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Achromatopsia is a congenital and stationary retinal disorder in which there is

normal rod function but absent or limited cone photoreceptor function associated with

photophobia and nystagmus. Mutations in CNGA3 and CNGB3 genes that encode the a -

and P-subunits of cone photoreceptor CNG channels respectively have been associated with

achromatopsia (Kohl et a l , 1998; W issinger et al., 2(X)1; Sundin et al., 2(XX); Kohl et al.,

2000). Indeed, when the importance o f the CNG channel in phototransduction is

considered, the phenotype of achromatopsia can be well explained as a result of mutations

in CNGA3 or CNGB3. Moreover, analysis of the homologous CNGA3 knockout-mouse

model shows complete absence of physiologically measurable cone function, a decrease in

the number o f cones in the retina, and morphological abnormalities o f the remaining cones (Biel etaL , 1999).

In agreement with previous studies which indicate that approximately 25% o f achromats have CNGA3 alterations (W issinger et a i, 2001), CNG A3 mutations were identified in seven o f the tw enty-five fam ilies studied (28%). This study has identified six previously unreported CNG A3 mutations associated with achromatopsia (Arg23Term, Gln251Term, Arg276Ser, Thr3(X)Met, Ile537fs and G ly603A rg). The distribution o f mutations found in this study is biased towards the terminal segments o f the gene and thus, the mutations are mainly confined to the functionally and structurally important central parts o f the CNG A3 polypeptide (figure 5.5). T w o previously identified comm on CNG A3

mutations in achromatopsia (W issinger et al., 2001) were also found in this panel o f patients: Arg491Trp was identified in one individual whilst two individuals had the Phe602Ile mutation. 60% o f the disease associated nucleotide alterations identified involved CG -dinucleotides which in part accounts for the high frequency o f substitutions at arginine codons (5/10).

Figure 5.5: Location o f CNG A3 mutations identified with respect to proposed topological model o f CNG A3 polypeptide.

Gln251Terin Thr300Met

\

P loop

ex tra c e llu la r in trac ellu la r

V i

\ \ f

—

>

S1 S2 S3 84 85

86

Arg278Trp Arg491Trp [276Ser Arg23Term He537fs Phe602Leu Gly603Arg Arg624His COOH

cGMP-binding site

Six of the families for whom putative disease-causing mutations were found had

homozygous nucleotide changes (families 10, 11, 12, 15, 20 and 24). In one of these cases

(family 11), mutation analysis of parental CNGA3 sequence demonstrated corresponding

heterozygous alterations. This confirms true homozygosity in the affected offspring as

opposed to a genotype involving a heterozygous change and a deletion. In the other

homozygous cases no parental information was available although a large deletion in one

allele of the whole gene could be ruled out for family 15 by observation o f heterozygosity

at the M etl 12Ile polymorphic site.

The simplest explanation o f genotype for those affected individuals for whom single

heterozygous mutations were found (RM 15, RM 18 and RM35) is that the second mutation

was missed by sequencing or is present in a yet unidentified exon, promotor or intron.

Alternatively, in these cases there may be a large structural alteration of the second allele

which it may be possible to detect by Southern blotting. A dominant effect of these single

heterozygous mutations can be ruled out in RM 18 and RM35 as there are unaffected

heterozygotes in their families (RM 17 and RM36 respectively). An additional explanation

might involve mutations in the CNG channel beta-subunit in addition to that found in

CNGA3.

The functional importance of the CNGA3 amino acid residues affected by mutation

is reflected by the high degree of evolutionary conservation. The majority of these residues

were highly conserved among vertebrate cone photoreceptors (human, mouse, chicken),

also among other vertebrate CNG channels (human rod and bovine olfactory epithelium)

and half were even conserved in the CNG channels o f Drosophila (table 5.6).

6/9 of the different mutations identified were missense mutations which indicates

that there is little tolerance for substitutions with respect to functional maintenance of the

channel polypeptide. This notion is supported by the high degree of evolutionary

conservation among CNG channel a-subunits as described above. Relatively little detail is

known, however, about the actual contribution of different portions of the CNG A3

polypeptide to cone photoreceptor CNG channel function and the majority of work to

elucidate the mechanism of CNG channel regulation in photoreceptors to date has involved

CN G A l and C N G B l of rod photoreceptors. It has been established, however, that

photoreceptor CNG channel gating is modulated by a number of factors whose action is

mediated by crucial segments and/or individual amino acids in each of the subunits:

• Nickle and zinc transition metal ions coordinate the histidine residues at

position 420 of adjacent subunits and increases sensistivity of the channel

by promoting the open state (Gordon and Zagotta, 1995).

• Sensitivity to cyclic nucleotides is decreased by serine/threonine and

tyrosine phosphoiylation. S577 of rod and S579 o f cone a-subunits and

Y498 of both a-subunits are involved in mediation of this effect (Gordon

et al., 1992; Molokanova et al., 1999). Déphosphorylation of Y498 has

the converse effect and is regulated by growth factors (Savchenko et al.,

2001

).

• The S6 transmembrane domain and flanking regions of the a-subunit are

involved in protein-protein interaction with protein tyrosine kinase which

decreases channel cyclic nucleotide sensitivity (Molokanova and Kramer,

2001).

• Phospholipid metabolites decrease channel sensitivity by interaction with

transmembrane domains (Crary et al., 2000).

It has been shown that the murine and bovine orthologues of the human CNGA3

gene are also expressed in non-retinal tissues, notably kidney, testis and the pineal gland

non-visual illnesses or complaints. If human CNGA3 is expressed in the pineal gland then

it appears not to be utilized in a manner similar to the retina since we know there is no light

detection by the mammalian pineal gland (Lucas et al. 1999).

Consideration of the results of a similar molecular genetic study o f the CNGB3 gene

in the same panel of achromats undertaken within the research group revealed that there

were only four families for whom no mutations in the cone CNG channel a - and P-subunits

could be found. The affected individuals in these families are appropriate for inclusion in

further studies to examine other candidate genes for achromatopsia. One of the families

(family 1) for whom CNGA3 and CNGB3 were excluded as disease genes is a large

consanguinous Pakistani family and was therefore suitable for a genome-wide linkage

screen of microsatellite markers to identify regions of homozygosity/autozygosity. This

study was undertaken in collaboration with Professor Eamonn R. Maher and Dr. Irene A.

Aligianis of the University of Birmingham. Results indicated significant linkage to a 16cM

autozygous region between markers D1S485 and D1S534 on chromosome lp l3 . GNAT2,

the gene that codes for cone photoreceptor a-transducin which is the G protein responsible

for coupling the cone pigments to cGMP-phosphodiesterase in phototransduction (section

1.7), was identified as a positional candidate in this interval. Screening of this gene in the

family by direct sequence analysis demonstrated a frameshift mutation (nt. C.842_843 ins

TCAG) that segregated with disease (Aligianis et al., 2002). At the time of completion of

the study mutations in GNAT2 had not been described previously for achromatopsia.

Subsequently, Kohl et al. (2002) published similar findings in five independent families

with achromatopsia. The mutations identified all differ from that found in family 1 and

each other although all result in premature translation termination and in mutant

polypeptides that lack considerable portions of the carboxy terminus.

It is noteworthy that the three genes now known to be involved in achromatopsia

encode crucial components of the cone phototransduction cascade. This indicates that all

three classes of cone photoreceptor utilize a common cone-specific cGMP-gated channel

and a common a-transducin subunit and that there is conservation of the phototransduction

process in cones. This observation also implies that, since not all achromats can be

accounted for by mutations in these three genes, other candidate genes for achromatopsia

may be found in other cone-specific components of the phototransduction cascade (table

5.9). In addition any genes that are expressed specifically in cones or are upregulated in

Chapter 6

- Cone-Rod Dystrophy 7

-

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