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

reported. Since their description, these observations have been largely dismissed or overlooked.2,3 In 1992, Moller opened a debate about the existence of these isolated recessively inherited simple optic atrophy and concluded that: “a very clear-cut well documented pedigree has yet to be published”.1

Here, we report a large multiplex consanguineous family segregating a true isolated autosomal recessive optic atrophies and the mapping of the disease-causing gene on chromosome 8q21-q22 at the ROA1 locus.

2. MATERIALS AND METHODS

A large multiplex consanguineous family of French origin affected with isolated autosomal ROA was ascertained through the Ophthalmologic Consultation of the “Hôpital des Enfants Malades” of Paris (Figure 4.3). All family members underwent general and ophthalmologic examinations and the mitochondrial DNA was analysed to exclude the diagnosis of Leber Optic Neuropathy (LHON [MIM 535500]).

A genome-wide search for homozygosity was undertaken with 382 pairs of fluorescent oligonucleotides of the Genescan Linkage Mapping Set, Version II (Perkin Elmer Cetus) under conditions recommended by the manufacturer. The polymorphic markers have an average spacing of 10 cM. Amplified fragments were electrophoresed and analysed on an automatic sequencer (ABI 3100, Applied Biosystems, Foster City, USA). Linkage analyses were performed using M-LINK and LINKMAP of the 5.1 version of the Linkage program.5,6 (gene frequency ~1/1000, penetrance = 1; allele frequencies available from the CEPH database).

All candidate genes at the ROA1 locus were screened by direct sequencing.

3. RESULTS

3.1. Clinical Evaluation of Isolated ROA1 Family

Four out of the five sibs of the ROA1 family (Figure 4.3) were affected with an isolated, early-onset but slowly progressive optic neuropathy. Between 2 and 6 years of age, all patients complained of a visual impairment that could not be corrected by glasses. At age 10, fundus examination, fluorescein angiography, visual field testing, colour vision analysis and electrophysiological recordings of individual V2, led to the diagnosis of optic atrophy without retinal degeneration. The three younger sibs displayed the same phenotype (Figures 4.1 and 4.2). In all affected sibs, the progression of the disease was very slow (visual acuity ranging from 1/10 to 2/10 for distant vision at 36 < age of patients < 45 years old). Moderate photophobia and dyschromatopsia with red-green confusion was noted. None of the four patients had nystagmus. Both parents (IV1 and IV2), and V1 underwent complete ophthalmologic exploration and no symptom of optic neuropathy was noted. A normal ocular pressure was measured for all members of the family, as well as a normal cup/disc ratio of the optic disk. Neurological examination failed to reveal any developmental failure, pyramidal or extra pyramidal signs, ataxia or hearing loss. Furthermore, no alopecia, diabetes or any malformation was noted, allowing the exclusion of all known syndromes associated with ROA.

Figure 4.1. Visual field and colour vision testings in normal control and patient V4 (age = 32 years).

A) Dynamic perimetry using a Goldman apparatus to test the visual field. The visual field of the patient is almost normal, except for the existence of a relative and partial scotoma at II1. B) Colour vision was tested using the D- 15 Farnsworth’s panel. The circling line on the left indicates that the normal individual was able to harmoniously classify the D colour chips 15 while the patient made a disorganized classification with confusion in the red-green axis.

The four patients were born to healthy second-cousins (Figure 4.3). This consanguinity associated with i) the existence of a common affected ancestor, ii) the absence of any affected individual in the common branch, ruling out autosomal dominant inheritance with incomplete penetrance, iii) the transmission of the disease gene through four healthy men (II2, III2, III3, and IV3), ruling out X-linked inheritance and iv) the affected male-female ratio of 1, strongly support the conclusion of autosomal recessive inheritance.

3.2. Primary Mapping of the Disease-Causing Gene

The 10-cM genome-wide search in the ROA1 multiplex family showed only one chromosomal region in which informative markers were found to be homozygous in all four patients. Indeed, all patients shared a common homozygote haplotype on chromosome 8q21q22. One recombination event detected in V2 as well as a loss of homozygosity detected in all patients allowed to define a critical homozygote region between D8S1702 and D8S1794

4. DISCUSSION

In 1992, Moller pointed out that the survey on causes of blindness in children reported by Fraser and Friedmann in 19677 and the review over 18 years of registration of visually impaired Danish children did not mention any recessive form of simple optic atrophy and concluded that “a clear-cut, well-documented pedigree had yet to be published”.4 Here, we describe a family with an unambiguous isolated ROA. In contrast to the few cases formerly reported with a very early and severe form of isolated ROA, the four patients of this family were affected with an early-onset but moderately progressive form of the disease. The clinical phenotype was strikingly different from AOD (no central scotoma, red-green dyschromatopsia).

A genome-wide search for homozygosity led to the identification of a single homozygous region on chromosome 8q21-q22 (ROA1 locus). This result excluded linkage and thus allelism of ROA1 with all known syndromic ROA (see OMIM).

Among the genes of the [D8S1702-D8S1794] 12 Mb physical interval, CNGB3 was first considered. Indeed, although the phenotype of all patients was strikingly different from that described in total colour blindness, the report of severe dyschromatopsia close to achromatopsia in the few known cases of isolated ROA2,3 prompted us to search for mutations in this gene but no mutation was found.

Subsequently, we screened all genes encoding mitochondrial proteins owing to the view that known hereditary optic neuropathies genes are either mitochondrial (LHON) or encode proteins with high mitochondrial targeting (OPA1, OPA3, TIMM8A [MIM 300356]). Thus we first decided to screen genes known to encode mitochondrial protein such as DECR1 and PDP and proteins potentially targeted to the mitochondria, using the prediction programs MITOPROT and PSORT. The exclusion of these candidate genes prompted us to screen all known genes lying within the interval and subsequently several hypothetical genes. Since no mutation was found, we decided to screen several genes lying within the haploidenticial region defined by the D8S1794 and D8S1699 loci following the hypothesis that, in this family, the common ancestor might be affected with ROA and could carry two different mutations segregating through each branch of the family. No mutation was found and further studies are now required i) to identify the causative gene in this family and ii) to delineate the role of this locus in small non-consanguineous families affected with ROA in which linkage analysis can not be conclusive.

Finally, one can expect that the identification of the ROA1 gene and other genes involved in ROA will allow the dissection of molecular mechanisms underlying optic neuropathies to aid genetic counselling and to develop rational therapeutic tools.

5. 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 FB.

6. ELECTRONIC DATABASE INFORMATION

MITOPROT, Prediction of mitochondrial targeting sequences; http://ihg.gsf.de/ihg/ mitoprot.html OMIM, Online Mendelian Inheritance in Man; http://www.ncbi.nlm.nih.gov/

entrez/query.fcgi?db=OMIMPSORT, Prediction of Protein Sorting Signals and Localization Sites in Amino Acid Sequences; http://psort.nibb.ac.jp/; UCSC, Human Genome Project Working Draft, http://genome.ucsc.edu

7. REFERENCES

1.F. Barbet, S. Hakiki, C. Orssaud, S. Gerber, I. Perrault, S. Hanein, D. Ducroq, J-L. Dufier, A. Munnich, J. Kaplan., and J-M. Rozet, A third locus for dominant optic atrophy on chromosome 22q, J Med Genet. 42:e001 (2005)

2.J. François, Affections du nerf optique; in Masson (eds), L’hérédité en ophtalmologie. Paris, 581-606 (1958)

3.S. Merin, Inherited diseases of the optic nerve, Dekker M (eds): Inherited eye diseases, Diagnosis and clinical management. New York, 323-344 (1991)

4.H. U. Moller, Recessively inherited, simple optic atrophy–does it exist? (Letter), Ophthalmic Paedia Genet. 13:31-32 (1992)

5.G. M. Lathrop, and J. M. Lalouel, Easy calculations of lod scores and genetic risks on small computers, Am J Hum Genet. 36:460-5 (1984)

6.E. S. Lander, and D. Botstein, Homozygosity mapping: a way to map human recessive traits with the DNA of inbred children, Science. 236:1567-70 (1987)

7.G. R. Fraser., and A. I. Friedmann, The Causes of Blindness in Childhood. A Study of 776 Children with Severe Visual Handicaps, Johns Hopkins Press 1 (1967)

interacting with RPGR. Furthermore, using RCC1-like domain of RPGR as bait, two-hybrid screening suggested that the RLD is a key region for this interactions. A RPGR-deficient mouse model for XLRP was created by gene knockout with homologous recombination.25 In that study, exons 6-8 were knocked out, and the authors did not detect any RPGR expression in variant organs of the knockout mice through RT-PCR or Northern blot. It indicated that transcripts lacking of exons 6-8 within RCC1-like domain was insufficient for the normal function of RPGR in the retina.

3. MACRODELETION OF RCC1-LIKE DOMAIN IN XLRP

So far, six deletions larger than one kilobase within RPGR gene were reported10,11,13,26-28 (Table 5.1). Before the cloning of the RPGR gene, Roepman et al screened 30 unrelated patients with XLRP and identified one deletion spinning exons 15, ORF15 and exon 15a. Meindl et al identified another deletion from IVS13 to IVS15 which the exon 14-15 were absent. Other studies confirmed two deletions within RCC1-like domain, exons 8-l0 and part of exon 8. Here, we describe a XLRP family with novel macrodeletion of the RPGR gene spanning from exon 1 to exon 11. Two affected males had severe symptoms with early onset and rapid deterioration. In the beginning, exons 1-11 could not be amplified by PCR while other exons were amplified successfully. To determine the suspected deletion, southern blot analysis was performed with a probe from the normal PCR products of exon 2 to exon 11, respectively. However, the result appeared normal. A Macrodeletion less than 2 M base was suspected and the two sides of the suspected deletion were amplified with designed primer sets. The deletion spanning the RCC1-like domain was confirmed with direct PCRsequencing. The deletion start-point was located 79 bp in front of exon 1, and the end-point was 42 bp downstream of exon 11 (Figure 5.2). We did not detect the pattern of the expression in the two patients, but it suggested the deletion of the RCC1-like domain could be involved in Japanese XLRP. It is suspected that transcripts lacking RLD would lose combining sites with PDE-delta or RPGRIP1, and that RLD is essential for the normal function of RPGR in the human retina.

4. SUMMARY

Clinical research into mutations of the RPGR gene showed that lack of either the RCC1like domain of the ORF15 causes X-linked retinitis pigmentosa. Thus, the ORF15 and

Table 5.1. Deletions in RPGR Gene.