ORIGINAL ARTICLE Identification of an Alu-repeat-mediated deletion of OPTN upstream region in a patient with a complex ocular phenotype Kala F. Schilter1,2, Linda M. Reis1, Elena A. Sorokina1 & Elena V. Semina1,2 1Department of Pediatrics and Children’s Research Institute, Medical College of Wisconsin, Milwaukee, Wisconsin 53226 2Department of Cell Biology, Neurobiology and Anatomy, Medical College of Wisconsin, Milwaukee, Wisconsin 53226 Keywords Abstract Alu-mediated recombination, anterior segment dysgenesis, CCDC3, OPTN Genetic causes of ocular conditions remain largely unknown. To reveal the molecular basis for a congenital ocular phenotype associated with glaucoma we Correspondence performed whole-exome sequencing (WES) and whole-genome copy number Elena V. Semina, Department of Pediatrics analyses of patient DNA. WES did not identify a causative variant. Copy num- and Children’s Research Institute, Medical ber variation analysis identified a deletion of 10p13 in the patient and his unaf- College of Wisconsin, 8701 Watertown Plank fected father; the deletion breakpoint contained a single 37-bp sequence that is Road, Milwaukee, WI 53226. normally present in two distinct Alu repeats separated by ~181 kb. The deletion Tel: 414 955 4996; Fax: 414 955 6329; E-mail: [email protected] removed part of the upstream region of optineurin (OPTN) as well as the upstream sequence and two coding exons of coiled-coil domain containing 3 Funding Information (CCDC3); analysis of the patient’s second allele showed normal OPTN and This work was supported by the National CCDC3 sequences. Studies of zebrafish orthologs identified expression in the Institutes of Health award R01EY015518 and developing eye for both genes. OPTN is a known factor in dominant adult- funds provided by the Children’s Hospital of onset glaucoma and Amyotrophic Lateral Sclerosis (ALS). The deletion elimi- Wisconsin (EVS), 1UL1RR031973 from the nates 98 kb of the OPTN upstream sequence leaving only ~1 kb of the proxi- Clinical and Translational Science Award (CTSA) program. mal promoter region. Comparison of transcriptional activation capability of the 3 kb normal and the rearranged del(10)(p13) OPTN promoter sequences dem- Received: 12 February 2015; Revised: 5 May onstrated a statistically significant decrease for the deleted allele; sequence 2015; Accepted: 8 May 2015 analysis of the entire deleted region identified multiple conserved elements with possible cis-regulatory activity. Additional screening of CCDC3 indicated that Molecular Genetics & Genomic Medicine heterozygous loss-of-function alleles are unlikely to cause congenital ocular dis- 2015; 3(6): 490–499 ease. In summary, we report the first regulatory region deletion involving doi: 10.1002/mgg3.159 OPTN, caused by Alu-mediated nonallelic homologous recombination and possibly contributing to the patient’s ocular phenotype. In addition, our data indicate that Alu-mediated rearrangements of the OPTN upstream region may represent a new source of affected alleles in human conditions. Evaluation of the upstream OPTN sequences in additional ocular and ALS patients may help to determine the role of this region, if any, in human disease. Background number of genes have been found to play a role in these disorders, including CYP1B1 (MIM: 601771), LTBP2 Anterior segment dysgenesis of the eye results from (MIM: 602091), MYOC (MIM: 601652), FOXC1 (MIM: abnormal embryonic development and is often associated 601090), PAX6 (MIM: 607108), and PITX2 (MIM: with congenital or early-onset glaucoma (Idrees et al. 601542) (Khan 2011; Reis and Semina 2011), many cases 2006). Pediatric glaucoma is difficult to manage with are still awaiting molecular diagnosis. most affected individuals requiring surgical intervention; Copy number variation (CNV) includes deletions or some degree of vision loss is commonly observed (Khitri duplications of various sizes that are identified in compari- et al. 2012). Anterior segment dysgenesis and pediatric son to a reference genome and can be inherited or occur glaucoma are highly heterogeneous conditions. While a de novo (Feuk et al. 2006). Three major mechanisms for 490 ª 2015 The Authors. Molecular Genetics & Genomic Medicine published by Wiley Periodicals, Inc. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. K. F. Schilter et al. Deletion of OPTN Upstream Region in Ocular Disease CNV in the human genome include nonallelic homologous Amplification of the deleted allele and sequencing of the recombination typically mediated by low-copy repeats or deletion breakpoints was performed using the primers highly homologous repetitive sequences like Alu and LINE, indicated in Table S2. The resultant product was cloned nonhomologous end-joining, and the Fork Stalling and into pCRII TOPO vector (Life Technologies) and Template Switching models (Gu et al. 2008). CNV alleles sequenced. were found to be associated with both dominant and, Whole-exome sequencing was undertaken through when coupled with another deleterious mutation of a gene Perkin Elmer, Inc (Branford, CT) using Agilent Sure Select within the region, recessive phenotypes (Lesnik Oberstein v4+UTR for exome capture and analyzed as previously et al. 2006; Pieras et al. 2011). It is widely accepted that described (Reis et al. 2013). Data were evaluated for muta- CNVs, especially deletion alleles, often result in affected tions in 203 genes known to be involved in ocular develop- phenotypes or increase an individual’s susceptibility for the ment (Schilter et al. 2013) through the Geospiza GeneSifter disease (Feuk et al. 2006; Beckmann et al. 2007; Stranger Analysis program hosted through Perkin Elmer Bioinfor- et al. 2007; Liu et al. 2011a). A surprisingly extensive vari- matics. The entire exome was analyzed using the SNP & ability in copy number has been identified in the human Variation Suite (SVS; Golden Helix, Bozeman, MT) as pre- genome and was proposed to represent a major source of viously described (Deml et al. 2014; Weh et al. 2014). inter-individual genetic diversity, possibly underlying the incomplete penetrance and variable expressivity of many Gene sequencing inherited Mendelian disorders as well as variation in phe- notypic expression associated with more complex disease Complete sequence for the coding regions of the OPTN (Beckmann et al. 2007). Copy number variation analysis (NM_001008211.1) and CCDC3 (NM_031455.3) genes was instrumental to the discoveries of regulatory regions/ was obtained for the proband utilizing the primers indi- mutations associated with disease phenotypes (Lauderdale cated in Table S2. Sequencing of CCDC3 was undertaken et al. 2000; Volkmann et al. 2011). in an additional 115 patients affected with ocular disor- In this paper, we present identification and character- ders and 183 (90 Caucasian, 93 Hispanic) controls. ization of a deletion involving the OPTN (MIM: 602432) Sequences were reviewed manually and using Mutation and CCDC3 (NM_031455.3) genes in a patient with a Surveyor (SoftGenetics, State College, PA). All mutations congenital ocular phenotype involving glaucoma. were confirmed by independent sequencing reactions using new PCR products. Variants of interest were Methods reviewed for their frequency in dbSNP (http:// www.ncbi.nlm.nih.gov/projects/SNP/), Exome Variant Server (EVS; http://evs.gs.washington.edu/EVS/), and Human subjects ExAC Browser (http://exac.broadinstitute.org). The human study was approved by the Institutional Review Board of the Children’s Hospital of Wisconsin Expression studies in zebrafish and informed consent was obtained from every subject and/or legal guardian, as appropriate. All experiments were executed in agreement with the guidelines described by the Institutional Animal Care and Use Committee at the Medical College of Wisconsin. The Copy number variation and WES analyses zebrafish optn (NM_001100066.2), ccdc3a (chromosome Copy number variation analysis via Affymetrix Genome- 4; NM_001025510), and ccdc3b (chromosome 11; Wide Human SNP Array 6.0 (Santa Clara, CA) was under- NM_001020570) constructs were generated using the PCR taken as previously described with custom region analysis products obtained with the primers indicated in Table S2. for RefSeq genes (NCBI build GRCh37/hg19) and 203 In situ hybridization was performed as previously genes known to be involved in ocular development includ- described (Liu and Semina 2012). ing 200 kb of potential regulatory regions (Schilter et al. 2013). The Database of Genomic Variants (http://pro- Reported constructs and luciferase assays jects.tcag.ca/variation/) was used as a control population along with 30 unaffected in-house controls; a control pop- OPTN wild-type promoter sequence was amplified with ulation specifically matched to the patient’s Trinidad and the primers indicated in Table S2 to generate a 3049-bp Tobago ancestry was not available. Predesigned and cus- product. This fragment was first inserted into a pCRII- tom-designed TaqMan probes (Life Technologies, Carls- TOPO plasmid and then subcloned into a pGL4.10[luc2] bad, CA) (Table S1) were utilized for independent vector (Promega, Madison, WI) using KpnI (located within verification/confirmation of the copy number states. the pCRII polylinker) and AvrII (located within OPTN ª 2015 The Authors. Molecular Genetics & Genomic Medicine published by Wiley Periodicals, Inc. 491 Deletion of OPTN Upstream Region in Ocular Disease