Abolished interaction of NUB1 with mutant AIPL1 involved in Leber congenital amaurosis

Constructs based on the pSUPER vector [Science 296 (2002) 550] and encoding small interfering RNAs specific for the Type I, Type II, or Type III isozymes of mammalian (rat) hexokinase were prepared. Transfection of Chinese hamster ovary and HeLa cells with these vectors resulted in selective depletion of the respective isozymes. A Zeocin marker was incorporated into the modified pSUPER vector, permitting isolation of stably transfected cell lines selectively depleted of the respective isozyme.     

Gene discovery and prevalence in inherited retinal dystrophies

Inherited retinal dystrophies are Mendelian neurodegenerative conditions classified as pigmentary retinopathies, macular dystrophies and others. Over a 21-year period, from 1990 to 2011, we have screened in Montpellier 107 genes in 609 families and have identified a causal mutation in 68.5% of them. Following a gene candidate approach, we established that RPE65, the isomerohydrolase of the visual cycle, is responsible for severe childhood blindness (Leber congenital amaurosis or early onset retinal dystrophy). In an ongoing study, we screened the genes in a series of 283 families with dominant retinitis pigmentosa and we have estimated that 80% of the families have a mutation in a known gene. A similar study is currently undergoing for autosomal recessive retinitis pigmentosa. Finally, we have identified IMPG1 as a responsible gene for rare cases of macular vitelliform dystrophy with a dominant or recessive inheritance.     

IMP Dehydrogenase-Linked Retinitis Pigmentosa

Many retinal diseases are caused by mutations in photoreceptor-specific proteins. However, retinal disease can also result from mutations in widely expressed proteins. One such protein is inosine monophosphate dehydrogenase type 1 (IMPDH1), which catalyzes a key step in guanine nucleotide biosynthesis and also binds single-stranded nucleic acids. The pathogenic IMPDH1 mutations are in or near the CBS domains and do not affect enzymatic activity. However, these mutations do decrease the affinity and specificity of single-stranded nucleic acid binding. These observations suggest that IMPDH1 has a previously unappreciated role in RNA metabolism that is crucial for photoreceptor function.     

Development of hybrid viral vectors for gene therapy

Adenoviral, retroviral/lentiviral, adeno-associated viral, and herpesviral vectors are the major viral vectors used in gene therapy. Compared with non-viral methods, viruses are highly-evolved, natural delivery agents for genetic materials. Despite their remarkable transduction efficiency, both clinical trials and laboratory experiments have suggested that viral vectors have inherent shortcomings for gene therapy, including limited loading capacity, immunogenicity, genotoxicity, and failure to support long-term adequate transgenic expression. One of the key issues in viral gene therapy is the state of the delivered genetic material in transduced cells. To address genotoxicity and improve the therapeutic transgene expression profile, construction of hybrid vectors have recently been developed. By adding new abilities or replacing certain undesirable elements, novel hybrid viral vectors are expected to outperform their conventional counterparts with improved safety and enhanced therapeutic efficacy. This review provides a comprehensive summary of current achievements in hybrid viral vector development and their impact on the field of gene therapy.     

Characterization of Leber Congenital Amaurosis-associated NMNAT1 Mutants

Leber congenital amaurosis 9 (LCA9) is an autosomal recessive retinal degeneration condition caused by mutations in the NAD⁺ biosynthetic enzyme NMNAT1. This condition leads to early blindness but no other consistent deficits have been reported in patients with NMNAT1 mutations despite its central role in metabolism and ubiquitous expression. To study how these mutations affect NMNAT1 function and ultimately lead to the retinal degeneration phenotype, we performed detailed analysis of LCA-associated NMNAT1 mutants, including the expression, nuclear localization, enzymatic activity, secondary structure, oligomerization, and promotion of axonal and cellular integrity in response to injury. In many assays, most mutants produced results similar to wild type NMNAT1. Indeed, NAD⁺ synthetic activity is unlikely to be a primary mechanism underlying retinal degeneration as most LCA-associated NMNAT1 mutants had normal enzymatic activity. In contrast, the secondary structure of many NMNAT1 mutants was relatively less stable as they lost enzymatic activity after heat shock, whereas wild type NMNAT1 retains significant activity after this stress. These results suggest that LCA-associated NMNAT1 mutants are more vulnerable to stressful conditions that lead to protein unfolding, a potential contributor to the retinal degeneration observed in this syndrome.     

Rescue of Enzymatic Function for Disease-associated RPE65 Proteins Containing Various Missense Mutations in Non-active Sites

Over 70 different missense mutations, including a dominant mutation, in RPE65 retinoid isomerase are associated with distinct forms of retinal degeneration; however, the disease mechanisms for most of these mutations have not been studied. Although some mutations have been shown to abolish enzyme activity, the molecular mechanisms leading to the loss of enzymatic function and retinal degeneration remain poorly understood. Here we show that the 26 S proteasome non-ATPase regulatory subunit 13 (PSMD13), a newly identified negative regulator of RPE65, plays a critical role in regulating pathogenicity of three mutations (L22P, T101I, and L408P) by mediating rapid degradation of mutated RPE65s via a ubiquitination- and proteasome-dependent non-lysosomal pathway. These mutant RPE65s were misfolded and formed aggregates or high molecular complexes via disulfide bonds. Interaction of PSMD13 with mutant RPE65s promoted degradation of misfolded but not properly folded mutant RPE65s. Many mutations, including L22P, T101I, and L408P, were mapped on non-active sites. Although their activities were very low, these mutant RPE65s were catalytically active and could be significantly rescued at low temperature, whereas mutant RPE65s with a distinct active site mutation could not be rescued under the same conditions. Sodium 4-phenylbutyrate and glycerol displayed a significant synergistic effect on the low temperature rescue of the mutant RPE65s by promoting proper folding, reducing aggregation, and increasing membrane association. Our results suggest that a low temperature eye mask and sodium 4-phenylbutyrate, a United States Food and Drug Administration-approved oral medicine, may provide a promising “protein repair therapy” that can enhance the efficacy of gene therapy by reducing the cytotoxic effect of misfolded mutant RPE65s.     

First 3D-Structural Data of Full-Length Guanylyl Cyclase 1 in Rod-Outer-Segment Preparations of Bovine Retina by Cross-Linking/Mass Spectrometry

The rod-outer-segment guanylyl cyclase 1 (ROS-GC1) is a key transmembrane protein for retinal phototransduction. Mutations of ROS-GC1 correlate with different retinal diseases that often lead to blindness. No structural data are available for ROS-GC1 so far. We performed a 3D-structural analysis of native ROS-GC1 from bovine retina by cross-linking/mass spectrometry (XL-MS) and computational modeling. Absolute quantification and activity measurements of native ROS-GC1 were performed by MS-based assays directly in bovine retina samples. Our data present the first 3D-structural analysis of active, full-length ROS-GC1 derived from bovine retina. We propose a novel domain organization for the intracellular domain ROS-GC1. Our XL-MS data of native ROS-GC1 from rod-outer-segment preparations of bovine retina agree with a dimeric architecture. Our integrated approach can serve as a blueprint for conducting 3D-structural studies of membrane proteins in their native environment.     

Exome sequencing identifies RDH12 compound heterozygous mutations in a family with severe retinitis pigmentosa

Objective

Retinitis pigmentosa (RP) is the most prevalent type of inherited retinal degeneration and one of the commonest causes of genetically determined visual dysfunction worldwide. To date, approximately 35 genes have been associated with nonsyndromic autosomal recessive RP (arRP), however the small contribution of each gene to the total prevalence of arRP and the lack of a clear genotype–phenotype correlation complicate the genetic analysis in affected patients. Next generation sequencing technologies are powerful and cost-effective methods for detecting causative mutations in both sporadic and familial RP cases.

Methods

A Mexican family with 5 members affected from arRP was studied. All patients underwent a complete ophthalmologic examination. Molecular methods included genome-wide SNP homozygosity mapping, exome sequencing analysis, and Sanger-sequencing confirmation of causal mutations.

Results

No regions of shared homozygosity among affected subjects were identified. Exome sequencing in a single patient allowed the detection of two missense mutations in the RDH12 gene: a c.446T>C transition predicting a novel p.L149P substitution, and a c.295C>A transversion predicting a previously reported p.L99I replacement. Sanger sequencing confirmed that all affected subjects carried both RDH12 mutations.

Conclusions

This study adds to the molecular spectrum of RDH12-related retinopathy and offers an additional example of the power of exome sequencing in the diagnosis of recessively inherited retinal degenerations.     

Rhodopsin p.N78I dominant mutation causing sectorial retinitis pigmentosa in a pedigree with intrafamilial clinical heterogeneity

Objective

The purpose of this study was to determine the molecular basis of retinitis pigmentosa (RP) in a 4 affected sib-family segregating this retinal phenotype.

Methods

Affected sibs underwent complete ophthalmologic examination including funduscopic inspection, electroretinogram, fluorescein angiography, visual field measurement, and optical coherence tomography. Both parents were deceased after their sixties and were reported with no visual handicap. Molecular analysis included direct nucleotide sequencing of the rhodopsin gene (RHO), at chromosome 3q21–q24, in DNA from a total of 4 affected sibs. A total of 200 ethnically matched alleles were included as mutation controls.

Results

Sector RP was clinically documented in this family. Wide phenotypic variability was observed with visual acuities ranging from 20/20 to 20/200 and variable funduscopic appearance. Molecular analysis disclosed a c.233A>T mutation at RHO exon 1, predicting a missense p.N78I substitution.

Conclusions

Even though RP can be caused by mutations in a variety of genes, the RHO gene was chosen to be investigated in this RP family since it has been previously associated to sector disease. This case exemplifies the value of guiding RP molecular analysis based on funduscopic features.