CNGA3 mutations in hereditary cone photoreceptor disorders

Abstract

We recently showed that mutations in the CNGA3 gene encoding the alpha-subunit of the cone photoreceptor cGMP-gated channel cause autosomal recessive complete achromatopsia linked to chromosome 2q11. We now report the results of a first comprehensive screening for CNGA3 mutations in a cohort of 258 additional independent families with hereditary cone photoreceptor disorders. CNGA3 mutations were detected not only in patients with the complete form of achromatopsia but also in incomplete achromats with residual cone photoreceptor function and (rarely) in patients with evidence for severe progressive cone dystrophy. In total, mutations were identified in 53 independent families comprising 38 new CNGA3 mutations, in addition to the 8 mutations reported elsewhere. Apparently, both mutant alleles were identified in 47 families, including 16 families with presumed homozygous mutations and 31 families with two heterozygous mutations. Single heterozygous mutations were identified in six additional families. The majority of all known CNGA3 mutations (39/46) are amino acid substitutions compared with only four stop-codon mutations, two 1-bp insertions and one 3-bp in-frame deletion. The missense mutations mostly affect amino acids conserved among the members of the cyclic nucleotide gated (CNG) channel family and cluster at the cytoplasmic face of transmembrane domains (TM) S1 and S2, in TM S4, and in the cGMP-binding domain. Several mutations were identified recurrently (e.g., R277C, R283W, R436W, and F547L). These four mutations account for 41.8% of all detected mutant CNGA3 alleles. Haplotype analysis suggests that the R436W and F547L mutant alleles have multiple origins, whereas we found evidence that the R283W alleles, which are particularly frequent among patients from Scandinavia and northern Italy, have a common origin.

Figure 1

Genomic structure of the CNGA3 gene and sequences of newly identified exons. A, Schematic genomic map of the CNGA3 gene covered by two finished BAC sequences (AC013751 and AC010134) and additional PAC clones isolated from the RPCI1 library and a chromosome 2–specific LLNL library (AI-2-D12). The CNGA3 gene is composed of nine exons, including the newly identified exons 0 and 2b. The locations of STR and SNP markers used for haplotype analysis are indicated at the top. B, Nucleotide sequence of exons 0 and 1 and flanking sequences. cDNA sequences are boxed and are shown in uppercase letters, whereas flanking 5′ genomic and intron sequences are given in lowercase letters. Note that the extent of exon 0 was deduced from the longest 5′ RACE clone. A 38-bp segment conserved between the human CNGA3 gene and the 5′ UTR of the orthologous bovine cDNA is highlighted by a gray box. C, Nucleotide sequence of the alternatively spliced exon 2b and flanking intron sequences. The exon sequence is boxed and is shown in uppercase letters, with the deduced in-frame amino acid sequence shown above it as single-letter codes.

Figure 2

Location of the mutations with respect to the proposed topological model of the CNGA3 polypeptide including the six transmembrane helices (S1–S6), the ion pore, and the cGMP-binding domain (modified according to Zagotta and Siegelbaum [1996]).

Figure 3

Haplotypes of CNGA3 mutant chromosomes. The origins of patients are given in the second column: D = Germany; DK = Denmark; GR = Greece; HT = Haiti; I = Italy; N = Norway; NL = The Netherlands; PK = Pakistan; S = Sweden; TK = Turkey; and US = United States. Similar haplotypes for a given mutation (which probably reflect a common origin) are boxed, and deviations within a group of similar haplotypes are represented by blank haplotype segments. The dark-shaded segment for CHRO180 represent a second-order haplotype deviation. The vertical line marked by an arrowhead represents the relative position of the mutations, between SNP 215+151T→C and D2S2311. Alleles of STR markers are given as fragment sizes.