Ординатура / Офтальмология / Английские материалы / Retinal Degeneration Disease_Hollyfield, Anderson, LaVail_1999
.pdfxxii |
|
CONTENTS |
68. |
LOCALIZATION OF THE INSULIN RECEPTOR AND |
|
|
PHOSPHOINOSITIDE 3-KINASE IN DETERGENT-RESISTANT |
|
|
MEMBRANE RAFTS OF ROD PHOTORECEPTOR OUTER |
|
|
SEGMENTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . |
. . . 491 |
Raju V.S. Rajala, Michael H. Elliott, Mark E. McClellan, and Robert E. Anderson |
||
69. |
MERTK ACTIVATION DURING RPE PHAGOCYTOSIS IN VIVO |
|
|
REQUIRES avb5 INTEGRIN . . . . . . . . . . . . . . . . . . . . . . . . . . . |
. . . 499 |
Silvia C. Finnemann and Emeline F. Nandrot |
|
|
70. |
PHOTORECEPTOR RETINOL DEHYDROGENASES. An attempt to |
|
|
characterize the function of Rdh11 . . . . . . . . . . . . . . . . . . . . . . . . |
. . . 505 |
Anne Kasus-Jacobi, David G. Birch, and Robert E. Anderson |
|
|
71. |
PIGMENT EPITHELIUM-DERIVED GROWTH FACTOR |
|
|
INHIBITS FETAL BOVINE SERUM STIMULATED |
|
|
VASCULAR ENDOTHELIAL GROWTH FACTOR |
|
|
SYNTHESIS IN CULTURED HUMAN RETINAL PIGMENT |
|
|
EPITHELIAL CELLS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . |
. . . 513 |
Piyush C. Kothary, Rhonda Lahiri, Lynn Kee, Nitin Sharma, Eugene Chun, |
|
|
Angela Kuznia, and Monte A. Del Monte |
|
|
72. |
THE RETINAL PIGMENT EPITHELIUM APICAL MICROVILLI |
|
|
AND RETINAL FUNCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . |
. . . 519 |
Vera L. Bonilha, Mary E. Rayborn, Sanjoy K. Bhattacharya, Xiarong Gu, |
|
|
John S. Crabb, John W. Crabb, and Joe G. Hollyfield |
|
|
73. |
UPREGULATION OF TRANSGLUTAMINASE IN THE GOLDFISH |
|
|
RETINA DURING OPTIC NERVE REGENERATION . . . . . . . . |
. . . 525 |
Kayo Sugitani, Toru Matsukawa, Ari Maeda, and Satoru Kato |
|
|
74. |
SURVIVAL SIGNALING IN RETINAL PIGMENT EPITHELIAL |
|
|
CELLS IN RESPONSE TO OXIDATIVE STRESS. |
|
|
Significance in retinal degenerations . . . . . . . . . . . . . . . . . . . . . . . |
. . . 531 |
Nicolas Bazan |
|
|
INDEX . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . |
541 |
PART I
MOLECULAR GENETICS AND CANDIDATE GENES
CHAPTER 1
GENETIC FACTORS MODIFYING CLINICAL EXPRESSION OF AUTOSOMAL DOMINANT RP
Stephen P. Daiger1, Suma P. Shankar1, Alice B. Schindler1,
Lori S. Sullivan1, Sara J. Bowne1, Terri M. King2, E. Warick Daw3, Edwin M. Stone4, and John R. Heckenlively5
1. INTRODUCTION
Factors modifying clinical expression of inherited diseases are likely to be complex, involving genetic factors, environmental factors and stochastic effects. One way to reduce the complexity is to focus on individuals who share a dominant mutation identical by descent, thus eliminating variability in the underlying mutation and variation in cis to the mutation. A further simplification is to limit analysis to a single, extended family, which may reduce, though not eliminate, environmental effects.
Autosomal dominant retinitis pigmentosa (adRP) offers a number of opportunities for such studies. We are investigating factors which modify clinical features consequent to an Arg677ter stop mutation in the RP1 gene, a mutation which causes adRP with variable age- of-onset, progression and end stage consequences, that is, variable severity (Berson et al., 2002; Jacobson et al., 2000; Sullivan et al., 1999).
The RP1 locus was mapped by linkage testing to 8q13 in a large, extended adRP family, RP01, largely situated in southeastern Kentucky (Blanton et al., 1991; Field et al., 1982). Subsequently, the RP1 gene was identified by positional cloning and mutation screening in RP01 and additional adRP families (Guillonneau et al., 1999; Pierce et al., 1999; Sullivan et al., 1999). Mutations in the RP1 gene account for approximately 4% of adRP families in the United States. The RP1 Arg677ter mutation, found in RP01 and other families, accounts for approximately 1⁄2 of the total (Bowne et al., 1999).
The RP1 gene codes for a novel protein, 2,156 amino acids in length, with high sequence similarity over the first 10% of the amino terminus to doublecortin, a protein
1 Human Genetics Ctr and Dept. of Ophthalmology, Univ. of Texas, Houston, TX, USA; 2 Dept. of Internal Medicine, Univ. of Texas, Houston, TX, USA; 3 Dept. of Epidemiology, Univ. of Texas MD Anderson Cancer Center, Houston, TX, USA; 4 Dept. of Ophthalmology, Univ. of Iowa, IA, USA; 5 Kellogg Eye Ctr, Univ. of Michigan, Ann Arbor, MI, USA. Corresponding author: S. P. Daiger, E-mail: stephen.p.daiger@uth.tmc.edu.
3
4 |
S.P. DAIGER ET AL. |
involved in cortical folding, but with very little sequence similarity elsewhere. The protein is photo-receptor-specific; localizes to the rod interconnecting cilium; stabilizes disc architecture in outer segments; and is most likely involved in ciliary matrix assembly and function (Gao et al., 2002; Liu et al., 2002; Liu et al., 2003; Liu et al., 2004; Pierce et al., 2004).
The Arg677ter mutation is in the final, 4th exon of the gene and probably escapes nonsense-mediated decay. If so, the product of the mutant message would be a severely truncated protein, retaining the doublecortin domain, but lacking most of the remaining sequence. Thus the pathophysiology probably involves a dominant negative or gain of function effect, rather than haploinsufficiency.
Factors modifying simple mendelian diseases, such as retinitis pigmentosa, may be just as complex as factors contributing to multifactorial diseases such as age-related macular degeneration (Haider et al., 2002; Nadeau, 2003). However, for dominant-acting diseases one factor is particularly likely: the “wild type” allele in trans to the mutation. For example, alleles in trans to alpha-spectrin mutations modify the severity of elliptocytisis (Gratzer, 1994; Randon et al., 1994). Also, of direct relevance to retinitis pigmentosa, variability in expression of the allele in trans to disease-causing mutations in PRPF31 modify penetrance of the RP11 form of adRP (McGee et al., 1997; Vithana et al., 2003). This is particularly relevant to RP1 because several polymorphic amino acid substitutions exist at the locus which result in at least 6 distinct amino acid haplotypes, i.e., distinct proteins, in Caucasian populations (Bowne et al., 1999). These protein haplotypes in trans are potential modifiers of clinical expression of the RP1 Arg677ter mutation.
2. CLINICAL AND MOLECULAR CHARACTERIZATION OF RP01
A published genealogy traces retinitis pigmentosa in the RP01 family to a single individual born in 1803 (Breeding, 1982). Over 100 living, affected family members have been identified by pedigree reconstruction (Blanton et al., 1991). The family has been the subject of clinical and genetic studies for more that 30 years, including field studies in Kentucky and ophthalmic evaluations at the Jules Stein Eye Institute, UCLA (Field et al., 1982; Heckenlively et al., 1982; Lehmer et al., 1992).
RP01 has classical type 2 adRP with late onset of night blindness, usually by the third decade of life, and slow progression. Characteristic findings include diffuse retinal pigmentation, progressive decrease in recordable ERGs, and concentric visual field loss. Fundoscopic findings include retinal atrophy, bone spicule-like pigment deposits, and vascular attenuation.
The family also shows substantial within-family variability. For example, three members, examined in their 50s, had only small areas of regional pigmentation while two of these had children with typical RP. Early changes in the children included focal depigmented spot atrophy with pigmentation of the edge and abnormal RPE granularity. By contrast, typical late changes in adults vary from diffuse atrophy with pavingstone-type changes to typical bone spicule pigmentation. Further, at least two instances of a “skipped generation” have been documented in RP01.
We have DNA samples, and in many cases, lymphoblastoid cell lines, from 96 members of the family. Of these, 66 have the Arg677ter mutation. Of the 66, we have at least minimal clinical data on 44 and detailed clinical records, including fundus photographs, on 35. For
1. GENETIC FACTORS MODIFYING CLINICAL EXPRESSION OF adRP |
5 |
linkage and segregation analysis we use a pedigree structure with 150 individuals, including two inbreeding loops (Blanton et al., 1992).
3. DO GENETIC FACTORS PLAY A ROLE?
To answer this question we assigned an age-adjusted affectation status to the 44 individuals with at least minimal records and tested for segregation of severity using the nonparametric pedigree analysis program Loki (Daw et al., 1999; Daw et al., 1999a; Heath, 1997). Loki uses an iterated, Monte Carlo Markoff-chain approach to estimate the extent to which genetic differences play a role in variation of a trait (severity in this case) and the potential number of contributing genes. Based on this evaluation, 36% of the differences in severity in RP01 can be explained by variation at the RP1 locus (other than the mutation itself ) and 18% can be explained by unlinked loci. For the unlinked loci, Loki estimated that there is an 80% probability that 6 or fewer loci are involved, and that one of these loci may account for at least 9% of the total variation (Figure 1.1; unpublished). These results are provisional, given the “soft” nature of the clinical phenotype, but they support the likelihood that genetic differences play a role in the variability of adRP caused by the Arg677ter mutation.
4. POLYMORPHIC AMINO ACID HAPLOTYPES AT THE RP1 LOCUS
Numerous non-pathogenic amino acid substitutions have been reported at the RP1 locus (Berson et al., 2002; Bowne et al., 1999; Sohocki et al., 2001; Sullivan et al., 1999). Of
Figure 1.1. Number of genes estimated to contribute to RP1 Arg677ter severity based on Loki analysis in RP01.
6 |
S.P. DAIGER ET AL. |
Table 1.1. Polymorphic amino acid substitutions and haplotypes in RP1.
Substitution |
Frequencies |
Arg872His (CGT Æ CAT) |
75%, 25% |
Asn985Tyr (AAT Æ TAT) |
54%, 46% |
Ala1670Thr (GCA Æ ACA) |
78%, 22% |
Ser1691Pro (TCT Æ CCT) |
77%, 23% |
Cys2033Tyr (TGT Æ TAT) |
46%, 54% |
Haplotype |
Frequencies |
1: Arg – Tyr – Ala – Ser – Tyr |
41% |
2: Arg – Asn – Ala – Ser – Cys |
30% |
3: His – Asn – Thr – Pro – Cys |
26% |
4: Arg – Tyr – Ala – Ser – Cys |
1% |
5: His – Tyr – Ala – Ser – Tyr |
1% |
6: His – Asn – Ala – Pro – Cys |
1% |
|
|
these, at least 5 have high heterozygosity, that is, the lesser allele has a frequency of 10% or greater (Table 1.1). Although there is considerable linkage disequilibrium between these alleles, 6 distinct amino acid haplotypes were found in 100 unrelated CEPH parents, using offspring genotypes to reconstruct haplotypes, as shown in Table 1.1 (Sohocki et al., 2001; and unpublished). These polymorphic protein haplotypes are potential modifiers, in trans, of the Arg677ter mutation.
5. DO THE HAPLOTYPES IN TRANS PLAY A ROLE?
To test this possibility, we determined the protein haplotype in trans in each of the 66 individuals carrying the Arg677ter mutation. Since a sequence of approximately 3 megabases on 8q13 is tracking with disease in the family, i.e., without recombination, there is effectively no variation in cis to the mutation (Blanton et al., 1992). (Not all Arg677ter mutations descend from a common ancestor [Bowne et al., 2002; Schwartz et al., 2003]). The haplotype in cis to the mutation in RP01 is haplotype 2 in Table 1.1; thus the allele in trans can be determined deductively from each individual’s genotype.
For this analysis we evaluated fundus photographs and clinical findings for the 33 individuals with detailed records. As dependent variables we considered age-of onset, other agebased landmarks, professionally graded fundus photographs and a composite, age-adjusted severity score. The independent variables in this case were the polymorphic amino acid alleles in trans, tested individually, and the protein haplotypes. Analytic methods included parametric significance testing and analysis of variance, and Kaplan-Meier survival modeling, implemented using the SAS statistical package (Allen, 1995).
In summary, a few combinations of dependent variables with independent variables show significant association, but are not significant if corrected for multiple comparisons. However, one of the significant associations, based on survival analysis, shows a protective effect of haplotype 3 in females (Figure 1.2). Because of the preliminary nature of the analysis, this should be taken as simply suggestive, but it is consistent with the Loki analysis and justifies further research.
1. GENETIC FACTORS MODIFYING CLINICAL EXPRESSION OF adRP |
7 |
Figure 1.2. Kaplan-Meier survival analysis, RP1 protein haplotype 3 versus severity in males and females.
6. ACKNOWLEDGMENTS
Supported by grants from the Foundation Fighting Blindness and the Hermann Eye Fund, and by grants EY07142 and EY14170 from the National Eye Institute - National Institutes of Health.
7. REFERENCES
Allen, P. D., 1995, “Survival Analysis Using the SAS System: A Practical Guide”, SAS Publishing, New York. Berson, E. L., Grimsby, J. L., Adams, S. M., McGee, T. L., Sweklo, E., Pierce, E. A., Sandberg, M. A., Dryja,
T. P., 2002, Clinical features and mutations in patients with dominant retinitis pigmentosa-1 (RP1), Invest. Ophthalmol. Vis. Sci. 42:2217-2224.
Breeding, C. “Partial Ison Genealogy, 1650-1982”. Private printing, copyright 1982, C. Breeding, Charleston, IN, USA.
Blanton, S. H., Heckenlively, J. R., Cottingham, A. W., Friedman, J., Sadler, L. A., Wagner, M., Friedman, L. H., Daiger, S. P., 1991, Linkage mapping of autosomal dominant retinitis pigmentosa (RP1) to the pericentric region of human chromosome 8, Genomics 11:857-873.
Bowne, S. J., Daiger, S. P., Hims, M. W., Sohocki, M. M., Malone, K. A., McKie, A. B., Heckenlively, J. R., Birch, D. R., Inglehearn, C. F., Bhattacharya, S. S., Bird, A., Sullivan, L. S., 1999, Mutations in the RP1 gene causing autosomal dominant retinitis pigmentosa, Hum. Mol. Genet. 11:2121-2128.
8 S.P. DAIGER ET AL.
Daw, E. W., Heath, S. C., Wijsman, E. M., 1999, Multipoint oligogenic analysis of age-at-onset data with applications to Alzheimer disease pedigrees. Am. J. Hum. Genet. 64:839-851.
Daw, E. W., Kumm, J., Snow, G. L., Thompson, E. A., Wijsman, E. M., 1999a, Monte Carlo Markov chain methods for genome screening, Genet. Epidemiol. 17:133-138.
Field, L. L., Heckenlively, J. R., Sparks, R. S., Garcia, C. A., Farson, C., Zedalis, D., Sparkes, M. C., Crist, M., Tideman, S., Spence, M. A., 1982, Linkage analysis of five pedigrees affected with typical autosomal dominant retinitis pigmentosa, J. Med. Genet. 19:266-270.
Gao, J., Cheon, K., Nusinowitz, S., Liu, Q., Bei, D., Atkins, K., Azimi, A., Daiger, S. P., Farber, D. B., Heckenlively, J. R., Pierce, E. A., Sullivan, L. S., Zuo, J., 2002, Progressive photoreceptor degeneration, outer segment dysplasia and rhodopsin mis-localization in mice with targeted disruption of the retinitis pigmentosa-1 (Rp1) gene, Proc. Natl Acad. Sci. USA 99:5698-5703.
Gratzer, W., 1994, Human genetics. Silence speaks in spectrin. Nat. 372:620-621.
Guillonneau, X., Piriev, N. I., Danciger, M., Kozak, C. A., Cideciyan, A. V., Jacobson, S. G., Farber, D. B., 1999, A nonsense mutation in a novel gene is associated with retinitis pigmentosa in a family linked to the RP1 locus, Hum. Mol. Genet. 8:1541-1546.
Haider, N. B., Ikeda, A., Naggert, J. K., Nishina, P. M., 2002, Genetic modifiers of vision and hearing, Hum. Mol. Genet. 10:1195-1206.
Heath, S. C., 1997, Markov chain Monte Carlo segregation and linkage analysis for oligogenic models, Am. J. Hum. Genet. 61:748-760.
Heckenlively, J. R., Pearlman, J. T., Sparkes, R. S., Spence, M. A., Zedalis, D., Field, L., Sparkes, M., Crist, M., Tideman, S., 1982, Possible assignment of a dominant retinitis pigmentosa gene to chromosome 1, Ophthalmic Res. 14:46-53.
Jacobson, S. G., Cideciyan, A. V., Iannaccone, A., Weleber, R. G., Fishman, G. A., Maguire, A. M., Affatigato, L. M., Bennett, J., Pierce, E. A., Danciger, M., Farber, D. B., Stone, E. M., 2000, Disease expression of RP1 mutations causing autosomal dominant retinitis pigmentosa, Invest. Ophthalmol. Vis. Sci. 41:1898-1908.
Lehmer, J. M., Heckenlively, J. R., Stone, E. M., Kimura, A. E., Blanton, S. H., Daiger, S. P., 1992, Clinical characterization of chromosome 8 autosomal dominant retinitis pigmentosa (UCLA-RP01), Invest. Ophthal. Vis. Sci., 33:1396.
Liu, Q., Lyubarsky, A., Skalet, J. H., Pugh Jr, E. N., Pierce E. A., 2003, RP1 is required for the correct stacking of outer segment discs, Invest. Ophthalmol. Vis. Sci. 44:4171-4183.
Liu, Q., Zhou, J., Daiger, S. P., Farber, D. B., Heckenlively, J. R., Smith, J. E., Sullivan, L. S., Zuo, J., Milam, A. H., Pierce, E. A., 2002, Identification and subcellular localization of the RP1 protein in human and mouse photoreceptors, Invest. Ophthal. Vis. Sci. 43:22-32.
Liu, Q., Zuo, J., Pierce, E. A., 2004, The retinitis pigmentosa 1 protein is a photoreceptor microtubule-associated protein, J. Neurosci. 24:6427-6436.
McGee, T. L., Devoto, M., Ott, J., Berson, E. L., Dryja, T. P., 1997, Evidence that the penetrance of mutations at the RP11 locus causing dominant retinitis pigmentosa is influenced by a gene linked to the homologous RP11 allele, Am. J. Hum. Genet. 61:1059-1066.
Nadeau J., 2003, Modifier genes and protective alleles in humans and mice, Curr. Opin. Genet. Dev. 3:290-295. Pierce, E. A., Quinn, T., Meehan, T., McGee, T. L., Berson, E. L., Dryja, T. P., 1999, Mutations in a gene encoding a new oxygen-regulated photoreceptor protein cause dominant retinitis pigmentosa, Nat. Genet. 22:48-
254.
Randon, J., Boulanger, L., Marechal, J., Garbarz, M., Vallier, A., Ribeiro, L., Tamagnini, G., Dhermy, D., Delaunay, J., 1994, A variant of spectrin low-expression allele alpha-LELY carrying a hereditary elliptocytosis mutation in codon 28, Brit. J. Haemat. 88:534-540.
Schwartz, S. B., Aleman, T. S., Cideciyan, A. V., Swaroop, A., Jacobson, S. G., Stone, E. M., 2003, De novo mutation in the RP1 gene (Arg677ter) associated with retinitis pigmentosa, Invest. Ophthalmol. Vis. Sci. 44:35933597.
Sohocki, M. M., Daiger, S. P., Bowne, S. J., Rodriquez, J. A., Northrup, H., Heckenlively, J. R., Birch, D. G., MintzHittner, H., Ruiz, R. S., Lewis, R. A., Saperstein, D. A., Sullivan, L. S. 2001, Prevalence of mutations causing retinitis pigmentosa and other inherited retinopathies, Hum. Mutat. 17:42-51.
Sullivan, L. S., Heckenlively, J. R., Bowne, S. J., Zuo, J., Hide, W. A., Gal, A., Denton, M., Inglehearn, C. F., Blanton, S. H., Daiger, S. P., 1999, Mutations in a novel retina-specific gene cause autosomal dominant retinitis pigmentosa, Nat. Genet. 22:248-251.
Vithana, E. N., Abu-Safieh, L., Pelosini, L., Winchester, E., Hornan, D., Bird, A. C., Hunt, D. M., Bustin, S. A., Bhattacharya, S. S., 2003, Expression of PRPF31 mRNA in patients with autosomal dominant retinitis pigmentosa: a molecular clue for incomplete penetrance?, Invest. Ophthalmol. Vis. Sci. 44:4204-4109.
CHAPTER 2
DISEASE-ASSOCIATED VARIANTS OF THE RODDERIVED CONE VIABILITY FACTOR (RdCVF) IN LEBER CONGENITAL AMAUROSIS
Rod-derived cone viability variants in LCA
Sylvain Hanein1, Isabelle Perrault1, Sylvie Gerber1, Hélène Dollfus2,
Jean-Louis Dufier3, Josué Feingold1, Arnold Munnich1,
Shomi Bhattacharya4, Josseline Kaplan1, José-Alain Sahel5,
Jean-Michel Rozet1, and Thierry Leveillard5
1. INTRODUCTION
Leber congenital amaurosis (LCA) is the most early and severe form of all inherited retinal dystrophies, responsible for congenital blindness. The genetic heterogeneity of LCA has been accepted for a long time but it turned out to be largely higher than all odds. So far, 11genes have been mapped on human chromosomes and eight identified. i) the retinal specific guanylate cyclase gene (GUCY2D, retGC1; 17p13.1; LCA1; MIM 600179), ii) the gene encoding the 65-kD protein specific to the retinal pigment epithelium (RPE65; 1p31; LCA2; MIM180069), iii) the cone-rod homeobox-containing gene (CRX; 19q13.3; LCA7; MIM 60225), iv) the gene encoding the arylhydrocarbon receptor interacting protein-like 1 (AIPL1; 17p13.1; LCA; MIM 604392), v) the gene encoding the retinitis pigmentosa GTPase regulator-interacting protein 1 (RPGRIP1; 14q11; LCA6; MIM 605446), vi) the human homologue of the drosophila melanogater crumbs gene (CRB1; 1q31; LCA8; MIM 604210), vii) the gene encoding the tubby-like protein 1 (TULP1; 6q21.3; LCA10; MIM 602280), viii) the retinol dehydrogenase 12 (RDH12; 14q24; LCA11; MIM 608830), ix) LCA3 (14q24; MIM 604232), x) LCA5 (6q11-16; MIM 604537) and xi) LCA9 (1p36; MIM608553).
1 Unité de Recherches sur les Handicaps Génétiques de l’Enfant. Hôpital Necker - Enfants Malades, 149 rue de Sèvres, 75743 Paris Cedex 15, France. Email: kaplan@necker.fr; 2 Clinique Ophtalmologique, Hopitaux Universitaires de Strasbourg, Strasbourg, France; Service d’ophtalmologie, Hôpital Necker, France; 4 Departments of Molecular Genetics and Visual Science, Institute of Ophthalmology, London, United Kingdom. Moorfields Eye Hospital, London, United Kingdom; 5 Laboratoire de physiopathologie cellulaire et moléculaire de la rétine Inserm U592, Paris, France.
9
10 S. HANEIN ET AL.
Interestingly, all LCA genes hitherto identified are involved in strikingly different physiologic pathways resulting in a wide physiopathologic variety.
So far, 50% of all cases have been related to a known disease-causing gene. If in 60% of these cases, the affection develops like a congenital severe cone-rod dystrophy, in about 40% of them, the disease appear to develop as a severe yet progressive rod-cone dystrophy and may represent the extremity of a spectrum of severity of retinitis pigmentosa (RP), (Hanein et al., 2004).
On the other hand, it has been shown that factors secreted from rods are an essential requirement for cone viability (Mohand-Saïd et al., 1998). One such trophic factor has just been identified by expression cloning and named rod-derived cone viability factor (RdCVF). RdCVF is a novel protein specifically expressed by photoreceptors. By immunodepletion with specific antibodies it has been demonstrated that RdCVF is required for cone rescue in mouse retinal cultured explants. The injection of recombinant RdCVF is able to prevent 40% of cones from degeneration in the rd1 mouse over a period of two weeks. These results provide a biochemical basis for the previously described paracrine interaction between rod and cone photoreceptors that appears to play a key role in maintaining cone cell viability.
The aim of the present study was to look for mutations in LCA patients in RdCVF, by screening the thioredoxin-like 6 gene which encode this novel protein (TXNL6; Leveillard et al., 2004).
2. MATERIAL AND METHODS
2.1. Patients Panel
A total of 200 unrelated patients were either seen at the Ophthalmo-Genetics Center of Necker–Enfants Malades Hospital or sent to the laboratory by referent ophthalmologists or geneticists from France or other countries worldwide.
Our inclusion criteria were: i) severe impairment of visual function detected at birth or during the first months of life with pendular nystagmus, roving eye movements, eye poking, inability to follow light or objects and normal fundus, ii) extinguished ERG, iii) exclusion of ophthalmologic or systemic diseases sharing features with LCA. Detailed clinical data were required for each patient ie i) age and mode of onset, ii) light behaviour since birth, iii) natural history of the visual impairment since the first months of life including the subjective impressions of parents, iv) refraction data, v) ophthalmologic findings (anterior chamber and fundus), vi) visual acuity (if measurable) and vii) electrophysiology recordings. The course of the disease was determined by interviewing the patients or their parents and a pedigree was established.
Genomic DNA was extracted from whole blood or immortalized lymphoblast cell lines of patients using standard methods. When a mutation was identified we examined the parents and other family members when available in both sporadic and familial cases.
2.2. Controls Panel
Genomic DNA obtained from 125 unrelated healthy individuals and a cohort of 55 patients affected with Stargardt (STGD) disease were used as a control panel for molecular studies.