CNGB3 mutations account for 50% of all cases with autosomal recessive achromatopsia

Achromatopsia is a congenital, autosomal recessively inherited disorder characterized by a lack of color discrimination, low visual acuity (<0.2), photophobia, and nystagmus. Mutations in the genes for CNGA3, CNGB3, and GNAT2 have been associated with this disorder. Here, we analyzed the spectrum and prevalence of CNGB3 gene mutations in a cohort of 341 independent patients with achromatopsia. In 163 patients, CNGB3 mutations could be identified. A total of 105 achromats carried apparent homozygous mutations, 44 were compound (double) heterozygotes, and 14 patients had only a single mutant allele. The derived CNGB3 mutation spectrum comprises 28 different mutations including 12 nonsense mutations, eight insertions and/or deletions, five putative splice site mutations, and three missense mutations. Thus, the majority of mutations in the CNGB3 gene result in significantly altered and/or truncated polypeptides. Several mutations were found recurrently, in particular a 1 bp deletion, c.1148delC, which accounts for over 70% of all CNGB3 mutant alleles. In conclusion, mutations in the CNGB3 gene are responsible for approximately 50% of all patients with achromatopsia. This indicates that the CNGB3/ACHM3 locus on chromosome 8q21 is the major locus for achromatopsia in patients of European origin or descent.     

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.     

AAV-mediated human CNGB3 restores cone function in an all-cone mouse model of CNGB3 achromatopsia

Complete congenital achromatopsia is a devastating hereditary visual disorder. Mutations in the CNGB3 gene account for more than 50% of all known cases of achromatopsia. This work investigated the efficiency of subretinal (SR) delivered AAV8 (Y447, 733F) vector containing a human PR2.1 promoter and a human CNGB3 cDNA in Cngb3^?/?/Nrl^?/? mice. The Cngb3^?/?/Nrl^?/? mouse was a cone-dominant model with Cngb3 channel deficiency, which partially mimicked the all-cone foveal structure of human achromatopsia with CNGB3 mutations. Following SR delivery of the vector, AAV-mediated CNGB3 expression restored cone function which was assessed by the restoration of the cone-mediated electroretinogram (ERG) and immunohistochemistry. This therapeutic rescue resulted in long-term improvement of retinal function with the restoration of cone ERG amplitude. This study demonstrated an AAV-mediated gene therapy in a cone-dominant mouse model using a human gene construct and provided the potential to be utilized in clinical trials.     

Long-term and age-dependent restoration of visual function in a mouse model of CNGB3-associated achromatopsia following gene therapy

Mutations in the CNGB3 gene account for >50% of all known cases of achromatopsia. Although of early onset, its stationary character and the potential for rapid assessment of restoration of retinal function following therapy renders achromatopsia a very attractive candidate for gene therapy. Here we tested the efficacy of an rAAV2/8 vector containing a human cone arrestin promoter and a human CNGB3 cDNA in CNGB3 deficient mice. Following subretinal delivery of the vector, CNGB3 was detected in both M- and S-cones and resulted in increased levels of CNGA3, increased cone density and survival, improved cone outer segment structure and normal subcellular compartmentalization of cone opsins. Therapy also resulted in long-term improvement of retinal function, with restoration of cone ERG amplitudes of up to 90% of wild-type and a significant improvement in visual acuity. Remarkably, successful restoration of cone function was observed even when treatment was initiated at 6 months of age; however, restoration of normal visual acuity was only possible in younger animals (e.g. 2-4 weeks old). This study represents achievement of the most substantial restoration of visual function reported to date in an animal model of achromatopsia using a human gene construct, which has the potential to be utilized in clinical trials.     

Mapping of a novel locus for achromatopsia (ACHM4) to 1p and identification of a germline mutation in the alpha subunit of cone transducin (GNAT2)

Design and methods: A large consanguineous Pakistani family containing six subjects with autosomal recessive complete achromatopsia was ascertained. After excluding linkage to the two known achromatopsia genes (CNGA3 and CNGB3), a genome wide linkage screen was undertaken.

Results: Significant linkage was detected to a 12 cM autozygous segment between markers D1S485 and D1S2881 on chromosome 1p13. Direct sequence analysis of the candidate gene GNAT2 located within this interval identified a frameshift mutation in exon 7 (c842_843insTCAG; M280fsX291) that segregated with the disease.

Conclusions: The GNAT2 gene codes for cone α-transducin, the G protein that couples the cone pigments to cGMP-phosphodiesterase in phototransduction. Although cone α-transducin has a fundamental role in cone phototransduction, mutations in GNAT2 have not been described previously. Since mutations in the CNGA3 gene may cause a variety of retinal dystrophies (complete and incomplete achromatopsia and progressive cone dystrophy), GNAT2 mutations may also prove to be implicated in other forms of retinal dystrophy with cone dysfunction.

     

Clinical and genetic investigation of a large Tunisian family with complete achromatopsia: identification of a new nonsense mutation in GNAT2 gene

Complete achromatopsia is a rare autosomal recessive disease associated with CNGA3, CNGB3, GNAT2 and PDE6C mutations. This retinal disorder is characterized by complete loss of color discrimination due to the absence or alteration of the cones function. The purpose of the present study was the clinical and the genetic characterization of achromatopsia in a large consanguineous Tunisian family. Ophthalmic evaluation included a full clinical examination, color vision testing and electroretinography. Linkage analysis using microsatellite markers flanking CNGA3, CNGB3, GNAT2 and PDE6C genes was performed. Mutations were screened by direct sequencing. A total of 12 individuals were diagnosed with congenital complete achromatopsia. They are members of six nuclear consanguineous families belonging to the same large consanguineous family. Linkage analysis revealed linkage to GNAT2. Mutational screening of GNAT2 revealed three intronic variations c.119-69G>C, c.161+66A>T and c.875-31G>C that co-segregated with a novel mutation p.R313X. An identical GNAT2 haplotype segregating with this mutation was identified, indicating a founder mutation. All patients were homozygous for the p.R313X mutation. This is the first report of the clinical and genetic investigation of complete achromatopsia in North Africa and the largest family with recessive achromatopsia involving GNAT2; thus, providing a unique opportunity for genotype-phenotype correlation for this extremely rare condition.     

Mutations in the gene PDE6C encoding the catalytic subunit of the cone photoreceptor phosphodiesterase in patients with achromatopsia

Biallelic PDE6C mutations are a known cause for rod monochromacy, better known as autosomal recessive achromatopsia (ACHM), and early‐onset cone photoreceptor dysfunction. PDE6C encodes the catalytic α′‐subunit of the cone photoreceptor phosphodiesterase, thereby constituting an essential part of the phototransduction cascade. Here, we present the results of a study comprising 176 genetically preselected patients who remained unsolved after Sanger sequencing of the most frequent genes accounting for ACHM, and were subsequently screened for exonic and splice site variants in PDE6C applying a targeted next generation sequencing approach. We were able to identify potentially pathogenic biallelic variants in 15 index cases. The mutation spectrum comprises 18 different alleles, 15 of which are novel. Our study significantly contributes to the mutation spectrum of PDE6C and allows for a realistic estimate of the prevalence of PDE6C mutations in ACHM since our entire ACHM cohort comprises 1,074 independent families.     

Cone cGMP-gated channel mutations and clinical findings in patients with achromatopsia, macular degeneration, and other hereditary cone diseases

Unrelated patients with achromatopsia, macular degeneration with onset under age 50 years, cone degeneration or dysfunction, cone-rod degeneration, or macular malfunction were screened for mutations in the three genes known to be associated with achromatopsia: the GNAT2 gene encoding the alpha subunit of cone transducin and the CNGA3 and CNGB3 genes encoding the alpha and beta subunits of the cone cGMP-gated cation channel. We found no examples of patients with GNAT2 mutations. Out of 36 achromats, 12 (33%) had mutations in CNGA3 (13 different mutations including five novel mutations) and 12 (33%) had mutations in CNGB3 (six different mutations including four novel mutations). All achromats with CNG mutations had residual, presumably cone function as determined by computer-averaged 30-Hz electroretinograms (ERGs). There was considerable variability in acuity and color vision, with most patients having acuities of 20/200-20/400 and complete absence of color perception, and others having acuities of 20/25-20/40 and some color vision. Two pseudodominant achromatopsia cases were uncovered, both with CNGA3 mutations, including one family in which some compound heterozygotes with achromatopsia mutations were clinically unaffected. We found two novel CNGB3 changes in three patients with juvenile macular degeneration, a phenotype not previously associated with mutations in the cone channel subunits. These patients had subnormal acuity (20/30-20/60), normal to subnormal color vision, and normal to subnormal full-field cone ERG amplitudes. Our results indicate that some patients with channel protein mutations retain residual foveal cone function. Based on our findings, CNGB3 should be considered as a candidate gene to be evaluated in patients with forms of cone dysfunction, including macular degeneration.     

A locus for autosomal recessive achromatopsia on human chromosome 8q

Autosomal recessive achromatopsia is a rare disorder characterized by total absent color vision, nystagmus, photophobia, and visual impairment, frequently leading to 'legal blindness'. The primary defect is at the photoreceptor level, with retinal cones being absent or defective. The first locus for this disorder was mapped to chromosome 2q11. Here, we confirm the genetic mapping of a locus discovered in our studies of a kindred with Irish ancestry, but no known consanguinity, in which 5 of 12 children are affected. We have mapped the locus in this disorder in this family to chromosome 8q. Available data now narrow the region containing the putative gene to 1.2 cM.     

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.     

Genetic mapping of the copper toxicosis locus in Bedlington terriers to dog chromosome 10, in a region syntenic to human chromosome region 2p13- p16

Abnormal hepatic copper accumulation is recognized as an inherited disorder in man, mouse, rat and dog. The major cause of hepatic copper accumulation in man is a dysfunctional ATP7B gene, causing Wilson disease (WD). Mutations in the ATP7B genes have also been demonstrated in mouse and rat. The ATP7B gene has been excluded in the much rarer human copper overload disease non-Indian childhood cirrhosis, indicating genetic heterogeneity. By investigating the common autosomal recessive copper toxicosis (CT) in Bedlington terriers, we have identified a new locus involved in progressive liver disease. We examined whether the WD gene ATP7B was also causative for CT by investigating the chromosomal co-localization of ATP7B and C04107, using fluorescence in situ hybridization (FISH). C04107 is an anonymous microsatellite marker closely linked to CT. However, BAC clones containing ATP7B and C04107 mapped to the canine chromosome regions CFA22q11 and CFA10q26, respectively, demonstrating that WD cannot be homologous to CT. The copper transport genes CTR1 and CTR2 were also excluded as candidate genes for CT since they both mapped to canine chromosome region CFA11q22. 2-22.5. A transcribed sequence identified from the C04107-containing BAC was found to be homologous to a gene expressed from human chromosome 2p13-p16, a region devoid of any positional candidate genes.      

A missense mutation in PIK3R5 gene in a family with ataxia and oculomotor apraxia

Autosomal recessive ataxias are heterogeneous group of disorders characterized by cerebellar atrophy and peripheral sensorimotor neuropathy. Molecular characterization of this group of disorders identified a number of genes contributing to these overlapping phenotypes. Ataxia with oculomotor apraxia type 2 (AOA2) is an autosomal recessive form of ataxia caused by mutations in the SETX gene. We report on a consanguineous family with autosomal recessive inheritance and clinical characteristics of AOA2, and no mutations in the SETX gene. We mapped the AOA locus in this family to chromosome 17p12-p13. Sequencing of all genes in the refined region identified a homozygous missense mutation in PIK3R5 that was absent in 477 normal controls. Our characterization of the PIK3R5 protein and findings suggest that it may play a role in the development of the cerebellum and vermis.     

Evidence for genetic heterogeneity in Best''s vitelliform macular dystrophy.

Best's vitelliform macular dystrophy is an early onset, autosomal dominant macular degeneration. Linkage analysis has previously mapped a disease locus in this disorder to the pericentromeric region of chromosome 11. We examined two families, one of German and one of Irish origin, both affected with this disorder. The Irish family (BTMD1) showed strong evidence for linkage to the previously reported locus on chromosome 11. Linkage of the disease locus to the same region of chromosome 11 has been significantly excluded in the German family (Fam E), thereby providing evidence of locus heterogeneity in this clinically unique condition.     

A locus for spondylocarpotarsal synostosis syndrome at chromosome 3p14

Spondylocarpotarsal synostosis syndrome is a rare autosomal recessive disorder characterised by vertebral fusions, frequently manifesting as an unsegmented vertebral bar, as well as fusions of the carpal and tarsal bones.

In a study of three consanguineous families and one non-consanguineous family, linkage analysis was used to establish the chromosomal location of the disease gene. Linkage analysis localised the disease gene to chromosome 3p14. A maximum lod score of 6.49 (q = 0) was obtained for the marker at locus D3S3532 on chromosome 3p. Recombination mapping narrowed the linked region to the 5.7 cM genetic interval between the markers at loci D3S3724 and D3S1300. A common region of homozygosity was found between the markers at loci D3S3724 and D3S1300, defining a physical interval of approximately 4 million base pairs likely to contain the disease gene.

Identification of the gene responsible for this disorder will provide insight into the genes that play a role in the formation of the vertebral column and joints.

     

Identification of the Syrian hamster cardiomyopathy gene

The BIO14.6 hamster is a widely used model for autosomal recessive cardiomyopathy. These animals die prematurely from progressive myocardial necrosis and heart failure. The primary genetic defect leading to the cardiomyopathy is still unknown. Recently, a genetic linkage map localized the cardiomyopathy locus on hamster chromosome 9qa2.1-b1, excluding several candidate genes. We now demonstrate that the cardiomyopathy results from a mutation in the delta-sarcoglycan gene that maps to the disease locus. This mutation was completely coincident with the disease in backcross and F2 pedigrees. This constitutes the first animal model identified for human sarcoglycan disorders.      

Congenital generalized lipodystrophy: profile of the disease and gender differences in two siblings

Congenital generalized Lipodystrophy (BSCL) or Berardinelli-Seip syndrome (Mendelian inheritance in man, catalog no. 269700) is a rare autosomal recessive syndrome characterized by paucity of body fat since birth and insulin resistance. The pathophysiology of this condition is unclear, but defects in insulin function and impaired adipogenesis have been described as important factors in the etiology of the disease. Recently, two gene loci have been identified to harbor the mutations causing this disorder: BSCL1 mapped to human chromosome 9q34 (1, 2) and BSCL2 mapped to human chromosome 11q13 (1, 3). This report describes the natural history of the disease in two siblings (female and male) of Lebanese origin who have mutations in the BSCL2 locus (669delGTATC).     

Mutations in HPRP3, a third member of pre-mRNA splicing factor genes, implicated in autosomal dominant retinitis pigmentosa.

Retinitis pigmentosa (RP), the commonest form of inherited retinal dystrophies is a clinically and genetically heterogeneous disorder. It is characterized by progressive degeneration of the peripheral retina leading to night blindness and loss of peripheral visual field. RP is inherited either in an autosomal dominant, autosomal recessive or X-linked mode. A locus (RP18) for autosomal dominant RP was previously mapped by linkage analysis in two large pedigrees to chromosome 1p13-q21. The human HPRP3 gene, the orthologue of the yeast pre-mRNA splicing factor (PRP3), localizes within the RP18 disease interval. The recent identification of mutations in human splicing factors, PRPF31 and PRPC8, led us to screen HPRP3 as a candidate in three chromosome 1q-linked families. So far, two different missense mutations in two English, a Danish family and in three RP individuals have been identified. Both mutations are clustered within a two-codon stretch in the 11th exon of the HPRP3 gene. Interestingly, one of the mutations (T494M) is seen repeatedly in apparently unlinked families raising the possibility of a mutation hot spot. This has been confirmed by haplotype analysis using SNPs spanning the HPRP3 gene region supporting multiple origins of the mutation. The altered HPRP3 amino acids, which are highly conserved in all known HPRP3 orthologues, indicate a major function of that domain in the splicing process. The identification of mutations in a third pre-mRNA splicing factor gene further highlights a novel mechanism of photoreceptor degeneration due to defects in the splicing process.