A Novel Mutation in the M1S1 Gene Responsible for Gelatinous Droplike Corneal Dystrophy

Abstract

purpose. To identify the genetic defect in the M1S1 gene causing gelatinous droplike corneal dystrophy (GDLD) in an Estonian family.

methods. DNA was extracted from members of a GDLD-affected family and control persons. Polymerase chain reaction followed by direct sequencing was used to detect mutations in the M1S1 gene. Sequencing results were confirmed with restriction analysis.

results. Sequencing of the M1S1 gene revealed a novel mutation and a common polymorphism. All patients with GDLD were found to be homozygous for the insertion of nucleotide C in position 520 in M1S1. The mutation leads to formation of truncated protein. The mutation was excluded in 103 normal, unaffected individuals. Very close to the location where the mutation was identified in the M1S1 gene, a single-nucleotide polymorphism (518A/C) was found, changing aspartic acid to alanine at codon 173.

conclusions. The data indicate that mutation ins520C in the M1S1 gene is the primary cause of GDLD in the family studied.

Gelatinous droplike corneal dystrophy (GDLD; On-line Mendelian Inheritance in Man number 204870), first described by Nakaizumi, ¹ is a form of primary amyloidosis of the cornea leading to blindness. Clinical manifestations usually appear in the first decade of life, and symptoms include foreign-body sensation, photophobia, lacrimation, and blurred vision. In later stages of the disease, accumulation of gelatinous masses in subepithelium and anterior stroma causes loss of vision, and in most cases recurrent lamellar keratoplasty is required. ² GDLD is an autosomal recessive disorder with highest frequency in the Japanese population (incidence 1:300,000). ^{2 3} Recently, a gene responsible for GDLD was localized to a 2.6-centimorgan (cM) interval on chromosome 1p by linkage analysis. ⁴ Focusing further studies on the critical 1p region, M1S1 was identified as a gene that was mutated in all patients with GDLD. ⁵  

The M1S1 gene consists of a single exon that encodes monoclonal antibody–defined, gastrointestinal tumor-associated antigen, formerly known as TROP2 and GA733-1. ⁶ The M1S1 protein is a monomeric cell surface glycoprotein expressed in the cornea, multistratified epithelia, and trophoblasts and at high levels in most carcinomas. ^{5 7 8} The physiological function of M1S1 is not well understood, but it has been suggested that the protein can act as a calcium signal transducer. ⁸ Four mutations causing GDLD were found in the M1S1 gene: (1) Q118X, a C→T transition replacing a glutamine at codon 118 with a stop codon; (2) Q207X, a C→T substitution replacing a glutamine at codon 207 with a stop codon; (3) S170X, a C→A transition changing from serine to a stop at codon 170; and (4) 632delA, a deletion of A at nucleotide 632. ⁵ All four mutations generate an early stop codon and lead to synthesis of a truncated protein. Using protein expression analysis, perinuclear aggregation of the mutated, truncated protein has been revealed, whereas the normal protein was distributed diffusely in the cytoplasm with a homogenous or fine granular pattern. ⁵  

To date, mutational analysis of GDLD has been performed only in the Japanese population. Herein, we report a novel mutation of GDLD found in a white family. A common polymorphism in the M1S1 gene was also found. 

Materials and Methods

Patients and Control Subjects

An Estonian pedigree with three patients with GDLD was examined. Diagnosis of GDLD was based on characteristic clinical appearance and was confirmed by histopathologic and electron microscopy findings after keratoplasty. The study group consisted of 18 members of the pedigree and 103 unrelated control individuals. The tenets of the Declaration of Helsinki were followed. Informed consent was obtained, and the protocol for human experimentation was approved by the Ethics Review Committee on Human Research, University of Tartu. 

Molecular Analysis

Genomic DNA was extracted from peripheral blood by standard procedures. A 681-bp DNA fragment of the M1S1 gene (nucleotides [nt] 103-783) was amplified using primers GDLD3F and GDLD5R. ⁵ The products were sequenced directly with a kit (BigDye Terminator Cycle Sequencing Kit; PE-Applied Biosystems, Foster City, CA) using the primers that were used for PCR. Sequencing reactions were analyzed on a gene analyzer (Prism 310; PE-Applied Biosystems). All products were sequenced on both strands. 

The presence of a mutation and a polymorphism found by sequencing was confirmed using restriction analysis. DNA segments of M1S1, generated by PCR using the primers described, were digested with three restriction enzymes—Eco47I, HinII, and CfrI (all from Fermentas AB, Vilnius, Lithuania)—and subjected to polyacrylamide gel electrophoresis. 

Results

Direct sequencing revealed a novel GDLD mutation and a common polymorphism in the M1S1 gene. An insertion of C at nucleotide 520 (520insC) was found in the family with GDLD, which resulted in completely different amino acid sequences from codon 174 to a premature stop codon at 216 (Fig. 1) . All patients with GDLD were homozygous for the mutation (Fig. 2) . Analysis of DNA from other family members revealed that the mother of the affected individuals was heterozygous for the mutation; the father’s DNA was not available. All the patients’ descendants were found to be homozygous for insertion of C at nucleotide 520. Among the 103 healthy people tested in the mutation screening, no carriers of the 520insC mutation were detected. 

A common single nucleotide polymorphism (518A/C) was found in the M1S1 gene, very close to the position where the mutation was discovered. Nucleotide substitution A→C at position 518 of the M1S1 gene changes codon 173 from GAC to GCC, which replaces aspartic acid with alanine (Fig. 1) . 

Restriction enzyme analysis in all members of the GDLD-affected family and 103 unrelated control individuals confirmed the results obtained by direct sequencing. Because the mutation and polymorphism were very close to each other, three enzymes (Eco47I, HinII, and CfrI) with overlapping restriction sites were used. The 520insC mutation in the M1S1 gene created a restriction site for enzyme Eco47I in those individuals who had A at the position of 518. Restriction endonuclease HinII was specific to wild-type allele with A at position 518. For CfrI an additional restriction site was present in DNA fragments amplified from individuals with the wild-type allele and nucleotide C at position 518 (Fig. 3) . 

Genotype frequencies for 518A/C polymorphism in the M1S1 gene were as follows: 81.5% of individuals from the control population were A/A homozygotes, 17.5% were A/C heterozygotes, and 1.0% were C/C homozygotes. All members of the family with GDLD who were available for molecular analysis had the 518A/A genotype. 

Discussion

This is the first report of a mutation analysis performed in white patients with GDLD. All patients with GDLD in Estonia were found to be homozygous for 520insC in the M1S1 gene and the mutation was well cosegregated with the phenotype in the GDLD pedigree, whereas no M1S1 mutations were found in the control population. Thus, the data suggest that ins502C in the M1S1 gene is responsible for GDLD in Estonia. The mutation found in Estonian patients has not been described in the Japanese population, where the disease has the highest frequency, and relatively many patients have been genotyped. It can be speculated that the mutation may also be found in other patients with GDLD among European descendants, because it was found in a family from Dago Island (off the west coast of Estonia), whose settlement had close connections with other countries around the Baltic Sea in the Middle Ages. 

M1S1 is a cell surface phosphoglycoprotein and a substrate for protein kinase C, the Ca²⁺-dependent protein kinase. The phosphorylation occurs on serine 303 in the cytoplasmic domain of the protein. ⁹ M1S1 transduces an intracellular calcium signal, and it has been hypothesized that M1S1 may function as a cell surface receptor, for which the physiological ligand has not been identified. ⁸ The carboxyl-terminus of M1S1 possesses a phosphatidylinositol 4,5-bis phosphate (PIP-2)–binding consensus sequence, which regulates binding to plasma membrane or to other cytosolic proteins. The mutation detected by us, as well as other mutations described so far, leads to formation of truncated protein, resulting in loss of the transmembrane domain of M1S1, the serine phosphorylation site, and the PIP2-binding site. ⁵ The truncated gene product triggers process of amyloid formation in the cornea. The exact mechanism of amyloid deposition remains to be investigated. Although M1S1 is expressed at high levels by human multistratified epithelia, no amyloid formation in tissues other than cornea has been found in patients with GDLD. 

A single-nucleotide polymorphism, 518A/C, in the M1S1 gene changes amino acid residue encoded by codon 173 from aspartic acid to alanine. Determining whether the change has any effect on biological properties of the protein is the object of our further studies. The close position of the 518A/C polymorphism and the ins502C mutation in the M1S1 gene must be considered when designing the molecular analysis method for mutation detection. 

 Supported by Estonian Science Foundation Research Grant 4349.

 Submitted for publication March 29, 2001; revised July 16, 2001; accepted July 26, 2001.

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Figure 1.

 

Part of the sequence of the M1S1 gene. Sequences of (A) M1S1 obtained from a patient with GDLD who had an insertion of C at position of 520 of the gene. Nucleotide A is seen in the polymorphic locus of the M1S1 gene at position 518; (B) a wild-type allele from a control subject with nucleotide A in the polymorphic site of the M1S1 gene; and (C) a wild-type allele from a control subject with nucleotide C at the polymorphic site of the gene.

Figure 2.

 

Pedigree of GDLD-affected family. All genotyped patients with the disease were homozygous for the mutation ins520C (M/M). All carriers having the ins520C in only one chromosome (wt/M) had no clinical signs of GDLD. Genotypes for persons found to have wild-type alleles in both chromosomes are designated wt/wt.

Figure 3.

 

Restriction analysis of the M1S1 gene. (A) Amplified M1S1 gene digested with Eco47I. The mutation ins520C creates a recognition sequence for Eco47I, if there is a nucleotide A in the position of 518, amplified DNA (681 bp) is cut into 416-bp and 266-bp fragments by the restriction enzyme. Lane 1: homozygote for the haplotype 520insC/518A; lane 2: haplotype 520insC/518A in one chromosome; lane 3: haplotype 520insC/518A absent. (B) Amplified fragments of the M1S1 gene digested with Hin1I. Wild-type allele with 518A is cut by the enzyme. A DNA fragment of 264 bp indicates the presence of haplotype wt/518A. Lane 1: homozygote for the haplotype wt/518A; lane 2: haplotype wt/518A present in one chromosome; lane 3: haplotype wt/518A absent. (C) Amplified fragments of the M1S1 gene digested with CfrI. The presence of a 244-bp DNA fragment after digestion with CfrI allows identification of the haplotype wt/518C. Lane 1: homozygote for the haplotype wt/518C; lane 2: haplotype wt/518C present in one chromosome; lane 3: haplotype wt/518C absent.