The landscape of submicroscopic structural variants at the OPN1LW/OPN1MW gene cluster on Xq28 underlying blue cone monochromacy

To perform a detailed clinical and psychophysical assessment of the members of three British families affected with blue cone monochromatism (BCM), and to determine the molecular basis of disease in these families. Affected and unaffected members of three families with BCM were examined clinically and underwent electrophysiological and detailed psychophysical testing. Blood samples were taken for DNA extraction. The strategy for molecular analysis was to amplify the coding regions of the long wavelength-sensitive (L) and middle wavelength-sensitive (M) cone opsin genes and the upstream locus control region by polymerase chain reaction, and to examine these fragments for mutations by direct sequencing. We have confirmed the reported finding of protan-like D-15 arrangements of patients with BCM. In addition, we have demonstrated that the Mollon-Reffin (MR) Minimal test is a useful colour-discrimination test to aid in the diagnosis of BCM. Affected males were shown to fail the protan and deutan axes, but retained good discrimination on the tritan axis of the MR test, a compelling evidence for residual colour vision in BCM. This residual tritan discrimination was also readily detected with HRR plates. In two families, psychophysical testing demonstrated evidence for progression of disease. In two pedigrees, BCM could be linked to unequal crossovers within the opsin gene array that resulted in a single 5'-L/M-3' hybrid gene, with an inactivating Cys203Arg mutation. The causative mutations were not identified in the third family. The MR test is a useful method of detecting BCM across a wide range of age groups; residual tritan colour discrimination is clearly demonstrated and allows BCM to be distinguished from rod monochromatism. BCM is usually classified as a stationary cone dysfunction syndrome; however, two of our families show evidence of progression. This is the first report of progression associated with a genotype consisting of a single 5'-L/M-3' hybrid gene carrying an inactivating mutation. We have confirmed that the Cys203Arg inactivating mutation is a common sequence change in blue cone monochromats.

PurposeTo perform a phenotypic assessment of members of three British families with blue cone monochromatism (BCM), and to determine the underlying molecular genetic basis of disease.MethodsAffected members of three British families with BCM were examined clinically and underwent detailed electrophysiological and psychophysical testing. Blood samples were taken for DNA extraction. Molecular analysis involved the amplification of the coding regions of the long (L) and medium (M) wave cone opsin genes and the upstream locus control region (LCR) by polymerase chain reaction (PCR). Gene products were directly sequenced and analyzed.ResultsIn all three families, genetic analysis identified that the underlying cause of BCM involved an unequal crossover within the opsin gene array, with an inactivating mutation. Family 1 had a single 5′-L–M-3′ hybrid gene, with an inactivating Cys203Arg (C203R) mutation. Family 3 had an array composed of a C203R inactivated 5′-L–M-3′ hybrid gene followed by a second inactive gene. Families 1 and 3 had typical clinical, electrophysiological, and psychophysical findings consistent with stationary BCM. A novel mutation was detected in Family 2 that had a single hybrid gene lacking exon 2. This family presented clinical and psychophysical evidence of a slowly progressive phenotype.ConclusionsTwo of the BCM-causing family genotypes identified in this study comprised different hybrid genes, each of which contained the commonly described C203R inactivating mutation. The genotype in the family with evidence of a slowly progressive phenotype represents a novel BCM mutation. The deleted exon 2 in this family is not predicted to result in a shift in the reading frame, therefore we hypothesize that an abnormal opsin protein product may accumulate and lead to cone cell loss over time. This is the first report of slow progression associated with this class of mutation in the L or M opsin genes in BCM.

PurposeBlue cone monochromacy (BCM), a congenital X-linked retinal disease caused by mutations in the OPN1LW/OPN1MW gene cluster, is under consideration for intravitreal gene therapy. Difficulties with near vision tasks experienced by these patients prompted this study of reading performance as a potential outcome measure for a future clinical trial.MethodsClinically and molecularly diagnosed patients with BCM (n = 17; ages 15–63 years) and subjects with normal vision (n = 22; ages 18–72 years) were examined with the MNREAD acuity chart for both uniocular and binocular conditions. Parameters derived from the measurements in patients were compared with normal data and also within the group of patients. Intersession, interocular and between-subject variabilities were determined. The frequent complaint of light sensitivity in BCM was examined by comparing results from black text on a white background (regular polarity) versus white on black (reverse polarity) conditions.ResultsMNREAD curves of print size versus reading speed were right-shifted compared with normal in all patients with BCM. All parameters in patients with BCM indicated abnormal reading performance. Intersession variability was slightly higher in BCM than in normal, but comparable with results previously reported for other patients with maculopathies. There was a high degree of disease symmetry in reading performance in this BCM cohort. Reverse polarity showed better reading parameters than regular polarity in 82% of the patients.ConclusionsMNREAD measures of reading performance in patients with BCM would be a worthy and robust secondary outcome in a clinical trial protocol, given its dual purpose of quantifying macular vision and addressing an important quality of life issue.Translational RelevanceAssessment of an outcome for a clinical trial.

Blue cone monochromacy (BCM) is an infrequent X-linked retinal disorder typified by poor central visual acuity and color discrimination, early onset of nystagmus, variable degrees of myopia and astigmatism, and a nearly normal retinal appearance. The physiologic functions of rods and blue cones are preserved. The regional location of the genetic mutation causing BCM has been unknown. We have applied the modern molecular techniques of analysis of restriction fragment length polymorphisms to three multigenerational kindreds in which BCM is segregating. Significant linkage is established to two DNA markers, DXS15 and DXS52, each of which maps to the vicinity of Xq28. Regional localization of the locus for BCM has the potential to improve carrier detection and to provide antenatal diagnosis in families at risk for the disease.

Novel therapeutic approaches for treating inherited retinal degenerations (IRDs) prompt a need to understand which patients with impaired vision have the anatomical potential to gain from participation in a clinical trial. We used supervised machine learning to predict foveal function from foveal structure in blue cone monochromacy (BCM), an X-linked congenital cone photoreceptor dysfunction secondary to mutations in the OPN1LW/OPN1MW gene cluster. BCM patients with either disease-associated large deletion or missense mutations were studied and results compared with those from subjects with other forms of IRD and various degrees of preserved central structure and function. A machine learning technique was used to associate foveal sensitivities and best-corrected visual acuities to foveal structure in IRD patients. Two random forest (RF) models trained on IRD data were applied to predict foveal function in BCM. A curve fitting method was also used and results compared with those of the RF models. The BCM and IRD patients had a comparable range of foveal structure. IRD patients had peak sensitivity at the fovea. Machine learning could successfully predict foveal sensitivity (FS) results from segmented or un-segmented optical coherence tomography (OCT) input. Application of machine learning predictions to BCM at the fovea showed differences between predicted and measured sensitivities, thereby defining treatment potential. The curve fitting method provided similar results. Given a measure of visual acuity (VA) and foveal outer nuclear layer thickness, the question of how many lines of acuity would represent the best efficacious result for each BCM patient could be answered. We propose that foveal vision improvement potential in BCM is predictable from retinal structure using machine learning and curve fitting approaches. This should allow estimates of maximal efficacy in patients being considered for clinical trials and also guide decisions about dosing.

Blue cone monochromacy and gene therapy

Blue cone monochromatism (BCM) is an X-linked recessive cone dysfunction disorder caused by mutations in the OPN1LW/OPN1MW gene cluster, encoding long (L)- and middle (M)-wavelength-sensitive cone opsins. Here, we report on the unusual clinical presentation of BCM caused by a novel mutation in the OPN1LW gene in a young man. We describe in detail the phenotype of the proband, and the subclinical morpho-functional anomalies shown by his carrier mother. At a clinical level, the extensive functional evaluation demonstrated in the proband the M/L cone affection and the sparing of S-cone function, distinctive findings of BCM. Interestingly, spectral-domain optical coherence tomography showed the presence of foveal hypoplasia with focal irregularities of the ellipsoid layer in the foveal area, reported to be associated with some cases of cone-rod dystrophy and achromatopsia. At a molecular level, we identified the novel mutation c.427T > C p.(Ser143Pro) in the OPN1LW gene and the common missense mutation c.607T > C (p.Cys203Arg) in the OPN1MW gene. In addition, we discovered the c.768-2_769delAGTT splicing variant in the GPR143 gene. To our knowledge, this is the first case of foveal hypoplasia in a BCM patient and of mild clinical affection in a female carrier caused by the concomitant effect of variants in OPN1LW/OPN1MW and GPR143 genes, thus as the result of the simultaneous action of two independent genetic defects.

X-linked blue cone monochromatism (BCM) has to be differentiated from x-linked cone dystrophy and autosomal recessive rod monochromatism. In nine male patients with congenital cone dysfunction (one family, six single cases; age range: 9-55 years), mutations in the red/green opsin gene cluster were confirmed. Clinical findings were analyzed retrospectively. In one family and three single cases, a single red-green hybrid gene was found carrying a Cys203Arg mutation. Two patients had multiple opsin genes, a red/green hybrid gene and at least one green pigment gene, all carrying the Cys203Arg mutation. In one patient, a large deletion of the locus control region and parts of the red pigment gene were detected. Two patients (ages: 45 and 55 years) complained about progression. Two patients presented with nystagmus. Refractive errors (+8.0 and -11.0 D) and visual acuity were variable (0.05-0.3). Only four patients had a visual acuity > or = 0.1. In two patients, visual acuity could be improved using blue filter glasses. Four of five patients > or = 25 years had dystrophic alterations in the macula. Severe color vision defects and relative central scotoma were present in all patients. In the electroretinogram, residual cone responses were detected in 2/8 patients. Hybrid red/green opsin genes carrying the Cys203Arg mutation are a frequent cause of BCM in German patients. Molecular genetic evaluation is mandatory for adequate diagnosis of patients since from the clinical data only two patients were diagnosed as having BCM. In the other patients, either rod monochromatism or cone-rod dystrophy could not be excluded with certainty. The patients should be cautioned that macular dystrophy may develop in adults older than 30 years.

The human X chromosome contains ∼ 1600 genes, about 15% of which have been associated with a specific genetic condition, mainly affecting males. Blue cone monochromacy (BCM) is an X-linked condition caused by a loss-of-function of both the OPN1LW and OPN1MW opsin genes. The cone opsin gene cluster is composed of 2-9 paralogs with 99.8% sequence homology and is susceptible to deletions, duplications, and mutations. Current diagnostic tests employ polymerase chain reaction (PCR)-based technologies; however, alterations remain undetermined in 10% of patients. Furthermore, carrier testing in females is limited or unavailable. High-resolution X chromosome-targeted CGH microarray was applied to test for rearrangements in males with BCM and female carriers from three unrelated families. Pathogenic alterations were revealed in all probands, characterized by sequencing of the breakpoint junctions and quantitative real-time PCR. In two families, we identified a novel founder mutation that consisted of a complex 3-kb deletion that embraced the cis-regulatory locus control region and insertion of an additional aberrant OPN1MW gene. The application of high-resolution X-chromosome microarray in clinical diagnosis brings significant advantages in detection of small aberrations that are beyond the resolution of clinically available aCGH analysis and which can improve molecular diagnosis of the known conditions and unravel previously unrecognized X-linked diseases.

Purpose A far East asian family with 4 affected maternal cousin males with congenital nystagmus, low vision and dyschromatopsia was investigated for a genetic cause after informed consent. Blue cone monochromacy is a rare form of X‐linked visual handicap with dyschromatopsia. Methods Family members had ophthalmologic examination including visual acuity, fundoscopy , slit lamp, biomicroscopy,colour vision testing and ERG and VEP recordings.DNA analysis of the composition of the cone ospin gene cluster was performed by PCR and PCR/RFLP as well as direct sequencing of LWS opsin gene. Results A novel nonsense Mutation in the single Long wave sensitive opsin gene was identified in all affected males and carrier females. The variability of the phenotype as well as the added role of parental myopia transmission in the phenotype will be discussed. Conclusion This is the first reported molecular diagnosis of blue cone monochromacy in the Asian population.The compound effect of dominantly inherited myopia offers insight of the effect of the added mutational load in these patients.

X-linked cone dysfunction disorders such as Blue Cone Monochromacy and X-linked Cone Dystrophy are characterized by complete loss (of) or reduced L- and M- cone function due to defects in the OPN1LW/OPN1MW gene cluster. Here we investigated 24 affected males from 16 families with either a structurally intact gene cluster or at least one intact single (hybrid) gene but harbouring rare combinations of common SNPs in exon 3 in single or multiple OPN1LW and OPN1MW gene copies. We assessed twelve different OPN1LW/MW exon 3 haplotypes by semi-quantitative minigene splicing assay. Nine haplotypes resulted in aberrant splicing of ≥20% of transcripts including the known pathogenic haplotypes (i.e. ‘LIAVA’, ‘LVAVA’) with absent or minute amounts of correctly spliced transcripts, respectively. De novo formation of the ‘LIAVA’ haplotype derived from an ancestral less deleterious ‘LIAVS’ haplotype was observed in one family with strikingly different phenotypes among affected family members. We could establish intrachromosomal gene conversion in the male germline as underlying mechanism. Gene conversion in the OPN1LW/OPN1MW genes has been postulated, however, we are first to demonstrate a de novo gene conversion within the lineage of a pedigree.

To examine the involvement of the long (L) and middle (M) wavelength-sensitive cone opsin genes in cone-dominated phenotypes. Clinical and molecular analyses included family history, color vision testing, full-field electroretinography (ERG), linkage analysis, and mutation detection. Eighteen families were recruited that had X-linked retinal disease characterized by cone impairment in which affected males usually had nystagmus, reduced visual acuity, normal to subnormal rod ERG, and reduced or extinguished cone ERG responses. A search for mutations in the L-M pigment gene array revealed disease-causing mutations in six families. In two of them, novel mutations were identified: a large deletion affecting both opsin genes and a single L opsin gene harboring a likely pathogenic mutation, p.Val120Met. A third family carried a single hybrid gene with the p.Cys203Arg mutation. Patients from the three remaining families carried a single opsin gene harboring two similar rare haplotypes. Although the phenotype of members in one of the families was compatible with blue cone monochromacy (BCM), patients from the two other families, who shared an identical haplotype, had only reduced or even normal full-field cone ERGs, but maculopathy was evident. Novel and known mutations affecting the L-M opsin gene array were identified in families with X-linked cone-dominated phenotypes. The results show that different mutations in this gene array can cause a variety of phenotypes, including BCM, cone dystrophy, and maculopathy. Males with X-linked cone-dominated diseases should be routinely analyzed for mutations in the L-M opsin gene array.

Rod monochromacy is a rare condition in vertebrates characterized by the absence of cone photoreceptor cells. The resulting phenotype is colourblindness and low acuity vision in dim-light and blindness in bright-light conditions. Early reports of xenarthrans (armadillos, sloths and anteaters) suggest that they are rod monochromats, but this has not been tested with genomic data. We searched the genomes of Dasypus novemcinctus (nine-banded armadillo), Choloepus hoffmanni (Hoffmann's two-toed sloth) and Mylodon darwinii (extinct ground sloth) for retinal photoreceptor genes and examined them for inactivating mutations. We performed PCR and Sanger sequencing on cone phototransduction genes of 10 additional xenarthrans to test for shared inactivating mutations and estimated the timing of inactivation for photoreceptor pseudogenes. We concluded that a stem xenarthran became an long-wavelength sensitive-cone monochromat following a missense mutation at a critical residue in SWS1, and a stem cingulate (armadillos, glyptodonts and pampatheres) and stem pilosan (sloths and anteaters) independently acquired rod monochromacy early in their evolutionary history following the inactivation of LWS and PDE6C, respectively. We hypothesize that rod monochromacy in armadillos and pilosans evolved as an adaptation to a subterranean habitat in the early history of Xenarthra. The presence of rod monochromacy has major implications for understanding xenarthran behavioural ecology and evolution.

Cones are responsible for daylight, central, high acuity and color vision. Three proteins found in human cones, i.e. long-wavelength (L)-, middle-wavelength (M)-, and short-wavelength sensitive (S)-opsins, are responsible for red, green and blue color recognition, respectively. Human blue cone monochromacy (BCM) is characterized by functional loss of both L- and M-cone opsins due to mutations in the OPN1LW/OPN1MW gene cluster on the X chromosome. BCM patients, who rely on their vision from only S-cones and rods, suffer severely reduced visual acuity and impaired color vision. Recent studies show that there is sufficient cone structure remaining in the central fovea of BCM patients to consider AAV-mediated gene augmentation therapy. In contrast, mouse retina has only two opsins, S-opsin and M-opsin, but no L-opsin. We generated an M-opsin knockout mouse (Opn1mw−/−) expressing only S-opsin as a model for human BCM. We show that recombinant M-opsin delivered by AAV5 vectors rescues M-cone function in Opn1mw−/− mice. We also show that AAV delivered M-opsin localizes in the dorsal cone outer segments, and co-localizes with S-opsin in the ventral retina. Our study demonstrates that cones without M-opsin remain viable and respond to gene augmentation therapy, thereby providing proof-of-concept for cone function restoration in BCM patients.

Gene Conversion between Red and Defective Green Opsin Gene in Blue Cone Monochromacy