Genetics of Meesmann corneal dystrophy: a novel mutation in the keratin 3 gene in an asymptomatic family suggests genotype-phenotype correlation

Abstract

Purpose: Juvenile epithelial corneal dystrophy of Meesmann (MCD, OMIM 122100) is a dominantly inherited disorder characterized by fragility of the anterior corneal epithelium and intraepithelial microcyst formation. Although the disease is generally mild and affected individuals are often asymptomatic, some suffer from recurrent erosions leading to lacrimation, photophobia, and deterioration in visual acuity. MCD is caused by mutations in keratin 3 (KRT3) or keratin 12 (KRT12) genes, which encode cornea-specific cytoskeletal proteins. Seventeen mutations in KRT12 and two in KRT3 have been described so far. The purpose of this study was to investigate the genetic background of MCD in a Polish family.

Methods: We report on a three-generation family with MCD. Epithelial lesions characteristic for MCD were visualized with slit-lamp examination and confirmed by in vivo confocal microscopy. Using genomic DNA as a template, all coding regions of KRT3 and KRT12 were amplified and sequenced. Presence of the mutation was verified with restriction endonuclease digestion.

Results: In the proband, direct sequencing of the polymerase chain reaction (PCR) product from amplified coding regions of KRT3 and KRT12 revealed a novel 1493A>T heterozygous missense mutation in exon 7 of KRT3, which predicts the substitution of glutamic acid for valine at codon 498 (E498V). Using PCR-Restriction Fragment Length Polymorphism (RFLP) analysis, the mutation was demonstrated to segregate with the disease (four affected members, three non-affected) and to be absent in 100 controls from the Polish population, indicating that it is not a common polymorphism.

Conclusions: Location of the E498V mutation emphasizes the functional relevance of the highly conserved boundary motifs at the COOH-terminus of the alpha-helical rod domain in keratin 3 (K3).

Figure 1

Slit-lamp photography of proband (III-1). This image demonstrates the microcystic appearance of the corneal epithelium.

Figure 2

Confocal microscopy image of proband’s cornea. This image shows the presence of hyperreflective material within the intraepithelial cysts.

Figure 3

Identification of the heterozygous point mutation E498V in exon 7 of KRT3 in the Polish MCD family. A: DNA sequencing. Electropherograms from bidirectional sequencing of KRT3 exon 7 in the proband showed a 1493 A>T (GAG>GTG) heterozygous mutation, predicting the amino aid change E498V. B: Pedigree of the studied family. The arrow indicates the proband (III-1). C: PCR-RFLP analysis. The KRT3 E498V mutation creates a recognition site for HphI. The presence of this restriction site is seen to cosegregate with MCD in this family. Upon digestion, the full sized 304 bp product is cut into bands of 260 bp and 44 bp (the latter not visible on the figure). DNA molecular weight markers are shown on the left (lane 1). The heterozygous E498V mutation (lanes 3–6) was detected in all affected family members (I-2, II-2, III-1, and III-2). The homozygous normal allele, represented by the 304 bp band (lane 2, 7, and 8), was found in unaffected family members (II-1, II-3, and III-3).

Figure 4

Schematic drawing of K3 and K12 structure with assigned positions of the published mutations. Keratins are composed of three main parts, the central α-helical rod domain, which is divided into four subdomains (1A, 1B, 2A, and 2B), and the two non-helical variable domains (V1 and V2) at each end [3]. All three mutations within KRT3 localize exclusively in the boundary motif of the 2B subdomain. Among the mutations in KRT12, 11 were found in the 1A subdomain and six in the 2B subdomain (see also Table 2).