Retina International's Scientific Newsletter
	Topic of the Month Cloning of the X-linked juvenile retinoschisis gene by Bernhard Weber

X-linked juvenile retinoschisis (RS) also known as sex linked retinoschisis, congenital vitreous veils and cystic detachment of the retina is a vitreoretinal dystrophy that only affects males. The descriptive term retinoschisis refers to a superficial splitting in the retina between the nerve fibre and ganglion cell layer. Generally, retinoschisis develops early in life and may already be present at birth. Characteristically, the foveal cysts are arranged in a radial pattern centered in the fovea while in approximately 50% of patients bilateral schisis occurs in the periphery mostly in the infero-temporal quadrant (see Figure 1a,b,c; in Nature Genetics 17: 164-170, 1997).
With an estimated prevalence of 1:15.000 to 1:30.000 RS is a more common cause of juvenile macular degeneration affecting more than 20.000 individuals in Europe alone.
During the first five years of life the progression of the disease is often rapid but eventually may slow down. In almost all cases visual acuity is impaired with the severity of the disease ranging from moderate to severe. Most RS patients are legally blind in their fifth or sixth decade of life. Up to now there is no satisfactory therapy.
A typical electrophysiological abnormality in RS patients can be demonstrated by an electroretinographic b wave / a wave ratio smaller 1 in the dark-adapted eye and suggests that the primary defect in RS may be located in the retinal Müller cells. These specialized glial cells of the retina serve a variety of functions including the clearance of K⁺ ions from the extracellular space. A defect in this function may be responsible for the ERG irregularities. In addition, the Müller cells are thought to support neurite outgrowth and the establishment of neuronal connections which seem to be impaired in RS patients. RS might therefore be considered a disorder of impaired retinal development rather than dystrophic processes. A more detailed description of the RS pathology is given in OMIM

Chromosomal localization and cloning of the RS gene locus

The RS gene was assigned to the distal short arm of the X-chromosome some 15 years ago. Subsequently, many laboratories have contributed to the refinement of the disease locus to a 900 kb interval in Xp22.2 between polymorphic markers at DXS418 and DXS999 (see figure 2 in Nature Genetics 17: 164-170, 1997).
As a first step towards the identification and cloning of the disease causing gene several YAC (yeast artificial chromosomes) contigs were constructed spanning the minimal candiate region. As YAC clones have been shown to be instable and chimeric their analysis is often difficult. Consequently, several groups have converted the YAC contig into overlapping PAC (P1-derived artificial chromosome) clones.

Sequencing of the entire RS locus at the Sanger Center, Cambridge

One of the many advantages of the PAC vector sytem is its immediate utilization for direct sequencing strategies. Towards this end, the Sanger Center in collaboration with the group of D. Trump (Addenbrooke's Hospital, UK) initiated the complete sequencing of the 900 kb RS locus. Finally, at the beginning of 1997 the first sequences were publically available via internet.
The availability of large regions of genomic sequences is most helpful for identifying expressed sequence tags stored electronically in dbEST databases

Identification of a retina-specific cDNA

Extensive database analyses have finally revealed several EST clones derived from a retinal cDNA library (e.g. ys86e07.r1). Detailed sequence comparisons suggested that the partial cDNA clones should be part of a single transcript.
Northern blot hybridizations confirmed our assumption and demonstrated the presence of two transcripts in retinal tissue with 1.1 kb and 3.1 kb in size, respectively (see figure 4 in Nature Genetics 17: 164-170, 1997).
The different transcript sizes were found to be due to alternative usage of polyA addition sites. There was no detectable expression in lung, cerebellum, retinal pigment epithelium, heart, brain, placenta, liver, skeletal muscle, kidney, pancreas and lymphocytes. This tissue-specific expression profile made this transcript an excellent candidate for the RS gene.

Identifying mutations in RS patients

Consequently, we determined the complete genomic organization and designed oligonucleotide primer pairs flanking the 6 exons of the gene (see figure 2 in Nature Genetics 17: 164-170, 1997).
Nine unrelated RS patients were then analyzed for mutational changes in the coding sequences of the gene by PCR-based SSCP. All analyzed patients were found to carry mutations that should impair gene function (see table 3 in Nature Genetics 17: 164-170, 1997). In addition, the mutations were shown to segregate with the disease in 7 large multigeneration RS families where additional DNA samples were available. These results strongly suggest that the transcript identified and termed XLRS1 indeed is the gene associated with RS pathology.

What is the function of XLRS1 ?

Although the cloning of the RS gene has been a first major step it now will be crucial to learn more about the functional properties of XLRS1 to finally understand the pathogenesis of RS and to eventually develop novel therapies that could slow down or even cure the disease. We need to know exactly where in the retina the XLRS1 protein is localized. This will help us to identify those cells that should intimately be involved in the disease process. We also need to understand the physiological functions of XLRS1 in those particular cells. This may be an even bigger challenge.
A first clue may come from protein databases searches that show a highly homologous region between XLRS1 and several proteins that were characterized in a variety of species spanning a phylogenetic distance from the slime mold, Dictyostelium discoideum, to man, Homo sapiens (see figure 5 in Nature Genetics 17: 164-170, 1997).
This discoidin motif is thought to take part in phospholipid bindings and therefore, to play an important role in cell-cell interaction. If RS pathology is a consequence of disrupted cell-cell adhesion than the central and periperal schisis observed early in the disease development may be a primary event directly due to loss of XLRS1 function. However, such a scenario seems rather simplistic as the role of the Müller cells in the disease process still remains unclear.

Future studies

An alternative approach to study the physiological functions of XLRS1 in more detail requires the generation of an animal model that carries a specific human RS mutation. For example, the targeted disruption of the mouse Xlrs1 gene will result in a so-called "knock-out" mouse that should simulate some of the identified human mutations that lead to a truncated XLRS1 protein (see table 3 in Nature Genetics 17: 164-170, 1997).
The mouse models will make it feasible to specifically analyse expression in early development. In addition, experimental intervention including drug applications, surgery, or gene therapeutical approaches, can easily be studied in the RS mice.

Has the cloning of the XLRS1 gene immediate consequences for RS patients?

Yes, now that the gene causing RS is known direct DNA diagnostic tests can be offered to RS families. DNA testing may be of help to confirm a clinical diagnosis but also be of great interest for female members of RS pedigrees to clarify the carrier status.
For further information on DNA testing please contact Bernhard H. F. Weber via e-mail (bweb@biozentrum.uni-wuerzburg.de)..

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