Identification of Novel Mutations in Patients with Leber Congenital Amaurosis and Juvenile RP by Genome-wide Homozygosity Mapping with SNP Microarrays Anneke I. den Hollander,1 Irma Lopez,2 Suzanne Yzer,3 Marijke N. Zonneveld,1 Irene M. Janssen,1 Tim M. Strom,4,5 Jayne Y. Hehir-Kwa,1 Joris A. Veltman,1 Maarten L. Arends,1 Thomas Meitinger,4,5 Maria A. Musarella,6 L. Ingeborgh van den Born,3 Gerald A. Fishman,7 Irene H. Maumenee,8 Klaus Rohrschneider,9 Frans P. M. Cremers,1 and Robert K. Koenekoop2 PURPOSE. Leber congenital amaurosis (LCA) and juvenile retini- RESULTS. Ten homozygous mutations, including seven novel tis pigmentosa (RP) cause severe visual impairment early in life. mutations, were identified in the CRB1, LRAT, RPE65, and Thus far, mutations in 13 genes have been associated with TULP1 genes in 12 patients. Ten patients were from consan- autosomal recessive LCA and juvenile RP. The purpose of this guineous marriages, but in two patients no consanguinity was study was to use homozygosity mapping to identify mutations reported. In 10 of the 12 patients, the causative mutation was in known LCA and juvenile RP genes. present in the largest or second largest homozygous segment METHODS. The genomes of 93 consanguineous and nonconsan- of the patient’s genome. guineous patients with LCA and juvenile RP were analyzed for CONCLUSIONS. Homozygosity mapping using SNP microarrays homozygous chromosomal regions by using SNP microarrays. identified mutations in a significant proportion (30%) of con- This patient cohort was highly selected, as mutations in the sanguineous patients with LCA and juvenile RP and in a small known genes had been excluded with the LCA mutation chip, number (3%) of nonconsanguineous patients. Significant ho- or a significant number of LCA genes had been excluded by mozygous regions which did not map to known LCA or juve- comprehensive mutation analysis. Known LCA and juvenile RP nile RP genes and may be instrumental in identifying novel genes residing in the identified homozygous regions were disease genes were detected in 33 patients. (Invest Ophthal- analyzed by sequencing. Detailed ophthalmic examinations mol Vis Sci. 2007;48:5690–5698) DOI:10.1167/iovs.07-0610 were performed on the genotyped patients. eber congenital amaurosis (LCA) represents a group of severe retinal dystrophies, characterized by congenital From the 1Department of Human Genetics, Nijmegen Centre for L Molecular Life Sciences, Radboud University Nijmegen Medical Centre, blindness, nystagmus, and a nondetectable electroretinogram Nijmegen, the Netherlands; the 2McGill Ocular Genetics Center, (ERG). LCA is generally inherited in an autosomal recessive McGill University Health Center, Montreal, Quebec, Canada; 3The manner and is genetically heterogeneous. To date, mutations in Rotterdam Eye Hospital, Rotterdam, The Netherlands; the 4Institute of 10 genes (AIPL1, CEP290, CRB1, CRX, GUCY2D, LCA5, Human Genetics, German Science Foundation National Research Cen- RDH12, RD3, RPE65, and RPGRIP1) and two loci (1p36 and ter for Environment and Health, Munich-Neuherberg, Germany; the 14q24) were found to be associated with autosomal recessive 5Institute of Human Genetics, Technical University, Munich, Germany; LCA.1–5 Mutations in each of these 10 genes have been iden- the 6Department of Ophthalmology, State University of New York, 7 tified in 0.1% to 20% of patients with LCA. Comprehensive New York; the Department of Ophthalmology, University of Illinois at mutation screening of all known LCA genes in a large cohort Chicago, Chicago, Illinois; the 8Wilmer Eye Institute, John Hopkins University, Baltimore, Maryland; and the 9Department of Ophthalmol- has not yet been performed, but it may currently be possible to ogy, University of Heidelberg, Heidelberg, Germany. determine the genetic basis of disease in approximately 60% of Supported by the Netherlands Organisation for Scientific Research LCA cases. Grants 916.56.160 (AIdH) and 917.66.363 (JAV); the Foundation Fight- LCA exhibits significant clinical overlap with juvenile reti- ing Blindness (AIdH, GAF); Flieringa Stichting/Stichting Wetenschap- nitis pigmentosa (RP). However, juvenile RP is considered pelijk Onderzoek Oogziekenhuis (AIdH, FPMC, LIvdB); Landelijke milder and does not have congenital onset of visual impair- Stichting voor Blinden en Slechtzienden (AIdH, FPMC); Algemene ment. Patients with juvenile RP usually experience night blind- Nederlandse Vereniging ter Voorkoming van Blindheid (AIdH, FPMC); ness within the first years of life, progress to a gradual loss of Foundation Fighting Blindness Canada (RKK, FPMC); Fonds de la Re- peripheral visual field, and eventually lose central vision. Mu- cherche en Sante´ du Que´bec (RKK); the Grant Healthcare Foundation tations in CRB1, RDH12, and RPE65 can cause both juvenile Chicago (GAF); and the Knights Templar Foundation Chicago (GAF). 6–8 Submitted for publication May 24, 2007; revised July 30, 2007; RP and LCA. In addition, mutations in LRAT, MERTK, and accepted October 1, 2007. TULP1 can cause juvenile RP, which in some cases may be 9–11 Disclosure: A.I. den Hollander, None; I. Lopez, None; S. Yzer, clinically diagnosed as LCA. None; M.N. Zonneveld, None; I.M. Janssen, None; T. M. Strom, Patients affected by a recessive disease and born of a con- None; J.Y. Hehir-Kwa, None; J.A. Veltman, None; M.L. Arends, sanguineous union are likely to be homozygous for the disease- None; T. Meitinger, None; M.A. Musarella, None; L.I. van den causing mutation and for polymorphisms in the region sur- Born, None; G.A. Fishman, None; I.H. Maumenee, None; K. Rohr- rounding this mutation.12 Homozygosity mapping is therefore schneider, None; F.P.M. Cremers, None; R.K. Koenekoop, None an effective method for locating the responsible gene in con- The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked “advertise- sanguineous families. In rare recessive diseases, as in LCA, the ment” in accordance with 18 U.S.C. §1734 solely to indicate this fact. frequency of homozygous mutations can also be relatively high Corresponding author: Anneke I. den Hollander, Department of Hu- in patients with no apparent consanguinity, due to a (distant) man Genetics, Radboud University Nijmegen Medical Centre, PO Box common ancestor who cannot be traced in the available family 9101, 6500 HB Nijmegen, The Netherlands; [email protected]. history. The average size of a homozygous region inherited Investigative Ophthalmology & Visual Science, December 2007, Vol. 48, No. 12 5690 Copyright © Association for Research in Vision and Ophthalmology Downloaded from tvst.arvojournals.org on 09/30/2021 IOVS, December 2007, Vol. 48, No. 12 Novel Mutations in Leber Congenital Amaurosis 5691 from a common ancestor living 10 generations ago measures SNP Microarrays 10 cM, a region that can readily be detected with high-density DNA samples for SNP analysis were purified (QIAamp DNA Mini Kit; SNP microarrays.13 Qiagen, Valencia, CA). Most of the consanguineous patients were In this study we analyzed 93 patients (82 isolated patients genotyped for 11,555 SNPs (GeneChip Human Mapping 10K Array; and 4 small families, each with 2 to 4 affected sibs) with LCA Affymetrix, Santa Clara, CA). French-Canadian patients were geno- and juvenile RP for homozygous regions using SNP microar- typed for 116,204 SNPs (GeneChip Human Mapping 100K Set, con- rays. Thirty-three patients were born from consanguineous sisting of a 50K Hind and a 50K Xba array; Affymetrix). The remaining marriage or in populations with a high degree of consanguin- patients were genotyped for 262,000 SNPs (GeneChip Mapping 250K eous unions. For 60 patients, mainly from The Netherlands or Nsp Array; Affymetrix). In addition, seven consanguineous patients Germany or of French Canadian descent, no consanguinity was who showed only a few or no homozygous regions with the 10K array reported. The patients included in the study represent a highly were also analyzed at higher resolution with the 250K array. Array selected patient group, as known mutations in the known LCA experiments were performed according to protocols provided by the genes had been excluded with the LCA mutation chip, or a manufacturer. Arrays were scanned and genotypes were called as significant number of LCA genes had been excluded by com- described.17 prehensive mutation analysis. Known LCA and juvenile RP The 10K SNP genotypes were analyzed for homozygous regions genes residing in homozygous regions were analyzed for mu- (ExcludeAR sheet; Excel, Microsoft, Redmond, WA).18 Chromosomal tations. This approach successfully identified homozygous mu- segments were accepted as homozygous if they contained Ն39 con- tations in 12 patients, including seven novel mutations in the secutive homozygous SNPs, since the likelihood that this would occur CRB1, RPE65, and TULP1 genes. In most patients, the disease- by chance is less than 1:100.18 These segments correspond to regions causing mutation was present in the longest or second longest of (on average) 8 Mb and larger. The 100K and 250K SNP data were homozygous segment of the patient’s genome. Significant ho- analyzed with the software package CNAG,19 and chromosomal seg- mozygous regions were detected in 33 consanguineous and ments were accepted to be homozygous if the loss-of-heterozygosity nonconsanguineous patients, which did not map to known (LOH) score was Ն15. The LOH score is a measure for the likelihood LCA or juvenile RP genes and may be instrumental to identify of a stretch of SNPs to be homozygous based on the population SNP novel LCA genes. In fact, we have recently demonstrated the allele frequencies. An LOH score of Ն15 corresponds to regions of (on effectiveness of using these homozygous regions to identify average) 4 Mb and larger. new LCA genes. One family with four affected siblings was instrumental in identifying the CEP290 gene as an important Mutation Analysis cause of LCA, and two unrelated patients with LCA recently allowed us to identify the LCA5 gene.3,5 Known LCA and juvenile RP genes that resided in homozygous chro- mosomal segments were analyzed for mutations.