Positional cloning in Xp22 : towards the isolation of the gene involved in
X-linked retinoschisis
Vosse, E. van de
Citation
Vosse, E. van de. (1998, January 7). Positional cloning in Xp22 : towards the isolation of the
gene involved in X-linked retinoschisis. Retrieved from https://hdl.handle.net/1887/28328
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Corrected Publisher’s Version
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Licence agreement concerning inclusion of doctoral thesis in the
Institutional Repository of the University of Leiden
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Author: Vosse, Esther van de
Title: Positional Cloning in Xp22 : towards the isolation of the gene involved in X-linked
retinoschisis
Summary
SUMMARY
The mammalian X chromosomes have several interesting features, including that they are one of the sex determining chromosomes, contain regions of homology with the Y chromosome (for instance in the pseudoautosomal regions), and display X inactivation. The human X chromosome is also known to contain many disease genes. The region we have focused on (Xp22.1-p22.2) contains amongst others the genes for X-linked juvenile retinoschisis (RS) and keratosis follicularis spinulosa decalvans (KFSD). RS is a slowly progressive retinal degeneration that causes a decrease in acuity and visual field and can result in total blindness. KFSD is a rare disorder that causes a range of symptoms of which hyperkeratosis of specific skin areas and absence of facial hair are its most striking clinical features.
In order to construct a physical map of the Xp22.1-p22.2 region, we screened Y AC libraries with markers and clones from this region and assembled the obtained Y ACs into a contig (overlapping set) based on Alu PCR fingerprinting and marker content. This Y AC contig spans
a region of 4.5 to 5Mb from the marker DXS451 to DXS414 (Chapter 2.1). Using this YAC contig we ordered several new markers that we subsequently used to refine the candidate regions for RS and KFSD by analysis of recombinants. Based on the length of the Y ACs in the Y AC contig we estimated that the candidate region for KFSD -between the markers DXS7161 and DXS1226- was 1 Mb and the candidate region for RS -between the markers DXS418 and DXS999- was 600 kb. However, when more markers became available between DXS418 and DXS999 it appeared that the key Y AC in the region (y939H7, 1.3 Mb in length) had a large deletion between these markers. To obtain the full-length YAC, we analysed many individual colonies from the original culture and found a ,stable<clone that contains all known markers between DXS418 and DXS999 and has a length of2.5 Mb. With this stable clone we performed YAC fragmentation. The yeast colony was transformed with a plasmid (pBP108/ADE2) that contained a yeast telomere, the ADE2 gene and an Alu repeat sequence. Homologous
recombination between theAlu's in the YAC and theAlu in the plasmid generated fragmented
Y ACs that could be selected for through the ADE2 gene, now present in the Y ACs. The
experiments resulted in a panel of fragmented YACs ranging in size from 170 kb to 2.4 Mb. This panel facilitated the construction of a large-scale restriction map and allowed binning of clones in the region (Chapter 2.2). The RS candidate region could then be estimated to be 1 Mb in size, almost twice the size of the original estimation.
Summar
We chose ex on trapping as method to isolate candidate genes for the diseases localised to this region and to construct a transcriptional map. We begarLwith the (plasmid-based) pSPL3 exon trap vector and the first transcripts we isolated were part of PHKA2, a known gene in the region. To improve the speed and efficiency of exon trapping, we constructed a new, cosmid-based, exon trap vector (sCOGH) to analyse larger stretches of DNA (up to 40 kb), thereby leaving more of the genomic context in tact, and allowing the isolation of multiple exons in one product. We first tested the system by exon trapping cosmids that contained up to seven exons of a known gene, DMD. Analysis of the ex on trap products proved that the exons were correct! y spliced (Chapter 3).
We subcloned key Y ACs from the Xp22.1-p22.2 region in the sCOGH exon trap vector and used the resulting cosmids in exon trap experiments. We generated many exon trap products, one of which was characterised extensively and proved to contain part of the SCMLl gene (Chapter 4.1), a gene that was simultaneously isolated by D. Trump (Cambridge, U.K) using pSPL3 exon trapping. We also contributed to the analysis of PPEF, another gene in the RS candidate region that was found using pSPL3 exon trapping (Chapter 4.2). This gene has a homologue in
Drosophila (rdgC) that plays a role in the prevention of light induced retinal degeneration and therefore seemed a strong candidate for RS. However, PPEF is not expressed in the mammalian eye, suggesting a different function for the human homologue.
We tested two candidate genes, PPEF and Txp3, in RS patients to search for mutations causing the disease, but no mutations were found using several techniques (Chapter 5). This makes it unlikely that either Qf these two genes is involved in RS. The newly identified SCMLl gene has not been extensively tested for mutations in RS patients. Based on P AC clones provided by the Retinoschisis Consortium the entire RS Cillldidate region has been sequenced, allowing
in silica identification of putative genes from the region. The identification of the RS gene therefore seems not far off. Although several genes have been isolated in the KFSD candidate region recently, none of these genes have been tested in KFSD patients so far.
