Centro Andaluz de Biología Molecular y Medicina Regenerativa

Retinal degeneration: from genetics to therapy

Research areas: 

-Disease gene mapping and gene identification of inherited eye diseases;
-Functional characterization of novel eye related genes and to study the impact of mutations on protein function;
-To understand the molecular basis of the disease process and develop novel approaches for treatment of patients with eye diseases.

Research area description:

Molecular Genetics defines a field of study where in the absence of a detectable biochemical defect (true for a majority of inherited diseases), a reverse genetics approach allows the chromosomal assignment and isolation of the disease causing genes. Advent of recombinant DNA technology led to the cloning of chromosome specific DNA markers in the early 1980s. The ability to detect restriction fragment length polymorphisms (RFLPs) in the Human Genome, permitted the use of these markers as genetic tools for linkage studies in families segregating for a particular disease trait. Inherited retinal dystrophies are a major cause of incurable blindness in the Western World. Amongst these a significant proportion is accounted for by a clinically heterogeneous group of diseases collectively known as Retinitis pigmentosa (RP).

RP is initially characterized by night blindness and progressive loss of peripheral vision due to the loss of rod photoreceptor cells.

As the disease progresses cone cells in the central retina are also involved, often leading to total blindness in later stages of the disease.

Based on pedigree analysis, all three modes of Mendelian inheritance (Autosomal dominant, Autosomal recessive and X-linked) have been observed. Decades of research into the biochemical basis of the disease failed to identify even a single causative factor. For this purpose clinically well characterized large pedigrees are essential and a number of ophthalmic departments nationally and internationally have been central to this programme. Positional cloning has subsequently led to the identification and characterization of many of the genes for inherited eye diseases:

  • With the help of recombinant DNA technology, development of X-chromosome specific probes allowed successful mapping of the X-linked form of RP (Ref 1) and laid the foundations of ophthalmic genetic research. Linkage studies demonstrated the existence of at least two genes for XLRP (RP2 and RP3) which eventually facilitated the isolation of these genes through a positional cloning approach.
  • In 1989 the first locus for autosomal dominant retinitis pigmentosa (ADRP) has mapped to chromosome 3q24, in the same interval as the gene for the rod-cell specific photopigment rhodopsin.
  • The first European mutation identified (Ref 2) was a small deletion of the rhodopsin gene which may have originated as a result of replication slippage during meiosis due to a 3bp repeating motif (CAT) present at the site of the mutation. At this point it became essential to develop a rapid and simple mutation detection method (Ref 3) which would allow large-scale screening of ADRP patients. Our studies indicated that up to 30% of all ADRP patients carry a rhodopsin gene mutation. A positional candidate approach soon implicated peripherin/rds in ADRP however, our studies demonstrated that the same gene is also involved in macular dystrophies (Ref 4), making it the first example of two distinct clinical phenotypes resulting from mutations in the same gene. This led to the re-examination of the peripherin/rds cellular localisation data which confirmed its expression in rod as well as cone cells, providing a possible explanation of the spectrum of retinal diseases associated with this gene.
  • A novel locus for ADRP has identified in a large 9-generation family on chromosome 7p (Ref 5) which was soon followed by the identification of a dominant cone-rod dystrophy (CORD2) locus (Ref 6) on chromosome 19q. A positional cloning approach was initiated which subsequently resulted in the identification of the CORD2 gene known as CRX (Ref 7). The gene contains a homeobox sequence (typical of most transcription factors), and has been shown to be involved in the regulation of photoreceptor specific genes such as rhodopsin, IRBP and arrestin. Apart from identifying two additional loci for ADRP on chromosome 17q and 19q, linkage studies in a large Cone dystrophy family highlighted the guanylate cyclase activating protein 1 (GCAP1) as a possible candidate gene on chromosome 6p. Soon a Y99C mutation was identified and biochemical studies in-vitro indicated that even in the presence of high levels of Ca (usually a suppressor of GCAP1 activity) GCAP1 remains active (Ref 8) leading to a possible constitutive activation of ret GC1 and raised levels of cGMP production. In animal models, high levels of intracellular cGMP have been associated with retinal degeneration and may explain the biological basis of the disease in our Cone-dystrophy family.
  • More recent work allowed the mapping of a further new locus for ADRP on chromosome 14 followed by the identification of a S50T mutation in a second transcription factor gene NRL (Ref 9). Transient co-transfection experiments, in-vitro, demonstrated consistent over stimulation of the rhodopsin promoter as a functional impact of the mutant protein in presence of CRX. Transgenic animals over-expressing wild type rhodopsin are known to display retinal degeneration. This may account for the phenotype seen in the chromosome 14-linked ADRP family. The clinical heterogeneity seen in patients with retinal degeneration has been corroborated by the finding of extensive genetic heterogeneity.
  • As more and more genes are identified, understanding the biochemical basis and functional consequences of the mutant protein may truly revolutionize the ‘Post-Genome’ era and ultimately lead to the development of more rationale gene based therapies. Subretinal injection of an adeno-associated viral construct containing a functional copy of the peripherin gene allowed complete ultrastructural and functional rescue of photoreceptor cells in the retinal degeneration slow (rds) mouse (Ref 10). As stated earlier, peripherin has been implicated in a variety of human retinopathies and the gene therapy based rescue of a mouse model provides a great deal of hope to patients suffering from degenerative disease of the retina.
  • Other notable achievements have been in the identification of genes for retinitis pigmentosa (PRPF3, PRPF31 and TOPORS, Ref 11), cone(GCAP1), cataract (connexins 46 and 50 and MIP), anterior segment dysgenesis/glaucoma (FOXC1) and dominant optic atrophy (OPA1). Recently, a previously unreported gene, EYS at the locus RP25, mutated in recessive retinitis pigmentosa was identified using a positional cloning approach (Ref.12). Information about the established function of insect orthologs suggests that EYS may possess similar functions in maintaining the integrity of the photoreceptor cells in human retina.

1. Close genetic linkage between X linked retinitis pigmentosa and a restriction fragment length polymorphism identified by recombinant DNA probe L1.28. Bhattacharya, S.S., Wright, A.F., Clayton, J.F., Price, W.H., Phillips, C.I., McKeown, C.M.E., Jay, M.R., Bird, A.C., Pearson, P.L., Southern, E.M. and Evans, H.J. (1984): Nature, 309: 253 255.

2. A three base pair deletion in the rhodopsin gene in a family with autosomal dominant retinitis pigmentosa. Inglehearn, C.F., Bashir, R., Lester, D.H., Jay, M.R., Bird, A.C. and Bhattacharya, S.S. (1991). Am. J. Hum. Genet. 48: 26 30.

3. Rapid detection of single base mismatches as heteroduplexes on hydrolink gels. Keen, J., Lester, D.H., Inglehearn, C.F., Curtis, A. and Bhattacharya, S.S. (1991). Trends in Genetics, 7: 5.

4. Mutations in the human retinal degeneration slow (RDS) gene can cause either retinitis pigmentosa or macular dystrophy. Wells, J., Wroblewski, J., Keen, T.K., Inglehearn, C.F., Jubb, A., Eckstein, M., Jay, M.R., Arden, G.B., Bhattacharya, S.S., Fitzke, F.W., Bird, A.C. (1993). Nature Genetics, 3: 213-218.

5. A new locus for autosomal dominant Retinitis Pigmentosa (adRP) on chromosome 7p. Inglehearn, C.F, Carter, S.A., Keen, T.J., Lindsey, J., Stephenson, A.M., Bashir, R., Al-Maghtheh, M., Moore, A.T., Jay, M.R., Bird, A.C. and Bhattacharya, S.S. (1993). Nature Genetics, 4: 51-53.

6. Genetic linkage of cone-rod dystrophy to chromosome 19q and evidence for segregation distortion. Evans, .E., Fryer, A.F., Inglehearn, C.F., Duvalloung, J., Whittaker, J., Gregory, C.Y., Ebenezer, N., Hunt, D. and Bhattacharya, S.S. (1994). Nature Genetics, 6: 210-213.

7. Cone-Rod Dystrophy due to mutations in a novel photreceptor-specific homeobox gene (CRX) essential for maintenance of the photoreceptor. Freund, C.L., Gregory-Evans, C.Y., Furukawa, T., Papaioannou, M., Looser, J., Ploder, L., Bellingham, J., Ng, D., Herbrick, J.S., Duncan, A., Scherer, S.W., Tsui, L., Loutradis-Anagnostou, A., Jacobson, S.G., Cepko, C.L., Bhattacharya, S.S. and McInnes, R.R. (1997). Cell, 91: 543-553.

8. GCAP1(Y99C) Mutant is constitutively active in autosomal dominant cone dystrophy. Sokal, I., Li, N., Surgucheva, I., Warren, M.J., Payne, A.M., Bhattacharya, S.S., Baehr, W. and Palczewski, K. (1998). Molecular Cell, 2: 129-133.

9. Mutation in NRL is associated with autosomal dominant retinitis pigmentosa. Bessant, D.A.R., Payne, A.M., Mitton, K.P., Wang, Q-L., Swain, P.K., Plant, C., Bird, A.C., Zack, D.J., Swaroop, A. and Bhattacharya, S.S. (1999). Nature Genetics, 21: 355-356.

10. Restoration of photoreceptor ultrastructure and function in retinal degeneration slow mice by gene therapy. Ali, R.R., Sarra, G.M., Stephens, C., Alwis, M.D., Bainbridge, J.W., Munro, P.M., Fauser, S., Reichel, M.B., Kinnon, C., Hunt, D.M., Bhattacharya, S.S. and Thrasher, A.J. (2000). Nature Genetics, 25: 306-310.

11. Mutations in TOPORS Cause Autosomal Dominant Retinitis Pigmentosa with Perivascular RPE Atrophy. Chakarova, C.F., Papaioannou, M.G., Khanna, H., Lopez, I., Waseem, N., Shah, A., Theis, T., Friedman, J., Maubaret, C., Bujakowska, K., Veraitch, B., Abd El-Aziz, M.M., Prescott, Q., Parapuram, S., Bickmore, W.A., Munro, P.M/G/, Gal. A/, Hamel, C.P., Marigo, V., Ponting, C.P., Wissinger, B., Zrenner, E., Matter, K., Swaroop, A., Koenekoop, R.K. and Bhattacharya, S.S. (2007). Am J Hum Genet, 81: 1098-1103.

12. EYS, encoding an ortholog of Drosophila spacemaker, is mutated in autosomal recessive retinitis pigmentosa. Abd El-Aziz, MM, Barragan,I, O’Driscoll1, CA, Goodstadt, L, Prigmore, E, Borrego,S, Mena, M, Pieras,JI, El-Ashry, MF, Abu Safieh, L, Shah, A, Cheetham, ME, Carter, NP, Chakarova, C, Ponting, CP, Bhattacharya, SS & Antinolo,G. (2008) Nature Genetics 40:1285-1287.

Senior Researchers:
  • Fco Javier Díaz-Corrales
  • Dr. Berta De La Cerda Haynes
  • Dr. Helena Lucena Padros
  • Ana Belen Garcia Delgado
  • Miriam Ruiz Ballester
  • María Lourdes Valdés Sánchez