Author: veronica tomatis
Date: 14/03/2013



Achromatopsia is an autosomal recessive genetic disorder affecting the cone cells of the eye. It may be complete, in which the individual can only perceive black, white, or shade of gray, or incomplete, in which the individual has a residual amount of colour vision.


Autosomal recessive achromatopsia is a rare disorder with an estimated prevalence of less than 1:30,000.
Parental consanguinity is common in certain geographical regions. On the island of Pingelap in the eastern Caroline Islands in Micronesia, the prevalence of achromatopsia is between 4% and 10%, derived from the founder mutation p.Ser435Phe in CNGB3.


Achromatopsia is characterized by a reduced visual acuity pendular nystagmus an increased sensitivity to light (photophobia), a small central scotoma eccentric fixation and reduced or complete loss of color discrimination. All individuals with achromatopsia (called achromats) have impaired color discrimination along all three color vision axes, each one corresponding to the three cone classes: the protan or long-wavelength-sensitive cone axis (red), the deutan or middle-wavelength-sensitive cone axis (green), and the tritan or short-wavelength-sensitive cone axis (blue). Most individuals have complete achromatopsia with total lack of function of all three types of cones. Rarely, individuals have incomplete achromatopsia, in which one or more cone types may be partially operating. The symptoms are similar to those of individuals with complete achromatopsia, but generally less severe. Hyperopia is common. Nystagmus develops during the first few weeks after the birth followed by increased sensitivity to bright light. Best visual acuity varies with the seriousness of the disease; it is 20 out of 200 or less in complete achromatopsia and may be as high as 20/80 in incomplete achromatopsia. Visual acuity is usually stable over time while both nystagmus and sensitivity to bright light may improve slightly. Although the fundus is usually normal, macular changes and vessel narrowing may be present in some affected individuals.


Clinical diagnosis

The clinical diagnosis of achromatopsia is based on the presence of typical clinical findings:
* Reduced visual acuity
* Pendular nystagmus
* Increased sensitivity to light
* Small central scotoma
* Eccentric fixation
* Reduced or complete loss of color discrimination

Molecular Genetic Testing
Genes: the following four genes are associated with autosomal recessive a chromatopsia
CNGB3 ~50% of affected individuals
CNGA3 ~25% of affected individuals
GNAT2 ~1% of affected individuals
PDE6C ~1% of affected individuals
Other loci: an additional locus (ACHM1) had been assigned to chromosome 14 as a result of a single case of maternal uniparental isodisomy of chromosome 14.
Clinical testing: targeted mutation analysis for the most common mutation in CNGB3 is available on a clinical basis. The 1-bp deletion c.1148delC accounts for more than the 70% of all mutant CNGB3 alleles. The analysis of the CNGA3, CNGB3, GNAT2, and PDE6C is available on a clinical basis.


Each known cause of congenital achromatopsia is due to malfunction of the retinal phototransduction pathway. Specifically, recessive forms of achromatopsia result from the inability of cone photoreceptors to properly respond to a light stimulus because of hyperpolarization.

Normal visual pathway:

Phototransduction phototrasdution is a process by which light is converted into electrical signals in the rod cells, cone cells and photosensitive ganglion cells of the retina of the eye.
The visual cycle is the biological conversion of photons into an electrical signal in the retina. This process occurs via G-protein coupled receptors called opsins which contain the chromophore 11-cis retinal. 11-cis retinal is covalently linked to the opsin receptor via Schiff base forming retinylidene protein. When struck by photons, 11-cis retinal undergoes photoisomerization to all-trans retinal. This changes the conformation of the opsin GPCR leading to signal transduction cascades which causes closure of cyclic GMP-gated cation channel, and hyperpolarization of the photoreceptor cell.
Following isomerization and release from the opsin protein, all-trans retinal is reduced to all-trans retinol and travels back to the retinal pigment epithelium to be "recharged". It is first esterified by lecithin-retinol acyltransferase (LRAT) and then converted to 11-cis retinol by the isomerohydrolase RPE65. While the isomerase activity of RPE65 has been shown, it is still uncertain whether it acts as hydrolase too. Finally, it is oxidized to 11-cis retinal before traveling back to the rod outer segment, where it is again conjugated to an opsin to form new functional visual pigment (rhodopsin).

The photoreceptor cells involved in vision are the rods and cones. These cells contain a chromophore (11-cis retinal, the aldehyde of Vitamin A1 and light-absorbing portion) bound to cell membrane protein, opsin. Rods deal with low light level and do not mediate color vision. Cones, on the other hand, can code the color of an image through comparison of the outputs of the three different types of cones. Each cone type responds best to certain wavelengths, or colours, of light because each type has a slightly different opsin. The three types of cones are L-cones, M-cones and S-cones that respond optimally to long wavelengths (reddish color), medium wavelengths (greenish color), and short wavelengths (bluish color) respectively. Humans have a trichromatic visual system consisting of three unique systems, rods, mid and long-wave-length sensitive (red and green) cones and short wavelength sensitive (blue) cones.

Process: to understand the photoreceptor's behaviour to exposure to light intensities, it is necessary to understand the roles of different currents.
There is an ongoing outward potassium current through nongated K+selective channels. This outward current tends to hyperpolarize the photoreceptor at around -70 mV (the equilibrium potential for K+).
There is also an inward sodium current carried by cGMP-gated sodium channels. This so-called 'dark current' depolarizes the cell to about-40 mV.
A high density of Na+-K+ pumps enables the photoreceptor to maintain a steady intracellular concentration of Na+ and K+.

• In the dark
Photoreceptor cells are “strange” because they are depolarized in the dark, meaning that light hyperpolarizes and switches off these cells. This 'switch off' activates the next cell and sends an excitatory signal down the neural pathway.
In the dark, cGMP levels are high and keep cGMP-gated sodium channels open allowing a steady inward current, called the dark current. This dark current keeps the cell depolarized at about -40 mV. The depolarization of the cell membrane opens voltage-gated calcium channels. An increased intracellular concentration of Ca2+ causes vesicles containing special chemicals, called neurotransmitters, to merge with the cell membrane, therefore releasing the neurotransmitter into the synaptic cleft, an area between the end of one cell and the beginning of another neuron. The neurotransmitter released is glutamate, a neurotransmitter whose receptors are often excitatory.

• In the light
1. A light photon interacts with the retinal in a photoreceptor cell: the retinal undergoes isomerisation, changing from the 11-cis to all-trans configuration.
2. Retinal no longer fits into the opsin binding site.
3. Opsin therefore undergoes a conformational change to metarhodopsin II.
4. Metarhodopsin II is unstable and splits, yielding opsin and all-trans retinal.
5. The opsin activates the regulatory protein transducin. This causes transducin to dissociate from its bound GDP, and bind GTP, then the alpha subunit of transducin dissociates from the beta and gamma subunits, with the GTP still bound to the alpha subunit.
6. The alpha subunit-GTP complex activates phosphodiesterase.
7. Phosphodiesterase breaks down cGMP to 5'-GMP. This lowers the concentration of cGMP and therefore the sodium channels close.
8. Closure of the sodium channels causes hyperpolarization of the cell due to the ongoing potassium current.
9. Hyperpolarization of the cell causes voltage-gated calcium channels to close.
10. As the calcium level in the photoreceptor cell drops, the amount of the neurotransmitter glutamate that is released by the cell drops too. This is because calcium is required for the glutamate-containing vesicles to fuse with cell membrane and release their contents.
11. A decrease in the amount of glutamate released by the photoreceptors causes depolarization of on-centre bipolar cells (rod and cone on bipolar cells) and hyperpolarization of cone off-centre bipolar cells.


In achromatopsia:

Achromatopsia has been associated with the mutation in four genes that play an essential role in the cone phototrasdution pathway. The first two genes discovered were CNGA3 (ACHM2 locus on chromosome 2q11) and CNGB3 (ACHM3 locus on chromosome 8q21), which respectively encode the α and β subunits of the cGMP-gated channel present in the photoreceptors. More recently, a third gene, GNAT2 was implicated in achromatopsia and was mapped to locus ACHM4 on chromosome 1p13. This gene codes for the α subunit of the cone photoreceptor transducing G-protein in the phototrasdution pathway. The fourth gene implicated is PDE6C (ACHM5) encodes the α-prime subunit of cone phosphodiesterase.
In particular: 25% of all ACHM patients carry mutation in CGNA3, (especially in patient from Middle East, them are called ACHM2), 45-50% carry mutation in CGNB3 (especially in European patients, in which prevalence is 87%, them are called ACHM3) whereas only few families have been reported to have GNAT2 (ACHM4) or PDE6C (ACHM5) mutations.

CNGA3 mutations in two United Arab Emirates families with achromatopsia (december 2007)

Cyclic nucleotide-gated ion channels:

INTRODUTION: cyclic nucleotide-gated ion channels (also known as CNG channels) are ion channels that operate in response to the binding of cyclic nucleotides. CNG channels are nonselective cation channels that are found in the membranes of various tissue and cell types, and are significant in the sensory transduction as well as in the cellular development. Their function can be the result of a combination of the binding of cyclic nucleotides (cGMP and cAMP) and either a depolarization or a hyperpolarization event. Initially discovered in the cells that make up the retina of the eye, CNG channels have been found in many different cell types across both the animal and the plant kingdoms. CNG channels have a very complex structure with various subunits and domains that play a critical role in their function.
They are directly activated by cyclic nucleotides, and approximately 4 cyclic nucleotides are needed to activate each channel. CNG channels are non-selective and allow many alkali ions to flow into or out of a cell expressing CNG channels on its membrane. This flow of ions can result in either depolarization or hyperpolarization. CNG channels can be activated by cAMP or cGMP exclusively, or sometimes by a combination of both cNMPs, and some channels are more selective than others.

STRUCTURE: a CNG channel consists of four subunits around a central pore. Each protein subunit consists of 6 transmembrane segments (S1-S6), a P-loop, an intracellular amino terminal region, and a carboxy terminal region. The P-loop and S6 segments are around the pore, which plays a role in ion conduction. There is a cyclic nucleotide binding domain (CNBD) and a connection region to the S6 segment in the carboxy terminal. There is a post-CNDB region in the amino terminal.
The structure of the pore is similar to other ion channels that contain P-loops. The P-loop enters the membrane of the pore from the extracellular side and exits to the intracellular side. The P-loop enters as an alpha helix and exits as an uncoiled strand. Helices cover the inner membrane line the channel. These also form a 6 helix bundle that signifies the entrance. In order to open the pore, a conformational change must occur in the inner 6 helix bundle.
The cyclic nucleotide-binding domain is an intracellular domain located in the C-terminus region and has a similar sequence to other cyclic nucleotide-binding proteins. The domain is believed to be made up of a β-pleated sheet and two α-helices. The β-pleated sheet is made up of eight antiparallel strands. The α helices are named the B and C helices. A ligand initially binds to the β-pleated sheet, and through allosteric regulation causes the movement to an α-helix toward the β-pleated sheet. The α-helix is flexible in closed channels. When an α-helix of a CNGA1 subunit is in close proximity to another α-helix, they form intersubunit disulfide bonds. This occurs mainly in closed channels, inhibiting movement of the α-helix towards the β-pleated sheet. When a ligand binds to the β-pleated sheet, this bound cyclic nucleotide stabilizes the movement of the α-helix toward the β-pleated sheet in each subunit, pulling the α-helices away from each other.



Cone vision mediated by CNG channel activation is essential for central and color vision and visual acuity. Naturally occurring mutations in CNGA3 and CNGB3 are associated with achromatopsia and progressive cone dystrophy in particular mutations in CNGA3 and CNGB3 account for 70% of all mutations found in achromatopsia patients. Nearly 50 mutations have been identified in the CNGA3 subunit. Among these mutations, the R277C and R283W substitutions are identified as the two most frequently occurring mutations. The R218C and R224W mutations appear to cause loss of channel function by impairing the channel cellular trafficking and plasma membrane targeting.


Normal allelic variants: CNGA3 consists of eight coding exons. Only a few normal allelic variants and some rare variants are observed; most occur within non-coding regions or do not result in an amino acid substitution.
Normal gene product: The polypeptide is 694 amino acids long and has a size of 78.8 kd. An alternatively spliced exon that extends the open reading frame by an additional 55 amino acids has been reported. CNGA3 encodes the cyclic nucleotide-gated cation channel alpha 3 (the alpha subunit of the cone photoreceptor cGMP-gated cation channel [CNG]). In vitro expression experiments have shown that alpha subunits on CNG channels alone are able to form functional homo-oligomeric channels, yet their biophysical properties differ from those of heteromeric native CNG channels consisting of two alpha and two beta subunits.
Pathologic allelic variants: More than 70 different mutations have been reported. The vast majority of mutations are missense (<80%). Only a few nonsense mutations, insertions, and deletions have been observed. Several studies of patient affected from ACHM2 evidence a mutation in CGNA3, in particular the DNA analysis revealed a change of nucleotide G to T at position 822 in exon 8. This nucleotide change results in the substitution of a serine residue for an arginine residue. The arginine residue at this position is conserved among the CGN alpha subunit of rods and olfactory neurons.


Normal allelic variants: CNGB3 consists of 18 coding exons. Only a few polymorphisms and rare variants are observed; most occur within non-coding regions or do not result in an amino acid substitution.
Normal gene product: The polypeptide is 809 amino acids long. CNGB3 encodes for cyclic nucleotide-gated cation channel beta 3 (the beta subunit of the cone photoreceptor cGMP-gated cation channel). In vitro expression experiments showed that beta subunits alone are not able to form functional homo-oligomeric channels; they are therefore thought to be modulatory subunits. Functional cone CNG channels consist of two alpha and two beta subunits.
Pathologic allelic variants: More than 40 different mutations have been reported. The vast majority are nonsense mutations, frame-shift deletions and insertions, and putative splice site mutations. Only a few missense mutations (~10%) have been observed. One, resulting in the p.Ser435Phe mutant protein, causes "Pingelapese blindness" in achromats originating from the island of Pingelap in Micronesia. The recurrent single base-pair deletion c.1148delC is the most common mutation underlying achromatopsia worldwide, accounting for approximately 70% of all CNGB3 disease-causing alleles and approximately 40% of all achromatopsia-associated alleles and results from a founder effect. The deletion results in a frameshift and premature termination of the protein. This frameshift should give rise to non-sense mediated decay of the mutant RNA, and it can therefore be considered a loss of function mutation.

Novel CNGA3 and CNGB3 mutations in two Pakistani families with a chromatopsia (april 2010)


As shown in figure, the intracellular calcium response to cGMP (100 μM) stimulation was completely abolished in cells expressing the R218C mutant. The response in R224W mutant was lowered to less than 10% (as analyzed at 300 sec after cGMP stimulation) of the wild type response. The deficiency of the mutant channels was also confirmed by electrophysiological recordings. Patch clamp shows that no current was recorded in cells expressing the mutants, leading to the conclusion that the R218C and R224W mutations abolished the channel activity. While the physiological assays indicated abolished or severely reduced responses, Western blot analysis revealed similar levels of mutant expression when compared to wild type expression. These data together indicate channel deficiency, not expression levels, as that which contributes to loss of cone function in achromatopsia patients.

Using immunofluorescence labelling it was determined cellular localization of the wild type and mutant channel subunits in cells. The scientists observed that cells expressing the R218C and R224W mutants showed apparent cytosol aggregation of the immunofluorescent signal. The Quantification immunofluorescence intensity analysis showed that cells expressing the mutants displayed the decreased plasma membrane labelling compared to cells expressing the wild type subunit. This data indicates the mislocalization of mutants is a contributing factor to decreased cone function.

Analysing the effects of co-expression of the mutant channel subunits with the wild type channel on the channel activity, the scientist found that it did not affect the wild type channel’s activity. The calcium responses to cGMP stimulation in cells co-expressing the wild type with R218C or R224W mutants were almost indistinguishable from those in cells expressing the wild type channel alone. This data indicates that the wild type channels do not form heterodimers with mutant channels. Otherwise it indicates that the wild type/ mutant heterodimers do form and that the wild type channels are dominant in the complex.

The other two genes implicated in achromatopsia are GNAT2 and PDE6C, but their mutations are responsible for the disease of only about 2% of the patients.

Molecular Pathogenesis of Achromatopsia Associated with Mutations in the Cone Cyclic Nucleotide-Gated Channel CNGA3 Subunit (June 2008)


Normal allelic variants: GNAT2 consists of eight coding exons. Only a few polymorphisms and rare variants are observed; most occur within non-coding regions or do not result in an amino acid substitution.
Normal gene product: The polypeptide is 354 amino acids long. GNAT2 encodes for guanine nucleotide-binding protein G(t), alpha-2 subunit (the cone-specific alpha subunit of transducin), and a heterotrimeric G protein that couples to the cone photopigments.
Pathologic allelic variants: Only nine different disease-associated mutations (one nonsense mutation, six frame-shifting deletions and/or insertions, one large deletion of exon 4, and a mutation c.461+24G>A activating a cryptic splice site and resulting in frame-shift and PTC) sorted in seven independent families have been described up to date .


Normal allelic variants: PDE6C consists of 22 coding exons. Several polymorphisms and rare variants are observed; most occur within non-coding regions or do not result in an amino acid substitution.
Normal gene product: The PDE6C transcript of 3,307 bp encodes for the phosphodiesterase 6C, cGMP-specific, cone, alpha-prime; PDE6C. This alpha’-subunit of the cone-specific phosphodiesterase consists of 858 amino acid residues.
Pathologic allelic variants: Up to date fifteen different mutations in PDE6C in eight independent families have been described: six missense and three nonsense mutations, a 2bp-duplication, and five mutations affecting splicing.

Clinical utility gene card for: achromatopsia


The successful restoration of visual function with recombinant adeno-associated virus (rAAV)-mediated gene replacement therapy in animals and humans with an inherited disease of the retinal pigment epithelium has ushered in a new era of retinal therapeutics. For many retinal disorders, however, targeting of therapeutic vectors to mutant rods and/or cones will be required. In many studies, the primary cone photoreceptor disorder achromatopsia served as the ideal translational model to develop gene therapy directed to cone photoreceptors. The scientist demonstrate that rAAV-mediated gene replacement therapy with different forms of the human red cone opsin promoter led to the restoration of cone function and day vision in two canine models of CNGB3 achromatopsia, a neuronal channelopathy that is the most common form of achromatopsia in man. The robustness and stability of the observed treatment effect was mutation independent, but promoter and age dependent. Subretinal administration of rAAV5–hCNGB3 with a long version of the red cone opsin promoter in younger animals led to a stable therapeutic effect for at least 33 months. The results hold promise for future clinical trials of cone-directed gene therapy in achromatopsia and other cone-specific disorders.

Gene therapy rescues cone function in congenital achromatopsia(april 2010)

(Veronica Tomatis e Nasari Elena)

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