Association of Missense Variants in VSX2 With a Peculiar Form of Congenital Stationary Night Blindness Affecting All Bipolar Cells.

Importance: Congenital stationary night blindness (CSNB) is an inherited stationary retinal disorder that is clinically and genetically heterogeneous. To date, the genetic association between some cases with CSNB and an unusual complex clinical picture is unclear. Objective: To describe an unreported CSNB phenotype and the associated gene defect in 3 patients from 2 unrelated families. Design, Setting, and Participants: This retrospective case series was conducted in 2021 and 2022 at a national referral center for rare ocular diseases. Data for 3 patients from a cohort of 140 genetically unsolved CSNB cases were analyzed clinically and genetically. Exposures: Complete ocular examination including full-field electroretinography and multimodal fundus imaging (spectral-domain optical coherence tomography, color, infrared reflectance, and short-wavelength autofluorescence photographs) were performed. The gene defect was identified by exome sequencing and confirmed by Sanger sequencing and co-segregation analysis in 1 family. Screening was performed for genetically unsolved CSNB cases for VSX2 variants by direct Sanger sequencing. Main Outcomes and Measures: Ocular and molecular biology findings. Results: The series included 3 patients whose clinical investigations occurred at ages in the early 30s, younger than 12 years, and in the mid 40s. They had nystagmus, low stable visual acuity, and myopia from birth and experienced night blindness. Two older patients had bilateral lens luxation and underwent lens extraction. Full-field electroretinography revealed an electronegative Schubert-Bornschein appearance, combining characteristics of incomplete and complete CSNB, affecting the function of rod and cone ON- and OFF-bipolar cells. Exome sequencing and co-segregation analysis in a consanguineous family with 2 affected members identified a homozygous variant in VSX2. Subsequently, screening of the CSNB cohort identified another unrelated patient harboring a distinct VSX2 variant. Conclusions and Relevance: This case series revealed a peculiar pan-bipolar cell retinopathy with lens luxation associated with variants in VSX2. Clinicians should be aware of this association and VSX2 added to CSNB diagnostic gene panels.

Whole Locus Sequencing Identifies a Prevalent Founder Deep Intronic RPGRIP1 Pathologic Variant in the French Leber Congenital Amaurosis Cohort.

Leber congenital amaurosis (LCA) encompasses the earliest and most severe retinal dystrophies and can occur as a non-syndromic or a syndromic disease. Molecular diagnosis in LCA is of particular importance in clinical decision-making and patient care since it can provide ocular and extraocular prognostics and identify patients eligible to develop gene-specific therapies. Routine high-throughput molecular testing in LCA yields 70%-80% of genetic diagnosis. In this study, we aimed to investigate the non-coding regions of one non-syndromic LCA gene, RPGRIP1, in a series of six families displaying one single disease allele after a gene-panel screening of 722 LCA families which identified 26 biallelic RPGRIP1 families. Using trio-based high-throughput whole locus sequencing (WLS) for second disease alleles, we identified a founder deep intronic mutation (NM_020366.3:c.1468-128T>G) in 3/6 families. We employed Sanger sequencing to search for the pathologic variant in unresolved LCA cases (106/722) and identified three additional families (two homozygous and one compound heterozygous with the NM_020366.3:c.930+77A>G deep intronic change). This makes the c.1468-128T>G the most frequent RPGRIP1 disease allele (8/60, 13%) in our cohort. Studying patient lymphoblasts, we show that the pathologic variant creates a donor splice-site and leads to the insertion of the pseudo-exon in the mRNA, which we were able to hamper using splice-switching antisense oligonucleotides (AONs), paving the way to therapies.

Loss of Function of RIMS2 Causes a Syndromic Congenital Cone-Rod Synaptic Disease with Neurodevelopmental and Pancreatic Involvement.

Congenital cone-rod synaptic disorder (CRSD), also known as incomplete congenital stationary night blindness (iCSNB), is a non-progressive inherited retinal disease (IRD) characterized by night blindness, photophobia, and nystagmus, and distinctive electroretinographic features. Here, we report bi-allelic RIMS2 variants in seven CRSD-affected individuals from four unrelated families. Apart from CRSD, neurodevelopmental disease was observed in all affected individuals, and abnormal glucose homeostasis was observed in the eldest affected individual. RIMS2 regulates synaptic membrane exocytosis. Data mining of human adult bulk and single-cell retinal transcriptional datasets revealed predominant expression in rod photoreceptors, and immunostaining demonstrated RIMS2 localization in the human retinal outer plexiform layer, Purkinje cells, and pancreatic islets. Additionally, nonsense variants were shown to result in truncated RIMS2 and decreased insulin secretion in mammalian cells. The identification of a syndromic stationary congenital IRD has a major impact on the differential diagnosis of syndromic congenital IRD, which has previously been exclusively linked with degenerative IRD.

Description of Two Siblings with Apparently Severe CEP290 Mutations and Unusually Mild Retinal Disease Unrelated to Basal Exon Skipping or Nonsense-Associated Altered Splicing.

CEP290 mutations cause a spectrum of ciliopathies, including Leber congenital amaurosis. Milder retinal diseases have been ascribed to exclusion of CEP290 mutant exons through basal exon skipping (BES) and/or nonsense-associated altered splicing (NAS). Here, we report two siblings with some preserved vision despite biallelism for presumably severe CEP290 mutations: a maternal splice site change in intron 18 (c.1824 + 3A > G) and a paternal c.6869dup (p.Asn2290Lysfs∗6) in exon 50 that introduces a premature termination codon (PTC) within the same exon. Analyzing mRNAs from fibroblasts of the two siblings, we detected no BES or NAS which could have enabled the production of PTC-free CEP290 isoforms from the paternal allele. In contrast, we reveal partial alteration of exon 18 donor splice site, allowing the transcription of some correctly spliced CEP290 mRNAs from the maternal allele which likely account for the mild retinal disease. This observation adds further variability to the mechanisms underlying CEP290 pleiotropy.

Genetic Deciphering of Early-Onset and Severe Retinal Dystrophy Associated with Sensorineural Hearing Loss.

The specific association of Leber congenital amaurosis (LCA) or early-onset severe retinal dystrophy (LCA-like) with sensorineural hearing loss (SHL) is uncommon. Recently, we ascribed some of these distinctive associations to dominant and de novo mutations in the ß-tubulin 4B isotype-encoding gene (TUBB4B), providing a link between a sensorineural disease and anomalies in microtubules behavior. Here, we report 12 sporadic cases with LCA/SHL or LCA-like/SHL and no TUBB4B mutation. Trio-based whole exome sequencing (WES) identified disease-causing mutations in 5/12 cases. Four out of five carried biallelic mutations in PEX1 (1/4) or PEX6 (3/4), involved in peroxisome biogenesis disorders from Zellweger syndrome characterized by severe neurologic and neurosensory dysfunctions, craniofacial abnormalities, and liver dysfunction to Heimler syndrome associating SHL, enamel hypoplasia of the secondary dentition, nail abnormalities, and occasional retinal disease. Upon reexamination, the index case carrying PEX1 mutations, a 4-year-old girl, presented additional symptoms consistent with Zellweger syndrome. Reexamination of individuals with PEX6 mutations (1/3 unavailable) revealed normal nails but enamel hypoplasia affecting one primary teeth in a 4-year-old girl and severe enamel hypoplasia of primary teeth hidden by dental prosthesis in a 50-year-old male, describing a novel PEX6-associated disease of the Zellweger/Heimler spectrum. Finally, hemizygosity for a CACNA1F mutation was identified in an 18-year-old male addressed for LCA/SHL, redirecting the retinal diagnosis to congenital stationary night blindness (CSNB2A). Consistent with the pure CSNB2A retinal involvement, SHL was ascribed to biallelic mutations in another gene, STRC, involved in nonprogressive DFNB16 deafness.

AON-Mediated Exon Skipping to Bypass Protein Truncation in Retinal Dystrophies Due to the Recurrent CEP290 c.4723A > T Mutation. Fact or Fiction?

Mutations in CEP290 encoding a centrosomal protein important to cilia formation cause a spectrum of diseases, from isolated retinal dystrophies to multivisceral and sometimes embryo-lethal ciliopathies. In recent years, endogenous and/or selective non-canonical exon skipping of mutant exons have been documented in attenuated retinal disease cases. This observation led us to consider targeted exon skipping to bypass protein truncation resulting from a recurrent mutation in exon 36 (c.4723A > T, p.Lys1575*) causing isolated retinal ciliopathy. Here, we report two unrelated individuals (P1 and P2), carrying the mutation in homozygosity but affected with early-onset severe retinal dystrophy and congenital blindness, respectively. Studying skin-derived fibroblasts, we observed basal skipping and nonsense associated-altered splicing of exon 36, producing low (P1) and very low (P2) levels of CEP290 products. Consistent with a more severe disease, fibroblasts from P2 exhibited reduced ciliation compared to P1 cells displaying normally abundant cilia; both lines presented however significantly elongated cilia, suggesting altered axonemal trafficking. Antisense oligonucleotides (AONs)-mediated skipping of exon 36 increased the abundance of the premature termination codon (PTC)-free mRNA and protein, reduced axonemal length and improved cilia formation in P2 but not in P1 expressing higher levels of skipped mRNA, questioning AON-mediated exon skipping to treat patients carrying the recurrent c.4723A > T mutation.

Basal exon skipping and nonsense-associated altered splicing allows bypassing complete CEP290 loss-of-function in individuals with unusually mild retinal disease.

CEP290 mutations cause a spectrum of ciliopathies from Leber congenital amaurosis type 10 (LCA10) to embryo-lethal Meckel syndrome (MKS). Using panel-based molecular diagnosis testing for inherited retinal diseases, we identified two individuals with some preserved vision despite biallelism for presumably truncating CEP290 mutations. The first one carried a homozygous 1 base pair deletion in Exon 17, introducing a premature termination codon (PTC) in Exon 18 (c.1666del; p.Ile556Phefs*17). mRNA analysis revealed a basal exon skipping (BES) of Exon 18, providing mutant cells with the ability to escape protein truncation, while disrupting the reading frame in controls. The second individual harbored compound heterozygous nonsense mutations in Exon 8 (c.508A>T, p.Lys170*) and Exon 32 (c.4090G>T, p.Glu1364*), respectively. Some CEP290 lacking Exon 8 were detected in mutant fibroblasts but not in controls whereas some skipping of Exon 32 occurred in both lines, but with higher amplitude in the mutant. Considering that the deletion of either exon maintains the reading frame in either line, skipping in mutant cells likely involves nonsense-associated altered splicing alone (Exon 8), or with BES (Exon 32). Skipping of PTC-containing exons in mutant cells allowed production of CEP290 isoforms with preserved ability to assemble into a high molecular weight complex and to interact efficiently with proteins important for cilia formation and intraflagellar trafficking. In contrast, studying LCA10 and MKS fibroblasts we show moderate to severe cilia alterations, providing support for a correlation between disease severity and the ability of cells to express shortened, yet functional, CEP290 isoforms.

Whole-genome sequencing in patients with ciliopathies uncovers a novel recurrent tandem duplication in IFT140.

Ciliopathies represent a wide spectrum of rare diseases with overlapping phenotypes and a high genetic heterogeneity. Among those, IFT140 is implicated in a variety of phenotypes ranging from isolated retinis pigmentosa to more syndromic cases. Using whole-genome sequencing in patients with uncharacterized ciliopathies, we identified a novel recurrent tandem duplication of exon 27-30 (6.7 kb) in IFT140, c.3454-488_4182+2588dup p.(Tyr1152_Thr1394dup), missed by whole-exome sequencing. Pathogenicity of the mutation was assessed on the patients' skin fibroblasts. Several hundreds of patients with a ciliopathy phenotype were screened and biallelic mutations were identified in 11 families representing 12 pathogenic variants of which seven are novel. Among those unrelated families especially with a Mainzer-Saldino syndrome, eight carried the same tandem duplication (two at the homozygous state and six at the heterozygous state). In conclusion, we demonstrated the implication of structural variations in IFT140-related diseases expanding its mutation spectrum. We also provide evidences for a unique genomic event mediated by an Alu-Alu recombination occurring on a shared haplotype. We confirm that whole-genome sequencing can be instrumental in the ability to detect structural variants for genomic disorders.

Mutations in TUBB4B Cause a Distinctive Sensorineural Disease.

Leber congenital amaurosis (LCA) is a neurodegenerative disease of photoreceptor cells that causes blindness within the first year of life. It occasionally occurs in syndromic metabolic diseases and plurisystemic ciliopathies. Using exome sequencing in a multiplex family and three simplex case subjects with an atypical association of LCA with early-onset hearing loss, we identified two heterozygous mutations affecting Arg391 in ß-tubulin 4B isotype-encoding (TUBB4B). Inspection of the atomic structure of the microtubule (MT) protofilament reveals that the ß-tubulin Arg391 residue contributes to a binding pocket that interacts with α-tubulin contained in the longitudinally adjacent αß-heterodimer, consistent with a role in maintaining MT stability. Functional analysis in cultured cells overexpressing FLAG-tagged wild-type or mutant TUBB4B as well as in primary skin-derived fibroblasts showed that the mutant TUBB4B is able to fold, form αß-heterodimers, and co-assemble into the endogenous MT lattice. However, the dynamics of growing MTs were consistently altered, showing that the mutations have a significant dampening impact on normal MT growth. Our findings provide a link between sensorineural disease and anomalies in MT behavior and describe a syndromic LCA unrelated to ciliary dysfunction.

LEBER CONGENITAL AMAUROSIS WITH LARGE RETINAL PIGMENT CLUMPS CAUSED BY COMPOUND HETEROZYGOUS MUTATIONS IN KCNJ13.

PURPOSE: To describe a patient with mutations in KCNJ13 presenting particular clinical features. METHODS: Standard ophthalmic examination, fundus autofluorescence, spectral domain optical coherence tomography, full-field electroretinography. The 3 exons of KCNJ13 were polymerase chain reaction amplified and Sanger sequenced. PATIENTS: A 31-year-old man with Leber congenital amaurosis. RESULTS: Patient had nystagmus since childhood, best-corrected visual acuity limited to 20/400 OD and 20/200 OS, and had cataracts extracted in both eyes. There were clumpy pigment deposits mostly in macular area, causing an uneven line of retinal pigment epithelium on spectral domain optical coherence tomography. In retinal parts devoid of pigment deposits around the optic disk and in periphery, retinal thickness was increased and hyperreflective formations were present either in the inner nuclear layer or in the outer nuclear layer. The patient was compound heterozygous for new mutations in KCNJ13 which encodes the Kir 7.1 potassium channel, c.314G>T (p.Ser105Ile) in exon 2 and c.655C>T (p.Gln219*) in exon 3. Both mutations were absent from databases. CONCLUSION: KCNJ13 mutations are responsible for early-onset retinal dystrophy, featuring remarkable clumpy pigment deposits at the level of the retinal pigment epithelium, suggesting dysfunction and disorganization of this tissue. Parts of the retina remain relatively preserved anatomically but are increased in thickness. This distinct fundus appearance should help in identifying the "KCNJ13 retinal dystrophy" to orient the molecular diagnosis.

A mutation in VPS15 (PIK3R4) causes a ciliopathy and affects IFT20 release from the cis-Golgi.

Ciliopathies are a group of diseases that affect kidney and retina among other organs. Here, we identify a missense mutation in PIK3R4 (phosphoinositide 3-kinase regulatory subunit 4, named VPS15) in a family with a ciliopathy phenotype. Besides being required for trafficking and autophagy, we show that VPS15 regulates primary cilium length in human fibroblasts, as well as ciliary processes in zebrafish. Furthermore, we demonstrate its interaction with the golgin GM130 and its localization to the Golgi. The VPS15-R998Q patient mutation impairs Golgi trafficking functions in humanized yeast cells. Moreover, in VPS15-R998Q patient fibroblasts, the intraflagellar transport protein IFT20 is not localized to vesicles trafficking to the cilium but is restricted to the Golgi. Our findings suggest that at the Golgi, VPS15 and GM130 form a protein complex devoid of VPS34 to ensure the IFT20-dependent sorting and transport of membrane proteins from the cis-Golgi to the primary cilium.

Nonsyndromic Retinal Dystrophy due to Bi-Allelic Mutations in the Ciliary Transport Gene IFT140.

PURPOSE: Mutations in the ciliary transporter gene IFT140, usually associated with a severe syndromic ciliopathy, may also cause isolated retinal dystrophy. A series of patients with nonsyndromic retinitis pigmentosa (RP) due to IFT140 was investigated in this study. METHODS: Five probands and available affected family members underwent detailed phenotyping including retinal imaging and electrophysiology. Whole exome sequencing was performed on two probands, a targeted sequencing panel of 176 retinal genes on a further two, and whole genome sequencing on the fifth. Missense mutations of IFT140 were further investigated in vitro using transient plasmid transfection of hTERT-RPE1 cells. RESULTS: Eight affected patients from five families had preserved visual acuity until at least the second decade; all had normal development without skeletal manifestations or renal failure at age 13 to 67 years (mean, 42 years; median, 44.5 years). Bi-allelic mutations in IFT140 were identified in all families including two novel mutations: c.2815T > C (p.Ser939Pro) and c.1422_23insAA (p.Arg475Asnfs*14). Expression studies demonstrated a significantly reduced number of cells showing localization of mutant IFT140 with the basal body for two nonsyndromic mutations and two syndromic mutations compared with the wild type and a polymorphism. CONCLUSIONS: This study highlights the phenotype of nonsyndromic RP due to mutations in IFT140 with milder retinal dystrophy than that associated with the syndromic disease.

Intravitreal Injection of Splice-switching Oligonucleotides to Manipulate Splicing in Retinal Cells.

Leber congenital amaurosis is a severe hereditary retinal dystrophy responsible for neonatal blindness. The most common disease-causing mutation (c.2991+1655A>G; 10-15%) creates a strong splice donor site that leads to insertion of a cryptic exon encoding a premature stop codon. Recently, we reported that splice-switching oligonucleotides (SSO) allow skipping of the mutant cryptic exon and the restoration of ciliation in fibroblasts of affected patients, supporting the feasibility of a SSO-mediated exon skipping strategy to correct the aberrant splicing. Here, we present data in the wild-type mouse, which demonstrate that intravitreal administration of 2'-OMePS-SSO allows selective alteration of Cep290 splicing in retinal cells, including photoreceptors as shown by successful alteration of Abca4 splicing using the same approach. We show that both SSOs and Cep290 skipped mRNA were detectable for at least 1 month and that intravitreal administration of oligonucleotides did not provoke any serious adverse event. These data suggest that intravitreal injections of SSO should be considered to bypass protein truncation resulting from the c.2991+1655A>G mutation as well as other truncating mutations in genes which like CEP290 or ABCA4 have a mRNA size that exceed cargo capacities of US Food and Drug Administration (FDA)-approved adeno-associated virus (AAV)-vectors, thus hampering gene augmentation therapy.

IFT81, encoding an IFT-B core protein, as a very rare cause of a ciliopathy phenotype.

BACKGROUND: Bidirectional intraflagellar transport (IFT) consists of two major protein complexes, IFT-A and IFT-B. In contrast to the IFT-B complex, all components of IFT-A have recently been linked to human ciliopathies when defective. We therefore hypothesised that mutations in additional IFT-B encoding genes can be found in patients with multisystemic ciliopathies. METHODS: We screened 1628 individuals with reno-ocular ciliopathies by targeted next-generation sequencing of ciliary candidate genes, including all IFT-B encoding genes. RESULTS: Consequently, we identified a homozygous mutation in IFT81 affecting an obligatory donor splice site in an individual with nephronophthisis and polydactyly. Further, we detected a loss-of-stop mutation with extension of the deduced protein by 10 amino acids in an individual with neuronal ceroid lipofuscinosis-1. This proband presented with retinal dystrophy and brain lesions including cerebellar atrophy, a phenotype to which the IFT81 variant might contribute. Cultured fibroblasts of this latter affected individual showed a significant decrease in ciliated cell abundance compared with controls and increased expression of the transcription factor GLI2 suggesting deranged sonic hedgehog signalling. CONCLUSIONS: This work describes identification of mutations of IFT81 in individuals with symptoms consistent with the clinical spectrum of ciliopathies. It might represent the rare case of a core IFT-B complex protein found associated with human disease. Our data further suggest that defects in the IFT-B core are an exceedingly rare finding, probably due to its indispensable role for ciliary assembly in development.

Mutations in DOCK7 in individuals with epileptic encephalopathy and cortical blindness.

Epileptic encephalopathies are increasingly thought to be of genetic origin, although the exact etiology remains uncertain in many cases. We describe here three girls from two nonconsanguineous families affected by a clinical entity characterized by dysmorphic features, early-onset intractable epilepsy, intellectual disability, and cortical blindness. In individuals from each family, brain imaging also showed specific changes, including an abnormally marked pontobulbar sulcus and abnormal signals (T2 hyperintensities) and atrophy in the occipital lobe. Exome sequencing performed in the first family did not reveal any gene with rare homozygous variants shared by both affected siblings. It did, however, show one gene, DOCK7, with two rare heterozygous variants (c.2510delA [p.Asp837Alafs(∗)48] and c.3709C>T [p.Arg1237(∗)]) found in both affected sisters. Exome sequencing performed in the proband of the second family also showed the presence of two rare heterozygous variants (c.983C>G [p.Ser328(∗)] and c.6232G>T [p.Glu2078(∗)]) in DOCK7. Sanger sequencing confirmed that all three individuals are compound heterozygotes for these truncating mutations in DOCK7. These mutations have not been observed in public SNP databases and are predicted to abolish domains critical for DOCK7 function. DOCK7 codes for a Rac guanine nucleotide exchange factor that has been implicated in the genesis and polarization of newborn pyramidal neurons and in the morphological differentiation of GABAergic interneurons in the developing cortex. All together, these observations suggest that loss of DOCK7 function causes a syndromic form of epileptic encephalopathy by affecting multiple neuronal processes.

Longitudinal burn scar quantification.

Quantitative studies of the clinical recovery of burn scars are currently lacking. Previous reports validate the objective, precise, diagnostic capabilities of high-frequency ultrasound to measure thickness, the Cutometer(®) to measure pliability and the Mexameter(®) to measure erythema and pigmentation of scars. Thus, we prospectively quantified clinical characteristics of patient-matched, after burn hypertrophic scar (HSc), donor site scar (D) and normal skin (N) using these instruments. One investigator measured 3 sites (HSc, D, N) in 46 burn survivors at 3, 6, and 12 months after-burn. A mixed model regression analysis, adjusting p-values for multiplicity of testing, was used to compare means among sites and time points. Participants were 41.2±13.5 years old, 87% males, predominantly Caucasian, with an average of 19.5% body surface area burned. HSc thickness decreased significantly between 3 and 6, 6 and 12, and 3 and 12 months (all p<0.0001), but remained thicker than D and N skin (all p<0.0001). Pliability differed significantly between HSc, D and N sites at all time points (all p<0.0001), with HSc and D increasing between 3 and 12 months (p<0.05) but not reaching normal. HSc and D sites were significantly more erythematous than normal skin (p<0.05) at 3 and 6 months but D sites approached normal by 12 months. The only time points at which pigmentation significantly differed were the HSc and D sites at 6 months. Thickness, pliability, erythema and pigmentation of N skin remained similar over the 12 months. We found that post-burn HSc thickness, pliability and erythema differed significantly from D and N skin at 3, 6, and 12 months and does not return to normal by 12 months after-injury; however, significant improvements towards normal can be expected. Donor sites are redder than normal skin at 3 and 6 months but can be expected to return to normal by 12 months. Although the color of HSc and D sites change markedly with time these color changes are primarily due to changes in redness of the site, not melanin in this primarily Caucasian population.

A homozygous PDE6D mutation in Joubert syndrome impairs targeting of farnesylated INPP5E protein to the primary cilium.

Joubert syndrome (JS) is characterized by a distinctive cerebellar structural defect, namely the << molar tooth sign >>. JS is genetically heterogeneous, involving 20 genes identified to date, which are all required for cilia biogenesis and/or function. In a consanguineous family with JS associated with optic nerve coloboma, kidney hypoplasia, and polydactyly, combined exome sequencing and mapping identified a homozygous splice-site mutation in PDE6D, encoding a prenyl-binding protein. We found that pde6d depletion in zebrafish leads to renal and retinal developmental anomalies and wild-type but not mutant PDE6D is able to rescue this phenotype. Proteomic analysis identified INPP5E, whose mutations also lead to JS or mental retardation, obesity, congenital retinal dystrophy, and micropenis syndromes, as novel prenyl-dependent cargo of PDE6D. Mutant PDE6D shows reduced binding to INPP5E, which fails to localize to primary cilia in patient fibroblasts and tissues. Furthermore, mutant PDE6D is unable to bind to GTP-bound ARL3, which acts as a cargo-release factor for PDE6D-bound INPP5E. Altogether, these results indicate that PDE6D is required for INPP5E ciliary targeting and suggest a broader role for PDE6D in targeting other prenylated proteins to the cilia. This study identifies PDE6D as a novel JS disease gene and provides the first evidence of prenyl-binding-dependent trafficking in ciliopathies.

TMEM126A is a mitochondrial located mRNA (MLR) protein of the mitochondrial inner membrane.

BACKGROUND: Hereditary optic neuropathies (HONs) are a heterogeneous group of disorders that affect retinal ganglion cells (RGCs) and axons that form the optic nerve. Leber's Hereditary Optic Neuropathy and the autosomal dominant optic atrophy related to OPA1 mutations are the most common forms. Nonsyndromic autosomal recessive optic neuropathies are rare and their existence has been long debated. We recently identified the first gene responsible for these conditions, TMEM126A. This gene is highly expressed in retinal cellular compartments enriched in mitochondria and supposed to encode a mitochondrial transmembrane protein of unknown function. METHODS: A specific polyclonal antibody targeting the TMEM126A protein has been generated. Quantitative fluorescent in situ hybridization, cellular fractionation, mitochondrial membrane association study, mitochondrial sub compartmentalization analysis by both proteolysis assays and transmission electron microscopy, and expression analysis of truncated TMEM126A constructs by immunofluorescence confocal microscopy were carried out. RESULTS: TMEM126A mRNAs are strongly enriched in the vicinity of mitochondria and encode an inner mitochondrial membrane associated cristae protein. Moreover, the second transmembrane domain of TMEM126A is required for its mitochondrial localization. CONCLUSIONS: TMEM126A is a mitochondrial located mRNA (MLR) that may be translated in the mitochondrial surface and the protein is subsequently imported to the inner membrane. These data constitute the first step toward a better understanding of the mechanism of action of TMEM126A in RGCs and support the importance of mitochondrial dysfunction in the pathogenesis of HON. GENERAL SIGNIFICANCE: Local translation of nuclearly encoded mitochondrial mRNAs might be a mechanism for rapid onsite supply of mitochondrial membrane proteins.

Union makes strength: a worldwide collaborative genetic and clinical study to provide a comprehensive survey of RD3 mutations and delineate the associated phenotype.

Leber congenital amaurosis (LCA) is the earliest and most severe retinal degeneration (RD), and the most common cause of incurable blindness diagnosed in children. It is occasionally the presenting symptom of multisystemic ciliopathies which diagnosis will require a specific care of patients. Nineteen LCA genes are currently identified and three of them account for both non-syndromic and syndromic forms of the disease. RD3 (LCA12) was implicated as a LCA gene based on the identification of homozygous truncating mutations in two LCA families despite the screening of large cohorts of patients. Here we provide a comprehensive survey of RD3 mutations and of their clinical expression through the screening of a cohort of 852 patients originating worldwide affected with LCA or early-onset and severe RD. We identified three RD3 mutations in seven unrelated consanguineous LCA families - i.e., a 2 bp deletion and two nonsense mutations - predicted to cause complete loss of function. Five families originating from the Southern Shores of the Mediterranean segregated a similar mutation (c.112C>T, p.R38*) suggesting that this change may have resulted from an ancient founder effect. Considering the low frequency of RD3 carriers, the recurrence risk for LCA in non-consanguineous unions is negligible for both heterozygote and homozygote RD3 individuals. The LCA12 phenotype in our patients is highly similar to those of patients with mutant photoreceptor-specific guanylate cyclase (GUCY2D/LCA1). This observation is consistent with the report of the role of RD3 in trafficking of GUCYs and gives further support to a common mechanism of photoreceptor degeneration in LCA12 and LCA1, i.e., inability to increase cytoplasmic cGMP concentration in outer segments and thus to recover the dark-state. Similar to LCA1, LCA12 patients have no extraocular symptoms despite complete inactivation of both RD3 alleles, supporting the view that extraocular investigations in LCA infants with RD3 mutations should be avoided.