to 1.15 mmol/l and TRP decreased further to 73.1%.

A/IV-2, brother, age 7 years: Additional laboratory findings include Ca 2.52 mmol/l (2.40- 2.65), P 1.15 mmol/l (1.00-1.8), iCa 1.28 mmol/l (1.10-1.35), Crea 44 µmol/l, suppressed iPTH of 1.27 pmol/l (1.60-6.90), 25(OH)D 77 nmol/l, 1,25(OH)2D 130 pmol/l, uCa/crea ratio 0.65 mol/mol (<0.85), TRP 95.3%. A/IV-3, sister, age 24/12 year: Additional clinical information: 75-90th percentile for W and 25- 50th percentile for H, HC 10-25th percentile. Additional laboratory results were; Ca 2.41 mmol/l (2.4-2.65), ion. Ca 1.31 mmol/l, low P 0.87 mmol/l, Mg 0.75 mmol/l (0.73-1.0), Crea 24 µmol/l, 25(OH)D 40 nmol/l, 1,25(OH)2D 99 pmol/l (48-160). ALP improved to 243 U/l under treatment with 20 mg P/kg/d and calcitriol 11.4 ng/kg/d, but her hypercalciuria persisted with a uCa/Crea ratio of 1.36 mol/mol (<1.00). Her hypercalciuria worsened to 2.2 mol/mol after accidentally adding 2500 IU cholecalciferol/day to her regimen, and 25(OH)D and 1,25(OH)2D increased to 168 nmol/l and 255 pmol/l, respectively, as a result of which all vitamin D analogs were stopped. A/IV-4, brother, age 1 year: Additional labs were Ca 2.39 mmol/l (2.4-2.65), P 1.26 mmol/l (1.00-1.8), a low ion. Ca of 0.93 mmol/l (1.1-1.35), elevated ALP at 320 IU/l (<300), Crea 31 µmol/l (20-65), iPTH 2.54 pmol/l (1.6-6.9), 25(OH)D 19 nmol/l, 1,25(OH)2D 90 pmol/l, uCa/Crea 0.52 mol/mol (<1.27), TRP 92.5%. A/III-1, father, age 34 years: H was 185.0 cm, additional labs were Ca 2.39 mmol/l (2.1-2.55), P 1.02 mmol/l (0.84-1.45), Crea 92 µmol/l (59-104), ALP 71 U/l (40-130), uCa/Crea 0.19 mol/mol (<0.62), TRP 89%, no glucosuria, proteinuria, or aminoaciduria, and his renal US was normal. A/III-2, mother, age 29 years: H was 158.0 cm, her laboratory studies were: Ca 2.31 mmol/l (2.1-2.55), P 0.86 mmol/l (0.84-1.45), Crea 59 µmol/l (45-84), ALP 72 U/l (35-105), uCa/Crea 0.38 mol/mol (<0.62), TRP 91.4%, no glucosuria, proteinuria, or aminoaciduria.

The index case in kindred B (Fig. 1 B), B/II-2, the younger of two children from non- consanguineous Caucasian parents, presented at 11 years of age with an elevated ALP of 675 IU/L (reference range 50-350) incidentally found during investigation for vasovagal syncope. He previously fractured his left femur in a skateboard park at age 6 years, sustained a greenstick fracture of his left forearm at age 8 years, and a displaced left radial fracture at age 12 years while snowboarding. His P was slightly reduced at 0.94 mmol/L (1.20-1.74) and his 25(OH)D was 44 nmol/L (51-250). No further action was taken for a two-year period apart from monitor- ing his ALP, which fluctuated between 650-820 U/L until age 13 years when he was referred to one of us for investigation of his persistently elevated ALP, bone pain and recurrent fractures. Repeat blood tests at this time showed an ALP of 769 U/L despite a meanwhile improved 25(OH)D level of 71 nmol/L, but his serum P was low 0.90 mmol/L (1.20-1.74), 1,25(OH)2D was elevated 419 pmol/L (60-158), while Ca, Crea, and iPTH levels were within normal limits. TmP/GFR of 0.98 mmol/L (0.77-1.29) and TRP 84% were inappropriate in the setting of hypophosphatemia. Calcium excretion was at the upper limit of normal with an uCa/Crea ratio of 0.507-0.706 mol/mol (<0.73) and 24hr Ca excretion was 4.8 mmol/day (1-7). His bone mineral densitometry showed a total hip BMD z-score of -3.6, spine BMD z-score of -1.6, and whole body bone mineral z-score of -6.1. On examination B/II-2 appeared well. He was not dysmorphic and had no birthmarks. He had been growing adequately; his latest height was 162.3cm (75th

2 percentile) with a weight of 53.9kg (77th percentile). He had normal sclerae and dentition, hy- permobile fingers, hyperextensible knees, bilateral pes planus and calcaneovalgus. His spine was straight. General clinical examination was otherwise unremarkable, he was normotensive, and renal ultrasound was unremarkable. The parents (B/I-1 and I-2) and an older brother (B/II-1) were well and revealed normal biochemistries as shown in Figure 1B, with the exception of an elevated 1,25(OH)2D level for father and brother of 218 and 172 pmol/L (60.00-158.00), respectively. There is no family history of renal stones. The index case of kindred C (Fig. 1 C), C/III-1, presented at age 5 years, following an earlier history of nephrolithiasis at age 3, managed with low calcium intake and hydration. Upon refer- ral to endocrinology, a more complete evaluation was performed revealing hypophosphatemia of 1 mmol/L (1.39-1.74). His 1,25(OH)2D was also elevated at 343.20 pmol/L (52-163.80), as was his urine Ca/Crea ratio at 1.90 mol/mol (<0.66). TmP/GFR was 0.74, and TRP was low at 76%(>90%). His remaining biochemical findings were within normal limits: 25(OH)D 84.86 nmol/L (24.96-169.73), serum Ca 2.40 mmol/L (2.13-2.63), serum Crea 44.23 umol/L (17.63- 52.88). His kidney stones required lithotripsy at age 4, since he had experienced mild back pain. He had had fracture of the radius and ulna after a fall but was otherwise healthy. Radiographs at that time revealed osteopenia and widened growth plates suggestive of “early rickets.” His physical examination revealed a normal child at the 10th centile for both height and weight. A DXA scan revealed a lumbar spine BMD Z score of -1.9 (at age 5). His father, C/II-2, age 37, developed renal stones at age 32, and reported intermittent symptoms, but was otherwise healthy. His labs were: Ca 2.38 mmol/L (2.13-2.63), P 1.03 mmol/L (0.87-1.45), Crea 88.14 mmol/L (70.512- 114.58), iPTH 12.94 pmol/L (5.30-34.99), 1,25(OH)2D 135.20 pmol/L (52-163.80), Urine Ca/Crea 0.33 mol/mol (<0.66), TmP/GFR at 0.86 mmol/L (0.89-1.08), TRP of 85% (>90%). The mother, C/II-1, age 32 years was healthy. Her biochemical findings upon presentation were: Ca 2.45 mmol/L (2.13-2.63), P 1.13 mmol/L (0.87-1.45), Crea 70.51 mmol/L (52.88-96.95), iPTH 13.47 pmol/L (5.30-34.99), 1,25(OH)2D 67.60 pmol/L (52-163.80), U-Ca/U-Crea 0.36 mol/mol (<0.66), TmP/GFR 1.08 mmol/L (0.89-1.08), and a TRP of 90% (>90%). His sister, C/III-2, 3 years of age, carried a diagnosis of juvenile rheumatoid arthritis, although this was mild. She had one episode of hematuria but no evidence of renal stones. Her biochemical findings upon presentation were: serum Ca 2.43 mmol/L (2.13-2.63), serum P 1.39 mmol/L (1.39-1.74), Crea 44.07 mmol/L (8.81-35.26), iPTH 11.03 pmol/L (5.30-34.99), 1,25(OH)2 vitamin D of 122.20 pmol/L (52-163.8), urinary Ca/Crea of 0.46 mol/mol (<0.66), a TmP/GFR of 1.42, and a TRP of 92% (>90%). A younger sister (1 yr of age) was reportedly normal. A paternal grandmother had a history of kidney stones. No further information is available. The index case of kindred D (Table 2 B), D/I-1, first presented at age 27 years 6 months to one of us. Her medical history started at 12 months of age when she developed bilateral genua valga and rickets, which required several years of bracing both lower extremities to prevent further valgus deformities. At age 11 she sustained a hairline fracture of the left elbow as well as a greenstick fracture of the right wrist. No laboratory studies are available from until age 16.6 years, when her uCa/Crea was elevated to 1.38 mol/mol (<0.74) and TRP was 88% (<90). At presentation age 27 she reportedly had asymptomatic hypercalciuria, P was reduced to 0.78 mmol/L (0.81-1.45) and 1,25(OH)2 was elevated at 228 pmol/L (35-140). All other biochemical findings were normal: Ca 2.29 mmol/L (2.12-2.54), Crea 51 µmol/L (60-124), 25(OH)D 48.6 nmol/L (40-130), iPTH 0.74 pmol/L (1.06-5.83), ALP 240 (U/L) (50-276). Physical examination at age 27 showed a height of 157.5 cm, a slightly enlarged thyroid gland but was otherwise nor-

3 mal. There is no family history of rickets or any other bone disease. Follow up biochemical findings at age 41 off treatment with phosphate supplements showed hypophosphataemia 0.73 mmol/L (0.80-1.50) and low 25(OH)D of 72.2 nmol/L (80.0-200.0), hypercalciuria of 9.48 mmol/d (2.50-7.50), uCa/Crea 0.92 mol/mol (<0.45) and TRP 58%, while the remaining biochemical findings were normal: Ca of 2.51 mmol/L (2.10-2.55), iPTH 0.74 pmol/L (1.1-5.81), 25(OH)2D 123 pmol/L (55-190), ALP 56 U/L (30-115). The proband’s son, D/II-2, age 16 months, H 78.5 cm (just below 50th percentile), W 9.1 kg (<3rd percentile, but less than 1 standard deviation below) suffers from Poland Syndrome (acquired hypoplasia of the pectoralis mus- cle) but is otherwise asymptomatic with respect to skeletal development and there is no history of renal stones. His available biochemical findings include Ca 2.42 mmol/L (2.20-2.60), P 1.53 mmol/L (1.39-1.74), a low Crea of 12 µmol/L (20-60), ALP 194 U/L (40-390), a depressed 25(OH)D of 46.2 nmol/L (80-200), 1,25(OH)2D 172 pmol/L (55-190). The proband’s daughter, D/II-1 was likewise well until age 9 years and renal or bone malformations are absent, but further studies are unavailable. The index case of kindred E (Table 2 B), E/II-2, presented at age 6 with kidney stones and nephrocalcinosis. At age 131/12 years his H: 155 cm, W: 48 kg (50-75th percentile for weight and th 25-50 percentile for height) His u-Ca/Crea ratio was 1.03 mol/mol (<0.74) and 1,25(OH)2D 437 pmol/L (26-156) were elevated, while the remaining biochemical findings included normal Ca 2.33 mmol/L (2.20-2.60), P 1.1 mmol/L (1.29-1.68), Crea 55 umol/L (35.26-61.70), 25(OH)D 119 nmol/L (25-125), iPTH 0.63 pmol/L (1.06-6.89), ALP 966 U/L (150-900), TmP/GFR 1.18 (0.77-1.29), and TRP was 107% (>90). Ultrasound and CT scan of his kidneys confirmed nephrocalcinosis, a 1.5 cm large cyst in the left kidney. On phosphate supplements (300-600 mg 5 times daily) he had no further episodes of kidney stones, and his DEXA-scan, which reportedly showed low bone density initially, but normalized 4 years after start with oral phosphate (Z-score -0.1 for spine and hip). At age 24 he developed gallstone pancreatitis, which resolved after cholecystectomy and papillotomy of the pancreatic duct. The proband’s mother, E/I-1, age 53 years, H 174 cm, W 76.9 kg, has a history of kidney stones/nephrocalcinosis, rheumatoid arthritis, Sjögren Syndrome, hyperparathyroidism and thyrotoxicosis and underwent thyro-parathyroidectomy. Her biochemical findings were within normal limits including Ca 2.14 mmol/L (2.20-2.60), P 1.1 mmol/L (0.6-1.5), Crea 74 mmol/L (52.88-96.95), iPTH 1.27 pmol/L (1.06-6.89), ALP 240 U/L (50-276), u-Ca/Crea ratio 0.50 mol/mol (<0.45), 25(OH)D 62 nmol/L (25-125), 1,25(OH)2D 104 pmol/L (26-156), TmP/GFR was 0.9 (0.77-1.29), and TRP was 90% (>90%). The proband’s father, E/I-2, age 56 years, H 175 cm, W 80 kg, is healthy, with no renal abnormalities, bone pain, or fractures. Ca 2.22 mmol/L (2.20-2.60), P 0.8 mmol/L (0.6-1.5), Crea 103 U/L (70.51-114.58), iPTH 3.92 pmol/L (1.06-6.89), ALP 210 U/L (50-276), u-Ca/Crea ratio 0.11 mol/mol (<0.45). 25(OH)D 63 nmol/L (25-125), 1,25(OH)2D 146 pmol/L (26-156), TmP/GFR was 0.7 (0.77-1.29), and TRP was 87% (>90%). The proband’s sister, E/II-1, H 166.5 cm, W 59.2 kg, has malignant melanoma diagnosed age 24, no history of kidney stones, rickets, bone pain, or fractures. Her biochemical findings were normal Ca 2.58 mmol/L (2.20-2.60), P 1.0 mmol/L (0.6-1.5), Crea 72 mmol/L (52.88-96.95), iPTH 0.45 pmol/L (1.06-6.89), ALP 510 U/L (150-900), u-Ca/Crea 0.67 mol/mol (<0.74). 25(OH)D 99 nmol/L (25-125), 1,25(OH)2D 119 pmol/L (26-156), TmP/GFR was 0.77 (0.77-1.29), and TRP was 77%(>90??%). The proband’s paternal uncle has recurrent kidney stones. Also her paternal great-grandfather (deceased at 93 years) had kidney stones.

Case F presented at age 6 with pyelonephritis, subsequent evaluation showed hypercalciuria 6- 10 mg/Kg/day, low P 1.11 mmol/L (1.16-1.91), but normal Ca 2.57 mmol/L (2.40-2.65), Crea 33.49 umol/L (17.63-44.07), PTH 1.38 pmol/L (1.60-6.90). Renal ultrasound showed grade 2-3 nephrocalcinosis. Therapy at the time consisted in oral potassium citrate (Shohl’ssolution). At age 6.7 her height was 118.4cm (P43), weight 20.7 kg (P28), at the time laboratory studies showed Crea 44.07 umol/L (17.63-44.07), P 1.23 mmol/L (1.16-1.91), S-Ca 2.6 mmol/l (2.40- 2.65), 25(OH)D 24.25 nmol/L (62.4-199.68), 1,25(OH)2-D 174 pmol/L (62.4-223.6), PTH 0.4 pmol/L (1.60-6.90), u-Ca/Crea 1.51-1.56 mol/mol (<0.85), uOxalat/Crea 50mg/g (24-56), uCitrat/Crea 1905mg/g (>180). At this age hydrochlorothiazide 12.5 mg bid was added. Repeat laboratory studies showed iCa 1.26 mmol/L (1.15-1.33), Ca 2.5-2.6 mmol/L (2.40-2.65), P 1.09- 1.29 mmol/L (1.16-1.91), u-Ca/Crea 1.11 mol/mol (<0.85), c-terminal FGF23 (cFGF23) 21-28 kRU/l (26-110), 25-OH-D 65 nmol/L (62.4-199.68), 1,25-OH2-D 209 pmol/L(62.4-223.6), PTH 0.74 pmol/L (1.60-6.90). Her mother was found to have hypercalciuria 4.7 mg/kg/d (<4) but no evidence was seen for nephrocalcinosis on ultrasound. Father and sister had no laboratory abnormalities and there was no family history for symptomatic nephrolithiasis. There is, however, a family history of osteoporosis in mother`s mother, mother`s aunt and their mother (mothers grandmother). Case G presented age 11 with renal colic due to a right urethral-bladder junction stone. Her weight was 30.7kg (P21), height 142.5cm (P58). Subsequent ultrasonographic evaluation showed grade 2-3 nephrocalcinosis, Ca 2.63mmol/l (2.40-2.65), P 1.26 mmol/L (1.16-1.91), Crea 88.14 umol/L (35.26-61.69), suppressed iPTH <0.3 pmol/L (1.60-6.90), 25-OH-D 45 nmol/L (62.4- 199.68), elevated 1,25-OH2-D 283 pmol/L (62.4-223.6) and uCa/Crea 0.86 (<0.85). A 24h-urine collection showed a Ca excretion of 3.8 mmol (1-7), uOxalate/Crea 33 mg/g (24-56), uCitrate/Crea 417 mg/g (>180). Treatment consisted of lithotripsy and pig-tail placement. Stone analysis showed calcium-oxalate (95% Weddellit, 5% Whewellit). Subsequently her renal function improved. Repeat laboratory analyses showedCa 2.6 mmol/l (2.40-2.65), P 1.22 mmol/L (1.16-1.91), Crea 70.5 umol/L (35.26-61.69). However, she continued to have elevated uCa/Crea 1.48 (<0.85) and low TmP/GFR = 1.17 mmol/L (1.22-1.61). Since she had low U- citrate 183mg/l, therapy with 8.8 mmol potassium citrate bid was started and laboratory studies three months later showed: Ca 2.6 mmol/l (2.40-2.65), P 1.19 mmol/L (1.16-1.91), Crea 70.5 umol/L (35.26-61.69), ALP 358 U/l (215-476), suppressed iPTH 0.63 pmol/L (1.60-6.90), cFGF23 was 28-33kRU/l (26-110), 25-OH-D 56.75 nmol/L (62.4-199.68), 1,25-OH2-D 153 pmol/L (62.4-223.6), uCa/Crea 1.44 (<0.85), uOxalate/Crea 28 mg/g (24-56), uCitrate/Crea 666 mg/g (>180), TmP/GFR = 1.04 mmol/L (1.22-1.61). Laboratory followup 12 months later showed: Ca 2.5mmol/l (2.40-2.65), P 1.19 mmol/L (1.16-1.91), Crea 79.3 umol/L (35.26-61.69), ALP 307 U/l (178-526), iPTH 1.47 pmol/L (1.60-6.90), 25-OH-D 33.75 nmol/L (62.4-199.68), 1,25-OH2-D 153 pmol/L (62.4-223.6), bone age was 11 ½ years and ultrasound showed un- changed nephrocalcinosis. Her family history was negative for nephrolithiasis. A maternal second degree cousin reportedly had vitamin D deficient rickets. Nucleotide sequence analysis of the SLC34A3/NPT2c gene of kindreds C-E, and case F Sequence analysis of five additional cases with HHRH or IH revealed previously described mutations in SLC34A3/NPT2c (see detailed case descriptions in Supplemental Results and Supple- mental Fig. S-1). Three heterozygous sequence variations were found in the proband of kindred C (C/III-1): c.413C>T(p.S138F), c.1304delG, c.1579_81del(p.L527del). The frameshift mutation

5 c.1304delG was inherited from the father, while c.413C>T(p.S138F) was inherited from the mother. Both parents carry the deletion c.1579_81del(p.L527del); c.1579_81del(p.L527del) and c.413C>T(p.S138F) were previously described in an unrelated kindreds with HHRH by us 1 and c.413C>T(p.S138F) was subsequently annotated as a rare single nucleotide polymorphism (rs141734934, MAF 0.001 in 4352 chromosomes)) in dbSNP 2. c.1304delG was also found in an unrelated individual 3 and introduces 45 novel amino acids followed by premature termination of the NPT2c protein after residue p.A434 (TPQTGCSAPCRSPSSTSSST- WPASCCGTWCLHCGCPSRWPGTSGW-Stop). Additional genotype information of the index cases C/II-1 includes a novel heterozygous single nucleotide polymorphism c.558G>A(p.Q186Q). Proband D/I-1 was found to carry a homozygous missense mutation, c.1402C>T (p.R468W), which was previously described in a compound heterozygous HHRH case 4. Proband E/II-2 was found to be homozygous for a previously described mutation c.575C>T (p.S192L) 1, 4 that she inherited from her two parents. Case F was found to carry two known SLC34A3/NPT2c mutations: c.575C>T (p.S192L) 1, 4 and c. 1093+41_1094-15del (g.2615_2699del) 5.

Supplemental Figure S-1A: Clinical and biochemical characteristics of kindreds A, B Supplemental Figure S-1B: Clinical and biochemical characteristics of kindreds C-G. Laboratory findings in patients with hereditary hypophosphatemic rickets with hypercalciuria (HHRH) or idiopathic hypercalciuria (IH) caused by novel SLC34A3 mutations. All abnormal values are shown in bold. Circles denote females, squares denote men. Black symbols indicate affected individuals, who presented with hypophosphatemia, hypercalciuria, elevated 1,25(OH)2 vitamin D levels, and skeletal findings consistent with rickets, while gray symbols indicate affected individuals, who had one or more of the above biochemical abnormalities and nephrocalcinosis. Open symbols indicate healthy individuals. Abbreviations are as follows: ALP, alkaline phosphatase; uCa/Cr, urinary calcium/creatinine ratio; PTH; parathyroid hormone; TmP/GFR, maximum tubular phosphate reabsorption per glomerular filtrate; TRP, tubular phosphate reabsorption; 1,25(OH)2D, 1,25(OH)2 vitamin D; 25(OH)D, 25(OH) vitamin D. The normal range for TRP is >90% in the setting of hypophosphatemia. Supplemental Figure S-2: Biochemical and renal phenotype of frequent SLC34A3/NPT2c mutations.

Legend: Representation of biochemical and renal phenotype (1=nephrocalcinosis, 2=stones, 3=nephrocalcinosis and stones) of individual mutations, which were observed in two or more individuals affecting one allele (het) or both alleles (hom). See Methods and legends of Fig. 2 and 3 for abbreviations, normalization and RVs of biochemical data and Table S-1 for the data table. Supplemental Table S-1: SLC34A3/NPT2c mutations and nucleotide sequence variants of of the index cases of kindreds A and B, and of case G

Sample Exon/ Flanking Sequence Nomenclature dbSNP/1000 primers Kit AT Band Intron genome size °C (bp), detection

A/II-1 Intron ccccctg- Hom.c.1336- rs28368709 12 gaacccac(g/c)ctcgttcttcttg 549G>C cc

Exon TTCTTCAAC- Hom.c.1369G>A Novel mis- 52, 276 Ex.1 55 267, 13 CTGGCC(G/A)GCATC (p.G457S) sense muta- seq. CTGCTGTGGT tion

Intron GAcgggcagtt- Hom.c.1800+14A> rs28591989 13 gctg(a/c)gcagaccgccccac C c

B/II-2 5'UT ctcccgcccc(g/a)tgtctcctcc Hom.c.1-804G>A rs9777338 R

Intron tgtgggtg- Het.c.560+27_561- Known 734- Ex.1 55 208+17 6 gaagggctgggc(tggggctgca 38del intronic 735 9 bp gtgg- (g.1440-1469del) deletion 6 cagccccagcccgggc/del tggggctgcagtgg- cagccccagcccgggc)ccccc cacct

Intron ccccctg- Hom.c.1336- rs28368709 12 gaacccac(g/c)ctcgttcttcttg 549G>C cc

Exon ATCCAC- Het.c.1357delTTC Novel mi- 41-276 Ex.1 55 571, 13 TTCTTC(TTC/ del- (p. F453del) crodeletion seq. TTC)AACCTGGCC

Exon TGGCAGGGGG- Hom.c.1538A>T rs28542318 13 CATGG(A/T)GCTGGC (p.E513V) CGCTGTCGG

3'UT Gcagtt- Hom.c.1800+14A> rs28591989 R gctg(a/c)gcagaccgcc C

Case G GTGGCTG- Het.c.367delC Novel dele- GACTGGT(C/delC)ATT tion GGCGTGCTGGTC

CTTTCAGCGGCT(C/T) Het. Known GGCGGTGCACGG- c.575C>T(p.S192L) missense GAT mutation 4.

Supplemental Table S-2: Review of current and previously published HHRH kindreds. Legend: Comprehensive table of all the available information collated from published and unpublished cases of HHRH and their families. Conversion into SI units is given where mass units where initially reported. Conversion factors were taken from www.endmemo.com/medical/unitconvert/, with the following exceptions: PTH conversion factor was taken from www.parathyroid-forum.co.uk/, and the TmP-GFR conversion factor was taken from www.mayomedicallaboratories.com/test-catalog/Clinical+and+interpretive/. For Ca/Crea, where the Mayo Clinic RVs (www.mayomedicallaboratories.com/test- catalog/Clinical+and+interpretive/) were used after conversion to SI using the factor 0.25/0.07626 (=3.278) was used to convert these to SI units where the paper presents the biochemical data in mass units. Where age-specific RVs were missing from the papers and for Braithwaite et al. 7, the Mayo Clinic age-adjusted RVs were used. For TmP/GFR, the RVs from the Alon & Hellerstein paper (1994) were used when not provided in the original publication. The daughter of index II-1 was subsequently genotyped [c.145C>T(p.Q49*)], and her biochemical lab values were included in the systematic meta-analysis 8.

Supplemental Table S-3: Clustal-W sequence alignment of novel SLC34A3 mutations (c.1357delTTC (p.F453del); c.G1369A (p.G457S)).

(p.F453del) c.1357delTTC

(p.G457S) c.G1369A

ENSOGAP00000000671/1-638 YPLTLGSNIGT-TTTAILAALASPR--EKLSSSFQIALCHFFFNISGI-LWYPVPCTR- ENSMGAP00000013100/1-674 YPLTLGANIGT-TTTAILAALASPG--STLKYSLQIALCHFFFNVSGI-ILFYPLPFTR- ENSP00000442397/1-599 YPLLLGSNIGT-TTTALLAALASPA--DRMLSALQVALIHFFFNLAG-ILLWYLVPALR- ENSAMEP00000019187/1-641 YPLTLGSNIGT-TTTAILAALASPR--EKLSSSFQIALCHFFFNISGI-LLWYPVPCTR- ENSACAP00000006271/1-622 YPLTLGSNMGT-TTTAILAALASPG--EKLASAFQIALCHFFFNLSGI-LLWYPLPCSR- ENSOANP00000015354/1-519 YPLTLGSNIGT-TTTAILAALASPG--DKLASAFQVGRCHFFFNISGI-LLWYPLPCTR- ENSCAFP00000028785/1-591 YPLFLGSNIGT-TTTALLAALASPA--DMLLSALQVALIHFFFNLAG-ILLWYVVPVLR- ENSCAFP00000024164/1-694 YPLTLGSNIGT-TTTAILAALASPG--NTLRSSLQIALCHFFFNISGI-LLWYPIPFTR- ENSCPOP00000003644/1-638 YPLTLGSNIGT-TTTAILAALASPR--EKLSSSFQIALCHFFFNISGI-LLWYPLPCMR- ENSMEUP00000010653/1-640 YPLTLGSNIGT-TTTAILAALASPR--EKLSSAFQIALCHFFFNISGI-LLWYPLPCTR- ENSGACP00000002260/1-595 YPLTLGSNIGT-TTTAILAAMASPG--ETLANSLQIALCHFFFNIMGI-LLWYPIPFTR- ENSMODP00000038338/1-529 YPLILGSNIGT-STITILAALSGPR--NALRKSFQVTLCHFLFNLFG-IILWYPIPGMR- ENSPTRP00000027486/1-690 YPLTLGSNIGT-TTTAILAALASPG--NALRSSLQIALCHFFFNISGI-LLWYPIPFTR- ENSEEUP00000007494/1-562 YPLFLGSNIGT-TTTAVXXXXXXXX--XXXXXXXXVALIHFFFNLAG-ILLWYVVPVLR- ENSFALP00000009130/1-620 YPLTLGSNIGT-TTTAILAALASPG--DKLASSFQIALCHFFFNISGI-LLWYPLPFTR- ENSPVAP00000007920/1-601 YPLLLGSNVGT-TTTALLAALASPS--RTLHFAIQVALIHFFFNVTG-IVLWYVVPVLR- ENSMGAP00000004094/1-668 YPLTLGSNIGT-TTTAILAALASPG--DKLASSFQIALCHFFFNISGI-LLWYPLPFTR- ENSTBEP00000007042/1-305 YPLFLGSNIGT-TTTALLAALASPS--AMLPSAVQVALIHFFFNLAG-IVLWYVVPVLR- ENSMPUP00000012027/1-639 YPLTLGSNIGT-TTTAILAALASPR--EKLSSSFQIALCHFFFNISGI-LLWYPVPCTR- ENSXMAP00000017507/1-643 YPLSLGSNIGT-TTTAILAAMASPG--DRLANALQIALVHFLFNISGI-ILWYPIPFTR- ENSCJAP00000012722/1-639 YPLTLGSNIGT-TTTAILAALASPR--EKLSSAFQIALCHFFFNISGI-LLWYPVPCTR- ENSSHAP00000005319/1-697 YPLTLGSNIGT-TTTAILAALASPG--STLHNSLQISLCHFFFNVSGI-ILWYPVPFMR- ENSDARP00000123811/1-525 YPLTLGSNIGT-TTTAILAGLASPG--DKLAAAFQVALCHFIFNILGI-LLWYPVPHTR- ENSTRUP00000004594/1-118 ------ENSMLUP00000019963/1-603 YPLTLGANIGT-TTTAIMAALASPG--KTLRSSLQIALCHFFFNITGI-LLWYPVPFTR- ENSPTRP00000043446/1-349 YPLLLGSNIGT-TTTALLAALASPA--DRMLSALQVALIHFFFNLAG-ILLWYLVPALR- ENSGACP00000021060/1-609 YPLSLGSNIGT-TTTAILAAMASPG--DTLGNALQIALVHFLFNISGI-ILWYPIPFTR- ENSCINP00000017272/1-603 YPLTLGANIGT-TTTSILAALAQ----DEIANALQIAFCHFFFNIFG-IIIWYPIPFMRN ENSOANP00000010689/1-696 YPLTLGSNIGT-TTTAILAALASPG--STLKNSLQISLCHFFFNISGI-LLWYPIPFTR- ENSMODP00000021461/1-621 YPLFLGSNIGT-TTTALLAALASPA--DMLLSAVQVSLIHLFFNLAG-ILLWYLVPTLR- ENSP00000321424/1-639 YPLTLGSNIGT-TTTAILAALASPR--EKLSSAFQIALCHFFFNISGI-LLWYPVPCTR- ENSPPYP00000016368/1-690 YPLTLGSNIGT-TTTAILAALASPG--NALRSSLQIALCHFFFNISGI-LLWYPIPFTR- ENSGMOP00000006752/1-564 YPLTLGSNIGT-TTTALLAALASPG--DQLAPALQIALCHLFFNVLGI-LLWYPIPATR- ENSPCAP00000005941/1-683 YPLTLGSNIGT-TTTSILAALASPG--NTLKSSLQIALCHFFFNISGI-LLWYPIPFTR- ENSPVAP00000008764/1-701 YPLTLGANIGT-TTTAIMAALASPG--NTLKSSLQIALCHFFFNITGI-LLWYPIPFTR- ENSETEP00000011673/1-591 YPLFLGSNIGT-TTTALLAALASPS--NMLLDALQVALIHFFFNVAG-ILLWYVVPALR- ENSCINP00000011241/1-583 YPLTLGANIGT-TTTGIFTALASEP--ERLEYSLQLALVHLFFNISG-ILLWYPIPLMRR ENSTGUP00000009608/1-675 YPLTLGANIGT-TTTAILAALASPG--STLKYSLQIALCHFFFNVSGI-ILFYPIPYTR- ENSDORP00000008855/1-593 XXXXXXXXXXX-XXXXXXXXXXXXX--XXXXXXXXVALIHFFFNLAGI-LLWYTVPVLR- ENSMICP00000013235/1-391 ------ENSMICP00000015680/1-671 YPLTLGSNIGT-TTTAILAALASPG--NTLRSALQXXXXXXXXXXXXX-XXXXXXXXXX- ENSNLEP00000011130/1-551 YPLLLGSNIGT-TTTALLAALASPA--DRMLSALQVALIHFFFNLAG-ILLWYLVPALR- ENSGGOP00000027403/1-689 YPLTLGSNIGT-TTTAILAALASPG--NALRSSLQIALCHFFFNISGI-LLWYPIPFTR- ENSXMAP00000008745/1-651 YPLTLGSNIGT-TATALLAALASPG--NKIVASIQIALCHLLFNVFGI-LLWYPVPFMR- ZK563.2/1-573 YPLVLGSNIGT-TFSGVLAAFSTDP--SRFEKALHMAMCQVIYNIIG-TCLFYIVPCTRK

ENSECAP00000000385/1-541 YPLFLGSNIGT-TTTALLAALASPA--DMLLSAVQGALSSFLFGSPSSLPQGAPVP---- ENSMLUP00000008643/1-639 YPLTLGSNIGT-TTTAILAALASPR--EKLSSAFQIALCHFFFNISGI-LLWYPVPCTR- ENSSHAP00000011947/1-666 YPLTLGSNIGT-TTTAILAALASPG--STLRNSLQIALCHFFFNVSGI-LLWYPVPFTR- ENSSHAP00000012725/1-215 ------ENSCJAP00000024604/1-689 YPLTLGSNIGT-TTTAILAALASPG--SSIKSSLQIALCHFFFNISGI-LLWYPIPFTR- ENSMUSP00000059138/1-637 YPLTLGSNIGT-TTTAILAALASPR--EKLSSSFQIALCHFFFNISGI-LLWYPLPCTR- ENSDARP00000103592/1-681 YPLTLGSNIGT-TTTAILAAMASPG--ETLANSLQISLCHFFFNIAGI-LLWYPIPFTR- ENSDNOP00000007326/1-478 XXXXXXXXXXX-XXXXXXXXXXXPG--NTLKSSL------ENSDARP00000023797/1-631 YPLSLGSNIGT-TTTAILAAMASPG--ETLGNSLQIALVHLFFNLSGI-LLWYPIPITR- ENSMODP00000038321/1-518 YPLILGSNIGT-TTTAILAALASPA--STLQDSLQIALCHLFFNIFG-VLLWYPIPFLR- ENSSTOP00000011627/1-646 YPLTLGSNIGT-TTTAILAALASPG--NTLKSSLQIALCHFFFNISGI-LLWYPIPFTR- ENSORLP00000020360/1-639 YPLTLGSNIGT-TTTSILAAMASPG--ETLSNSLQIALCHFFFNIFGI-IIWYPLPFMR- ENSCSAVP00000015160/1-583 YPLTLGANIGT-TTTGIFTALASEP--ARLEYSLQLALVHLFFNLSG-IVLWYPVPILRR ENSLAFP00000018716/1-695 YPLTLGSNIGT-TTTSILAALASPG--NTLKSSLQIALCHFFFNVSGI-LLWYPIPFTR- ENSTGUP00000015042/1-368 YPLTLGANIGT-TTTAILAALASPG--STLKYSLQIALCHFFFNVSGI-ILFYPIPYTR- ENSCSAVP00000010664/1-548 YAVTVGANMGT-TLTAVLAALATGN-----SNALQLAFCHFFFNING-FLLWYPLPFMRR ENSTTRP00000005760/1-637 YPLTLGANIGT-TTTAILAALASPG--STLKSSLQIALCHFFFNISGI-LLWYPIPFTR- ENSPTRP00000030034/1-521 YPLTLGSNIGT-TTTAILAALASPR--EKLSSAFQIALCHFFFNISGI-LLWYPVPA-R- ENSOGAP00000005733/1-689 YPLTLGSNIGT-TTTAILAALASPG--NTLRSSLQIALCHFFFNVSGI-LLFYPIPFTR- ENSGACP00000023831/1-585 YPLTLGSNMGT-TATALLAALASPG--NKLAAAMQIAFCHLFFNVFGI-LLWYPLPVTR- ENSMEUP00000000243/1-614 YPLTLGSNIGT-TTTAILAALASPG--NTLQSSLQIALCHFFFNITGV-ILWYPVPYMR- ENSONIP00000016391/1-665 YPLTLGSNIGT-TATALLAALASPG--NKLAAAIQIALCHLFFNVSGI-LLWYPLPFMR- ENSXETP00000050672/1-599 YPLFLGSNIGT-TTTAVLAALASPA--DALSNSVQVAFIHLFFNLTG-LVLWYVVPFLR- ENSMODP00000005525/1-655 YPLTLGSNIGT-TTTAILAALASPR--EKLSSAFQIALCHFFFNISGI-LLWYPIPCTR- ENSFCAP00000024487/1-693 YPLTLGSNIGT-TTTAILAALASPG--NTLRSSLQIALCHFFFNISGI-LLWYPIPFTR- ENSONIP00000003907/1-642 YPLTLGSNIGT-TTTSILAAMASPG--ETLDNSLQIALCHFFFNIFGI-VLWYPIPFMR- ENSFCAP00000001426/1-639 YPLTLGSNIGT-TTTAILAALASPR--EKLSSAFQIALCHFFFNISGI-LLWYPVPCTR- ENSMUSP00000006638/1-601 YPLFLGSNIGT-TTTALLAALASPA--DMLIFAVQVALIHFFFNLAG-ILLWYLVPVLR- ENSAMEP00000017332/1-602 YPLFLGSNVGT-TTTALLAALASPA--DMLLSALQVALIHFFFNVAG-ILLWYVVPILR- ENSGGOP00000009414/1-639 YPLTLGSNIGT-TTTAILAALASPR--EKLSSAFQIALCHFFFNISGI-LLWYPVPCTR- ENSCPOP00000008789/1-279 ------ENSLAFP00000009867/1-671 YPLTLGSNIGT-TTTSILAALASPG--NTMKSSLQIALCHFFFNVSGI-LLWYPIPLTR- ENSDARP00000123760/1-645 YPLTLGSNIGT-TTTAILAALASPK--EKLPAACQIALCHFYFNIFGI-LLWYPFPLTR- ENSRNOP00000050889/1-695 YPLTLGSNIGT-TTTAILAALASPG--NTLRSSLQIALCHFFFNISGI-LLWYPIPFTR- ENSSTOP00000007510/1-639 YPLTLGSNIGT-TTTAILAALASPK--EKLSSSFQIALCHFFFNISGI-LLWYPLPCTR- ENSPPYP00000022223/1-395 ------ALQVALIHFFFNLAG-ILLWYLVPALR- ENSONIP00000022669/1-655 YPLCLGSNIGT-TTTAILAAMASPG--ETLADALQIALVHFLFNISGI-ILWYPIPFTR- ENSCSAVP00000017377/1-567 YPLTLGSNIGT-TTTSILAALAQ----DEIANSLQIAFCHFFFNIFG-IAIWYPIPFMRN ENSMLUP00000000634/1-696 YPLTLGANIGT-TTTAIMAALASPG--KTLRSSLQIALCHFFFNITGI-LLWYPIPFTR- ENSFCAP00000024839/1-589 YPLFLGSNIGT-TTTALLAALASPA--DMLLSALQVALIHFFFNLAG-ILLWYVVPVLR- ENSSHAP00000010351/1-373 YPLTLGSNIGT-TTMAILAALASPG--STLEEFLQIALCHFFFNLSGV-LLWY-VPFMR- ENSP00000371483/1-690 YPLTLGSNIGT-TTTAILAALASPG--NALRSSLQIALCHFFFNISGI-LLWYPIPFTR- ENSCINP00000017268/1-531 YPLTLGANIGT-TTTGIFAALASDP--DRLEYSLQIALVHLFFNVSG-IALWYPVPFLRR ENSLAFP00000011155/1-512 YPLTLGSNIGT-TTTAILAALASPG--NTLKSSLQIALCHFFFNVSGI-LLWYPIPIIR- ENSMMUP00000027823/1-690 YPLTLGSNIGT-TTTAILAALASPG--NTLRSSLQIALCHFFFNISGI-LLWYPIPFTR- ENSECAP00000014904/1-639 YPLTLGSNIGT-TTTAILAALASPR--EKLSSAFQIALCHFFFNISGI-LLWYPVPCTR- ENSLACP00000014496/1-661 YPLTLGSNIGT-TTTAILAAMASPG--ETLTNSLQIALCHFFFNISGI-LLWYPVPITR- ENSDORP00000004969/1-471 YPLTLGSNIGT-TTTAILAALASPR--EKLSSSFQ------ENSDNOP00000010295/1-484 YPLTLGSNIGT-TATAILAALTSPP--EKLASAFQIALCHFFFNISGI-LLWYPVPCTR- ENSCPOP00000016205/1-682 YPLTLGSNIGT-TTTAIMAALASPG--NTLISSIQIALCHFFFNISGI-LLWYPIPFTR- ENSOGAP00000005297/1-598 YPLFLGSNIGT-TTTALLAALASPA--DMMLSAVQVALIHFFFNLAG-ILLWYLVPALR- ENSAMEP00000019136/1-693 YPLTLGSNIGT-TTTAILAALASPG--NALRSSLQIALCHFFFNISGI-ILWYPIPFTR- ENSORLP00000006343/1-600 YPLTLGSNIGT-TATALLAALASPG--NKLAAAIQIALCHLFFNIFGI-LLWYPIPFMR- ENSCINP00000022041/1-570 YPLTLGANIGT-TTTSILAALAQ----DEIANALQIAFCHFFFNIFG-IIIWYPIPFMRN ENSACAP00000014198/1-675 YPLTLGSNIGT-TTTAILAALASPG--SRLKYSLQIALCHFFFNISGI-LLWYPIPFMR- ENSMMUP00000026048/1-639 YPLTLGSNIGT-TTTAILAALASPK--EKLFSAFQIALCHFFFNISGI-LLWYPVPCTR- ENSEEUP00000009764/1-639 XXXXXXXXXXX-XXXXXXXXXXXXX--XXXXXXXXIAFCHFFFNISGI-LLWYPVPCLR- ENSGALP00000023177/1-674 YPLTLGANIGT-TTTAILAALASPG--STLKYSLQIALCHFFFNVSGI-ILFYPLPFTR- ENSGMOP00000012872/1-556 YPLTLGSNIGT-TTTAILAAMAAPG--ETLANSLQIALCHFFFNIMGI-MLWYPIPFTR- ENSVPAP00000002189/1-402 YPLTLGANLGT-TTTAILAALASPG--STLRSSIQ------ENSNLEP00000015536/1-600 YPLTLGSNIGT-TTTAILAALASPR--EKLSSAFQIALCHFFFNISGI-LLWYPVPCTR- ENSMODP00000000391/1-706 YPLTLGSNIGT-TTTAILAALASPG--NTLQDSLQIALCHFFFNISGI-LLWYPIPFTR- ENSAPLP00000010975/1-470 YPLTLGSNIGT-TTTAILAALASPG--DKLASSFQIALCHFFFNISGI-LLWYPLPFTR- ENSOCUP00000002559/1-691 YPLTLGSNIGT-TTTAILAALASPG--NTLRSSLQIALCHFFFNISGI-LLWYPIPFTR- ENSTTRP00000014558/1-599 YPLFLGSNIGT-TTTALLAALASPS--DMLISAVQVALIHFFFNLAG-ILLWYVVPVLR-

ENSMUSP00000092380/1-697 YPLTLGSNIGT-TTTAILAALASPG--NTLRSSLQIALCHFFFNISGI-LLWYPIPFTR- ENSPMAP00000003321/1-586 YPLTLGSNIGT-TTTALLAALASPG--ETLNNALQIALCHFFFNISGI-ILWYPIPQLR- ENSFALP00000009572/1-675 YPLTLGANIGT-TTTAILAALASPG--STLKYSLQIALCHFFFNVSGI-ILFYPIPYTR- ENSPCAP00000005454/1-388 XXXXXXXXXXX-XXXXXXXXXXXXX--XXXXXXXXIALCHFFFNISGI-LLWYPIPFTR- ENSMPUP00000016619/1-693 YPLTLGSNIGT-TTTAILAALASPG--NTLRSSLQIALCHFFFNISGI-MLWYPIPFTR- ENSTGUP00000000400/1-502 YPLTLGSNIGT-TTTAILAALASPG--DKLASSFQIALCHFFFNISGI-LLWYPLPFTR- ENSTRUP00000003108/1-212 ----LGSNIGT-TATSVLAAMASPG--DRRANALQISLVHFLFNISGI-LLWYPVPCTR- ENSXMAP00000013891/1-643 YPLTLGSNIGT-TTTAILAAMASTG--DKLGNSLQIALCHFFFNIMGI-LLWYPIPFMR- ENSTNIP00000022970/1-600 YPLTLGSNIGT-TTTSILAAMASPG--ETLANSLQIALCHFFFNIFGI-VIWYPLPFMR- ENSRNOP00000031764/1-637 YPLTLGSNIGT-TTTAILAALASPR--EKLSSSFQIALCHFFFNISGI-LLWYPLPCTR- ENSOCUP00000002416/1-642 YPLTLGSNIGT-TTTAILAALASPR--EKLSSSFQIALCHFFFNISGI-LLWYPLPCTR- ENSSHAP00000009641/1-569 YPLLLGSNIGT-TTTALLAALASPA--DKLLSAVQVSLIHVFFNMAG-ILLWYLVPALR- ENSOPRP00000002451/1-601 YPLTLGSNIGT-TTTAILAALASPR--EKLASAFQIALCHFFFNISGI-LLWYPLPCMR- ENSGALP00000004850/1-651 YPLTLGSNIGT-TTTAILAALASPG--DKLASSFQIALCHFFFNISGI-LLWYPLPFTR- ENSXETP00000063986/1-676 YPLTLGSNIGT-TTTAILAALASPG--ETLQNSVQIALCHFFFNISGI-IIWYPIPFMR- ENSETEP00000004829/1-591 YPLTLGSNIGT-TTTAILAALASPK--EKLPSSVQIALCHFFFNISGI-LLWYPVPCTR- ENSTTRP00000009075/1-626 YPLTLGANIGT-TTTAILAALASPR--EKLSSAFQIALCHFFFNISGI-LLWYPVPCTR- ENSLACP00000002539/1-596 YPLCLGSNLGT-TTTAILAALASPS--TTLNSAIQVALIHLFFNLSG-LALWYVVPILR- ENSSARP00000008226/1-663 YPLTLGSNIGT-TTTAIMAALASPG--NTLRSSLQIALCHFFFNISGI-LLWYPIPFTR- ENSMODP00000000362/1-363 YPLTLGSNIGTTTTTAILAALASPG--NTLQDSLQVRMH------ENSTRUP00000034707/1-643 YPLTLGSNIGT-TTTSILAAMASPG--DTLSDSLQIALCHFIFNIMGI-LIWYPIPFMR- ENSBTAP00000027121/1-639 YPLTLGSNIGT-TTTAILAALASPR--EKLSSAFQIALCHFFFNISGI-LLWYPMPCTR- ENSLAFP00000008447/1-639 YPLTLGSNIGT-TTTAILAALASPR--EKLPSAFQIALCHFFFNISGI-LLWYPVPCTR- ENSOPRP00000001272/1-685 YPLTLGANIGT-TTTAILAALASPG--NTLKSSLQIALCHFFFNVSGI-LLWYPIPFTR- ENSXMAP00000012278/1-531 YPLTLGSNLGT-TGTALLAALASRA--DKLAAATQVALCHFFFNLFGI-LLWYPIPATR- ENSMMUP00000015960/1-600 YPLLLGSNIGT-TTTALLAALASPA--DRMLSALQVALIHFFFNLAG-ILLWYLVPALR- ENSMICP00000009955/1-518 YPLFLGSNIGT-TTTALLAALASPA--DMMLSAVQVALIHFFFNLAG-ILLWYLVPAMR- ENSRNOP00000014060/1-601 YPLFLGSNIGT-TTTALLAALASPA--DTLLFAVQVALIHFFFNLAG-ILLWYLVPVLR- ENSMEUP00000014025/1-569 YPLFLGSNVGT-TTTTLLAALASPA--DMLLSAVQVSLIHLFFNMAGI-LLWYIVPAFR- ENSTRUP00000002317/1-145 ------ENSEEUP00000010001/1-650 YPLTLGSNIGT-TTTAILAALASPG--DTMKSSLQIALCHLSFNVTGL-LLWYPIPFTR- ENSPPYP00000018005/1-616 YPLTLGSNIGT-TTTAILAALASPR--EKLSSAFQIALCHFFFNISGI-LLWYPVPCTR- ENSBTAP00000054408/1-578 VPTTVTIREGH-ASQGA-----QAP--EALPPSLQVALIHFFFNLAG-ILLWYVVPILR- ENSMPUP00000015003/1-602 YPLLLGSNIGT-TTTALLAALASPA--DMLLSALQVALIHFFFNLAG-ILLWYVVPVLR- ENSSSCP00000009335/1-671 YPLMLGANIGT-TTTAILAALASPG--DTLKSALQIALCHFFFNVTGI-VLWYPIPFAR- ENSSHAP00000017419/1-642 YPLTLGSNIGT-TTTAILAALASPK--EKLSSAFQIALCHFFFNISGI-LLWYPLPCTR- ENSPVAP00000013085/1-639 YPLTLGSNIGT-TTTAILAALASPR--EKLSSAFQIALCHFFFNISGI-LLWYPVPCLR- ENSECAP00000011338/1-693 YPLTLGSNIGT-TTTAILASLASPG--NTLKSSLQIALCHFFFNISGI-LLWYPIPFTR- ENSORLP00000005426/1-631 YPLSLGSNIGT-TTTAILAAMASPG--ETLANALQIALVHFLFNISGI-VLWYPIPFTR- ENSPCAP00000007551/1-624 YPLTLGSSIGT-TTTAILASLVSPT--KKLSSAFQIALCHFFYNILGI-LLWYPVPGMC- ENSCSAVP00000015098/1-558 YPLTLGANIGT-TTTGIFTALASEP--ARLEYSLQLALVHLFFNLSG-IVLWYPIPILRR ENSGMOP00000005546/1-607 YPLSLGANIGT-TTTAILAAMASPG--ETLGNALQIALVHFLFNISGI-ILWYPIPFTR- ENSBTAP00000002023/1-693 YPLTLGANIGT-TTTAILAALASPG--STLKSSLQIALCHFFFNISGI-ILWYPIPFTR- ENSNLEP00000020480/1-712 YPLTLGSNIGT-TTTAILAALASPG--NALRSSIQIALCHFFFNISGI-LLWYPIPFTR- ENSGGOP00000027080/1-590 YPLLLGSNIGT-TTTALLAALASPA--DRMLSALQVALIHFFFNLAG-ILLWYLVPALR- ENSAPLP00000007956/1-622 YPLTLGANIGT-TTTAILAALASPG--STLKYSLQIALCHFFFNVSGI-ILFYPLPFTR- ENSTNIP00000008400/1-581 FGLPLGLLPGS-PTLSILLPIYSLYSLSVLSKTSKIALVHFLFNISGI-LLWYPVPFTR- ENSDORP00000015148/1-609 YPLTLGSNIGT-TTTAILAALASPG--NTLKSSLQIALCHFFFNISGI-LLWYPIPFTR- ENSTBEP00000011496/1-366 ------ENSCINP00000001618/1-539 YAVTVGANMGT-TLTAVLAALATGN-----SNALQLAFCHFFFNISG-FVIWYPIPFMRR ENSLAFP00000006358/1-235 YPLFLGSNIGT-TTTALLAALATPS--NMLLSAVQVALIHFLFNLAG-ILLWYVVPALR- ENSPCAP00000004466/1-598 YPLFLGSNIGT-TATALLAALASPA--DMLLSAVQVALIHFLFNLAG-ILLWYTVPALR- ENSSTOP00000012949/1-602 YPLFLGSNIGT-TTTALLAALASPA--DMLLFAVQVALIHFLFNLAG-ILLWYLVPILR- ENSCJAP00000051496/1-446 YPLLLGSNIGT-TTTALLAALASPA--DRIFSALQV------ENSMICP00000004615/1-516 YPLTLGSNIGT-TTTAILAALASPG--NTLRSALQIALCHFFFNISGI-LLWYPIPFTR- ENSCAFP00000024163/1-639 YPLTLGSNIGT-TTTAILAALASPR--EKLSSAVQIALCHFFFNISGI-LLWYPVPCTR- ENSPSIP00000018384/1-676 YPLTLGSNIGT-TTTAILAALASPG--STLKYSLQIALCHFFFNISGI-ILWYPIPFTR-

Supplemental Table S-4: List of primers

Primer Orientation Location Sequence 22 Reverse Intron 12 AGGAGGTCTCAAGGGAGGAGA 32 Forward Intron 4 GAGGGCCAGCCAGGGACA 33 Reverse Exon 13 TCCAGAGAATGGAGCCAGAC 46 Forward Exon 12 CAGGGCTGACCCAGCATC 52 Forward Intron 12 CATCCACTTCTTCTTCAACCTG 53 Reverse Exon 13 TCCAGAGAATGGAGCCAGAC 57 Forward Intron 5 GGGTGTCAGGCTGGCGGC 87 Reverse Exon 7 AGCATGGTGGCTGCTAAGC 276 Reverse Exon 13 CGCCGCTGCAGGACAGTAAC 734 Forward Exon 6 GTGTCAACGTAGGCACATCC 735 Reverse Exon 7 CAGTTGAAGATCCCGTGCAC

REFERENCES

1. Bergwitz, C, Roslin, NM, Tieder, M, Loredo-Osti, JC, Bastepe, M, Abu-Zahra, H, Frappier, D, Burkett, K, Carpenter, TO, Anderson, D, Garabedian, M, Sermet, I, Fujiwara, TM, Morgan, K, Tenenhouse, HS, Juppner, H: SLC34A3 mutations in patients with hereditary hypophosphatemic rickets with hypercalciuria predict a key role for the sodium- phosphate cotransporter NaPi-IIc in maintaining phosphate homeostasis. Am J Hum Genet, 78: 179-192, 2006. 2. Wheeler, DL, Barrett, T, Benson, DA, Bryant, SH, Canese, K, Chetvernin, V, Church, DM, DiCuccio, M, Edgar, R, Federhen, S, Geer, LY, Helmberg, W, Kapustin, Y, Kenton, DL, Khovayko, O, Lipman, DJ, Madden, TL, Maglott, DR, Ostell, J, Pruitt, KD, Schuler, GD, Schriml, LM, Sequeira, E, Sherry, ST, Sirotkin, K, Souvorov, A, Starchenko, G, Suzek, TO, Tatusov, R, Tatusova, TA, Wagner, L, Yaschenko, E: Database resources of the National Center for Biotechnology Information. Nucleic Acids Res, 34: D173-180, 2006. 3. Ichikawa, S, Tuchman, S, Padgett, LR, Gray, AK, Baluarte, HJ, Econs, MJ: Intronic deletions in the SLC34A3 gene: A cautionary tale for mutation analysis of hereditary hypophosphatemic rickets with hypercalciuria. Bone, in press, 2013. 4. Lorenz-Depiereux, B, Benet-Pages, A, Eckstein, G, Tenenbaum-Rakover, Y, Wagenstaller, J, Tiosano, D, Gershoni-Baruch, R, Albers, N, Lichtner, P, Schnabel, D, Hochberg, Z, Strom, TM: Hereditary Hypophosphatemic Rickets with Hypercalciuria Is Caused by Mutations in the Sodium-Phosphate Cotransporter Gene SLC34A3. Am J Hum Genet, 78: 193-201, 2006. 5. Ichikawa, S, Sorenson, AH, Imel, EA, Friedman, NE, Gertner, JM, Econs, MJ: Intronic Deletions in the SLC34A3 Gene Cause Hereditary Hypophosphatemic Rickets with Hypercalciuria. J Clin Endocrinol Metab, 91: 4022-4027, 2006. 6. Yu, Y, Sanderson, SR, Reyes, M, Sharma, A, Dunbar, N, Srivastava, T, Juppner, H, Bergwitz, C: Novel NaPi-IIc mutations causing HHRH and idiopathic hypercalciuria in several unrelated families: Long-term follow-up in one kindred. Bone, 50: 1100-1106, 2012. 7. Braithwaite, V, Pettifor, JM, Prentice, A: Novel SLC34A3 mutation causing hereditary hypophosphataemic rickets with hypercalciuria in a Gambian family. Bone, 53: 216-220, 2013. 8. Page, K, Bergwitz, C, Jaureguiberry, G, Harinarayan, CV, Insogna, K: A patient with hypophosphatemia, a femoral fracture, and recurrent kidney stones: report of a novel mutation in SLC34A3. Endocr Pract, 14: 869-874, 2008.

III I

II IV

Kindred A B Individual A/III-1 A/III-2 A/IV-1 (index) A/IV-2 A/IV-3 A/IV-4 B/II-2 (index) B/II-1 B/I-1 B/I-2 age 34 y, 9 m 29 y, 2 m 9 y, 1 m 7 y 2 y, 4 m 1 y 13 y 15 y adult adult 1.28 1.28 1.31 0.93 ND ion. Ca (mmol/L) (1.10-1.35) (1.10-1.35) (1.10-1.35) (1.10-1.35) ND 2.39 2.31 2.40 2.52 2.41 2.39 2.40 2.42 2.35 2.34 serum calcium (mmol/L) (2.10-2.55) (2.10-2.55) (2.40-2.65) (2.40-2.65) (2.40-2.65) (2.40-2.65) (2.40-2.65) (2.38-2.63) (2.10-2.65) (2.10-2.65) serum phosphate 1.02 0.86 0.77 1.15 0.87 1.26 0.90 1.27 0.95 1.02 (mmol/L) (0.84-1.45) (0.84-1.45) (1.00-1.80) (1.00-1.80) (1.00-1.80) (1.00-1.80) (1.20-1.74) (1.20-1.74) (0.80-1.90) (0.80-1.90) 1.48 1.27 1.06 2.55 1.70 3.30 5.90 6.20 serum iPTH (pmol/L) (1.59-6.89) (1.59-6.89) (1.59-6.89) (1.59-6.89) (1.59-6.89) (1.59-6.89) (1.00-7.00) (1.00-7.00) 55.00 77.00 40.00 19.00 71.00 78.00 73.00 51.00 serum 25(OH)D (nmol/L) (62.40-199.68) (62.40-199.68) (62.40-199.68) (62.40-199.68) (51.00-250.00) (51.00-250.00) (51.00-250.00) (51.00-250.00)

serum 1,25(OH)2D 155.00 130.00 99.00 90.00 419.00 218.00 172.00 149.00 (pmol/L) (48.00-160.00) (48.00-160.00) (48.00-160.00) (48.00-160.00) (60.00-158.00) (60.00-158.00) (60.00-158.00) (60.00-158.00) 92.00 59.00 39.00 44.00 24.00 31.00 60.00 75.00 90.00 75.00 serum creatinine (µmol/L) (59.00-104.00) (45.00-84.00) (33.00-68.00) (33.00-56.00) (20.0-42.00) (20.00-65.00) (30.50-61.01) (38.13-68.63) (61.01-99.14) (61.01-99.14) 0.19 0.38 0.86 0.65 1.35 0.52 0.71 0.31 0.30 0.12 U-Ca/Crea (mol/mol) (<0.62) (<0.62) (<0.85) (<0.85) (<1.00) (<1.27) (<0.73) (<0.73) (<0.45) (<0.45) 0.98 TmP/GFR (mmol/L) (0.77-1.29) 89.00 91.40 82.00 95.30 87.00 92.50 84.00 85.00 77.00 89.00 TRP(%) (>90.00) (>90.00) (>90.00) (>90.00) (>90.00) (>90.00) (>90.00) (>90.00) (>90.00) (>90.00) 71.00 72.00 725.00 342.00 492.00 320.00 699.00 157.00 71.00 68.00 serum ALP (U/L) (40.00-130.00) (35.00-105.00) (86.00-300.00) (69.00-300.00) (104.00-300.00) (104.00-300.00) (182.00-587.00) (138.00-511.00) (50.00-350.00) (50.00-350.00) bone involvement N N Y N Y N Y N N N renal involvement N N Y Y N N N N N N c.1369G>A (p.G457S) + + + + + + + + + + c.560+27_561-38del + + c.1357delTTC (p. F453del) + + + I

II I

III II

Kindred C D E Case F Case G Individual C/II-1 C/II-2 C/III-1 (index) C/III-2 D/I-1(index) D/II-1 E/II-2 (index) E/II-1 E/I-1 E/I-2 F/II-1 G/II-1 age adult adult 4 y, 11 m 3 y 27 y, 7 m 12 m 13 y 15 y 45 y 42 y 5 y, 9 m 10 y, 3 m 1.27 ion. Ca (mmol/L) (1.15-1.33) 2.45 2.38 2.40 2.43 2.42 2.33 2.58 2.22 2.14 2.57 2.6 Ca (mmol/L) (2.13-2.63) (2.13-2.63) (2.13-2.63) (2.13-2.63) (2.20-2.60) (2.2-2.6) (2.2-2.6) (2.2-2.6) (2.2-2.6) (2.4-2.65) (2.4-2.65) 1.13 1.03 1.00 1.39 0.775 1.59 1.1 1 0.8 1.1 1.11 1.26 P (mmol/L) (0.87-1.45) (0.87-1.45) (1.39-1.74) (1.39-1.74) (0.81-1.45) (1.39-1.74) (1.29-1.68) (1.13-1.58) (0.81-1.45) (0.81-1.45) (1.20-1.74) (1.20-1.74) 13.47 12.94 11.03 0.74 0.64 0.46 3.92 1.27 1.38 0.32 PTH (pmol/L) (5.30-34.99) (5.30-34.99) (5.30-34.99) (1.06-5.83) (1.06-6.89) (1.06-6.89) (1.06-6.89) (1.06-6.89) (1.59-6.89) (1.59-6.89) 84.86 48.6 70 119 99 63 62 24.21 44.928 25(OH)D (nmol/L) (24.96-169.73) (40-130) (80-120) (25-125) (25-125) (25-125) (25-125) (62.4-199.68) (62.4-199.68) 67.60 135.20 343.20 122.20 178 172 436.8 119.6 145.6 104 174.2 283.4 1,25(OH) D (pmol/L) 2 (52.00-163.80) (52.00-163.80) (52.00-163.80) (52.00-163.80) (35-140) (55-190) (26.00-156.00) (26.00-156.00) (26.00-156.00) (26.00-156.00) (62.4-223.6) (62.4-223.6) 51 12 55 72 103 74 28.979 76.26 Crea (µmol/L) (60-124) (20-60) (30.50-53.38) (30.50-53.38) (45.76-83.89) (61.01-99.14) (15.25-38.13) (30.50-53.38) 0.31 0.28 1.64 0.39 0.92 1.03 0.67 0.11 0.5 1.56 2.75 Ca/Crea (mol/mol) (<0.57) (<0.57) (<1.00) (<1.00) (<0.45) (<0.74) (<0.74) (<0.45) (<0.45) (<0.85) (<0.85) 1.08 0.86 0.74 1.42 1.18 0.77 0.7 0.9 1.08 TmP/GFR (mmol/L) (0.89-1.08) (0.89-1.08) ND ND (0.77-1.29) (0.77-1.29) (0.77-1.29) (0.77-1.29) (1.17-1.54) 90.00 85.00 76.00 92.00 88 107 77 88 82 92 TRP(%) (>90.00) (>90.00) (>90.00) (>90.00) (>90.00) (>90.00) (>90.00) (>90.00) (>90.00) (>90.00) 74 223 966 510 210 240 358 ALP (U/L) (39-117) (40-390) (150-900) (150-900) (50-276) (50-276) (215-476) bone involvement N N Y N Y N N N N N N N renal involvement N Y Y N N N Y N N Y Y Y c.413C>T + + + c.1304delG + + c.1579_81del + + + c.575C>T + + + + + + g.2615_2699del + c.367delC + c.1402C>T + + + het.c.1579_1581del (p.L527del) het.c.1579_1581del (p.L527del) hom.c.1369G>A (p.G457S) hom.c.1369G>A (p.G457S) hom.c.1369G>A (p.G457S) hom.c.1369G>A (p.G457S) het.c.1369G>A (p.G457S) het.c.1369G>A (p.G457S) hom.c.1402C>T (p.R468W) het.c.1402C>T (p.R468W) (OC) het.c.1402C>T (p.R468W) (OC) het.g.4225_50del het.g.4225_50del het.g.4225_50del het.g.4225_50del het.g.4225_50del het.g.4225_50del hom.g.2259_2359del hom.g.2259_2359del hom.g.2259–2359del het.g.2259_2359del het.g.2259_2359del het.g.2259_2359del het.g.2259_2359del het.g.2259_2359del (OC) hom.c.586G>A (p.G196R) hom.c.586G>A (p.G196R) het.c.586G>A (p.G196R) het.c.586G>A (p.G196R) het.c.586G>A (p.G196R) het.c.586G>A (p.G196R) het.c.586G>A (p.G196R) het.c.586G>A (p.G196R) het.c.586G>A (p.G196R) Hom.c.575C>T (p.S192L) het.c.575C>T (p.S192L) het.c.575C>T (p.S192L) het.c.575C>T (p.S192L) het.c.575C>T (p.S192L) Het.c.575C>T (p.S192L) Het.c.575C>T (p.S192L) het.c.560+27_561-38del (g.1440-1469del) het.c.560+27_561-38del (g.1440-1469del)

Het.c.560+27_561-38del (g.1440-1469del) hom.c.503C>T (p.S168F) hom.c.503C>T (p.S168F) hom.c.503C>T (p.S168F) het.c.503C>T (p.S168F) het.c.503C>T (p.S168F) het.c.503C>T (p.S168F) hom.c.448+5G>A het.c.448+5G>A het.c.448+5G>A het.c.448+5G>A het.c.448+5G>A (OC) het.c.418G>A (p.G78R) het.c.418G>A (p.G78R) het.c.418G>A (p.G78R) hom.c.1764C>G (p.Y588*) het.c.1764C>G (p.Y588*) het.c.1764C>G (p.Y588*) het.c.1764C>G (p.Y588*) het.c.1690C>T het.c.1690C>T het.c.145C>T (p.Q49*) het.c.145C>T (p.Q49*) het.c.145C>T (p.Q49*) het.c.1402C>T (p.R468W) het.c.1402C>T (p.R468W) Het.c.1357delTTC (p.F453del) Het.c.1357delTTC (p.F453del) hom.c.1046_47delTG het.c.1045_46delTG het.c.1045_46delTG het.c.1046_47delTG het.c.1046_47delTG -8 -6 -4 -2 0 2 4 -6 -4 -2 0 2 4 0 50 100 1500 200 400 600 8000 501001500 100 200 3000 1 2 3 serum P (z-score)serum Ca (z-score) iPTH (%UL) 1,25D (pmol/L) TRP (%) U-Ca/Crea (%UL) renal dx