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Genetics  |   September 2014
Mutations in the Zinc Finger Protein Gene, ZNF469, Contribute to the Pathogenesis of Keratoconus
Author Affiliations & Notes
  • Andrea L. Vincent
    Department of Ophthalmology, New Zealand National Eye Centre, University of Auckland, Auckland, New Zealand
    Eye Department, Greenlane Clinical Centre, Auckland District Health Board, Auckland, New Zealand
  • Charlotte A. Jordan
    Department of Ophthalmology, New Zealand National Eye Centre, University of Auckland, Auckland, New Zealand
    Eye Department, Greenlane Clinical Centre, Auckland District Health Board, Auckland, New Zealand
  • Murray J. Cadzow
    Department of Biochemistry, University of Otago, Dunedin, New Zealand
  • Tony R. Merriman
    Department of Biochemistry, University of Otago, Dunedin, New Zealand
  • Charles N. McGhee
    Department of Ophthalmology, New Zealand National Eye Centre, University of Auckland, Auckland, New Zealand
    Eye Department, Greenlane Clinical Centre, Auckland District Health Board, Auckland, New Zealand
  • Correspondence: Andrea L. Vincent, Department of Ophthalmology, FMHS, University of Auckland, Private Bag 92019, Auckland, New Zealand; a.vincent@auckland.ac.nz
Investigative Ophthalmology & Visual Science September 2014, Vol.55, 5629-5635. doi:10.1167/iovs.14-14532
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      Andrea L. Vincent, Charlotte A. Jordan, Murray J. Cadzow, Tony R. Merriman, Charles N. McGhee; Mutations in the Zinc Finger Protein Gene, ZNF469, Contribute to the Pathogenesis of Keratoconus. Invest. Ophthalmol. Vis. Sci. 2014;55(9):5629-5635. doi: 10.1167/iovs.14-14532.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose.: Mutations in the zinc finger protein gene ZNF469 cause recessive brittle cornea syndrome, characterized by spontaneous corneal perforations. Genome-wide association studies (GWAS) have implicated common variants in this gene as a determinant for central corneal thickness (CCT). We investigated the contribution of ZNF469 in a sample set of keratoconus patients.

Methods.: Forty-three patients with keratoconus (49% Māori or Pacific [Polynesian]) were recruited. If a family history was present, family members were recruited. Participants underwent comprehensive examination, and a DNA sample was collected. Mutational analysis of ZNF469 was undertaken using Sanger sequencing, including an ancestrally matched Polynesian control population. Bioinformatic databases of exome variation and protein prediction software were used to determine presence and frequency and the pathogenicity for each observed change.

Results.: Fourteen nonsynonymous missense single nucleotide polymorphisms (SNPs) were observed in ZNF469. Of the 43 probands, at least one probable disease-causing variant was detected in 20 (46%) (16/32 sporadic, 4/11 familial) and two variants in 5 (11.6%) (3/32 sporadic, 2/11 familial). Only heterozygous changes segregated with disease. Three “deleterious” changes observed in the Polynesian controls were removed from analysis; therefore pathogenic variants occurred in 10/43 (23.3%).

Conclusions.: Rare missense mutations in ZNF469, predicted to be pathogenic, occurred heterozygously, at a frequency of 23% in a keratoconus population. ZNF469 is associated with CCT in GWAS and is therefore likely to play a role in the synthesis and/or organization of corneal collagen fibers. The pathogenic changes observed either genetically predispose toward a “thin” cornea, which then becomes keratoconic, or are directly pathogenic.

Introduction
Keratoconus is a progressive corneal dystrophy leading to a characteristic pattern of corneal thinning and bulging (ectasia) with induced irregular myopic astigmatism. It may be markedly asymmetrical, and onset is usually in the teens, with noticeable change occurring during puberty. The incidence of keratoconus within the general population is approximately 1 in 2000, 1 with no predilection for either sex. 
Keratoconus is a major indication for corneal transplantation in the Western world, constituting 41% of all keratoplasties performed in New Zealand via the New Zealand National Eye Bank over a 10-year period 2 and 67% in the pediatric population (<14 years). 3 Comparatively, keratoconus was the clinical indication in 28.8% in a French series 4 and in 11.4% to 15.4% in two US series. 5,6 This represents a disproportionate percentage of transplantation for keratoconus in New Zealand compared with the rest of the developed world. 
Many multigenerational families are identified that show autosomal dominant (AD) inheritance; of affected New Zealand Māori and Pacific individuals, 31% report a positive family history of keratoconus. 7 One study looking at corneal topographic values in teenagers showed a far higher incidence of suspected keratoconus in Māori/Pacific students compared with European Caucasian students (26.9% vs. 12.9% P = 0.0014). 8 Other investigators characterizing the mode of inheritance have reported sporadic autosomal recessive 9 and AD inheritance. 10 However, other studies have established linkage by using a model of inheritance that lies somewhere between these two. 11  
Brittle cornea syndrome (BCS) is an autosomal recessive condition in which a thin cornea spontaneously perforates with minimal trauma, resulting in poor ocular survival. Cases in the literature arise from 1913, with early descriptions including brittle corneas, blue sclerae, keratoglobus, and keratoconus, although it is difficult to exclude an Ehlers-Danlos phenotype from the descriptors. 12 An association with red hair was subsequently observed in consanguineous pedigrees of Arabic origin, 13 Greek origin, 14 and among Jews of Tunisian origin, 15 which aided identification of mutations in the ZNF469 gene in patients with BCS in 2008 16 and has been further replicated. 1720 A second transcription factor gene, PRMD5, has also had mutations demonstrated in BCS. 21,22  
Recently, genome-wide association studies (GWAS) have established a role for common variants in ZNF469 in the control of central corneal thickness (CCT) and therefore as a potential risk factor for glaucoma, 2327 and very recently keratoconus. 28 A number of other genes are implicated in keratoconus, and these are summarized in a recent review article. 29 Although there is evidence for a strong genetic contribution to keratoconus and there has been identification of specific and significant causative genes, most genes identified to date account for only a minority of cases. 
Given the association of pathogenic mutations in ZNF469 causing autosomal recessive BCS, and the mounting evidence that ZNF469 plays a role in determination or regulation of CCT, this is an ideal candidate gene for the keratoconus phenotype. The high incidence and familial aggregation of cases in the Polynesian population suggest a mode of inheritance that could be AD with reduced penetrance, or autosomal recessive with pseudodominance assuming a high carrier frequency of potentially pathogenic mutations given a small migratory population and founder effect. This study examines whether ZNF469 is a candidate gene in the pathogenesis of keratoconus in a New Zealand population, including people of Polynesian ancestry. 
Methods
Patient Recruitment
Patients were recruited from the Department of Ophthalmology, Greenlane Clinical Centre, Auckland District Health Board, with a clinical diagnosis of keratoconus, and were reviewed in the University Clinic, Department of Ophthalmology, University of Auckland. The protocol of this study adhered to the tenets of the Declaration of Helsinki with Institutional Ethics and Māori Research Review Board approval (Ministry of Health NTX/06/12/161 and ADHB A+3657). Family members were recruited where possible when a positive family history was reported. 
Clinical
All subjects underwent extensive clinical examination including Snellen visual acuity, autorefraction, corneal topography, and pachymetry using a combined Placido/slit-scanning elevation tomography system (Orbscan II; Bausch & Lomb Surgical, Rochester, NY, USA) and/or Pentacam Schiempflug analysis (Oculus, Wetzlar, Germany), slit-lamp examination and photography, and laser scanning in vivo confocal microscopy (IVCM) using the HRTII (Heidelberg Retina Tomograph II, Rostock Corneal Module [RCM]; Heidelberg Engineering GmbH, Heidelberg, Germany). 
DNA Collection
Following informed consent, biological samples (peripheral venous blood or saliva specimen) were collected for DNA extraction using the salt extraction method from blood 30 and according to the manufacturer's instructions for saliva kits (Oragene; DNAGenotek, Ottawa, ON, Canada). For controls, two populations were used. Deoxyribonucleic acid samples were collected from randomly selected and ancestrally matched individuals attending the Department of Ophthalmology who did not exhibit any clinical evidence of corneal abnormality in terms of appearance or topographic parameters. The other control population with self-reported Māori ancestry was recruited as previously described with ethical approval from a regional Ethics Committee (OTA/99/11/098). 31  
Mutational Analysis of ZNF469
Deoxyribonucleic acid samples were screened for mutations in all coding exons of ZNF469 (NM_001127464.1) and PRMD5 (NM_018699.2) including intron–exon boundaries; further details of primers and polymerase chain reaction (PCR) conditions are provided in Supplementary Table S1. Following column purification with HighPure PCR purification kit (Roche Diagnostic, Mannheim, Germany), the product was sequenced directly according to protocols accompanying the ABI BigDye terminator kit v3.1 (Applied Biosystems, Inc., Foster City, CA, USA). Bidirectional sequencing of amplicons was undertaken on an ABI 3700 prism genetic analyzer (Applied Biosystems, Inc). Nucleotide sequences were compared with the published ZNF469 and PRMD5 sequences using CodonCode Aligner (CodonCode Corporation, Centreville, MA, USA) and polymorphic variation data in electronic databases to determine pathogenicity. Thirty-one Polynesian (New Zealand Māori = 14, Samoan = 15, Tongan = 1, Niuean = 1) control individuals (62 alleles) underwent full sequencing of the coding regions of ZNF469. A further 15 control participants with self-reported Māori ancestry had exomes sequenced at 50× depth by Illumina technology using an Agilent (Santa Clara, CA, USA) exome enrichment kit. Sequence data were aligned, analyzed, and managed as previously described. 32  
To determine the frequency of the ZNF469 variants detected in the familial cases (p.E316K, p.R2129K, p.A2475E, p.R2879H), in addition to the Polynesian controls, a further 70 New Zealand European controls (140 alleles) were screened. Screening for the detected ZNF469 sequence variants used HRMA on the RotorGene6000 (Corbett Life Sciences, San Francisco, CA, USA), using the High Resolution Melting Master kit (Roche Diagnostic). Further details of primers are provided in Supplementary Material S1. Each HRMA reaction included a positive and negative control based on sequencing confirmation. Any sample on the melt curve that produced an equivocal reading was subject to further PCR and sequencing to confirm or exclude the presence of the sequence variation. 
For the sequence variants, homology and predicted destruction or creation of exonic splicing enhancers, or effects on splicing, were evaluated using a variety of publicly available software. PolyPhen2 (http://genetics.bwh.harvard.edu/pph2/) and SIFT (available in the public domain at http://sift.jcvi.org/) analysis were used to predict the impact of the missense variants on protein structure and function, with Gene Splicer and Human Splicing Finder 33 for splicing signal analysis. Factors considered for determining potential pathogenicity of unclassified variants were positive family segregation, an allele frequency of <1/100 control chromosomes, homology, and/or bioinformatic prediction of biological significance. For an unclassified variant to remain in the list as a possible disease-causing variant, the criteria included present in patient population, absent in Polynesian control population, absent in population databases of human variation (Exome Variant Server [EVS; available in the public domain at http://evs.gs.washington.edu/EVS] or 1000 Genomes [1000G; available in the public domain at http://browser.1000genomes.org]), or with an allele frequency of less than 1/100 in European control chromosomes studied here, prediction of probably/possibly damaging or damaging/deleterious in at least one of the protein prediction software programs. 
For three changes (p.R2129K, p.A2475E, and p.G3415V), both protein prediction algorithms suggested that the change was deleterious/pathogenic; however, the alleles were present in the Polynesian control population and databases of human variation. Similarly, p.G3256R was not present in any population databases or in the control population, but was calculated to be benign or tolerated in terms of protein pathogenicity. These four variants were nevertheless included in the initial analysis. 
Results
Forty-tree probands were recruited: 32 were isolated sporadic cases and the remaining 11 were probands of families with more than one affected member. Demographic details are provided in Table 1. The pedigrees are shown in Figure 1. Of the families, all were Polynesian except for one (FYJ1 European). 
Figure 1
 
Pedigrees of familial keratoconus, with ZNF469 variants by genotyped individuals. Filled symbols, affected; unfilled symbols, clinically unaffected; diagonal line, deceased; plus sign, wild-type allele; asterisk, examined and DNA sample collected.
Figure 1
 
Pedigrees of familial keratoconus, with ZNF469 variants by genotyped individuals. Filled symbols, affected; unfilled symbols, clinically unaffected; diagonal line, deceased; plus sign, wild-type allele; asterisk, examined and DNA sample collected.
Table 1
 
Demographics of the Keratoconus Probands at Recruitment: Familial and Sporadic
Table 1
 
Demographics of the Keratoconus Probands at Recruitment: Familial and Sporadic
Disease Number Age, y (Range) Sex, Female:Male Ethnicity (%) Familial (%) Sporadic (%)
Keratoconus 43 41.5 (15–83) 22:21 11 (25) 32 (75)
Caucasian 18 (42) 1 (2) 17 (40)
Polynesian 21 (49) 10 (23) 11 (25)
Indian 4 (9) 0 4 (9)
ZNF469
Thirty-seven variants were observed in ZNF469, as listed in Supplementary Table S1, but these were all missense changes, with no indel, frame shift, or nonsense changes. Of the 37 variants observed, synonymous single nucleotide polymorphisms (SNPs) were excluded from further investigation, and nonsynonymous SNPs that were either absent in population databases and/or predicted to be damaging on at least one protein prediction algorithm were included for further investigation. This left 14 variants, but a further 4 were excluded as both protein prediction algorithms were benign and/or the frequency in both the case and control population was very high. Ten possibly deleterious nonsynonymous missense SNPs therefore remained, 6 of which were not previously documented in any population-based genetic variant database (Table 2). The locations of the observed variants are illustrated in Figure 2
Figure 2
 
Schematic of ZNF469 protein domains, with location of variants described in keratoconus cohort (Reference Transcript ZNF46-201 ENST00000437464).
Figure 2
 
Schematic of ZNF469 protein domains, with location of variants described in keratoconus cohort (Reference Transcript ZNF46-201 ENST00000437464).
Table 2
 
Nonsynonymous SNPs Identified
Table 2
 
Nonsynonymous SNPs Identified
Nucleotide Protein Cases Ancestry rs Number Polynesian Controls MAF Polynesian Controls Caucasian Controls EVS 1000G PolyPhen2 SIFT
c.946G>A p.E316L 1 fam Māori - 0/92 0% 0/140 Absent Absent Prob dam Tol
c.1697C>T p.A566V 3 Caucasian, n = 2
Indian
rs181785233 0/92 0% Absent T0.005 Poss dam Tol
c.6386G>A p.R2129K 8 Tonga n, = 2
Samoan, n = 3
Caucasian, n = 2
Māori
rs13334190 13/92 14.13% 0/140 Absent A0.063 Poss dam Del
c.6796G>A p.G2266A 1 Caucasian 0/92 0.00% Absent Absent Benign Tol
c.7424C>A p.A2475E 4, including  2 fam Family:
1 Caucasian,
1 Māori
Sporadic: Caucasian, Indian
rs141218390 2/92 2.1% 6/140 5.6 T0.056 Prob dam Dam
c.8246A>T p.D2749V 1 Caucasian rs3812954 1/92 1.08% 0/140 (4.3%) 5.6 T0.056 Poss dam Del
c.8636G>A p.R2879H 1 fam Tongan - 0 0.00% Absent Absent Poss dam Dam
c.9616C>T p.P3206L 1 Caucasian 0 0.00% Absent Absent Benign Dam
c.9766G>A p.G3256R 1 Caucasian 0 0.00% Absent Absent Benign Tol
c.10244g>T p.G3415V 3, 1 fam Family: CI Māori Sporadic:
Māori, n = 2
-rs140056980 12/92 13.04% Absent T0.010 Benign Dam
In the 43 cases, at least one of these possibly deleterious/rare variants in ZNF469 was detected in 20 (46%). In 5 cases, two variants were observed (11.6%—three compound heterozygotes, one homozygote, and one case with two changes presumed to be in cis); all these had a severe phenotype using the Collaborative Longitudinal Evaluation of Keratoconus (CLEK) classification 34 (Table 3). For the sporadic cases, 13/32 had one change and 3/32 had two changes. Of the familial cases, 2/11 probands had one change and 2/11 had two changes, although only heterozygous changes segregated with disease. 
Table 3
 
Characteristics of Individuals With Two Changes in ZNF469 (Homozygous, Compound Heterozygous, and in cis)
Table 3
 
Characteristics of Individuals With Two Changes in ZNF469 (Homozygous, Compound Heterozygous, and in cis)
Family Mutations Ethnicity CLEK29 Classification Proband CLEK29 Classification Sibling Pedigree
1 1 fam p.E316K NZ Māori Severe OU Severe OU TAR1
p.A2475E in cis
2 1 fam p.R2129K Tongan Severe OU Severe/moderate TUT1
p.R2879H
3 p.G2266A Caucasian Severe OU
p.D2749V
4 p.A566V Caucasian Severe OU
p.R2129K
5 p.G3415V NZ Māori Severe OU
p.G3415V
Three of the observed variants, although predicted to be deleterious, appeared at a frequency greater than 1% in the Polynesian controls and/or in the EVS or 1000G databases. Removing these three variants (p.R2129K, p.A2475E, and p.G3415V) left one change present in 10 probands (23.2%), but only one compound heterozygote (1/43 2.3%). 
PRMD5
PRMD5 mutational analysis was undertaken in 30 sporadic cases. Two of the sporadic cases included in the ZNF469 investigation had insufficient DNA remaining for analysis. 
Sequence variants observed were synonymous, except for one change, observed heterozygously in three Māori cases (Supplementary Table S2), p.S356T. SIFT predicted that this change would be tolerated, although it was called possibly damaging by PolyPhen2. Because of the low yield of changes in PRMD5, further testing was not undertaken in the familial cases. 
Discussion
The association of ZNF469 in the determination of central corneal curvature and thickness in the normal population, paired with the BCS phenotype with recessive mutations, makes ZNF469 an excellent candidate gene to study in a keratoconus population. As a candidate it also makes biological sense, as functionally ZNF469 is considered to play a role in the synthesis and/or organization of corneal collagen fibers in conjunction with PRMD5. 22 Recent work shows downregulation of key extracellular matrix (ECM) component genes [clusterin [CLU], glypican-6 [GPC6], procollagen C-endopeptidase enhancer 2 [PCOLCE2], and thrombospondin [THBS1]) in ZNF mutant fibroblasts. 35  
Rare missense variants in ZNF469, predicted to be pathogenic, were detected in the heterozygous state at a frequency of up to 46% in our keratoconus population, and two variants were observed in 11.6% (three compound heterozygotes, one homozygote, one with two changes in cis). Patients with two changes generally had a severe keratoconus phenotype as defined by the CLEK classification 34 (Table 3), all requiring corneal transplantation by the age of 20. Three of the observed variant changes were also present in the control Polynesian population and the population databases of human variation, although at very different frequencies. Removing these changes from analysis, 23% of patients still had one probable pathogenic variant. 
In the initial analysis we included three changes (p.R2129K, p.A2475E, and p.G3415V) observed in both the keratoconus population and the control Polynesian population, which were predicted by one or both protein prediction algorithms to be damaging (Table 2). p.G3415V occurred in the homozygous state in one patient with a severe keratoconus phenotype and in the heterozygous state in two patients. 
The p.R2129K variant was also found in combination with a second presumed pathogenic variant in two patients, both with a severe keratoconus phenotype, and the p.A2475E variant occurred in association with the p.E316K variant in two Tongan brothers with a severe phenotype (Fig. 1, Ped TAR1); however, nonsegregation suggests that the changes occur in cis. 
The presence of the p.G3415V allele in the heterozygous state in 13% of the Polynesian control population, as well as the p.R2129K in 14%, is a relatively high frequency. A likely explanation is that this is due to a founder effect given the migratory history of the Polynesian population. Ensembl (available in the public domain at http://www.ensembl.org/) frequency data for the p.R2129K are 6% in all populations, but range from 2% in the Asian compared with 15% in the African population (with the highest rate in the subpopulation American of African Ancestry in the SW USA and 16% in the Yoruba). p.G3415V is not present in most populations but is observed in the Asian population (4%–6% in Han Chinese). 
Thus p.R2129K is common, with highest prevalence in Polynesians and African Americans. The normal range of CCT in the Polynesians is not reported, but Haseltine et al. 36 showed a clear ethnic variation; CCT in African Americans was lower (529.3 μm) than in Hispanic people (544.7 μm, P = 0.008) and Europeans (549.9 μm, P < 0.001). While it is feasible that common variants such as p.R2129K influence not only keratoconus but also CCT, this variant was not significant in any of the previously reported GWAS studies of CCT. 2327  
Although these three variants (p.R2129K, p.A2475E, and p.G3415V) were excluded in the final analysis because of the high frequency in control populations, a sound argument for leaving them in the analysis could potentially be made. However, we have documented, discussed, and removed them as the frequency in controls would render them as polymorphisms. 
Although anecdotally, it is widely believed that keratoconus is more prevalent and aggressive in New Zealand, especially in the Māori and Pacific Island population, exact figures are not available. 7,8 However, keratoconus is the leading indication for corneal transplantation in both adults and children in New Zealand. 37,38 It is plausible that genetic factors are responsible for the ethnic predisposition to keratoconus in New Zealand, and the increased frequency of these probable deleterious ZNF469 variants may go some way toward explaining the higher incidence of keratoconus in the Polynesian population with the appropriate precipitating environmental stimulus. The lack of segregation may be explained by an inheritance pattern of AD with reduced penetrance, or the frequency of the common variant(s) if an environmental component to disease manifestation is not universally present. 
Brittle cornea syndrome has a spectrum of nonocular features including red hair, 12 which informed the ZNF469 gene identification 16,39 and has been replicated in other individuals since. 1720 The extraocular manifestations reported in association with BCS include scoliosis and reduced bone mineral density in the range of osteopenia and osteoporosis, as well as abnormal dentition (ranging from hypomineralization to mandibular crowding) and cardiac anomalies. Of note, the spectrum of phenotype includes absence of extraocular features, 19 individuals without corneal fragility, 20 and a phenotype more consistent with Ehlers-Danlos. 17  
One of the limitations of this study was that although all patients were extensively characterized from an ocular perspective and a full medical history was taken, we did not specifically ask or examine for the BCS extraocular features described. Therefore subclinical abnormalities in bone mineral density or abnormal dentition would not have been detected by us. Unfortunately, it is not possible to retrospectively systemically phentoype these patients. Another limitation with this study was the relatively small number of individuals comprising the control Polynesian population. Genetic variation in Māori and Polynesian individuals is not represented in any population variation databases; this emphasizes one of the current limitations of these public databases, for which allele frequencies are not available for minority ancestral groups. 
The nature of the mutations in ZNF469 previously described in association with the BCS phenotype are also more severe than observed in this cohort. The mutations in BCS described to date are deletions or duplications resulting in frame shifts with premature termination of the protein, 17,21,39 nonsense, 21 or missense and are located in the fourth of the five zinc finger domains. 18 This contrasts with the missense variants we report, predominantly occurring heterozygously and not directly located within the ZNF domains. This may also contribute to the ocular findings and, indeed, to extraocular features (if present) being at the mild end of the BCS spectrum. 
Burkitt Wright et al. 22 demonstrated mutations in PRMD5 in BCS and noted a milder corneal phenotype, but still reduced CCT and keratoconus in heterozygous carriers of PRMD5 mutations compared to those in homozygous mutation carriers. The authors suggested that this may be due to a dosage effect. In this population, only one nonsynonymous PRMD5 change of unknown significance was observed in three individuals. 
No clear functional studies of ZNF469 mutants are available to clearly document the pathophysiological process causing these changes. However, the high frequency of at least one potentially pathogenic variant in Polynesians compared with a control population variant is surprising. 
In conclusion, ZNF469 is a strongly associated factor in determination of CCT with recessive loss of function mutations causing BCS. This study, demonstrating an increased frequency of heterozygous, pathogenic changes in a keratoconus cohort of sporadic and familial cases of mixed ethnicities, suggests that ZNF469 contributes to the pathogenesis of keratoconus. The higher allele frequency of some of these changes in a control Polynesian population either excludes them as pathogenic or may partly account for the higher frequency of keratoconus within this population. These findings suggest that the pathogenic changes observed may genetically predispose toward a thin cornea, which given the right environmental stimulus may become keratoconic; or, alternatively, it is feasible that the changes are directly pathogenic and that a dosage effect predisposes to keratoconus. 
Replication of these data in other population cohorts may contribute to understanding the findings, as will further clarification of the role of the ZNF469 gene product in the development and regulation of normal corneal collagen structure and function. 
Supplementary Materials
Acknowledgments
We thank Amanda Richards and Bryan Hay for excellent technical assistance. Referring Ophthalmologists. 
Supported by the Save Sight Society of New Zealand, the Maurice and Phyllis Paykel Trust, and the Auckland Medical Research Foundation. 
Disclosure: A.L. Vincent, None; C.A. Jordan, None; M.J. Cadzow, None; T.R. Merriman, None; C.N. McGhee, None 
References
Rabinowitz YS. Keratoconus. Surv Ophthalmol . 1998; 42: 297–319. [CrossRef] [PubMed]
Cunningham WJ Brookes NH Twohill HC Trends in the distribution of donor corneal tissue and indications for corneal transplantation: the New Zealand National Eye Bank Study 2000-2009. Clin Experiment Ophthalmol . 2012; 40: 141–147. [CrossRef] [PubMed]
Patel HY Ormonde S Brookes NH Moffatt LS McGhee CN. The indications and outcome of paediatric corneal transplantation in New Zealand: 1991-2003. Br J Ophthalmol . 2005; 89: 404–408. [CrossRef] [PubMed]
Legeais JM Parc C d'Hermies F Pouliquen Y Renard G. Nineteen years of penetrating keratoplasty in the Hotel-Dieu Hospital in Paris. Cornea . 2001; 20: 603–606. [CrossRef] [PubMed]
Cosar CB Sridhar MS Cohen EJ Indications for penetrating keratoplasty and associated procedures, 1996-2000. Cornea . 2002; 21: 148–151. [CrossRef] [PubMed]
Dobbins KR Price FW Jr Whitson WE. Trends in the indications for penetrating keratoplasty in the midwestern United States. Cornea . 2000; 19: 813–816. [CrossRef] [PubMed]
Jordan CA Zamri A Wheeldon C Patel DV Johnson R McGhee CN. Computerized corneal tomography and associated features in a large New Zealand keratoconic population. J Cataract Refract Surg . 2011; 37: 1493–1501. [CrossRef] [PubMed]
Owens H Gamble GD Bjornholdt MC Boyce NK Keung L. Topographic indications of emerging keratoconus in teenage New Zealanders. Cornea . 2007; 26: 312–328. [CrossRef] [PubMed]
Wang Y Rabinowitz YS Rotter JI Yang H. Genetic epidemiological study of keratoconus: evidence for major gene determination. Am J Med Genet . 2000; 93: 403–409. [CrossRef] [PubMed]
Tyynismaa H Sistonen P Tuupanen S A locus for autosomal dominant keratoconus: linkage to 16q22.3-q23.1 in Finnish families. Invest Ophthalmol Vis Sci . 2002; 43: 3160–3164. [PubMed]
Fullerton J Paprocki P Foote S Mackey DA Williamson R Forrest S. Identity-by-descent approach to gene localisation in eight individuals affected by keratoconus from north-west Tasmania, Australia. Hum Genet . 2002; 110: 462–470. [CrossRef] [PubMed]
Zlotogora J BenEzra D Cohen T Cohen E. Syndrome of brittle cornea, blue sclera, and joint hyperextensibility. Am J Med Genet . 1990; 36: 269–272. [CrossRef] [PubMed]
Stein R Lazar M Adam A. Brittle cornea. A familial trait associated with blue sclera. Am J Ophthalmol . 1968; 66: 67–69. [CrossRef] [PubMed]
Gregoratos ND Bartsocas CS Papas K. Blue sclerae with keratoglobus and brittle cornea. Br J Ophthalmol . 1971; 55: 424–426. [CrossRef] [PubMed]
Ticho U Ivry M Merin S. Brittle cornea, blue sclera, and red hair syndrome (the brittle cornea syndrome). Br J Ophthalmol . 1980; 64: 175–177. [CrossRef] [PubMed]
Abu A Frydman M Marek D Pras E Nir U Reznik-Wolf H. Deleterious mutations in the Zinc-Finger 469 gene cause brittle cornea syndrome. Am J Hum Genet . 2008; 82: 1217–1222. [CrossRef] [PubMed]
Al-Owain M Al-Dosari MS Sunker A Shuaib T Alkuraya FS. Identification of a novel ZNF469 mutation in a large family with Ehlers-Danlos phenotype. Gene . 2012; 511: 447–450. [CrossRef] [PubMed]
Christensen AE Knappskog PM Midtbo M Brittle cornea syndrome associated with a missense mutation in the zinc-finger 469 gene. Invest Ophthalmol Vis Sci . 2009; 51: 47–52. [CrossRef] [PubMed]
Khan AO Aldahmesh MA Alkuraya FS. Brittle cornea without clinically-evident extraocular findings in an adult harboring a novel homozygous ZNF469 mutation. Ophthalmic Genet . 2012; 33: 257–259. [CrossRef] [PubMed]
Khan AO Aldahmesh MA Mohamed JN Alkuraya FS. Blue sclera with and without corneal fragility (brittle cornea syndrome) in a consanguineous family harboring ZNF469 mutation (p.E1392X). Arch Ophthalmol . 2010; 128: 1376–1379. [CrossRef] [PubMed]
Aldahmesh M Mohamed J Alkuraya F. A novel mutation in PRDM5 in brittle cornea syndrome. Clin Genet . 2011; 81: 198–199. [CrossRef] [PubMed]
Burkitt EM Wright HL Spencer Daly SB Mutations in PRDM5 in brittle cornea syndrome identify a pathway regulating extracellular matrix development and maintenance. Am J Hum Genet . 2011; 88: 767–777. [CrossRef] [PubMed]
Hoehn R Zeller T Verhoeven VJ Population-based meta-analysis in Caucasians confirms association with COL5A1 and ZNF469 but not COL8A2 with central corneal thickness. Hum Genet . 2012; 131: 1783–1793. [CrossRef] [PubMed]
Lu Y Dimasi DP Hysi PG Common genetic variants near the Brittle Cornea Syndrome locus ZNF469 influence the blinding disease risk factor central corneal thickness. PLoS Genet . 2010; 6: e1000947. [CrossRef] [PubMed]
Ulmer M Li J Yaspan BL Genome-wide analysis of central corneal thickness in primary open-angle glaucoma cases in the NEIGHBOR and GLAUGEN consortia. Invest Ophthalmol Vis Sci . 2012; 53: 4468–4474. [CrossRef] [PubMed]
Vitart V Bencic G Hayward C New loci associated with central cornea thickness include COL5A1, AKAP13 and AVGR8. Hum Mol Genet . 2010; 19: 4304–4311. [CrossRef] [PubMed]
Vithana EN Aung T Khor CC Collagen-related genes influence the glaucoma risk factor, central corneal thickness. Hum Mol Genet . 2010; 20: 649–658. [CrossRef] [PubMed]
Lu Y Vitart V Burdon KP Genome-wide association analyses identify multiple loci associated with central corneal thickness and keratoconus. Nat Genet . 2013; 45: 155–163. [CrossRef] [PubMed]
Burdon KP Vincent AL. Insights into keratoconus from a genetic perspective. Clin Exp Optom . 2013; 96: 146–154. [CrossRef] [PubMed]
Miller SA Dykes DD Polesky HF. A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Res . 1988; 16: 1215. [CrossRef] [PubMed]
Hollis-Moffatt JE Xu X Dalbeth N Role of the urate transporter SLC2A9 gene in susceptibility to gout in New Zealand Maori, Pacific Island, and Caucasian case-control sample sets. Arthritis Rheum . 2009; 60: 3485–3492. [CrossRef] [PubMed]
Roberts RL Wallace MC Wright DF Frequency of CYP2C9 polymorphisms in polynesian people and potential relevance to management of gout with benzbromarone. Joint Bone Spine . 2013; 81: 160–163. [CrossRef] [PubMed]
Desmet FO Hamroun D Lalande M Collod-Beroud G Claustres M Beroud C. Human Splicing Finder: an online bioinformatics tool to predict splicing signals. Nucleic Acids Res . 2009; 37: e67. [CrossRef] [PubMed]
Wagner H Barr JT Zadnik K. Collaborative Longitudinal Evaluation of Keratoconus (CLEK) Study: methods and findings to date. Cont Lens Anterior Eye . 2007; 30: 223–232. [CrossRef] [PubMed]
Rohrbach M Spencer HL Porter LF ZNF469 frequently mutated in the brittle cornea syndrome (BCS) is a single exon gene possibly regulating the expression of several extracellular matrix components. Mol Genet Metab . 2013; 109: 289–295. [CrossRef] [PubMed]
Haseltine SJ Pae J Ehrlich JR Shammas M Radcliffe NM. Variation in corneal hysteresis and central corneal thickness among black, hispanic and white subjects. Acta Ophthalmol . 2012; 90: e626–e631. [CrossRef] [PubMed]
Edwards M Clover GM Brookes N Pendergrast D Chaulk J McGhee CN. Indications for corneal transplantation in New Zealand: 1991-1999. Cornea . 2002; 21: 152–155. [CrossRef] [PubMed]
Patel HY Brookes NH Moffatt L The New Zealand National Eye Bank study 1991-2003: a review of the source and management of corneal tissue. Cornea . 2005; 24: 576–582. [CrossRef] [PubMed]
Abu A Frydman M Marek D Mapping of a gene causing brittle cornea syndrome in Tunisian Jews to 16q24. Invest Ophthalmol Vis Sci . 2006; 47: 5283–5287. [CrossRef] [PubMed]
Figure 1
 
Pedigrees of familial keratoconus, with ZNF469 variants by genotyped individuals. Filled symbols, affected; unfilled symbols, clinically unaffected; diagonal line, deceased; plus sign, wild-type allele; asterisk, examined and DNA sample collected.
Figure 1
 
Pedigrees of familial keratoconus, with ZNF469 variants by genotyped individuals. Filled symbols, affected; unfilled symbols, clinically unaffected; diagonal line, deceased; plus sign, wild-type allele; asterisk, examined and DNA sample collected.
Figure 2
 
Schematic of ZNF469 protein domains, with location of variants described in keratoconus cohort (Reference Transcript ZNF46-201 ENST00000437464).
Figure 2
 
Schematic of ZNF469 protein domains, with location of variants described in keratoconus cohort (Reference Transcript ZNF46-201 ENST00000437464).
Table 1
 
Demographics of the Keratoconus Probands at Recruitment: Familial and Sporadic
Table 1
 
Demographics of the Keratoconus Probands at Recruitment: Familial and Sporadic
Disease Number Age, y (Range) Sex, Female:Male Ethnicity (%) Familial (%) Sporadic (%)
Keratoconus 43 41.5 (15–83) 22:21 11 (25) 32 (75)
Caucasian 18 (42) 1 (2) 17 (40)
Polynesian 21 (49) 10 (23) 11 (25)
Indian 4 (9) 0 4 (9)
Table 2
 
Nonsynonymous SNPs Identified
Table 2
 
Nonsynonymous SNPs Identified
Nucleotide Protein Cases Ancestry rs Number Polynesian Controls MAF Polynesian Controls Caucasian Controls EVS 1000G PolyPhen2 SIFT
c.946G>A p.E316L 1 fam Māori - 0/92 0% 0/140 Absent Absent Prob dam Tol
c.1697C>T p.A566V 3 Caucasian, n = 2
Indian
rs181785233 0/92 0% Absent T0.005 Poss dam Tol
c.6386G>A p.R2129K 8 Tonga n, = 2
Samoan, n = 3
Caucasian, n = 2
Māori
rs13334190 13/92 14.13% 0/140 Absent A0.063 Poss dam Del
c.6796G>A p.G2266A 1 Caucasian 0/92 0.00% Absent Absent Benign Tol
c.7424C>A p.A2475E 4, including  2 fam Family:
1 Caucasian,
1 Māori
Sporadic: Caucasian, Indian
rs141218390 2/92 2.1% 6/140 5.6 T0.056 Prob dam Dam
c.8246A>T p.D2749V 1 Caucasian rs3812954 1/92 1.08% 0/140 (4.3%) 5.6 T0.056 Poss dam Del
c.8636G>A p.R2879H 1 fam Tongan - 0 0.00% Absent Absent Poss dam Dam
c.9616C>T p.P3206L 1 Caucasian 0 0.00% Absent Absent Benign Dam
c.9766G>A p.G3256R 1 Caucasian 0 0.00% Absent Absent Benign Tol
c.10244g>T p.G3415V 3, 1 fam Family: CI Māori Sporadic:
Māori, n = 2
-rs140056980 12/92 13.04% Absent T0.010 Benign Dam
Table 3
 
Characteristics of Individuals With Two Changes in ZNF469 (Homozygous, Compound Heterozygous, and in cis)
Table 3
 
Characteristics of Individuals With Two Changes in ZNF469 (Homozygous, Compound Heterozygous, and in cis)
Family Mutations Ethnicity CLEK29 Classification Proband CLEK29 Classification Sibling Pedigree
1 1 fam p.E316K NZ Māori Severe OU Severe OU TAR1
p.A2475E in cis
2 1 fam p.R2129K Tongan Severe OU Severe/moderate TUT1
p.R2879H
3 p.G2266A Caucasian Severe OU
p.D2749V
4 p.A566V Caucasian Severe OU
p.R2129K
5 p.G3415V NZ Māori Severe OU
p.G3415V
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