Purpose
To investigate the clinical and genetic features of late-onset Fuchs corneal dystrophy (FCD) on Tangier, an island in the Chesapeake Bay with an isolated population of approximately 500 individuals.
Design
Observational, cross-sectional study.
Methods
A total of 156 individuals born to inhabitants of Tangier Island volunteered to undergo ophthalmic evaluation. Medical history was ascertained prior to examination. All participants underwent anterior segment examination with slit-lamp biomicroscopy. Retroillumination photographs were acquired from affected individuals and the disease severity was compared with individuals from large families ascertained previously. Genomic DNA samples were investigated for the presence of the recently identified risk allele rs613872, an intronic variant of TCF4 .
Results
Of the 148 examined individuals who were at least 30 years of age, 32 showed the classical symptoms of late-onset FCD (21.6%), providing a minimum prevalence of 11% among individuals over the age of 50 years. Severity was significantly lower compared to 51 cases from unlinked families, among individuals either 50 to 70 or above 70 years of age ( P = .05 and P = .01, respectively). Retroillumination photography analyses were suggestive of mild severity when compared with the disease phenotype associated with FCD1 – and FCD2 -linked families. The rs613872 variant was associated with a higher affectation rate ( P = .01), while the wild-type allele was correlated with a higher proportion of subclinical disease ( P = .01).
Conclusions
In this study population in Tangier, late-onset FCD manifests clinically with a mild phenotype and increased prevalence. The rs613872 variant correlates with increased affectation and a clinical disease phenotype.
Fuchs corneal dystrophy (FCD) is a progressive, hereditary degenerative condition of the posterior cornea associated with endothelial cell loss, thickening of Descemet membrane, and focal excrescences termed guttae. The phenotype was first described by the Austrian ophthalmologist Ernst Fuchs in 1910. Hereditary transmission was suggested by Clegg in 1915 and subsequent studies confirmed a familial component to disease. FCD is considered to affect 4% of the United States population above the age of 40 years and is more prevalent among women.
Recent studies have identified 4 genetic loci, FCD1 , FCD2 , FCD3 , and FCD4 , on chromosome 13, 18, 5, and 9 respectively; as well as 2 causally associated genes, TCF8 and SLC4A11 . Of these, the FCD2 locus appears to be the most common contributor, with approximately 40% of large families in our cohort mapping to this locus. A TCF4 intronic variant at chromosome map location 18q21, rs613872, was recently associated with increased odds of developing FCD, and was independently confirmed by our group and associates.
The extent to which the genetic susceptibility to FCD may be modifiable by environmental risk factors has yet to be elucidated; height, weight, smoking status, and ultraviolet light exposure have been postulated as potential factors but no strong, repeatable correlation has been found. Determination of risk factors requires appropriately powered studies of populations at risk for developing FCD.
Here we describe the study of FCD among an isolated population on Tangier, an island in Virginia with a population of over 500 related individuals and a known presence of disease after an inhabitant presented to our clinic for management of FCD.
Methods
Subjects
Following Descemet stripping endothelial keratoplasty of a 50-year-old man with classic signs of late-onset FCD, we traveled to Tangier to examine 8 first- and second-degree relatives of the patient. We acquired a comprehensive pedigree of the island population, and thus the family pedigree was expanded to include the population of Tangier. A total of 156 individuals born to inhabitants of the island volunteered to undergo ophthalmic evaluation. Blood samples of approximately 10 to 15 mL were collected from all willing participants, and genomic DNA was extracted from white blood cells (Gentra Puregene Blood Kit; Qiagen Inc, Valencia, California, USA).
To determine the number of current inhabitants, we used the most recent published government estimate of 535. We then used the most recent US Census data (2000 at time of study), which publishes figures stratified by age range, to develop an estimate of the percentage of individuals above 50.
Examination
Medical history, including medications and comorbidities, was asked of each study participant prior to examination. We inquired into height, weight, and smoking status, factors that have been previously associated with FCD. All participants underwent anterior segment examination by an ophthalmologist using a Haag-Streit 900 slit-lamp biomicroscope (Haag-Streit International, Koeniz, Switzerland).
To grade severity, we used a scale described previously by Krachmer and associates. This scale includes 6 levels of severity: negative, defined as up to 12 central guttae; grade 1, greater than 12 central nonconfluent guttae; grade 2, 1 to 2 mm of central confluent guttae; grade 3, 2 to 5 mm of central confluent guttae; grade 4, more than 5 mm of central confluent guttae; and grade 5, the addition of corneal stromal or epithelial edema to grade 4 findings. We defined positive affectation as a minimum of 1 eye with grade 1 severity in this scale. In this early stage, localization of guttae within the central 5-mm zone distinguishes lesions from Hassall-Henle bodies, small extrusions of Descemet membrane that may appear in the periphery with age. Confluency represents an increase in the surface density of guttae such that excrescences appear adjacent to one another, either with direct slit-beam illumination or with corneal retroillumination.
Disease severity was compared with 51 cases from large families ascertained previously and of undetermined genetic linkage, acquired through similar testing in other geographic locations. To quantify and further assess severity, we then used slit-lamp retroillumination photography as described previously to develop a profile of the age-severity relationship of FCD on the island and compare it to previously reported cohorts with FCD.
Single Nucleotide Polymorphism Genotyping
We interrogated genomic DNA samples of all individuals for rs613872, an intronic TCF4 single nucleotide polymorphism (SNP), as described previously. Briefly, polymerase chain reaction (PCR) was performed in 5-μL volumes containing 10 ng of genomic DNA, 2.5 μL of ABI Taqman SNP genotyping master mix, and 0.125 μL of ABI Taqman genotyping assay mix (Applied Biosystems, Foster City, California, USA). Reactions for this SNP were amplified independently in a 9700 thermocycler (Applied Biosystems, Foster City, California, USA). The cycling parameters consisted of 2 minutes incubation at 50 C and denaturation at 95 C for 10 minutes followed by 40 cycles of 10 seconds at 95 C, and 1 minute elongation at 72 C with a final 10-minute extension at 72 C. Amplified products were analyzed for the enrichment of specific alleles in an ABI 7900HT Sequence Detection System (Applied Biosystems).
Exclusion Analyses
All of the known late-onset FCD loci were excluded using closely spaced short tandem repeat (STR) markers. Primer sequences for all the STR markers are as described previously. PCR was completed in the GeneAmp 9700 PCR System (Applied Biosystems). Briefly, each reaction was carried out in a 5-μL mixture containing 40 ng genomic DNA, various combinations of 10 μM fluorescently labeled primer pairs, 0.5 μL 10× PCR Buffer (Applied Biosystems), 0.5 μL 10 mM dNTP mix, 2.5 mM MgCl 2 , and 0.2 U Taq DNA polymerase (Applied Biosystems). Initial denaturation was carried out for 5 minutes at 95 C, followed by 10 cycles of 15 seconds at 94 C, 15 seconds at 55 C, and 30 seconds at 72 C and then 20 cycles of 15 seconds at 89 C, 15 seconds at 55 C, and 30 seconds at 72 C. The final extension was performed for 10 minutes at 72 C and followed by a final hold at 4 C. PCR products from each DNA sample were pooled and mixed with a loading cocktail containing HD-400 size standards (Applied Biosystems). The resulting PCR products were separated in an ABI 3100 DNA sequencer and analyzed using GENESCAN 4.0 software packages (Applied Biosystems).
Results
A total of 156 individuals participated in this study, which included approximately half (107/212; 50.4%) of the predicted island population over 50 years of age. Mean and median ages were 57, with range of 18 to 87 years. Of 148 individuals at least 30 years of age, 32 were affected (21.6%). Independent of severity, the affectation rate did not significantly increase with age in the study cohort, with age-dependent affectation rates of 19.5% (8/41; 30 to 49 years old), 22.9% (16/70; 50 to 69 years old), and 21.6% (8/37; 70 years or older). Therefore, a more modest estimate, developed by considering all untested individuals to be unaffected, predicts that at least 11% of all individuals over 50 on the island would be positive. Participants included 92 women and 64 men. Although our studies are de facto limited by the size of the Tangier population, male subjects appeared to be less commonly affected (9/64; 14.1%) than female (23/91; 25.3%); this finding, however, did not reach statistical significance ( P < .07). We observed no difference in age between the 2 groups ( P < .47).
Severity
As evidenced by histologic analysis of Descemet membrane provided by the proband through endothelial keratoplasty, guttate changes observed during examination were consistent with common late-onset FCD. Severity increased with age, with age-dependent mean Krachmer scores of 1.44 (30 to 50 years old), 2.04 (50 to 70 years old), and 2.50 (70 years or older). Severity in both older groups represented a significant increase relative to the 30- to 49-year-old group ( P = .03 and P = .001, respectively); only 1 individual progressed to transplantation. Severity was mild and significantly decreased relative to 51 cases from unlinked families, in both the 50- to 69-year-old ( P < .05) and 70 and older ( P < .02) categories. Assessment of retroillumination photography reveals an increase of 10% annually ( P < .001). Comparisons with previously reported eyes associated with the FCD1 and FCD2 locus confirm a milder severity, which develops later in life ( Figure 1 ) . Specifically, at age 60, the maximum age in the FCD1 sample, the number of guttae in affected eyes associated with FCD1 is 39.6 times higher ( P < .001) and FCD2 is 13.5 times higher ( P < .001) than in eyes associated with Tangier. There was no difference between left and right eyes ( P = .27).
Risk Factors
Next, we assessed our sample for previously identified factors associated with FCD. Average height was decreased significantly among patients with FCD by t test ( P = .005). After stratifying for sex, this correlation approached but did not reach statistical significance among women ( P = .06) or men ( P = .37). Individuals with FCD also appeared to exhibit decreased weight ( P = .03); stratification by sex revealed no significant difference between affected and unaffected female subjects ( P = .57) and a difference that fell short of significance among male subjects ( P = .06). Approximately 28% (7/25) of affected individuals demonstrated a history of regularly smoking tobacco, compared to 24.8% (26/105) of unaffected subjects; there was no correlation with FCD affectation by Fisher exact test ( P = .46). The most common medical comorbidities among the sample population were hypertension (54/156; 35%), diabetes (25/156; 16%), and dyslipidemia (21/156; 13%), none of which correlated with disease status.
Genetics
Initially, we excluded all 4 known late-onset FCD loci ( FCD1 , FCD2 , FCD3 , FCD4 ) using closely spaced STR markers, as described in Methods. Next, we interrogated genomic DNA samples for the rs613872 SNP in TCF4 , which revealed that the minor allele is present in Hardy-Weinberg equilibrium in this island population, in a relatively high frequency (0.37) nearing previously reported cohorts of cases. Individuals homozygous for the minor allele comprised a minority (17%) of this isolated population compared to individuals heterozygous (40%) or homozygous (43%) for the wild-type allele. We observed the lowest affectation rate among individuals homozygous for the wild-type allele, of which 8.8% were affected. In contrast, subjects demonstrated a significantly higher affectation rate when heterozygous (24.5% affected, P = .02) or homozygous (31.8% affected, P < .02) for the minor allele. There was no interaction between rs613872 status and sex ( P < .64).
We also investigated whether presence of the minor allele would correlate with a difference in clinical severity. Among positive cases, defined by at least 12 central guttae in 1 eye, individuals demonstrated no significant increase in Krachmer grading if heterozygous ( P = .31) or homozygous ( P = .48) for the minor allele compared to wild-type individuals, consistent with previous reports. Since FCD on Tangier Island is manifested by a particularly mild phenotype, we also investigated whether subclinical, or mildest, disease severity may be modified in its presentation by the minor allele. We particularly inquired into individuals with documented guttae but fewer than 12 in number. Indeed, among individuals considered clinically negative, we observed that a significantly greater proportion of wild-type individuals demonstrated subclinical signs of FCD than those with the intronic variant ( P = .01; Figure 2 ) .
