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APPENDIX

Visual Dysfunction Caused by Mutant CNG Channel Genes

Mutations in cyclic nucleotide-gated (CNG) channel genes can cause the malfunction and degeneration of photoreceptors and, concomitantly, partial or complete blindness. Mutations in the genes encoding the CNG channel subunits of rod photoreceptors— CNGA1 and CNGB1—account for a few percent of recessive retinitis pigmentosa (RP) [1–3]. RP is characterized by night blindness, a progressive loss of the peripheral visual field, and eventual loss of central vision, resulting in blindness. These symptoms reflect early dysfunction and degeneration of rod photoreceptors, followed by a slower degeneration of cone photoreceptors that proceeds from the periphery to the center of the visual field (for review, see [4, 5]). Mutations in the genes for the CNG channel subunits of cones— CNGA3 and CNGB3—can cause achromatopsia. Achromatopsia is a recessive, nonprogressive disease resulting from the dysfunction of cone photoreceptors. Symptoms include absence of color vision, light sensitivity (photophobia), and poor visual acuity. The prevalence of achromatopsia has been estimated as 1:30,000 (for review, see [6, 7]), with approximately 20–30% of all cases caused by mutations in CNGA3 and 40–50% caused by mutations in CNGB3 [8]. In some instances, mutations in CNGA3 or CNGB3 result in cone dystrophy, a disease similar to achromatopsia but characterized by the progressive loss of cone function and sometimes progressive loss of rod function [9].

The steps that lead to rod or cone degeneration and, in particular, the role of CNG channel subunits in this process are not well understood. The recent success, however, in the identification of CNG channel mutants involved in visual diseases and the study of their defects by heterologous expression have provided insight into the molecular basis of photoreceptor dysfunction. The results of molecular genetic analyses of patients with

Fig. A1. Mutations in cyclic nucleotide-gated (CNG) channel subunits associated with photo receptor dysfunction. See text for details. Underlined amino acid substitutions are found in patients with residual cone function. cNMP nucleoside 3′,5′-cyclic monophosphate, del deletion, Fs frame shift mutation, GARP glutamic acid-rich protein.

RP, achromatopsia, and cone dystrophy, as well as the in vitro experiments with the respective mutant CNG channel subunits are discussed next.

Mutations in CNGA1 and CNGB1 Associated with Retinitis Pigmentosa

Currently, six different mutant CNGA1 alleles have been identified in RP patients [1, 2] (Fig. A1). Three of these mutant alleles carry stop codons that terminate translation within the cytoplasmic N-terminus of CNGA1 (R28stop, E76stop, and K139stop). Another mutant CNGA1 allele is deleted in most of the protein-coding region and does not encode functional channels (not shown in figure). The remaining two mutant alleles encode either a single amino acid substitution (S316F) or an amino acid substitution and a truncation C-terminal to the cNMP-binding site (R654D-stop). Dryja

and collaborators expressed CNGA1S316F and CNGA1R654D-stop in human embryonic kidney cells and found that these mutants form few functional homomeric channels

[1]; cyclic guanosine monophosphate (cGMP)-dependent currents carried by single or

few CNG channels were detected only in 3 of 85 (CNGA1S316F) or 1 of 83 membrane patches (CNGA1R654D-stop). Similar results were obtained with the bovine CNGA1R654D-stop homolog (CNGA1R656D-stop) expressed in Xenopus oocytes by Mallouk and collaborators

[10]. Coexpression of CNGA1R656D-stop with CNGB1 did not rescue impaired channel

trafficking. Surprisingly, neither truncation of the C-terminus (R656stop) nor the substitution R656D alone impaired surface expression. Apparently, the C-terminal five amino

acids of CNGA1R656D-stop (KLKQD) generate a novel signal that causes retention in the endoplasmic reticulum (ER). Indeed, substitution of the C-terminal five amino acids of

wild-type CNGA1 for KLKQD produces a mutant with impaired surface expression. Furthermore, Mallouk and coworkers provided evidence that the C-terminal sequence of CNGA1R656D-stop serves as a specific retention signal. Replacement of two key residues,

leucine (L) or aspartate (D), in the C-terminal sequence KLKQD of CNGA1R656D-stop restored normal surface expression. This strong influence of single residues at the very

C-terminus argues against the possibility that the mutations in CNGA1R656D-stop just cause improper protein folding.

In contrast to the results reported by Dryja and Mallouk and their collaborators, Trudeau and Zagotta observed normal surface expression and channel formation of the

human CNGA1 mutant CNGA1R654D-stop in Xenopus oocytes [11]. Impaired trafficking to the membrane occurs only when CNGA1R654D-stop is coexpressed with CNGB1. Using a biochemical pull-down assay, Trudeau and Zagotta demonstrated stable binding of

a peptide representing the C-terminus of CNGA1 (amino acids 609–693) to a peptide that represents the region between the glutamic acid-rich protein (GARP) domain and the first transmembrane segment of CNGB1 (amino acids 677–764). Removing the C-terminal portion from the CNGA1 peptide, which corresponds to amino acids 657–693 in wild-type CNGA1, abolished this binding. Trudeau and Zagotta concluded from their results that the C-terminal amino acids 657–693 of CNGA1—which are deleted in R654D-stop—mask an ER retention signal within the amino acids 677–764 of CNGB1. In coexpression experiments with CNGB1 deletion mutants, this retention signal was confined to a segment of ten amino acids that precedes the first transmembrane segment of CNGB1 (YQFPQSIDPL). At present, it is not clear how the contradictory observations reported by Dryja and Mallouk and their collaborators on the one hand and by Trudeau and Zagotta on the other hand can be reconciled.

Only a single mutation in the CNGB1 gene has yet been associated with RP [3]. The mutation results in the substitution of a highly conserved glycine for valine (G993V). This glycine is thought to reside in a turn between two β strands (β2 and β3) that contribute to a β-roll inside which the ligand binds [12]. The substitution of valine for glycine at this site might impair the relative orientation of the two β strands and result in a nonfunctional cNMP-binding site.

Mutations in CNGA3 and CNGB3 Associated with Cone Dysfunction

Genetic screens identified 57 mutant CNGA3 alleles that are associated with achromatopsia or cone dystrophy [9, 13–16]. The majority of these alleles (45) cause single amino acid substitutions, indicating that there is little tolerance for sequence variations in CNGA3 with respect to cone function. A genomic region that includes the CNGB3 gene was identified as a locus for achromatopsia in people from Pingelap, an atoll of Micronesia [17, 18]. About 5% of the Pingelap population is affected by achromatopsia, also known as Pingelapese blindness (OMIM 262300). This story gained notoriety as the subject of Oliver Sacks’ book, The Island of the Colorblind [19]. The prevalence of the disease traces back to 1775, when a typhoon decimated the population of Pingelap,

Fig. A2. Achromatopsia affects color vision and visual acuity. A sunset as it might be seen by a person with normal color vision (A), by two sisters who confuse desaturated colors due to an incomplete form of achromatopsia (B), or by a person with complete achromatopsia (C).

leaving only a handful of survivors who repopulated the island. Genetic analysis identified a missense mutation in the CNGB3 gene (S435F) as the genetic basis of Pingelapese achromatopsia [20, 21].

Other than the Pingelap mutation, 15 additional mutant CNGB3 alleles have been associated with cone dysfunction [13, 15, 20–22]. Eight of these mutant alleles encode CNGB3 subunits lacking the cNMP-binding site due to stop-codon or frame-shift mutations. The remaining mutated CNGB3 alleles encode missense mutations in the p region or the cNMP-binding site, and one allele harbors a splice-site mutation in intron 13 (not shown in figure).

Complete achromats are likely to carry mutant CNGA3 or CNGB3 alleles encoding nonfunctional channel proteins. Indeed, several mutant CNGA3 subunits associated with complete achromatopsia fold improperly and fail to reach the plasma membrane in heterologous expression systems [23, 24]. The fact that CNGA3 forms homomeric channels in heterologous expression systems raises the possibility that patients carrying CNGB3 null alleles do have cone CNG channels composed of CNGA3 only. In rod photoreceptors, however, the B subunit is essential for proper targeting of CNG channels to the outer segment [25]. Similarly, the lack of CNGB3 might cause low levels of CNGA3 in cone outer segments and thereby prevent cone function. In this respect, it would be informative to examine cone photoreceptors in dogs with naturally occurring mutations in CNGB3 [26].

In contrast to complete achromats, incomplete achromats are expected to carry at least one CNGA3 and one CNGB3 allele that allow some degree of cone function. Similarly, patients with cone dystrophy are likely to carry mutants that permit cone function until the cells degenerate. Alleles found exclusively in incomplete achromats or patients with cone dystrophy are underlined in Fig. A1. The in vitro characterization of CNGA3 or CNGB3 mutants encoded by these alleles might help scientists understand why the maintenance and degree of cone function varies among patients with impaired cone function.

We analyzed the molecular basis of an incomplete form of achromatopsia, with considerable cone function, present in two sisters who both express the mutants

CNGA3T224R and CNGA3T369S [27]. The sisters are able to discriminate saturated but not desaturated colors. Figure A2 gives an example of how the sisters would view colorful

scenery. Detailed clinical characterization of the sisters, including psychophysical and electroretinographical analyses, showed that their cone system is characterized by