Keratoconus and Posterior Polymorphous Corneal Dystrophy via the VSX1 Gene

  • Summary and Pricing
  • Clinical Features and Genetics
  • Citations
  • Methods
  • Ordering/Specimens
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Test Code Test Copy GenesIndividual Gene PriceCPT Code Copy CPT Codes
1676 VSX1$650.00 81479 Add to Order
Targeted Testing

For ordering targeted known variants, please proceed to our Targeted Variants landing page.

Turnaround Time

The great majority of tests are completed within 18 days.

Clinical Sensitivity

A VSX1 screening detected causative mutations in 3.1% of KC patients (42/1350) (De Bonis et al. 2011). Another VSX1 mutational analysis detected mutations in 8.7% of Italian KC patients (Bisceglia 2005). Clinical sensitivity cannot be precisely estimated in PPCD patients as it is a rare form of corneal dystrophy. Analytical sensitivity should be high because all mutations reported are detectable by this method. No gross deletions or duplications have been reported so far (Human Gene Mutation Database).

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Deletion/Duplication Testing via aCGH

Test Code Test Copy GenesIndividual Gene PriceCPT Code Copy CPT Codes
600 VSX1$690.00 81479 Add to Order
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Turnaround Time

The great majority of tests are completed within 28 days.

Clinical Features

Keratoconus (KC; kerato meaning cornea and conus meaning cone) is a common form of corneal dystrophy. Clinically it is characterized by noninflammatory bilateral progressive protrusion of the cornea and central stroma thinning. These often result in high myopia, irregular astigmatism and impairment of visual acuity. Usually it is evident in the second decade of life and eventually stabilizes in the third and fourth decades. Estimated prevalence of KC in the general population is approximately 1: 2,000. KC is most commonly an isolated disorder, though several reports describe it as syndromic and seen in Ehlers-Danlos, Marfan, Apert, Noonan, and Down syndromes (Rabinowitz 1998; Bisceglia 2005; Espandar and Meyer 2010). KC treatment ranges from simple spectacle correction to corneal transplantation (Krachmer et al. 1984).

Posterior polymorphous corneal dystrophy (PPCD) is rare autosomal dominant disorder of the cornea, which is clinically characterized as a nonprogressive disorder affecting both the corneal endothelium (CE) and Descemet’s membrane. IN PPCD, affected patient's CE is often multi-layered due to the presence of desmosomes, tonofilaments, and microvilli. These abnormal cells retain their ability to divide and spread to the trabecular meshwork. 40% of affected patients develop glaucoma necessitating keratoplasty (Cibis et al. 1977; Gwilliam 2005).


KC is clinically and genetically heterogeneous. Both autosomal recessive and dominant (AR and AD) inheritance patterns have been described. AD inheritance is frequently reported in families, showing incomplete penetrance and variable expressivity. Mutations in the VSX1 (visual system homeobox 1, also known as RINX) gene have been associated with two distinct autosomal dominantly inherited corneal dystrophies; KC and PPCD (described in the clinical features). The VSX1 gene, which is located on chromosome 20p11.2 (Gwilliam 2005), is a member of the “paired-like” homeodomain transcription factors. Members of this family are shown to be essential for craniofacial and eye development (Vincent et al. 2013). VSX1, encodes a pair-like homeodomian protein, expressed in the inner nuclear layer of the adult retina (Hayashi et al. 2000). VSX1 protein has been reported to regulate cone bipolar cell differentiation during embryonic development. VSX1 mutations may cause impaired photopic vision (Ohtoshi et al. 2004; Wheeler et al. 2012). So far, about 15 mutations (Missense/nonsense, splicing and small indels) have been reported in VSX1 that are involved in KC and PPCD (Human Gene Mutation Database).

Testing Strategy

This test involves bidirectional DNA Sanger sequencing of all coding exons and ~ 20 bp of flanking noncoding sequence of VSX1 gene. We will also sequence any single exon (Test #100) in family members of patients with a known mutation or to confirm research results.

Indications for Test

All patients with symptoms suggestive of Keratoconus or Posterior polymorphous corneal dystrophy are candidates.


Official Gene Symbol OMIM ID
VSX1 605020
Inheritance Abbreviation
Autosomal Dominant AD
Autosomal Recessive AR
X-Linked XL
Mitochondrial MT


Name Inheritance OMIM ID
Keratoconus 1 148300
Polymorphous Corneal Dystrophy 122000


Genetic Counselors
  • Bisceglia L. 2005. VSX1 Mutational Analysis in a Series of Italian Patients Affected by Keratoconus: Detection of a Novel Mutation. Investigative Ophthalmology & Visual Science 46: 39–45. PubMed ID: 15623752
  • Cibis GW, Krachmer JA, Phelps CD, Weingeist TA. 1977. The clinical spectrum of posterior polymorphous dystrophy. Arch. Ophthalmol. 95: 1529–1537. PubMed ID: 302697
  • De Bonis P, Laborante A, Pizzicoli C, Stallone R, Barbano R, Longo C, Mazzilli E, Zelante L, Bisceglia L. 2011. Mutational screening of VSX1, SPARC, SOD1, LOX, and TIMP3 in keratoconus. Molecular vision 17: 2482. PubMed ID: 21976959
  • Espandar L, Meyer J. 2010. Keratoconus: Overview and Update on Treatment. Middle East Afr J Ophthalmol 17: 15–20. PubMed ID: 20543932
  • Gwilliam R. 2005. Posterior Polymorphous Corneal Dystrophy in Czech Families Maps to Chromosome 20 and Excludes the VSX1 Gene. Investigative Ophthalmology & Visual Science 46: 4480–4484. PubMed ID: 16303937
  • Hayashi T, Huang J, Deeb SS. 2000. RINX(VSX1), a novel homeobox gene expressed in the inner nuclear layer of the adult retina. Genomics 67: 128–139. PubMed ID: 10903837
  • Human Gene Mutation Database (Bio-base).
  • Krachmer JH, Feder RS, Belin MW. 1984. Keratoconus and related noninflammatory corneal thinning disorders. Surv Ophthalmol 28: 293–322. PubMed ID: 6230745
  • Ohtoshi A, Wang SW, Maeda H, Saszik SM, Frishman LJ, Klein WH, Behringer RR. 2004. Regulation of Retinal Cone Bipolar Cell Differentiation and Photopic Vision by the CVC Homeobox Gene< i> Vsx1. Current biology 14: 530–536. PubMed ID: 15043821
  • Rabinowitz YS. 1998. Keratoconus. Surv Ophthalmol 42: 297–319. PubMed ID: 9493273
  • Vincent AL, Jordan C, Sheck L, Niederer R, Patel DV, McGhee CNJ. 2013. Screening the visual system homeobox 1 gene in keratoconus and posterior polymorphous dystrophy cohorts identifies a novel variant. Mol. Vis. 19: 852–860. PubMed ID: 23592923
  • Wheeler J, Hauser MA, Afshari NA, Allingham RR, Liu Y. 2012. The genetics of keratoconus: a review. Microscopy (Oxford, England). PubMed ID: 23795306
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Bi-Directional Sanger Sequencing

Test Procedure

Nomenclature for sequence variants was from the Human Genome Variation Society (  As required, DNA is extracted from the patient specimen.  PCR is used to amplify the indicated exons plus additional flanking non-coding sequence.  After cleaning of the PCR products, cycle sequencing is carried out using the ABI Big Dye Terminator v.3.0 kit.  Products are resolved by electrophoresis on an ABI 3730xl capillary sequencer.  In most cases, sequencing is performed in both forward and reverse directions; in some cases, sequencing is performed twice in either the forward or reverse directions.  In nearly all cases, the full coding region of each exon as well as 20 bases of non-coding DNA flanking the exon are sequenced.

Analytical Validity

As of March 2016, we compared 17.37 Mb of Sanger DNA sequence generated at PreventionGenetics to NextGen sequence generated in other labs. We detected only 4 errors in our Sanger sequences, and these were all due to allele dropout during PCR. For Proficiency Testing, both external and internal, in the 12 years of our lab operation we have Sanger sequenced roughly 8,800 PCR amplicons. Only one error has been identified, and this was due to sequence analysis error.

Our Sanger sequencing is capable of detecting virtually all nucleotide substitutions within the PCR amplicons. Similarly, we detect essentially all heterozygous or homozygous deletions within the amplicons. Homozygous deletions which overlap one or more PCR primer annealing sites are detectable as PCR failure. Heterozygous deletions which overlap one or more PCR primer annealing sites are usually not detected (see Analytical Limitations). All heterozygous insertions within the amplicons up to about 100 nucleotides in length appear to be detectable. Larger heterozygous insertions may not be detected. All homozygous insertions within the amplicons up to about 300 nucleotides in length appear to be detectable. Larger homozygous insertions may masquerade as homozygous deletions (PCR failure).

Analytical Limitations

In exons where our sequencing did not reveal any variation between the two alleles, we cannot be certain that we were able to PCR amplify both of the patient’s alleles. Occasionally, a patient may carry an allele which does not amplify, due for example to a deletion or a large insertion. In these cases, the report contains no information about the second allele.

Similarly, our sequencing tests have almost no power to detect duplications, triplications, etc. of the gene sequences.

In most cases, only the indicated exons and roughly 20 bp of flanking non-coding sequence on each side are analyzed. Test reports contain little or no information about other portions of the gene, including many regulatory regions.

In nearly all cases, we are unable to determine the phase of sequence variants. In particular, when we find two likely causative mutations for recessive disorders, we cannot be certain that the mutations are on different alleles.

Our ability to detect minor sequence variants, due for example to somatic mosaicism is limited. Sequence variants that are present in less than 50% of the patient’s nucleated cells may not be detected.

Runs of mononucleotide repeats (eg (A)n or (T)n) with n >8 in the reference sequence are generally not analyzed because of strand slippage during PCR and cycle sequencing.

Unless otherwise indicated, the sequence data that we report are based on DNA isolated from a specific tissue (usually leukocytes). Test reports contain no information about gene sequences in other tissues.

Deletion/Duplication Testing via Array Comparative Genomic Hybridization

Test Procedure

Equal amounts of genomic DNA from the patient and a gender matched reference sample are amplified and labeled with Cy3 and Cy5 dyes, respectively. To prevent any sample cross contamination, a unique sample tracking control is added into each patient sample. Each labeled patient product is then purified, quantified, and combined with the same amount of reference product. The combined sample is loaded onto the designed array and hybridized for at least 22-42 hours at 65°C. Arrays are then washed and scanned immediately with 2.5 µM resolution. Only data for the gene(s) of interest for each patient are extracted and analyzed.

Analytical Validity

PreventionGenetics' high density gene-centric custom designed aCGH enables the detection of relatively small deletions and duplications within a single exon of a given gene or deletions and duplications encompassing the entire gene. PreventionGenetics has established and verified this test's accuracy and precision.

Analytical Limitations

Our dense probe coverage may allow detection of deletions/duplications down to 100 bp; however due to limitations and probe spacing this cannot be guaranteed across all exons of all genes. Therefore, some copy number changes smaller than 100-300 bp within a targeted large exon may not be detected by our array.

This array may not detect deletions and duplications present at low levels of mosaicism or those present in genes that have pseudogene copies or repeats elsewhere in the genome.

aCGH will not detect balanced translocations, inversions, or point mutations that may be responsible for the clinical phenotype.

Breakpoints, if occurring outside the targeted gene, may be hard to define.

The sensitivity of this assay may be reduced when DNA is extracted by an outside laboratory.

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Ordering Options

myPrevent - Online Ordering
  • The test can be added to your online orders in the Summary and Pricing section.
  • Once the test has been added log in to myPrevent to fill out an online requisition form.
  • A completed requisition form must accompany all specimens.
  • Billing information along with specimen and shipping instructions are within the requisition form.
  • All testing must be ordered by a qualified healthcare provider.


(Delivery accepted Monday - Saturday)

  • Collect 3 ml -5 ml (5 ml preferred) of whole blood in EDTA (purple top tube) or ACD (yellow top tube). For Test #500-DNA Banking only, collect 10 ml -20 ml of whole blood.
  • For small babies, we require a minimum of 1 ml of blood.
  • Only one blood tube is required for multiple tests.
  • Ship blood tubes at room temperature in an insulated container. Do not freeze blood.
  • During hot weather, include a frozen ice pack in the shipping container. Place a paper towel or other thin material between the ice pack and the blood tube.
  • In cold weather, include an unfrozen ice pack in the shipping container as insulation.
  • At room temperature, blood specimen is stable for up to 48 hours.
  • If refrigerated, blood specimen is stable for up to one week.
  • Label the tube with the patient name, date of birth and/or ID number.


(Delivery accepted Monday - Saturday)

  • Send in screw cap tube at least 5 µg -10 µg of purified DNA at a concentration of at least 20 µg/ml for NGS and Sanger tests and at least 5 µg of purified DNA at a concentration of at least 100 µg/ml for gene-centric aCGH, MLPA, and CMA tests, minimum 2 µg for limited specimens.
  • For requests requiring more than one test, send an additional 5 µg DNA per test ordered when possible.
  • DNA may be shipped at room temperature.
  • Label the tube with the composition of the solute, DNA concentration as well as the patient’s name, date of birth, and/or ID number.
  • We only accept genomic DNA for testing. We do NOT accept products of whole genome amplification reactions or other amplification reactions.


(Delivery preferred Monday - Thursday)

  • PreventionGenetics should be notified in advance of arrival of a cell culture.
  • Culture and send at least two T25 flasks of confluent cells.
  • Some panels may require additional flasks (dependent on size of genes, amount of Sanger sequencing required, etc.). Multiple test requests may also require additional flasks. Please contact us for details.
  • Send specimens in insulated, shatterproof container overnight.
  • Cell cultures may be shipped at room temperature or refrigerated.
  • Label the flasks with the patient name, date of birth, and/or ID number.
  • We strongly recommend maintaining a local back-up culture. We do not culture cells.
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