Genetic variants in the FOXO1 and ZNF469 genes are associated with keratoconus in Sweden: a case-control study.

BACKGROUND: Keratoconus (KC) is characterized by pathological thinning and bulging of the cornea that may lead to visual impairment. The etiology of sporadic KC remains enigmatic despite intensive research in recent decades. The purpose of this study was to examine the relationship between previously highlighted genetic variants associated with KC and sporadic KC in a Swedish cohort. METHODS: A total of 176 patients (age 16-70 years) with sporadic KC diagnosed by Scheimpflug-topography (Pentacam) were included. The control group (n = 418; age 70 years) was a subsample originating from the Gothenburg H70 Birth Cohort Studies of ageing. Extraction of DNA from blood samples was performed according to standard procedures, and genotyping was performed using competitive allele specific PCR (KASP) technology. A total of 11 single nucleotide polymorphisms (SNPs) were selected for analysis. RESULTS: Statistically significant associations (p = 0.005) were found between the SNPs rs2721051 and rs9938149 and sporadic KC. These results replicate earlier research that found associations between genetic variants in the FOXO1 and BANP-ZNF469 genes and sporadic KC in other populations. CONCLUSION: Genetic variations in the FOXO1 and BANP-ZNF469 genes may be involved in the pathogenesis of sporadic KC.

Repeated Same-Day Versus Single Tomography Measurements of Keratoconic Eyes for Analysis of Disease Progression.

PURPOSE: Corneal tomography is used to assess progression of keratoconus and to direct clinical decisions regarding corneal cross-linking. The purpose of this study was to analyze the variability of repeated Scheimpflug-tomography (Pentacam Classic; Oculus, Wetzlar, Germany) measurements of keratoconic eyes in a clinical setting and to assess the validity of such measurements as a clinical decision-making tool. METHODS: Eighty keratoconic eyes of 45 patients (age range 16-32 years) were examined at baseline and after follow-up periods of 3 to 6 months using 3 consecutive tomography measurements at each visit. Minimum corneal thickness and anterior sagittal curvature map parameters were studied [simulated keratometry (K) astigmatism (SimKast); maximum simulated K-reading (SimKmax); average SimK (SimKave); maximum K-readings on the 3-mm (Kmax3) and 5-mm (Kmax5) rings; and maximum K-reading (Kmax)]. RESULTS: When comparing the first measurements at the first and second visits, respectively, 9% to 20% of eyes were classified as progressive depending on which parameter was chosen. Using the average of 3 consecutive measurements at each visit, 5% to 19% of eyes were classified as progressive. An increase in the SD of 3 consecutive measurements of SimKast (SD_SimKast) at the first visit of 1 diopter makes true progression of keratoconus 3.6 times more likely (odds ratio = 3.6; 95% confidence interval: 0.846-16.027; area under the curve = 0.70). CONCLUSIONS: The approach used to analyze progression in keratoconus, that is, single versus repeated measurements, may confer a great impact on the decision to perform corneal cross-linking treatment or not.

Validation of Catquest-9SF-A Visual Disability Instrument to Evaluate Patient Function After Corneal Transplantation.

PURPOSE: Catquest-9SF is a 9-item visual disability questionnaire developed for evaluating patient-reported outcome measures after cataract surgery. The aim of this study was to use Rasch analysis to determine the responsiveness of Catquest-9SF for corneal transplant patients. METHODS: Patients who underwent corneal transplantation primarily to improve vision were included. One group (n = 199) completed the Catquest-9SF questionnaire before corneal transplantation and a second independent group (n = 199) completed the questionnaire 2 years after surgery. All patients were recorded in the Swedish Cornea Registry, which provided clinical and demographic data for the study. Winsteps software v.3.91.0 (Winsteps.com, Beaverton, OR) was used to assess the fit of the Catquest-9SF data to the Rasch model. RESULTS: Rasch analysis showed that Catquest-9SF applied to corneal transplant patients was unidimensional (infit range, 0.73-1.32; outfit range, 0.81-1.35), and therefore, measured a single underlying construct (visual disability). The Rasch model explained 68.5% of raw variance. The response categories of the 9-item questionnaire were ordered, and the category thresholds were well defined. Item difficulty matched the level of patients' ability (0.36 logit difference between the means). Precision in terms of person separation (3.09) and person reliability (0.91) was good. Differential item functioning was notable for only 1 item (satisfaction with vision), which had a differential item functioning contrast of 1.08 logit. CONCLUSIONS: Rasch analysis showed that Catquest-9SF is a valid instrument for measuring visual disability in patients who have undergone corneal transplantation primarily to improve vision.

Clinical outcome of repeat penetrating keratoplasty.

PURPOSE: To compare the clinical outcome of regrafts with first grafts. METHODS: Two-year outcome data were obtained from the Swedish Cornea Transplant Register for patients undergoing penetrating keratoplasty between 2001 and 2008. Only data from the 3 centers with follow-up return rates >75% were included. The survival and visual outcome of regrafts with the original diagnoses of keratoconus, Fuchs endothelial dystrophy (FED), or bullous keratopathy (BK) were compared with first grafts for the same diagnoses by univariate and logistic regression methods. RESULTS: For keratoconus, the failure rate increased 3-fold in regrafts compared with first grafts (ie, 17% vs. 6%; P = 0.002) and doubled in FED regrafts (33% vs. 15%; P = 0.001). In BK, the failure rate was already high in first grafts, and the increase in failure of regrafts was minimal (P = 0.9). Visual acuity was also worse in regrafts compared with first grafts, mainly in the keratoconus and FED patients. In the keratoconus group, visual acuity with preferred correction was ≥0.5 in 69% of first grafts compared with only 55% in regrafts (P = 001). In FED, 52% of first grafts but only 19% of regrafts achieved visual acuity ≥ 0.5 (P = 0.001). The visual outcome of regrafts in BK was poor but little different from first grafts where fewer than 20% achieved visual acuity ≥ 0.5. CONCLUSIONS: This analysis confirmed the poorer survival of regrafts where the original indication was keratoconus or FED. In addition, visual outcome was also worse than in the first grafts. However, the outcomes of regrafts in BK were similar to first grafts.

Donor and recipient endothelial cell population of the transplanted human cornea: a two-dimensional imaging study.

Purpose. To elucidate the pattern of donor and recipient endothelial cell populations in transplanted human corneas and determine the degree to which donor endothelial cells survive in the graft. Methods. Thirty-six corneal grafts were collected from recipients of opposite sex to the donor, at the time of retransplantation for various indications. Cells from the endothelial side of the grafts were harvested, preserving their relative location on the endothelium. Fluorescence in situ hybridization of the sex chromosomes enabled each cell to be identified as donor- or recipient-derived. Images of the graft endothelium were assembled, to depict the pattern of cell population of the graft, and the proportion of donor cells present was estimated. Results. Endothelial cells of donor origin were found in 26 of 36 grafts (72.2%)-in one case, up to 26 years after transplantation. The proportion of donor endothelium ranged from 2% to 99%; however, there was no significant correlation of this proportion with postoperative time (P = 0.19). The mean annual rate of donor cell loss correlated negatively with the time to graft failure by endothelial decompensation (P = 0.002). Endothelial images indicated a highly variable pattern of recipient cell repopulation of the graft. A tendency toward donor cell retention in transparent, successful grafts was noted; however, this feature alone was not a reliable indicator of long-term graft transparency. Conclusions. Two-dimensional imaging of the corneal graft endothelium revealed a variable pattern and extent of donor and recipient cell population, indicating the highly dynamic nature of the corneal endothelium after transplantation.

Ten-year follow-up of graft survival and visual outcome after penetrating keratoplasty in Sweden.

PURPOSE: To determine factors influencing graft survival and visual outcome 10 years after penetrating keratoplasty. METHODS: Ten-year follow-up data were obtained from a cohort of patients that represented 20% of corneal transplants in Sweden between 1996 and 1998. Multiple regression analyses (logistic and linear) were performed on graft survival and visual outcome (visual acuity and astigmatism). RESULTS: Of the initial 242 patients receiving a corneal transplant during 1996-1998, 140 were available at 10 years. The majority of patients lost to follow-up had the indication bullous keratopathy and many were deceased. Overall, 71% of transplants available for follow-up at 10 years were still functioning, with keratoconus showing the best result (88%) and bullous keratopathy the worst (48%). Complications during the first 2 postoperative years reduced the percentage of functioning grafts at 10 years from 84% to 50%. The visual acuity was influenced by indication and postoperative complications. The change in Snellen lines between preoperative and 10-year visual acuity for the individual patients also depended on indication and postoperative complications. Most of the changes occurred during the first 2 postoperative years. The astigmatism at 10 years was also affected by postoperative complications and in addition by the amount of astigmatism at 2 years. CONCLUSION: Graft survival and visual outcome at 10 years depended on indication and postoperative complications. Most improvement of vision occurred during the first 2 years and was predictive of the 10-year visual outcome.

Survival of donor-derived cells in human corneal transplants.

PURPOSE: To determine the fate of donor epithelial, stromal, and endothelial cells after corneal transplantation in humans. METHODS: Fifty-two transplanted corneal buttons were explanted over a 2-year period from patients who required regrafting and had received corneas from donors of opposite sex. Fluorescence in situ hybridization of the sex chromosomes of the epithelial, stromal, and endothelial cells was performed in histologic sections prepared from each freshly explanted graft. Fluorescence microscopy was subsequently used to determine the origin of cells in the graft (donor or recipient) and to quantify the relative proportion of donor and recipient cells of each corneal cell type. RESULTS: As early as 3 months after transplantation, donor epithelial cells were completely replaced by recipient epithelium in all corneal buttons examined. Donor stromal and endothelial cells, however, were found in all 52 buttons, with 4% to 95% of stromal cells and 6% to 95% of endothelial cells being of donor origin. No significant correlation between donor cell proportion and the age of the graft could be found. Donor-derived cells were found in significant numbers up to 32 years after transplantation. Eight corneas in this study were transparent, compensated grafts, and a similar long-term survival of donor stromal and endothelial cells was found in these cases. CONCLUSIONS: Although donor epithelial cells are promptly replaced, a high proportion of donor stromal and endothelial cells can survive within the corneal transplant in the long-term. The proportion of surviving donor cells is highly variable; however, the source of this variability remains unknown.

Corneal oedema after cataract surgery: predisposing factors and corneal graft outcome.

PURPOSE: Pseudophakic bullous keratopathy (PBK) is one of the main indications for corneal transplantation. Graft survival and visual outcome in this group are often poorer than for other indications. The aim of this study was to find risk factors for developing corneal oedema after cataract surgery and factors that influence the subsequent survival of the graft and the visual outcome. METHODS: We carried out an observational, retrospective cohort study using data from the Swedish Cornea Transplant Register and patient medical records. A total of 273 patients whose indication for corneal transplantation was corneal oedema after cataract surgery were included in the study. Multiple logistic regression analysis and, where appropriate, univariate analyses were applied. RESULTS: A total of 43% of the patients developed persistent corneal oedema immediately after cataract surgery, the main risk factors for which were phacoemulsification and pre-existing endothelial disease. Almost a third (32%) of the transplants for PBK failed within 2 years, for which rejection and other postoperative complications increased the risk. Half (50%) the patients had visual acuity < or = 0.1 at 2 years after keratoplasty. Comorbidity, increasing duration of the bullous keratopathy and increasing age affected the visual outcome negatively. CONCLUSIONS: Phacoemulsification was a risk factor for immediate persistent corneal oedema after cataract surgery, although it did not increase the overall risk of developing PBK. However, transplants for immediate PBK had a better survival rate than those for later onset PBK. Shorter duration of PBK and intraocular lens exchange at the time of penetrating keratoplasty increased the likelihood of good visual acuity.

Corneal transplant rejection rate and severity after endothelial keratoplasty.

PURPOSE: To examine the corneal transplant rejection rate and the rate of graft failure subsequent to rejection in the first 2 years after endothelial keratoplasty (EK) and to compare this to background data for similar cases of penetrating keratoplasty (PK). METHODS: Anonymized data from consecutive cases of EK [deep lamellar (DLEK) or Descemet stripping (DSEK)] for Fuchs corneal endothelial dystrophy or pseudophakic bullous keratopathy operated on before August 2004 were collected in 4 participating centers. The number and timing of rejection episodes and cases of subsequent graft failure were recorded, together with the time to cessation of topical steroid medication. Two-year postoperative findings were compared with background data for similar patients undergoing PK recorded in the Swedish Corneal Transplant Registry. RESULTS: Rejection episodes were less frequent after EK than PK (P = 0.035). Fifteen (7.5%) of 199 EK cases had a rejection episode in the first 2 years after surgery versus 92 (13%) of 708 PK cases. Graft failure after rejection in EK may have been less frequent than in PK (P = 0.063), with only 1 (6.7%) case of rejection after EK versus 26 (28.3%) cases of rejection after PK proceeding to graft failure. A strong trend toward continued use of low-dose topical steroid medication was observed in the EK group. Eighty percent of EK patients were still taking topical steroid medication 2 years after surgery, whereas topical steroid medication had been ceased within a year of surgery in almost all PK patients in the comparator group. CONCLUSIONS: Corneal transplant rejection is less frequent and may be less severe after EK than after PK. It is not yet clear whether these apparent differences are simply a product of relatively prolonged postoperative topical steroid cover in EK patients.

Astigmatism and the impact of relaxing incisions after penetrating keratoplasty.

PURPOSE: To determine the impact of relaxing incisions for correcting postoperative astigmatism following penetrating keratoplasty. METHODS: Data were collected through the Swedish Corneal Transplant Register. Of the 1161 grafts with complete 2-year follow-up, 131 underwent relaxing incisions. Stepwise multiple regression was used to determine the factors that influenced the extent of astigmatism in diopters (D) (square root transformed). The change in astigmatism brought about by relaxing incisions was evaluated both by subtraction (ie, ignoring angle) and vector analysis. RESULTS: The overall mean astigmatism was 4.56 D (95% confidence interval [CI]: 4.40-4.73, n = 1161). The final regression model explained only a small proportion of the overall variability of the data (< 5%). There was a slight increase in postoperative astigmatism with recipient age (P = .025), and two of the seven participating clinics achieved lower levels of astigmatism (P = .001 and P = .036, respectively). In patients who underwent relaxing incisions, astigmatism was reduced from 8.40 D (95% CI: 8.0-9.0, n = 131) to 3.80 D (95% CI: 3.5-4.3). The mean difference by subtraction was 4.50 D (95% CI: 4.0-5.0, P < .001, paired t test). Vector analysis showed the overall reduction of astigmatism due to surgery to be 7.90 D (95% CI: 7.2-8.7). Compared with grafts with no refractive surgery, a trend was noted that suggested corrected visual acuity was improved following relaxing incisions. CONCLUSIONS: Relaxing incisions were found to be a safe and effective method for reducing postoperative astigmatism and may improve visual acuity.

Longterm survival of transplanted human corneal epithelial cells and corneal stem cells.

PURPOSE: To investigate the survival of donor-derived epithelial cells in conventional penetrating keratoplasty (PKP) and in homologous penetrating central limbal keratoplasty (HPCLK). METHODS AND PATIENTS: Epithelial cells from 26 eyes of 26 patients were analysed. All cases were sex-mismatched (i.e. the transplant and patient were of different genders). At suture removal more than 1 year post surgery, epithelial cells were obtained by gently wiping the removed sutures on glass slides. The cell samples were analysed using fluorescent in situ hybridization (FISH) of the sex chromosomes. This technique makes it possible to allocate the origin of each cell nucleus to either the donor or the recipient. RESULTS: All 19 conventional PKPs were clear and seven had donor-derived epithelial cells at suture removal. Five of the seven HPCLK grafts were clear at the time of investigation (365-1355 days post surgery), and donor-derived epithelial cells were found in two grafts. CONCLUSION: Harvesting cells from removed sutures in combination with FISH enables the clinical study of cell survival in corneal transplants without jeopardizing functioning grafts. From the limited sample investigated, the following tentative conclusions can be made. Donor-derived epithelial cells can remain in conventional PKP for over 1 year. In combined stem cell and corneal grafts (HPCLK), donor-derived epithelial cells may also be retrieved at 1 year or beyond following surgery but the correlation between their presence and a remaining clear graft is uncertain.

Longterm survival of transplanted human corneal epithelial cells and corneal stem cells.

PURPOSE: To investigate the survival of donor-derived epithelial cells in conventional penetrating keratoplasty (PKP) and in homologous penetrating central limbal keratoplasty (HPCLK). METHODS AND PATIENTS: Epithelial cells from 26 eyes of 26 patients were analysed. All cases were sex-mismatched (i.e. the transplant and patient were of different genders). At suture removal more than 1 year post surgery, epithelial cells were obtained by gently wiping the removed sutures on glass slides. The cell samples were analysed using fluorescent in situ hybridization (FISH) of the sex chromosomes. This technique makes it possible to allocate the origin of each cell nucleus to either the donor or the recipient. RESULTS: All 19 conventional PKPs were clear and seven had donor-derived epithelial cells at suture removal. Five of the seven HPCLK grafts were clear at the time of investigation (365--1355 days post surgery), and donor-derived epithelial cells were found in two grafts. CONCLUSION: Harvesting cells from removed sutures in combination with FISH enables the clinical study of cell survival in corneal transplants without jeopardizing functioning grafts. From the limited sample investigated, the following tentative conclusions can be made. Donor-derived epithelial cells can remain in conventional PKP for over 1 year. In combined stem cell and corneal grafts (HPCLK), donor-derived epithelial cells may also be retrieved at 1 year or beyond following surgery but the correlation between their presence and a remaining clear graft is uncertain.

Time-lapse recordings of human corneal epithelial healing.

PURPOSE: The aim of this study was to design an experimental set-up for the study of human corneal epithelial wound healing in a controlled in vitro situation. METHODS: A time-lapse set-up was used. This allowed for pictures to be captured with a magnification ranging from x 80 to x 1800. Pictures were captured at 1-min intervals during the observation period, which lasted up to 4 days. Human corneal tissue was obtained from the Eye Bank or from surgery. A small, rounded lesion was produced in the corneal epithelium with a miniature drill. The specimens were placed in a mini-incubator; the camera focused on the epithelial lesion and continuously observed using the time-lapse set-up. RESULTS: The healing process of human corneal epithelium could be followed for several days. The initial healing response could be divided into a slow, a rapid and a consolidating phase. The first two phases lasted about 12 hours, and by then, epithelial cells covered the lesion. Depending on the origin of the tissue and the placement of the lesion, variations in the healing response could be seen. CONCLUSION: The time-lapse technique makes it possible to study epithelial wound healing over time at the cellular level. Data collected in this way can fill the gap between in vivo studies, where, by nature, human wound healing studies are restricted, and cell culture techniques, where cellular responses in many cases differ from the in vivo situation.

[Growing waiting list for corneal transplantation]. / Växande väntelista för kornealtransplantation.

New corneal storage techniques and legislative changes have transformed conditions for corneal grafting in Sweden. Since 1995, the Swedish Corneal Surgery Society has registered the number of grafts, the size of waiting lists for surgery as well as changes in the capacity of the banks. Since 1995 the number of procedures per year has declined while the waiting list has grown. The required number of procedures is 650 rather than the 448 performed in 1999. The decline is due to a lack of donor corneas, priority accorded to other surgery, efforts to adapt contact lenses to keratoconus, and further indications for excimer laser surgery. The four established corneal banks as well as two opening in 2001 will increase the number of grafts available for surgery.

Survival of transplanted human corneal stem cells. Case report.

PURPOSE: To report a case history involving long-term survival of transplanted human corneal stem cells. METHODS: A male patient with severe bilateral chemical burns received six corneal transplants, all of which failed. He subsequently received combined corneal transplants and stem cell transplants, which have remained clear for 3 and 4 years respectively. One of the donors was female. We studied the gender of the epithelial cells of the cheek of the patient and of the two grafts using fixation and fluorescent in situ hybridization (FISH) analyses. RESULTS: In the graft from the female donor, 30% of the epithelial cells were of female origin. All the epithelial cells from the cheek and the other graft were of male origin. CONCLUSION: This case demonstrates that transplanted human corneal stem cells can survive and replicate in the long-term (3 years) without systemic immunosuppression. The case also indicates that a minority (30%) of healthy transplanted epithelial cells is enough to present a clear graft with a clinically healthy ocular surface.