A ninth locus (RP18) for autosomal dominant retinitis pigmentosa maps in the pericentromeric region of chromosome 1
A ninth locus ( RP18 ) for autosomal dominant retinitis pigmentosa maps in the pericentromeric region of chromosome 1Su Ying Xu1, Marianne Schwartz2, Thomas Rosenberg3 and Andreas Gal1,*
1Institut für Humangenetik, Universitäts-Krankenhaus Eppendorf, Butenfeld 32, D-22529 Hamburg, Germany, 2Section of Clinical Genetics, Department of Pediatrics, Rigshospitalet, Copenhagen, Denmark and 3National Eye Clinic for the Visually Impaired, Hellerup, Denmark
Received April 16, 1996;Revised and Accepted May 24, 1996
We studied a large Danish family of seven generations in which autosomal dominant retinitis pigmentosa (adRP), a heterogeneous genetic form of retinal dystrophy, was segregating. After linkage had been excluded to all known adRP loci on chromosomes 3q, 6p, 7p, 7q, 8q, 17p, 17q and 19q, a genome screening was performed. Positive lod scores suggestive of linkage with values ranging between Z = 1.58-5.36 at [theta] = 0.04-0.20 were obtained for eight loci on proximal 1p and 1q. Close linkage without recombination and a maximum lod score of 7.22 at [theta] = 0.00 was found between the adRP locus (RP18) in this family and D1S498 which is on 1q very near the centromere. Analysis of multiply informative meioses suggests that in this family D1S534 and D1S305 flank RP18 in interval 1p13-q23. No linkage has been found to loci from this chromosomal region in six other medium sized adRP families in which the disease locus has been excluded from all known chromosomal regions harbouring an adRP gene or locus suggesting that there is (at least) one further adRP locus to be mapped in the future.
Retinitis pigmentosa (RP) designates a frequent but heterogeneous genetic form of retinal dystrophy affecting ~1 in 3000 people and responsible for the visual handicap of ~1.5 million individuals worldwide (for references and review, see 1 ,2 ). The condition can be inherited as an autosomal dominant, autosomal recessive or X-linked trait, whereas a considerable portion are simplex cases in which the pattern of inheritance remains undetermined. Typical clinical symptoms of RP include night blindness, reduced visual acuity and progressive loss of peripheral vision leading to blindness usually after the fourth decade. Ophthalmological examinations show constricted visual fields, attenuated vessels and abnormal accumulation of pigment in the retina.
Autosomal dominant retinitis pigmentosa (adRP) accounts for ~20% of all RP cases and is characterized by significant allelic and non-allelic heterogeneity (for references and review, see 1 ,3 ). Eight loci for adRP have been mapped genetically to date on chromosomes 3q, 6p, 7p, 7q, 8q, 17p, 17q and 19q (for review, see 4 and references therein, and 5 ). Of the eight corresponding adRP genes, those on 3q and 6p encode rhodopsin (for review, see 1 and references therein) and peripherin (for review, see 6 and references therein), respectively. Here we report the mapping of a further adRP locus (RP18) on the pericentric region of human chromosome 1.
We studied a large Danish adRP family of seven generations (Fig. 1 ). Clinical diagnosis was based on a history of night blindness, typical ocular fundus with peripheral bone spicule formation and severe constriction of the retinal arterioles, and progressive visual field defects beginning as mid-peripheral ring scotomas. Disease manifestation was rather uniform, with both sexes equally affected. Twelve subjects had one or several eye examinations performed at the National Eye Clinic, including full field electroretinography (ERG) and dark adaptometry a.m. Goldmann Weekers. Many of the patients claimed that night blindness was present `from birth' on. Nevertheless, pathological dark adaptation starting with a prolonged mesopic phase and elevation of the fully dark-adapted threshold did not occur until the end of the first decade. Atrophy of the retinal pigment epithelium with typical bone spicule hyperpigmentation was also observed from the second half of the first decennium. From the beginning of the second decade, the ERG showed abolished rod responses and strongly attenuated cone responses with prolonged implicit times. ERG was extinguished from the beginning of the third decade if measured by conventional techniques. Visual field defects progressed during the third and fourth decades, leaving the central area relatively spared. Accordingly, visual acuity remained above 0.3 in the majority of patients for the first three decades. Most of the affected subjects had myopia in the range of -2 to -4 diopters, but a few cases with slight hypermetropia or high myopia were also encountered. Some of the patients developed a moderate photophobia; glaucoma was diagnosed in three patients in their sixties. Complicating cataracts were observed as fine subcapsular opacifications which did not require lensectomy. Some of the older patients were totally blind from the age of 80.
The data presented in this communication strongly suggest that a gene implicated in autosomal dominant RP maps in the pericentromeric region of chromosome 1. On a recent integrated map including physical and genetic data (8 ), D1S534 and D1S305, the two loci flanking RP18, are placed <30 Mb apart, which defines the maximal size of the critical region and corresponds to about one-ninth of the total length of chromosome 1.
RP18 represents the ninth locus for the autosomal dominant form of RP assigned by linkage analysis and confirms the extreme non-allelic genetic heterogeneity of the trait. It is not yet known what proportion of patients with RP have mutations in the corresponding gene on chromosome 1. Both linkage studies on families and screening of larger collections of individual patients suggest that mutations of rhodopsin and peripherin account for, respectively, ~25 and 5% of all adRP cases (for review see, respectively, 1 and 6 and references therein). In contrast, the majority of the remaining seven adRP loci have each been assigned in a single large family or have been detected in a few families (for references and review, see 1 ,4 ,12 ). We have found no linkage to the corresponding marker loci on chromosome 1 in six other medium sized adRP families in which the disease locus has been excluded from all known chromosomal regions harbouring an adRP gene or locus (unpublished results). Therefore, it is likely that there is-at least-one further adRP locus to be mapped in the future.
We have found two recombinants between GNAI3 and RP18 which practically excludes the possibility that a mutation in GNAI3 is the primary genetic cause of the disease in this family. As both recombinants were in individuals affected by RP, phenotypic misclassification due to reduced penetrance can also be rejected.
GNAT2 encodes the [alpha]-subunit of a retina-specific transducin expressed exclusively in cone photoreceptors (9 ). Transducin is a heterotrimeric ([alpha],[beta],[gamma]) protein whereas the [alpha]-subunit defines both the specificity for interaction with the receptor and the effector function. Stimulated receptor activates transducin/the [alpha]-subunit by catalysing the exchange of the [alpha]-bound GDP for GTP and the dissociation of the [alpha]-subunit from the [beta]- and [gamma]-subunits (for review, see 11 and 13 and references therein). As RP affects primarily the rod photoreceptors, we consider that the probability is low that the disease segregating in the Danish family is due to a mutation in GNAT2, although formal proof is missing. Independent support for this assumption comes from the observation that mammalian G protein [alpha]-subunit genes belong to a multigene family thought to be generated by a series of genome duplications during evolution. A recent model hypothesizes that GNAT2 and GNAI3 evolved from a single ancestral gene by a subsequent tandem duplication (11 ), i.e. that the two genes are physically linked. A similar pair of G protein [alpha]-subunit genes (GNAT1 and GNAI2) has been found on chromosome 3p21 (11 ). Given the fact that in the Danish family GNAI3 maps ~20 cM from RP18 and under the assumption of close physical proximity of GNAI3 and GNAT2 in 1p13 on one hand and in view of the expression of GNAT2 in cone photoreceptor cells on the other hand, our data suggest that GNAT2 is a very unlikely candidate for the gene mutated and responsible for the adRP in the Danish family described here.
Molecular genetic and biological analysis of other candidate genes from this chromosomal region (8 ), e.g. KCNA3 (potassium voltage-gated channel) or NGFB (nerve growth factor) will be carried out to prove or disprove their role in the pathogenesis of this form of retinal dystrophy.
Medical files on affected members of the seven-generation kindred studied here (Fig. 1 ) were retrieved from the Danish Retinitis Pigmentosa Register (14 ). Autosomal dominant inheritance was documented by several instances of male-to-male transmission. Penetrance seems to be complete as no case of a skipped generation was observed.
The whole study was carried out in accordance with the Helsinki Declaration II and approved in advance both by the Danish National Register Authority (Registertilsynet) and the Scientific Ethical Committee (KF 01-226/93). Informed, written consent was obtained from all participants.
Blood samples for linkage analyses were obtained from 20 patients, eight unaffected relatives older than 21 and therefore considered as not affected, three unaffected relatives below the age of 20 and thus classified with disease phenotype unknown, and six spouses. Experimental procedures for genotyping were carried out following standard protocols. Detection of allelic fragments was done by silver staining essentially as described elsewhere (15 ). Linkage analysis was performed with the Liped program (16 ).
For PCR amplification of a 293 bp DNA segment from the 5' end of the GNAI3 gene, oligonucleotide primers GNAI3-5'F (5'-GCCA-CCGCCCAGCAATAGAC-3') and GNAI3-5'R (5'-CCCGACCAGACCCCGTTCAG-3') were designed based on published sequence data (10 ). SSCP analysis and direct sequencing of PCR products of aberrant electrophoretic mobility were carried out as described elsewhere (15 ). Single-base substitutions were found at nucleotide positions -72 (T -> C) and -61 (C -> T). The T/C and C/T changes result in gain of a recognition site for BanI and BpuI and allelic cleavage products of 293/273 + 20 and 293/260 + 33 bp, respectively. Allele frequencies for linkage analysis were calculated from the genotypes of eight spouses in the family.
The experimental work described here was financially supported by the Deutsche Forschungsgemeinschaft. The Danish Retinitis Pigmentosa Register received grants from the Danish Association of the Blind (DBS) whereas blood sampling was financially supported by Cykelhandler P. Th. Rasmussens og Hustru Alma Rasmussens Mindelegat.
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*To whom correspondence should be addressed
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