Ординатура / Офтальмология / Английские материалы / Retinal Degeneration Disease_Hollyfield, Anderson, LaVail_1999

Table 2.1. Sequences of forward and reverse primers used for the mutational screening of the TXNL6 gene.

2.3. Mutational Screening of TXNL6

Mutational screening of TXNL6 gene was performed on genomic DNA from the patients using primers designed to flank the splice junctions of each coding exon (Table 2.1). After standard PCR amplification (conditions available on request), products were screened for mutations using denaturing high-pressure liquid chromatography (DHPLC). heteroduplex formation was induced by heat denaturation of PCR products at 94°C for 10 min, followed by gradual reannealing from 94°C to 25°C over 30 min. DHPLC analysis was performed with the WAVE DNA fragment analysis system [Transgenomic, Cheshire, UK]. PCR products were eluted at a flow rate of 0.9 ml/min with a linear acetonitrile gradient. the values of the buffer gradients (buffer A, 0.1 M triethylammoniumacetate; buffer B, 0.1 M triethylammoniumacetate/25% acetonitrile), start and end points of the gradient, and melting temperature predictions were determined by the WAVEMAKER software (Transgenomic, Cheshire, UK). Optimal run temperatures were empirically determined.

PCR fragments displaying DHPLC abnormal profiles were further sequenced using the Big Dye Terminator Cycle Sequencing Kit v3 (ABI Prism, Applied Biosystems, Foster City, USA on a 3100 automated sequencer).

2.4. Mutation Nomenclature

We have chosen to number the A of the start codon (ATG) of the TXNL6 cDNA sequence (Genbank accession number BC014127) as nucleotide 1.

2.5. Statistical Test

Comparison of the genotype and allele frequencies in patients and controls were performed by the Fisher’s exact test (two sided).

3. RESULTS

3.1. Mutational Screening of the TXNL6 Gene

Sequence analysis of the 2 TXNL6 exons allowed to identify eleven variant alleles (9/11 different) in 7/200 unrelated LCA patients excluding all eight known LCA genes and in 2/56 other patients for whom the genetic screening of LCA genes is still ongoing. Single or compound heterozygosity for non-conservative amino acid substitutions was identified in seven patients (3 consanguineous) and two patients (1 consanguineous), respectively (Table 2.2). For both compound heterozygous patients, the variants were inherited from healthy parents and co-segregated with the disease.

* Indicate consanguinity of the parents of LCA patients. S: sporadic case, F: familial case. A of the start codon (ATG) of the cDNA sequences of TXNL6 (Genbank accession numbers BC014127) as nucleotide 1.

Table 2.3. Sequence changes in 125 control individuals and 55 STGD patients.

A of the start codon (ATG) of the TXNL6 cDNA sequence (Genbank accession number BC014127) as nucleotide 1.

All 11 variant alleles resulted from non-conservative amino acid substitutions. None of them was found either in 125 unaffected control individuals or in 55 patients affected with typical Stargardt disease (Table 2.3).

3.2. TXNL6 Variants in LCA

The proportions of TXNL6 variant alleles in LCA patients versus controls and STGD patients were compared by the Fisher exact test. A statistically significant difference in TXNL6 genotype frequencies was evidenced between LCA patients and control individuals: PLCA = 9/200 vs Pcontrols = 0/125, p = 0.024. This difference was even more significant when STGD patients were added to the control populations: PLCA = 10/200 vs Pcontrols+STGD patients = 0/180, p = 0.006. TXNL6 allele frequencies were also compared: PLCA

= 0/250, p = 0.009 and PLCA = 10/400 vs Pcontrols+STGD patients = 0/360,

None of the 9 different variants identified in LCA patients was found in a control population of 125 healthy individuals and 55 patients affected with Stargardt disease supporting the involvement of these alterations in LCA.

The identification of compound heterozygous variants in a LCA patient born to nonconsanguineous parents was consistent with a recessive inheritance. However, four patients born to consanguineous were either compound heterozygous (1/4) or single heterozygous (3/4) for TXNL6 substitutions. This observation ruled out a simple recessive transmission and supported the view that TXNL6 variants may act as modifiers of the phenotype or may be disease-causing mutations in a multiallelic mode of inheritance.

The absence of variants in control individuals and STGD patients makes the hypothesis of a modifier role of TXNL6 in LCA less likely than a possible multiallelic inheritance. From this point of view, one has to mention that a triallelism have been demonstrated in Bardet-Biedl syndrome by the identification of several patients harbouring two mutations in the BBS2 gene and one mutation in the BBS6 gene and some asymptomatic individuals carrying two BBS2 gene mutations (Katsanis et al., 2002). These data could be related to the microarray-based mutation analysis of all LCA genes (>260 mutations) in large cohorts of LCA patients showing that i) more than two (expected) variants in a substantial fraction of patients and that ii) the third allele segregated with a more severe disease phenotype in several families (Allikmets et al., 2004). Along the same lines, it is worth noting that all nine patients harbouring TXNL6 variants are affected with a severe form of congenital conerod dystrophy according to the description recently by Hanein et al. (2004).

In conclusion, our data suggest that TXNL6 variants may be associated to 7.5% of LCA patients in our series. Further experiments are now necessary to confirm this hypothesis by showing, for instance, that the TXNL6 variants identified in LCA patients are responsible for a significant reduction of the capacity of the RdCVF protein to maintain cone cell viability.

6. ACKNOWLEDGEMENTS

This work was supported by the Associations Retina France and Valentin Haüy. We thank the National Eye Institute for the travel award provided to SH.

7. ELECTRONIC DATA BASES

Online Mendelian inheritance in Man: http://www4.ncbi.nlm.nih.gov/OMIM/

8. REFERENCES

Hanein, S., Perrault, I., Gerber, S., Tanguy, G., Barbet, F., Ducroq, D., Calvas, P., Dollfus, H., Hamel, C., Lopponen, T., Munier, F., Santos, L., Shalev, S., Zafeiriou, D., Dufier, J.L., Munnich, A., Rozet, J.M., Kaplan, J., 2004, Leber congenital amaurosis: comprehensive survey of the genetic heterogeneity, refinement of the clinical definition, and genotype-phenotype correlations as a strategy for molecular diagnosis. Hum Mutat 23(4):306-17.

Kaplan, J., Bonneau, D., Frezal, J., Munnich, A., Dufier, J.L., 1990, Clinical and genetic heterogeneity in retinitis pigmentosa. Hum Genet 86:635-42.

14 S. HANEIN ET AL.

Katsanis, N., Eichers, E.R., Ansley, S.J., Lewis, R.A., Kayserili, H., Hoskins, B.E., Scambler, P.J., Beales, P.L., Lupski, J.R., 2002. BBS4 is a minor contributor to Bardet-Biedl syndrome and may also participate in triallelic inheritance. Am J Hum Genet 71(1):22-9.

Leveillard, T., Mohand-Said, S., Lorentz, O., Hicks, D., Fintz, A.C., Clerin, E., Simonutti, M., Forster, V., Cavusoglu, N., Chalmel, F., Dolle, P., Poch, O., Lambrou, G., Sahel, J.A., 2004, Identification and characterization of rod-derived cone viability factor. Nat Genet 36(7):755-9.

Mohand-Said, S., Deudon-Combe, A., Hicks, D., Simonutti, M., Forster, V., Fintz, A.C., Leveillard, T., Dreyfus, H., Sahel, J.A., 1998, Normal retina releases a diffusible factor stimulating cone survival in the retinal degeneration mouse. Proc Natl Acad Sci U S A 7;95(14):8357-62.

Allikmets, R.L., Zernant, J., Perrault, I., den Hollander, A., Dharmaraj, S., F.P., Cremers, M., Kaplan, J., Koenekoop, R.K., Maumenee., I., 2004, Multiallelic inheritance and/or modifier alleles in leber congenital amaurosis: analysis with the lca disease chip, ARVO Meeting (2444/B79).

2. MATERIAL AND METHODS

The minimal criteria for inclusion of patients (n = 195) were a severe impairment of visual function since birth or the first months of life, a normal fundus and an unrecordable ERG.

Among the families included in this study, 141/195 were simplex (29 consanguineous) and 54/195 were multiplex (26 consanguineous). Most of them hailed from Europe and North Africa.3

Prior to the mutational screening, the linkage status of familial cases and sporadic consanguineous patients was determined using a set of highly polymorphic markers specific to all known LCA loci. LCA genes were subsequently screened using combination of denaturing high-pressure liquid chromatography (DHPLC) and direct sequencing as described elsewhere.3,4

3. RESULTS

3.1. Spectrum of LCA Genes Mutations

In most cases, the genetic studies were not informative. Hints of linkage pointed to a LCA locus in only 13/52 familial cases and 6/27 consanguineous sporadic cases. One hundred seventy-four disease alleles were identified in 93/195 unrelated patients. The frequency of mutations varied greatly from gene to another, the GUCY2D gene being by far the most frequently involved in the disease. In this gene, most mutations were clustered in exons 2, 15 and 17 and two were shown to result from a founder effect: the 2000-3000 years

Table 3.2. LCA genes involved in other retinal dystrophies.

been evidenced between LCA and other retinal dystrophies. Furthermore, a high prevalence of null alleles has been evidenced in our series of LCA patients: (in decreasing order, CRX non included) 83.3% in RPGRIP1, 66.7% in AIPL1, 62.7% in GUCY2D, 57.9% in RPE65, 50% in RDH12, 36.1% in CRB1 and 33.3% in TULP1. Therefore, we propose that the LCA phenotype is consistently accounted for by the knocking-out of one of these genes while moderate mutations are responsible for less severe progressive retinitis pigmentosa or conerod dystrophy with onset ranging from infancy to adulthood.

4. DISCUSSION

To date, mutations in 11 genes encoding proteins involved in strikingly different physiologic pathways have been shown to cause LCA. The genotyping of 195 unrelated LCA patients enabled us to determine the prevalence of each genetic subtype. Mutations were identified in 93/195 patients: GUCY2D (21.2%), CRB1 (10%), RPE65 (6.1%), RPGRIP1 (4.5%), RDH12 (4.1%), AIPL1 (3.4%), TULP1 (1.7%) and CRX (0.6%).

The high prevalence of GUCY2D mutations in our series might be explained by the identification of several population-specific mutations accounting for 19/38 (50%) of patients with mutation in this gene3,6 (c.387delC, p.Phe565Ser, 620delC and p.Ser448X in 14 North African families; c.2943delG in 5 Finish families).

Whatever the gene (CRX apart), most patients (86/93, >92.5%) were found to be homozygous (n = 50) or compound heterozygous (n = 34) for mutations. Only seven patients were single heterozygous (RPGRIP1, n = 3; RPE65, n = 3; GUCY2D, n = 1; respectively). It is likely that the second mutation lie in unscreened regions of the genes (promoter region, intragenic sequences, 3’ untranslated regions).

For about 48% the patients the disease gene remained to be identified. Considering that genome wide search for homozygosity in large multiplex and consanguineous families failed to identify a major locus, it is likely that many disease genes accounting for a small proportion of patients have to be identified.

The growing number of LCA genes leads to growing difficulties in genotyping patients. However, genotyping remains essential prior to any therapeutic approach and it is thus necessary to identify criteria to direct genetic analyses. The survey of the molecular pathology in LCA enabled us to identify hot spots mutations as well as population-specific mutations that might be search in first intention. But more importantly, this study allowed identifying sound genotype-phenotype correlations as a main criterion to select genes to screen in priority. Indeed, we not only confirmed the subdivision of LCA into two main forms (types I

Figure 3.2. Decision-making flowchart for the molecular diagnosis of LCA.

and II) but we also subdivided each of them into distinct clinical subtypes on the basis of the progression course the disease, the refraction error, the severity of the visual deficiency, and the aspect of the retina. Each clinical subtype was specific to one or two LCA genes.

The clinical description of the congenital severe stationary cone-rod dystrophy form of the disease (LCA type I) appeared to be consistent with the traditional definition of LCA. Conversely, the boundary between LCA and early onset severe retinal dystrophy was unclear when the second subtype of the less severe and progressive rod-cone dystrophy form of the disease was considered (LCA type II, subtype II; Figure 3.1). This notion led to the idea that some LCA cases might represent the extreme end of severity in the clinical spectrum of RP.

Altogether these findings allowed us to draw a decisional flowchart to direct the genotyping of selected LCA genes (Figure 3.2). This flowchart is undoubtedly helpful to lighten the molecular diagnosis in a remarkably genetically heterogeneous condition in which linkage analyses turned out to be little useful to guide the molecular diagnosis in patients (genome identity often found by random, several homozygous loci in consanguineous cases despite informative markers).

5. ELECTRONIC DATABASE INFORMATION

OMIM: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=OMIM

6. ACKNOWLEDGEMENTS

This work was supported by the Associations Retina France and Valentin Haüy. We thank the National Eye Institute for the Travel Award provided to SH.

7. REFERENCES

1.Kaplan, J., Bonneau, D., Frezal, J., Munnich, A., Dufier, J.L., 1990, Clinical and genetic heterogeneity in retinitis pigmentosa. Hum Genet 86:635-42.

2.Perrault, I., Rozet, J.M., Ghazi, I., Leowski, C., Bonnemaison, M., Gerber, S., Ducroq, D., Cabot, A., Souied, E., Dufier, J.L., Munnich, A., Kaplan, J. 1999. Different functional outcome of retGC1 and RPE65 gene mutations in Leber congenital amaurosis. Am. J. Hum. Genet 64:1225-8.

3.Hanein, S., Perrault, I., Gerber, S., Tanguy, G., Barbet, F., Ducroq, D., Calvas, P., Dollfus, H., Hamel, C., Lopponen, T., Munier, F., Santos, L., Shalev, S., Zafeiriou, D., Dufier, J.L., Munnich, A., Rozet, J.M., Kaplan, J., 2004, Leber congenital amaurosis: comprehensive survey of the genetic heterogeneity, refinement of the clinical definition, and genotype-phenotype correlations as a strategy for molecular diagnosis. Hum Mutat 23(4):306-17.

4.Perrault, I., Hanein, S., Gerber, S., Barbet, F., Ducroq, D., Dollfus, H., Hamel, C., Dufier, J.L., Munnich, A., Kaplan, J., Rozet, J.M., 2004, Retinal dehydrogenase 12 (RDH12) mutations in leber congenital amaurosis. Am J Hum Genet 75(4):639-46.

5.Perrault, I., Hanein, S., Gerber, S., Barbet, F., Dufier, J-L., Munnich, A., Rozet, J-M., Kaplan, J., 2003, Evidence of autosomal dominant Leber congenital amaurosis (LCA) underlain by a CRX heterozygote null allele. J Med Genet 40:E90.

6.Hanein, S., Perrault, I., Olsen, P., Lopponen, T., Hietala, M., Gerber, S., Jeanpierre, M., Barbet, F., Ducroq, D., Hakiki, S., Munnich, A., Rozet, J.M., Kaplan, J., 2002, Evidence of a founder effect for the RETGC1 (GUCY2D) 2943DelG mutation in Leber congenital amaurosis pedigrees of Finnish origin. Hum Mutat 20:322-3.

7.Rozet J.M., Perrault I., Gerber S., Hanein S., Barbet F., Ducroq D., Souied E., Munnich A., Kaplan J. Complete abolition of the retinal-specific guanylyl cyclase (retGC-1) catalytic ability consistently leads to leber congenital amaurosis (LCA). Invest Ophthalmol Vis Sci. 2001 May;42(6):1190-2.

8.Perrault, I., Hanein, S., Gerber, S., Lebail, B., Vlajnik, P., Barbet, F., Dufier, J-L., Munnich, A., Kaplan, J. and Rozet, J-M., 2005, A novel mutation in the GUCY2D gene responsible for an early onset severe RP different from the usual GUCY2D-LCA phenotype. Hum Mutat in press.