A novel arginine substitution mutation in 1A domain and a novel 27 bp insertion mutation in 2B domain of keratin 12 gene associated with Meesmann's corneal dystrophy

Abstract

Aim: To determine the disease causing gene defects in two patients with Meesmann's corneal dystrophy.

Methods: Mutational analysis of domains 1A and 2B of the keratin 3 (K3) and keratin 12 (K12) genes from two patients with Meesmann's corneal dystrophy was performed by polymerase chain reaction amplification and direct sequencing.

Results: Novel mutations of the K12 gene were identified in both patients. In one patient a heterozygous point mutation (429A-->C = Arg135Ser) was found in the 1A domain of the K12 gene. This mutation was confirmed by restriction digestion. In the second patient a heterozygous 27 bp duplication was found inserted in the 2B domain at nucleotide position 1222 (1222ins27) of the K12 gene. This mutation was confirmed by gel electrophoresis. The mutations were not present in unaffected controls.

Conclusion: Novel K12 mutations were linked to Meesmann's corneal dystrophy in two different patients. A missense mutation replacing a highly conserved arginine residue in the beginning of the helix initiation motif was found in one patient, and an insertion mutation, consisting of a duplication of 27 nucleotides, was found before the helix termination motif in the other.

Figure 1

Clinical photographs of affected family members. (A) Slit lamp photograph of proband of family A. Red reflex retroillumination demonstrates the numerous translucent corneal epithelilal microcysts characteristic of Meesmann’s dystrophy. (B) Slit lamp photograph of proband of family B. Iris retroillumination reveals the diffuse pattern of corneal epithelial involvement characteristic of family B.

Figure 2

Mutational analysis of families A and B. (A) Left side of panel shows normal sequence of K12 exon 1 from unaffected member of family A. Right side of panel shows mutant sequence of K12 exon 1 from affected member of family A, representing a heterozygous A→C transversion (arrow) at the third position of codon 135 (boxed sequence), resulting in an arginine to serine replacement (R135S). (B) Pedigree of family A, which had no previous evidence of family history of disease before the proband (II-1, marked by arrow). The mutation R135S created a novel restriction enzyme site (Blp I). Upon digestion, the PCR product was visualised as two distinct bands. Unaffected family members and 50 control individuals produced a single band, and were screened using this digest. (C) Left side of panel shows normal sequence of K12 exon 6 from unaffected member of family B. Right side of panel shows mutant sequence of K12 exon 6 from affected member of family B. Sequence begins to diverge at nucleotide 1222 (arrow), which represents an insertion of duplicated nucleotides (boxed sequence) from 27 base pairs earlier (1222ins27). (D) Pedigree of family B, which shows proband (II-3, marked by arrow), affected family members, and unaffected family members. The mutation 1222ins27 created a PCR product that consisted of a normal product and a slightly longer mutant product. These products were visualised on a polyacrylamide gel at 109 bp (normal product, bottom band), at 136 bp (mutant product, middle band), and a heteroduplex of the two products (top band). Affected family members produced the same gel pattern. Unaffected family members and 50 control individuals produced a single band, and were screened in this manner. (E) Nucleotide sequence data of family B mutant K12 exon 6, with duplication of sequence first seen at position 1222. Normal sequences indicated in black, mutant sequences in red. Regions of duplication are in capital letters. Insertions of duplications into mutant sequences are boxed. (F) Amino acid sequences. Duplication of sequence first seen at codon position 400.