AIM To study a kindred with Meesmann's corneal dystrophy (MCD) to determine if a mutation within the cornea specific K3 or K12 genes is responsible for the disease phenotype.
METHODS Slit lamp examination of the cornea in four members of the kindred was carried out to confirm the diagnosis of MCD. The region encoding the helix initiation motif (HIM) of the K12 polypeptide was polymerase chain reaction (PCR) amplified from genomic DNA derived from affected individuals in the kindred. PCR products generated were subjected to direct automated sequencing. Restriction enzyme analysis employingBan I was used to confirm the presence of the mutation in affected individuals of the family.
RESULTS Sequencing of the K12 gene in an affected individual from the family revealed a novel heterozygous missense mutation (413A→C), predicting the substitution of a proline for a glutamine at codon 130 (Q130P) in the HIM of the K12 protein. The mutation was excluded from 50 normal, unaffected individuals by restriction enyzme analysis and was therefore unlikely to be a common polymorphism.
CONCLUSION A novel missense mutation in the K12 gene leads to MCD in a German kindred. Missense mutations have now been identified within the region encoding the helix initiation motif of the K12 protein in eight of 11 MCD kindreds analysed at the molecular level.
- Meesmann's corneal dystrophy
- dominant negative mutation
- corneal epithelium
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Meesmann's corneal dystrophy (MCD) (OMIM 122100) is an autosomal dominantly inherited disorder affecting the corneal epithelium of both eyes.1-3 It manifests in early childhood and is characterised by myriads of intraepithelial microcysts of variable distribution and density. The microcysts are best seen by retroillumination and slit lamp examination.4 5 Further clinical signs of the disease are recurrent punctate erosions with lacrimation and photophobia. Symptoms include blurred vision and foreign body sensation. Histologically, the corneal epithelium appears irregularly thickened and contains numerous vacuolated suprabasal cells.6-8 In addition, there is intraepithelial cyst formation with accumulated periodic acid Schiff (PAS) positive material. By electron microscopy, epithelial cells have been shown to contain fibrogranular material often referred to as “peculiar substance”.6 7 9
Keratins K3 and K12 are specifically expressed within all cell layers of the adult central corneal epithelium10-13 and are regarded as markers of advanced corneal epithelial differentiation.14 15 K5 and K14 are also expressed within the corneal epithelium, though to a lesser extent than either K3 or K12.15 16 Comparative studies of autosomal dominantly inherited blistering disorders of human epidermis and other epithelial tissues have provided ample evidence that pathogenic keratin gene mutations exert dominant negative effects on keratin filament assembly which result in intracellular keratin aggregation.17-19In vitro biochemical assays20 and protein expression experiments in cultured cells21 have shown that mutations in keratin rod domains exert powerful dominant negative effects. Lack of a functional keratin cytoskeleton appears to critically reduce the mechanical resilience of the affected epithelial cells and tissues.22
Mice have been genetically engineered in which the K12 gene has been ablated.16 The corneal epithelium of these mice is very fragile and easily removed by wiping the surface of the eye with a microsponge.16 No mutations within the K3 or K12 genes have yet been found to underlie a recessive corneal epithelial disease in humans. However, this research group recently found the first dominant negative mutations within the K3 and K12 genes in MCD patients.4 Further heterozygous missense mutations in the K12 gene have since been reported.5 23 24
Here we investigated a southern German family with MCD and detected a novel mutation within the region encoding the highly conserved helix initiation motif of the K12 protein. In addition we review the mutations published in the K3 and K12 genes to date.
Materials and methods
We investigated a four generation family with MCD from southern Germany. The family history indicated that seven of 18 living family members were affected. The pedigree was consistent with an autosomal dominant mode of inheritance (Fig 1). A full ophthalmological check up including slit lamp examination was performed in four affected family members. Three of them showed typical signs of MCD. Their corneal surfaces were studded with numerous intraepithelial microcysts, as shown in Figure 2. Irregularly shaped areas devoid of microcysts were noted in all affected corneas (Fig 2b). One of the patients (II-1) showed additional changes in the form of curved grey lines (not shown). All of the patients complained about foreign body sensation and recurrent painful erosions accompanied by photophobia, lacrimation, and blurred vision. Signs of MCD were noted in early childhood and tended to progress with age of patient. No further ocular abnormalities were noted.
SEQUENCING THE HELIX INITIATION MOTIF OF THE K12 GENE
Most of exon 1 of the K12 gene, incorporating the HIM of the K12 protein, was amplified from the proband and an unaffected individual in the family using the primers K12e1.L (5′ ATG GAT CTC TCC AAC AAC ACC ATG 3′) and K12e1.R (5′ GTA ATC GCT CTG TGA AGC ATC TGC 3′). “Touchdown” polymerase chain reaction (PCR) conditions were used, consisting of: 94°C for 5 minutes for 1 cycle; 94°C for 30 seconds; 65/63/61°C for 45 seconds; 72°C for 45 seconds for two cycles at each annealing temperature; 94°C for 30 seconds; 58°C for 45 seconds; 72°C for 45 seconds for 30 cycles; and 72°C for 5 minutes for one cycle. PCR products were purified using QIAquick columns (Qiagen, Chatsworth, USA) and sequenced with both forward and reverse primers, using the ABI PRISM Ready Reaction System (Perkin-Elmer, Foster City, CA, USA). Sequencing ladders were analysed on an ABI 377 automated sequencer.
Mutation 413A→C did not alter any known restriction enzyme site and so a mismatch primer was designed which in conjunction with the mutation, generated a Ban I site. PCR amplification was carried out using the mismatch primer K12M3 (5′ CTG GAT CAG AAA AAG AAA CGG TGC 3′) (mismatches underlined), with K12e1.R (as above), in Promega PCR buffer supplemented with 1 mM magnesium chloride and the following programme: 94°C for 5 minutes for one cycle; 94°C for 30 seconds, 50°C for 30 seconds, 72°C for 1 minute for 35 cycles; and 72°C for 5 minutes for one cycle. Resultant PCR products were each digested overnight with 4 U ofBan I enzyme. An additional band was generated in members of the family carrying the mutation (Fig 3C). Fifty normal unrelated individuals were also screened using thisBan I restriction enzyme digest for presence of the mutation (data not shown).
Sequencing of the region encoding the helix initiation motif of the K12 gene in an affected individual in the family revealed a heterozygous nucleotide substitution 413A→C (Fig 3A). This is predicted to substitute a proline residue for glutamine at codon 130 (Q130P). Sequencing in the reverse direction confirmed the presence of the heterozygous missense mutation in an affected individual (not shown). DNA from an unaffected individual from the family was also sequenced and did not contain the 413A→C nucleotide substitution (Fig3B). The mutation did not alter any known restriction enzyme site and so a mismatch primer was designed in order to generate a novel restriction site upon amplification of the mutant allele. This restriction fragment analysis showed that all affected individuals within the kindred harboured the mutation, whereas the unaffected individuals did not (Fig 3C). This test was used to exclude the mutation from 50 normal unrelated people (data not shown).
Meesmann's corneal dystrophy is an autosomal dominant disorder of the corneal epithelium. Its clinical and morphological characteristics were first reported in a family from northern Germany more than 60 years ago.1-3 Previously, we demonstrated genetic linkage to the type I keratin locus on chromosome 17q and the first K12 mutation,4 in the descendants of the original Meesmann kindred.2 25 Simultaneously, we described linkage to the type II keratin cluster on chromosome 12q and the first mutation in K3 in an Irish MCD family.4
Here, we have studied an MCD kindred from southern Germany and identified a novel heterozygous mutation within the region encoding the helix initiation motif of the K12 protein, Q130P. The 10 MCD mutations published to date are shown in Table 1 and the positions of these mutations in the K3 and K12 proteins is shown schematically in Figure4. From the small number of mutations reported to date, the 1A domain of K12, where the mutation reported here is located, is emerging as a hot spot for MCD mutations.
The helix initiation and termination motifs are highly conserved regions of the keratin proteins and have been found to have an important role in normal filament assembly.26-29 Numerous mutations have been found within these regions underlying a wide range of epithelial diseases.17-19 From Table 1, it can be seen that the majority of published mutations involve substitution of an amino acid of a different group to the one which is present within the normal polypeptide. For example, the mutation E509K4involves the substitution of a basic amino acid (lysine) for an acidic one (glutamic acid).
The novel Q130P mutation described in this paper involves the substitution of a polar amino acid of molecular weight 146 Da (glutamine) by an apolar one of 115 Da (proline). Proline substitutions are especially disruptive to α helical tertiary structures in proteins.30 This is because the nitrogen atom in proline which is involved in peptide bond formation is contained within a ring structure, which is not the case for other amino acids. In the case of intermediate filament proteins, proline mutations within the conserved ends of the α helical rod domain have been shown to have more dramatic effects on filament structure and assembly in cultured cells than similar mutations contained more centrally within the rod domain.31 This is the second instance of a proline substitution at the fifth residue of the α helical 1A region within a type I keratin. The substitution of a proline for a glutamine residue at codon five of the 1A domain (Q122P) has been previously reported in the K16 gene in a case of pachyonychia congenita.32
We would like to thank the patients and their family for participation in this study. Thanks also to Carrie M Coleman, Epithelial Genetics Group, for genomic DNA extraction and Andrew J Cassidy, Molecular Genetics Analysis Facility, Department of Molecular and Cellular Pathology, Ninewells Medical School, Dundee, for DNA sequencing. WHIM and LDC are funded by a Wellcome Trust senior research fellowship (to WHIM) and this work was also supported by the Dystrophic Epidermolysis Bullosa Research Association (DEBRA) UK (to WHIM).
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