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Clustering and frequency of mutations in the retinal guanylate cyclase (GUCY2D) gene in patients with dominant cone-rod dystrophies

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Editor—Guanylate cyclase (retGC-1) is a key enzyme in the recovery phase of phototransduction in both cone and rod photoreceptor cells.1 Upon excitation by a photon of light, an enzymatic cascade of events occurs which leads to the hydrolysis of cGMP and the closure of the cGMP gated cation channels. This results in hyperpolarisation of the plasma membrane and the generation of a signal higher up in the visual pathway. Upon closure of the ion channels, the cytosolic levels of Ca2+ decrease because export by the Na+, K+, Ca2+exchanger continues. This reduced Ca2+ concentration results in the activation of retGC by activating proteins (GCAPs) and the increased conversion of GTP to cGMP, thus restoring the level of cGMP in the photoreceptors to their dark level.

Mutations in GUCY2D, the gene encoding retGC-1, are a cause of Leber congenital amaurosis (LCA1), a recessive condition which manifests itself either at birth or during the first few months of life as total or near total blindness.2 3 Recently, we identified mutations inGUCY2D in four British families with autosomal dominant cone-rod dystrophy (ADCORD).4Subsequent to this, mutations in this gene were shown to be responsible for ADCORD in a French,5 a Swiss,6 and a Norwegian7 family. In all seven families, the mutations are either in the same or in adjacent codons in a highly conserved region of the protein. In our four families and in the Swiss and Norwegian families, mutations were found in either codon 837 or 838,4 6 7 whereas codons 837-839 each encode for an amino acid substitution in the French family.5

In order to determine whether ADCORD arising from mutations inGUCY2D are restricted to these codons and how important these mutations are to autosomal retinal disease in general, we have screened an additional group of unrelated patients diagnosed with autosomal dominant macular dystrophy or autosomal dominant cone or cone-rod dystrophy.



The coding exons of GUCY2D were amplified using the intronic primers and annealing temperatures essentially as described previously2 4 and subjected to heteroduplex analysis.8 All fragments exhibiting band shifts were directly sequenced using the PRISMTM Ready Reaction Sequencing Kit (Perkin Elmer PE Biosystems), and the products were visualised on an ABI Model 373 DNA sequencer.


One of each primer pair was end labelled with 10 μCi of [γ-32P]ATP using polynucleotidyl kinase for 30 minutes at 37°C , followed by 10 minutes at 65°C. PCR was carried out using 1.5 mmol/l MgCl2, 0.2 mmol/l dNTP mix, KCl buffer, 0.05 U/ml Taq polymerase (Bioline), 0.1 mmol/l of each primer, and 0.1-0.2 μg of genomic DNA. The amplification protocol was 94°C for three minutes, followed by 35 cycles at 94°C for 30 seconds, 56°C for 30 seconds, and 72°C for 30 seconds. The resulting products were visualised on a 6% polyacrylamide/urea denaturing gel. The gel was dried down at 80°C under vacuum and autoradiographed over x ray film overnight. The DISLAMB program9 was used to obtain an estimate of linkage disequilibrium.


A group of 40 patients, 27 with autosomal dominant macular dystrophy and 13 with autosomal dominant cone or cone-rod dystrophy, was screened for mutations in all exons ofGUCY2D. This group was drawn from the same panel that was used in our original study4 and is composed of unrelated patients with autosomal dominant macular dystrophies or cone or cone-rod dystrophies attending a Medical Retina Clinic at Moorfields Eye Hospital, London, UK. From this screen, three additional probands with mutations in GUCY2D were identified. Of these, two have the identical R838C substitution to that previously reported4 and one has a novel G2586A transition in codon 838, resulting in an R838H substitution (fig 1). In addition, a re-examination of our CORD6 family has shown a second mutation, a C2585A transversion again in codon 838 that results in the substitution of arginine by serine (fig 1). This mutation is in the adjacent codon to the originally reported E837D substitution.4 This is therefore a second example of a GUCY2Ddisease allele carrying multiple mutations. In total, five of our families carry a C to T change in codon 838, one family has a G to A change in codon 838, and one family has a double mutation in codons 837 and 838. All these mutations were confirmed by restriction enzyme digestion, since all cause the loss of aHhaI site. None of these changes were observed in 50 ethnically matched controls. In each case, the diagnosis was confirmed as cone-rod dystrophy4 10 (D Bessant, personal communication). Excluding the original CORD6 family, the 90 unrelated patients screened in this and the previous study therefore yielded a total of six ADCORD patients with mutations in codon 838 of the GUCY2D gene. The above mutations, together with all previously reported mutations,4-7 are summarised in table1.

Figure 1

Sequence of exon 13 of retGC1. Heterozygous mutations in adjacent codons of the original CORD6 family to give the Glu837Asp and Arg838Ser substitutions, and in family 8 to give the Arg838His substitution are shown.

Table 1

Dominant cone-rod mutations in the GUCY2D gene

Haplotype analysis was used to investigate whether there is evidence for relatedness among the five families with the R838C substitution (table 2). In order to determine the haplotype of the disease chromosome, additional family members were sought. However, family 5 could not be extended beyond the original proband; the disease associated alleles for markers D17S1881 and D17S1852 could not therefore be fully resolved. All families show some commonality for marker alleles adjacent to the GUCY2D gene; families 2, 3, 5, and 6 share allele 5 at D17S960, families 2, 4, 6, and possibly 5 share allele 2 at D17S1796, and families 3 to 6 share allele 5 at D17S1881. However, although family 3 shares the same allele as families 4, 5, and 6 at D17S1881, it is unlikely that this is part of a founder haplotype since it would require a double crossover within a very short map interval. An estimate of the likelihood of linkage disequilibrium was obtained from the DISLAMB program9 by using allele frequencies obtained from 20 unrelated “married in” subjects in the families. This is significant at the 5% probability level only for D17S960; the lower estimates of λ and p for the other markers reflect in part the common occurrence of the disease associated alleles in the “married in” subjects.

Table 2

Microsatellite markers in the vicinity of the GUCY2D gene

During our extensive sequence analysis of theGUCY2D gene, a number of single nucleotide polymorphisms (SNPs) were identified as follows: a silent C220A transversion in exon 2, coding G227A (A52S) and G227T (A52T) changes in exon 2 (the G227T transversion has been previously reported as a possible sequence polymorphism2), a silent G2182A transition in exon 10, a coding T2418A (L783H) transversion in exon 12, a silent G2589A transition in exon 13, a G to A transition in intron 17, and a T insertion in intron 19. Unfortunately, in each of our R838C disease families, the more common nucleotide was present at each position. These SNPs do not therefore help to resolve the ancestry of the R838C mutations.


In this and our previous study,4 the panel of patients with autosomal dominant disease was drawn at random from unrelated subjects who had received the diagnosis of cone-rod, cone, or macular dystrophy. Our previous study examined 50 members of this panel and identified three probands with an R838C mutation in the GUCY2D gene. In this follow up study of a further 40 patients, three additional patients with mutations in this codon have been identified, two with an R838C substitution and one with an R838H substitution.

The clinical phenotypes in the families with single (R838C or R838H) and double (E837D, R838S) mutations have been reported in detail elsewhere.4 10 11 In summary, the cone-rod dystrophy exhibited by the single mutation patients is less severe than that in the original CORD6 family with the double mutation, with mild variation in disease severity in the R838C families. In all cases, photophobia with decreased visual acuity and loss of colour vision is present from early childhood. However, during the early phases of the disorder when visual acuity is still good, a marked reduction in visual function in bright light is characteristically present. Fundoscopic abnormalities are confined to the central macula with increasing central atrophy with age. Electrophysiological testing showed a marked loss of cone function with only minimal rod involvement in the single mutation families. This contrasts with expression in the CORD6 family where moderate to severe rod involvement is present.11 Different mutations in this region of the GUCY2D gene can result therefore in differing severities of cone-rod dystrophy, especially with regard to the involvement of the scotopic system.

Pooling across our two studies, a conservative estimate of the overall frequency of mutations in codon 838 ofGUCY2D among autosomal dominant patients with macular, cone, or cone-rod dystrophy is therefore 6.7%, although this rises to 23% if only the three new mutations found among the 13 cone and cone-rod dystrophy patients examined in this study are considered. It is important to emphasise that these two frequencies are estimates of the relative contribution that mutations in this codon make to the total frequency of autosomal dominant cone-rod disease in the population and that this conclusion is valid irrespective of the presence or absence of a founder effect for the R838C mutations. Whether such a founder effect is present is unclear from the present data. There is evidence for linkage disequilibrium between the disease allele and one of the flanking markers (D17S960) although, since the disease associated allele is relatively common (28%), this renders the test of association less powerful, and the situation is not further resolved by a number of SNPs scattered through theGUCY2D gene, since none was informative in our five families. Where a founder effect has been clearly established, for example for Sorsby's fundus dystrophy,12 a highly significant disease associated haplotype covering 3 cM of the chromosomal region surrounding the disease gene was present. In contrast, the disease associated haplotype for the R838C mutations covers <0.2 cM. This indicates that either the R838C mutations have arisen separately from each other or that a single mutation occurred in a much more distant ancestor than the common mutation for Sorsby's fundus dystrophy, with a consequent wider distribution in the population. Furthermore and again irrespective of the presence or absence of a common ancestor for our R838C families, the occurrence of the R838C mutation in a presumably unrelated Norwegian family,7 the R838H mutations in one of our British families and in a Swiss family,6 and the multiple mutations in codon 838 and adjacent codons in the original CORD6 family,4 as well as in a French family,5 all identify this codon as particularly mutation prone.

Two other dominantly inherited diseases have been associated with mutation prone regions: recurring C to T and G to A transitions were found in adjacent nucleotides within theMYH7 gene in hypertrophic cardiomyopathy13 and recurring G to A transitions and G to C transversions were found at the same nucleotide within theFGFR3 gene in achondroplasia.14The recurring DNA transitions at these two loci are situated at CpG dinucleotides and a study of nucleotide substitution rates15 has confirmed the high mutability of CpG sequences. The spontaneous deamination of methylated cytosine, its relatively slow repair in mammalian cells, and the production of an intermediate susceptible to deamination in the enzymatic process by which cytosine itself is methylated, are all mechanisms which make CpG sequences preferential targets for spontaneous mutation.16 17 It is perhaps significant therefore that the C to T transitions and C to A transversions found in codons 838 and 839 of the GUCY2D gene all occur within a CpG dinucleotide (fig 2). What remains unclear is the mechanism responsible for the generation of multiple mutations in this region of exon 13 of the GUCY2Dgene.

Figure 2

Nucleotide and amino acid substitutions in exon 13 of GUCY2D associated with autosomal dominant cone-rod dystrophy.

Recessive mutations in GUCY2D are a relatively common cause of LCA. However, the widespread distribution of LCA mutations (including missense, frameshift, and splice site changes) throughout the gene18 contrasts with the clustering of ADCORD mutations to codons 837, 838, and 839 encoded by exon 13. To date, no LCA mutations have been localised to exon 13. The causative ADCORD mutations may, however, be even more restricted since the E837D substitution present in patients with double (the original CORD6 family) and triple mutations5 would appear by itself to have little effect on enzyme activity in vitro.19 In contrast, the changes in codon 838, R838H, R838C, or R838S, have all been shown to alter the sensitivity of the protein to Ca2+inhibition via interactions with GCAPs.19-21 Substitution at this site may be the critical change therefore in all cases so far reported, in causing ADCORD rather than recessive LCA. The effect of this dominant mutation is a change in function (altered Ca2+ sensitivity) whereas the recessiveLCA1 mutations may represent loss of activity.22

There have been reports of other retinal dystrophies mapping to regions of chromosome 17p which overlap with the position of theGUCY2D gene. These include two dominant cone dystrophies and dominant central areolar choroidal dystrophy, diseases which exhibit degeneration primarily of the cone-rich macular region only.23-25 As yet, there have been no reports ofGUCY2D mutations in these disorders, despite screening this gene in patients with central areolar choroidal dystrophy.26


We thank the patients for their cooperation in this study. This work was supported by the Wellcome Trust (grant numbers 041905 and 053405) and the Medical Research Council (grant number G9301094). We would also like to thank the Wellcome Trust for a Major Equipment Grant for the sequencing facility (grant number 039283).