View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by RERO DOC Digital Library Human Molecular Genetics, 2014, Vol. 23, No. 20 5527–5535 doi:10.1093/hmg/ddu253 Advance Access published on June 3, 2014 Enrichment of pathogenic alleles in the brittle cornea gene, ZNF469, in keratoconus Judith Lechner1,{, Louise F. Porter2,{, Aine Rice3, Veronique Vitart4, David J. Armstrong5, Daniel F. Schorderet6,7,8, Francis L. Munier6,9, Alan F. Wright4, Chris F. Inglehearn3, Graeme C. Black2, David A. Simpson1, Forbes Manson2,{ and Colin E. Willoughby10,{,∗ 1Centre for Vision and Vascular Science, Queen’s University Belfast, Belfast BT12 6BA, UK, 2Institute of Human Development, The University of Manchester, Manchester Academic Health Science Centre, Central Manchester University Hospitals NHS Foundation Trust, Manchester M13 9WU, UK, 3Leeds Institute of Molecular Medicine, St. James’s University Hospital, University of Leeds, Leeds LS9 7TF, UK, 4MRC Human Genetics Unit, Institute of Genetics and Molecular Medicine, University of Edinburgh, Western General Hospital, Edinburgh EH4 2XU, UK, 5Department of Ophthalmology, Royal Victoria Hospital, Belfast BT12 6BA, UK, 6IRO - Institute for Research in Ophthalmology, Sion, Switzerland, 7Department of Ophthalmology, University of Lausanne, Lausanne, Switzerland, 8Faculty of Life Sciences, Ecole Polytechnique Fe´de´rale de Lausanne, Lausanne, Switzerland, 9Jules-Gonin Eye Hospital, Lausanne, Switzerland and 10Department of Eye and Vision Science, Institute of Ageing and Chronic Disease, University of Liverpool, Liverpool L69 3GA, UK Received February 4, 2014; Revised May 6, 2014; Accepted May 19, 2014 Keratoconus, a common inherited ocular disorder resulting in progressive corneal thinning, is the leading indi- cation for corneal transplantation in the developed world. Genome-wide association studies have identified common SNPs 100 kb upstream of ZNF469 strongly associated with corneal thickness. Homozygous mutations in ZNF469 and PR domain-containing protein 5 (PRDM5) genes result in brittle cornea syndrome (BCS) Types 1 and 2, respectively. BCS is an autosomal recessive generalized connective tissue disorder associated with ex- treme corneal thinning and a high risk of corneal rupture. Some individuals with heterozygous PRDM5 mutations demonstrate a carrier ocular phenotype, which includes a mildly reduced corneal thickness, keratoconus and blue sclera. We hypothesized that heterozygous variants in PRDM5 and ZNF469 predispose to the development of isolated keratoconus. We found a significant enrichment of potentially pathologic heterozygous alleles in ZNF469 associated with the development of keratoconus (P 5 0.00102) resulting in a relative risk of 12.0. This enrichment of rare potentially pathogenic alleles in ZNF469 in 12.5% of keratoconus patients represents a sig- nificant mutational load and highlights ZNF469 as the most significant genetic factor responsible for keratoco- nus identified to date. INTRODUCTION annually in the USA (2). Keratoconus usually arises in the Keratoconus (MIM 148300), a common bilateral, progressive teenage years and presents a significant health burden in corneal thinning disorder (1), is the leading indication for work-age adults. The minimum incidence is 1 in 2000, but it is corneal transplantation in the developed world, accounting for much more common in some ethnic groups (1,3). There is 25% of the 2500 corneal transplants performed annually in the strong evidence for a heritable component in the development UK and a similar proportion of the 32 000 grafts performed of keratoconus (4,5). Most studies describe autosomal dominant ∗To whom correspondence should be addressed at: Department of Eye and Vision Science, Institute of Ageing and Chronic Disease, Faculty of Health and Life Sciences, University of Liverpool, 3rd floor, UCD Building, Daulby Street, Liverpool L69 3GA, UK. Tel: +44 1517064070; Email: c.willoughby@ liverpool.ac.uk †The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors. ‡These authors contributed equally. # The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: [email protected] 5528 Human Molecular Genetics, 2014, Vol. 23, No. 20 inheritance, with incomplete penetrance or variable expressivity patients with keratoconus. This analysis failed to identify (4). However, in a genetic modelling study in a multi-ethnicity any pathogenic variants, although eight known SNPs were population a major recessive genetic defect was the most parsi- detected (rs146268537, rs74320998, rs343192, rs17051264, monious (6), although no recessive loci for keratoconus have rs34666716, rs12499000, rs75893420 and rs55774575). We been described to date. therefore proceeded to Sanger sequence ZNF469 in the original The progressive corneal thinning associated with keratoconus cohort increased with additional keratoconus cases (total [mean central corneal thickness (CCT) 450–500 mm] (7) results number of cases ¼ 112) of unrelated European patients with in myopia and irregular corneal astigmatism. In healthy humans, keratoconus from three study centres (Belfast, Leeds and Lau- CCT is a normally distributed quantitative trait with a mean sanne). Sequence variants detected by Sanger sequencing were of 536 + 31 mm(8), which has an estimated heritability up classified as potentially pathogenic alleles by filtering using (i) to 95% (9). Genome-wide association studies (GWAS) in the ethnically matched population-specific control data from 784 healthy European and Asian populations have identified individuals (outlined in the Materials and Methods); (ii) the CCT-associated loci, with common SNPs upstream of zinc data from dbSNP (Build 137), the May 2012 release of the finger 469 (ZNF469 [MIM 612078]) the most strongly asso- 1000 Genomes (1KG) Project and the Exome Variant Server ciated with CCT (10–14). Mutations in three genes (ZNF469, (EVS), NHLBI Exome Sequencing Project (ESP), with no COL5A1 and COL8A2) close or within these identified loci allele having a minor allele frequency (MAF) .0.1% and (iii) are responsible for rare Mendelian conditions that affect the classified as damaging using the Sorting Intolerant from Tolerant corneal structure: brittle corneal syndrome, Ehlers–Danlos syn- (SIFT) programme as outlined in the Materials and Methods and drome and posterior polymorphous corneal dystrophy, respect- Figure 2. ively (10–12). From these stringently filtered sequencing data, 12 potentially Brittle cornea syndrome (BCS) is an autosomal recessive gen- pathogenic non-synonymous heterozygous alleles were detected eralized connective tissue disorder associated with extreme in the keratoconus cohort (Table 1) and two in-frame deletions: corneal thinning (220–450 mm) and a high risk of corneal c.2904_2909delGTCGGG; p.Ser969_Gly970del and c.9011_ rupture (15,16). Homozygous mutations in ZNF469 and PR 9025delTTCCCGGGAACACCC; p.Leu3004_Thr3008del. In domain-containing protein 5 [PRDM5 (MIM 614161)] genes the keratoconus cohort following filtering, there remained 15 non- result in BCS Type 1 [BCS1 (MIM 229200)] (17) and BCS synonymous classified as tolerated by SIFT which were absent Type 2 [BCS2 (MIM 614170)] (15), respectively. Some indivi- from control data and had an MAF ,0.1% (Table 2). On the duals with heterozygous PRDM5 mutations demonstrate a basis of poor conservation, six of these variants were classified carrier ocular phenotype which includes a mildly reduced CCT as polymorphisms leaving nine variants of unknown significance. (480–505 mm), keratoconus and blue sclera (15). In one We detected 34 non-synonymous and 33 synonymous variants family with BCS2, there was a relationship between the severity that were observed in both cases and controls, had an MAF and age of onset of keratoconus and PRDM5 mutational status. ≥0.1% or were common variants, and were deemed non- Family members with a homozygous PRDM5 mutation (dele- pathogenic (Supplementary Material, Table S1). Overall, this tion of exons 9–14) developed early and severe keratoconus, study identified 34 novel variants (29 non-synonymous and 5 syn- whereas one heterozygous family member developed keratoco- onymous) that have been submitted to the NCBI dbSNP (Supple- nus which was clinically milder with a later onset (15). The rela- mentary Material, Table S2). The severity of keratoconus was tionship between the degree of CCT reduction and the presence graded using the Amsler–Krumeich classification (18,19), and of homozygous or heterozygous mutations in PRDM5 suggested 10 individuals had grade Stage III or above indicating severe a dosage effect (15). Although none of the CCT-associated loci disease (illustrated in Fig. 1). Stages III and IV usually require sur- have been mapped to PRDM5, a common SNP (rs10518367) gical approaches for visual rehabilitation, and three individuals which is 70 kb upstream of PRDM5 has been associated with required corneal transplantation. CCT in the European population at the significance level of As there was the possibility that the alleles identified in the P ¼ 8.9 × 1025. keratoconus subjects represented chance events, we Sanger Given that rare ZNF469 and PRDM5 homozygous mutations sequenced the complete coding sequence of ZNF469 (13 result in the extreme corneal thinning disorder (BCS), and that 203 bp) in 96 unaffected and unrelated European control there was evidence of a carrier ocular phenotype (keratoconus samples (192 chromosomes)