Ординатура / Офтальмология / Английские материалы / Recent Advances in Retinal Degeneration_LaVail, Hollyfield, Anderson _2008

Fig. 4 Goldmann visual fields (left) and grayscale automated monochromatic perimetry plots obtained under dark-adapted (DA) conditions to 500 nm stimuli (rod-mediated sensitivity loss plots) and under light-adapted (LA) conditions to 600 nm stimuli on white background (L/M conemediated sensitivity loss plots) and to 440 nm stimuli on yellow background (S-cones) for the right (OD, top) and left (OS, bottom) eyes of subject IV:7. Patches of absolute and relative scotomas were evident, paralleled by areas of sensitivity losses at corresponding loci at all wavelengths. More severe and more widespread dysfunction was seen for S-cones

normal rodand cone-mediated sensitivities were identified in the macular region, corresponding to the area of preservation of the I4e target, and in the far periphery. In the remainder of the visual field, dissociation in the severity of rod- (i.e., ≤1.0 log unit) and L/M cone-mediated (i.e., ≥2.0 log unit) sensitivity losses at corresponding loci was also observed

3.2 Female Carriers

Female carriers were either entirely symptom-free subjectively and disease-free to visual function testing (subject V:5), or showed late-onset mild patchy and asymmetric RP with corresponding regional cone>rod areas of dysfunction (subject IV:7, Fig. 4) and cone>rod amplitude losses to ERG testing (Fig. 2). In subject IV:7, monochromatic perimetry revealed that S-cones were significantly more severely affected than L/M-cones. Systemic manifestations were seen also in carriers (Iannaccone et al., 2003).

4 Discussion

Males carrying the G173R RPGR mutation have early-onset sizable reduction in both rodand cone-mediated function, although rod function can still be measured and displays regional variability. These findings indicate that, at least in association with this specific mutation, rod function is still partially preserved in RPGR- associated phenotypes within the first decade of life. On the other hand, although

cone function remains overall better preserved than that of rods, cone disease can be already severe within the retina of children with the G173R mutation, and can exceed that of rods at corresponding loci. This suggests that rods and cones may undergo degeneration in the presence of this mutation as a result of events intrinsic to each cell type and, quite possibly, independent of one another. This is different than what is observed, e.g., in many rhodopsin mutants, in which cone disease tends to follow rod disease (Cideciyan et al., 1998; Iannaccone et al., 2006). This observation underscores the important function that RPGR has not only in rods but also in cones. It remains to be determined if this pattern of rod-cone dissociation is observed also in other RPGR-associated phenotypes.

Female carriers presented with late-onset, cone>rod disease. Comparison between light-adapted thresholds conducted at 440 nm (S-cones) vs. 600 nm (L/M cones) revealed that S-cones are significantly more affected than L/M-cones, with very few loci with normal thresholds to 440 nm stimuli, even when many corresponding loci retained entirely normal L/M-cone function. This also suggests that S-cones may be more vulnerable to defective RPGR function than L/M-cones. We could not obtain 440 nm thresholds in males to verify whether this pattern would occur also in males, or whether it may be unique to females, who have an opposite (i.e., cone>rod) pattern of dysfunction.

The existence of families affected with complex phenotypes resembling closely yet distinct from Usher syndrome has now been well documented. This RPGR- linked syndrome can be associated only with mild to moderate sensorineural hearing loss, as is the case of genuine Usher syndrome type II; with recurrent upper respiratory tract infections; or both (reviewed in Iannaccone et al., 2003, 2004).

Mutations affecting the RPGR RCC1-like domain account for all families with RPGR-linked pseudo-Usher phenotypes reported to date, and likely for earlier reports of a possible X-linked (type-IV) Usher syndrome (Davenport et al., 1978; Gorlin et al., 1979; Grondhal, 1987; Tamayo et al., 1991; Baldellou Vázquez et al., 1993). It cannot be presently excluded that more severe instances of hearing loss, possibly also congenital in nature, may also occur, mimicking Usher syndrome type I. Based on our findings, we propose that males with presumed Usher syndrome who test negative for mutations in Usher genes should be carefully evaluated for the possible presence of any of the aforementioned systemic manifestations and screened for mutations in the RPGR gene.

Acknowledgments Supported by: RPB, New York, NY (unrestricted grants to UTHSC and UM Depts. Ophthalmology, and individual awards to AI, MMJ and AS); NEI grants EY07961 and EY07003 (AS); and FFB, Hunt Valley, MD (AS).

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Mutation in the PYK2-Binding Domain

of PITPNM3 Causes Autosomal Dominant Cone Dystrophy (CORD5) in Two Swedish Families

Linda Köhn, Konstantin Kadzhaev, Marie S. I. Burstedt, Susann Haraldsson, Ola Sandgren, and Irina Golovleva

1 Introduction

Progressive cone or cone-rod dystrophies (CORDs) characterized by a defective cone function demonstrate abnormalities in cone-mediated electroretinogram (ERG) components. The presenting symptoms are defective color vision, impaired central visual acuity and sensitivity to light (Small and Gehrs, 1996; van Ghelue et al., 2000). The inheritance patterns for CORDs are autosomal dominant, autosomal recessive and X-linked (Michaelides et al., 2005a). The preservation of rod function in CORDs can differ both between and within families and depends on the disease causing mutation within a gene (Small and Gehrs, 1996; Michaelides et al., 2005b).

A variant of autosomal dominant cone dystrophy was mapped to 17p12-p13 in a Swedish family (Balciuniene et al., 1995) (CORD5, MIM 600977). Two other studies, one from the UK and one from the USA also showed linkage of dominant cone dystrophy to 17p (Small et al., 1996; Kelsell et al., 1997). The disorder in the British family, designated as CORD6 (MIM 601777) was caused by mutations in the GUCY2D gene (Kelsell et al., 1998). In the USA family, the disease mapped to 17p12-p13 was later reported to be caused by the same mutation as in the British CORD6 family, and therefore, it was concluded that CORD5 and CORD6 is the same disease (Small 1996; Udar et al., 2003). However, in CORD5 patients of Swedish origin there were no indications of GUCY2D mutations (van Ghelue et al., 2000).

Besides GUCY2D, another gene, AIPL1 (MIM 604392) at 17p13 is associated with autosomal dominant CORD (Sohocki et al., 2000). Other variants of autosomal dominant CORDs are caused by mutations in RIM1 (6q14) (MIM 606629), peripherin/RDS (MIM 179605) (6p21.2), GUCA1A (6p21.1) (MIM 600364) and CRX (19q13) (MIM 602225) genes.

Here, we report on fine mapping of the CORD5 locus and a mutation in PITPNM3 associated with CORD5 in two Swedish families.

I. Golovleva

Medical and Clinical Genetics, Department of Medical Biosciences, Umeå University, S-901 85 Umeå, Sweden, Tel: 46 907856820, Fax: 46 90 128163

e-mail: Irina.Golovleva@vll.se

Twelve microsatellite markers in the proximity to D17S938 situated approximately 2 cM apart were used for genotyping. The raw data collected on a 3730 xl DNA analyzer were analyzed with ABI Prism GeneMapper Software v3.0 (Applied Biosystems).

Two-point linkage analyses were done using the FASTLINK implementation of the LINKAGE program package. CORD5 was analyzed as an autosomal dominant trait, with a disease allele frequency of 0.01 using five age-dependent liability classes (Balciuniene et al., 1995).

For bidirectional sequencing of PITPNM3 (MIM 608921), coding and adjacent intronic sequences of 19 exons were amplified from genomic DNA. Primers designed with Primer3 software (http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_ www.cgi) can be found in Köhn et al (2007).The sequencing reactions were separated on a 3730 xl DNA analyzer (Applied Biosystems). Sequences were compared with a reference sequence of the PITPNM3 gene (ENSG00000091622) available on the http://www.ensembl.org.

3 Results

3.1 Linkage and Haplotype Analysis

Fine mapping of the CORD5 locus revealed significant LOD scores at θ = 0 for D17S945 to D17S1828 with a maximum of 12.67 at D17S938 (Fig. 2A, Table 1). The reconstructed haplotypes in both families (Fig. 1) confirmed segregation of CORD5 with markers D17S678, D17S938, D17S1881, D17S720 and D17S1844. In family 151 the CORD locus was limited by one recombination event for D17S945 in three affected individuals and in family 152 recombination events at D17S1828 and D17S1854 were detected. Thus, the CORD5 locus was narrowed down from 26.9 cM to 14.3 cM including flanking markers. In this region at least three genes of interest were present: AIPL1, an aryl-hydrocarbon interacting protein-like 1; GUCY2D, guanylate cyclase 2D, both known to be mutated in cone rod dystrophy and Leber congenital amaurosis; and PITPNM3, a membrane associated phosphatidylinositol transfer protein 3, a human homologue of the Drosophila retinal degeneration B (rdgB). AIPL1 and GUCY2D were excluded by SSCP and direct sequencing.

3.2 Mutation Analysis

The human rdgB homolog, PITPNM3 contains 20 exons and encodes a protein belonging to the phosphatidyl-inosytol transfer protein (PITP) family. PITPNM3 was proposed to be a gene causing retinal degenerations (Lev et al., 1999; Lev, 2001) based on linkage of several retinal disorders to 17p12-p13. Sequence analysis of 19 exons revealed a transversion c.1878G>C (NM_031220) in exon 14 resulting

Fig. 2 Genomic structure of the PITPNM3 gene and segregation of Q626H with CORD5. A – schematic representation of the genomic region in proximity to the D17S938 marker. Exon 2 in PITPNM3 encodes a Ca+2-binding domain, six transmembrane domains spread over exons 6, 7, 10, 11, 12 and exons 14 to 20 encode PYK2-binding domain. The PITP domain characteristic for this family is missing in PITPNM3. Q626H in exon 14 is marked with an asterisk. B – c.1878G>C (p.Q626H) detected by direct sequencing. C – segregation of CORD5 and the Q626H mutation in family 151. Filled symbols indicate affected individuals, while empty symbols indicate unaffected. D – partial protein sequence alignment of PYK-2 binding domain between mammalian PITPNM3 (http://www.ebi.ac.uk/clustalw/). Position 626 is marked in bold

in substitution of a glutamine at position 626 by a histidine (p.Q626H) (Fig. 2B). PCR-RFLP analysis using MaeII endonuclease showed consistent segregation of the Q626H in all affected individuals in family 151 (Fig. 2C).

Three individuals from family 152 with unknown disease history shared the same haplotype as all affected individuals (Fig. 1) and also had the Q626H mutation.

Table 1 Two-point LOD scores between markers on chromosome 17p13 and CORD5

a marker location is based on the human genome assembly Build 35

Shared haplotypes and presence of the Q626H mutation in both families indicate that these families have a common descent (Fig. 1).

The Q626H mutation was absent on 322 control chromosomes of ethnically matched healthy individuals and in 140 individuals affected with autosomal dominant or recessive forms of retinitis pigmentosa. Residue 626 in PITPNM3 is located in a PYK2-binding domain (Lev et al., 1999), which is evolutionary conserved in mammals (Fig. 2D).

4 Discussion

Presence of the Q626H mutation in Swedish CORD5 patients, its absence on healthy chromosomes and the fact that PITPNM3 is a Drosophila retinal degeneration homologue causing light-induced retinal degeneration and severely impaired ERG (Harris and Stark, 1977) indicate a potential pathogenic role of the PITPNM3 gene.

In humans PITPNM3 is expressed in the brain, spleen and ovary (Lev et al., 1999). pl-RdgB, a zebrafish homologue, is mainly expressed in inner segments of cone photoreceptors (Elagin et al., 2000), while in rat retina PITPNM3 is found through all cell layers but mainly in Muller cells (Tian and Lev, 2002).

Members of the PITPfamily contain a phosphatidylinositol transfer domain (PIT), an acidic region/Ca+2 binding domain, six transmembrane domains, the DDHD and a C-terminal. PITPs participate in phospholipase C-mediated inositol signaling, ATP-dependent Ca+2– activated secretion, lipid metabolism, trafficking from Golgi-membranes and exocytosis (Lev, 2004). An importance of a PIT domain was shown in rdgB mutant flies with light-dependent retinal degeneration which was rescued by transfer of the PIT-domain (Milligan et al., 1997). However, transgenic expression of zebrafish p1-RdgB lacking a PIT domain in Drosophila rdgB2 null mutant improved photoreceptor survival but did not show any effect on ERG (Elagin et al., 2000) suggesting that PIT-domain is necessary for light response and function of others protein domains prevents photoreceptors degeneration.

Recently it was shown that the C-terminal region of the PITPNM3 is necessary for the interaction with protein tyrosine kinase PYK2 (Lev et al., 1999; Lev, 2004). We showed that the Q626H mutation in PITPNM3 is located in PYK-2 binding domain, therefore one obvious expectation might be that the mutation abolishes or modifies the interaction with PYK2 in humans. Further screening for additional PITPNM3 mutations in both familiar and isolated cases and studies on model organisms carrying the Q626H mutation will clarify the mechanisms by which mutations in the PYK-2-binding domain result in defective vision.

In summary, this study adds one more gene on 17p, which causes retinal degeneration. We provide evidence that CORD5 in Swedish patients is a distinct clinical entity and describe the first disease causing mutation within the PYK2 – binding domain in the PITP family.

Acknowledgments This study was supported by grants from Visare Norr and University Hospital of Umeå.

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Elagin, V.A., Elagina, R. B., Doro, C. J., Vihtelic, T. S., and Hyde, D. R., 2000, Cloning and tissue localization of a novel zebrafish RdgB homolog that lacks a phospholipid transfer domain, Vis Neurosci. 17:303.

van Ghelue, M., Eriksen, H. L., Ponjavic, V., Fagerheim, T., Andreasson, S., Forsman-Semb, K., Sandgren, O., Holmgren, G., and Tranebjaerg, L., 2000, Autosomal dominant cone-rod dystrophy due to a missense mutation (R838C) in retinal guanylate cyclase gene (RETGC-1) is associated with considerable variation, Ophthalmol Genet. 21:197.

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Kelsell, R. E., Evans, K., Gregory, C. Y., Moore, A. T., Bird, A. C., and Hunt, D. M., 1997, Localization of a gene for dominant cone-rod dystrophy (CORD6) to chromosome 17p, Hum Molec Genet. 6:597.

Kelsell, R. E., Gregory-Evans, K., Payne, A. M., Perrault, I., Kaplan, J., Yang, R. B., Garbers, D. L., Bird, A. C. A., Moore, T., and Hunt, D. M., 1998, Mutations in the retinal guanylate cyclase (RETGC-1) gene in dominant cone-rod dystrophy, Hum Molec Genet. 7:1179.

Köhn, L., Kadzhaev, K., Burstedt, M. S. I., Haraldsson, S., Hallberg, B., Sandgren, O., and Golovleva, I., 2007, Mutation in the PYK2-binding domain of PITPNM3 causes autosomal dominant cone dystrophy (CORD5) in two Swedish families, Eur J Hum Genet. 15:664.

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Michaelides, M., Holder, G. E., Bradshaw, K., Hunt, D. M., and Moore, A. T., 2005b, Conerod dystrophy, intrafamilial variability, and incomplete penetrance associated with the R172W mutation in the peripherin/RDS gene, Ophthalmol. 112:1592.

Milligan, S. C., Alb, J. G., Elagina, R. B., Bankaitis, V. A., and Hyde, D. R., 1997, The phosphatidylinositol transfer protein domain of Drosophila retinal degeneration B protein is essential for photoreceptor cell survival and recovery from light stimulation, J Cell Biol. 139:351.

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Identification and Characterization of Genes

Expressed in Cone Photoreceptors

Mehrnoosh Saghizadeh, Novrouz B. Akhmedov, and Debora B. Farber

1 Introduction

Most human hereditary retinal degenerations can be classified as rod-cone degenerations (such as retinitis pigmentosa), cone-rod degenerations (exemplified by some cone dystrophies), and diseases affecting cones exclusively (i.e., cone degenerations)(Krill, 1977; Hamel, 2007; Simunovic and Moore, 1998). In these disorders the disease process is often difficult to analyze because its time-course is slow (many years) and tissues for morphologic, biochemical or molecular biology studies are not always available. Use of animal models of retinal degeneration and advanced biotechnology have helped to elucidate the cause of some of these diseases and the mechanisms by which mutated genes lead to blindness. In general, when there is widespread degeneration of rods, regardless of the selective or not selective expression of the gene product in these cells, there is a concomitant loss of cones. In contrast, molecular defects affecting cone photoreceptors result in diseases either manifesting subsequent loss of rod-mediated vision or just loss of cones without progressive and generalized involvement of rods. Since cones, like rods, have their own unique set of genes and proteins, it is still not clear why mutations in any of the rodor cone-specific genes can lead to degeneration of the other type of photoreceptor.

Cone dystrophies are usually associated with a panretinal loss of cones that often affects the macula resulting in loss of central vision and day blindness. Although cone gene products have a major contribution to vision, only few genes expressed in cones have been studied in detail. A complete understanding of cone-associated retinal diseases will be achieved only after the function of most of the uncharacterized genes in cone cells has been elucidated. This is why for many years we have been interested in the isolation and characterization of genes expressed in cone photoreceptors. To carry out this task, we initially took advantage of the adult cd

M. Saghizadeh

Jules Stein Eye Institute, David Geffen School of Medicine and Molecular Biology Institute, UCLA, Los Angeles, California 90095, Tel: 310-206-6935, Fax: 818-986-7400

e-mail: nooshs@ucla.edu