LCA is a severe, inherited retinal dystrophy of childhood characterized by severe visual loss at or near birth, nystagmus, and a pigmentary retinopathy. This genetically heterogeneous disorder renders infants with little or no retinal photoreceptor function. In this report, we characterize two novel, de novo CRX mutations in two unrelated patients that lead to presumed autosomal dominant LCA.
CRX is a homeodomain protein expressed in photoreceptor cells, regulating vital photoreceptor differentiation and integrity (Chen, et al., 1997
; Furukawa, et al., 2002
; Furukawa, et al., 1997
). Mutations have been described in patients with a variety of presentations including autosomal dominant LCA, dominant cone-rod dsytrophy, and retinitis pigmentosa (Freund, et al., 1997
; Freund, et al., 1998
; Sohocki, et al., 1998
). As with all transcription factors, CRX is thought to interact with other proteins such as the neural leucine zipper transcription factor NRL in vivo
to activate tissue-specific gene transcription. We demonstrate that different mutations of CRX can not only differentially affect CRX function alone, but that they may also have different effects on transactivation in the presence of a co-activating factor such as NRL. Specifically, the K88N mutant protein not only has little intrinsic transactivation capability, but it also interferes with the independent ability of NRL to activate transcription—an effect akin to a classic “dominant negative effect”, where a mutant protein interferes with the normal function of a co-expressed wild-type protein. Because co-expression of K88N with wild-type NRL results in a reduction in steady state levels of both proteins, we posit that the mutant protein is capable of binding NRL, but is recognized as misfolded by cells and targeted for degradation. In contrast, co-expression of Ile138fs48 CRX with wild-type NRL does not drastically alter the steady state level of either protein. While the ability of Ile138fs48 CRX to transactivate at the rhodopsin promoter is reduced compared to wild-type CRX, this mutant protein does not appear to drastically interfere with the ability of NRL to independently activate transcription. These results agree with previous observations by Mitton et al., who found that CRX-NRL interactions were mostly localized to the homeodomain and the immediate surrounding sequences (Mitton, et al., 2000
It is difficult to make a clear correlation between genotype and phenotype in our two patients. Although Patient#2 initially had better acuity than Patient#1—at least by preferential looking tests—her subsequent macular atrophy seems to have reduced her acuity by the time a formal measurement could be made. It is theoretically possible that this mutant protein, by allowing NRL to independently regulate retinal gene expression, would lead to a less severe phenotype, at least initially.
Consistent with this hypothesis, Koenekoop et al. have described visual improvement in a child with a similar frameshift mutation in CRX
(Koenekoop, et al., 2002
). To date, nearly all the reported CRX
mutations that cause LCA are expected to frameshift the CRX protein, usually downstream of the homeodomain protein. Missense mutations in the homeodomain and elsewhere have been largely associated with cone-rod dystrophy. As suggested by our molecular modeling, the disruption of a critical hydrogen bond in the p.K88N mutant protein—which is energetically stronger than the electrostatic interactions disrupted by two CORD-causing mutations—may help explain why Patient #1 had more severe phenotype. Consistent with the observations of Fei and Hughes, our point mutation at amino acid 88 causes some of the CRX protein to localize to the cytoplasm, as well as the normal, nuclear localization (Fei and Hughes, 2000
). Somewhat surprisingly, the frameshift mutation we describe has similar cytoplasmic and nuclear localization, which differs from these authors’ findings using CRX deletion constructs in HEK-290 cells. The reason for this difference may be a difference in the cell type studied and/or differences caused by the novel C-terminal protein sequence created by the Ile138fs48 mutation.
Recent gene therapy trials of one genetic form of LCA caused by mutations in the RPE65
gene have been very encouraging (Bainbridge, et al., 2008
; Cideciyan, et al., 2008
; Hauswirth, et al., 2008
; Maguire, et al., 2009
; Maguire, et al., 2008
). Because RPE65
causes autosomal recessive LCA that is likely due to loss of protein function and some outer nuclear layer is preserved anatomically (Jacobson, et al., 2008
), it is an excellent choice for a gene replacement strategy in humans. The clinical situation in patients with CRX
mutations may be more complex. First, adult patients with cone-rod dystrophy due to CRX
mutation have dramatic thinning of the outer nuclear layer, suggesting that the target cells may rapidly degenerate (Jacobson, et al., 1998
). Our OCT results confirm that this thinning is present early in life. However, it is somewhat encouraging that a band of reflectivity consistent with some preservation of an outer nuclear layer (although not photoreceptor outer segments) was observed in Patient #2. Pronounced nystagmus and the age of the child limit our ability to make more detailed observations using OCT at this time. A second consideration in designing a therapy for dominant, CRX-mediated LCA is that it is not clear that the mechanism of degeneration is purely due to haploinsufficiency/loss-of-protein function. Our data suggest that some mutations in CRX may have an effect akin to a dominant negative effect on other retinal transcription factor genes, such as NRL. Chen et al., in their analysis of multiple CRX point mutations and frameshift mutations found no specific correlation between residual activity of CRX and disease severity (Chen, et al., 2002
). Swaroop et al. have reported a family with a homeodomain missense mutation, R90W, that causes a relatively mild, adult-onset cone dysfunction syndrome when heterozygous and a severe, LCA phenotype when homozygous(Swaroop, et al., 1999
). In fact, Silva et al. have reported a frameshift mutation in CRX upstream of the homeodomain region that is found in both normal subjects and patients with LCA, suggesting that haploinsufficiency for CRX function isn’t necessarily pathogenic (Silva, et al., 2000
). In order to clarify the pathogenesis of dominant CRX mutations in vivo, “knock-in” of specific CRX mutations in mouse models may need to be compared with the CRX knockout mouse. If dominant-negative-like effects or other, complex mechanisms are responsible for disease in humans, specific knock-down of the mutant CRX allele may be more beneficial than replacing CRX function.