Peggy Reuter

Mutations in CNGA3 impair trafficking or function of cone cyclic nucleotide-gated channels, resulting in achromatopsia

CNGA3 encodes the A‐subunit of the cone photoreceptor cyclic nucleotide‐gated (CNG) channel, which is a crucial component of the phototransduction cascade in cone outer segments. Mutations in the CNGA3 gene have been associated with complete and incomplete forms of achromatopsia (ACHR), a congenital, autosomal recessively inherited retinal disorder characterized by lack of color discrimination, reduced visual acuity, nystagmus, and photophobia. Here we report the identification of three novel CNGA3 missense mutations in ACHR patients: c.682G>A (p.E228 K), c.1315C>T (p.R439W), and c.1405G>A (p.A469 T), and the detailed functional analyses of these new as well as five previously reported mutations (R283Q, T291R, F547L, G557R, and E590 K), in conjunction with clinical data of patients carrying these mutations, to establish genotype–phenotype correlations. The functional characterization of mutant CNGA3 channels was performed with calcium imaging and patch clamp recordings in a heterologous HEK293 cell expression system. Results were corroborated by immunostaining and colocalization experiments of the channel protein with the plasma membrane. Several mutations evoked pronounced alterations of the apparent cGMP sensitivity of mutant channels. These functional defects were fully or partially compensated by coexpressing the mutant CNGA3 subunit with the wild‐type CNGB3 subunit for channels with the mutations R439W, A469 T, F547L, and E590 K. We could show that several mutant channels with agonist dose–response relationships similar to the wild‐type exhibited severely impaired membrane targeting. In addition, this study presents the positive effect of reduced cell culture temperature on surface expression and functional performance of mutant CNG channels with protein folding or trafficking defects. Hum Mutat 0,1–9;, 2008.

Functional analysis of human CNGA3 mutations associated with colour blindness suggests impaired surface expression of channel mutants A3R427C and A3R563C

Mutations in the CNGA3 gene have been associated with complete and incomplete forms of total colour blindness (achromatopsia), a disorder characterized by reduced visual acuity, lack of colour discrimination, photophobia and nystagmus. CNGA3 encodes the A‐subunit of the cone photoreceptor cyclic nucleotide‐gated (CNG) channel, an essential component of the phototransduction cascade. Here we report the identification of three new CNGA3 mutations in patients with achromatopsia. To assess the pathogenicity of these newly identified and four previously reported mutations, mutant CNGA3 channels were heterologously expressed in a human embryonic kidney cell line (HEK293 cells) and functionally analysed using calcium imaging. Channels with the mutations R427C and R563C showed a response in imaging experiments and were subsequently characterized in‐depth with the patch‐clamp technique. The mutant channels were analysed as homooligomers and also as heterooligomers with the wild‐type B‐subunit present in native channels. Overall, cyclic guanosine monophosphate (cGMP) maximum currents of mutant channels were profoundly reduced in homo‐ and heteromers. Treatment with the chemical chaperone glycerol effectively increased macroscopic currents, presumably by enhancing surface expression of mutant channels as confirmed by immunocytochemistry. These results suggest decreased channel density in the cell membrane due to impaired folding or trafficking of the channel protein as the main pathogenic effect of the mutations R427C and R563C. Moreover, A3R427C homomers showed distinctly increased cGMP and cyclic adenosine monophosphate (cAMP) sensitivities as well as cAMP fractional currents that were raised to over 90% of cGMP maximum currents. Co‐expression of A3R427C with the B3 subunit compensated for most of these aberrant properties, apart from the reduced cGMP maximum currents.

Dissecting the pathogenic mechanisms of mutations in the pore region of the human cone photoreceptor cyclic nucleotide‐gated channel

The CNGA3 gene encodes the A3 subunit of the cone photoreceptor cyclic nucleotide‐gated (CNG) channel, an essential component of the phototransduction cascade. Certain mutations in CNGA3 cause autosomal recessive achromatopsia, a retinal disorder characterized by severely reduced visual acuity, lack of color discrimination, photophobia, and nystagmus. We identified three novel mutations in the pore‐forming region of CNGA3 (L363P, G367V, and E376K) in patients diagnosed with achromatopsia. We assessed the expression and function of channels with these three new and two previously described mutations (S341P and P372S) in a heterologous HEK293 cell expression system using Western blot, subcellular localization on the basis of immunocytochemistry, calcium imaging, and patch clamp recordings. In this first comparative functional analysis of disease‐associated mutations in the pore of a CNG channel, we found impaired surface expression of S341P, L363P, and P372S mutants and reduced macroscopic currents for channels with the mutations S341P, G367V, and E376K. Calcium imaging and patch clamp experiments after incubation at 37°C revealed nonfunctional homo‐ and heteromeric channels in all five mutants, but incubation at 27°C combined with coexpression of the B3 subunit restored residual function of channels with the mutations S341P, G367V, and E376K. Hum Mutat 31:830–839, 2010.

Homozygous missense variant in the human CNGA3 channel causes cone-rod dystrophy.

We assessed a large consanguineous Pakistani family (PKAB157) segregating early onset low vision problems. Funduscopic and electroretinographic evaluation of affected individuals revealed juvenile cone-rod dystrophy (CRD) with maculopathy. Other clinical symptoms included loss of color discrimination, photophobia and nystagmus. Whole-exome sequencing, segregation and haplotype analyses demonstrated that a transition variant (c.955T>C; p.(Cys319Arg)) in CNGA3 co-segregated with the CRD phenotype in family PKAB157. The ability of CNGA3 channel to influx calcium in response to agonist, when expressed either alone or together with the wild-type CNGB3 subunit in HEK293 cells, was completely abolished due to p.Cys319Arg variant. Western blotting and immunolocalization studies suggest that a decreased channel density in the HEK293 cell membrane due to impaired folding and/or trafficking of the CNGA3 protein is the main pathogenic effect of the p.Cys319Arg variant. Mutant alleles of the human cone photoreceptor cyclic nucleotide-gated channel (CNGA3) are frequently associated with achromatopsia. In rare cases, variants in CNGA3 are also associated with cone dystrophy, Leber’s congenital amaurosis and oligo cone trichromacy. The identification of predicted p.(Cys319Arg) missense variant in CNGA3 expands the repertoire of the known genetic causes of CRD and phenotypic spectrum of CNGA3 alleles.

CNGB3 mutation spectrum including copy number variations in 552 achromatopsia patients

Achromatopsia is a rare autosomal recessive cone disorder characterized by color vision defects, photophobia, nystagmus, and severely reduced visual acuity. The disease is caused by mutations in genes encoding crucial components of the cone phototransduction cascade (CNGA3, CNGB3, GNAT2, PDE6C, and PDE6H) or in ATF6, involved in the unfolded protein response. CNGB3 encoding the beta subunit of the cyclic nucleotide‐gated ion channel in cone photoreceptors is the major achromatopsia gene. Here, we present a comprehensive spectrum of CNGB3 mutations and their prevalence in a cohort of 1074 independent families clinically diagnosed with achromatopsia. Of these, 485 (45.2%) carried mutations in CNGB3. We identified a total of 98 different potentially disease‐causing CNGB3 variants, 58 of which are novel. About 10% of patients with CNGB3 mutations only harbored a single heterozygous variant. Therefore, we performed quantitative real‐time PCR in 43 of such single heterozygotes in search of the missing allele, followed by microarray‐based comparative genomic hybridization and breakpoint mapping. We discovered nine different heterozygous copy number variations encompassing one to 10 consecutive exons in 16 unrelated patients. Moreover, one additional patient with a homozygous CNGB3 deletion encompassing exons 4−18 was identified, highlighting the importance of CNV analysis for this gene.

Olfactory Dysfunction in Patients With CNGB1-Associated Retinitis Pigmentosa

Importance Co-occurrence of retinitis pigmentosa (RP) and olfactory dysfunction may have a common genetic cause. Objective To report olfactory function and the retinal phenotype in patients with biallelic mutations in CNGB1, a gene coding for a signal transduction channel subunit expressed in rod photoreceptors and olfactory sensory neurons. Design, Setting, and Participants This case series was conducted from August 2015 through July 2017. The setting was a multicenter study involving 4 tertiary referral centers for inherited retinal dystrophies. Participants were 9 patients with CNGB1-associated RP. Main Outcomes and Measures Results of olfactory testing, ocular phenotyping, and molecular genetic testing using targeted next-generation sequencing. Results Nine patients were included in the study, 3 of whom were female. Their ages ranged between 34 and 79 years. All patients had an early onset of night blindness but were usually not diagnosed as having RP before the fourth decade because of slow retinal degeneration. Retinal features were characteristic of a rod-cone dystrophy. Olfactory testing revealed reduced or absent olfactory function, with all except one patient scoring in the lowest quartile in relation to age-related norms. Brain magnetic resonance imaging and electroencephalography measurements in response to olfactory stimulation were available for 1 patient and revealed no visible olfactory bulbs and reduced responses to odor, respectively. Molecular genetic testing identified 5 novel (c.1312C>T, c.2210G>A, c.2492+1G>A, c.2763C>G, and c.3044_3050delGGAAATC) and 5 previously reported mutations in CNGB1. Conclusions and Relevance Mutations in CNGB1 may cause an autosomal recessive RP–olfactory dysfunction syndrome characterized by a slow progression of retinal degeneration and variable anosmia or hyposmia.

An early nonsense mutation facilitates the expression of a short isoform of CNGA3 by alternative translation initiation

Abstract The cyclic nucleotide‐gated (CNG) channel ‐ composed of CNGA3 and CNGB3 subunits ‐ mediates the influx of cations in cone photoreceptors after light stimulation and thus is a key element in cone phototransduction. Mutations in CNGA3 and CNGB3 are associated with achromatopsia, a rare autosomal recessive retinal disorder. Here, we demonstrate that the presence of an early nonsense mutation in CNGA3 induces the usage of a downstream alternative translation initiation site giving rise to a short CNGA3 isoform. The expression of this short isoform was verified by Western blot analysis and DAB staining of HEK293 cells and cone photoreceptor‐like 661W cells expressing CNGA3‐GST fusion constructs. Functionality of the short isoform was confirmed by a cellular calcium influx assay. Furthermore, patients carrying an early nonsense mutation were analyzed for residual cone photoreceptor function in order to identify a potential role of the short isoform to modify the clinical outcome in achromatopsia patients. Yet the results suggest that the short isoform is not able to compensate for the loss of the long isoform leaving the biological role of this variant unclear. HighlightsAlternative translation initiation results in a short isoform of CNGA3.This CNGA3 isoform retains functional activity as observed by a calcium influx assay.The short CNGA3 isoform is expressed in cone photoreceptor‐like 661W cells.The expression of this CNGA3 isoform was also observed in injected zebrafish larvae.

Erbliche Ionenkanalerkrankungen der Netzhaut

ZusammenfassungRetinale Ionenkanalerkrankungen sind klinisch und genetisch sehr heterogen. Die bisher identifizierten krankheitsassoziierten Ionenkanäle umfassen zyklisch nukleotidgesteuerte (CNG-)Kanäle, spannungsgesteuerte Kalium- und Kalziumkanäle, einen einwärtsrektifizierenden Kaliumkanal, einen kalziumaktivierten Chloridkanal und den transienten Rezeptorpotenzialionenkanal TRPM1. Dieses breite Spektrum spiegelt sich auch in der resultierenden Pathophysiologie wieder. Mutationen in retinalen Ionenkanälen können die Detektion von Lichtreizen bzw. deren Umwandlung in ein elektrisches Signal oder die Weiterleitung des Signals von den Fotorezeptoren zu nachgeschalteten Neuronen beeinträchtigen. Einige Erkrankungen werden auch durch Mutationen in Ionenkanälen, die im retinalen Pigmentepithel lokalisiert sind, hervorgerufen. Dieses ist mit seinen unterstützenden Aufgaben für eine normale Netzhautfunktion essenziell.AbstractRetinal channelopathies are clinically and genetically heterogeneous, and are caused by mutations in genes for a variety of ion channels such as cyclic nucleotide-gated channels, voltage-gated potassium and calcium channels, an inwardly rectifying potassium channel, a calcium-dependent chloride channel and the TRPM1 channel. This broad spectrum of disease-associated ion channels is also reflected in the diversity of pathophysiological consequences. Mutations in retinal ion channels may affect phototransduction, thereby impairing the detection of light or interfere with the transmission of the stimulus from the photoreceptor to second-order neurons. Ion channels located in the retinal pigment epithelium, which supports normal retina function, can also be affected in some diseases.

Development of a pharmacorphore model for pharmacological chaperones targeting mutant trafficking-deficient CNG channels

Complete colorblindness (achromatopsia) is caused by autosomal recessively inherited mutations in the retinal phototransduction pathway, predominantly in the CNGA3- and CNGB3-subunit of the cyclic nucleotide-gated (CNG) channels in cone photoreceptors. CNGA3, which is mutated in about 25% of the achromatopsia patients, mainly harbors missense mutations which frequently impair the folding and/or trafficking of the mutant CNGA3-channels [1]. Pharmacological chaperones stabilizing the folding of the mutant protein may be used to overcome this folding-/trafficking-deficiency. More than 50 compounds were evaluated in their ability to restore signal transduction using a calcium imaging-based bioassay utilizing the CNGA3-mutant E228K [2]. With this data we created several pharmacophore models using Schrodinger Phase [3], which describe the chemical features of potential pharmacological chaperones targeting achromatopsia. We used several approaches leading to different pharmacophore hypotheses: a) Training with the complete set of experimental data (see Figure ​Figure11) Figure 1 Pharmacophore depicting potential features of CNG channel-chaperones. b) Training with only dihydropyridines since this group showed the highest experimental activity, and c) Training with a data set excluding dihydropyridines. Our in-house database TueScreen, which includes ZINC12 [4], was screened to identify potentially active compounds. As a result, several potential molecule classes could be found that may be useful as pharmacological chaperones to improve folding/trafficking of mutant CNG-channels. We will experimentally validate these predictions in a calcium imaging-based bioassay.

Identification And Analysis Of CNGA3 And CNGB3 From Zebrafish

Cyclic nucleotide-gated (CNG) channels are a crucial component of the phototransduction cascade in vertebrate photoreceptors. The opening and closure of these channels and consequently the influx of sodium and calcium ions into the photoreceptor outer segment is directed by the intracellular light-dependent cGMP level. Cone CNG channels are heterooligomers consisting of two A3- and two B3-subunits, which are encoded by the CNGA3 and the CNGB3 gene. In both genes mutations have been identified, which can lead to a dysfunction of the CNG channels in cone photoreceptors. In humans this results in the autosomal-recessively inherited disease achromatopsia (color blindness).In order to characterize CNG channels in zebrafish, which possess four morphologically and physiologically distinct classes of cones, we have identified two homologous candidate genes for CNGA3 and two for CNGB3 by in silico database analyses. All four genes as well as a splice variant of CNGA3-1 have been cloned and were heterologously expressed in HEK293 cells. Subsequently, the zebrafish CNG channels were functionally characterized by calcium imaging and patch-clamp measurements.The retinal expression of all four genes has been confirmed by RT-PCR. In silico analyses revealed, that the two CNGA3 candidates are located at two different locations in the zebrafish genome and are presumably a result of the whole genome duplication as it is known for several genes in zebrafish. In contrast to that, the two CNGB3 candidates are located in a tandem as a result of an additional gene duplication event. ZfCNGA3-1 and zfCNGA3-2 have 62 % identity with the human CNGA3 protein. ZfCNGB3-1 has 43 % and zfCNGB3-2 has 49 % identity with human CNGB3.