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

Blue cone monochromacy (BCM) is an X-linked retinal disorder characterized by low vision, photoaversion, and poor color discrimination. BCM is due to the lack of long-wavelength-sensitive and middle-wavelength-sensitive cone photoreceptor function and caused by mutations in the OPN1LW/OPN1MW gene cluster on Xq28. Here, we investigated the prevalence and the landscape of submicroscopic structural variants (SVs) at single-base resolution in BCM patients. We found that about one-third (n = 73) of the 213 molecularly confirmed BCM families carry an SV, most commonly deletions restricted to the OPN1LW/OPN1MW gene cluster. The structure and precise breakpoints of the SVs were resolved in all but one of the 73 families. Twenty-two families-all from the United States-showed the same SV, and we confirmed a common ancestry of this mutation. In total, 42 distinct SVs were identified, including 40 previously unreported SVs, thereby quadrupling the number of precisely mapped SVs underlying BCM. Notably, there was no "region of overlap" among these SVs. However, 90% of SVs encompass the upstream locus control region, an essential enhancer element. Its minimal functional extent based on deletion mapping in patients was refined to 358 bp. Breakpoint analyses suggest diverse mechanisms underlying SV formation as well as in one case the gene conversion-based exchange of a 142-bp deletion between opsin genes. Using parsimonious assumptions, we reconstructed the composition and copy number of the OPN1LW/OPN1MW gene cluster prior to the mutation event and found evidence that large gene arrays may be predisposed to the occurrence of SVs at this locus.

Novel OPN1LW/OPN1MW Exon 3 Haplotype-Associated Splicing Defect in Patients with X-Linked Cone Dysfunction.

Certain combinations of common variants in exon 3 of OPN1LW and OPN1MW, the genes encoding the apo-protein of the long- and middle-wavelength sensitive cone photoreceptor visual pigments in humans, induce splicing defects and have been associated with dyschromatopsia and cone dysfunction syndromes. Here we report the identification of a novel exon 3 haplotype, G-C-G-A-T-T-G-G (referring to nucleotide variants at cDNA positions c.453, c.457, c.465, c.511, c.513, c.521, c.532, and c.538) deduced to encode a pigment with the amino acid residues L-I-V-V-A at positions p.153, p.171, p.174, p.178, and p.180, in OPN1LW or OPN1MW or both in a series of seven patients from four families with cone dysfunction. Applying minigene assays for all observed exon 3 haplotypes in the patients, we demonstrated that the novel exon 3 haplotype L-I-V-V-A induces a strong but incomplete splicing defect with 3-5% of residual correctly spliced transcripts. Minigene splicing outcomes were similar in HEK293 cells and the human retinoblastoma cell line WERI-Rb1, the latter retaining a cone photoreceptor expression profile including endogenous OPN1LW and OPN1MW gene expression. Patients carrying the novel L-I-V-V-A haplotype presented with a mild form of Blue Cone Monochromacy or Bornholm Eye Disease-like phenotype with reduced visual acuity, reduced cone electroretinography responses, red-green color vision defects, and frequently with severe myopia.

Relatively mild blue cone monochromacy phenotype caused by various haplotypes in the L- and M-cone opsin genes.

Purpose: Blue cone monochromacy (BCM) is an X-linked retinopathy caused by mutations in the red and green cone opsin genes. The aim of this study was to establish the clinical, genetic, and electrophysiological characteristics of a specific form of BCM. Methods: Patients harboring mutations in the OPN1LW/OPN1MW genes underwent a full clinical examination, including ocular examination, color vision, full-field electroretinography, color fundus and autofluorescence photography, and optical coherence tomography. Genetic analysis was performed using whole-exome sequencing, duplex PCR, PCR/restriction fragment length polymorphism, and Sanger sequencing. IBM SPSS Statistics v. 21.0 was used for the data analysis. Results: Twenty-five patients harboring various haplotypes in exon 3 of the OPN1LW/OPN1MW genes were recruited. They showed a milder incomplete phenotype of BCM than the typical BCM control group. They presented significantly better visual acuity (logarithm of the minimum angle of resolution [logMAR] 0.48 ± 0.26 vs. 1.10 ± 0.54; p < 0.0001) and a highly myopic refraction (-7.81 ± 5.81 D vs. -4.78 ± 5.27 D; p = 0.0222) compared with the BCM control group. The study group had higher 30-Hz cone flicker responses (28.60 ± 15.02 µv; n = 24), whereas the BCM group had none (0.66 ± 2.12 µv; n = 21; p < 0.0001). The Lanthony 15-HUE desaturated test was variable for the exon 3 haplotype group, with a tendency toward the deutan-protan axis. Conclusions: The present study included genetic and clinical data from the largest cohort of patients with exon 3 haplotypes that were previously shown to cause missplicing of the OPN1LW and OPN1MW genes. Analysis of the clinical data revealed better best-corrected visual acuity, more severe myopia, and higher 30-Hz cone flicker responses in the patients with exon 3 haplotypes than in those with typical BCM.

Comprehensive variant spectrum of the CNGA3 gene in patients affected by achromatopsia.

Achromatopsia (ACHM) is a congenital cone photoreceptor disorder characterized by impaired color discrimination, low visual acuity, photosensitivity, and nystagmus. To date, six genes have been associated with ACHM (CNGA3, CNGB3, GNAT2, PDE6C, PDE6H, and ATF6), the majority of these being implicated in the cone phototransduction cascade. CNGA3 encodes the CNGA3 subunit of the cyclic nucleotide-gated ion channel in cone photoreceptors and is one of the major disease-associated genes for ACHM. Herein, we provide a comprehensive overview of the CNGA3 variant spectrum in a cohort of 1060 genetically confirmed ACHM patients, 385 (36.3%) of these carrying "likely disease-causing" variants in CNGA3. Compiling our own genetic data with those reported in the literature and in public databases, we further extend the CNGA3 variant spectrum to a total of 316 variants, 244 of which we interpreted as "likely disease-causing" according to ACMG/AMP criteria. We report 48 novel "likely disease-causing" variants, 24 of which are missense substitutions underlining the predominant role of this mutation class in the CNGA3 variant spectrum. In addition, we provide extensive in silico analyses and summarize reported functional data of previously analyzed missense, nonsense and splicing variants to further advance the pathogenicity assessment of the identified variants.

Paternal Uniparental Isodisomy of Chromosome 2 in a Patient with CNGA3-Associated Autosomal Recessive Achromatopsia.

Achromatopsia (ACHM) is a rare autosomal recessively inherited retinal disease characterized by congenital photophobia, nystagmus, low visual acuity, and absence of color vision. ACHM is genetically heterogeneous and can be caused by biallelic mutations in the genes CNGA3, CNGB3, GNAT2, PDE6C, PDE6H, or ATF6. We undertook molecular genetic analysis in a single female patient with a clinical diagnosis of ACHM and identified the homozygous variant c.778G>C;p.(D260H) in the CNGA3 gene. While segregation analysis in the father, as expected, identified the CNGA3 variant in a heterozygous state, it could not be displayed in the mother. Microsatellite marker analysis provided evidence that the homozygosity of the CNGA3 variant is due to partial or complete paternal uniparental isodisomy (UPD) of chromosome 2 in the patient. Apart from the ACHM phenotype, the patient was clinically unsuspicious and healthy. This is one of few examples proving UPD as the underlying mechanism for the clinical manifestation of a recessive mutation in a patient with inherited retinal disease. It also highlights the importance of segregation analysis in both parents of a given patient or especially in cases of homozygous recessive mutations, as UPD has significant implications for genetic counseling with a very low recurrence risk assessment in such families.

Multiexon deletion alleles of ATF6 linked to achromatopsia.

Achromatopsia (ACHM) is an autosomal recessive disease that results in severe visual loss. Symptoms of ACHM include impaired visual acuity, nystagmus, and photoaversion starting from infancy; furthermore, ACHM is associated with bilateral foveal hypoplasia and absent or severely reduced cone photoreceptor function on electroretinography. Here, we performed genetic sequencing in 3 patients from 2 families with ACHM, identifying and functionally characterizing 2 mutations in the activating transcription factor 6 (ATF6) gene. We identified a homozygous deletion covering exons 8-14 of the ATF6 gene from 2 siblings from the same family. In another patient from a different family, we identified a heterozygous deletion covering exons 2 and 3 of the ATF6 gene found in trans with a previously identified ATF6 c.970C>T (p.Arg324Cys) ACHM disease allele. Recombinant ATF6 proteins bearing these exon deletions showed markedly impaired transcriptional activity by qPCR and RNA-Seq analysis compared with WT-ATF6. Finally, RNAscope revealed that ATF6 and the related ATF6B transcripts were expressed in cones as well as in all retinal layers in normal human retina. Overall, our data identify loss-of-function ATF6 disease alleles that cause human foveal disease.

Deep-intronic variants in CNGB3 cause achromatopsia by pseudoexon activation.

Our comprehensive cohort of 1100 unrelated achromatopsia (ACHM) patients comprises a considerable number of cases (~5%) harboring only a single pathogenic variant in the major ACHM gene CNGB3. We sequenced the entire CNGB3 locus in 33 of these patients to find a second variant which eventually explained the patients' phenotype. Forty-seven intronic CNGB3 variants were identified in 28 subjects after a filtering step based on frequency and the exclusion of variants found in cis with pathogenic alleles. In a second step, in silico prediction tools were used to filter out those variants with little odds of being deleterious. This left three variants that were analyzed using heterologous splicing assays. Variant c.1663-1205G>A, found in 14 subjects, and variant c.1663-2137C>T, found in two subjects, were indeed shown to exert a splicing defect by causing pseudoexon insertion into the transcript. Subsequent screening of further unsolved CNGB3 subjects identified four additional cases harboring the c.1663-1205G>A variant which makes it the eighth most frequent CNGB3 variant in our cohort. Compound heterozygosity could be validated in ten cases. Our study demonstrates that whole gene sequencing can be a powerful approach to identify the second pathogenic allele in patients apparently harboring only one disease-causing variant.

Mutation spectrum and clinical investigation of achromatopsia patients with mutations in the GNAT2 gene.

Achromatopsia (ACHM) is a hereditary cone photoreceptor disorder characterized by the inability to discriminate colors, nystagmus, photophobia, and low-visual acuity. Six genes have been associated with this rare autosomal recessively inherited disease, including the GNAT2 gene encoding the catalytic α-subunit of the G-protein transducin which is expressed in the cone photoreceptor outer segment. Out of a cohort of 1,116 independent families diagnosed with a primary clinical diagnosis of ACHM, we identified 23 patients with ACHM from 19 independent families with likely causative mutations in GNAT2, representing 1.7% of our large ACHM cohort. In total 22 different potentially disease-causing variants, of which 12 are novel, were identified. The mutation spectrum also includes a novel copy number variation, a heterozygous duplication of exon 4, of which the breakpoint matches exactly that of the previously reported exon 4 deletion. Two patients carry just a single heterozygous variant. In addition to our previous study on GNAT2-ACHM, we also present detailed clinical data of these patients.

Accessory heterozygous mutations in cone photoreceptor CNGA3 exacerbate CNG channel-associated retinopathy.

Mutations in CNGA3 and CNGB3, the genes encoding the subunits of the tetrameric cone photoreceptor cyclic nucleotide-gated ion channel, cause achromatopsia, a congenital retinal disorder characterized by loss of cone function. However, a small number of patients carrying the CNGB3/c.1208G>A;p.R403Q mutation present with a variable retinal phenotype ranging from complete and incomplete achromatopsia to moderate cone dysfunction or progressive cone dystrophy. By exploring a large patient cohort and published cases, we identified 16 unrelated individuals who were homozygous or (compound-)heterozygous for the CNGB3/c.1208G>A;p.R403Q mutation. In-depth genetic and clinical analysis revealed a co-occurrence of a mutant CNGA3 allele in a high proportion of these patients (10 of 16), likely contributing to the disease phenotype. To verify these findings, we generated a Cngb3R403Q/R403Q mouse model, which was crossbred with Cnga3-deficient (Cnga3-/-) mice to obtain triallelic Cnga3+/- Cngb3R403Q/R403Q mutants. As in human subjects, there was a striking genotype-phenotype correlation, since the presence of 1 Cnga3-null allele exacerbated the cone dystrophy phenotype in Cngb3R403Q/R403Q mice. These findings strongly suggest a digenic and triallelic inheritance pattern in a subset of patients with achromatopsia/severe cone dystrophy linked to the CNGB3/p.R403Q mutation, with important implications for diagnosis, prognosis, and genetic counseling.

Phenotype Variations Caused by Mutations in the RP1L1 Gene in a Large Mainly German Cohort.

Purpose: Mutations in the retinitis pigmentosa-1-like-1 (RP1L1) gene are the major cause of autosomal dominant occult macular dystrophy (OCMD), while recessive mutations have been linked to autosomal recessive retinitis pigmentosa (arRP). We present the clinical phenotype of a large German OCMD cohort, as well as four RP patients. Methods: A total of 42 OCMD patients (27 families) and 4 arRP patients (3 families) with genetically confirmed mutations in RP1L1 were included. Genomic DNA was analyzed by targeted analysis of the c.133C>T;p.R45W mutation for all RP or macular dystrophy-related genes. All patients underwent ophthalmologic examination including psychophysical tests, electrophysiology, fundus autofluorescence (FAF), and spectral domain optical coherence tomography (SD-OCT). Follow-up time was up to 12 years. Results: In 25 OCMD index patients genomic testing revealed the heterozygous mutation c.133C>T;p.R45W in RP1L1; one patient was homozygous for the mutation. Two OCMD patients displayed the variants c.3599G>A;p.G1200D and c.2849G>A;p.R950H, respectively, in a heterozygous state. All OCMD patients showed characteristic clinical findings and typical microstructural photoreceptor changes. Two arRP patients displayed the novel homozygous mutations c.3022C>T;p.Q1008* and c.1107G>A;p.W369*, respectively, while two RP-siblings carried the two heterozygous mutations c.455G>A;p.R152Q and c.5959C>T;p.Q1987*, the first also being novel. All arRP cases were mild with disease onset ≈30 years and preserved ERG-responses. Conclusions: OCMD phenotype showed consistent clinical findings including classical microstructural changes on SD-OCT. An important hallmark of RP1L1-related OCMD is the dominant family history with reduced penetrance. Furthermore, novel mutations in association with arRP were identified, outlining the complexity of the protein.

Multimodal imaging including semiquantitative short-wavelength and near-infrared autofluorescence in achromatopsia.

Multimodal imaging provides insights into phenotype and disease progression in inherited retinal disorders. Congenital achromatopsia (ACHM), a cone dysfunction syndrome, has been long considered a stable condition, but recent evidence suggests structural progression. With gene replacement strategies under development for ACHM, there is a critical need for imaging biomarkers to define progression patterns and follow therapy. Using semiquantitative plots, near-infrared (NIR-AF) and short-wavelength autofluorescence (SW-AF) were explored and correlated with clinical characteristics and retinal structure on optical coherence tomography (OCT). In sixteen ACHM patients with genetic confirmation (CNGA3, n = 8; CNGB3, n = 7; PDE6C, n = 1), semiquantitative plots allowed the detailed analysis of autofluorescence patterns, even in poorly fixating eyes. Twelve eyes showed perifoveal hyperautofluorescent rings on SW-AF, and 7 eyes had central hypoautofluorescent areas on NIR-AF, without association between these alterations (P = 0.57). Patients with central NIR-AF hypoautofluorescence were older (P = 0.004) and showed more advanced retinal alterations on OCT than those with normal NIR-AF (P = 0.051). NIR-AF hypoautofluorescence diameter was correlated to patient age (r = 0.63, P = 0.009), size of ellipsoid zone defect on OCT (r = 0.67, P = 0.005), but not to the size of SW-AF hyperautofluorescence (P = 0.27). These results demonstrate the interest of NIR-AF as imaging biomarker in ACHM, suggesting a relationship with age and disease progression.

CNGB3 mutation spectrum including copy number variations in 552 achromatopsia patients.

Achromatopsia is a rare autosomal recessive cone disorder characterized by color vision defects, photophobia, nystagmus, and severely reduced visual acuity. The disease is caused by mutations in genes encoding crucial components of the cone phototransduction cascade (CNGA3, CNGB3, GNAT2, PDE6C, and PDE6H) or in ATF6, involved in the unfolded protein response. CNGB3 encoding the beta subunit of the cyclic nucleotide-gated ion channel in cone photoreceptors is the major achromatopsia gene. Here, we present a comprehensive spectrum of CNGB3 mutations and their prevalence in a cohort of 1074 independent families clinically diagnosed with achromatopsia. Of these, 485 (45.2%) carried mutations in CNGB3. We identified a total of 98 different potentially disease-causing CNGB3 variants, 58 of which are novel. About 10% of patients with CNGB3 mutations only harbored a single heterozygous variant. Therefore, we performed quantitative real-time PCR in 43 of such single heterozygotes in search of the missing allele, followed by microarray-based comparative genomic hybridization and breakpoint mapping. We discovered nine different heterozygous copy number variations encompassing one to 10 consecutive exons in 16 unrelated patients. Moreover, one additional patient with a homozygous CNGB3 deletion encompassing exons 4-18 was identified, highlighting the importance of CNV analysis for this gene.

De novo intrachromosomal gene conversion from OPN1MW to OPN1LW in the male germline results in Blue Cone Monochromacy.

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.

Novel C8orf37 mutations cause retinitis pigmentosa in consanguineous families of Pakistani origin.

PURPOSE: To investigate the molecular basis of retinitis pigmentosa in two consanguineous families of Pakistani origin with multiple affected members. METHODS: Homozygosity mapping and Sanger sequencing of candidate genes were performed in one family while the other was analyzed with whole exome next-generation sequencing. A minigene splicing assay was used to confirm the splicing defects. RESULTS: In family MA48, a novel homozygous nucleotide substitution in C8orf37, c.244-2A>C, that disrupted the consensus splice acceptor site of exon 3 was found. The minigene splicing assay revealed that this mutation activated a cryptic splice site within exon 3, causing a 22 bp deletion in the transcript that is predicted to lead to a frameshift followed by premature protein truncation. In family MA13, a novel homozygous null mutation in C8orf37, c.555G>A, p.W185*, was identified. Both mutations segregated with the disease phenotype as expected in a recessive manner and were absent in 8,244 unrelated individuals of South Asian origin. CONCLUSIONS: In this report, we describe C8orf37 mutations that cause retinal dystrophy in two families of Pakistani origin, contributing further data on the phenotype and the spectrum of mutations in this form of retinitis pigmentosa.

Spectral-domain optical coherence tomography staging and autofluorescence imaging in achromatopsia.

IMPORTANCE Evidence is mounting that achromatopsia is a progressive retinal degeneration, and treatments for this condition are on the horizon. OBJECTIVES To categorize achromatopsia into clinically identifiable stages using spectral-domain optical coherence tomography and to describe fundus autofluorescence imaging in this condition. DESIGN, SETTING, AND PARTICIPANTS A prospective observational study was performed between 2010 and 2012 at the Edward S. Harkness Eye Institute, New York-Presbyterian Hospital. Participants included 17 patients (aged 10-62 years) with full-field electroretinography-confirmed achromatopsia. MAIN OUTCOMES AND MEASURES Spectral-domain optical coherence tomography features and staging system, fundus autofluorescence and near-infrared reflectance features and their correlation to optical coherence tomography, and genetic mutations served as the outcomes and measures. RESULTS Achromatopsia was categorized into 5 stages on spectral-domain optical coherence tomography: stage 1 (2 patients [12%]), intact outer retina; stage 2 (2 patients [12%]), inner segment ellipsoid line disruption; stage 3 (5 patients [29%]), presence of an optically empty space; stage 4 (5 patients [29%]), optically empty space with partial retinal pigment epithelium disruption; and stage 5 (3 patients [18%]), complete retinal pigment epithelium disruption and/or loss of the outer nuclear layer. Stage 1 patients showed isolated hyperreflectivity of the external limiting membrane in the fovea, and the external limiting membrane was hyperreflective above each optically empty space. On near infrared reflectance imaging, the fovea was normal, hyporeflective, or showed both hyporeflective and hyperreflective features. All patients demonstrated autofluorescence abnormalities in the fovea and/or parafovea: 9 participants (53%) had reduced or absent autofluorescence surrounded by increased autofluorescence, 4 individuals (24%) showed only reduced or absent autofluorescence, 3 patients (18%) displayed only increased autofluorescence, and 1 individual (6%) exhibited decreased macular pigment contrast. Inner segment ellipsoid line loss generally correlated with the area of reduced autofluorescence, but hyperautofluorescence extended into this region in 2 patients (12%). Bilateral coloboma-like atrophic macular lesions were observed in 1 patient (6%). Five novel mutations were identified (4 in the CNGA3 gene and 1 in the CNGB3 gene). CONCLUSIONS AND RELEVANCE Achromatopsia often demonstrates hyperautofluorescence suggestive of progressive retinal degeneration. The proposed staging system facilitates classification of the disease into different phases of progression and may have therapeutic implications.

Homozygosity mapping reveals new nonsense mutation in the FAM161A gene causing autosomal recessive retinitis pigmentosa in a Palestinian family.

PURPOSE: Retinitis pigmentosa (RP) is a heterogenous group of inherited retinal degenerations caused by mutations in at least 45 genes. Recently, the FAM161A gene was identified as the causative gene for RP28, an autosomal recessive form of RP. METHODS: We performed a clinical and molecular genetic study of a consanguineous Palestinian family with two three siblings affected with retinitis pigmentosa. DNA samples were collected from the index patient, his father, his affected sister, and two non-affected brothers. DNA sample from the index was subjected to high resolution genome-wide SNP array. Assuming identity-by-descent in this consanguineous family we applied homozygosity mapping to identify disease causing genes. RESULTS: The index patient reported night blindness since the age of 20 years, followed by moderate disease progression with decrease of peripheral vision, the development of photophobia and later on reduced central vision. At the age of 40 his visual acuity was counting fingers (CF) for both eyes, color discrimination was not possible and his visual fields were severely constricted. Funduscopic examination revealed a typical appearance of advanced RP with optic disc pallor, narrowed retinal vessels, bone-spicule like pigmentary changes in the mid-periphery and atrophic changes in the macula. His younger affected brother (37 years) was reported with overall milder symptoms, while the youngest sister (21 years) reported problems only with night vision. Applying high-density SNP arrays we identified several homozygous genomic regions one of which included the recently identified FAM161A gene mutated in RP28-linked autosomal recessive RP. Sequencing analysis revealed the presence of a novel homozygous nonsense mutation, c.1003C>T/p.R335X in the index patient and the affected sister. CONCLUSION: We identified an RP28-linked RP family in the Palestinian population caused by a novel nonsense mutation in FAM161A. RP in this family shows a typical disease onset with moderate to rapid progression into severe visual impairment including central vision in the index and overall milder symptoms in the younger brother and sister.

Five novel CNGB3 gene mutations in Polish patients with achromatopsia.

PURPOSE: To identify the genetic basis of achromatopsia (ACHM) in four patients from four unrelated Polish families. METHODS: In this study, we investigated probands with a clinical diagnosis of ACHM. Ophthalmologic examinations, including visual acuity testing, color vision testing, and full-field electroretinography (ERG), were performed in all patients (with the exception of patient p4, who had no ERG). Direct DNA sequencing encompassing the entire coding region of the CNGB3 gene, eight exons of the GNAT2 gene, and exons 5-7 of the CNGA3 gene was performed. Segregation analysis for the presence and independent inheritance of two mutant alleles was performed in the three families available for study. RESULTS: All patients showed typical achromatopsia signs and symptoms. Sequencing helped detect causative changes in the CNGB3 gene in all probands. Eight different mutations were detected in the CNGB3 gene, including five novel mutations: two splice site mutations (c.1579-1G>A and c.494-2A>T), one nonsense substitution (c.1194T>G), and two frame-shift mutations (c.393_394delGCinsTCCTGGTGA and c.1366delC). We also found three mutations: one splice site (c.1578+1G>A) and two frame-shift deletions that had been previously described (c.819_826del and c.1148delC). All respective parents were shown to be heterozygous carriers for the mutation detected in their children. CONCLUSIONS: The present study reports five novel mutations in the CNGB3 gene, and thus broadens the spectrum of probably pathogenic mutations associated with ACHM. Together with molecular data, we provide a brief clinical description of the affected individuals.

Large deletions of the KCNV2 gene are common in patients with cone dystrophy with supernormal rod response.

Cone dystrophy with supernormal rod response (CDSRR) is considered to be a very rare autosomal recessive retinal disorder. CDSRR is associated with mutations in KCNV2, a gene that encodes a modulatory subunit (Kv8.2) of a voltage-gated potassium channel. In this study, we found that KCNV2 mutations are present in a substantial fraction (2.2-4.3%) of a sample of 367 independent patients with a variety of initial clinical diagnoses of cone malfunction, indicating that CDSRR is underdiagnosed and more common than previously thought. In total, we identified 20 different KCNV2 mutations; 15 of them are novel. A new finding of this study is the substantial proportion of large deletions at the KCNV2 locus that accounts for 15.5% of the mutant alleles in our sample. We determined the breakpoints and size of all five different deletions, which ranged between 10.9 and 236.8 kb. Two deletions encompass the entire KCNV2 gene and one also includes the adjacent VLDLR gene. Furthermore, we investigated N-terminal amino acid substitution mutations for its effect on interaction with Kv2.1 using yeast two-hybrid technology. We found that these mutations dramatically reduce or abolish this interaction suggesting a lack of assembly of heteromeric Kv channels as one underlying pathomechanism of CDSRR.

A clinically complex form of dominant optic atrophy (OPA8) maps on chromosome 16.

Dominant optic atrophy (DOA) is genetically heterogeneous and pathogenic mutations have been identified in the OPA1 and OPA3 genes, both encoding for mitochondrial proteins. We characterized clinical and laboratory features in a large OPA1-negative family with complicated DOA. Search for mitochondrial dysfunction was performed by studying muscle biopsies, fibroblasts, platelets and magnetic resonance (MR) spectroscopy. Genetic investigations included mitochondrial DNA (mtDNA) analysis, linkage analysis, copy number variation (CNV) analysis and candidate gene screening. Optic neuropathy was undistinguishable from that in OPA1-DOA and frequently associated with late-onset sensorineural hearing loss, increases of central conduction times at somato-sensory evoked potentials and various cardiac abnormalities. Serum lactic acid after exercise, platelet respiratory complex activities, adenosine triphosphate (ATP) content in fibroblasts and muscle phosphorus MR spectroscopy all failed to reveal a mitochondrial dysfunction. However, muscle biopsies and their mtDNA analysis showed increased mitochondrial biogenesis. Furthermore, patient's fibroblasts grown in the galactose medium were unable to increase ATP content compared with controls, and exhibited abnormally high rate of fusion activity. Genome-wide linkage revealed a locus on chromosome 16q21-q22 with a maximum two-point LOD score of 8.84 for the marker D16S752 and a non-recombinant interval of ∼ 6.96 cM. Genomic screening of 45 genes in this interval including several likely candidate genes (CALB2, CYB5B, TK2, DHODH, PLEKHG4) revealed no mutation. Moreover, we excluded the presence of CNVs using array-based comparative genome hybridization. The identification of a new OPA locus (OPA8) in this pedigree demonstrates further genetic heterogeneity in DOA, and our results indicate that the pathogenesis may still involve mitochondria.

Dissecting the pathogenic mechanisms of mutations in the pore region of the human cone photoreceptor cyclic nucleotide-gated channel.

The CNGA3 gene encodes the A3 subunit of the cone photoreceptor cyclic nucleotide-gated (CNG) channel, an essential component of the phototransduction cascade. Certain mutations in CNGA3 cause autosomal recessive achromatopsia, a retinal disorder characterized by severely reduced visual acuity, lack of color discrimination, photophobia, and nystagmus. We identified three novel mutations in the pore-forming region of CNGA3 (L363P, G367V, and E376K) in patients diagnosed with achromatopsia. We assessed the expression and function of channels with these three new and two previously described mutations (S341P and P372S) in a heterologous HEK293 cell expression system using Western blot, subcellular localization on the basis of immunocytochemistry, calcium imaging, and patch clamp recordings. In this first comparative functional analysis of disease-associated mutations in the pore of a CNG channel, we found impaired surface expression of S341P, L363P, and P372S mutants and reduced macroscopic currents for channels with the mutations S341P, G367V, and E376K. Calcium imaging and patch clamp experiments after incubation at 37 degrees C revealed nonfunctional homo- and heteromeric channels in all five mutants, but incubation at 27 degrees C combined with coexpression of the B3 subunit restored residual function of channels with the mutations S341P, G367V, and E376K.