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Fourteen Monogenic Genes Account for 15% of Nephrolithiasis/Nephrocalcinosis

† †‡ ‡ Jan Halbritter,* Michelle Baum,* Ann Marie Hynes, Sarah J. Rice, David T. Thwaites, Zoran S. Gucev,§ Brittany Fisher,* Leslie Spaneas,* Jonathan D. Porath,* Daniela A. Braun,* | † Ari J. Wassner, Caleb P. Nelson,¶ Velibor Tasic,§ John A. Sayer, and Friedhelm Hildebrandt***

*Division of , Department of , |Division of , Department of Medicine, and ¶Department of , Boston Children’s Hospital, Harvard , Boston, Massachusetts; †Institute of Genetic Medicine, International Centre for Life and ‡Epithelial Research Group, Institute for Cell and Molecular Biosciences, Faculty of Medical Sciences, Newcastle University, Newcastle upon Tyne, United Kingdom; §Medical Faculty Skopje, University Children’s Hospital, Skopje, Macedonia; and **Howard Hughes Medical Institute, Chevy Chase, Maryland

ABSTRACT Nephrolithiasis is a prevalent condition with a high morbidity. Although dozens of the overall population of stone formers. monogenic causes have been identified, the fraction of single-gene disease has not been Furthermore, absence of a positive family well studied. To determine the percentage of cases that can be molecularly explained by history, which will be the rule in recessive mutations in 1 of 30 known kidney stone genes, we conducted a high-throughput genes and may be frequent in dominant mutation analysis in a cohort of consecutively recruited patients from typical kidney stone genes with incomplete penetrance, often clinics. The cohort comprised 272 genetically unresolved individuals (106 children and leads to the false assumption that a mono- 166 adults) from 268 families with nephrolithiasis (n=256) or isolated nephrocalcinosis genic cause is unlikely. We, therefore, (n=16). We detected 50 likely causative mutations in 14 of 30 analyzed genes, leading hypothesized that monogenic causes of to a molecular diagnosis in 14.9% (40 of 268) of all cases; 20 of 50 detected mutations NL/NC account for a high percentage of were novel (40%). The cystinuria gene SLC7A9 (n=19) was most frequently mutated. The stone formers. For most individuals with percentage of monogenic cases was notably high in both the adult (11.4%) and pediatric NL/NC, mutation analysis for a causative cohorts (20.8%). Recessive causes were more frequent among children, whereas domi- geneticdefect has notbeen accessibleso far. nant disease occurred more abundantly in adults. Our study provides an in-depth analysis With the advent of massively parallel se- of monogenic causes of . We suggest that knowledge of the mo- quencing techniques and high-throughput lecular cause of nephrolithiasis and nephrocalcinosis may have practical implications and library preparation, mutation analysis of might facilitate personalized treatment. multiple genes in large cohorts has not only become technically feasible but also, J Am Soc Nephrol 26: 543–551, 2015. doi: 10.1681/ASN.2014040388 cost-effective.7 To determine the percentage of mono- genic causes in kidney stone disease, we Nephrolithiasis (NL) is a highly prevalent autosomal-dominant, autosomal-recessive, 5,6 condition affecting up to 10% of individ- or X-linked transmission. The ability Received April 18, 2014. Accepted June 18, 2014. uals in the Western world.1 It is associated to detect the causative mutation(s) in a fi Published online ahead of print. Publication date with signi cant morbidity because of col- monogenic disease gene is of great diagnos- available at www.jasn.org. icky pain, the necessity of surgical proce- tic and potentially therapeutic importance, dures, and progression to CKD.2 NL and because there is an almost deterministic Correspondence: Dr.JohnA.Sayer,Instituteof – Genetic Medicine, International Centre for Life, related conditions, such as nephrocalci- cause effect relationship in monogenic Newcastle University, Central Parkway, Newcastle nosis (NC), share a well recognized heri- disease. upon Tyne NE1 3BZ, UK, or Prof. Friedhelm Hildebrandt, tability, emphasized by the fact that up to However, the contribution of mono- Division of Nephrology, Boston Children’sHospital,300 Longwood Avenue, Boston, MA 02115. Email: john. two thirds of hypercalciuric stone formers genic disorders to the overall prevalence of [email protected] or friedhelm.hildebrandt@ have relatives with NL.3–5 There are at NL has never been studied comprehen- childrens.harvard.edu least 30 genes that have been shown to sively. Hence, data are lacking on the Copyright © 2015 by the American Society of cause single-gene forms of NL/NC by frequency of monogenic forms of NL in Nephrology

J Am Soc Nephrol 26: 543–551, 2015 ISSN : 1046-6673/2603-543 543 544

Table 1. Twenty-nine molecular diagnoses established in six dominant genes in a cohort of 272 individuals (268 families) with NL/NC COMMUNICATION BRIEF Clinical Genetic Practical Implication Gene/ Nucleotide Amino Acid Evolutionary Age of Stone State PPh2 Ref. Diagnosis Diagnosis (Because of Genetic Individual Change Change Conservation Onset (yr) Analysis ora fteAeia oit fNephrology of Society American the of Journal (Prescreening) (Postscreening) Diagnosis) SLC7A9 B208–21 c.313G.A p.Gly105Arg Het 0.998 D. melanog. 9 1 Cystine CU+NL ► CU, type B — JAS-C8a c.313G.A p.Gly105Arg Het 0.998 D. melanog. 9 17 CaOx Idiopathic NL ► CU, type B Quantify urinary cystine JAS-E5 c.313G.A p.Gly105Arg Het 0.998 D. melanog. 9 17 Ca CU+NL/NC ► CU, type B Hydration and urinary alkalinization F1029–21a,b c.313G.A p.Gly105Arg Het 0.998 D. melanog. 9 6 ND Idiopathic NC ► CU, type B Quantify urinary cystine, www.jasn.org c.544G.A p.Ala182Thr Het 0.056 D. melanog. 10 hydration, and urinary alkalinization JAS-D30 c.313G.A p.Gly105Arg Het 0.998 D. melanog. 9 25 Cystine CU+NL ► CU, type B — c.614del p.Lys205Argfs*59 Het ——Novel JAS-D31 c.411_412del p.Pro139Leufs*69 Het ——10 28 Cystine CU+NL ► CU, type B — JAS-D47 c.411_412del p.Pro139Leufs*69 Het ——10 45 Cystine CU+NL ► CU, type B — JAS-D34 c.411_412del p.Pro139Leufs*69 Het ——10 35 Cystine CU+NL ► CU, type B — JAS-D28 c.544G.A p.Ala182Thr Hom 0.056 D. melanog. 10 30 Cystine CU+NL ► CU, type B — JAS-F41a c.544G.A p.Ala182Thr Het 0.056 D. melanog. 10 60 ND HC+NL ► CU, type B Quantify urinary cystine JAS-F50a c.544G.A p.Ala182Thr Het 0.056 D. melanog. 10 50 ND Idiopathic NL ► CU, type B Quantify urinary cystine JAS-D57 c.544G.A p.Ala182Thr Het 0.056 D. melanog. 10 7 Cystine CU+NL ► CU, type B — c.614dup p.Asn206Glufs*3 Het ——11 JAS-F87a c.614dup p.Asn206Glufs*3 Het ——11 19 CaPO HC+NL ► CU, type B Quantify urinary cystine JAS-G10 c.614dup p.Asn206Glufs*3 Het ——11 43 Cystine CU+NL ► CU, type B — JAS-D29 c.671C.T p.Ala224Val Hom 0.999 S. cerevisae 12 15 Cystine CU+NL ► CU, type B — JAS-D55 c.671C.T p.Ala224Val Het 0.999 S. cerevisae 12 26 Cystine CU+NL ► CU, type B — JAS-F19a c.671C.T p.Ala224Val Het 0.999 S. cerevisae 12 28 ND HC+NL ► CU, type B Quantify urinary cystine c.1369T.C p.Tyr457His Het 0.059 D. rerio Novel B114–21 c.814del p.Val272Cysfs*6 Het ——13 6 Cystine CU+NL ► CU, type B — c.997C.T p.Arg333Trp Het 0.999 D. melanog. 14 JAS-E9 c.1353C.A p.Tyr451* Het ——Novel 26 Cystine CU+NL ► CU, type B — c.1400–2A.G39 splice Het ——Novel ADCY10 JAS-F8 c.1263C.A p.Tyr421* Het ——Novel 10 ND Idiopathic HC ► Idiopathic HC Monitor urinary Ca JAS-F68 c.1282G.A p.Asp428Asn Het 0.996 C. intestinalis Novel 50 CaOx Idiopathic HC+NC ► Idiopathic HC Monitor urinary Ca mScNephrol Soc Am J JAS-F29 c.4477del p.Leu1493Serfs*24 Het ——Novel 40 ND Idiopathic NL ► Idiopathic HC Monitor urinary Ca SLC2A9 B179–21a c.1343C.T p.Pro448Leu Het 0.997 D. melanog. Novel 6 ND Idiopathic NL ► RHUC2 Recheck serum/urinary uric acid B230–21a c.1419+1G.A59 splice Het — — Novel 7 ND Idiopathic HC+NL ► RHUC2 Recheck serum/urinary 26: uric acid 543 – 5,2015 551, www.jasn.org BRIEF COMMUNICATION

analyzed, in a cohort of 272 genetically unresolved individuals (268 families) (Supplemental Table 1), all coding exons and splice sites of 30 known NL/NC- causing genes with a defined Online — Mendelian Inheritance in Man (OMIM) Diagnosis) phenotype (Supplemental Table 2 and (Because of Genetic Practical Implication Concise Methods). Participants were re- prevention of bone disease (osteoporosis/fractures) uric acid prevention of bone disease (osteoporosis/fractures) cruited consecutively and without prior preselection at three typical kidney stone clinics on the basis of diagnosed NL or NC as outlined in Concise Meth- ods. We applied a high-throughput mu- Genetic Diagnosis tation analysis technique that we recently (Postscreening) Primary dRTA Correct acidosis NPHLOP2 Screen family members at risk, RHUC1 Recheck serum/urinary RHUC1 NPHLOP2 Screen family members at risk, developed.7,8 As a result, we show that ► ► ► ► ► causative mutations in known NL/NC- causing genes are present in around 15% of these 268 families, and we suggest

; RHUC2, renal hypouricemia type 2; NPHLOP2, hypophosphatemic possible therapeutic and prophylactic

Clinical implications for some of these genetic Diagnosis fi (Prescreening) ndings.

; CU, cystinuria; CaOx, oxalate; CaPO, calcium phosphate; Hom, By targetedresequencingof381 cod- ing exons of 30 genes known to cause Ciona intestinalis , autosomal-dominant, autosomal-recessive, Stone Analysis or X-linked NL/NC if mutated, we iden- tified 12,589 single-nucleotide variants

C. intestinalis and 1246 deletion-insertion variants in ; Age of

Onset (yr) 336 individuals (272 genetically unre- Drosophila melanogaster

, solved individuals and 64 controls). To 15 5 ND Idiopathic HC+NL 16 17 Ca Idiopathic NL

Ref. distinguish benign variants from disease- Novel 29 ND HC+NC Novel 25 CaOx HU+NL Novel 17 ND Idiopathic NL Danio rerio , causing mutations, we evaluated each

D. melanog. variant individually on the basis of strict D. rerio

; criteria as described in Concise Methods. Overall, 162 (1.2%) variants met our Evolutionary D. rerio D. rerio S. cerevisae D. melanog. Conservation criteria for being likely disease-causing variants, 139 of which were confirmed by —— Sanger sequencing (86%), whereas the others represented low-representation ar- tifacts of multiplex PCR. Afterexclusion of State PPh2 Saccharomyces cerevisae , single heterozygous variants in recessive genes and nonsegregating variants and subtraction of variants from duplicates

S. cerevisae and positive controls, a total of 50 most

Change likely disease-causing variants remained Amino Acid splice site Het

9 (Supplemental Table 3). As a result, we made a causal molec- C5

C p.Glu906Gln Het 0.597 ular genetic diagnosis that is likely to T p.Arg434Cys Het 0.235 . A p.Glu225Lys Het 0.816 C p.Leu144Pro Het 0.999 . .

. explain the disease phenotype in 40 .

21, both heterozygous mutations are on the same allele. unrelated individuals in our total cohort Change – Nucleotide of 268 families with NL/NC (14.9%) c.673G c.888+2T c.1300C HumVar (http://genetics.bwh.harvard.edu/pph2/); Het, heterozygous; – (Tables 1 and 2). Pathogenic mutations Continued a a a were detected in the following six dom- 21 21 12 – – – inant disease genes: SLC7A9 (19 fami-

Gene/ lies), ADCY10 (3 families), SLC2A9 B224 JAS-E8 c.2716G B109 B155 JAS-F98 c.431T Individual For individual F1029 Molecular genetic diagnosis added new aspects to the previously established clinical diagnosis (11 of 29=37.9%). SLC9A3R1 SLC9A3R1 SLC4A1 Table 1. PPh2, Polyphen2 a b SLC22A12 homozygous; ND, no data; HC, ; nephrolithiasis/osteoporosis-2; HU, hypouricemia; RHUC1, renal hypouricemia type 1; dRTA, distal . (2 families), (2 families),

J Am Soc Nephrol 26: 543–551, 2015 Genetics of Kidney Stone Formers 545 546 Table 2. Twelve molecular diagnoses established in eight recessive genes in a cohort of 272 individuals (268 families) with NL/NC RE COMMUNICATION BRIEF Clinical Genetic Practical Implication Gene/ Nucleotide Amino Acid Evolutionary Age of Stone State PPh2 Ref. Diagnosis Diagnosis (Because of Genetic Individual Change Change Conservation Onset (yr) Analysis (Prescreening) (Postscreening) Diagnosis) ora fteAeia oit fNephrology of Society American the of Journal SLC3A1 JAS-B21a c.1354C.T p.Arg452Trp Hom 1 S. cerevisae 17 16 Cystine CU+NL ► CU, type A — JAS-B22a c.1354C.T p.Arg452Trp Hom 1 S. cerevisae 17 16 Cystine CU+NL ► CU, type A — JAS-G7 c.1400T.C p.Met467Thr Hom 0.289 D. melanog. 18 55 Cystine CU+NL ► CU, type A — ATP6V1B1 B214–21b c.481G.A p.Glu161Lys Hom 0.005 S. cerevisae 19 6 ND NL ► dRTA, deafness Prompt audiometry, screen

family members at risk www.jasn.org CLCN5c B111–21 c.344G.A p.Trp115* Hem ——Novel 8 ND Dent disease ► Dent disease/NL — type 1 B167–21b,d c.1009G.A p.Glu337Lys Hem 0.987 S. cerevisae Novel 11 ND FHHNC/PH ► Dent disease/NL ESRD, no recurrence of disease type 1 in RTX, screen family members at risk CLDN16 JAS-C1d c.445C.T p.Arg149* Hom ——20 20 ND FHHNC+NL ► FHHNC ESRD, no recurrence of disease in RTX, screen family members at risk B178–21 c.453G.T p.Leu151Phe Hom 0.985 C. intestinalis 21 1 ND FHHNC ► FHHNC — CYP24A1 B223–21b c.428_430del p.Glu143del Hom — D. rerio 22 12 ND FHHNC ► (Infantile) HC/NL Infantile HC: not restricted to infancy (12 yr), monitor levels and serum Ca, avoid vitamin D AGXT B106–21 c.416_418del p.Val139del Het — X. tropicalis Novel 12 CaOx PH1 ► PH1 Definite clinical diagnosis was c.846+1G.T59 splice site Het ——Novel obtained by liver ; invasive biopsy could have been avoided SLC34A1 B168–21b,e c.271_291del p.Val91_Ala97del Het ——Novel 0.4 ND NC+PH ► NPHLOP1/FS Recheck serum/urinary c.1534C.T p.Arg512Cys Het 1 C. elegans Novel phosphate, screen family

mScNephrol Soc Am J members at risk, prevention of bone disease (osteoporosis/fractures) 26: 543 – 5,2015 551, www.jasn.org BRIEF COMMUNICATION

SLC22A12 (2 families), and SLC4A1

D. (1 family) (Table 1, Supplemental Table 4). ; Furthermore, we identified pathogenic mutations in the following eight reces- sive disease genes: SLC3A1 (two fami- lies), CLCN5 (two families), CLDN16 Ciona intestinalis

Diagnosis) ATP6V1B1 , (two families), (one family), CYP24A1 AGXT

; ND, no data; dRTA, distal (one family), (one fam- (Because of Genetic Practical Implication ily), SLC34A1 (one family), and phosphate and vitamin D, prevention of bone disease (osteoporosis/fractures) SLC34A3 (one family) (Table 2, Supple- C. intestinalis mental Table 4). No pathogenic mutations were identi- fied in the genes APRT, ATP6V0A4, CA2, CASR, CLCNKB, CLDN19, FAM20A, Genetic Diagnosis Drosophila melanogaster GRHPR HNF4A HOGA1 HPRT1 , , , , , (Postscreening) HHRH Monitor serum/urinary KCNJ1, OCRL, SLC12A1, VDR,and

► XDH

TX, renal transplantation; . In total, 20 of 50 mutations (40%) detected were novel pathogenic variants, D. melanog. which have not been previously reported (Human Gene Mutation Database; http://

Clinical 9–22 Diagnosis www.hgmd.cf.ac.uk/ac/index.php).

(Prescreening) Inthepediatricsubgroupofindividuals ; NPHLOP1, hypophosphatemic nephrolithiasis/osteoporosis-1; FS, Fanconi ; CU, cystinuria; with age of onset before 18 years (defined by age at first stone or age at diagnosis of Stone Analysis NC), we identified a causative mutation in 20.8% (22 of 106) of individuals (Figure 1A). In contrast, in the adult cohort (de- 41.6%).

Age of fi $ 5 ned by age of onset 18 years), delete- Caenorhabditis elegans Onset (yr) , Saccharomyces cerevisae

, rious variants were detected in 11.4% (19 of 166) of individuals (Figure 1A). The Ref. Novel 18 ND Idiopathic NL Novel distribution of median age of onset across C. elegans ; 14 genes, in which causative mutations S. cerevisae were found, correlated with the mode of inheritance as follows: although four of six dominant genes were associated with Evolutionary C. elegans C. savigny Conservation adult manifestation ($18 years), muta- Xenopus tropicalis

, tions in recessive genes were mainly iden- tified in probands with an age of onset before 18 years (Figure 1B).

State PPh2 SLC7A9 X. tropicalis For the gene ,whichwas found most commonly mutated in this cohort, there was no correlation between

; HHRH, hereditary hypophosophatemic rickets with hypercalciuria. age of onset and allelic strength (truncat- ing versus missense variant) (Supple- Change

Amino Acid mental Figure 1A). Almost one third of individuals with SLC7A9 mutations manifested within the age range of A p.Arg485His Het 0.981 T p.Ile529Phe Het 0.12 . . $18–30 years, with a median at 26 years

Caenorhabditis savigny (Figure 1). However, there was a geno- , Change – Nucleotide

21 and JAS-C1 both reached ESRD in their 20s and 30s. type phenotype correlation in that indi- HumVar (http://genetics.bwh.harvard.edu/pph2/); Hom, homozygous; c.1585A c.1454G – ; HC, hypercalciuria; Het, heterozygous; – viduals with two pathogenic variants of Continued

b SLC7A9 21, segregation analysis shows compound heterozygosity. C. savigny (8 of 19 unrelated individuals) – is located on the X chromosome (X-linked recessive mode of inheritance). showed a significantly earlier manifesta- Danio rerio , Gene/ tion compared with individuals with JAS-F43 Individual Molecular genetic diagnosis added new aspects to the previouslyIndividuals established B167 clinical diagnosis (5 of 12 For B168 Note that JAS-B21 and JAS-B22 are siblings (same family); all other listed individuals are unrelated. CLCN5 SLC34A3 Table 2. PPh2, Polyphen2 a b c d e rerio syndrome; renal tubular acidosis; Hem, hemizygous; FHHNC, familial hypomagnesemia with hypercalciuria and nephrocalcinosis; PH, primary ; R only one pathogenic variant (mean: 18

J Am Soc Nephrol 26: 543–551, 2015 Genetics of Kidney Stone Formers 547 BRIEF COMMUNICATION www.jasn.org

versus 30 years) (Supplemental Figure 1B). For approximately 60% of individ- uals, the identified genetic diagnosis confirmed the previously obtained clin- ical diagnosis. In approximately 40% of cases, however, the genetic diagnosis contributed additional etiologic and diag- nostic information to what was clinically suspected, suggesting practical implica- tions (Tables 1 and 2). We here examined an international cohort of 272 typical kidney stone for- mers for the presence of mutations in 30 genes that cause NL/NC if mutated. We identified 50 (20 novel) pathogenic al- lelesin14differentgenesin40of268 families (14.9%). This work, to the best of our knowl- edge, is the most extensive genetic screen- ing of known NL/NC-causing genes in a combined cohort of pediatric and adult individuals with kidney stones and/or NC. The overall percentage of families with pathogenic mutations exceeds the general assumption of a relatively small contribu- tion of monogenic causes to the general population of stone formers. Remarkably, in almost 21% of the pediatric cohort and 11% of the adult cohort, we identified causative mutations in 1 of 14 genes. The fact that we did not find mutations in the remaining 16 genes suggests that muta- tions in those genes are less prevalent. Although it is generally assumed that around 85% of causative mutations in monogenic disorders reside within coding regions and adjacent splice sites,23 copy number variations and deleterious deep intronic variants are undetectable with the screening approach that we applied to this study. These limitations as well as Figure 1. Distribution of established molecular genetic causes of NL/NC. (A) Percentage of population genetic factors may have led to subjects with identified molecular diagnoses acrossgroupedages of onset. Thispercentage false negatives and a selection bias in re- is significantly higher in the pediatric cohort (age of onset,18 years) compared with the gards to the distribution of molecular di- adult cohort (age of onset$18 years): 20.8% versus 11.4%. *P#0.05. In A and B, the green agnoses in this study cohort. rectangles indicate six individuals with heterozygous SLC7A9 mutations enriched within SLC7A9 was, by far, the most preva- the age group $18–30 years (6 of 19=31.6%). (B) Distribution of age of onset across mu- lent disease-causing gene in our cohort, tated causative genes. Genes with a dominant mode are annotated in black, whereas fi † with a median age of rst stone at 26 genes with a recessive mode are annotated in red. For SLC2A9, SLC22A12,andSLC7A9, years (Figure 1B). This finding is in line detected mutations were primarily dominant, although both modes of inheritance have with retrospective data derived from been reported. Detected mutations in four of six genes with a median onset $18 years are in dominant genes (upper right quadrant as indicated by dashed lines), whereas six of eight stone composition analysis showing the genes with a median onset of disease ,18 years are in recessive genes (lower left quadrant predominance of cystinuria as the major as indicated by dashed lines). monogenic cause of stone disease in the adult population.24 Presence of two pathogenic variants in SLC7A9 led to

548 Journal of the American Society of Nephrology J Am Soc Nephrol 26: 543–551, 2015 www.jasn.org BRIEF COMMUNICATION earlier manifestation in our cohort (Sup- genetic diagnoses contributed a new as- (7348 samples), 272 of which represented ge- plemental Figure 1B). Whether this find- pect to the previously established clinical netically unresolved individuals from 268 dif- ing is because of recessive inheritance or diagnosis, suggesting practical implica- ferent families with NL/NC. The remaining 64 the presence of two mutations on the tions, such as avoiding vitamin D samples comprised 33 unaffected controls same allele could not be fully investigated (CYP24A1), initiating audiometry (negative controls), 7 duplicate samples, 9 for reasons of incomplete allelic segrega- (ATP6V1B1), or excluding the risk of re- samples from individuals who, in retrospect, tion data. Interestingly, in six individu- currence in renal transplants (CLCN5 or did not match any of the inclusion criteria after als with mutated SLC7A9,clinicaldata CLDN16) (Tables 1 and 2). thorough clinical re-evaluation, and 15 sam- did not raise the suspicion of cystinuria, An excellent example of individual- ples from individuals with established molec- and three of them exhibited calcium- ized on the basis of molecular ular diagnoses in any 1 of 30 known NL/NC containing kidney stones (JAS-C8, JAS-E5, geneticdiagnostics was previouslyshown genes (positive controls). Study participants’ and JAS-F87) (Tables 1 and 2). There are for individuals with primary hyperoxaluria ethnicities were as follows: western and north- recent reports showing similar find- type 1 caused by AGXT mutations, where ern Europe (n=174), eastern Europe (n=69), ings of calcium-based stones in indi- pyridoxine sensitivity is associated with the the United States (European American; viduals with heterozygous SLC7A9 presence of a distinct allele (Gly170Arg).30 n=18), Asia (n=6), the Middle East (n=3), mutations.25,26 Additionally, Martins Furthermore, invasive and potentially southern Europe (n=1), and South America et al.27 showed that cystine promotes harmful procedures, such as a diagnos- (n=1) (Supplemental Table 1). There were calcium oxalate crystal formation in tic liver biopsy in individuals with sus- 171 men (62.9%) and 101 women (37.1%) in vitro, implying that cystinuria may pected primary hyperoxaluria type 1, the study; 106 individuals were diagnosed be- be a risk factor for calcium oxalate cal- can be circumvented after diagnostic fore 18 years of age (pediatric cohort; 39.0%), culi. In conclusion, these data empha- gene panels become part of the clinical and 166 individuals were diagnosed as adults size the need to screen urine for the repertoire.31 The use of such a broad ge- (adult cohort; 61.0%). presence of excess dibasic amino acids, netic screening device may also help including cystine, in all stone formers, raise awareness of extremely rare disor- Mutation Analysis of Known NL/NC- even if the molecular genetic diagnosis ders and be beneficial in cases of atypical Causing Genes has been established.28 Although cys- clinical presentation or hindered standard Mutation analysis was carried out on DNA tinuria is the most common clinically diagnostics because of advanced CKD. extracted according to standard methods from known genetic diagnosis, in some of peripheral blood or saliva obtained from the these individuals, only broad genetic study participants. Targeted amplification of screening revealed the etiology, affect- CONCISE METHODS 428ampliconsatatimewasperformedby ing their future treatment and the pre- multiplexed PCR using Fluidigm Access-Array ventative measures that could then be Human Participants technology followed by barcoding and next instituted. We obtained blood samples and pedigrees after generation resequencing on an Illumina MiSeq Admittedly, for ADCY10, in which we receiving informed consent from individuals platform as previously established by our found three putatively deleterious alleles, with reported NL/NC. The study was approved group.7,8 Sanger DNA sequencing was further the evidence for single-gene causation is by the Institutional Review Boards of the conducted for single-mutation confirmation. controversial. The OMIM phenotype Boston Children’s Hospital (BCH) and the All coding exons and adjacent splice sites of implies susceptibility to absorptive hy- Newcastle and North Tyneside Research Ethics the following 30 genes that are known to percalciuria. This notion is on the basis Committee. Participants were included from cause monogenic forms of NL/NC if mutated ofasinglecasecontrolstudybyReed typical kidney stone clinics in a consecutive (defined by an OMIM phenotype) were et al.29 and has never been confirmed in manner over a time period of 2 years at the screened: ADCY10, AGXT, APRT, ATP6V0A4, larger-sized cohorts. University of Newcastle and University Clinic ATP6V1B1, CA2, CASR, CLCN5, CLCNKB, Nevertheless, our study supports the Skopje and 5 months at the BCH. Enrollment CLDN16, CLDN19, CYP24A1, FAM20A, conception that NL/NC is genetically in the study was on the basis of the follow- GRHPR, HNF4A, HOGA1, HPRT1, KCNJ1, broadly heterogeneous. We provide evidence ing clinical diagnoses by an investigator ne- OCRL, SLC12A1, SLC22A12, SLC2A9, that the role ofmonogeniccausesaccounts phrologist: NL of any kind (n=256) or isolated SLC34A1, SLC34A3, SLC3A1, SLC4A1, for a significantly higher percentage of NC (n=16) (Supplemental Table 1). Of 256 SLC7A9, SLC9A3R1, VDR,andXDH (Supple- kidney stone formers than generally individuals with NL, 11 individuals also mental Table 2). suspected, especially in the adult popu- showed NC on renal and/or com- lation. Most importantly, identification puted tomography scan. Excluded from the Sensitivity of Mutation Detection of the monogenic causes of NL/NC may study were subjects with a kidney stone-related To calculate the sensitivity of mutation de- not only have important prognostic disorder known to be secondary to drugs tection, we included 15 DNA samples with 18 implications but also lead to therapeutic (i.e., vitamin D) or primary systemic disease (pri- known mutations in single-gene cases of consequences (Tables 1 and 2): in .40% mary ). In total, mutation NL/NC as positive controls. Overall, 17 of of the cases in our study, the molecular analysis was conducted in 336 DNA samples 18 alleles were redetected (sensitivity: 94.4%).

J Am Soc Nephrol 26: 543–551, 2015 Genetics of Kidney Stone Formers 549 BRIEF COMMUNICATION www.jasn.org

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Karim Z, Gérard B, Bakouh N, Alili R, Leroy C, Beara-Lasic L, Anglani F, Milliner DS, Palsson effect on the protein level; or (3) the given Beck L, Silve C, Planelles G, Urena-Torres P, R: Hereditary causes of kidney stones and Grandchamp B, Friedlander G, Prié D: disease-causing allele is supported by func- . Pediatr Nephrol 28: NHERF1 mutations and responsiveness of – tional data. 1923 1942, 2013 renal parathyroid hormone. NEnglJMed Exclusion criteria: (1) allele is present in 7. Halbritter J, Diaz K, Chaki M, Porath JD, 359: 1128–1135, 2008 healthy controls of the Exome Variant Server Tarrier B, Fu C, Innis JL, Allen SJ, Lyons RH, 16. 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Mohebbi N, Vargas-Poussou R, Hegemann SC, andtheirfamilies.WethankP.Hoggfor technical point mutations, and survey of the frequency Schuknecht B, Kistler AD, Wüthrich RP, Wagner support. of mutations within the SLC3A1 and SLC7A9 CA: Homozygous and compound heterozygous genes in a cohort of 172 cystinuric Italian mutations in the ATP6V1B1 gene in patients S.J.R.,D.T.T.,andJ.A.S. are supportedby the patients. Mol Genet Metab 99: 42–52, 2010 with renal tubular acidosis and sensorineural Northern Counties Kidney Research Fund. 10. Feliubadaló L, Font M, Purroy J, Rousaud F, hearing loss. Clin Genet 83: 274–278, 2013 F.H. is an Investigator of the Howard Hughes Estivill X, Nunes V, Golomb E, Centola M, 20. Simon DB, Lu Y, Choate KA, Velazquez H, Medical Institute, a Doris Duke Distinguished Aksentijevich I, Kreiss Y, Goldman B, Pras M, Al-Sabban E, Praga M, Casari G, Bettinelli A, Clinical Scientist, and the Warren E. Grupe Kastner DL, Pras E, Gasparini P, Bisceglia L, Colussi G, Rodriguez-Soriano J, McCredie D, Beccia E, Gallucci M, de Sanctis L, Ponzone Milford D, Sanjad S, Lifton RP: Paracellin-1, a renal Professor of . This research was A, Rizzoni GF, Zelante L, Bassi MT, George tight junction protein required for paracellular supported by National Institutes of Health AL Jr., Manzoni M, De Grandi A, Riboni M, Mg2+ resorption. Science 285: 103–106, 1999 Grant R01-DK088767 (to F.H.) and March of Endsley JK, Ballabio A, Borsani G, Reig N, 21. Weber S, Hoffmann K, Jeck N, Saar K, Dimes Foundation Grant 6FY11-241. Fernández E, Estévez R, Pineda M, Torrents Boeswald M, Kuwertz-Broeking E, Meij II, D, Camps M, Lloberas J, Zorzano A, Palacín Knoers NV, Cochat P, Suláková T, Bonzel KE, M; International Cystinuria Consortium: Non- Soergel M, Manz F, Schaerer K, Seyberth HW, type I cystinuria caused by mutations in Reis A, Konrad M: Familial hypomagnesaemia DISCLOSURES SLC7A9, encoding a subunit (bo,+AT) of with hypercalciuria and nephrocalcinosis maps None. rBAT. Nat Genet 23: 52–57, 1999 to chromosome 3q27 and is associated with

550 Journal of the American Society of Nephrology J Am Soc Nephrol 26: 543–551, 2015 www.jasn.org BRIEF COMMUNICATION

mutations in the PCLN-1 gene. Eur J Hum diagnostiques et thérapeutiques. Nephrol Ther Clin Endocrinol Metab 84: 3907–3913, Genet 8: 414–422, 2000 4: 231–255, 2008 1999 22. Schlingmann KP, Kaufmann M, Weber S, Irwin 25. Cupisti A, Farnesi I, Armillotta N, Francesca F: 30. Monico CG, Rossetti S, Olson JB, Milliner DS: A, Goos C, John U, Misselwitz J, Klaus G, Staghorn cystine stone in a 72-year-old re- Pyridoxine effect in type I primary hyper- Kuwertz-Bröking E, Fehrenbach H, Wingen current calcium stone former. Clin Nephrol 78: oxaluria is associated with the most common – – AM, Güran T, Hoenderop JG, Bindels RJ, 76 80, 2012 mutant allele. Kidney Int 67: 1704 1709, Prosser DE, Jones G, Konrad M: Mutations in 26. Elkoushy MA, Andonian S: Characterization 2005 of patients with heterozygous cystinuria. 31. Rumsby G, Williams E, Coulter-Mackie M: CYP24A1 and idiopathic infantile hypercal- Urology 80: 795–799, 2012 Evaluation of mutation screening as a first cemia. N Engl J Med 365: 410–421, 2011 27. Martins MC, Meyers AA, Whalley NA, Rodgers line test for the diagnosis of the primary hy- 23. Choi M, Scholl UI, Ji W, Liu T, Tikhonova IR, AL: Cystine: A promoter of the growth and ag- peroxalurias. Kidney Int 66: 959–963, 2004 Zumbo P, Nayir A, Bakkaloglu A, Ozen S, gregation of calcium oxalate crystals in normal Sanjad S, Nelson-Williams C, Farhi A, Mane undiluted human urine. JUrol167: 317–321, S, Lifton RP: Genetic diagnosis by whole 2002 exome capture and massively parallel DNA 28. Sayer JA, Moochhala SH, Thomas DJ: The See related editorial, “The Search for Monogenic sequencing. Proc Natl Acad Sci U S A 106: medical management of urolithiasis. Br J Causes of Kidney Stones,” on pages 507–510. 19096–19101, 2009 MedSurgUrol3: 87–95, 2010 24. Jungers P, Joly D, Blanchard A, Courbebaisse 29. Reed BY, Heller HJ, Gitomer WL, Pak CY: This article contains supplemental material online at M, Knebelmann B, Daudon M: Lithiases rénales Mapping a gene defect in absorptive hy- http://jasn.asnjournals.org/lookup/suppl/doi:10.1681/ héréditaires monogéniques: Récents acquis percalciuria to chromosome 1q23.3-q24. J ASN.2014040388/-/DCSupplemental.

J Am Soc Nephrol 26: 543–551, 2015 Genetics of Kidney Stone Formers 551

A ] yr Age of onset (NL/NC) [

B *

] yr Age of onset (NL/NC) [

Figure S1. Correlation between median age of onset and mutated alleles detected in SLC7A9. (A) Five truncating (red) and three missense alleles (black) sorted by median age of onset. Note that there is no correlation between the presence of a truncating variant and median age of onset in the affected individuals. (B) In contrast, the presence of two mutated variants in SLC7A9 results in a significantly earlier age of onset compared to 1 mutated variant (*p=0.029, unpaired Student’s t-test). NL, nephrolithiasis; NC, nephrocalcinosis; yr, years. Supplemental Table 1. Cohort characteristics of 272 individuals from 268 different families with NL/NC

General / Age of onset Gender Ethnicity / Origin

Inclusion Total Median # of pediatric # of adult Con- Female Male Western / Eastern EA Asia Middle Southern South criteriona # age of individuals individuals sang- Northern Europe / East Europe America / onset (age of onset (age of onset uinity Europe Balkans Hispanic (range - <18 yr) •18 yr) yr)

30 NL 256 91 165 4 95 161 170 62 17 4 1 1 1 (1-81)

8 NC 16 15 1 2 6 10 4 7 1 2 2 0 0 (1-46)

28 106 166 6 101 171 174 69 18 6 3 1 1 Total # 272 (1-81) (39.0%) (61.0%) (2.2%) (37.1%) (62.9%) (64.0%) (25.4%) (6.6%) (2.2%) (1.1%) (0.4%) (0.4%)

aNote that, the inclusion criteria nephrocalcinosis and hypercalcuria refer to isolated conditions without a reported history of stone disease. Another 11 individuals, who primarily presented with kidney stone disease also showed nephrocalcinosis on renal ultrasound or CT scan. Abbreviations: EA, European American; NC, isolated nephrocalcinosis; NL, nephrolithiasis; yr, years

Supplemental Table 2. Thirty genes, known to cause monogenic forms of NL/NC that were included in the study.

Gene Gene Name Accession # Disease entity MIM-Phenotype Mode Coding Ref. Symbola # Exons

1

1 ADCY10/SAC adenylate cyclase 10 (soluble) NM_018417.4 Idiopathic (absorptive) hypercalciuria, susceptibility 143870 AD 32

2

2 AGXT alanine-glyoxylate aminotransferase NM_000030.2 Primary hyperoxaluria, type 1 259900 AR 11

Adenine phosphoribosyltransferase deficiency, 3

3 adenine phosphoribosyltransferase NM_000485.2 614723 AR 5 APRT APRT ATPase, H+ transporting, lysosomal V0 4

4 NM_020632.2 dRTA 602722 AR 20 ATP6V0A4 subunit a4 ATPase, H+ transporting, lysosomal 5

5 NM_001692.3 distal renal tubular acidosis (dRTA) with deafness 267300 AR 14 ATP6V1B1 56/58kDa, V1 subunit B1 6

6 CA2 carbonic anhydrase II NM_000067.2 Osteopetrosis + d/pRTA 259730 AR 7

Hypocalcemia with / 7

7 calcium-sensing receptor NM_001178065.1 601198 AD 6 CASR hypocalcemia, autosomal dominant 8

8 CLCN5 chloride channel, voltage-sensitive 5 NM_001127898.3 Dent disease / Nephrolithiasis, type 1 300009 / 310468 XR 14

9

9 CLCNKB chloride channel, voltage-sensitive Kb NM_000085.4 Bartter syndrome, type 3 607364 AR 19

Familial hypomagnesemia with hypercalciuria & 10

10 claudin 16 NM_006580.3 248250 AR 5 CLDN16 nephrocalcinosis, FHHNC Familial hypomagnesemia with hypercalciuria & 11

11 claudin 19 NM_001123395.1 248190 AR 4 CLDN19 nephrocalcinosis with ocular abnormalities cytochrome P450, family 24, subfamily 1,25-(OH) D-24 hydroxylase deficiency , infantile 12

12 NM_000782.4 143880 AR 11 CYP24A1 A, polypeptide 1 Hypercalcemia family with sequence similarity 20, Enamel-Renal syndrome, amelogenesis imperfect 13

13 NM_017565.3 204690 AR 12 FAM20A member A and nephrocalcinosis glyoxylate reductase/hydroxypyruvate 14

14 NM_012203.1 Primary hyperoxaluria, type 2 260000 AR 9 GRHPR reductase MODY + + Nephrocalcinosis 15

15 hepatocyte nuclear factor 4, alpha NM_000457.4 125850 AD 1 HNF4A (p.R76W) 16

16 HOGA1 4-hydroxy-2-oxoglutarate aldolase 1 NM_138413.3 Primary hyperoxaluria, type 3 613616 AR 7

hypoxanthine Kelley-Seegmiller syndrome, partial HPRT 17

17 NM_000194.2 300323 XR 9 HPRT1 phosphoribosyltransferase 1 deficiency, HPRT-related gout potassium inwardly-rectifying channel, 18

18 NM_000220.4 Bartter syndrome, type 2 241200 AR 2 KCNJ1 subfamily J, member 1 19

19 OCRL oculocerebrorenal syndrome of Lowe NM_000276.3 Lowe syndrome / Dent disease 2 309000 / 300555 XR 24

20

20 SLC12A1 solute carrier family 12, member 1 NM_000338.2 Bartter syndrome, type 1 601678 AR 27 solute carrier family 22 (organic 21

21 NM_144585.3 Renal hypouricemia, RHUC1 220150 AD/AR 10 SLC22A12 anion/urate transporter), member 12 solute carrier family 2 (facilitated 22

22 NM_001001290.1 Renal hypouricemia, RHUC2 612076 AD/AR 13 SLC2A9 glucose transporter), member 9 solute carrier family 34 (sodium Hypophosphatemic nephrolithiasis/osteoporosis-1, 23

23 NM_003052.4 612286 / 613388 AD/AR 13 SLC34A1 phosphate), member 1 NPHLOP1 / Fanconi renotubular syndrome 2 solute carrier family 34 (sodium 24

24 NM_001177316.1 Hypophosphatemic rickets with hypercalciuria 241530 AR 12 SLC34A3 phosphate), member 3 solute carrier family 3 (cystine, dibasic and neutral amino acid transporters, 25

25 NM_000341.3 Cystinuria, type A 220100 AR 10 SLC3A1 activator of cystine, dibasic and neutral amino acid transport), member 1 solute carrier family 4, anion exchanger, Primary distal renal tubular acidosis, dominant / 26

26 member 1 (erythrocyte membrane NM_000342.3 179800 / 611590 AD/AR 19 SLC4A1 recessive protein band 3, Diego blood group) solute carrier family 7 (glycoprotein- 27

27 SLC7A9 associated amino acid transporter light NM_014270.4 Cystinuria, type B 220100 AD/AR 12 chain, bo,+ system), member 9 solute carrier family 9, subfamily A Hypophosphatemic nephrolithiasis/osteoporosis-2, 28

28 (NHE3, cation proton antiporter 3), NM_004252.4 612287 AD 6 SLC9A3R1 NPHLOP2 member 3 regulator 1 vitamin D (1,25- dihydroxyvitamin D3) 29

29 NM_000376.2 Idiopathic hypercalciuria 277440 AD 11 VDR receptor 30

30 XDH xanthine dehydrogenase NM_000379.3 Xanthinuria, type 1 278300 AR 36 381 aGene symbols are underlined whenever putatively causative mutations were detected in the present study. For HNF4A the MIM-phenotype number denotes MODY type 1, as occurrence of Fanconi syndrome and NC has only been shown in the presence of a specific allele (p.R76W).

Abbreviations: AD, autosomal dominant; AR, autosomal recessive; NC, isolated nephrocalcinosis; NL, nephrolithiasis; Ref., reference; XR, x- chromosomal recessive.

REFERENCES

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Paracellin-1, a renal tight junction protein required for paracellular Mg2+ resorption. Science 285, 103-6 (1999). 11. Konrad, M. et al. Mutations in the Tight-Junction Gene Claudin 19 (CLDN19) Are Associated with Renal Magnesium Wasting, Renal Failure, and Severe Ocular Involvement. The American Journal of Human Genetics 79, 949-957 (2006). 12. Schlingmann, K.P. et al. Mutations in CYP24A1 and Idiopathic Infantile Hypercalcemia. New England Journal of Medicine 365, 410-421 (2011). 13. Jaureguiberry, G. et al. Nephrocalcinosis (Enamel Renal Syndrome) Caused by Autosomal Recessive FAM20A Mutations. Nephron Physiology 122, 1-6 (2012). 14. Cramer, S.D., Ferree, P.M., Lin, K., Milliner, D.S. & Holmes, R.P. The Gene Encoding Hydroxypyruvate Reductase (GRHPR) is Mutated in Patients with Primary Hyperoxaluria Type II. Hum Mol Genet 8, 2063-2069 (1999). 15. Hamilton, A.J. et al. The H1)$ 5: PXWDWLRQ FDXVHV DW\SLFDO GRPLQDQW )DQFRQL V\QGURPH LQ DGGLWLRQ WR D ȕ FHOO SKHQRW\SH Journal of 51, 165-169 (2014). 16. Belostotsky, R. et al. Mutations in DHDPSL Are Responsible For Primary Hyperoxaluria Type III. The American Journal of Human Genetics 87, 392-399 (2010). 17. Davidson BL, T.S., Van Antwerp M, Gibbs DA, Watts RW, Kelley WN, Palella TD. Identification of 17 independent mutations responsible for human hypoxanthine-guanine phosphoribosyltransferase (HPRT) deficiency. American journal of human genetics 48(1991). 18. Simon, D.B. et al. Genetic heterogeneity of Bartter's syndrome revealed by mutations in the K+ channel, ROMK. Nat Genet 14, 152-6 (1996). 19. Reilly DS, L.R., Ledbetter DH, Nussbaum RL. Tightly linked flanking markers for the Lowe oculocerebrorenal syndrome, with application to carrier assessment. American Journal of Human Genetics 42(1988). 20. Simon, D.B. et al. Bartter's syndrome, hypokalaemic alkalosis with hypercalciuria, is caused by mutations in the Na-K-2Cl cotransporter NKCC2. Nat Genet 13, 183-8 (1996). 21. Enomoto, A. et al. Molecular identification of a renal urate-anion exchanger that regulates blood urate levels. Nature 417, 447-452 (2002). 22. Matsuo, H. et al. Mutations in Glucose Transporter 9 Gene SLC2A9 Cause Renal Hypouricemia. The American Journal of Human Genetics 83, 744-751 (2008). 23. Prié, D. et al. Nephrolithiasis and Osteoporosis Associated with Hypophosphatemia Caused by Mutations in the Type 2a Sodium±Phosphate Cotransporter. New England Journal of Medicine 347, 983-991 (2002). 24. Lorenz-Depiereux, B. et al. Hereditary Hypophosphatemic Rickets with Hypercalciuria Is Caused by Mutations in the Sodium-Phosphate Cotransporter Gene SLC34A3. The American Journal of Human Genetics 78, 193-201 (2006). 25. Calonge, M.J. et al. Cystinuria caused by mutations in rBAT, a gene involved in the transport of cystine. 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Supplemental Table 3. Coverage and variant statistics for 336 DNA samples and 428 amplicons representing the coding exons of 30 genes known to be mutated in NL/NC/HC.

Coverage statistics a

Total # Percentage Median coverage per individual 261x - - individuals with coverage 0 0 0.0% - individuals with coverage <10x 1 0.3% - individuals with coverage <20x 1 0.3% - individuals with coverage <100x 4 1.2% Median coverage per amplicon 264x - - amplicons with coverage 0 9 2.1% - amplicons with coverage <10x 14 3.3% - amplicons with coverage <20x 21 4.9% - amplicons with coverage <50x 35 8.2% a Median coverage per individual/sample was 261x. One individual/sample failed to undergo multiplex amplification (0.3%), highlighted in grey. Median coverage per amplicon reached 264x, with 14 amplicons (3.3%) showing less than 10x coverage in total (highlighted in grey). These amplicons were FAM20A-exon1_1, FAM20A-exon1_2, SLC9A3R1-exon1_1, SLC9A3R1- exon1_2, CA2-exon1, CA2-exon3, APRT-exon1, SLC34A3-exon2, SLC34A3-exon9, OCRL- exon1, CLCNKB-exon20, HPRT1-exon1, HPRT1-exon4, and VDR-exon2. Note that among failed exons, the first coding exon of a gene was highly overrepresented, presumably due to generally greater GC-content.

Variant statistics b

Total # Percentage All variants 13,835 100% SNV 12,589 91% DIV 1,246 9% Retained deleterious variants after filtering 162 1.2% Sanger confirmed variants 139 1.0% Disease causing variants 50 0.4% b Total number of variant calls (grey) in 336 DNA samples and percentage of SNV/DIV calls after mapping to the concatenated reference sequence of 30 NL/NC/HC genes. The variant ILOWHULQJ SURFHVV LV GHVFULEHG LQ WKH µ0HWKRGV¶ section. Abbreviations: SNV, single nucleotide variant; DIV, deletion/insertion variant. Supplemental Table S4. Clinical characteristics of 41 individuals (40 families) with newly established genetic/molecular diagnoses and their allele frequency compared to the general population.

Gene, CKD- Urine Serum Imaging Other Ethnicity Nucleotide AA EVS (All alleles) Individuala stage abnormalities abnormalities (US/CT/KUB) change change (ESRD at age) SLC7A9 B208-21 1 F\VWLQHĹ - - - Albanian c.313G>A p.Gly105Arg AA=0/AG=4/GG=6499 CaOx stone, 6 mm normal urinary JAS-C8 1 - uric DFLGĹ White British c.313G>A p.Gly105Arg AA=0/AG=4/GG=6499 calculus RUJ Ox + Ca2+, CU not quantified urinary tract JAS-E5 1 F\VWLQHĹ - NC White British c.313G>A p.Gly105Arg AA=0/AG=4/GG=6499 infections solitary renal cyst, c.313G>A p.Gly105Arg AA=0/AG=4/GG=6499 F1029-21 1 - - increased - German echogenicity c.544G>A p.Ala182Thr AA=1/AG=42/GG=6460 cystine stones 5 c.313G>A p.Gly105Arg AA=0/AG=4/GG=6499 JAS-D30 F\VWLQHĹ - - including White British (45) staghorn calculi c.614del p.Lys205Argfs*59 A1A1=0/A1R=2/RR=6257 JAS-D31 1 F\VWLQHĹ - - cystine stones White British c.411_412del p.Pro139Leufs*69 A1A1=69/A1R=78/RR=6112 JAS-D47 1 F\VWLQHĹ - - cystine stones White British c.411_412del p.Pro139Leufs*69 A1A1=69/A1R=78/RR=6112 JAS-D34 1 F\VWLQHĹ - - cystine stones White British c.411_412del p.Pro139Leufs*69 A1A1=69/A1R=78/RR=6112 JAS-D28 1 F\VWLQHĹ - - cystine stones White British c.544G>A p.Ala182Thr AA=1/AG=42/GG=6460 renal calculus JAS-F41 1 &DĹ - seen on KUB, - White British c.544G>A p.Ala182Thr AA=1/AG=42/GG=6460 radioopaque cystine not JAS-F50 1 - uULFDFLGĹ radiolucent stone White British c.544G>A p.Ala182Thr AA=1/AG=42/GG=6460 quantified c.544G>A p.Ala182Thr AA=1/AG=42/GG=6460 JAS-D57 1 F\VWLQHĹ - - cystine stones White British c.614dup p.Asn206Glufs*3 A1A1=0/A1R=2/RR=6257 radioopaque JAS-F87 1 &DĹ - stones, large CaPO- stones White British c.614dup p.Asn206Glufs*3 A1A1=0/A1R=2/RR=6257 > 15 mm JAS-G10 1 F\VWLQHĹ - - cystine stones c.614dup p.Asn206Glufs*3 A1A1=0/A1R=2/RR=6257 JAS-D29 1 F\VWLQHĹ - - cystine stones White British c.671C>T p.Ala224Val - JAS-D55 1 F\VWLQHĹ - - cystine stones White British c.671C>T p.Ala224Val - tiny bilateral c.671C>T p.Ala224Val - JAS-F19 1 &DĹ PLOG PO-Ļ calculi - White British on KUB c.1369T>C p.Tyr457His CC=0/CT=2/TT=6499 bladder stone on European c.814del p.Val272Cysfs*6 - B114-21 1 F\VWLQHĹ - cystine stones US American c.997C>T p.Arg333Trp TT=0/TC=2/CC=6501 c.1353C>A p.Tyr451* - JAS-E9 1 F\VWLQHĹ - - cystine stones White British c.1400-2A>G 3' splice - ADCY10 JAS-F8 1 &DĹ - - - Polish c.1263C>A p.Tyr421* - radioopaque renal JAS-F68 1 &DĹ - calculus 6 x 4 mm CaOx stone White British c.1282G>A p.Asp428Asn AA=0/AG=1/GG=6502 ± suspected NC bilateral calculi normal urinary JAS-F29 1 - PO-Ļ on CT, 2 small White British c.4477del p.Leu1493Serfs*24 A1A1=0/A1R=3/RR=6257 Ca kidney cysts SLC2A9 B179-21 1 - - - - Albanian c.1343C>T p.Pro448Leu TT=0/TC=1/CC=6502 fractionary B230-21 1 XULFDFLGĹ uULFDFLGĹ - - Macedonian c.1419+1G>A 5' splice - &DĹ SLC9A3R1 normal serum B224-21 1 &DĹ - stone on US Macedonian c.673G>A p.Glu225Lys AA=0/AG=31/GG=6472 PO- Ulcerative European B109-21 1 - - stone on CT c.888+2T>C 5' splice site CC=0/CT=6/TT=6497 colitis American SLC22A12 no HC, serum CKĹ, muscle JAS-F98 1 - uULFDFLGĹ - White British c.431T>C p.Leu144Pro CC=0/CT=5/TT=6493 pains, CaOx stones reported Ca- European B155-12 1 - - - c.1300C>T p.Arg434Cys TT=0/TC=1/CC=6484 stones American SLC4A1 JAS-E8 1 &DĹ - NC - White British c.2716G>C p.Glu906Gln - SLC3A1 JAS-B21a 1 F\VWLQHĹ - - cystine stones White British c.1354C>T p.Arg452Trp no JAS-B22a 1 F\VWLQHĹ - - cystine stones White British c.1354C>T p.Arg452Trp no JAS-G7 1 F\VWLQHĹ - - cystine stones White British c.1400T>C p.Met467Thr CC=0/CT=27/TT=6476 ATP6V1B1 no known B214-21 1 &DĹ HCO3-Ļ - hearing Gypsy c.481G>A p.Glu161Lys AA=4/AG=294/GG=6205 problems CLCN5 B111-21 1 - - NC - Filipino c.344G>A p.Trp115* no 5 B167-21 R[DODWHĹ 0JĻ NC - Macedonian c.1009G>A p.Glu337Lys no (25) CLDN16 5 recurrent JAS-C1 &DĹ 0JĻ NC Arabian c.445C>T p.Arg149* no (38) stones B178-21 2 &DĹ 0JĻ NC - Macedonian c.453G>T p.Leu151Phe no CYP24A1 no FTT, no B223-21 1 &DĹ 0JĻ NC infantile onset Albanian c.428_430del p.Glu143del no (age 12 yr) AGXT CaOx stones, European c.416_418del p.Val139del no B106-21 1 R[DODWHĹ - - not responsive American to pyridoxin c.846+1G>T ¶VSOLFHVLWH no SLC34A1 R[DODWHĹ PO-Ļ, severe FTT, cow-milk c.271_291del p.Val91Ala97del7 A1A1=1/A1R=229/RR=6029 B168-21 1 (pyridoxine metabolic NC Macedonian sensitive) acidosis c.1534C>T p.Arg512Cys no SLC34A3

bilat. calculi on c.1454G>A p.Arg485His AA=0/AG=18/GG=6469 JAS-F43 1 - PO-Ļ - White British US c.1585A>T p.Ile529Phe TT=0/TA=15/AA=6341 aJAS-B21 and JAS-B22 are siblings (same family). All other listed individuals are unrelated.

Abbreviations: AA, amino acid; AKI, ; bilat., bilateral; Ca, calcium; CaOx, calcium oxalate; CaPO, calcium phosphate; CKD, chronic kidney disease; CT, computed tomography; CU, cystinuria; ESRD, end-stage renal disease; EVS, Exome variant sever (http://evs.gs.washington.edu/EVS/); FTT, failure to thrive; HC, hypercalciuria; HO, hyperoxaluria; HCO3-, bicarbonate; HU, hypouricemia; KUB, x-ray of kidney, ureter, and bladder; Mg2+, magnesium; NC, nephrocalcinosis; NL, nephrolithiasis; PO-, phosphate; PH, primary hyperoxaluria; RUJ, right pelvicalyceal-ureteric junction; RTX, renal transplant; US, ultrasound; yr, years.