Introduction

Complete achromatopsia (total colour blindness; ACHM3; OMIM 262300) is an autosomal recessive disease with an estimated incidence of 1 : 30 000,1 characterised by total inability to distinguish colours, reduced visual acuity, photophobia and nystagmus. The disease was genetically mapped to chromosome 2q11.2-q12 (ACHM2 locus)2,3 and chromosome 8q21-q22 (ACHM3 locus),4,5 which comprise CNGA3 and CNGB3 genes, respectively. Mutations in the genes for the α-subunit (CNGA3) and β-subunit (CNGB3) of the CNG channel expressed in cones are the primary cause of complete achromatopsia in families with diverse geographic or ethnic origin.6,7,8,9

Mutation analysis of affected members from a consanguineous family resident in a rural isolate in central Chile with high disease incidence (1 : 60),10 allowed us to exclude all previously reported CNGA3 or CNGB3 mutations in these subjects. Homozygosity of STRP markers for the ACHM3 locus prompted us to search for the underlying genetic defect in CNGB3. We identified two close nucleotide changes that segregate with the disease, a transition and a single nucleotide insertion. The frameshift insertion, that would cause premature translation termination, is likely responsible for complete achromatopsia in this kindred.

Materials and Methods

Subjects

Ten subjects of Hispanic descent from a rural isolate of 600 inhabitants were diagnosed with complete achromatopsia. These patients exhibited complete inability to discriminate colours, reduced visual acuity (<0,2), nistagmus, and photophobia. Clinical symptoms were evident in early childhood. Fundus examination revealed atrophic macular changes in two older patients. Electroretinographic examination carried in two patients showed reduced cone photopic flicker response.

Personal interviews and records from the local government were used to establish family relationships for 82 individuals and to construct the family pedigree accounted for in this study (Figure 1). Peripheral blood was obtained after informed consent from eight available subjects, five affected and three healthy relatives. This research project was approved by the Ethics Committee at INTA, Universidad de Chile.

Figure 1
figure 1

Pedigree of the family with complete achromatopsia. Genotyping data for 8q21-q22 STRP markers are given below individuals available for analysis. The markers are ordered from centromere to telomere. The arrow indicates the proband.

Genotype analysis with STRP markers

Genomic DNA isolated from white cells by standard phenol-chloroform procedure was used for genotyping with STRP markers D2S2333, D2S113 and D2S2264 on 2q11-q12 and D8S1119, D8S1707, D8S167, D8S273 on 8q21- q22. Marker amplification was carried out by PCR and analysed by denaturing polyacrylamide electrophoresis (see Appendix).

Linkage analysis

We used a modified version of the MLINK 4.03 programme11 compiled for analysis of pedigrees with up to seven consanguinity-loops. An autosomal recessive model of inheritance with no phenocopies and complete penetrance was assumed. The frequency of the disease-causing allele was set to 0.13, reflecting the approximate frequency of the disease in this community. To reduce the computational burden of multipoint linkage analysis, the entire 8q21-q22 haplotype was recoded into a single three-allele marker system, assuming no recombination among such markers.

Mutation screening

Coding exons and intron-exon boundaries were PCR-amplified using primers within flanking introns. At least three independent reactions per amplicon were pooled for sequence analysis using an ABI model 377 DNA sequencer. Mutations were verified on both DNA strands (see Appendix).

Results

Five affected subjects and three unaffected siblings (Figure 1) were genotyped to define whether the disease segregates with genetic markers for the ACHM2 or ACHM3 loci in this family. ACHM2 markers failed to display any haplotype-phenotype association. Conversely, all patients showed homozygosity for D8S167, D8S1707, D8S1119 and D8S273 markers spanning a 1.6 cM interval in 8q21-q22. Whereas, their unaffected siblings were heterozygous. Constructed haplotypes for chromosome 8q21 markers are shown in Figure 1.

Given that allele frequencies for these STRP markers are unknown in this population and that LOD score analysis may be influenced by incorrect specification of such frequencies,12 either equal allele frequency for each marker or allele frequencies drawn from public databases were assumed to compute LOD scores. The frequency of the linked haplotype (2-4-8-3) was set equal to the frequency of the least frequent allele in the haplotype. Under these conditions, LOD scores of 2.15 (equal allele frequency) and 2.74 (allele frequency from databases) were calculated.

Sequence analysis of the eighteen exons from the proband (Figure 1) revealed two nucleotide changes in CNGB3, a transition (c.488A>G) that results in a K to E substitution, and a single frameshift insertion (c.492_493insT) flanking an adenosine (A5) repeat in exon 4. All affected individuals were homozygous for both nucleotide changes; whereas heterozygotes displayed the mixed sequence pattern illustrated in Figure 2A. Two unaffected relatives, whose family relationships were not clearly established (consequently, not included in the pedigree), were also heterozygous for the described sequence modifications. The frameshift insertion, not detected in 100 control chromosomes, creates downstream consecutive nonsense codons at positions 178 and 179.

Figure 2
figure 2

(A) Electropherogram sections of exon 4 sequence around the A5 repeat for homozygous, heterozygous and wildtype subjects. (B) Folding model for cone CNG β-subunit. The arrow indicates the approximate position of the changes in protein sequence.

Discussion

Evidence for association of complete achromatopsia to the CNGB3 gene in this kindred was provided by homozygosity of all four markers spanning a 1.6 cM interval that encompasses the CNGB3 gene in all affected subjects. Unaffected siblings exhibited heterozygous haplotypes (Figure 1).

Sequencing analysis of CNGB3 exons in the proband lead to the identification of two close nucleotide changes in CNGB3. These sequence modifications were confirmed in all available individuals in this kindred. The c.488A>G transition causes a missense substitution (K148E) of an amino acid residue, conserved among human and mouse cone CNGB3 subunits, that is located in the protein amino terminus. However, CNGB1 subunits from human, mouse and bovine rods bear glutamic acid in this position. The frameshift insertion (c.492_493T) creates two consecutive nonsense codons in exon 5 that would cause premature termination of protein translation. The truncated polypeptide is only 177-amino acids long and lacks all membrane spanning segments, acccording to the folding model (Figure 2B). Therefore, it is highly likely that complete achromatopsia in this kindred arises from loss of CNGB3 function attributable to the single base insertion (c.492_493insT).

This family, with four consanguineous marriages, belongs to a community with a high inbreeding coefficient.10 Conceivably, the disease mutation was spread among family members and other inhabitants of the rural isolate due to consanguinity.