Gene

Cone dystrophy 3 (COD3) is a severe dominantly inherited retinal degeneration caused by missense mutations in GUCA1A, the gene encoding Guanylate Cyclase Activating Protein 1 (GCAP1). The role of GCAP1 in controlling cyclic nucleotide levels in photoreceptors has largely been elucidated using knock-out mice, but the disease pathology in these mice cannot be extrapolated directly to COD3 as this involves altered, rather than loss of, GCAP1 function. Therefore, in order to evaluate the pathology of this dominant disorder, we have introduced a point mutation into the murine Guca1a gene that causes an E155G amino acid substitution; this is one of the disease-causing mutations found in COD3 patients. Disease progression in this novel mouse model of cone dystrophy was determined by a variety of techniques including electroretinography (ERG), retinal histology, immunohistochemistry and measurement of cGMP levels. It was established that although retinal development was normal up to 3 months of age, there was a subsequent progressive decline in retinal function, with a far greater alteration in cone than rod responses, associated with a corresponding loss of photoreceptors. In addition, we have demonstrated that accumulation of cyclic GMP precedes the observed retinal degeneration and is likely to contribute to the disease mechanism. Importantly, this knock-in mutant mouse has many features in common with the human disease, thereby making it an excellent model to further probe disease pathogenesis and investigate therapeutic interventions.</p

Photoreceptor-specific protein expression levels in young <i>Guca1a</i>^COD3 ‘knock-in’ mice.

<p>Western blots from retinal homogenates taken from six week-old wild-type (WT), heterozygous (Het) and homozygous (Hom) mutant mice. Equally loaded protein extracts were blotted with anti-β-actin as a loading control. (<b>a</b>) α-GCAP1 antibody DSC-1 with the retinae from three eyes per genotype. (<b>b</b>) PDE6β (rod-specific), L/M opsin, Gα_T2 (GNAT2 - cone-specific α-transducin) and retGC (rod and cone) antibodies, with the retinae from a single eye per genotype. In all cases, there are no discernible differences in staining between genotypes.</p

Mutant GCAP1 leads to progressive loss of cone photoreceptors.

<p>(<b>a</b>) Representative retinal flat mounts taken from 5 month-old mice stained with PNA and anti-cone opsin antibody. There are fewer PNA-positive cone cells present in mutant compared to wild-type retinae, as well as a fewer opsin-expressing cells in mutant versus wild-type retinae. Quantitative analysis shows that there are significantly fewer cone opsin-expressing cells in mutant retinae than PNA-positive cells, although the labelling of both markers is significantly reduced in <i>Guca1a</i>^+/COD3 and <i>Guca1a</i>^COD3/COD3 mice as compared to wild-type littermates. (<b>b</b>) Representative retinal flatmounts taken from 12 month-old mice confirms progression of cone degeneration; in mutant mice, there are fewer opsin-positive cells at 12 months compared to wild-type littermates.</p

Characterization of a familial t(16;22) balanced translocation associated with congenital cataract leads to identification of a novel gene, TMEM114, expressed in the lens and disrupted by the translocation

Molecular characterization of chromosomal rearrangements is a powerful resource in identification of genes associated with monogenic disorders. We describe the molecular characterization of a balanced familial chromosomal translocation, t(16;22)(p13.3;q11.2), segregating with congenital lamellar cataract. This led to the discovery of a cluster of lens-derived expressed sequence tags (ESTs) close to the 16p13.3 breakpoint. This region harbors a locus associated with cataract and microphthalmia. Long-range PCR and 16p13.3 breakpoint sequencing identified genomic sequence in a human genome sequence gap, and allowed identification of a novel four-exon gene, designated TMEM114, which encodes a predicted protein of 223 amino acids. The breakpoint lies in the promoter region of TMEM114 and separates the gene from predicted eye-specific upstream transcription factor binding sites. There is sequence conservation among orthologs down to zebrafish. The protein is predicted to contain four transmembrane domains with homology to the lens intrinsic membrane protein, LIM2 (also known as MP20), in the PMP-22/EMP/MP20 family. TMEM114 mutation screening in 130 congenital cataract patients revealed missense mutations leading to the exchange of highly-conserved amino acids in the first extracellular domain of the protein (p.I35T;, p.F106L) in two separate patients and their reportedly healthy sibling and mother, respectively. In the lens, Tmem114 shows expression in the lens epithelial cells extending into the transitional zone where early fiber differentiation occurs. Our findings implicate dysregulation of expression of this novel human gene, TMEM114, in mammalian cataract formation. © 2007 Wiley-Liss, Inc

E155G mutation in GCAP1 causes photoreceptor degeneration.

<p>Photomicrographs of resin-embedded sections from (<b>a</b>) wild-type (<b>b</b>) <i>Guca1a</i>^+/COD3, and (<b>c</b>) <i>Guca1a</i>^COD3/COD3 mice at five months of age. There is a marked thinning of the outer nuclear layer (ONL) in mutant eyes as shown by the loss of photoreceptor nuclei. There is also an apparent shortening of outer segment length, particularly in the <i>Guca1a</i>^COD3/COD3 homozygous mice. Photoreceptor loss in <i>Guca1a</i>^+/COD3 and <i>Guca1a</i>^COD3/COD3 mice was quantified by counting photoreceptor nuclei in photomicrographs of sections from paraffin-embedded eyes taken at fixed positions around the optic nerve. At five months of age (<b>d</b>), there is 30% loss in <i>Guca1a</i>^+/COD3 and a 46% loss in <i>Guca1a</i>^COD3/COD3 mice. At 12 months of age (<b>e</b>), this has progressed to a 32% loss in <i>Guca1a</i>^+/COD3 and a 49% loss in <i>Guca1a</i>^COD3/COD3 mice. * indicates statistical significance at the 1% probability level. Note that in the lower panels of (a), PNA shows non-cone staining above the ONL and INL. INL inner nuclear layer, ONL outer nuclear layer, IS/OS inner segment/outer segment.</p

Impaired recovery from bright flash of <i>Guca1a</i>^COD3 ‘knock-in’ mice.

<p><b>T</b>he a-wave amplitude from a bright test flash, presented at varying times after a conditioning flash, is plotted as a proportion of the amplitude elicited by the conditioning flash. A value of 0 indicates the amplitude from the second flash is the same as that from the first; full recovery from a bright conditioning flash occurs within two seconds in wild-type mice, and the second flash elicits an amplitude half that of the first when the inter-stimulus interval is approximately 1000 ms. This recovery time is extended to 1600 ms in both heterozygous and homozygous knock-in mice, whilst the amplitude from the second flash fails to reach that elicited by the first flash during the time period of recording.</p

Generation of <i>Guca1a</i>^COD3 ‘knock-in’ mice with E155G mutation in GCAP1.

<p>(<b>a</b>) A vector targeting the endogenous <i>Guca1a</i> locus was constructed to include an A-to-G transversion at nucleotide position 19 in exon 4 of <i>Guca1a</i> (red circle), as well as a <i>loxP</i>-flanked (blue arrow heads) neomycin resistance gene within intron 3. Following <i>Cre</i>-mediated excision of the <i>Neo</i> selectable marker, the resulting locus contained the A-to-G change in exon 4 plus a residual 34 bp <i>loxP</i> sequence in intron 3. This was used to distinguish between mutant and native alleles by PCR – the position of the primers used is indicated by the small blue arrows on the <i>Cre</i>-deleted <i>Guca1a</i> locus. (<b>b</b>) PCR amplicons from wild-type, <i>Guca1a</i>^+/COD3 and <i>Guca1a</i>^COD3/COD3 mice (lanes 3, 4 and 5 respectively), with 1 kb DNA ladder (lane 1) and no-DNA control (lane 2). The wild type allele generates a band at 734 bp whereas the mutant allele generates a 768 bp band which includes the residual intronic <i>loxP</i> sequence. Wild-type mice have therefore a single band at 734 bp, homozygous <i>Guca1a</i>^COD3/COD3 mice have a single band at 768 bp, and heterozygous <i>Guca1a</i>^+/COD3 mice have both bands. (<b>c</b>) Sequence of wild type and targeted <i>Guca1a</i> allele showing A-to-G transversion at nucleotide position 19 of exon 4.</p

Electroretinography of <i>Guca1a</i>^COD3 ‘knock-in’ mice.

<p>(<b>a</b>) Representative ERG traces recorded at five months of age. Light-adapted, cone-mediated ERG (upper trace, right eye, lower trace, left eye) is reduced in <i>Guca1a</i>^+/COD3 and <i>Guca1a</i>^COD3/COD3 mice compared to wild-type littermates, as is the cone-mediated flicker response to 10- and 15-Hz stimuli (* and # respectively). (<b>b</b>) Representative ERG traces recorded at 12 months of age. There is a greater reduction in cone function in mutant mice, with light-adapted flash responses further attenuated and flicker responses almost extinguished. (<b>c</b>) Averaged photopic, cone-mediated b-wave amplitudes from wild-type, <i>Guca1a</i>^+/COD3 and <i>Guca1a</i>^COD3/COD3 mice recorded over a twelve month period from birth. There is a progressive loss of cone function over time in both heterozygous and homozygous mutant mice compared to wild-type littermates (<i>n = 8</i> per genotype). (<b>d</b>) ERG b-wave amplitudes from <i>Guca1a</i>^+/COD3 and <i>Guca1a</i>^COD3/COD3 mice plotted as a percentage of wild-type amplitudes over a twelve month period from birth.</p

Photopigment expression in ‘<i>Guca1a</i>^COD3 ‘knock-in’ mice.

<p>Representative cryosections from five month-old mice stained with DAPI and (<b>a</b>) a combined anti-L/M- and S-cone opsin antibody (red) and PNA (green), and (<b>b</b>) an anti-rhodopsin (rod opsin) antibody (red). For both, upper panels show fluorescent light micrographs, and lower panels show single slices from confocal microscopy. The expression of both cone and rod opsin appears similar in both <i>Guca1a</i>^+/COD3 and <i>Guca1a</i>^COD3/COD3 mice compared with wild-type littermates, although the rod opsin staining indicates a shortening of rod outer segments. PNA staining (green) in (<b>a</b>) indicates a reduced number of cones in the mutant retinae which is more pronounced in homozygous than in heterozygous mutant mice. INL inner nuclear layer, ONL outer nuclear layer, IS/OS inner segment/outer segment.</p