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

Abstract

purpose. Keratoconus is a noninflammatory corneal disorder that is clinically and genetically heterogeneous. Mutations in the VSX1 (visual system homeobox 1) gene have been identified for two distinct, inherited corneal dystrophies: posterior polymorphous corneal dystrophy and keratoconus. To evaluate the possible role of the VSX1 gene in a series of Italian patients, 80 keratoconus-affected subjects were screened for mutations.

methods. The diagnosis of keratoconus was made on the basis of clinical examination and corneal topography. The whole coding region and the exon–intron junctions of the VSX1 gene were analyzed by direct sequencing.

results. Three already-described changes, D144E, G160D, and P247R, and a novel L17P mutation were found in 7 of 80 unrelated patients (8.7%). Two undescribed intronic polymorphisms are also reported.

conclusions. Mutational analysis of the VSX1 gene in a series of Italian patients revealed one novel mutation and confirmed an important role played by this gene in a significant proportion of patients affected by keratoconus, when it is inherited as an autosomal dominant trait with variable expressivity and incomplete penetrance.

Keratoconus (Mendelian Inheritance in Man [MIM] 148300) is a frequent corneal dystrophy characterized by progressive conical protrusion of the cornea and noninflammatory central stroma thinning. It is a major indication for corneal transplantation in the Western world, ^{1 2} and its prevalence in the general population is approximately 1:2000. ^{3 4}  

The disease arises in the teenage years with progressive myopia and astigmatism. In nearly all patients, keratoconus is an isolated defect, but in other cases it may be a finding in syndromic conditions, as observed in Ehlers-Danlos, Marfan, Apert, Noonan, and Down syndromes. 

Most cases of keratoconus appear to be sporadic, but a positive family history has been documented in 6% to 10% of patients. ⁵ Both recessive and dominant patterns of inheritance have been described. ^{6 7 8} Autosomal dominant inheritance has more frequently been reported in families, showing incomplete penetrance and variable expressivity. Subtle videokeratographic anomalies have been reported among relatives of patients with keratoconus, allowing the detection of low-expressivity forms of keratoconus, usually referred to as subclinical or forme fruste keratoconus. ^{9 10 11} Multifactorial inheritance and a major gene model have also been proposed. ^{12 13} In some cases nongenetic causes have been postulated, such as eye rubbing or rigid contact lens wear, which could be responsible for keratoconus phenocopies. However, in these cases, mechanical trauma may behave only as a precipitating factor. ¹⁴ Two candidate genes, COL6A1, located on the telomeric region of chromosome 21, and matrix metalloproteinase-9 (MMP9), located at 20q11.2-q13.1, were excluded as causative genes by two different studies. ^{15 16} A locus for autosomal dominant keratoconus was mapped on 16q22.3-q23.1 by Tyynismaa et al. ¹⁷ in Finnish families. Recently, two further loci for autosomal dominant keratoconus have been reported: one associated with cataracts on the long arm of chromosome 15 and one at p14-q13 on chromosome 3. ^{18 19}  

Mutations in the VSX1 gene have been found in Canadian patients affected by either keratoconus or posterior polymorphous corneal dystrophy (PPCD) by Héon et al. ²⁰  

In this study, we report the results of a mutational analysis of the VSX1 gene performed in a series of unrelated Italian patients affected by keratoconus. The VSX1 reference sequence is GenBank accession no. AF176797 (http://www.ncbi.nlm.nih.gov/Genbank; provided in the public domain by the National Center for Biotechnology Information, Bethesda, MD). 

Methods

Patients

Eighty Italian patients affected by keratoconus were recruited and, after providing informed consent, enrolled in the study. The research adhered to the tenets of the Declaration of Helsinki. 

Fourteen participants with keratoconus belonged to families in which at least two keratoconus-affected subjects were present, whereas the other 66 patients were isolated cases. The patients were aged from 11 to 62 years; the first clinical diagnosis was made at ages ranging from 9 to 43 years. 

The diagnosis of keratoconus was made on the basis of clinical examination (corneal stromal thinning, Vogt’s striae, Fleischer’s ring, Munson’s sign), a history of penetrating keratoplasty for keratoconus, and corneal topography. In addition, corneal topographies of the relatives of patients carrying VSX1 mutations were evaluated with a corneal analysis system (model 2000; Eye Sys Laboratories, Houston, TX) to examine videokeratographic anomalies typical of clinical or subclinical keratoconus. According to the classification of Rabinowitz ²¹ and Levy et al., ⁹ a color-coded map with 0.5-D increments was used to define corneal shapes, and four indices: corneal power (K), inferior–superior dioptric asymmetry (I-S), astigmatism (Ast), and skewed radial axis (SRAX) were used to calculate KISA%, a single index that quantifies the irregular corneal shape and astigmatism typical of keratoconus with good clinical correlation. ^{9 10 11 12 13 14 15 16 17 18 19 20 21} After the results of several studies examining the relationship between the above-mentioned parameters and keratoconus, ^{9 10 11 12 13 14 15 16 17 18 19 20 21 -23} topography could be considered keratoconus-suspect if the KISA% index is higher than 60% and/or an AB/SRAX, J or inverted-J (Jinv) topographical pattern are present. 

One-hundred twenty-five subjects without ocular diseases were selected from the general population and used as the normal control. 

Mutational Search

DNA was extracted from peripheral blood by standard phenol-chloroform methodology and amplified in a final reaction volume of 25 μL by using 150 ng of genomic DNA, 10× PCR buffer with 15 mM MgCl₂, 200 μM each dNTPs, 0.10 μM primers, and 1.0 U DNA polymerase (AmpliTaq Gold; Applied Biosystems [ABI], Foster City, CA). PCR cycling conditions consisted of an initial denaturation step at 95°C for 12 minutes followed by 35 cycles of 94°C for 30 seconds, 58°C (exon 1), 59°C (exons 2, 4, 5), 62°C (exon 3) for 30 seconds, 72°C for 30 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 designed by using the Primer 3 program ²⁴ : exon 1 with 599-bp product 1 forward (F) (5′-CAGCTGATTGGAGCCCTTC-3′) and 1 reverse (R) (5′-CTCAGAGCCTAGGGGACAGG-3′); exon 2 with 393-bp product 2F (5′-GCACTAAAAATGCTGGCTCA-3′) and 2R (5′-GCCTCCTAGGAACTGCAGAA-3′); exon 3 with 419-bp product 3F (5′-CATTCAGAGGTGGGGTGTT-3′) and 3R (5′-TCTTGTGGTGCCTTCAGCTA-3′); exon 4 with 394-bp product 4F (5′-GATCATGCTCGGGAGAGAAG-3′) and 4R (5′-CGTTGCTTTGCTTTGGAAAT-3′); and exon 5 with 495-bp product 5F (5′-CCCCAGAGATAGGCACTGAC-3′) and 5R (5′-TGGACAATTTTTGTCTTTTGG-3′). All fragments were sequenced in both forward and reverse directions, according to dye terminator chemistry (Big Dye Terminator protocol; ABI) and analyzed on a DNA sequencer (model 3100 sequencer; ABI). 

Detection of the alleles D144E (substitution of the aspartate with the glutamate at codon 144), G160D (substitution of the glycine with the aspartate at codon 160), and P247R (substitution of the proline with the arginine at codon 247) in normal control subjects was performed by denaturing HPLC analysis performed on a nucleic acid fragment analysis system (Wave, 3500 HT; Transgenomic, Crewe, UK). 

The previously undescribed L17P mutation (substitution of the leucine with the proline at codon 17) was analyzed by amplification of a 238-bp fragment with primers 1F and 1yR (5′-CCCAGCAGGTCCGTGAT-3′), followed by endonuclease digestion with the BsaHI enzyme. 

Results

The entire VSX1 gene coding region and the exon–intron junctions were analyzed for mutations in a total of 80 unrelated Italian patients with keratoconus. Four nucleotide changes—c.323T→C, c.705C→G, c.752G→A, and c.1013_1014CG→GA—leading to the amino acid substitutions L17P, D144E, 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 as well as qualitative and quantitative videokeratographic analyses performed on subjects carrying the mutations, and their relatives are summarized in Table 1 . In addition, one already-published and two undescribed intronic polymorphisms were identified. 

Detection of New Sequence Variants

The L17P allele was found in three families. In family K1 (Fig. 1) , a heterozygous L17P mutation was detected in a subject affected by keratoconus and was inherited from his mother, whose corneal topography shows an astigmatic, keratoconus-suspect cornea, with a KISA% index of 76.9 in one eye (Table 1) . The mutation was not found in two normal brothers of the proband. 

Case K2-II:1 was 28 years old and could be considered a case of sporadic keratoconus, as his parents had a negative history (Fig. 2 , Fam. K2). Patient K3-II:1 (Fig. 2 , Table 1 ) showed the same mutation in a compound heterozygous state, along with the G160D allele, a mutation described in corneal PPCD. ²⁰ In this pedigree, both his mother and brother were heterozygous for the L17P and showed keratoconus-suspect videokeratography, with a J pattern in at least one eye and a high KISA% index (Table 1) . The mutation was not found in a normal relative. The father of the patient was clinically normal. 

The L17P mutation can be analyzed by PCR, since the T→C transition at nucleotide 323 creates a new BsaHI restriction site, leading to the formation of the 174- and 64-bp bands in the mutated allele after amplification with the 1F and 1yR primers, as described before (Fig. 1) . 

Two previously unreported intronic nucleotide changes, c.900+23A/G and c.900+84T/A, were found (Table 2) . 

Previously Reported Sequence Variants

The D144E allele was identified in two families (Fig. 2 , Table 1 ). In family K4 the D144E allele was found in a 58-year-old subject who underwent corneal transplantation for bilateral keratoconus. The patient was heterozygous for the mutation, which was transmitted to his 22-year-old son whose clinical examination showed with-the-rule astigmatism, but the subject was not available for videokeratographic testing. 

The same D144E allele was detected in a 12-year-old boy affected by unilateral keratoconus associated with a J topographical pattern and a high KISA% index in the fellow eye (Fam. K5, Fig. 2 , Table 1 ). The change was subsequently found in the father of the patient and also in his uncle and aunt. All these subjects had altered, keratoconus-suspect topographical indexes. 

The G160D allele was also detected in family K6, with a 27-year-old woman affected by bilateral keratoconus. The change was confirmed in her father and in a brother, whereas it was absent in two brothers (Table 1 , Fig. 2 , individuals II:1 and II:3). In all of them videokeratographic examination showed either a J pattern or a high KISA% index, or both, irrespective of the presence or not of the mutated G160D allele. 

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 change was also detected in the first son, who showed unilateral keratoconus and in the I:3 relative, who was said to be clinically normal but was not available for examination (Fig. 2 , Fam. K7). 

One already described polymorphism c.819A→G was also detected in 38 subjects (Table 2) . 

Discussion

The human VSX1 gene is a member of the CVC domain-containing paired-like class of homeoproteins. The paired-like subfamily of homeodomain proteins are implicated in craniofacial and ocular development. ²⁵ The VSX1 gene was mapped to chromosome 20 at p11-q11 and its expression in humans was detected in embryonic craniofacial, adult retinal, and adult corneal tissue. ^{24 25} The gene spans approximately 6.2 kb and contains five exons. ^{25 26} Moreover, it has been recently established that the homeobox gene Vsx1 controls the late off-center cone bipolar cell differentiation and visual signaling in mice. ²⁷  

In our study we identified one new and three already reported mutations in 7 of 80 patients with keratoconus (8.75%), enhancing the percentage published by Héon et al. ²⁰ All the changes in the VSX1 gene have been found only in families that were first examined because of corneal disease and not in a total of 402 control individuals (those in Héon et al. and our control subjects). Moreover, analysis of our series revealed novel phenotype–genotype correlations, mainly associated with keratoconus, that were not present in the cases reported by Héon et al. ²⁰  

Altogether, the new L17P mutation was found in three patients affected by clinical keratoconus, and in three relatives, all with keratoconus-suspect topographies. This association indicates a possible causative role of this new mutation for keratoconus, although in one case it was in a compound heterozygote along with the G160D allele. Even if the location of this change (amino acid at position 17) does not directly affect the homeodomain and the CVC domain, there are at least three lines of evidence that lead to the conclusion that this change is a mutation: (1) the substitution of the proline for the leucine represents an alteration of an amino acid that has been well conserved from zebrafish to humans (Table 3) , (2) keratoconus or keratoconus-suspect phenotypes segregate with the mutation in the pedigrees described herein, and (3) the L17P allele was not found in 125 normal control subjects. 

In our families, the P247R and G160D mutations were found in keratoconus patients, whereas in the series published by Héon et al., ²⁰ the same mutations were both identified in a compound heterozygous child who was affected by very severe PPCD. In the same family, relatives carrying the P247R change had no corneal consequences, whereas the G160D mutation seemed clearly to be pathogenic on its own, causing a moderate PPCD in heterozygous relatives. 

The P247R change in our study cosegregated with keratoconus in three subjects: two of them underwent corneal transplantation for bilateral keratoconus. This observation along with the absence of the change in 125 normal control individuals suggests a possible causative role in keratoconus. 

In contrast, our data do not fully support the proposal that G160D is causative of keratoconus, as in family K3 it was present in a compound heterozygote with the L17P mutation and in family K6 it was found in a patient with keratoconus, in two relatives with suspected keratoconus, but not in two additional relatives also with suspected keratoconus. Further observations are needed to clarify definitively the pathogenetic role of the G160D mutation. 

Both the G160D and P247R alleles were associated with mildly abnormal function of the inner retina on ERG examination, ²⁰ whereas in our study, the ERGs performed on the patients with keratoconus in the families K7 (I:2 and II:1) and K6 (II:2) were normal (data not shown). 

The D144E change was previously described in two siblings, both affected by PPCD and keratoconus and in one patient with glaucoma without corneal defects. ²⁰ Because of this, the change was considered a possible mutation. Our pedigrees, in which the D144E change segregates (Fam. K4) from an affected father who had undergone corneal transplantation, to an astigmatic son (not available for topography), and (Fam. K5) from a father with suspected keratoconus, to an affected child with two other relatives with keratoconus-suspect topography carrying the mutation, suggests that this change is a disease-causing mutation. 

All the discrepancies observed in the genotype–phenotype correlations between our cases and those reported by Hèon et al. ²⁰ may be only a consequence of the complex pathogenesis of keratoconus. In fact, the VSX1 gene may have a pleiotropic action among the tissues of the cornea leading to keratoconus, in some cases, and to PPCD or both, in others, as observed for the BIGH3 gene, which causes four distinct autosomal dominant corneal diseases. ²⁸ Similar findings have also been published for the paired box gene 6 (PAX6), involved in oculogenesis, which is responsible for aniridia and for other anterior segment malformations. ²⁹ Another explanation could be an interaction between VSX1 and other modifier genes. A further hypothesis might consider the curvature of the cornea as a polygenic trait. In this case VSX1 could act as a major gene along with other still unknown genetic factors. 

The videokeratographic studies in the relatives of patients with keratoconus carrying VSX1 mutations showed that keratoconus-suspect topographic patterns (if not keratoconus) were present in almost all cases (Figs. 1 2) . The opposite situation, that relatives without mutations did not show keratoconus-suspect topography, was also true, with the notable exception of family K6 (G160D change). Moreover, the KISA% in the individuals with and without mutations (mean ± SD, 105.62 ± 134.18 and 31.9 ± 35.67, respectively) was significantly different on the basis of the Mann-Whitney test (P = 0.018). These observations support and expand, at the genotype–phenotype correlation level, the results reported by other researchers, ^{9 21 22} describing suspect topographical patterns among unaffected relatives of the patients with keratoconus. The present study shows J and G shapes as the patterns mainly associated with VSX1 mutations, and confirms the KISA% as a potential tool for monitoring the irregular shapes and astigmatism of the cornea that, in familial forms of keratoconus, could represent low expressivity form of the disease. 

In conclusion, our study reports a new VSX1 mutation and confirms an important role played by this gene in a significant proportion of Italian patients affected by keratoconus. In our pedigrees, the disease is transmitted as an autosomal dominant trait with variable expressivity and incomplete penetrance. 

 Supported by the Italian Ministry of Health Ricerca Corrente 2002-2004.

 Submitted for publication May 13, 2004; revised September 28, 2004; accepted October 8, 2004.

 Disclosure: L. Bisceglia, None; M. Ciaschetti, None; P. De Bonis, None; P.A.P. Campo, None; C. Pizzicoli, None; C. Scala, None; M. Grifa, None; P. Ciavarella, None; N. Delle Noci, None; F. Vaira, None; C. Macaluso, None; L. Zelante, None

 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked “advertisement” in accordance with 18 U.S.C. §1734 solely to indicate this fact.

 

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/+	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	+/+	OD	Clinically normal
		OS
K1-II:2	L17P/+	OD	Keratoconus
		OS	Keratoconus
K1-II:3	+/+	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/+	OD	Keratoconus
		OS	Keratoconus
K3-I:2	L17P/+	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	+/+	OD	Clinically normal
		OS
K3-II:1	L17P/G160D	OD	Keratoconus
		OS	Keratoconus
K3-II:2	L17P/+	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/+	OD	Keratoconus
		OS	Corneal graft
K4-I:2	+/+	OD	Clinically normal
		OS
K4-II:1	D144E/+	OD	With-the-rule astigmatism (topography not available)
		OS
K5-I:1	+/+	OD	Clinically normal
		OS
K5-II:1	+/+	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/+	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/+	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/+	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/+	OD	46.37	1.50	3.02	27.0	1890.5	J
		OS	Keratoconus
K5-III:2	+/+	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/+	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	+/+	OD	Clinically normal
		OS
K6-II:1	+/+	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/+	OD	Keratoconus
		OS	Keratoconus
K6-II:3	+/+	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/+	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	+/+	OD	Clinically normal
		OS
K7-I:2	P247R/+	OD	Corneal graft
		OS	Corneal graft
K7-I:3	P247R/+	OD	Clinically normal (topography not available)
		OS
K7-II:2	P247R/+	OD	Corneal graft
		OS	Corneal graft
K7-II:1	P247R/+	OD	Keratoconus
		OS	44.31	0.22	0.53	30.0	51.7	J inv
K7-II:3	+/+	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 × Abs(I-S) × Ast × SRAX/3.

*  Corneal shape classification as reported by Levi et al. ⁹

Figure 1.

 

Molecular characterization of L17P mutation in family K1. (A) Pedigree and restriction endonuclease assay. (▪) Keratoconus-affected subject; (□) unaffected subjects; ( ) subject with keratoconus-suspect topography (KISA% > 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 T→C transition in the mutated allele.

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% > 60); ?, individual with unknown phenotype; +, wild-type; ND, not determined.

Table 2.

 

Outcome of VSX1 Screening in 80 Italian Patients Affected by Keratoconus

Localization	Sequence Change	Aminoacid Change	Individuals (n)
Exon 1	c.291 G→T	—	23 (heterozygous)
			2 (homozygous)
	c.323T→C	L17P*	3 (heterozygous)
Exon 2	c.705C→G	D144E	2 (heterozygous)
	c.752G→A	G160D	2 (heterozygous)
Exon 3	c.819A→G	—	32 (heterozygous)
			6 (homozygous)
Intron 3	c.900 + 23A/G*	—	23 (heterozygous)
			4 (homozygous)
	c.900 + 84T/A*	—	20 (heterozygous)
			6 (homozygous)
Exon 4	c.1013 1014CG→GA	P247R	1 (heterozygous)

 The L17P, D144E, G160D, and P247R changes were found in seven distinct pedigrees.

*  Previously unreported intronic nucleotide changes.

Table 3.

 

Summarized Sequences of VSX1 Functional Homologues

Amino Acid No:	1	17
Mutant Amino Acid		P
Human VSX1	MTGRDSLSDGRTSSRALVPG
Bovine VSX1	…..A…..AR….V.G
Mouse VSX1	…..G…A.SR….A.G
Zebrafish VSX1	….EEAT.EKPK-VK.Y.S
Cynops pyrr. Chx10-1	…..D.C..SSKGKV.V.N
Chicken CX10	…KAGAALAPSLPGKPK.D

 Sequence alignments were performed using the program Clustal (provided in the public domain by European Bioinformatics Institute, European Molecular Biology Laboratory, Heidelberg, Germany; available at http://www.ebi.ac.uk/clustalw/).

The authors thank the patients who participated in the study and colleague Pietro Stanziale for his technical assistance.