|Molecular Vision 2006;
Received 25 April 2005 | Accepted 13 April 2006 | Published 9 May 2006
Screening of the MERTK gene for mutations in Japanese patients with autosomal recessive retinitis pigmentosa
Toshitaka Itabashi, Miyuki Kawamura,
Makoto Tamai, Kohji Nishida
Department of Ophthalmology, Tohoku University School of Medicine, Sendai, Japan
Correspondence to: Yuko Wada, MD, Department of Ophthalmology, Tohoku
University School of Medicine, 1-1, Seiryo-machi, Aoba-ku, Sendai
980-77, Japan; Phone: 81-22-717-7294; FAX: 81-22-717-7298; emaill:
Dr. Tamai is now at the Department of Biofunctional Science, Tohoku University Biomedical Engineering Research Organization
Purpose: To determine whether mutations in the MERTK gene are present in Japanese patients with autosomal recessive retinitis pigmentosa (arRP).
Methods: The coding sequence of all 19 exons and the adjacent flanking intron sequences of the MERTK gene were directly sequenced in 96 unrelated Japanese patients with arRP.
Results: Seventeen sequence variants were found; six missense changes, three isocoding changes, and eight intron changes were also observed. One arRP patient had a novel homozygous Leu12Pro missense mutation in the MERTK gene.
Conclusions: Mutations in the MERTK gene are relatively rare in Japanese patients with arRP.
Royal College of Surgeons (RCS) rats have been extensively used as an animal model of a recessively inherited retinal degeneration. RCS rats have a genetic defect that leads to phagocytosis of photoreceptor outer segments by the retinal pigment epithelial cells that then results in retinal degeneration. In 2000, positional cloning showed that RCS rats had a deletion of the MERTK gene, which encodes a receptor tyrosine kinase . Subsequently, patients with retinal dystrophies were screened for mutations in the human ortholog of the MERTK gene, and three disease-causing mutations and 11 sequence variants were detected .
To date, there have been only three published investigations of mutational screening of the MERTK gene on patients with inherited retinal degeneration [2-4]. Interestingly, mutations in the MERTK gene have not been reported in Japanese patients with autosomal recessive retinitis pigmentosa (arRP).
The purpose of this study was to determine whether mutations in the MERTK gene were present in Japanese patients with arRP. To accomplish this, we screened 96 patients who were diagnosed to have arRP clinical findings.
After informing the patients on the purpose and procedures to be performed, a signed consent form was obtained from all patients.
Ninety-six DNA samples from 96 unrelated Japanese families with arRP were screened for mutations in the MERTK gene. All patients were previously evaluated for mutations in the arrestin, ABCA4, NR2ES, LRAT, TULP1, and RPE65 genes. Genomic DNA was isolated from leukocytes prepared from each patient's blood (10-15 ml) using the Gene Ball Genome Preparation Kit (TaKaRa, Kyoto, Japan). Patients were diagnosed with retinitis pigmentosa (RP) by clinical findings and electroretinographic characteristics. Patients with arRP were from families with several affected siblings and had no other family members with RP in the preceding generation, or were the affected offspring of a consanguineous mating of parents who had no history of RP. We also screened 200 chromosomes from 100 normal subjects for mutations of the MERTK gene.
Exons of the MERTK gene (exons 1-19) were individually amplified using polymerase chain reaction (PCR). For the screenings, 20 sets of primer pairs were prepared. Table 1 presents the primers used in amplification. For other exons, we used the same primers reported by Vollrath and colleagues . PCR products were directly sequenced (Model 3100; Applied Biosystems, Foster City, CA). The numbering of the bases of the cDNA sequence used was in accordance to the GenBank accession number NM_006343.
There were 17 sequence variants found by direct sequencing (GeneBank accession numbers, NM_006343; Table 2, Figure 1). Eleven of the 17 sequence variants (IVS1-18G->A, IVS2+68ins TTTATTTATTTA, IVS3+10C->T, IVS4+13T->C, IVS4+51G->T, IVS14-42A->G, IVS15-11C->A, IVS18-143T->C, Pro252Pro, Asn498Asn, and Ser627Ser) were isocoding or intronic changes. Thus, none of these changes affected the amino acid sequence of the MERTK protein. The intronic changes did not create or destroy any splice donor or acceptor sites with splice-site prediction software (NNSPLICE). These changes, except for IVS15-11C->A, were also detected in normal controls (Table 2).
Six missense changes were identified in this study; Leu12Pro, Cys115Gly, Asn118Ser, Arg466Lys, Ile518Val, and Arg909 His altered the amino acid sequence. Among these missense changes, Asn118Ser, Arg466Lys, and Ile518Val were detected with higher frequencies in patients with arRP, and with almost the same allele frequencies in normal controls (Table 2); we therefore considered that these changes to most likely be nonpathogenic.
Cys115Gly (TGC to GGC; c.480T->G) and Arg909His (CGC to CAC; c.2863G->A) were rare and were not observed in normal controls. Patients with a heterozygous Cys115Gly or Arg909His mutation did not have a second mutation in the MERTK gene. One patient with a homozygous Leu12Pro mutation was the child of a consanguineous union. Unfortunately his parents and siblings were deceased but were reported to be unaffected. We were not able to perform segregation analysis in this family. The Leu12Pro of the MERTK gene were not seen in 100 normal controls.
To date, only five pathogenic mutations in the MERTK gene have been reported in patients with arRP [2-4]. The purpose of this study was to determine the frequency and kinds of mutations of the MERTK gene in Japanese patients with arRP. We found 17 sequence variants, which included six missense changes, three isocoding changes, and eight intron changes in the MERTK gene. The Asn118Ser, Arg466Lys, and Ile518Val variants are the same variants that were reported by Gal et al. . The two rare sequence variants, Cys115Gly and Arg909His were not detected in 100 normal controls. The Cys115Gly mutation is inside the Ig-like domain and is conserved in humans, mice, and rats, which would suggest the possibility that it is a pathogenic mutation. The Arg909His mutation was of uncertain pathogenicity because it was outside the domain and not conserved (Figure 2). Alternatively, these two mutations may affect other than kinase function of the receptor. In this study, we were not able to find a second mutation in the MERTK gene for two patients associated with the Cys115Gly or Arg909His mutation.
We also considered two possibilities for the homozygous Leu12Pro mutation in the MERTK gene. First, although we could not perform the segregation analysis, a homozygous Leu12Pro mutation was found in one member of a consanguineous marriage. It was not detected in 100 normal controls, and was conserved in humans, mice, and rats (Figure 2). This would suggest that this mutation may be a pathogenic mutation in the MERTK gene. Second, the MERTK gene has 19 coding exons, and encodes four functional domains, such as immunoglobulin-like, fibronectin-like, transmembrane-like, and tyrosine kinase-like domains. All reported pathogenic mutations (Arg651X, 2070del AGGAC, Arg722X, and Arg844Cys) are inside the tyrosine kinase-like domain, and the IVS10-2A->G is at the splice acceptor site [2-4]. On the other hand, the Leu12Pro mutation is outside these domains. These results suggest that this mutation might be a rare sequence variant in Japanese patients with arRP.
Previous reports on mutational screenings for inherited retinal degeneration disclosed that the kinds and frequency of mutations were different in ethnic populations [5-7]. However, the pathogenic mutation in the MERTK gene appeared to be rare for patients in both Japanese and other ethnic populations.
In conclusion, our results provide evidence that mutations in the MERTK gene are relatively rare in a group of 96 Japanese arRP patients screened.
This study was supported in part by a grant from the Research Committee on Chorioretinal Degenerations and Optic Atrophy, the Ministry of Health, Labour and Welfare of the Japanese Government (TI), Tokyo, Japan, and a Grant-In-Aid for Scientific Research from the Ministry of Education, Science, and Culture of the Japanese Government (YW; A-14704044), Tokyo, Japan.
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