VSX1 Mutational Analysis in a Series of Italian Patients Affected by Keratoconus: Detection of a Novel Mutation

Luigi Bisceglia,1 Marilena Ciaschetti,2 Patrizia De Bonis,1 Pablo Alberto Perafan Campo,2 Costantina Pizzicoli,3 Costanza Scala,1 Michele Grifa,1 Pio Ciavarella,1 Nicola Delle Noci,3 Filippo Vaira,4 Claudio Macaluso,5 and Leopoldo Zelante1

PURPOSE. Keratoconus is a noninflammatory corneal disorder Most cases of keratoconus appear to be sporadic, but a that is clinically and genetically heterogeneous. Mutations in positive family history has been documented in 6% to 10% of the VSX1 (visual system homeobox 1) gene have been identi- patients.5 Both recessive and dominant patterns of inheritance fied for two distinct, inherited corneal dystrophies: posterior have been described.6–8 Autosomal dominant inheritance has polymorphous corneal dystrophy and keratoconus. To evalu- more frequently been reported in families, showing incom- ate the possible role of the VSX1 gene in a series of Italian plete penetrance and variable expressivity. Subtle videokerato- patients, 80 keratoconus-affected subjects were screened for graphic anomalies have been reported among relatives of pa- mutations. tients with keratoconus, allowing the detection of low- ETHODS expressivity forms of keratoconus, usually referred to as M . The diagnosis of keratoconus was made on the basis 9–11 of clinical examination and corneal topography. The whole subclinical or forme fruste keratoconus. Multifactorial inheritance and a major gene model have also been pro- coding region and the exon–intron junctions of the VSX1 gene 12,13 were analyzed by direct sequencing. posed. In some cases nongenetic causes have been postu- lated, such as eye rubbing or rigid contact lens wear, which RESULTS. Three already-described changes, D144E, G160D, and could be responsible for keratoconus phenocopies. However, P247R, and a novel L17P mutation were found in 7 of 80 in these cases, mechanical trauma may behave only as a pre- unrelated patients (8.7%). Two undescribed intronic polymor- cipitating factor.14 Two candidate genes, COL6A1, located on phisms are also reported. the telomeric region of chromosome 21, and matrix metallo- CONCLUSIONS. Mutational analysis of the VSX1 gene in a series of proteinase-9 (MMP9), located at 20q11.2-q13.1, were excluded Italian patients revealed one novel mutation and confirmed an as causative genes by two different studies.15,16 A locus for important role played by this gene in a significant proportion autosomal dominant keratoconus was mapped on 16q22.3- of patients affected by keratoconus, when it is inherited as an q23.1 by Tyynismaa et al.17 in Finnish families. Recently, two autosomal dominant trait with variable expressivity and incom- further loci for autosomal dominant keratoconus have been plete penetrance. (Invest Ophthalmol Vis Sci. 2005;46:39–45) reported: one associated with cataracts on the long arm of DOI:10.1167/iovs.04-0533 chromosome 15 and one at p14-q13 on chromosome 3.18,19 Mutations in the VSX1 gene have been found in Canadian eratoconus (Mendelian Inheritance in Man [MIM] 148300) patients affected by either keratoconus or posterior polymor- Kis a frequent corneal dystrophy characterized by progres- phous corneal dystrophy (PPCD) by Heónetal.20 sive conical protrusion of the cornea and noninflammatory In this study, we report the results of a mutational analysis central stroma thinning. It is a major indication for corneal of the VSX1 gene performed in a series of unrelated Italian transplantation in the Western world,1,2 and its prevalence in patients affected by keratoconus. The VSX1 reference se- the general population is approximately 1:2000.3,4 quence is GenBank accession no. AF176797 (http://www. The disease arises in the teenage years with progressive ncbi.nlm.nih.gov/Genbank; provided in the public domain by myopia and astigmatism. In nearly all patients, keratoconus is the National Center for Biotechnology Information, Bethesda, an isolated defect, but in other cases it may be a finding in MD). syndromic conditions, as observed in Ehlers-Danlos, Marfan, Apert, Noonan, and Down syndromes. METHODS

From the 1Medical Genetics Service and Ophthalmology Depart- Patients 2 ment, IRCCS-CSS Hospital, San Giovanni Rotondo (Fg), Italy; the Stu- Eighty Italian patients affected by keratoconus were recruited and, 3 dio Medico Associato, Chieti, Italy; the Department of Ophthalmol- after providing informed consent, enrolled in the study. The research ogy, University of Foggia, Foggia, Italy; the 4 Studio Oculistico, adhered to the tenets of the Declaration of Helsinki. Manfredonia (Fg), Italy; and the 5Department of Ophthalmology, Uni- versity of Parma, Parma, Italy. Fourteen participants with keratoconus belonged to families in Supported by the Italian Ministry of Health Ricerca Corrente which at least two keratoconus-affected subjects were present, 2002-2004. whereas the other 66 patients were isolated cases. The patients were Submitted for publication May 13, 2004; revised September 28, aged from 11 to 62 years; the ﬁrst clinical diagnosis was made at ages 2004; accepted October 8, 2004. ranging from 9 to 43 years. Disclosure: L. Bisceglia, None; M. Ciaschetti, None; P. De Bo- The diagnosis of keratoconus was made on the basis of clinical nis, None; P.A.P. Campo, None; C. Pizzicoli, None; C. Scala, None; examination (corneal stromal thinning, Vogt’s striae, Fleischer’s ring, M. Grifa, None; P. Ciavarella, None; N. Delle Noci, None; F. Vaira, Munson’s sign), a history of penetrating keratoplasty for keratoconus, None; , None; , None C. Macaluso L. Zelante and corneal topography. In addition, corneal topographies of the The publication costs of this article were defrayed in part by page VSX1 charge payment. This article must therefore be marked “advertise- relatives of patients carrying mutations were evaluated with a ment” in accordance with 18 U.S.C. §1734 solely to indicate this fact. corneal analysis system (model 2000; Eye Sys Laboratories, Houston, Corresponding author: Leopoldo Zelante, Medical Genetics Ser- TX) to examine videokeratographic anomalies typical of clinical or vice, IRCCS-CSS Hospital, I-71013 San Giovanni Rotondo, Italy; subclinical keratoconus. According to the classiﬁcation of Rabino- [email protected]. witz21 and Levy et al.,9 a color-coded map with 0.5-D increments was

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used to define corneal shapes, and four indices: corneal power (K), affected by keratoconus and was inherited from his mother, inferior–superior dioptric asymmetry (I-S), astigmatism (Ast), and whose corneal topography shows an astigmatic, keratoconus- skewed radial axis (SRAX) were used to calculate KISA%, a single index suspect cornea, with a KISA% index of 76.9 in one eye (Table that quantifies the irregular corneal shape and astigmatism typical of 1). The mutation was not found in two normal brothers of the keratoconus with good clinical correlation.9–21 After the results of proband. several studies examining the relationship between the above-men- Case K2-II:1 was 28 years old and could be considered a tioned parameters and keratoconus,9–21-23 topography could be con- case of sporadic keratoconus, as his parents had a negative sidered keratoconus-suspect if the KISA% index is higher than 60% history (Fig. 2, Fam. K2). Patient K3-II:1 (Fig. 2, Table 1) and/or an AB/SRAX, J or inverted-J (Jinv) topographical pattern are showed the same mutation in a compound heterozygous state, present. along with the G160D allele, a mutation described in corneal One-hundred twenty-five subjects without ocular diseases were PPCD.20 In this pedigree, both his mother and brother were selected from the general population and used as the normal control. heterozygous for the L17P and showed keratoconus-suspect videokeratography, with a J pattern in at least one eye and a Mutational Search high KISA% index (Table 1). The mutation was not found in a normal relative. The father of the patient was clinically normal. DNA was extracted from peripheral blood by standard phenol-chloro- 3 form methodology and amplified in a final reaction volume of 25 ␮Lby The L17P mutation can be analyzed by PCR, since the T C ϫ transition at nucleotide 323 creates a new BsaHI restriction using 150 ng of genomic DNA, 10 PCR buffer with 15 mM MgCl2, 200 ␮M each dNTPs, 0.10 ␮M primers, and 1.0 U DNA polymerase (Am- site, leading to the formation of the 174- and 64-bp bands in the pliTaq Gold; Applied Biosystems [ABI], Foster City, CA). PCR cycling mutated allele after amplification with the 1F and 1yR primers, conditions consisted of an initial denaturation step at 95°C for 12 as described before (Fig. 1). Two previously unreported intronic nucleotide changes, minutes followed by 35 cycles of 94°C for 30 seconds, 58°C (exon 1), ϩ ϩ 59°C (exons 2, 4, 5), 62°C (exon 3) for 30 seconds, 72°C for 30 c.900 23A/G and c.900 84T/A, were found (Table 2). seconds and ending with a final elongation step at 72°C for 7 minutes. The primer pairs to amplify each of the five coding VSX1 exons were Previously Reported Sequence Variants designed by using the Primer 3 program24: exon 1 with 599-bp product The D144E allele was identified in two families (Fig. 2, Table 1 forward (F) (5Ј-CAGCTGATTGGAGCCCTTC-3Ј) and 1 reverse (R) 1). In family K4 the D144E allele was found in a 58-year-old (5Ј-CTCAGAGCCTAGGGGACAGG-3Ј); exon 2 with 393-bp product 2F subject who underwent corneal transplantation for bilateral (5Ј-GCACTAAAAATGCTGGCTCA-3Ј) and 2R (5Ј-GCCTCCTAGGAACT- keratoconus. The patient was heterozygous for the mutation, GCAGAA-3Ј); exon 3 with 419-bp product 3F (5Ј-CATTCAGAGGT- which was transmitted to his 22-year-old son whose clinical GGGGTGTT-3Ј) and 3R (5Ј-TCTTGTGGTGCCTTCAGCTA-3Ј); exon 4 examination showed with-the-rule astigmatism, but the subject with 394-bp product 4F (5Ј-GATCATGCTCGGGAGAGAAG-3Ј) and 4R was not available for videokeratographic testing. (5Ј-CGTTGCTTTGCTTTGGAAAT-3Ј); and exon 5 with 495-bp product The same D144E allele was detected in a 12-year-old boy 5F (5Ј-CCCCAGAGATAGGCACTGAC-3Ј) and 5R (5Ј-TGGACAATTTTT- affected by unilateral keratoconus associated with a J topo- GTCTTTTGG-3Ј). All fragments were sequenced in both forward and graphical pattern and a high KISA% index in the fellow eye reverse directions, according to dye terminator chemistry (Big Dye (Fam. K5, Fig. 2, Table 1). The change was subsequently found Terminator protocol; ABI) and analyzed on a DNA sequencer (model in the father of the patient and also in his uncle and aunt. All 3100 sequencer; ABI). these subjects had altered, keratoconus-suspect topographical Detection of the alleles D144E (substitution of the aspartate with indexes. the glutamate at codon 144), G160D (substitution of the glycine with The G160D allele was also detected in family K6, with a the aspartate at codon 160), and P247R (substitution of the proline 27-year-old woman affected by bilateral keratoconus. The with the arginine at codon 247) in normal control subjects was per- change was confirmed in her father and in a brother, whereas formed by denaturing HPLC analysis performed on a nucleic acid it was absent in two brothers (Table 1, Fig. 2, individuals II:1 fragment analysis system (Wave, 3500 HT; Transgenomic, Crewe, UK). and II:3). In all of them videokeratographic examination The previously undescribed L17P mutation (substitution of the showed either a J pattern or a high KISA% index, or both, leucine with the proline at codon 17) was analyzed by amplification of irrespective of the presence or not of the mutated G160D a 238-bp fragment with primers 1F and 1yR (5Ј-CCCAGCAGGTCCGT- allele. GAT-3Ј), followed by endonuclease digestion with the BsaHI enzyme. The P247R allele was found at heterozygous status in a woman and her second son, both of whom underwent penetrating keratoplasty because of bilateral keratoconus. The same RESULTS change was also detected in the first son, who showed unilat- The entire VSX1 gene coding region and the exon–intron eral keratoconus and in the I:3 relative, who was said to be junctions were analyzed for mutations in a total of 80 unrelated clinically normal but was not available for examination (Fig. 2, Italian patients with keratoconus. Four nucleotide changes— Fam. K7). c.323T3C, c.705C3G, c.752G3A, and c.1013_1014CG3 One already described polymorphism c.819A3G was also GA—leading to the amino acid substitutions L17P, D144E, detected in 38 subjects (Table 2). G160D, and P247R respectively, were identified. None of the four changes were found in 125 normal control individuals. The sequence change identified in the VSX1 gene DISCUSSION as well as qualitative and quantitative videokeratographic anal- The human VSX1 gene is a member of the CVC domain- yses performed on subjects carrying the mutations, and their containing paired-like class of homeoproteins. The paired-like relatives are summarized in Table 1. In addition, one already- subfamily of homeodomain proteins are implicated in cranio- published and two undescribed intronic polymorphisms were facial and ocular development.25 The VSX1 gene was mapped identified. to chromosome 20 at p11-q11 and its expression in humans was detected in embryonic craniofacial, adult retinal, and adult Detection of New Sequence Variants corneal tissue.24,25 The gene spans approximately 6.2 kb and The L17P allele was found in three families. In family K1 (Fig. contains five exons.25,26 Moreover, it has been recently estab- 1), a heterozygous L17P mutation was detected in a subject lished that the homeobox gene Vsx1 controls the late off-

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TABLE 1. VSX1 Mutations, Quantitative and Qualitative Videokeratographic Parameters Evaluated on Keratoconic Patients and Their Relatives

Subject VSX1 Mutations Eye K Abs (I-S) Ast SRAX KISA% Corneal Shape*

K1-I:1 L17P/ϩ OD 45.25 0.20 1.70 15.0 76.9 G OS 45.10 0.05 1.78 13.0 17.4 F K1-II:1 ϩ/ϩ OD Clinically normal OS K1-II:2 L17P/ϩ OD Keratoconus OS Keratoconus K1-II:3 ϩ/ϩ OD 45.25 0.35 0.6 5.0 9.5 F OS 45.50 0.30 0.07 5.0 1.6 F K2-II:1 L17P/ϩ OD Keratoconus OS Keratoconus K3-I:2 L17P/ϩ OD 43.50 0.35 2.68 16.0 217.6 G OS 43.25 0.60 0.83 30.0 215.4 J K3-I:3 ϩ/ϩ OD Clinically normal OS K3-II:1 L17P/G160D OD Keratoconus OS Keratoconus K3-II:2 L17P/ϩ OD 42.75 1.25 0.80 40.0 570 J OS 42.25 0.20 0.53 5.0 7.5 F K4-I:1 D144E/ϩ OD Keratoconus OS Corneal graft K4-I:2 ϩ/ϩ OD Clinically normal OS K4-II:1 D144E/ϩ OD With-the-rule astigmatism (topography not available) OS K5-I:1 ϩ/ϩ OD Clinically normal OS K5-II:1 ϩ/ϩ OD 43.62 0.80 0.45 5.0 26.2 D OS 44.49 0.97 0.53 3.0 22.9 D K5-II:2 D144E/ϩ OD 43.21 0.15 1.27 30.0 82.3 G OS 44.19 0.23 1.33 7.0 31.5 G K5-II:3 D144E/ϩ OD 44.09 0.20 0.69 12.0 24.3 G OS 44.26 0.16 1.97 13.0 60.5 G K5-II:4 D144E/ϩ OD 43.51 0.19 1.20 9.0 29.8 G OS 44.20 0.28 2.03 11.0 92.1 G K5-III:1 D144E/ϩ OD 46.37 1.50 3.02 27.0 1890.5 J OS Keratoconus K5-III:2 ϩ/ϩ OD 44.89 0.27 0.60 1.0 2.4 F OS 43.70 0.60 0.70 1.0 6.1 F K6-I:1 G160D/ϩ OD 47.03 0.50 0.57 15.0 67.0 A OS 48.20 0.27 0.46 30.0 59.9 J K6-I:2 ϩ/ϩ OD Clinically normal OS K6-II:1 ϩ/ϩ OD 43.80 0.50 0.80 5.0 29.2 J OS 44.20 0.70 0.97 3.0 30.0 J K6-II:2 G160D/ϩ OD Keratoconus OS Keratoconus K6-II:3 ϩ/ϩ OD 42.86 2.0 0.59 5.0 84.3 J OS 43.63 1.50 0.70 7.0 106.89 J K6-II:4 G160D/ϩ OD 43.70 0.50 0.50 17.0 61.91 J OS 43.46 0.80 0.56 20.0 129.8 J K7-I:1 ϩ/ϩ OD Clinically normal OS K7-I:2 P247R/ϩ OD Corneal graft OS Corneal graft K7-I:3 P247R/ϩ OD Clinically normal (topography not available) OS K7-II:2 P247R/ϩ OD Corneal graft OS Corneal graft K7-II:1 P247R/ϩ OD Keratoconus OS 44.31 0.22 0.53 30.0 51.7 J inv K7-II:3 ϩ/ϩ OD Clinically normal OS

K, central corneal power; Abs (I-S), absolute value of inferior-superior value; Ast, regular corneal astigmatism; SRAX, skewed radial axis index; KISA%, K ϫ Abs(I-S) ϫ Ast ϫ SRAX/3. * Corneal shape classiﬁcation as reported by Levi et al.9

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FIGURE 1. Molecular characteriza- tion of L17P mutation in family K1. (A) Pedigree and restriction endonuclease assay. (f) Keratoconus-affected subject; (Ⅺ) unaffected subjects; (u) subject with keratoconus- suspect topography (KISA% Ͼ 60); M, 1-kb ladder molecular weight marker; ND, not digested. Note the heterozygous restriction pattern in subjects I:1 and II:2. (B) Partial exon 1 nucleotide sequence of VSX1 gene showing the T3C transition in the mutated allele.

center cone bipolar cell differentiation and visual signaling in In our families, the P247R and G160D mutations were mice.27 found in keratoconus patients, whereas in the series published In our study we identified one new and three already re- by Heón et al.,20 the same mutations were both identified in a ported mutations in 7 of 80 patients with keratoconus (8.75%), compound heterozygous child who was affected by very se- enhancing the percentage published by Heónetal.20 All the vere PPCD. In the same family, relatives carrying the P247R changes in the VSX1 gene have been found only in families that change had no corneal consequences, whereas the G160D were first examined because of corneal disease and not in a mutation seemed clearly to be pathogenic on its own, causing total of 402 control individuals (those in Heón et al. and our a moderate PPCD in heterozygous relatives. control subjects). Moreover, analysis of our series revealed The P247R change in our study cosegregated with kerato- novel phenotype–genotype correlations, mainly associated conus in three subjects: two of them underwent corneal trans- with keratoconus, that were not present in the cases reported 20 plantation for bilateral keratoconus. This observation along by Heónetal. with the absence of the change in 125 normal control individ- Altogether, the new L17P mutation was found in three uals suggests a possible causative role in keratoconus. patients affected by clinical keratoconus, and in three relatives, In contrast, our data do not fully support the proposal that all with keratoconus-suspect topographies. This association indicates a possible causative role of this new mutation for G160D is causative of keratoconus, as in family K3 it was keratoconus, although in one case it was in a compound present in a compound heterozygote with the L17P mutation heterozygote along with the G160D allele. Even if the location and in family K6 it was found in a patient with keratoconus, in of this change (amino acid at position 17) does not directly two relatives with suspected keratoconus, but not in two affect the homeodomain and the CVC domain, there are at least additional relatives also with suspected keratoconus. Further three lines of evidence that lead to the conclusion that this observations are needed to clarify definitively the pathogenetic change is a mutation: (1) the substitution of the proline for the role of the G160D mutation. leucine represents an alteration of an amino acid that has been Both the G160D and P247R alleles were associated with well conserved from zebrafish to humans (Table 3), (2) kera- mildly abnormal function of the inner retina on ERG examina- toconus or keratoconus-suspect phenotypes segregate with the tion,20 whereas in our study, the ERGs performed on the mutation in the pedigrees described herein, and (3) the L17P patients with keratoconus in the families K7 (I:2 and II:1) and allele was not found in 125 normal control subjects. K6 (II:2) were normal (data not shown).

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FIGURE 2. Segregation of VSX1 mutations in the analyzed pedigrees. Filled symbols: keratoconus-affected subjects; open symbols: unaffected subjects; gray symbols: subjects with keratoconus-suspect topography (KISA% Ͼ 60); ?, individual with unknown phenotype; ϩ, wild-type; ND, not determined.

The D144E change was previously described in two sib- pect topography carrying the mutation, suggests that this lings, both affected by PPCD and keratoconus and in one change is a disease-causing mutation. patient with glaucoma without corneal defects.20 Because of All the discrepancies observed in the genotype–phenotype this, the change was considered a possible mutation. Our correlations between our cases and those reported by He`on et pedigrees, in which the D144E change segregates (Fam. K4) al. 20 may be only a consequence of the complex pathogenesis from an affected father who had undergone corneal transplan- of keratoconus. In fact, the VSX1 gene may have a pleiotropic tation, to an astigmatic son (not available for topography), and action among the tissues of the cornea leading to keratoconus, (Fam. K5) from a father with suspected keratoconus, to an in some cases, and to PPCD or both, in others, as observed for affected child with two other relatives with keratoconus-sus- the BIGH3 gene, which causes four distinct autosomal domi-

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TABLE 2. Outcome of VSX1 Screening in 80 Italian Patients Affected by Keratoconus

Localization Sequence Change Aminoacid Change Individuals (n)

Exon 1 c.291 G3T — 23 (heterozygous) 2 (homozygous) c.323T3C L17P* 3 (heterozygous) Exon 2 c.705C3G D144E 2 (heterozygous) c.752G3A G160D 2 (heterozygous) Exon 3 c.819A3G — 32 (heterozygous) 6 (homozygous) Intron 3 c.900 ϩ 23A/G* — 23 (heterozygous) 4 (homozygous) c.900 ϩ 84T/A* — 20 (heterozygous) 6 (homozygous) Exon 4 c.1013 1014CG3GA P247R 1 (heterozygous)

The L17P, D144E, G160D, and P247R changes were found in seven distinct pedigrees. * Previously unreported intronic nucleotide changes.

nant corneal diseases.28 Similar findings have also been pub- Acknowledgments lished for the paired box gene 6 (PAX6), involved in oculo- genesis, which is responsible for aniridia and for other anterior The authors thank the patients who participated in the study and segment malformations.29 Another explanation could be an colleague Pietro Stanziale for his technical assistance. interaction between VSX1 and other modifier genes. A further hypothesis might consider the curvature of the cornea as a References polygenic trait. In this case VSX1 could act as a major gene along with other still unknown genetic factors. 1. Dobbins KR, Price EW Jr, Wilson WE. Trends in the indication for The videokeratographic studies in the relatives of patients penetrating keratoplasty in the midwestern United States. Cornea. with keratoconus carrying VSX1 mutations showed that kera- 2000;19:813–816. toconus-suspect topographic patterns (if not keratoconus) 2. Legeais JM, Parc C, D’hermies F, Pouliquen Y, Renard G. Nineteen years of penetrating keratoplasty in the Hotel-Dieu Hospital in were present in almost all cases (Figs. 1, 2). The opposite Paris. Cornea. 2001;20:603–606. situation, that relatives without mutations did not show kera- 3. Kennedy RH, Bourne WM, Dyer JA. A 48-year clinical and epide- toconus-suspect topography, was also true, with the notable miologic study of keratoconus. Am J Ophthalmol. 1986;101:267– exception of family K6 (G160D change). Moreover, the KISA% 273. in the individuals with and without mutations (mean Ϯ SD, 4. Rabinowitz YS. Keratoconus. In: Traboulsi E, ed. Genetic Disease 105.62 Ϯ 134.18 and 31.9 Ϯ 35.67, respectively) was signifi- of the Eye. New York: Oxford University Press; 1998:267–284. cantly different on the basis of the Mann-Whitney test (P ϭ 5. Rabinowitz YS. Keratoconus. Surv Ophthalmol. 1998;42:297–319. 0.018). These observations support and expand, at the geno- 6. Hamilton JB. Significance of heredity in ophthalmology: prelimi- type–phenotype correlation level, the results reported by other nary survey of hereditary eye diseases in Tasmania. Br J Ophthal- researchers,9,21,22 describing suspect topographical patterns mol. 1938;22:83–108. among unaffected relatives of the patients with keratoconus. 7. Falls HF, Allen AW. Dominantly inherited keratoconus: report of a The present study shows J and G shapes as the patterns mainly family. J Genet Hum. 1969;17:317–324. associated with VSX1 mutations, and confirms the KISA% as a 8. Ihalainen A. Clinical and epidemiological features of keratoconus: potential tool for monitoring the irregular shapes and astigma- genetic and external factors in the pathogenesis of the disease. tism of the cornea that, in familial forms of keratoconus, could Acta Ophthalmol. 1986;(suppl)178:1–66. represent low expressivity form of the disease. 9. Levy D, Hutchings H, Rouland JF, et al. Videokeratographic anomalies in familial keratoconus. Ophthalmology. 2004;111:867–874. In conclusion, our study reports a new VSX1 mutation and 10. Rabinowitz YS, Garbus J, McDonnell PJ. Computer-assisted corneal confirms an important role played by this gene in a significant topography in family members of keratoconus. Arch Ophthalmol. proportion of Italian patients affected by keratoconus. In our 1990;108:365–371. pedigrees, the disease is transmitted as an autosomal dominant 11. Salabert D, Cochener B, Mage F, Colin J. Keratoconus and familial trait with variable expressivity and incomplete penetrance. topographic corneal anomalies. J Fr Ophtalmol. 1994;17:646– 656. 12. Hallermann W, Wilson E. Genetische Betrachtungen ueber der TABLE 3. Summarized Sequences of VSX1 Functional Homologues Keratokonus. Klin Monatsbl Augenheilkd. 1977;170:906–908. 13. Wang Y, Rabinowitz YS, Rotter JI, Yang H. Genetic epidemiolog- Amino Acid No: 1 17 ical study of keratoconus: evidence for major gene determination. Mutant Amino Acid P Am J Med Genet. 2000;93:403–409. 14. Jafri B, Lichter H, Stulting RD. Asymmetric keratoconus attributed Human VSX1 MTGRDSLSDGRTSSRALVPG to eye rubbing. Cornea. 2004;23:560–564. Bovine VSX1 .....A.....AR....V.G 15. Rabinowitz YS, Maumenee IH, Lundergan MK, et al. Molecular Mouse VSX1 .....G...A.SR....A.G genetic analysis in autosomal dominant keratoconus. Cornea. Zebrafish VSX1 ....EEAT.EKPK-VK.Y.S 1992;11:302–308. Cynops pyrr. Chx10-1 .....D.C..SSKGKV.V.N 16. Fullerton J, Paprocki P, Foote S, Mackey DA, Williamson R, Forrest Chicken CX10 ...KAGAALAPSLPGKPK.D S,. Identity-by-descent approach to gene localisation in eight individuals affected by keratoconus from north-west Tasmania, Austra- Sequence alignments were performed using the program Clustal lia. Hum Genet. 2002;110:462–470. (provided in the public domain by European Bioinformatics Institute, 17. 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