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First molecular evidence for a de novo mutation inRS1 (XLRS1) associated with X linked juvenile retinoschisis
  1. Institut für Humangenetik, Biozentrum, Universität Würzburg, Am Hubland, 97074 Würzburg, Germany
  2. Abteilung für Kinderophthalmologie, Strabismologie und Ophthalmogenetik, Klinikum der Universität, 93042 Regensburg, Germany
  1. Dr Weber
  1. Institut für Humangenetik, Biozentrum, Universität Würzburg, Am Hubland, 97074 Würzburg, Germany
  2. Abteilung für Kinderophthalmologie, Strabismologie und Ophthalmogenetik, Klinikum der Universität, 93042 Regensburg, Germany
  1. Dr Weber

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Editor—Juvenile retinoschisis (RS, OMIM 312700) is an X linked recessive vitreoretinal disorder that variably affects visual acuity because of microcystic degeneration of the central retina.1 2 In approximately 50% of affected males, peripheral schisis may also occur. Major sight threatening complications include vitreal haemorrhages, retinal detachment, and neovascular glaucoma.3

Recently, the gene underlying RS, designatedRS1 (also calledXLRS1), was positionally cloned4 and more than 80 different mutations covering a wide mutational spectrum, including intragenic deletions, splice site, frameshift, nonsense, and missense mutations, were identified.4-7 Interestingly, missense mutations mainly cluster in exons 4 to 6 of the RS1 gene known to encode a highly conserved discoidin domain thought to be involved in cell-cell interactions on membrane surfaces.8

The high recurrence rate of some of the RS1mutations (for example, Glu72Lys in more than 34 patients from different ethnic backgrounds) suggests a significant de novo mutation rate in RS.8 In this report, we provide the first molecular evidence of a de novo RS1 mutation (Pro203Leu) in a Greek family. The Pro203Leu mutation is present in two brothers diagnosed with severe features of RS at the ages of 9 and 5 years, respectively. We show that the mother is a heterozygous carrier while neither of the maternal grandparents carry the Pro203Leu mutation. Haplotyping data from several polymorphic DNA loci flanking the RS1 gene confirm paternity and strongly suggest that the Pro203Leu mutation originated on the X chromosome of the maternal grandfather.

Two brothers were referred to one of the authors (BL) presenting with unclassified vitreoretinal degeneration in both eyes. By history, retinal detachment had been diagnosed in the right eye in the older (III.1) at the age of 9 months. At the age of 9 years, best corrected visual acuity was 20/200 in the right eye (RE) and 20/40 in the left eye (LE). Fundoscopy showed a bullous peripheral schisis and a flat schisis at the entire posterior pole with inner leaf hole formation in the RE. In the LE, a macular schisis with marked vitreous veils could be seen. Electroretinogram (ERG) recordings corresponding to the ISCEV Standard were consistent with the diagnosis of RS, that is, rod response was unrecordable in the RE and residual in the LE, there was a negative maximal response, and an unrecordable cone response in both eyes.

In the younger brother (III.2), bullous cyst-like retinal changes in both eyes had been diagnosed at the age of 1 year. Four years later fundoscopy showed a bullous retinal detachment in the inferotemporal retina of the RE including the macula with some cystic changes in the area of the inferior temporal vascular arcade. In the LE, only pigmentary abnormalities and whitish subretinal deposits consistent with a collapsed schisis could be seen. Best corrected visual acuity was light perception RE and 2/100 LE. Because of severe nystagmus and reduced compliance, the ERG was not recorded.

Fundus examination and ERG were normal in the mother (II.1) and maternal grandfather (I.2).

Genomic DNA from the members of the Greek family was extracted using standard techniques. Haplotyping was done using microsatellite markers 207F/R (DXS207), 389gt, 418F/R (DXS418), and RX324 (DXS443) closely flanking the RS locus (table 1).9-11Microsatellite marker 389gt was identified in PAC clone dJ389A20 as a (CA)30 dinucleotide repeat located 50 kb distal to DXS418 (genomic sequence available at The repeat sequences were PCR amplified in the presence of 32P-dCTP (3000 Ci/mmol) using flanking oligonucleotide primers and conditions as given in the references (table 1). To confirm paternity, an additional two highly polymorphic microsatellite markers at theATM locus on 11q23 and theBRCA1 locus on 17q21 (D17S855) were used (table 1).

Table 1

Polymorphic microsatellite markers used in the study

For mutational analysis, the six exons of theRS1 gene were PCR amplified from genomic DNA of patients III.1 and III.2 with intronic oligonucleotide primers flanking the respective coding exons and amplification conditions as described previously.4 Mutation detection was done by single stranded conformational analysis (SSCA). Amplification of the coding exons was carried out with Taqpolymerase (Gibco BRL) in a 25 μl volume in 1 × PCR buffer supplied by the manufacturer. PCR products were electrophoretically separated on a 6% non-denaturing polyacrylamide gel with or without 5% glycerol at 4°C. DNA fragments showing aberrant mobility shifts as well as the corresponding maternal and grandparental PCR products were directly sequenced using the Thermo Sequenase radiolabelled terminator cycle sequencing kit (Amersham, Life Science).

Prescreening by SSCA of the six coding exons of theRS1 gene showed a similar aberrant band shift in exon 6 in the two brothers III.1 and III.2 (fig 1 and data not shown). Direct sequencing of PCR products identified a C to T transition at nucleotide position 608 of the cDNA. This is predicted to result in a proline to leucine substitution at codon 203 (fig 1). Subsequently, sequencing of RS1 exon 6 was performed in the mother, II.1, as well as in both maternal grandparents, I.1 and I.2. The mother was heterozygous for the Pro203Leu mutation while neither of the maternal grandparents showed a mutational change (fig 1).

Figure 1

Analysis of a three generation Greek family with two cases of X linked juvenile retinoschisis (III.1 and III.2). Polymorphic markers at the ATM and the BRCA1 locus (D17S855) were used to confirm paternity. Haplotype analysis was performed using microsatellite markers DXS207, 389gt, DXS418, and DXS443 that closely flank the RS1 gene on Xp22.2. The order of markers is from telomere to centromere. Haplotypes associated with the Pro203Leu mutation in exon 6 of the RS1 gene are boxed. Note that the grandfather, I.2, shares the disease associated haplotype with his two grandsons III.1 and III.2 but does not carry the Pro203Leu mutation.

To confirm paternity, genotyping with polymorphic markers ATM/in45 and D17S855 localised within theATM and the BRCA1gene, respectively, were performed. Segregation of allelic markers is consistent with the grandfather, I.2, being the father of II.1 (fig 1). In addition, haplotype analysis was done with markers closely flanking the RS1 locus. A haplotype could be constructed (DXS207-389gt-DXS418-DXS443: 1-2-2-2) which is shared by the carrier mother, II.1, and her two sons, III.1 and III.2, and therefore should be associated with the Pro203Leu mutation (fig 1). The grandfather also carried the 1-2-2-2 haplotype indicating that the Pro203Leu mutation occurred on this haplotype.

Here, we describe a de novo missense mutation in the RS1 gene, Pro203Leu, that is associated with severe features of X linked juvenile retinoschisis (RS) in two Greek brothers. Although the mother is a heterozygous carrier neither of the maternal grandparents have the Pro203Leu mutation. Haplotype analysis with polymorphic markers closely flanking the RS1 locus provides strong evidence that the Pro203Leu mutation occurred de novo on the X chromosome of the maternal grandfather.

The proline residue at codon 203 of the RS1 protein is part of the evolutionarily highly conserved discoidin domain that is thought to be involved in cell-cell interaction on membrane surfaces.8Without exception, the proline residue is retained at this particular position throughout evolution in all proteins containing the discoidin motif.4 7 In addition, Pro203Leu mutations have independently been identified in affected subjects of three familial RS cases of French and Dutch origin but were not found on 100 additional normal X chromosomes.7 Together, this strongly suggests that, rather than a polymorphism, the Pro203Leu mutation represents an amino acid change that should severely affect protein function and therefore should be responsible for the RS phenotype in the two Greek brothers.

Haplotype analysis has shown that the maternal grandfather of the two Greek RS patients carries the haplotype that becomes disease associated in his daughter and his two grandsons. This provides strong evidence that the Pro203Leu mutation is in fact a de novo event. It should be pointed out that the Pro203Leu mutation occurred at a CpG dinucleotide (codon 203: CCG to CTG) which, if methylated at the genomic level, is known to be frequently involved in C→T transitions.16 We cannot exclude that the unaffected grandfather is a mosaic for the Pro203Leu mutation with the mutant genotype being present in one or more tissues, excluding the ocular tissues but including a precursor of the germ cells. Assuming such a situation in the grandfather, the mutation could be transferred to his daughter and would then be perceived as a de novo germinal mutation.

Besides the Greek family, we were able to analyse the segregation ofRS1 mutations in another four pedigrees where RS occurred in a single generation of large families. There was no further evidence of de novo events in the extended families. However, considering the small number of families tested, the present study supports an earlier notion that the new mutation rate in RS may be significant.7 Further segregation analyses in multigeneration families with “sporadic” or only a few cases of RS will be required to estimate more accurately the frequency of de novo mutations in X linked juvenile retinoschisis.


We thank the patients and their family for their kind cooperation. This work was supported by the Deutsche Forschungsgemeinschaft (We 1259/5-3 and Lo 457/3-1).


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