Daga A, Majmundar AJ, Braun DA, Gee HY, Lawson JA, Shril S, Jobst

Daga A, Majmundar AJ, Braun DA, Gee HY, Lawson JA, Shril S, Jobst-Schwan T, Vivante A, Schapiro D, Tan W, Warejko JK, Widmeier E, Nelson CP, Fathy HM, Gucev Z, Soliman NA, Hashmi S, Halbritter J, Halty M, Kari JA, El-Desoky S, Ferguson MA, Somers MJG, Traum AZ, Stein DR, Daouk GH, Rodig NM, Katz A, Hanna C, Schwaderer AL, Sayer JA, Wassner AJ, Mane S, Lifton RP, Milosevic D, Tasic V, Baum MA, Hildebrandt F. Whole exome sequencing frequently detects a monogenic cause in early onset nephrolithiasis and nephrocalcinosis. Kidney International 2018, 93(1), 204-213.

DOI link

https://doi.org/10.1016/j.kint.2017.06.025

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http://eprint.ncl.ac.uk/241812

Date deposited

01/03/2018

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12/10/2018

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This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International licence

Newcastle University ePrints | eprint.ncl.ac.uk Whole exome sequencing reveals a monogenic disease cause in 24.3% of patients with early onset urinary stone disease

Ankana Daga1*, Amar J. Majmundar1*, Daniela A. Braun1, Heon Yung Gee2, Jennifer A. Lawson1, Shirlee Shril1, Tilman Jobst-Schwan1, Asaf Vivante1, David Schapiro1, Weizhan Tan1, Caleb P. Nelson3, Hanan M. Fathy4, Velibor Tasic5, Neveen A. Soliman6, Seema Hashmi7, Jan Halbritter8, Michael A. Ferguson1, Michael J.G. Somers1, Avram Z. Traum1, Deborah R. Stein1, Ghaleb H. Daouk1, Nancy M. Rodig1, Avi Katz9, Christian Hanna9, Andrew L. Schwaderer10, John A. Sayer11, Ari J. Wassner12, Shrikant Mane13, 14,15, Richard P. Lifton13,14,15, Michelle A. Baum1, and Friedhelm Hildebrandt1 1 Division of Nephrology, Department of Medicine, Boston Children’s Hospital, Harvard Medical School, Boston, Massachusetts, USA 2Department of Pharmacology, Brain Korea 21 PLUS Project for Medical Sciences, Yonsei University College of Medicine, Seoul 3Division of Urology and General Pediatrics, Department of Medicine, Boston Children’s Hospital, Harvard Medical School, Boston, Massachusetts, USA 4Pediatric Nephrology Unit, Alexandria University, Alexandria, Egypt 5Medical Faculty Skopje, University Children’s Hospital, Skopje, Macedonia 6Department of Pediatrics, Center of Pediatric Nephrology & Transplantation, Kasr Al Ainy School of Medicine, Cairo University, Cairo, Egypt; 7Department of Pediatric Nephrology, Sindh Institute of Urology and Transplantation, SIUT, Karachi, Pakistan 8Division of Endocrinology and Nephrology, Department of Internal Medicine, University Clinic Leipzig, Leipzig 04103, Germany 9Division of Pediatric Nephrology, University of Minnesota, Minneapolis 10Division of Nephrology, Department of Pediatrics, Nationwide Children's Hospital/The Ohio State University 11Institute of Genetic Medicine, International Centre for Life, Newcastle University, Newcastle upon Tyne, United Kingdom 12Division of Endocrinology, Department of Medicine, Boston Children’s Hospital, Harvard Medical School, Boston, Massachusetts 12Department of Genetics, Yale University School of Medicine, New Haven, Connecticut, USA 13Yale Center for Mendelian Genomics, Yale University School of Medicine, New Haven, Connecticut, USA 14Howard Hughes Medical Institute, Chevy Chase, MD, USA

* These authors contributed equally to this work.

Correspondence should be addressed to: Friedhelm Hildebrandt, M.D. Division of Nephrology Boston Children's Hospital 300 Longwood Avenue Boston, Massachusetts 02115 Phone: +1 617-355-6129 Fax: +1 617-730-0365 Email: [email protected]

Running title: Exome sequencing detects monogenic causes of nephrolithiasis and nephrocalcinosis

ABSTRACT (272/250 words, Text 1459/1500 characters, without abbreviations, subheadings, references)

The incidence of nephrolithiasis (NL) continues to rise. However, its causes and mechanisms are not well understood. Previously, we revealed that a monogenic cause could be detected in 11.4% of individuals with adult-onset NL or nephrocalcinosis (NC) and in 16.7-20.8% of individuals with onset before 18 years of life using gene panel sequencing of 30 known genes. To overcome the limitation of panel sequencing being restricted to 30 genes, we here utilized whole exome sequencing (WES) rather than panel sequencing in 41 families, who presented before age 25 yrs with at least 1 renal stone or exhibited a renal ultrasound finding of NC to identify the underlying molecular genetic cause of disease. In 10 out of 41 (24.3%) families, we detected a monogenic causative mutation by WES. We detected a mutation in 5 recessive genes (GRHPR, AGXT, SLC12A1, ATP6V1B1, and CLDN19), 1 dominant gene (SLC9A3R1), and 1 gene (SLC34A1) with both recessive and dominant inheritance. 5 of the 14 different mutations were not previously described as disease causing. Median age of onset was significantly lower in patients in whom we detected a monogenic cause (3 yrs) vs. those without mutation detection (7 yrs) (Z score = 2.49, p < 0.05). In one family we detected a causative mutation in one of 117 genes that may represent phenocopies of NL- causing genes. In 7/11 patients the genetic diagnosis had specific implications for clinical management and primary and secondary prophylaxis. Thus, we established WES as an efficient approach towards a molecular genetic diagnosis in individuals with NL/NC manifesting before age 25 yrs, and confirmed a surprisingly high fraction of individuals (24.3%), in whom a monogenic cause of disease may be identified.

INTRODUCTION Nephrolithiasis (NL) is a highly prevalent condition affecting up to 10% of individuals worldwide (Scales, 2012). It is associated with high morbidity, high recurrence rate as well as high economic cost (Rule, 2009). Although NL is less common among children compared to adults, the incidence of NL and nephrocalcinosis (NC) in the pediatric age group has been rising over the past 10 years (Dwyer, 2012). The causes of NL are not well understood. Monogenic causes of NL were thought to be rare and restricted to rare tubulopathies and genetic syndromes. However, we recently tested the hypothesis that monogenic causes of NL account for a high percentage of stone formers, and revealed that a causative monogenic mutation can be detected in one of 30 NL genes in 20.8% of patients with early-onset of NL before age of 18 yrs (Halbritter, 2015); (Goldfarb, 2015). We subsequently confirmed the high detection rate of a molecular diagnosis in 16.7% of early-onset NL in a three-center cohort and found that important therapeutic and preventative measures may result from mutation detection (Braun, 2016). These previous studies employed exon sequencing in gene panels, rather than whole exome sequencing (WES). Because WES offers the opportunity to detect dozens of additional genes at a rather low cost and has not been applied to patients with NL, we here employ WES in 41 families (54 individuals) with one episode of stone or evidence of NC on renal ultrasound before the age of 25 yrs to identify monogenic causes in 30 NL/NC known genes, 117 phenocopy genes (30 known tubulopathy genes, 87 ciliopathy genes), and 17 hypothesized candidate genes. We confirm the surprisingly high rate of monogenic causative mutation detection, study genotype-phenotype correlations, and determine that molecular genetic diagnosis allows for finely tailored treatment plans that may prevent recurrent disease or delay progression to ESRD.

MATERIALS AND METHODS

Human subjects. The study was approved by the institutional review board (IRB) of Boston Children’s Hospital. After obtaining informed consent clinical data, pedigree information, and DNA samples were collected from subjects who had at least one occurrence of nephrolithiasis or demonstration of nephrocalcinosis on renal ultrasound manifesting before the age of 25 years. Subjects with a potential secondary cause of NL such as use of loop diuretics or hyperparathyroidism were excluded from the study. Whole exome sequencing was performed in 54 individuals from 41 families. 4 families were consanguineous, and 15 families were either sibling cases or with more than one affected child. The families were selected for study as follows. We enrolled 339 families affected with NL or NC from November 2013 to May 2016 (31 months). Of the 339 families, 239 families were previously screened by multiplex PCR of a 30-gene panel (reported in Halbritter, 2015; Braun, 2016). Of the remaining previously unscreened 100 families, we examined 41 families by WES. These families were a representative subset detected by the criteria of availability of multiple affected family members (15 families), or on availability of parents for trio analysis (13 families).

Whole exome sequencing and mutation calling. Whole exome sequencing (WES) and variant burden analysis were performed as previously described1, 10, 20. In brief, genomic DNA was isolated from blood lymphocytes and subjected to exome capture using Agilent SureSelect™ human exome capture arrays (Life technologies™) followed by next generation sequencing on the Illumina HighSeq™ sequencing platform. Sequence reads were mapped to the human reference genome assembly (NCBI build 3/hg19) using CLC Genomics Workbench™ (version 6.5.2) software (CLC bio, Aarhus, Denmark). Following alignment to the human reference genome, variants were filtered as previously described1, 21 and as summarized in Suppl. Fig. 1. In the first step variants with minor allele frequencies (MAF) >1% in the dbSNP (version 144) were excluded. In the second step, synonymous variants and intronic variants that were not located within splice site regions were excluded. In step 3, variants were evaluated for mutations in 30 known NL/NC genes (Suppl. Table 1). In step 4, remaining variants were ranked based on their probable impact on protein considering evolutionary

5 conservation among orthologs across phylogeny, as well as web-based prediction programs (PolyPhen-2, SIFT, and MutationTaster). In step 5, variants remaining were searched further evaluated by reviewing existing literature and determining phenotypic match. Clinician scientists and geneticists, who had knowledge of the clinical phenotypes and pedigree structure, as well as experience with exome evaluation performed mutation calling. Remaining variants were confirmed in original patient DNA by Sanger sequencing as previously described. Whenever parental DNA was available, segregation analysis was performed.

In a second evaluation process, variants in all 117 genes (Suppl. Table 2) that are known monogenic, recessive causes of renal ciliopathies (Gee, 2014) and may phenocopy the clinical presentation of NC/NL were systematically evaluated. In the third evaluation process, variants in 17 novel candidate genes for NL/NC (Gee, 2016) were systematically evaluated. These genes were ADCY6, CHD1, CLDN2, CLDN10, CLDN14, DMP1, ENPP1, FETUB, GALNT3, ITPKC, KL, MGP, ORAl1, PHEX, SLC13A2, SPP1, UMOD (Gee, 2016)

Web Resources UCSC Genome Browser, http://genome.ucsc.edu/cgi-bin/hgGateway; 1000 Genomes Browser, http://browser.1000genomes.org; Ensembl Genome Browser, http://www.ensembl.org; Exome Variant Server, http://evs.gs.washington.edu/EVS; Exome Aggregation Consortium, exac.broadinstitute.org; Online Mendelian Inheritance in Man (OMIM), http://www.omim.org; Clinvar https://www.ncbi.nlm.nih.gov/clinvar/ Biobase https://www.qiagenbioinformatics.com/products/human-gene-mutation Polyphen2, http://genetics.bwh.harvard.edu/pph2; Sorting Intolerant From Tolerant (SIFT), http://sift.jcvi.org/ MutationTaster http://www.mutationtaster.org/

RESULTS

We performed WES in 54 individuals from 41 families with nephrolithiasis and/or nephrocalcinosis on renal ultrasound who manifested before the age of 25 years. These families had isolated NL/NC or a combination of both along with or without hypercalciuria. Of the 54 individuals, 27 had isolated NL, 16 had isolated NC, and 11 had both NL/NC. No affected individual had hypercalciuria without NL or NC (Suppl. Fig. 2). When evaluating WES data for 30 genes (Suppl. Table 1) known to cause NL or NC when mutated, we identified a mutation in 10 of 41 families (24.3%) (Fig. 1) Recessive or dominant causative mutations were detected in 7 genes (Table 1). Pathogenic mutations were detected in 11 individuals from 7 families in 6 genes: GRHPR (2 individuals), AGXT (3 individuals), SLC12A1 (1 individual), ATP6V1B1 (1 individual), CLDN19 (3 individuals), SLC34A1 (1 individual) (Table 1, Part A). Pathogenic mutations were detected in 5 individuals in 2 dominant genes: SLC9A3R1 (1 individual) and SLC34A1 (4 individuals) (Table 1, Part B). SLC34A1 gene mutations may follow an autosomal recessive mode of inheritance causing infantile hypercalcemia (Konrad, 2015) or an autosomal dominant mode of inheritance causing nephrolithiasis (Prie, 2002). Of the 16 individuals in whom we detected causative mutations, 6 presented with NL and 10 presented with NC on a renal ultrasound (Fig. 2).

Five of 14 detected mutations (35.7%) were novel pathogenic variants that have not been previously reported in databases of human disease causing mutations. (https://portal.biobase-international.com/hgmd/pro/search_gene.php)

Detection rate of causative mutations was not different between sexes. (10/26 in males and 6/28 in females, p = 0.17). Median age of onset was significantly lower (Z score: 2.6, p= 0.009) in patients with a monogenic cause (3 yrs) versus those without (7 yrs) (Suppl. Fig 3). We evaluated our cohort for differences regarding disease (NL/NC) at presentation, age of onset of disease, and causative mutation detection (Fig. 3). We observed that individuals with NC presented before age 15 yrs, whereas individuals with NL presented from 0-25 years. Causative mutations were detected in 10/23 NC individuals and in 5/31 NL individuals (Fig. 3).

We evaluated age of onset of disease for each gene mutation detected in this cohort to the ones we previously published (Halbritter, 2015; Braun 2016) (Fig.4). Age of onset for mutations was very consistent for the following recessive genes SLC12A1 (age of onset < 1 yr), ATP6V1B1 (age of onset 5-7 yrs), SLC34A1 (age of onset was 0-3 yrs). Individuals with mutations in 2 dominant genes in all three cohorts presented after 1 year of age.

In 5 of the 15 families (33%) with multiple affected individuals included in our cohort we detected a causative genetic mutation, whereas in only 5 of the 32 (15.6%) families with single affected individuals a causative mutation was detected.

WES data of 31 unsolved families were evaluated for mutations in 117 genes known to represent phenocopies of NL or NC. This included genes causing renal ciliopathies and tubulopathy (Suppl. Table 2). These genes if mutated may cause phenocopies of NC as they cause increased echogenicity on renal US (Gee, 2014). Among these phenocopy genes (Suppl. Table 2), we detected a homozygous frameshift, previously reported mutation in CTNS gene (Besouw, MT) in one patient with nephrocalcinosis (Table 1, Part C). This homozygous CTNS mutation was located in a homozygous peak. (Suppl Fig. 4).

We generated a list of 17 hypothetical novel candidate genes (Gee, 2016) and evaluated WES data of 41 families for these genes, and detected no mutation.

DISCUSSION

MUTATION DETECTION PERCENTAGE

Here, we performed WES in 54 individuals from 41 families with NL or NC with onset before 25 years of age. We identified a causative mutation in 10 of 41 families (24.3%) in one of 30 genes known to cause monogenic NL/NC. Our results confirm our previous findings when identifying a monogenic cause of NL/NC in 15% of all subjects (20.8% children and 11% adults) in a separate mixed adult and pediatric cohort using targeted sequencing panels (Halbritter, 2015). Our findings are also similar to the previous panel based identification of a monogenic cause of NL/NC in 16.7% of children below the age of 18 years in a three-center cohort (Braun, 2016). We here choose WES because for an increasing number of potentially causative genes and phenocopies targeted sequencing panels have severe limitations.

So far, WES has not been applied to patients with NL/NC. As we identified the disease- causing mutation in about one quarter of families with NL/NC, our data show that WES is an efficient tool for molecular diagnostics in this disease group.

PHENOCOPIES We evaluated 31 families without a molecular diagnosis in 30 NL/NC genes for mutations in phenocopy genes. About 117 genes that are known to cause recessive renal ciliopathies or tubulopathies have been shown to represent phenocopies of NC because of renal US findings of increased echogenicity (Braun, 2016). In our cohort, in one family with NC and chronic kidney disease, we detected a mutation in a phenocopy gene (CTNS) that causes nephropathic cystinosis. This molecular genetic diagnosis was not suspected clinically since the affected individual did not present with all classic features of cystinosis disease at initial presentation. Therefore, in this respective individual, the molecular diagnosis differed from the initial clinical diagnosis, demonstrating that WES helps to distinguish between NL/NC and other kidney diseases that phenocopy the presentation of NC on renal ultrasound This can be particularly advantageous in very early stages of disease progression, in which additional

9 characteristic symptoms might not yet be present, as well as in very rare genetic syndromes. The ability to evaluate a large number of phenocopy genes underlines an additional benefit of WES. Mutation detection is not restricted to a limited subset of monogenic diseases that were suspected based on the clinical presentation, but allows taking a broader spectrum of monogenic causes into consideration.

In addition, we evaluated WES data for 17 hypothesized potential new NL/NC candidate genes in all 41 families, but did not detect any mutations (Gee, 2016).

HIGH MUTATION DETECTION RATE

The percentage of individuals with NL/NC, in whom a causative mutation was detected in this study and the two previous ones (Halbritter 2015, Braun 2016) may be subject to a certain bias toward severe cases, since individuals were recruited from specialized tertiary renal stone clinics.

GENOTYPE-PHENOTYPE CORRELATION Although, the sample size is limited in this study, our findings confirm that early onset and familial prevalence are indicators of inherited disease as we have shown previously (Halbritter, 2015). In this study, we showed that median age of onset (3 yrs) was lower in individuals with a molecular genetic diagnosis in comparison to those without a genetic diagnosis (7 yrs). Additionally, mutation detection rate was higher in families with multiple affected individuals (33% multiple affected vs 19% single affected).

None of the 5 individuals in whom a mutation in a dominant gene was identified presented before the age of 1 year. This is consistent with the finding that dominant monogenic causes typically manifest later in life as shown in our previous study (Braun, 2016). We also compared age of onset of disease for each specific mutation detected in this study to previous published data from Braun, 2016, Halbritter 2015 (Fig 4). For example, the median age of onset from all three studies was 6.5 yrs for ATP6V1B1 gene, which is higher than expected for mutations in ATP6V1B1, which causes dRTA and deafness. However, on further analysis we observed that all ATP6V1B1 mutations in 3 studies were missense mutations that may manifest later in life, in comparison to

10 truncating mutations, which present early in life. SLC34A1 We identified mutations in SLC34A1 in 5 individuals from 2 families (Table 1). The individual harboring recessive mutations exhibited nephrocalcinosis, hypercalcemia, hypercalciuria, and hypophosphatemia consistent with renal phosphate wasting. On the other hand, affected individuals bearing dominant mutations had isolated nephrocalcinosis or hypercalciuria. Upon reviewing all published pediatric cases with SLC34A1 mutations (Table S3), the phenotypic spectrum observed mirrors our findings. All cases exhibit nephrolithiasis or nephrocalcinosis. However, recessive cases typically exhibit hypercalcemia, hypercalciuria, and hypophosphatemia whereas dominant disease virtually never includes hypercalcemia (Table S3, S4). Moreover, there is also a dichotomy between reported recessive and dominant cases based on age of onset, as recessive cases typically present in infancy (median 0.25 years) whereas dominant cases tend to present later in childhood (median 3.5 years) (Table S4, Suppl. Fig 5).

CLINICAL IMPLICATIONS A molecular genetic diagnosis may have implications for both the affected individual and unaffected family members. As addressed in Table 2, following identification of certain causative mutations in affected individuals, the resulting knowledge might have important prophylactic implications for primary prophylaxis (stone recurrence) and secondary prophylaxis (loss of renal function) for affected and currently unaffected family members. Information resulting from mutation analysis will guide clinicians to monitor individuals for development of disease and to institute preventative treatment when possible. For example, individuals identified with recessive SLC34A1 mutations may benefit from phosphate supplementation to prevent hypercalcemia, hypercalciuria and nephrocalcinosis (Schlingmann, Konrad 2014). Similarly, individuals with recessive mutations in ATP6V1B1 and CLDN19 may benefit from hearing and ophthalmology testing respectively, and from early treatment for growth retardation and ESRD prevention. Individual (B969), who has a SLC9A3R1 mutation will benefit from close monitoring and treatment for hypophosphatemia. Recessive AGXT mutations detected in one sibling with severe hyperoxaluria and another sibling with a milder phenotype

(B1468) may allow early chronic kidney disease preventative care for the latter sibling. We also detected a recessive mutation in CTNS in an individual who presented with an incomplete cystinosis phenotype at time of presentation, which aided in providing disease specific treatment and delaying progression to ESRD. Therefore, we suggest “Practical Implications” (Table 2) for each gene in which we detected a likely disease- causing mutation

FUTURE DIRECTIONS/LIMITATIONS 30 of 41 families in our cohort (73%) remained without a molecular diagnosis after WES. In recessive monogenic diseases about 85% of all causative mutations are located within the coding sequence or the adjacent splice sites. The remaining 15% however, may represent genetic variants that are difficult to detect by WES such as complex deletion-insertion variants, copy-number variants, or may reside within a promotor and other intronic region. As WES can detect none of these variants, this technical limitation might explain why some cases remain without a molecular diagnosis after WES. In addition, causative mutations may be missed because the stringent criteria that we currently use to assess for deleteriousness may not include mild, hypomorphic mutations. Furthermore, WES might miss a subset of causative variants due to low coverage in the respective target region.

We here present WES as a rapid and reliable tool for molecular diagnostics in individuals with a history of at least one renal stone or NC on a renal ultrasound in 24% of affected individuals. This represents a major advance in providing a specific etiologic diagnosis in NL/NC, delaying progression to CKD/ESRD, and enabling personalization of the treatment plan for each stone former. CONCLUSION

ACKNOWLEDGEMENTS Leslie Speanas, Brittany Fisher, Kassandra Ammam We thank the physicians and the participating families for their contribution. F.H. is the Warren E. Grupe Professor of Pediatrics. This research was supported by grants from the National Institutes of Health DK1069274, DK1068306, and DK064614 to FH and 5U54HG006504 to RPL, and by the March of Dimes Foundation (6-FY11-241 to FH). H.Y.G. was supported by the ASN-NephCure Foundation grant.

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FIGURE LEGENDS AND TITELS

Figure 1. Flow diagram on detection of causative monogenic mutations by WES in 41 families with NL/ NC. A causative mutation was detected in 10 of 41 families (24.3%). A causative mutation was detected in the phenocopy gene (CTNS) in one patient with nephrocalcinosis.

Figure 2. Renal ultrasound images of patients with NC, in whom causative mutations were detected. (A) Image from B1437_21 (SLC34A1). (B) Image from B1121 (ATP6V1B1). (C) Image from B1301_21 (SLC34A1) (D) Image from A4283 (CLDN19). (E) Image from B785 (CTNS) (F) Image from B986_21 (SLC34A1).

Figure 3. Distribution of number of affected individuals with NL/NC by age of onset. In a total of 16/54 (30%) affected individuals, a causative mutation was detected. 6/16 (37.5%) affected individuals presented with NL, and 10/16 affected individuals (62.5%) presented with NC.

Figure 4. Distribution of age of first diagnosis of NL/NC for each mutated gene detected by WES in 16 individuals from 10 families with NL/NC in this paper (filled circles), and from 9 families in Braun, 2016 (hollow squares), and from 5 families in Halbritter, 2015 (hollow triangles). Medians are depicted as black horizontal lines. Gene symbols are given on the X-axis for dominant genes (red), and for recessive genes (black). Note that age of onset of disease for mutated recessive genes SLC12A1, ATP6V1B1, SLC34A1 is similar in all three publications. SLC12A1, Solute carrier family 12 (sodium/potassium/chloride transporter) member 1, SLC34A1, Solute carrier family 34 (Type II Sodium/Phosphate cotransporter) member 1; GRHPR, Glyoxalate Reducatse/Hydroxypyruvate Reducatse; AGXT, Alanine-Glyoxalate Aminotransferase; ATP6V1B1, ATPase, H+ transporting, lysosomal, 56/58-KD, V1 subunit B, isoform 1; SLC9A3R1, Solute Carrier Family 9, Member 3, Regulator 1; CLDN19, Claudin 19

Table 1, Part A: Monogenic causative mutations of 6 recessive genes detected in 7 of 41 families with NL/NC. Mutated genes and related disease (OMIM nomenclature) are shown in sub headers to the table

Nucleotide State Evolutionary Individual Amino acid change PPh2 Ref. Ethnicity (Sex) Age of Onset NL/NC change conservation GRHPR, Primary Hyperoxaluria Type II

B35-21 c.103del p.Asp35Thrfs*11 het NA NA Y (Cramer, 99) Caucasian (M) 0.08 NL/NC c.404+5G>A splice site het NA NA Y (Hopp, 2015) Caucasian (M) 0.08 NL/NC B35-22 c.103del p.Asp35Thrfs*11 het NA NA Y (Cramer, 99) Caucasian (M) 3 NL/NC c.404+5G>A splice site het NA NA Y (Hopp, 2015) Caucasian (M) 3 NL/NC

AGXT, Primary Hyperoxaluria Type I

B1468-21 c.1079G>C p.Arg360Pro het 1 C. elegans Novela Arabic(M) 6 NL/NC c.121G>A p.Gly41Arg het 0.99 D. melanogaster Y (Danpure, 99) Arabic (M) 6 NL/NC B1468-24 c.1079G>C p.Arg360Pro het 1 C. elegans Novela Arabic (F) 2.5 NC c.121G>A p.Gly41Arg het 0.99 D. melanogaster Y (Danpure, 99) Arabic (F) 2.5 NC

B1230_21 c.731T>C p.Ile244Thr hom 0.4 M. musculus Y (Von, 97) Egyptian (M) 1 NL

SLC12A1, Bartter Syndrome type 2

B917-21 c.769G>A p.Gly257Ser het 1 C. elegans Y (Adachi, 2007) Caucasian (M) 1 NL/NC c.1424G>A p.Cys475Tyr het 0.99 D. melanogaster Novel Caucasian (M) 1 NL/NC

ATP6V1B1, Renal Tubular acidosis with deafness

B1121 c.242T>C p.Leu81Pro Hom 1 C. elegans Y (Karet, 99) Macedonian (M) 1.7 NC

CLDN19, Hypomagnesemia/renal/ophthalmological abnormalities

A4283_21 c.535G>A p.Gly179Ser Hom 0.99 D. rerio Novel South Asian (F) 11 NC A4283_22 c.535G>A p.Gly179Ser Hom 0.99 D. rerio Novel South Asian (M) 5 NC A4283_23 c.535G>A p.Gly179Ser Hom 0.99 D. rerio Novel South Asian (M) 4 NC

SLC34A1, Infantile hypercalcemia/hypophosphatemia/nephrolithiasis

B1437-21 c.644+1G>A Splice site het NA NA Y (Schlingmann, Arabic (M) 3 NC c.1204G>C p.Gly402Arg het 1 D. rerio 2016) Arabic (M) 3 NC Novel

AGXT, Alanine-Glyoxalate aminotransferase; ATP6V1B1 (ATPase, H+ transporting, Lysosomal, 56/58-KD, V1 Subunit B, Isoform 1), BS1, Bartter syndrome Type 1; CKD, chronic kidney disease; CLDN19, CLAUDIN 19; DRTAD, distal renal tubular acidosis with progressive deafness; F, female; het, heterozygous; GRHPR Glyoxalate Reductase/Hydroxypyruvate Reducatse; Hom, homozygous; HOMG5, Hypomagnesemia 5, Renal, With Ocular Involvement; (OMIM # 248190), M, Male; NA, Not applicable; NC, nephrocalcinosis; NPHLOP1, NEPHROLITHIASIS/OSTEOPOROSIS, HYPOPHOSPHATEMIC, 1 (OMIM # 612286); NL, nephrolithiasis; PH, primary hyperoxaluria; PH1, primary hyperoxaluria type 1; PH2, primary hyperoxaluria type 2; PPh2, polyphen-2; RTA, renal tubular acidosis; SLC12A1 (Solute carrier family 12 , SLC34A1, Solute carrier family 34. aDifferent mutations at this position are reported Table 1, Part B: Monogenic causative mutations in 2 dominant genes in 3 of 41 families with NL/NC. Mutated genes and related diseases (OMIM nomenclature) are shown in sub-headers to table

Nucleotide Amino acid Sta Evolutionary Ethnicity Age of onset Individual PPh2 Reference NL/NC change change te conservation (Sex) (yr)

SLC34A1, Infantile Hypercalcemia/Hypophosphatemia/Nephrolithiasis

B1301_21 c.398C>T p.Ala133Val het 0.99 C. intestinalis Y (Lapointe, 06) Macedonian (F) 6.5 NC B1301_22 c.398C>T p.Ala133Val het 0.99 C. intestinalis Y (Lapointe, 06) Macedonian (M) 10.5 NC

B986_21 c.536T>C p.Leu179Pro het 1 C. intestinalis Novel Caucasian (F) 1.5 NC B986_22 c.536T>C p.Leu179Pro het 1 C. intestinalis Novel Caucasian (F) 1.5 NC

SLC9A3R1, Nephrolithiasis/osteoporosis

B969-21 c.673G>A p.Glu225Lys het 0.82 D. rerio Y (Karim,2008) Caucasian (F) 7 NL

F, female; het, Heterozygous; M, Male; NC, nephrocalcinosis; NL, nephrolithiasis; NPHLOP1, Nephrolithiasis/osteoporosis, hypophosphatemic, 1; NPHLOP2, Nephrolithiasis/osteoporosis, hypophosphatemic, 2; PPh2, polyphen-2 http://genetics.bwh.harvard.edu/pph2/; SLC9A3R1 (Solute carrier family 9, member 3, regulator 1); SLC34A1, Solute carrier family 34

Table 1, Part C. Monogenic causative mutations in 1 phenocopy gene in 1 of 41 families with NL/NC. Mutated gene and related diseases (OMIM nomenclature) are shown in sub-headers to table

Nucleotid Amino acid Evolutionary Ethnicity Age of Individual State PPh2 Reference NL/NC e change change conservation (Sex) onset (yr)

CTNS (Cystinosin)

B785 c.[828_82 p.[Thr277fs] HOM NA NA Besouw, 2012 Arabic (M) 2 NC 9insA]

HOM, homozygous; M, Male; NC, nephrocalcinosis; PPh2, polyphen-2 http://genetics.bwh.harvard.edu/pph2/

Table 2: Clinical implications following detection of a monogenic cause of NL/NC.

Gene Clinical Genetic diagnosis Practical implications (Family number) diagnosis (Post-screening) (following genetic diagnosis) (Pre- screening) ATP6V1B1 NC Distal renal tubular acidosis, GC, (B1121) deafness Hearing test, Metabolic correction, Growth retardation prevention, ESRD progression prevention SLC9A3R1 NL Nephrolithiasis, GC, (B969) osteoporosis, Screening for hypophosphatemia, hypophosphatemia Screening for abnormal bone density, Osteoporosis prevention CLDN19 NC Hypomagnesemia 5, renal GC, (A4283) with ocular disease Ophthalmology test, Treatment with magnesium

AGXT NL Primary hyperoxaluria GC, (B1468) Consider pyridoxine trial, Consider RNAi treatment study in for future stone prevention

SLC34A1 NC Hypercalcemia, infantile, GC, (B1427) type 2 Phosphate supplementation

CTNS NC Cystinosis GC, (B785) Cysteamine therapy, Metabolic correction, Growth retardation prevention, ESRD progression prevention

AGXT, Alanine-Glyoxalate Aminotransferase; ATP6V1B1, ATPase, H+ transporting, lysosomal, 56/58-KD, V1 Subunit B, Isoform 1; CLDN19, Claudin 19; CTNS, Cystinosis; GC, genetic counseling; NC, nephrocalcinosis; NL, nephrolithiasis, SLC34A1, solute carrier family 34; SLC9A3R1, solute carrier family 9, member 3, regulator 1

Diagnosis of NL, AND/OR NC on renal ultrasound: 41 families (100%)

Whole Exome Sequencing

Cases solved for Cases not solved for genes known to genes known to cause NL: cause NL: 10 families 31 families (24.3%) (75.6%)

Solved for Not solved for Recessive Dominant mutation in mutation in mutations mutations phenocopy phenocopy 7/10 3/10 gene gene families families 1 family 30 families (70%) (30%)

Figure 1 A B

C D

E F

Figure 2 Figure 3 Figure 4 Reject common SNP (MAF>1%) (Hg 19)

Reject synonymous or intronic variants not located in within splice site

Evaluation for all 30 genes known to cause Nephrolithiasis

Keep Likely deleterious for protein function Protein-truncating, highly conserved residue across phylogeny Predicted to be deleterious based on prediction scores

Genotype/Phenotype Match, Literature review

Accept causative known/novel mutation in known NL/NC genes SupplementaryPhenotype Figure 1: Variant Match, filtering process Literature for the identification review of the causative mutation in genes known to cause nephrolithiasis (NL). Schematic overview of the workflow that was used for filtering of WES data in all families. i) Exclusion of all variants that are present with a minor allele frequency (MAF)>1% in healthy control cohorts (1000 Genomes). ii) Exclusion of synonymous variants and all intronic variants that are not located within splice sites. iii) Keep all variants detected in known NL genes iv) Ranking of remaining variants based on their predicted likelihood to be deleterious for the function of the encoded protein. v) Reviewing literature and delineating whether the detected mutation matches the phenotype. Nephrolithiasis 22

5 8 3

Hypercalciuria

Nephrocalcinosis 12

Supplementary Fig 2. Description of cohort based on diagnosis. Number of patients who first presented with isolated NL = 2, isolated NC = 12. No patient had isolated hypercalciuria. Supplementary Figure 3: Median age of onset in individuals with a monogenic cause of NL (age 3 yrs) was significantly lower than in those without a monogenic cause (age 7 yrs). (Mann Whitney U test, Z score = 2.6, p = 0.009) A

B C

Supplementary Figure 4: (A) Homozygozity mapping of B785. The CTNS mutation was located in the homozygous peak of chromosome 17. (B) Sanger sequencing trace showing insertion of nucleotide A. (C) Renal ultrasound of B785 showing nephrocalcinosis Supplementary Figure 5. Age of onset versus mode of Mendelian SLC34A1 Inheritance in 30 individuals in whom a monogenic cause of nephrolithiais/nephrocalcinosis was detected. Supplementary Table 1. 30 genes that are known to cause monogenic forms of NL/NC and that were included in the study

MIM- Coding Gene Symbol Gene Name Accession # Disease entity Mode Reference Phenotype # Exons Alanine-glyoxylate Primary hyperoxaluria, type 1 1 AGXT NM_000030.2 259900 AR 11 Purdue, 1999 aminotransferase Adenine phosphoribosyltransferase Adenine 2 APRT NM_000485.2 deficiency, 614723 AR 5 Hidaka, 1987 phosphoribosyltransferase Urolithiasis (DHA stones), renal failure ATPase, H+ transporting, dRTA 3 ATP6V0A4 lysosomal V0 NM_020632.2 602722 AR 20 Smith, 2000 subunit a4 ATPase, H+ transporting, Distal renal tubular acidosis (dRTA) 4 ATP6V1B1 lysosomal NM_001692.3 with deafness 267300 AR 14 Karet, 1999 56/58kDa, V1 subunit B1 5 CA2 Carbonic anhydrase II NM_000067.2 Osteopetrosis + d/pRTA 259730 AR 7 Venta, 1991 Hypocalcemia with Bartter syndrome / 6 CASR Calcium-sensing receptor NM_001178065.1 601198 AD 6 Pearce, 1996 hypocalcemia, autosomal dominant Chloride channel, voltage- Dent disease / Nephrolithiasis, type 1 300009 / 7 CLCN5 NM_001127898.3 XR 14 Lloyd, 1996 sensitive 5 310468 Chloride channel, voltage- Bartter syndrome, type 3 8 CLCNKB NM_000085.4 607364 AR 19 Simon, 1997 sensitive Kb Familial hypomagnesemia with 9 CLDN16 Claudin 16 NM_006580.3 hypercalciuria & 248250 AR 5 Simon, 1999 nephrocalcinosis, FHHNC Familial hypomagnesemia with hypercalciuria & 10 CLDN19 Claudin 19 NM_001123395.1 248190 AR 4 Konrad, 2006 nephrocalcinosis with ocular abnormalities Cytochrome P450, family 24, 1,25-(OH) D-24 hydroxylase deficiency Schlingmann, 11 CYP24A1 subfamily NM_000782.4 ,infantile 143880 AR 11 2011 A, polypeptide 1 Hypercalcemia Family with sequence similarity Enamel-Renal syndrome, Jaureguiberry, 12 FAM20A 20, NM_017565.3 amelogenesis imperfecta 204690 AR 12 2012 member A and nephrocalcinosis Glyoxylate Primary hyperoxaluria, type 2 Cramer, 13 GRHPR reductase/hydroxypyruvate NM_012203.1 260000 AR 9 1999 reductase Hepatocyte nuclear factor 4, MODY + Fanconi syndrome + Hamilton, 14 HNF4A NM_000457.4 125850 AD 1 alpha Nephrocalcinosis 2014 4-hydroxy-2-oxoglutarate Primary hyperoxaluria, type 3 Belostotsky, 15 HOGA1 NM_138413.3 613616 AR 7 aldolase 1 2010 Hypoxanthine Kelley-Seegmiller syndrome, partial Davidson, 16 HPRT1 NM_000194.2 300323 XR 9 phosphoribosyltransferase 1 HPRTdeficiency, HPRT-related gout 1991 Potassium inwardly-rectifying Bartter syndrome, type 2 Simon, 17 KCNJ1 channel, NM_000220.4 241200 AR 2 1996 subfamily J, member 1 Oculocerebrorenal syndrome of Lowe syndrome / Dent disease 2 309000 / 18 OCRL NM_000276.3 XR 24 Reilly, 1988 Lowe 300555 Solute carrier family 12, member Bartter syndrome, type 1 19 SLC12A1 NM_000338.2 601678 AR 27 Simon, 1996 1 Solute carrier family 22 (organic Renal hypouricemia, RHUC1 Enomoto, 20 SLC22A12 anion/urate transporter), NM_144585.3 220150 AD/AR 10 2002 member 12 Solute carrier family 26 (sulfate Nephrolithiasis, calcium oxalate 21 SLC26A1 NM_213613 167030 AR 4 Gee, 2016 transporter), member 1 Solute carrier family 2 (facilitated Renal hypouricemia, RHUC2 22 SLC2A9 NM_001001290.1 612076 AD/AR 13 Matuso, 2008 glucose transporter), member 9 Hypophosphatemic Solute carrier family 34 (sodium nephrolithiasis/osteoporosis-1, 612286 / 23 SLC34A1 NM_003052.4 AD/AR 13 Prie, 2002 phosphate), member 1 NPHLOP1 / Fanconi renotubular 613388 syndrome 2 Hypophosphatemic rickets with Lorenz- Solute carrier family 34 (sodium 24 SLC34A3 NM_001177316.1 hypercalciuria 241530 AR 12 Depiereux, phosphate), member 3 2006 Solute carrier family 3 (cystine, Cystinuria, type A dibasic and neutral amino acid Calonge, 25 SLC3A1 transporters, NM_000341.3 220100 AR 10 1994 activator of cystine, dibasic and neutral amino acid transport), member 1 Solute carrier family 4, anion Primary distal renal tubular acidosis, exchanger, dominant / member 1 (erythrocyte recessive 179800 / 26 SLC4A1 NM_000342.3 AD/AR 19 Bruce, 1997 membrane 611590 protein band 3, Diego blood group) Solute carrier family 7 Cystinuria, type B (glycoproteinassociated Feliubadalo, 27 SLC7A9 NM_014270.4 220100 AD/AR 12 amino acid transporter light 1999 chain, bo,+ system), member 9 Solute carrier family 9, subfamily Hypophosphatemic 28 SLC9A3R1 A(NHE3, cation proton antiporter NM_004252.4 nephrolithiasis/osteoporosis 2, 612287 AD 6 Karim, 2008 3),member 3 regulator 1 NPHLOP2 Vitamin D (1,25- Idiopathic hypercalciuria 29 VDR dihydroxyvitamin D3) NM_000376.2 277440 AD 11 Scott, 1999 receptor 30 XDH Xanthine dehydrogenase NM_000379.3 Xanthinuria, type 1 278300 AR 36 Ichida,1997

Supplementary Table S2. Nephrocalcinosis phenocopy panel of 117 genes.

Disease Group Causative Genes Renal Ciliopathy NPHP1, INVS, NPHP3, NPHP4, IQCB1, CEP290, GLIS2, genes RPGRIP1L, NEK8, SDCCAG8, TMEM67, TTC21B, WDR19, ZNF423, CEP164, ANKS6, IFT172, CEP83, DCDC2, IFT81, TRAF3IP1, XPNPEP3, FAN1, PKHD1, INPP5E, TMEM216, AHI1, ARL13B, CC2D2A, KIF7, TCTN1, TMEM237, CEP41, TMEM138, C5orf42, TCTN3, TMEM231, CSPP1, PDE6D, KIAA0586, TCTN2, CEP104, KIAA0556, MKS1, B9D1, B9D2, KIF14, TMEM107, BBS1, BBS2, ARL6, BBS4, BBS5, MKKS, BBS7, TTC8, PTHB1, BBS10, TRIM32, BBS12, WDPCP, LZTFL1, BBIP1, IFT27, OFD1, DDX59, SCLT1, C2CD3, KIAA0753, IFT122, WDR35, IFT43, IFT80, DYNC2H1, NEK1, WDR60, IFT140, WDR34, CEP120, EVC, EVC2, IFT57, IFT52, ALMS1, ATXN10, POC1A, POC1B

Renal SLC41A1, HNF1B, BSND, CLCNKB, AQP2, AVP, AVPR2, REN, Tubulopathies CYP27B1, CYP2R1, HNF4A, SLC12A3, SLC4A4, UMOD, VDR, and WNK4, XDH, SLC2A2, SLC6A19, SLC7A7, EGF, NR3C2, CTNS, Endocrinopathies TRPM6, KCNJ10, FXYD2, WNK1, SCNN1A, SCNN1G, SCNN1B

References: 1. Vivante A and Hildebrandt F. Exploring the genetic basis of early-onset chronic kidney disease. Nature Reviews Nephrology. 12(3):133-46 (2016). 2. Braun DA and Hildebrandt F. Ciliopathies. Cold Spring Harbor Perspectives in Biology. pii: a028191 (2016). Table S3. Phenotypic spectrum of previousl reported SLC34A1 mutations and ovel mutations reported in this current study Subject Inheritance Age of onset (Yrs) NC/NL HP HCU HCE Reference F1.1 Recessive 0.05 NC YES YES YES 1 F1.2 Recessive 0.08 NC YES YES YES 1 F3.1 Recessive 0.16 NC YES YES YES 1 F12.1 Recessive 0.58 NC YES YES YES 1 F7.1 Recessive 0.83 NC - YES YES 1 F8.1 Recessive 0.08 NC YES YES YES 1 F11.1 Recessive 0.33 NC YES - YES 1 F14.1 Recessive 0.25 NC - YES YES 1 B1437 Recessive 3 NC YES YES YES Current F2.1 Recessive 0.16 NC YES YES YES 1 F6.1 Recessive 0.75 NC YES YES YES 1 F9.1 Recessive 0.25 NC YES - YES 1 F10.1 Recessive 0.25 NC YES YES YES 1 F13.1 Recessive 0.5 NC YES - - 1 F15.1 Recessive 1.5 NC - - - 1 JCEM1 Recessive 1.5 NC YES YES YES 2 JCEM2 Recessive 0.16 NC YES YES YES 2 PN1 Recessive 0 NC - - - 3 B168 Recessive 0.4 NC YES NA NA 4 F4.1 Recessive 0.16 NC YES YES YES 1 F5.1 Dominant 0.33 NC YES YES YES 1 B491 Dominant 4 NL YES YES - 5 B523 Dominant 3 NL YES YES - 5 B484 Dominant 3 NL YES - - 5 B610 Dominant 9 NL - YES - 5 B417 Dominant 7 NL YES YES - 5 B1301_21 Dominant 6.5 NC - YES - Current B1301_22 Dominant 10.5 NC - YES - Current B986_21 Dominant 1.5 NC - YES - Current B986_22 Dominant 1.5 NC - YES - Current Abbreviations: HCE, hypercalcemia; HCU, hypercalciuria; HP, hypophosphatemia; NA, no data available; NC, nephrocalcinosis; NL, nephrolithiasis

Table S4. Comparison of clinical features between recessive and dominant SLC34A1 cases.

Mode of inheritance/ Recessive Dominant Total Hypercalcemia HC 16 1 17 No HC 4 9 13 Total 20 10 30

Odds ratio 36, p = 0.0027 (chi square test)