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Keratoconus: Further Reading on Findings



"There are no problems... there are only solutions to problems" 


Once keratoconus is triggered (by whatever mechanism/s initially), a number of things have been observed. One of the earliest histological findings of keratoconus is disruption of the epithelial basement membrane. "Typical" keratoconus shows breaks in Bowman’s layer and central thinning, while "atypical" keratoconus lacks the Bowman’s layer breaks and has little central thinning. Abnormal levels of degradative protease activity and elevated levels of degradative enzymes, including lysosomal acid phosphatase (LAP) and cathepsin B, leads to a slow, progressive dissolution of Bowman’s layer and the epithelial basement membrane. The epithelium then comes into contact with the stroma, cytokines/growth factors are released and as a result, the cells begin to produce both abnormal extra cellular matrix (fibrosis-like structure, or scar tissue) and proteases.

Secondarily, the increased protease activity and decreased levels of protease inhibitors; such as alpha1-proteinase inhibitor (alpha 1-PI) and alpha 2-macroglobulin (alpha 2-M), also affects the structural framework of the corneal collagen especially in the epithelial layer. Proteoglycans which are specialised proteins found between the cells that contain complex sugars and glycoproteins which are proteins that contain some simple sugars result in damage to the integrity of the cornea.

Keratoconus is a disorder with local micro-environmental changes rather than a situation where the entire cornea is involved. Studies have shown that keratoconus corneas have focal, non-scarred regions with decreased amounts of various basement membrane components, while other regions are fibrotic and show increased deposition of stromal and basement membrane extra cellular matrices. This suggests to us that within a single keratoconus cornea there are areas of increased protease activity (thinning) and other areas of ongoing wound healing (scar tissue build-up).

Past and Present studies are finding interesting and unexpected results:

Hamilton (1938) claimed that certain of his pedigrees strongly supported autosomal recessive inheritance.

Irregular autosomal dominant inheritance was suggested by Falls and Allen (1969), who observed affected aunt and niece. The mother, who presumably transmitted the trait, had astigmatism and other features the authors interpreted as forme fruste of keratoconus. They cited several instances of multigeneration involvement including the family of Staehli (1925) with transmission through 3 generations.

From study of a large series, Hallermann and Wilson (1977) favored multifactorial inheritance but could not exclude isolated instances of dominant or recessive inheritance.

The studies of Hamilton (1938) were conducted in Tasmania where, in the coastal town of Burnie, keratoconus is present at a 5-fold increased incidence. Based on the assumption that individuals with keratoconus from this town are likely to be related through a founder effect,

Fullerton et al. (2002) conducted a 10-cM interval genome scan on 6 patients of undefined genetic relationship and 1 affected sib pair to identify commonly shared chromosomal segments for the elucidation of candidate gene loci. Analysis of allele sharing revealed 4 markers on 3 chromosomes where all 8 individuals shared a common allele on at least 1 chromosome and 13 markers where all but 1 patient shared common alleles. No excess of allele sharing was observed at any marker tested on chromosome 21, a suggested candidate chromosome for keratoconus because of the occurrence of keratoconus with a 150-fold  ncreased incidence in Down syndrome (Shapiro and France, 1985; van Allen ey al.,1999). Further analysis of positive loci revealed suggestive association at 20q12, where significant deviation in frequency of the allele D20S119 was observed. The nearby candidate gene matrix metalloproteinase-9 (MMP9), which is located at 20q11.2-q13.1, was excluded.
Ihalainen (1986) found multiple cases in 19 of 101 families studied in the north of Finland and in 5 of 58 families in the south. Mean family size was 4.9 in the north as compared with 3.5 in the south. In 24 of 28 multiplex families the pattern of inheritance was autosomal dominant. The disorder was inherited from the mother in 15 cases and from the father in 9. Incomplete penetrance was indicated. Corneal transplant was carried out in 65 of the 144 patients coming from the area served by Oulu University Central Hospital in Finland. Among 212 patients, 63% were male. Symptoms usually began in young adults. Pregnancy seemed to precipitate keratoconus in some instances.

Kennedy et al. (1986) found keratoconus in less than 6% of the relatives of affected probands.

Rabinowitz et al. (1992) studied members of 3 generations of a family in which the propositus had keratoconus. By biomicroscopy, keratoconus was detected in 8 of 15 family members with vertical transmission, consistent with autosomal dominant inheritance. Affected individuals displayed variable topographic features. Abortive 'nipple-type' cones were identified in some individuals by use of the computer-assisted videophotokeratoscope, and more advanced nipple-type cones were detected on biomicroscopy of other family members. Rabinowitz et al. (1992) selected COL6A1 as a candidate gene and by linkage analysis excluded this specific gene as well as the most telomeric region of chromosome 21 as the site of the mutation in this family. A subset of the cases of keratoconus may have autosomal dominant transmission with a
wide range in the level of expression.

Wang et al. (2000) conducted a family study to investigate genetic contributions to the development of keratoconus. The estimated prevalence in first-degree relatives was 3.34% (41/1,226), which is 15 to 67 times higher than that in the general population (0.23 to 0.05%). The correlation in sib and parent-offspring pairs (r = 0.30 and 0.22, respectively) was significantly greater than that in marital pairs (r = 0.14) and the latter was not significantly different from zero. Segregation analysis in 95 families did not reject a major gene model;
the most parsimonious model was autosomal recessive inheritance.

Nielsen et al. (2003) used gene microarrays to investigate differential gene expression in corneal epithelium from samples with and without keratoconus. Keratoconus epithelium appeared to be characterized by massive changes of the cytoskeleton, reduced extracellular matrix remodeling, altered transmembrane signaling, and modified cell-to-cell and cell-to-matrix interactions. Validation of gene expression with dChip analysis and real-time PCR indicated Gene Chip to be a valid technique for investigation of epithelium from single dissected corneal samples.

Ihalainen (1986) reviewed various conditions with which keratoconus is at times associated. Keratoconus is frequent in cases of amaurosis congenita of Leber.

Individuals with keratoconus are not candidates for LASIK (laser-assisted in situ keratomileusis) for correction of their myopia and/or astigmatism.

Jabbur et al. (2001) described the clinical course and histopathology of an individual with suspected keratoconus who underwent bilateral simultaneous LASIK. She required penetrating
keratoplasty due to progressively worsening vision from corneal ectasia after LASIK.

Classically, corneal allograft rejection was thought to be a Th1-mediated phenomenon. However, Th2-mediated allograft rejection has been reported in heart and kidney transplanted systems.

Hargrave et al. (2003) reviewed the records of 84 consecutive patients who underwent penetrating keratoplasty for keratoconus. Because an association between keratoconus and atopic disease had been documented in the literature and had been considered significant since 1937, careful attention was paid to the clinical history of atopy, in this study. Atopic patients have been shown to have a 'Th2 immune bias.' Of the 7 patients who rejected their corneal allografts, 4 had repeat penetrating keratoplasty. Of these 4 repeat corneal allografts, 3 showed eosinophilia when compared with rejected grafts in control (nonkeratoconic, nonatopic) patients. Atopic keratoconus patients had a mixed inflammatory cellular infiltrate in the rejected corneal tissue specimen with a significantly greater density of eosinophils compared with patients who did not have a preexisting Th2 bias. The histopathology was comparable to the authors' murine model of rejection in Th2 mice, characterized by a predominantly eosinophilic infiltrate when compared with wildtype (Th1) mice that had a predominantly mononuclear infiltrate in the rejected corneal graft bed.

Dogru et al. (2003) reviewed the ocular surface disease in keratoconus. Keratoconus patients showed disorders of tear quality, lowered tear film breakup time (BUT), squamous metaplasia of the corneal epithelium, and goblet cell loss, all of which seemed to relate to the extent of keratoconus progression.

Li et al. (2004) examined 778 patients with keratoconus and found that116 (14.9%) had clinically unilateral keratoconus at baseline. These 116 patients were followed for a period ranging from 6 months to 8 years. Approximately 50% of clinically normal fellow eyes progressed to keratoconus within 16 years. The greatest risk was during the first 6 years. Li et al. (2004) also described quantitative indices  and qualitative patterns that might predict this progression.

Lema and Duran (2005) determined the levels of a panel of inflammatory molecules and matrix metalloproteinases in the tears of patients with keratoconus. Patients with keratoconus had significantly higher levels of IL6, TNFA, and MMP9 than control subjects. The extent of the increase was associated with the severity of keratoconus. Lema and Duran (2005) suggested that the pathogenesis of keratoconus may involve chronic inflammatory events.

Heon et al. (2002) identified mutations in the VSX1 homeobox gene in patients with either keratoconus or posterior polymorphous corneal dystrophy. One of the mutations responsible for keratoconus altered the homeodomain and impaired DNA binding. Two sequence changes were associated with keratoconus and PPCD, respectively, and involved a region adjacent to the homeodomain.

Bisceglia et al. (2005) evaluated the role of the VSX1 gene in a series of 80 keratoconus-affected Italian subjects. They found 3 theretofore described missense changes and a novel mutation (148300.0005) in 7 of 80 unrelated patients (8.7%); they also found 2 undescribed intronic polymorphisms. The authors concluded that the VSX1 gene plays an important role in a significant proportion of patients affected by keratoconus inherited as an autosomal dominant trait with variable expressivity and incomplete penetrance.

Atilano et al. (2005) found that keratoconus-affected corneas showed a trend of lower mtDNA-to-nDNA ratio than did control corneas, had decreased cytochrome c oxidase subunit I (MTCO1; in areas of corneal thinning, and had significantly increased numbers of mtDNA deletions compared to control corneas. Atilano et al. (2005) suggested that increased oxidative stress and altered integrity of mtDNA may be related to each other, contributing to keratoconus pathogenesis.

Kenney et al. (2005) found that keratoconus corneas exhibited a 2.20-fold increase in catalase mRNA and 1.8-fold increase in enzyme activity; a 1.5-fold increase in cathepsis V/L2 mRNA and abnormal protein distribution; and a 1.8-fold decrease in TIMP1 mRNA and a 2.8-fold decrease in protein compared with normal (physiologic) corneas. Kenney et al. (2005) concluded that keratoconus corneas had elevated levels of cathepsins V/L2, which could stimulate hydrogen peroxide production, which, in turn, could upregulate catalase, an antioxidant enzyme. In addition, decreased TIMP1 and increased cathepsin V/L2 levels might play a role in the matrix degradation that is a hallmark of keratoconus corneas. These findings supported the hypothesis that keratoconus corneas undergo oxidative stress and tissue degradation.

Animal Model

Tachibana et al. (2002) established an inbred line of spontaneous mutant mice with keratoconus-affected corneas (SKC mice). The SKC mouse cornea resembled corneas of human eyes with keratoconus: both corneas were conical and showed similar changes, including apoptosis of keratocytes and increased expression of Fos protein. The SKC mouse phenotype was transmitted in an autosomal recessive manner, but it was observed almost exclusively in males. Female mice showed the phenotype when injected with testosterone, whereas male incidence of the phenotype diminished drastically when the mice were castrated. Linkage analysis localized a predisposition locus to a major histocompatibility complex (MHC) region on mouse chromosome 17 that includes the gene encoding' sex-limited protein,' or Slp. The authors proposed that the SKC mouse may be a potential model for a subset of human keratoconus.

 More recent findings 

Using reverse transcription and polymerase chain reaction (RT-PCR) to identify RNA types which are found either in greater quantity in normal tissue or in greater quantity in keratoconus tissues CD45 (leukocyte common antigen or T200), a transmembrane phosphotyrosine phosphatase (normally found associated with blood cells), is present in corneas. Further, there are more cells displaying CD45 within keratoconus corneas than normal. These cells may represent the source of some of the proteolytic enzymes reported to be associated with keratoconus. 

A gene product referred to as glucose regulated protein (GRP78) was found in greater quantity in normal corneas. GRP78 is a protein that aids or "chaperones" the folding and secretion of other proteins produced in cells. The reduction in GRP78 could affect the secretion and folding of the major structural proteins of the cornea which are reduced in the keratoconus cornea.

An increased expression of the transcription factor Sp1 was also demonstrated in keratoconus. In corneal epithelial cells, as in stromal cells, alpha 1-PI promoter activity was suppressed by cotransfection of pPacSp1. The LAP, cathepsin B, and alpha 2-M promoters were functional in corneal cells, whereas activities of these promoters were much lower in skin fibroblasts. Cotransfection experiments indicated that the up or down regulation of LAP, cathepsin B, and alpha 2-M observed in keratoconus-affected corneas was not mediated by Sp1. These results support the theory that the corneal epithelium, along with the stroma, is involved in keratoconus. An upstream role of Sp1 is indicated and the Sp1-mediated down-regulation of the alpha 1-PI gene may be a key event in the disease development.

The activation of matrix metalloproteinase-2 (MMP-2) could be a crucial pathogenic factor behind progressive and chronic diseases in which basement membranes are disrupted. Although MMP-2 of Mr 65,000 on SDS gelatin polyacrylamide gels is the major protease secreted by keratocytes of normal corneas, the keratocytes of early-phase keratoconic corneas secrete an additional zymographic activity of Mr 61,000. From their N-terminal amino acid sequences, both these proteins were shown to be conformers of proMMP-2. Treatment with SDS followed by protein fractionation was required to achieve in vitro activation of the MMP-2 secreted by normal corneal keratocytes. Treatment with SDS alone partially activated the enzyme produced by early-phase keratoconic corneal keratocytes. This procedure and autocatalysis yielded an enzyme of Mr 43,000 that selectively hydrolysed Type IV and denatured Type 1 collagen. The zymographic gelatinase activities of apparent Mr 65,000 and 61,000 are conformers of corneal proMMP-2. Activated enzyme, of Mr 43,000, is more readily generated from protein preparations of the culture media of early phase keratoconic corneal keratocytes than from protein preparations of the culture media of normal corneal keratocytes.

Recent analysis increases the database of genes expressed in the human cornea and provides insights into KC. KC6 is a novel gene of unknown function that shows cornea-preferred expression, whereas the suppression of transcripts for AQP5 provides the first clear evidence of a molecular defect identified in KC. (The absence of transcripts for the water channel protein Aquaporin 5).

The results of the on-going genome-wide linkage and refinement studies indicate that a major gene for KC, responsible for 50–60% of familial KC cases studied, is located within a 1.69 Mb region (one of two small regions, both <0.5 Mb) at 2p24. It is worth noting that there may be other genes involved, which has not been indentified yet, and other factors may play a part.

To sum up, there is some evidence that one form of keratoconus (KC1) can be caused by mutation in the VSX1 gene on chromosome 20. Other loci for keratoconus have been mapped to chromosomes 16q22.3-q23.1 (KC2), 3p14-q13 (KC3),and 2p24 (KC4). Also there is the APQ5 (missing Water Channels) finding as well.

Keratoconus is likely to be multi-factorial, although there may be one root cause, or different Keratoconus patients may have Keratoconus for different reasons. The short list of possible causes are: genetic, stress/trauma to the cornea, environmental, or it may be at cellular level - any of which may cause the trigger/s for the onset of Keratoconus and it's progression.



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