Prevalence of Monogenic Causes in Pediatric Patients with Nephrolithiasis Or Nephrocalcinosis

Article

Prevalence of Monogenic Causes in Pediatric Patients with Nephrolithiasis or Nephrocalcinosis

Daniela Anne Braun,* Jennifer Ashley Lawson,* Heon Yung Gee,*† Jan Halbritter,*‡ Shirlee Shril,* Weizhen Tan,* | Deborah Stein,* Ari J. Wassner,§ Michael A. Ferguson,* Zoran Gucev, Brittany Fisher,* Leslie Spaneas,* | Jennifer Varner,* John A. Sayer,¶ Danko Milosevic,** Michelle Baum,* Velibor Tasic, and Friedhelm Hildebrandt*††

Abstract Background and objectives Nephrolithiasis is a prevalent condition that affects 10%–15% of adults in their *Division of Nephrology, lifetime. It is associated with high morbidity due to colicky pain, the necessity for surgical intervention, and Department of sometimes progression to CKD. In recent years, multiple monogenic causes of nephrolithiasis and Medicine and §Division nephrocalcinosis have been identified. However, the prevalence of each monogenic gene in a pediatric renal of Endocrinology, stone cohort has not yet been extensively studied. Department of Medicine, Boston Children’s Hospital, Design, setting, participants, & measurements To determine the percentage of cases that can be explained Harvard Medical molecularly by mutations in one of 30 known nephrolithiasis/nephrocalcinosis genes, we conducted a School, Boston, high-throughput exon sequencing analysis in an international cohort of 143 individuals ,18 years of age, Massachusetts; † with nephrolithiasis (n=123) or isolated nephrocalcinosis (n=20). Over 7 months, all eligible individuals at Department of Pharmacology, Brain three renal stone clinics in the United States and Europe were approached for study participation. Korea 21 PLUS Project for Medical Sciences, Results We detected likely causative mutations in 14 of 30 analyzed genes, leading to a molecular diagnosis in Yon sei University 16.8% (24 of 143) of affected individuals; 12 of the 27 detected mutations were not previously described as disease College of Medicine, Seoul, Republic of causing (44.4%). We observed that in our cohort all individuals with infantile manifestation of nephrolithiasis or ‡ Korea; Division of nephrocalcinosis had causative mutations in recessive rather than dominant monogenic genes. In individuals Endocrinology/ who manifested later in life, causative mutations in dominant genes were more frequent. Nephrology, Department of Internal Conclusions We present the first exclusively pediatric cohort examined for monogenic causes of nephrolithiasis/ Medicine, University Clinic Leipzig, Leipzig, ║ nephrocalcinosis, and suggest that important therapeutic and preventative measures may result from Germany; Department mutational analysis in individuals with early manifestation of nephrolithiasis or nephrocalcinosis. of Pediatric Nephrology, Clin J Am Soc Nephrol 11: 664–672, 2016. doi: 10.2215/CJN.07540715 Medical Faculty Skopje, University Children’s Hospital, Skopje, Macedonia; ¶Institute of Introduction pediatric patients are more likely to have a monogenic Genetic Medicine, International Centre for The incidence of pediatric nephrolithiasis and nephro- cause of disease, mutational analysis is particularly Life, Newcastle calcinosis (NL/NC) has significantly increased over the relevant in this cohort. Furthermore, early detection of University, Newcastle last several decades (1). Furthermore, this condition is disease-causing mutations is of great importance in upon Tyne, United associated with high morbidity due to episodes of col- early-onset NL/NC because genetic diagnoses allow Kingdom; **Department icky pain, the necessity for surgical intervention, and fi of Pediatric Nephrology, nely tailored treatment plans that may prevent Dialysis and sometimes progression to CKD. Nephrolithiasis (NL) recurrent disease or progression to ESRD. Transplantation, Clinical and nephrocalcinosis (NC) share a recognized degree Until recently, mutation analysis for individuals Hospital Center Zagreb, of heritability. In twin studies, heritability accounted with NL/NC has not been widely accessible. How- University of Zagreb for nearly half of all NL/NC prevalence (2,3). Accord- ever, through the availability of high-throughput Medical School, Zagreb, Croatia; and ††Howard ing to the Online Mendelian Inheritance in Man multiplex PCR and next-generation sequencing, rapid Hughes Medical (OMIM) database, mutations in at least 30 genes can mutation analysis of multiple genes in large cohorts Institute, Chevy Chase, cause monogenic forms of NL/NC by autosomal re- has become an efficient and cost-effective screening Maryland cessive, autosomal dominant, or X-linked transmission. method (5–7). Recently, we studied 166 adults and 106 children with To identify the prevalence of monogenic causes in Correspondence: Dr. Friedhelm NC or NL and detected causative mutations in 11.4% early-onset NL/NC (onset before 18 years of age), we Hildebrandt, Division of of adult and 20.8% of early-onset cases (4). This result analyzed all coding exons and adjacent splice sites of Nephrology, Boston confirmed a significant occurrence of heritable NL/NC, 30 known NL/NC-causing genes with a defined Children’s Hospital, while also indicating that there are additional uniden- OMIM phenotype in a cohort of 143 children re- 300 Longwood Avenue, fi Boston, MA 02115. ti ed monogenic causes of NL/NC. cruited at three renal stone clinics with at least one Email: friedhelm. However, the contribution of monogenic causes of episode of NL or the presence of NC upon renal ul- hildebrandt@childrens. NL/NC has yet to be extensively studied. Because trasound before 18 years of age. This patient cohort harvard.edu

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had no patient overlap with the previously published All variants that were present in the homozygous state in cohort (4). healthy control cohorts (Exome Aggregation Consortium and Exome Variant Server) were excluded. The remaining variants were confirmed in original patient DNA by Sanger Materials and Methods sequencing. Whenever parental DNA was available, segre- Study Cohort gation analysis was performed. Final calling of variant This study was approved by the Institutional Review pathogenicity was performed by geneticists together with ’ Board of Boston Children s Hospital. Study inclusion cri- physician scientists, who had knowledge of the clinical fi fi teria were de ned as: rst clinical manifestation of NL phenotypes and pedigree structure. and/or presence of NC on renal ultrasound before 18 years of age. Individuals with conditions or medication Coverage Statistics that might have caused secondary renal stone disease were We achieved a median sequencing coverage of 1943 per excluded. To avoid selection bias, all patients seen at three 3 ’ individual, and 197 per amplicon. As previously pub- renal stone clinics (Boston Children s Hospital, University lished, median coverage values .203 are sufficient to ex- Clinic Skopje, and University Clinic Zagreb) over a finite – clude false-negative results in high-throughput exon period (February August 2014) were approached for sequencing (5,6). In this study, only eight of 143 individ- study participation. The exact percentage of individuals uals (5.7%), and 37 of 518 amplicons (7.1%) had a median who declined study participation was not recorded. After coverage ,203. This value is average for next-generation obtaining informed consent, clinical data, pedigree infor- sequencing; hence, this experiment fulfilled the necessary mation, and DNA samples were collected from 143 indi- quality criteria. viduals. All individuals were recruited after the conclusion of the study of Halbritter et al. (4), thereby excluding en- rollment overlap between the two studies. The cohort con- Web Resources Online resources used were as follows: sisted of 72 male and 71 female participants. Of these individuals, 123 had NL, 20 had NC based on renal ultra- UCSC Genome Browser, http://genome.ucsc.edu/cgi-bin/ sound, and three with NC also had reported NL. hgGateway; 1000 Genomes Browser, http://browser.1000genomes.org; Mutation Analysis Ensembl Genome Browser, http://www.ensembl.org; Mutation analysis was performed using a barcoded Exome Variant Server, http://evs.gs.washington.edu/EVS; multiplex PCR-based approach, as previously described Exome Aggregation Consortium, exac.broadinstitute.org; fi (5,6). We designed 518 target-speci c primers for 381 cod- OMIM, http://www.omim.org; ing exons and the adjacent splice sites of 30 genes that are PolyPhen-2, http://genetics.bwh.harvard.edu/pph2 (8); fi known monogenic causes of NL/NC (de ned by OMIM; SIFT, http://sift.jcvi.org (9); and www.ncbi.nlm.nih.gov/omim). The genes screened were: MutationTaster http://www.mutationtaster.org (10). ADCY10, AGXT, PRT, ATP6V0A4, ATP6V1B1, CA2, CASR, CLCN5, CLCNKB, CLDN16, CLDN19, CYP24A1, FAM20A, GRHPR, HNF4A, HOGA1, HPRT1, KCNJ1, OCRL, Results SLC12A1, SLC22A12, SLC2A9, SLC34A1, SLC34A3, We examined an international cohort of 143 individuals SLC3A1, SLC4A1, SLC7A9, SLC9A3R1, VDR,andXDH with early-onset NL or NC for the presence of mutations (Supplemental Tables 1 and 2). Amplicon sizes were chosen in 30 genes that cause NL/NC if mutated. We detected to range from 250 to 300 bp (primer sequences are avail- mutations in 14 of these 30 genes and established a able from the authors). The use of barcoded multiplex PCR molecular diagnosis likely to explain the disease phenotype (Fluidigm 48.48-Access Arrays system) allowed parallel in 24 of 143 unrelated individuals with NL/NC (16.8%) amplification of all 518 amplicons in 48 individuals at a (Tables 1 and 2). Pathogenic mutations were detected in time (5,6). Subsequently, the pooled libraries were se- nine recessive genes in 13 individuals: ATP6V1B1 (one in- quenced on an Illumina MiSeq instrument using the v2 dividual), ATP6V0A4 (one individual), CLDN16 (one indi- chemistry. Sequence reads were aligned to the human ref- vidual), CLDN19 (one individual), SLC3A1 (three erence sequence using CLC Genomics Workbench (CLC- individuals), CYP24A1 (two individuals), SLC12A1 (one bio, Aarhus, Denmark) (5). Prior to further evaluation, we individual), AGXT (one individual), and OCRL (two indi- excluded synonymous variants and variants that occur viduals) (Figure 1, Table 1). We also detected pathogenic with minor allele frequency .1% in the dbSNP (version mutations in five dominant genes in 11 individuals: 138) database. Remaining variants were validated as pre- ADCY10 (two individuals), SLC4A1 (one individual), viously described (7). Briefly, all variants that had previ- SLC9A3R1 (one individual), SLC34A1 (five individuals), ously been described in individuals with NL/NC were and VDR (two individuals) (Figure 1, Table 2). considered as likely to be disease causing. Novel variants No pathogenic mutations were detected in genes APRT, were ranked based on their likelihood to be deleterious for CA2, CASR, CLCN5, CLCNKB, FAM20A, GRHPR, HNF4A, the function of the encoded protein considering protein HOGA1, HPRT1, KCNJ1, SLC2A9, SLC22A12, SLC34A3, truncation and obligatory splice site mutations as likely SLC7A9,andXDH. Of the 27 detected mutations, 12 to be disease causing. For missense alleles, evolutionary (44.4%) were novel pathogenic variants that have not been conservation among orthologs across phylogeny, and bio- previously reported in databases of human disease-causing informatics prediction programs (PolyPhen-2 [8], SIFT [9], mutations. In this study, no additional functional studies and MutationTaster [10]) were taken into consideration. were performed to validate the pathogenicity of these alleles. 6 lnclJunlo h mrcnSceyo Nephrology of Society American the of Journal Clinical 666

Table 1. Molecular genetic diagnoses established in 13 of 143 (9.1%) individuals from 143 families with NL/NC in one of nine recessive or X-linked genes

Clinical Genetic Practical Gene [Protein] PPh2, Diagnosis Diagnosis Implication Nucleotide Amino Acid Zygosity Age of Stone (Individual Evolutionary Reference Sex NL/NC (Before (After (Following Change Change State Onset, yr Analysis with Mutation) Conservation Mutational Mutational Genetic Analysis) Analysis) Diagnosis)a

ATP6V1B1 c.242T.C p.Leu81Pro hom 1.00, X.t. 14 M 7 NL CaOx Incomplete dRTA, Hearing screen, [ATPase, H+ dRTA deafness monitor for transporting, ↑ K+, no lysosomal diagnostics 56/58 kDa, for secondary V1 subunit B1] causes, genetic (B482)b counseling ATP6V0A4 c.292–1G.A obligatory comp het N/A Novel F ,1 NL+NC N/A HC, HK, dRTA Monitor for ↑ K+, [ATPase, H+ splice dRTA, no diagnostics transporting, c.1346G.T p.Arg449Leu 1.00, X.t. 34 metabolic for secondary lysosomal V0 acidosis causes, genetic subunit a4] counseling (B329)c CLDN16 c.453G.T p.Leu151Phe hom 0.99, X.t. 17 M 13 NC N/A FHHNC FHHNC Monitor for [Claudin 16] tetany and (B604)b seizures, no diagnostics for secondary causes, genetic counseling CLDN19 c.59G.A p.Gly20Asp hom 0.99, D.r. 18 F 15 NL+NC N/A Medullary HOMG5 Evaluate for [Claudin 19] NC, HC, eye disease, (A4592)c HM monitor for NC, genetic counseling SLC3A1 c.647C.T p.Thr216Met hom 0.98, D.r. 35 F 2 NL Cystine Cystinuria, HO Cystinuria Very high ﬂuid [Cystine, dibasic c.647C.T p.Thr216Met hom 0.98, D.r. 35 M 1 NL Cystine Cystinuria Cystinuria intake, and neutral amino c.1094G.A p.Arg365Gln comp 0.94, D.r. 36 F 17 NL Cystine Cystinuria Cystinuria treatment acid transporters, c.1400T.C p.Met467Thr het 0.29, D.r. 24 with tiopronin activator of cystine, and potassium dibasic, and neutral amino citrate, acid transport] alkalinization (B425)b of urine, limit (B458)b animal protein (B499)c intake, genetic counseling CYP24A1 c.1186C.T p.Arg396Trp hom 1.00, D.r. 19 M 5 NL N/A NL HC/NL Avoidance of [Cytochrome P450, c.1147G.C p.Glu383Gln comp 1.00, D.r. Novel F 0.8 NC CaOx Idiopathic HC HC/NL exogenous family 24, subfamily c.428_430del p.Glu143del het N/A, D.r. 19 Vitamin D A, polypeptide 1] and extreme (B540)b sunlight, (B607)b monitor for hypercalcemia with peptic ulcers and pancreatitis, genetic counseling lnJA o eho 1 6–7,Arl 2016 April, 664–672, 11: Nephrol Soc Am J Clin Table 1. (Continued)

SLC12A1 c.2755G.C p.Asp919His hom 1.00, G.g. Novel F 0.3 NC N/A BS BS Treatment with [Sodium/potassium/ indomethacin chloride transporter] and electrolyte (B446)b substitution, genetic counseling AGXT c.508G.A p.Gly170Arg hom 1.00, D.r. 12 F 0.6 NC CaOx Primary PH1 Trial treatment [Alanine-gloxylate hyperoxaluria with pyridoxine, aminotransferase] ophthalmology (B424)b screen, cardiac screen, monitor for oxalates and tissue calciﬁcation, genetic counseling OCRL c.1484C.T p.Pro495Leu hem 1.00, D.r. 37 M 10 NL N/A LS LS/DD2 Consult with [Oculocerebrorenal c.2510G.A p.Arg837His hem 0.23, D.r. Novel M 7 NL CaOx Idiopathic HC LS/DD2 ophthalmology, syndrome of Lowe] monitor for (B422)b seizures, (B199)b genetic counseling

PPh2, Polyphen2-HumVar (http://genetics.bwh.harvard.edu/pph2/); NL, nephrolithiasis; NC, nephrocalcinosis; H+, proton; hom, homozygous; X.t., Xenopus tropicalis; M, male; CaOx, 667 al. et Braun Disease, Stone Urinary Early-Onset in Genes Monogenetic calcium oxalate; dRTA, distal renal tubular acidosis; ↑, increased; K+, potassium; comp het, compound heterozygous; F, female; n/a, not available; HC, hypercalciuria; HK, hypokalemia; FHHNC, familial hypomagnesemia with hypercalciuria and nephrocalcinosis; D.r., Dario rerio; HM, hypomagnesemia; HOMG5, hypomagnesemia 5, renal, with ocular involvement; HO, hyperoxaluria; N/A, not available; G.g., Gallus gallus; BS, Bartter syndrome; PH1, primary hyperoxaluria, type 1; hem, hemizygous; LS, Lowe syndrome; DD2, Dent disease 2. aPractical implications are based off the deﬁned Online Mendelian Inheritance of Man database phenotype (http://www.omim.org). bPatients derive from the Balkan region. cPatients are American. 6 lnclJunlo h mrcnSceyo Nephrology of Society American the of Journal Clinical 668

Table 2. Molecular genetic diagnoses established in 11 of 143 (7.7%) individuals from 143 families with NL/NC in one of ﬁve dominant genes

Genetic Practical Clinical Gene [Protein] PPh2, Diagnosis Implication Nucleotide Amino Acid Zygosity Age of Stone Diagnosis (Before (Individual with Evolutionary Reference Sex NL/NC (After (Following Change Change State Onset, yr Analysis Mutational mutation) Conservation Mutational Genetic Analysis) Analysis) Diagnosis)a

ADYC10 c.1052C.A p.Pro351His het 1.00, G.g Novel M 15 NL CaOx HC Absorptive HC Genetic [Adenylate c.758G.A p.Cys253Tyr het 0.69, M.m. Novel M 1.5 NL+NC N/A HC Absorptive HC counseling cyclase 10 (soluble)] (B580)b (B599)b SLC4A1 c.1766G.A p.Arg589His het 0.95, X.t. 25 F 11 NC N/A dRTA + cysts Primary dRTA Monitor for [Anion exchanger hereditary (Diego blood spherocytosis, group)] osteomalacia, (B280)b hypokalemia, and periodic paralysis, genetic counseling SLC9A3R1 c.328C.G p.Leu110Val het 0.10, M.m. 27 F 11 NL N/A HPh NPHLOP2 Monitor bone [NHE3, cation density, proton antiporter 3] genetic (B529)c counseling SLC34A1 c.458G.T p.Gly153Val het 1.00, D.r. Novel M 4 NL N/A HPh, NPHLOP1/FS Screen for [Type 2 sodium/ idiopathic HC deafness, phosphate c.398C.T p.Ala133Val het 0.99, D.r. 30 M 3 NL N/A HPh, HC NPHLOP1/FS genetic cotransporter] c.437C.T p.Pro146Leu het 0.95, D.r. Novel M 3 NL N/A HPh NPHLOP1/FS counseling (B491)c c.1367T.A p.Ile456Asn het 0.99, D.r. Novel F 9 NL CaOx, HSP, HC NPHLOP1/FS (B523)c CaPh (B484)c c.1348G.A p.Gly450Ser het 0.99, D.r. Novel M 7 NL CaOx HPh, NPHLOP1/FS (B610)b,d idiopathic HC (B417)c VDR c.260A.G p.Asn87Ser het 0.99, D.r. Novel M 12 NL N/A HPh VDDR2A Calcium [Vitamin D (1, 25- c.1207G.A p.Glu403Lys het 0.90, D.r. Novel F 4 NL N/A HPh VDDR2A supplements, dihydroxyvitamin genetic D3) receptor] counseling (B481)c (B447)c

PPh2, Polyphen2-HumVar (http://genetics.bwh.harvard.edu/pph2/); NL, nephrolithiasis; NC, nephrocalcinosis; het, heterozygous; G.g., Gallus gallus; M, male; CaOx, calcium oxalate; HC, hypercalciuria; M.m., Mus musculus; N/A, not available; X.t., Xenopus tropicalis; F, female; dRTA, distal renal tubular acidosis; HPh, hypophosphatemia; NPHLOP2, hypophosphatemic nephrolithiasis/osteoporosis, 2; D.r., Danio rerio; NPHLOP1, hypophosphatemic nephrolithiasis/osteoporosis, 1; FS, Fanconi syndrome; CaPh, calcium phosphate; HSP, Henoch-Schonlein purpura; VDDR2A, Vitamin D-dependent rickets, type 2A. aPractical implications are based off the deﬁned Online Mendelian Inheritance of Man database phenotype (http://www.omim.org). bPatients are American. cPatients derive from the Balkan region. dB610 is an adopted individual and biologic family information is unavailable. Clin J Am Soc Nephrol 11: 664–672, April, 2016 Monogenetic Genes in Early-Onset Urinary Stone Disease, Braun et al. 669

Figure 1. | Established molecular diagnoses in 24 of 143 (16.8%) individuals with nephrolithiasis/nephrocalcinosis (NL/NC). A ﬂow chart showing the distribution for molecular diagnoses of nephrolithiasis (NL) or nephrocalcinosis (NC), and for recessive and dominant inheritance. aBy renal ultrasound. b“Solved” denotes that two recessive or one dominant mutation(s) were/was detected that explain the disease phenotype of NL or NC.

To determine a possible correlation between sex and where most children with NL/NC are seen. Therefore, monogenic causes of disease, we analyzed the sex of the the cohort may have a certain bias toward severe cases molecularly solved individuals normalized to that of the and future studies might show that the percentage of cohort. The cohort consisted of 72 males and 71 females. monogenic causes in isolated NL/NC is lower than ob- Among individuals with pathogenic mutations, 13 were served in this study. male and 11 were female (Tables 1 and 2), resulting in no The monogenic causes of NL/NC reported in the significant difference in the detection of pathogenic muta- literature are very heterogeneous (11–28). This heterogene- tions between sexes. ity is evident in our cohort, in which causative mutations We observed that in the infantile subgroup of five were distributed among 14 of the 30 genes screened. This individuals ,1 year of age when the disease first manifes- finding demonstrates that broad genetic screening, as per- ted, all individuals (five of five) had pathogenic mutations formed in this study, is necessary to establish a molecular in a recessive gene (Figure 2), including ATP6V0A4, diagnosis in monogenic cases of early-onset NL/NC. SLC3A1, CYP24A1, SLC12A1,andAGXT. No individual We analyzed the age distribution of individuals in whom in this age group had a pathogenic mutation in a dominant we identified a causative mutation. Recessive monogenic gene (Figure 2). Among the subgroup of 19 individuals diseases typically manifest earlier in life than dominant .1 year, eight of 19 (42.1%) individuals had a pathogenic monogenic diseases (4) and our results reflect this finding mutation in a recessive gene (Figure 2), including for monogenic causes of NL/NC. As shown in Figure 2, all ATP6V1B1, CLDN16, CLDN19, SLC3A1, CYP24A1,or individuals with infantile manifestation of NL/NC in our OCRL. The remaining 11 of 19 (57.9%) individuals had a cohort harbored causative mutations in recessive rather pathogenic mutation in a dominant gene, including than dominant monogenic genes. In contrast, causative ADYC10, SLC4A1, SLC9A3R1, SLC34A1,orVDR (Figure 2). mutations in dominant monogenic genes were more fre- These results show that in our cohort recessive causes of quently observed in individuals in whom the disease man- diseaseweremorefrequentininfantile-onsetNL/NC, ifested later in life. whereas dominant causes were more frequent thereafter. In recessive cases, we observed a surprisingly high number of homozygous (ten of 13) as compared with Discussion compound heterozygous (three of 13) mutations. We Pediatric-onset NL and NC are often overlooked clinical therefore revisited all respective cases to specifically exclude conditions that frustrate both clinicians and families. In parental consanguinity. However, none of these children recent years, multiple monogenic causes of NL/NC have were knowingly born of consanguineous unions. Conse- been identified (11–28), but the prevalence of mutations in quently, at this time we cannot provide a satisfying expla- each monogenic gene in a pediatric NL/NC cohort has not nation for this phenomenon. yet been extensively studied. A major challenge of high-throughput mutational anal- Here, we performed mutational analysis in a cohort of ysis is to differentiate between pathogenic mutations and 143 individuals with NL or NC onset before 18 years of benign variants. We distinguished between alleles that had age. We sequenced the coding regions of 30 genes known previously been described in individuals with NL/NC, and to cause monogenic NL/NC, and identified a causative thus were likely to be causative, and novel variants. Novel mutation in 24 of 143 individuals (16.8%). This percentage variants were only considered as likely to be disease confirms the findings of Halbritter et al. who identified a causing if they were protein-truncating, affected highly monogenic cause of NL/NC in 20.8% of children in a sep- conserved amino acid residues, were predicted to be arate/nonoverlapping mixed adult and pediatric cohort damaging in bioinformatics prediction programs, and (4). However, participants for both studies were recruited were not present in the homozygous state in healthy in specialized renal stone clinics at tertiary care centers control individuals. 670 Clinical Journal of the American Society of Nephrology

Figure 2. | Distribution of established molecular genetic causes of nephrolithiasis/nephrocalcinosis (NL/NC). Number of individuals with molecular diagnoses grouped by age of onset, and genetic heritability (recessive versus dominant). Note that, in the infantile subgroup, recessive diagnoses are more frequent, whereas in patients .1 year at onset of disease dominant diagnoses are more frequent.

The gene SLC34A1 was originally reported as an auto- unaffected family members. In these situations we recom- somal-dominant disease gene (23). However, multiple mend that affected individuals initiate discussion on genetic groups later questioned the pathogenicity of single hetero- counseling and/or mutation analysis for healthy relatives at zygous alleles in SLC34A1 (29–31). At this point, the path- risk (Tables 1 and 2). Ultimately, information resulting from ogenicity of single heterozygous alleles in SLC34A1,as mutation analysis will guide clinicians to monitor individuals identified in five individuals with NL/NC in this study, for development of disease and to institute preventative cannot be clarified definitely. treatment when possible. Heritability has been suggested to account for nearly 50% Furthermore, consensus guidelines recommend standard of NL/NC cases (2,3). The study of Halbritter et al. has treatment for NL/NC, such as increased fluid intake, shown that known NL/NC genes account for 11.4% of limited sodium intake, treatment with thiazide diuretics, adult-onset NL/NC and 20.8% of early-onset NL/NC and potassium citrate therapy (33), that may not directly (4). These percentages indicate that many more NL/NC- address the pathophysiology of a particular molecular di- associated genes remain to be identified. Monogenic con- agnosis. For example, although standard measures may ditions, particularly recessive conditions, are commonly address NC associated with CLDN16 mutations, with a not appreciated as being of genetic origin because many definite molecular diagnosis clinicians will also know to appear as sporadic cases, since the parents will be healthy monitor for tetany and seizures, which have been reported heterozygous carriers of one mutated allele. However, for certain CLDN16 mutations (Table 1). Therefore, we sug- with approximately one in five NL/NC patients harboring gest “Practical Implications” (Tables 1 and 2) for each gene monogenic mutations, there are many indicators of inher- in which we detected a likely disease-causing mutation. ited disease that clinicians should be aware of. Examples of Inconclusion,wehaveshownthatmutationsina such indicators are: early onset, familial prevalence, familial heterogeneous array of genes can be identified in 16.8% consanguinity, multiple or recurrent stones, and NC. An in- of individuals with early-onset NL/NC, and we suggest depth discussion of when to suspect a genetic condition in that specific genetic diagnoses in such cases hold vast NL/NC is outside the scope of this publication, however, potential for personalization of treatment plans. Therefore, other groups have reviewed this in great detail (32). genetic screening should be implemented in the clinical A molecular genetic diagnosis has vast implications for practice of pediatric patients with NL or NC, because both affected individuals and unaffected family members. knowledge of the molecular diagnosis may change the As addressed in Tables 1 and 2, genetic screening of approach to prophylaxis and treatment. asymptomatic relatives may identify individuals carrying the same disease-causing mutation. Because molecular ge- Acknowledgments netic screening in healthy relatives of individuals with We thank the physicians and the participating families for their monogenic disease is generally discouraged, we initially re- contribution. F.H. is an Investigator of the Howard Hughes Medical frained from performing mutation analysis in healthy rela- Institute, a Doris Duke Distinguished Clinical Scientist, and the tives. However, following identification of certain causative Warren E. Grupe Professor of Pediatrics. mutations in affected individuals, the resulting knowledge This research was supported by grants from the National Insti- might have important prophylactic implications for currently tutes of Health (DK1069274, DK1068306, and DK064614 to F.H.), and Clin J Am Soc Nephrol 11: 664–672, April, 2016 Monogenetic Genes in Early-Onset Urinary Stone Disease, Braun et al. 671

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MIM- Coding Gene Symbol Gene Name Accession # Disease entity Mode Ref. Phenotype # Exons 1 ADCY10/SAC adenylate cyclase 10 (soluble) NM_018417.4 Hypercalciuria, Calcium oxalate nephrolithiasis 143870 AD 32 1

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

Adenine phosphoribosyltransferase deficiency, 3 APRT adenine phosphoribosyltransferase NM_000485.2 614723 AR 5 3 Urolithiasis (DHA stones), renal failure ATPase, H+ transporting, lysosomal V0 4 ATP6V0A4 NM_020632.2 dRTA 602722 AR 20 4 subunit a4 ATPase, H+ transporting, lysosomal Distal renal tubular acidosis (dRTA) with 5 ATP6V1B1 NM_001692.3 267300 AR 14 5 56/58kDa, V1 subunit B1 deafness 6 CA2 carbonic anhydrase II NM_000067.2 Osteopetrosis + d/pRTA 259730 AR 7 6

Hypocalcemia with Bartter syndrome / 7 CASR calcium-sensing receptor NM_001178065.1 601198 AD 6 7 hypocalcemia, autosomal dominant 300009 / 8 CLCN5 chloride channel, voltage-sensitive 5 NM_001127898.3 Dent disease / Nephrolithiasis, type 1 XR 14 8 310468 9 CLCNKB chloride channel, voltage-sensitive Kb NM_000085.4 Bartter syndrome, type 3 607364 AR 19 9

Familial hypomagnesemia with hypercalciuria & 10 CLDN16 claudin 16 NM_006580.3 248250 AR 5 10 nephrocalcinosis, FHHNC Familial hypomagnesemia with hypercalciuria & 11 CLDN19 claudin 19 NM_001123395.1 248190 AR 4 11 nephrocalcinosis with ocular abnormalities cytochrome P450, family 24, subfamily 1,25-(OH) D-24 hydroxylase deficiency , infantile 12 CYP24A1 NM_000782.4 143880 AR 11 12 A, polypeptide 1 Hypercalcemia family with sequence similarity 20, Enamel-Renal syndrome, amelogenesis 13 FAM20A NM_017565.3 204690 AR 12 13 member A imperfecta and nephrocalcinosis glyoxylate reductase/hydroxypyruvate 14 GRHPR NM_012203.1 Primary hyperoxaluria, type 2 260000 AR 9 14 reductase 15 HNF4A hepatocyte nuclear factor 4, alpha NM_000457.4 MODY + Fanconi syndrome + Nephrocalcinosis 125850 AD 1 15 16 HOGA1 4-hydroxy-2-oxoglutarate aldolase 1 NM_138413.3 Primary hyperoxaluria, type 3 613616 AR 7 16 hypoxanthine Kelley-Seegmiller syndrome, partial HPRT 17 HPRT1 NM_000194.2 300323 XR 9 17 phosphoribosyltransferase 1 deficiency, HPRT-related gout potassium inwardly-rectifying channel, 18 KCNJ1 NM_000220.4 Bartter syndrome, type 2 241200 AR 2 18 subfamily J, member 1 309000 / 19 OCRL oculocerebrorenal syndrome of Lowe NM_000276.3 Lowe syndrome / Dent disease 2 XR 24 19 300555 20 SLC12A1 solute carrier family 12, member 1 NM_000338.2 Bartter syndrome, type 1 601678 AR 27 20 solute carrier family 22 (organic 21 SLC22A12 NM_144585.3 Renal hypouricemia, RHUC1 220150 AD/AR 10 21 anion/urate transporter), member 12 solute carrier family 2 (facilitated 22 SLC2A9 NM_001001290.1 Renal hypouricemia, RHUC2 612076 AD/AR 13 22 glucose transporter), member 9 Hypophosphatemic nephrolithiasis solute carrier family 34 (sodium 612286 / 23 SLC34A1 NM_003052.4 /osteoporosis-1, AD/AR 13 23 phosphate), member 1 613388 NPHLOP1 / Fanconi renotubular syndrome 2 solute carrier family 34 (sodium 24 SLC34A3 NM_001177316.1 Hypophosphatemic rickets with hypercalciuria 241530 AR 12 24 phosphate), member 3 solute carrier family 3 (cystine, dibasic and neutral amino acid transporters, 25 NM_000341.3 Cystinuria, type A 220100 AR 10 25 SLC3A1 activator of cystine, dibasic and neutral amino acid transport), member 1 solute carrier family 4, anion exchanger, Primary distal renal tubular acidosis, dominant / 179800 / 26 SLC4A1 NM_000342.3 AD/AR 19 26 member 1 (erythrocyte membrane recessive 611590 protein band 3, Diego blood group) solute carrier family 7 (glycoproteinassociated 27 SLC7A9 NM_014270.4 Cystinuria, type B 220100 AD/AR 12 27 amino acid transporter light chain, bo,+ system), member 9 solute carrier family 9, subfamily A Hypophosphatemic nephrolithiasis/osteoporosis- 28 SLC9A3R1 (NHE3, cation proton antiporter 3), NM_004252.4 2, 612287 AD 6 28 member 3 regulator 1 NPHLOP2 vitamin D (1,25- dihydroxyvitamin D3) 29 VDR NM_000376.2 Idiopathic hypercalciuria 277440 AD 11 29 receptor 30 XDH xanthine dehydrogenase NM_000379.3 Xanthinuria, type 1 278300 AR 36 30 AR, autosomal recessive; AD, autosomal dominant; XR, X-linked recessive Genes in which mutations were detected are underlined.

REFERENCES

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15. Hamilton, A.J. et al. The HNF4A R76W mutation causes atypical dominant Fanconi syndrome in addition to a β cell phenotype. Journal of Medical Genetics 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. Nat Genet 6, 420-425 (1994). 26. Bruce, L.J. et al. Familial distal renal tubular acidosis is associated with mutations in the red cell anion exchanger (Band 3, AE1) gene. J Clin Invest 100, 1693-1707 (1997). 27. Feliubadalo, L. et al. Non-type I cystinuria caused by mutations in SLC7A9, encoding a subunit (bo,+AT) of rBAT. Nat Genet 23, 52-57 (1999). 28. Karim, Z. et al. NHERF1 Mutations and Responsiveness of Renal Parathyroid Hormone. New England Journal of Medicine 359, 1128-1135 (2008). 29. Scott, P. et al. Suggestive Evidence for a Susceptibility Gene Near the Vitamin D Receptor Locus in Idiopathic Calcium Stone Formation. Journal of the American Society of Nephrology 10, 1007-1013 (1999). 30. Ichida, K. et al. Identification of two mutations in human xanthine dehydrogenase gene responsible for classical type I xanthinuria. J Clin Invest 99, 2391- 2397 (1997).

Supplemental Table 2. Clinical characteristics of individuals with identified monogenic disease causes.

Compatible a Molecular Stone Familial Gene Individual Urine / Serum Diagnosis Analysis Inheritance Chemistry ADYC10 B580 Hypercalciuria Y ☑ N n/a CaOx AR AD ☑ ADYC10 B599 Hypercalciuria Y ☑ N n/a n/a AR AD ☑ dRTA Y ☑ N n/a SLC4A1 B280 n/a AR AD ☑ Hypocalcemia Y N ☑ n/a Hypophosphatemia Y ☑ N n/a SLC9A3R1 B529 n/a AR ☑ AD Hyperphosphaturia Y ☑ N n/a SLC34A1 B491 Hypophosphatemia Y ☑ N n/a n/a AR AD ☑ SLC34A1 B484 Hypophosphatemia Y ☑ N n/a n/a AR AD ☑ SLC34A1 B417 Hypophosphatemia Y ☑ N n/a CaOx AR AD ☑ b CaOx, SLC34A1 B610 Hypophosphatemia Y N ☑ n/a AR AD CaPh SLC34A1 B523 Hypophosphatemia Y ☑ N n/a n/a AR AD ☑ Hypocalcemia Y N ☑ n/a VDR B481 n/a AR ☑ AD Hypophosphatemia Y ☑ N n/a Hypocalcemia Y N ☑ n/a VDR B447 n/a AR ☑ AD Hypophosphatemia Y ☑ N n/a AGXT B424 Hyperoxaluria Y ☑ N n/a CaOx AR ☑ AD dRTA Y ☑ N n/a ATP6V1B1 B482 CaOx AR ☑ AD Deafness Y N n/a ☑

Hypokalemia Y ☑ N n/a

ATP6V0A4 B329 dRTA Y ☑ N n/a n/a AR ☑ AD

Metabolic acidosis Y ☑ N n/a

Hypomagnesemia Y ☑ N n/a

Hyperuricemia Y N ☑ n/a

CLDN16 B604 Hypermagnesiuria Y ☑ N n/a n/a AR ☑ AD

Hypercalciuria Y ☑ N n/a

Hypocitraturia Y ☑ N n/a

Hypomagnesemia Y ☑ N n/a

CLDN19 A4592 Hypercalciuria Y ☑ N n/a n/a AR ☑ AD

Hypermagnesuria Y N n/a ☑ Hypercalcemia Y ☑ N n/a CYP24A1 B607 n/a AR ☑ AD Hypercalciuria Y ☑ N n/a Hypercalcemia Y ☑ N n/a CYP24A1 B540 CaOx AR ☑ AD Hypercalciuria Y ☑ N n/a

Hypokalemia Y ☑ N n/a

Hypercalciuria Y ☑ N n/a

SLC12A1 B446 Hypochloremia Y ☑ N n/a n/a AR ☑ AD

Hyposthenuria Y ☑ N n/a

Metabolic alkalosis Y ☑ N n/a SLC3A1 B425 Cystinuria Y ☑ N n/a cystine AR ☑ AD SLC3A1 B458 Cystinuria Y ☑ N n/a cystine AR ☑ AD SLC3A1 B499 Cystinuria Y ☑ N n/a cystine AR ☑ AD

Hypercalciuria Y ☑ N n/a

Aminoaciduria Y ☑ N n/a

OCRL B422 pRTA Y ☑ N n/a n/a XR ☑ AD

Phosphoturia Y ☑ N n/a

Proteinuria Y ☑ N n/a

Hypercalciuria Y ☑ N n/a

Aminoaciduria Y N ☑ n/a

OCRL B199 pRTA Y N ☑ n/a CaOx XR ☑ AD

Phosphoturia Y N ☑ n/a

Proteinuria Y N ☑ n/a

AD, autosomal dominant; AR, autosomal recessive; CaOx, calcium oxalate; CaPh, calcium phosphate; dRTA, distal renal tubular acidosis; N, no; n/a, not available; pRTA, proximal renal tubular acidosis; XR, X-linked recessive; Y, yes aAutosomal dominant genes are underlined, autosomal recessive genes are not underlined, and X-linked recessive genes are double underlined. bB610 is an adopted individual, and biological family information is unavailable.