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Recurrent Missense (R197C) and Nonsense (Y89X) Mutations in the XLRS1 Gene in Families with X-Linked Retinoschisis
Biochemical and Biophysical Research Communications 256, 317–319 (1999) Article ID bbrc.1999.0323, available online at http://www.idealibrary.com on
Recurrent Missense (R197C) and Nonsense (Y89X) Mutations in the XLRS1 Gene in Families with X-Linked Retinoschisis Barkur S. Shastry,* ,1 Fielding J. Hejtmancik,† and Michael T. Trese‡ *Eye Research Institute, Oakland University, Rochester, Michigan 48309-4410; †National Eye Institute, Bethesda, Maryland 20892; and ‡Department of Ophthalmology, William Beaumont Hospital, Royal Oak, Michigan 48073
Received January 12, 1999
Congenital retinoschisis (RS) is a hereditary eye disorder characterized by intraretinal schisis and central and peripheral retinal lesion. The gene responsible for the X-linked retinoschisis (XLRS1) has recently been isolated and found to contain mutations in affected members of several families. In this communication, two families with X-linked RS were analyzed for possible disease-causing mutations by polymerase chain reaction amplification of exons followed by DNA sequencing. Our analyses reveal a missense mutation at codon 197 in exon 6 and a nonsense mutation in exon-4 of XLRS1 gene. These changes resulted in the replacement of a highly conserved arginine by a cysteine residue and introduced a premature termination signal at codon 89, respectively. These mutations, which are transmitted through three generations, cosegregated with the disease, and are not found in the unaffected family members and 150 normal X-chromosomes, are likely to be pathogenic in these families. © 1999 Academic Press
X-linked juvenile retinoschisis (RS) or congenital retinoschisis is an inherited congenital vitreoretinal disorder, resulting in poor, uncorrectable visual acuity. It is a bilateral progressive disease, having a variable clinical expression with complete penetrance (1). The disorder is characterized by intraretinal schisis, central (cart-wheel) and peripheral retinal lesions (2). In the majority of cases, however, the foveal schisis is reported to be the most common abnormality. According to a new classification, there are three types of retinoschisis: hereditary, degenerative and secondary (3). Among the he1 To whom correspondence should be addressed. Fax: 248-3702006.
reditary types, the more frequent form is the X-linked juvenile. In the X-linked families, the affected males show mild visual impairment until the 4th or 5th decade of life. The disease progresses thereafter, and in rare cases, complete blindness occurs from retinal detachment. The female carriers have normal vision, with no abnormalities in the retina and cannot be identified clinically. The cause of RS pathology is not known, but histopathological and electroretinographic studies suggested defects in retinal Muller cell function in the development of the disease (2). A large number of genetic linkage studies (4 –7) with microsatellite markers have localized the RS gene to the short arm of the X-chromosome. No locus heterogeneity has been reported among different sets of families (6). The gene responsible for RS has recently been isolated (8), and is found to be exclusively expressed in the retina. The mature protein shows sequence homology to discoidin-1, which is implicated in cell-cell interaction. Mutational analysis of RS families implied that the mutated gene can cause RS pathology. In this communication, we describe a missense and a nonsense mutation in two families segregating for X-linked RS. PATIENTS AND METHODS All available family members (affected and unaffected) underwent a complete ophthalmological evaluation at William Beaumont Hospital in Royal Oak, Michigan. The study was approved by the Institutional Review Board of Oakland University. All patients were informed of the purpose of the study and written consent were obtained. In the pedigrees (Fig. 1A and 2A) there are no male to male transmission; all affected individuals are males. The males are related to each other through unaffected females and the transmission is from affected man to carrier daughter, to affected grandsons, consistent with an X-linked pattern of inheritance. The fundus examination of all affected individuals showed foveal and peripheral schisis which are the characteristic features of RS.
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0006-291X/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.
Vol. 256, No. 2, 1999
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
RESULTS AND DISCUSSION The RS gene (XLRS1) contains six exons. The availability of oligonucleotide primers (8) permitted us to screen the RS family whose linkage has already been established (6). The sequencing of the XLRS1 gene (Fig. 1B) in the family (Family 1) identified a missense mutation in exon-6 at codon 197 (R197C) in all affected individuals, but not in unaffected males and 40 normal unrelated, randomly selected controls (60 normal X-chromosomes from our own laboratory and 90 con-
FIG. 1. (A) Pedigree of an RS family showing unaffected (open symbols) and affected (closed symbols) family members. Slashed symbols denote deceased individuals. Individuals examined are indicated by the X mark on the vertical and horizontal connecting bars. (B) Nucleotide sequence of the mutant part of exon-6 of the XLRS1 gene. The nucleotide sequence change in the patient is C to T, which results in the amino acid cysteine (TGC) instead of the normal arginine (CGC). The same mutation was segregated in all affected individuals.
We collected venous blood from several affected and unaffected individuals and the genomic DNA was extracted as described before (9). All exons of the RS gene were amplified by the polymerase chain reaction (PCR) using commercially synthesized primers, as reported by others (8). The PCR was carried out by using Applied Biosystems Taq DNA polymerase with 30 cycles of 1.5 min at 94°C, 1 min at 60°C, 2 min at 72°C in the manufacturer’s buffer containing 2– 4 units polymerase, 10pmole each of primer, 50mM each of the four deoxynucleotides, 1.5mM MgCl 2 and 10mM tris-HCl pH 8.3. The amplified products were cloned into a pT7 blue vector and sequenced with fluorescent primers using applied Biosystems Data analysis software, following the manufacturer’s protocol.
FIG. 2. (A) Pedigree of a second RS family analyzed in this study. (B) Nucleotide sequence of the mutant part of exon 4 of the XLRS1 gene. The nucleotide sequence change in the patient is T to A, which results in the termination codon (TAA) instead of the normal tyrosine residue (TAT). The rest of the symbols are the same as those in Fig. 1.
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Vol. 256, No. 2, 1999
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
trol chromosomes from the other laboratory). This mutation (C3 T) changed the encoded and highly conserved arginine residue to cysteine (based on the sequence change). Sequencing of exon-4 in family 2 (Fig. 2A and B) identified a T to A transversion in codon 89 resulting in a stop codon Y89X. This mutation is present in the second affected individual (IV-2) as well as in the heterozygous mother (III-3), but not in unaffected individuals. The corresponding mutant protein, if expressed, should result in a severely truncated polypeptide with 89 amino acids, rather than the normal 224. Although the role of the XLRS1 has not been clearly established, it is possible that the truncated protein is non-functional. It is also likely that no mutant protein would be expressed, since a premature termination codon has been reported to cause unstable, short-lived mRNA or reduction in the level of mRNA (10,11). Since the mutations are transmitted through three generations, segregate with the disease and are in the conserved motif of the protein, it is likely that these mutations are pathogenic in the families. Although the mothers (IV-4 in Family 1 and III-3 in Family 2), carried this mutation in one of their alleles, they had no visual impairment when examined. This not only confirms the recessive nature of the disease, but also suggests that 50% of the normal RS gene product is sufficient for the retinal structure and function. Although the effect of the structural alteration on the functioning of the protein is not understood, the amino aid change in the phylogenetically conserved carboxy terminus may result in an aberrant function, which may be the cause of RS in Family 1. The above sequence alterations have also been recently reported in other families (12), but at present, no genotype/ phenotype correlations can be made among these pa-
tients. In vitro studies on this mutant gene may shed some light on the molecular basis of this disorder. ACKNOWLEDGMENTS We thank the participants who kindly donated blood samples for the study. Our sincere thanks also to Dr. Frank J. Giblin of Oakland University for reading the manuscript. This work was supported in part by a grant from the Retinopathy of Prematurity Foundation (ROPARD) and a core grant for vision research from the NEI (EY05230).
REFERENCES 1. Forsius, H., Krause, U., and Helve, J. (1973) Can. J. Ophthalmol. 8, 385–393. 2. George, N. D. L., Yates, J. R. W., and Moore, A. T. (1996) Arch. Ophthalmol. 114, 274 –280. 3. Madjaru, B., Hilton, G. F., Brinton, D. A., and Lec, S. S. (1995) Retina 15, 282–285. 4. George, N. D. L., Payne, S. J., Bill, R. M., Barton, D. E., Moore, A. T., and Yates, J. R. W. (1996) J. Med. Genet. 33, 919 –922. 5. Pawar, H., Bingham, E. L., Lunetta, K. L., Segal, M., Richards, J. E., Bochnke, M., and Sieving, P. A. (1995) Hum. Hered. 45, 206 –210. 6. Shastry, B. S., Hejtmancik, F. J., Margherio, R. T., and Trese, M. T. (1996) Biochem. Biophys. Res. Comm. 220, 824 – 827. 7. Shastry, B. S., Hejtmancik, F. J., Rodriguez, A., Rodriguez, F., and Tamayo, M. L. (1997) J. Med. Genet. 34, 504 –506. 8. Sauer, C. G., Gehring, A., Warneke-Wittstock, R., Marquardt, A., Ewing, C. C., Gibson, A., Lorenz, B., Jurklies, B., and Weber, B. H. F. (1997) Nat. Genet. 17, 164 –170. 9. Bell, G. I., Karam, J. H., and Rutter, W. J. (1981) Proc. Nat. Acad. Sci. USA 78, 5759 –5763. 10. Kanniburgh, A. J., Maquat, L. E., Schedl, T., et al. (1982) Nucl. Acids Res. 10, 5421–5427. 11. Beserga, S. J., and Benz, E. J. (1988) Proc. Nat. Acad. Sci. USA 85, 2056 –2060. 12. The retinoschisis consortium (1998) Hum. Mol. Genet. 7, 1185– 1192.
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Recurrent Missense (R197C) and Nonsense (Y89X) Mutations in the XLRS1 Gene in Families with X-Linked Retinoschisis Barkur S. Shastry,* ,1 Fielding J. Hejtmancik,† and Michael T. Trese‡ *Eye Research Institute, Oakland University, Rochester, Michigan 48309-4410; †National Eye Institute, Bethesda, Maryland 20892; and ‡Department of Ophthalmology, William Beaumont Hospital, Royal Oak, Michigan 48073
Received January 12, 1999
Congenital retinoschisis (RS) is a hereditary eye disorder characterized by intraretinal schisis and central and peripheral retinal lesion. The gene responsible for the X-linked retinoschisis (XLRS1) has recently been isolated and found to contain mutations in affected members of several families. In this communication, two families with X-linked RS were analyzed for possible disease-causing mutations by polymerase chain reaction amplification of exons followed by DNA sequencing. Our analyses reveal a missense mutation at codon 197 in exon 6 and a nonsense mutation in exon-4 of XLRS1 gene. These changes resulted in the replacement of a highly conserved arginine by a cysteine residue and introduced a premature termination signal at codon 89, respectively. These mutations, which are transmitted through three generations, cosegregated with the disease, and are not found in the unaffected family members and 150 normal X-chromosomes, are likely to be pathogenic in these families. © 1999 Academic Press
X-linked juvenile retinoschisis (RS) or congenital retinoschisis is an inherited congenital vitreoretinal disorder, resulting in poor, uncorrectable visual acuity. It is a bilateral progressive disease, having a variable clinical expression with complete penetrance (1). The disorder is characterized by intraretinal schisis, central (cart-wheel) and peripheral retinal lesions (2). In the majority of cases, however, the foveal schisis is reported to be the most common abnormality. According to a new classification, there are three types of retinoschisis: hereditary, degenerative and secondary (3). Among the he1 To whom correspondence should be addressed. Fax: 248-3702006.
reditary types, the more frequent form is the X-linked juvenile. In the X-linked families, the affected males show mild visual impairment until the 4th or 5th decade of life. The disease progresses thereafter, and in rare cases, complete blindness occurs from retinal detachment. The female carriers have normal vision, with no abnormalities in the retina and cannot be identified clinically. The cause of RS pathology is not known, but histopathological and electroretinographic studies suggested defects in retinal Muller cell function in the development of the disease (2). A large number of genetic linkage studies (4 –7) with microsatellite markers have localized the RS gene to the short arm of the X-chromosome. No locus heterogeneity has been reported among different sets of families (6). The gene responsible for RS has recently been isolated (8), and is found to be exclusively expressed in the retina. The mature protein shows sequence homology to discoidin-1, which is implicated in cell-cell interaction. Mutational analysis of RS families implied that the mutated gene can cause RS pathology. In this communication, we describe a missense and a nonsense mutation in two families segregating for X-linked RS. PATIENTS AND METHODS All available family members (affected and unaffected) underwent a complete ophthalmological evaluation at William Beaumont Hospital in Royal Oak, Michigan. The study was approved by the Institutional Review Board of Oakland University. All patients were informed of the purpose of the study and written consent were obtained. In the pedigrees (Fig. 1A and 2A) there are no male to male transmission; all affected individuals are males. The males are related to each other through unaffected females and the transmission is from affected man to carrier daughter, to affected grandsons, consistent with an X-linked pattern of inheritance. The fundus examination of all affected individuals showed foveal and peripheral schisis which are the characteristic features of RS.
317
0006-291X/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.
Vol. 256, No. 2, 1999
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
RESULTS AND DISCUSSION The RS gene (XLRS1) contains six exons. The availability of oligonucleotide primers (8) permitted us to screen the RS family whose linkage has already been established (6). The sequencing of the XLRS1 gene (Fig. 1B) in the family (Family 1) identified a missense mutation in exon-6 at codon 197 (R197C) in all affected individuals, but not in unaffected males and 40 normal unrelated, randomly selected controls (60 normal X-chromosomes from our own laboratory and 90 con-
FIG. 1. (A) Pedigree of an RS family showing unaffected (open symbols) and affected (closed symbols) family members. Slashed symbols denote deceased individuals. Individuals examined are indicated by the X mark on the vertical and horizontal connecting bars. (B) Nucleotide sequence of the mutant part of exon-6 of the XLRS1 gene. The nucleotide sequence change in the patient is C to T, which results in the amino acid cysteine (TGC) instead of the normal arginine (CGC). The same mutation was segregated in all affected individuals.
We collected venous blood from several affected and unaffected individuals and the genomic DNA was extracted as described before (9). All exons of the RS gene were amplified by the polymerase chain reaction (PCR) using commercially synthesized primers, as reported by others (8). The PCR was carried out by using Applied Biosystems Taq DNA polymerase with 30 cycles of 1.5 min at 94°C, 1 min at 60°C, 2 min at 72°C in the manufacturer’s buffer containing 2– 4 units polymerase, 10pmole each of primer, 50mM each of the four deoxynucleotides, 1.5mM MgCl 2 and 10mM tris-HCl pH 8.3. The amplified products were cloned into a pT7 blue vector and sequenced with fluorescent primers using applied Biosystems Data analysis software, following the manufacturer’s protocol.
FIG. 2. (A) Pedigree of a second RS family analyzed in this study. (B) Nucleotide sequence of the mutant part of exon 4 of the XLRS1 gene. The nucleotide sequence change in the patient is T to A, which results in the termination codon (TAA) instead of the normal tyrosine residue (TAT). The rest of the symbols are the same as those in Fig. 1.
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Vol. 256, No. 2, 1999
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
trol chromosomes from the other laboratory). This mutation (C3 T) changed the encoded and highly conserved arginine residue to cysteine (based on the sequence change). Sequencing of exon-4 in family 2 (Fig. 2A and B) identified a T to A transversion in codon 89 resulting in a stop codon Y89X. This mutation is present in the second affected individual (IV-2) as well as in the heterozygous mother (III-3), but not in unaffected individuals. The corresponding mutant protein, if expressed, should result in a severely truncated polypeptide with 89 amino acids, rather than the normal 224. Although the role of the XLRS1 has not been clearly established, it is possible that the truncated protein is non-functional. It is also likely that no mutant protein would be expressed, since a premature termination codon has been reported to cause unstable, short-lived mRNA or reduction in the level of mRNA (10,11). Since the mutations are transmitted through three generations, segregate with the disease and are in the conserved motif of the protein, it is likely that these mutations are pathogenic in the families. Although the mothers (IV-4 in Family 1 and III-3 in Family 2), carried this mutation in one of their alleles, they had no visual impairment when examined. This not only confirms the recessive nature of the disease, but also suggests that 50% of the normal RS gene product is sufficient for the retinal structure and function. Although the effect of the structural alteration on the functioning of the protein is not understood, the amino aid change in the phylogenetically conserved carboxy terminus may result in an aberrant function, which may be the cause of RS in Family 1. The above sequence alterations have also been recently reported in other families (12), but at present, no genotype/ phenotype correlations can be made among these pa-
tients. In vitro studies on this mutant gene may shed some light on the molecular basis of this disorder. ACKNOWLEDGMENTS We thank the participants who kindly donated blood samples for the study. Our sincere thanks also to Dr. Frank J. Giblin of Oakland University for reading the manuscript. This work was supported in part by a grant from the Retinopathy of Prematurity Foundation (ROPARD) and a core grant for vision research from the NEI (EY05230).
REFERENCES 1. Forsius, H., Krause, U., and Helve, J. (1973) Can. J. Ophthalmol. 8, 385–393. 2. George, N. D. L., Yates, J. R. W., and Moore, A. T. (1996) Arch. Ophthalmol. 114, 274 –280. 3. Madjaru, B., Hilton, G. F., Brinton, D. A., and Lec, S. S. (1995) Retina 15, 282–285. 4. George, N. D. L., Payne, S. J., Bill, R. M., Barton, D. E., Moore, A. T., and Yates, J. R. W. (1996) J. Med. Genet. 33, 919 –922. 5. Pawar, H., Bingham, E. L., Lunetta, K. L., Segal, M., Richards, J. E., Bochnke, M., and Sieving, P. A. (1995) Hum. Hered. 45, 206 –210. 6. Shastry, B. S., Hejtmancik, F. J., Margherio, R. T., and Trese, M. T. (1996) Biochem. Biophys. Res. Comm. 220, 824 – 827. 7. Shastry, B. S., Hejtmancik, F. J., Rodriguez, A., Rodriguez, F., and Tamayo, M. L. (1997) J. Med. Genet. 34, 504 –506. 8. Sauer, C. G., Gehring, A., Warneke-Wittstock, R., Marquardt, A., Ewing, C. C., Gibson, A., Lorenz, B., Jurklies, B., and Weber, B. H. F. (1997) Nat. Genet. 17, 164 –170. 9. Bell, G. I., Karam, J. H., and Rutter, W. J. (1981) Proc. Nat. Acad. Sci. USA 78, 5759 –5763. 10. Kanniburgh, A. J., Maquat, L. E., Schedl, T., et al. (1982) Nucl. Acids Res. 10, 5421–5427. 11. Beserga, S. J., and Benz, E. J. (1988) Proc. Nat. Acad. Sci. USA 85, 2056 –2060. 12. The retinoschisis consortium (1998) Hum. Mol. Genet. 7, 1185– 1192.
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