BRIEF COMMUNICATION www.jasn.org Exome Sequencing Reveals Cubilin Mutation as a Single-Gene Cause of Proteinuria Bugsu Ovunc,*† Edgar A. Otto,* Virginia Vega-Warner,* Pawaree Saisawat,* Shazia Ashraf,* Gokul Ramaswami,* Hanan M. Fathy,‡ Dominik Schoeb,* Gil Chernin,* Robert H. Lyons,§ ʈ Engin Yilmaz,† and Friedhelm Hildebrandt* ¶ ʈ Departments of *Pediatrics and Human Genetics, §Department of Biological Chemistry and DNA Sequencing Core, and ¶Howard Hughes Medical Institute, University of Michigan, Ann Arbor, Michigan; †Department of Medical Biology, Hacettepe University, Ankara, Turkey; and ‡The Pediatric Nephrology Unit, Alexandria University, Alexandria, Egypt ABSTRACT In two siblings of consanguineous parents with intermittent nephrotic-range pro- tion is still unknown.7 This forbids the use of teinuria, we identified a homozygous deleterious frameshift mutation in the gene cohort studies for gene identification and ne- CUBN, which encodes cubulin, using exome capture and massively parallel re- cessitates the ability to identify disease-caus- sequencing. The mutation segregated with affected members of this family and ing genes in single families. We therefore was absent from 92 healthy individuals, thereby identifying a recessive mutation in combined whole genome homozygosity CUBN as the single-gene cause of proteinuria in this sibship. Cubulin mutations mapping with consecutive whole human ex- cause a hereditary form of megaloblastic anemia secondary to vitamin B12 defi- ome capture (WHEC) and massively par- ciency, and proteinuria occurs in 50% of cases since cubilin is coreceptor for both allel re-sequencing to overcome this lim- 6 the intestinal vitamin B12-intrinsic factor complex and the tubular reabsorption of itation. In this way we here identify a protein in the proximal tubule. In summary, we report successful use of exome homozygous deleterious frameshift mu- capture and massively parallel re-sequencing to identify a rare, single-gene cause tation within the cubilin gene (CUBN)as of nephropathy. a novel unexpected cause of proteinuria in two siblings with intermittent ne- J Am Soc Nephrol 22: 1815–1820, 2011. doi: 10.1681/ASN.2011040337 phrotic-range proteinuria. This ap- proach will likely be very useful to rap- idly solve cases of rare nephropathies. Protein-encoding exons constitute only drome,2 Kabuki syndrome,3 Schinzel- 1% of the human genome but harbor Giedion syndrome,4 Bartter syndrome,5 85% of mutations in single-gene disor- and nephronophthisis type 10.6 RESULTS ders.1 Among about 2800 Mendelian Nephrotic syndrome (NS) is a com- diseases, for which the causative gene mon kidney disease characterized by Two siblings (A 2410-21 and A2410-22) has been identified, mutations affect proteinuria, hypoalbuminemia, general- from family A2410 were discovered to the coding region or canonical splice ized edema, and hyperlipidemia. Identi- have proteinuria on routine examina- sites and thereby the function of the fication of recessive single-gene causes of encoded proteins 1. However, PCR am- NS has provided important insights into Received April 4, 2011. Accepted May 27, 2011. plification of thousands of candidate the pathogenesis of this enigmatic disor- Published online ahead of print. Publication date exons is costly and impractical and has der. Whereas NPHS2 mutations explain available at www.jasn.org. hampered discovery of single-gene dis- 10 to 25% of childhood NS, and two B.O. and E.A.O. contributed equally to this work. ease causes. Recently, re-sequencing of thirds of all NS in the first year of life can Correspondence: Dr. Friedhelm Hildebrandt, How- entire coding regions of the human ge- be explained by mutations in four genes ard Hughes Medical Institute, University of Michigan nome, the “exome,” with consecutive only (NPHS1, NPHS2, WT1, and Health System, 8220C MSRB III, 1150 West Medical massively parallel (MP) re-sequencing PLCE1),7 most other recessive causes of Center Drive, Ann Arbor, Michigan 48109-5646. 2 8–13 Phone: 734-615-7285 (office), 734-615-7895, -7896 has dramatically changed this situation. NS are very rare (1 to 3% of cases). (laboratories); Fax: 734-615-1386, -7770; E-mail: This approach has been successfully ap- Whereas single-gene causes of NS occur in [email protected] plied to identify single-gene causes of childhood and adolescence, in more than Copyright © 2011 by the American Society of rare diseases including Miller syn- about 70% of cases the causative gene muta- Nephrology J Am Soc Nephrol 22: 1815–1820, 2011 ISSN : 1046-6673/2210-1815 1815 BRIEF COMMUNICATION www.jasn.org tion. The amount of proteinuria in both aligned to the human reference genome gously in both parents (Figure 3). We siblings had been fluctuating, sometimes assembly hg19 (NCBI build 37) using then searched for additional CUBN reaching up to 2 g/d and then decreasing “CLC Genomics Workbench” software mutations in a large worldwide cohort without any treatment (see Patients in (CLC-bio, Aarhus, Denmark). About of 540 families with NS in whom we the Concise Methods section). Given con- 20.1 million of these reads (68%) had generated homozygosity mapping sanguinity of the parents of the two siblings matched the targeted exons contributing data by WGHM. Four affected individ- with proteinuria, we hypothesized that its to a median coverage of 11-fold (mean: uals (A155-21, A849-21, A1605-21, cause was a recessively inherited mutation. 14-fold). No coverage was obtained for and A2591-21) of these families Therefore, we performed linkage analysis about 2.2% of all 180,000 targeted exons. showed a genomic segment of Ͼ2Mb and homozygosity mapping in both affected CLC Genomics Workbench software was of homozygosity at the CUBN locus. siblings (A2410-21 and A2410-22) using Af- further used to call single nucleotide However, direct Sanger sequencing of fymetrix 250K SNP StyI arrays. Nonpara- variants or small insertions/deletions. By all exons of CUBN in these individuals metric LOD score analysis14 was calculated filtering for homozygous-only variants, did not yield any additional mutations for both siblings together, yielding seven we identified 1968 variants from the ref- (data not shown). segments of homozygosity by descent on erence sequence with at least fivefold se- chromosomes 3, 10, 14, 17, 21, and 22 quence coverage. Of these, only 48 were with a total cumulative genomic length not known single nucleotide polymor- DISCUSSION of 130 Mb (Figure 1). One segment of phisms, either reported in the database homozygosity on chromosome 10 coin- dbSNP130 or the 1000 genomes project. We here detected a novel 1-bp homozy- cided with PLCE1, a known locus for ne- When examining the segments of ho- gous deletion of the cubilin (CUBN) phrotic syndrome. However, Sanger re- mozygosity (132 Mb total), for which we gene as the cause of proteinuria in two sequencing of all exons and adjacent had initially generated a positional can- siblings with NS using a combined exon-intron boundaries of PLCE1 didate hypothesis by whole genome ho- strategy of homozygosity mapping and yielded no mutation in this gene. mozygosity mapping, only 11 nonsyn- WHEC with massively parallel re-se- We then performed whole human ex- onymous exonic variants remained. One quencing. The finding represented an ome capture in one sibling (A2410-22) of these changes was a novel 1-bp ho- unexpected cause of NS because inter- using NimbleGen 2.1M Human Exome mozygous deletion (c.8355delA) in exon mittent gross proteinuria is rare in pa- Array with consecutive massively parallel 53 of the gene CUBN (cubilin), resulting tients with CUBN mutations who are re-sequencing on two lanes of an Illu- in a frameshift and a predicted prema- known to have Imerslund-Grasbeck syn- mina-GAIIx sequencing platform to ture truncation of the encoded protein drome (IGS; OMIM ID #261100), a form identify the underlying disease-causing (p.S2785fsX19) (Figure 2). We con- of congenital megaloblastic anemia due mutation. Sequencing generated 29.5 firmed this mutation by exon-PCR and to vitamin B12 deficiency caused by a de- million single-end reads of 78 bases. Sanger sequencing homozygously in fect in the vitamin B12/intrinsic factor re- Quality-filtered sequence reads were both affected siblings and heterozy- ceptor (CUBN; OMIM ID #602997). Figure 1. Nonparametric lod (NPL) scores across the human genome in two siblings with intermittent gross proteinuria of consangui- nous family A2410 reveal four regions of homozygosity by descent. The x-axis shows Affymetrix 250K SNP StyI array SNP positions on human chromosomes concatenated from p-ter (left) to q-ter (right). Genetic distance is given in centimorgan (cM). Seven maximum NPL peaks (red circles) indicate candidate regions of homozygosity by descent. The arrow shows the region of homozygosity in chromosome 10, which includes CUBN. 1816 Journal of the American Society of Nephrology J Am Soc Nephrol 22: 1815–1820, 2011 www.jasn.org BRIEF COMMUNICATION Figure 2. The novel single-base homozygous deletion (c.8355delA) in exon 53 of the cubilin (CUBN) gene that causes nephrotic syndrome in family A2410. Note that the sequence shown depicts the plus strand of chromosome 10 and that CUBN is encoded on the reverse strand, therefore showing a deletion of a T (thymine) rather than A (adenine). The renal effects of genetic ablation of identified in a genome-wide association with IGS and CUBN mutation was re- cubilin in a mouse model has been re- study.16 ported. The patient was a 15-year-old girl ported previously.15 It was observed that