Prevalence of Monogenic Causes in Pediatric Patients with Nephrolithiasis Or Nephrocalcinosis
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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 664 Copyright © 2016 by the American Society of Nephrology www.cjasn.org Vol 11 April, 2016 Clin J Am Soc Nephrol 11: 664–672, April, 2016 Monogenetic Genes in Early-Onset Urinary Stone Disease, Braun et al. 665 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