Isabelle Meunier

Mutations in CNNM4 Cause Recessive Cone-Rod Dystrophy with Amelogenesis Imperfecta

Cone-rod dystrophies are inherited dystrophies of the retina characterized by the accumulation of deposits mainly localized to the cone-rich macular region of the eye. Dystrophy can be limited to the retina or be part of a syndrome. Unlike nonsyndromic cone-rod dystrophies, syndromic cone-rod dystrophies are genetically heterogeneous with mutations in genes encoding structural, cell-adhesion, and transporter proteins. Using a genome-wide single-nucleotide polymorphism (SNP) haplotype analysis to fine map the locus and a gene-candidate approach, we identified homozygous mutations in the ancient conserved domain protein 4 gene (CNNM4) that either generate a truncated protein or occur in highly conserved regions of the protein. Given that CNNM4 is implicated in metal ion transport, cone-rod dystrophy and amelogenesis imperfecta may originate from abnormal ion homeostasis.

Systematic Screening of BEST1 and PRPH2 in Juvenile and Adult Vitelliform Macular Dystrophies: A Rationale for Molecular Analysis

PURPOSE To evaluate a genetic approach of BEST1 and PRPH2 screening according to age of onset, family history, and Arden ratio in patients with juvenile vitelliform macular dystrophy (VMD2) or adult-onset vitelliform macular dystrophy (AVMD), which are characterized by autofluorescent deposits. DESIGN Clinical, electrophysiologic, and molecular retrospective study. PARTICIPANTS The database of a clinic specialized in genetic sensory diseases was screened for patients with macular vitelliform dystrophy. Patients with an age of onset less than 40 years were included in the VMD2 group (25 unrelated patients), and patients with an age of onset more than 40 years were included in the AVMD group (19 unrelated patients). METHODS Clinical, fundus photography, and electro-oculogram (EOG) findings were reviewed. Mutation screening of BEST1 and PRPH2 genes was systematically performed. MAIN OUTCOME MEASURES Relevance of age of onset, family history, and Arden ratio were reviewed. RESULTS Patients with VMD2 carried a BEST1 mutation in 60% of the cases. Seven novel mutations in BEST1 (p.V9L, p.F80V, p.I73V, p.R130S, pF298C, pD302A, and p.179delN) were found. Patients with VMD2 with a positive family history or a reduced Arden ratio carried a BEST1 mutation in 70.5% of cases and in 83% if both criteria were fulfilled. Patients with AVMD carried a PRPH2 mutation in 10.5% of cases and did not carry a BEST1 mutation. The probability of finding a PRPH2 mutation increased in the case of a family history (2/5 patients). Electro-oculogram was normal in 3 of 15 patients with BEST1 mutations and reduced in the 3 patients with PRPH2 mutations. CONCLUSIONS Age of onset is a major criterion to distinguish VMD2 from AVMD. Electro-oculogram is not as relevant because decreased or normal Arden ratios have been associated with mutations in both genes and diseases. A positive family history increased the probability of finding a mutation. BEST1 screening should be recommended to patients with an age of onset less than 40 years, and PRPH2 screening should be recommended to patients with an age of onset more than 40 years. For an onset between 30 and 40 years, PRPH2 can be screened if no mutation has been detected in BEST1. FINANCIAL DISCLOSURE(S) The author(s) have no proprietary or commercial interest in any materials discussed in this article.

Relative frequencies of inherited retinal dystrophies and optic neuropathies in Southern France: assessment of 21-year data management.

ABSTRACT Purpose: Inherited retinal dystrophies (IRDs) and inherited optic neuropathies (IONs) are rare diseases defined by specific clinical and molecular features. The relative prevalence of these conditions was determined in Southern France. Methods: Patients recruited from a specialized outpatient clinic over a 21-year period underwent extensive clinical investigations and 107 genes were screened by polymerase chain reaction/sequencing. Results: There were 1957 IRD cases (1481 families) distributed in 70% of pigmentary retinopathy cases (56% non-syndromic, 14% syndromic), 20% maculopathies and 7% stationary conditions. Patients with retinitis pigmentosa were the most frequent (47%) followed by Usher syndrome (10.8%). Among non-syndromic pigmentary retinopathy patients, 84% had rod-cone dystrophy, 8% cone-rod dystrophy and 5% Leber congenital amaurosis. Macular dystrophies were encountered in 398 cases (30% had Stargardt disease and 11% had Best disease). There were 184 ION cases (127 families) distributed in 51% with dominant optic neuropathies, 33% with recessive/sporadic forms and 16% with Leber hereditary optic neuropathy. Positive molecular results were obtained in 417/609 families with IRDs (68.5%) and in 27/58 with IONs (46.5%). The sequencing of 5 genes (ABCA4, USH2A, MYO7A, RPGR and PRPH2) provided a positive molecular result in 48% of 417 families with IRDs. Except for autosomal retinitis pigmentosa, in which less than half the families had positive molecular results, about 75% of families with other forms of retinal conditions had a positive molecular diagnosis. Conclusions: Although gene discovery considerably improved molecular diagnosis in many subgroups of IRDs and IONs, retinitis pigmentosa, accounting for almost half of IRDs, remains only partly molecularly defined.

Homozygous mutation in MERTK causes severe autosomal recessive retinitis pigmentosa

Purpose Gene identification in retinitis pigmentosa is a prerequisite to future therapies. Accordingly, autosomal recessive retinitis pigmentosa families were genotyped to search for causative mutations. Methods Members of a consanguineous Moroccan family had standard ophthalmologic examination, optical coherence tomography–3 scan, autofluorescence testing, and electroretinogram. Their DNA was genotyped with the 250K SNP microchip (Affymetrix) and homozygosity mapping was done. MERTK exons were polymerase chain reaction amplified and sequenced. Results Two sisters and one brother out of 6 siblings had rod cone dystrophy type of retinitis pigmentosa. Salient features were night blindness starting in early infancy, dot-like whitish deposits in fovea and macula with corresponding autofluorescent dots in youngest patients, decreased visual acuity, and cone responses higher than rod responses at electroretinogram. The patients were homozygous in regions from chromosomes 2 and 8, but only that of chromosome 2 was inherited from a common ancestor. Sequencing of the MERTK gene belonging to the chromosome 2 region showed that the 3 affected patients carried a novel homozygous mutation in exon 17, c.2323C>T, leading to p.Arg775X, while their unaffected brothers and sister, parents, and paternal grandfather were heterozygous. Conclusions MERTK mutations lead to severe retinitis pigmentosa with discrete dot-like autofluorescent deposits at early stages, which are a hallmark of this MERTK-specific dystrophy.

Mosaic synaptopathy and functional defects in Cav1.4 heterozygous mice and human carriers of CSNB2

Mutations in CACNA1F encoding the α1-subunit of the retinal Cav1.4 L-type calcium channel have been linked to Cav1.4 channelopathies including incomplete congenital stationary night blindness type 2A (CSNB2), Åland Island eye disease (AIED) and cone-rod dystrophy type 3 (CORDX3). Since CACNA1F is located on the X chromosome, Cav1.4 channelopathies are typically affecting male patients via X-chromosomal recessive inheritance. Occasionally, clinical symptoms have been observed in female carriers, too. It is currently unknown how these mutations lead to symptoms in carriers and how the retinal network in these females is affected. To investigate these clinically important issues, we compared retinal phenotypes in Cav1.4-deficient and Cav1.4 heterozygous mice and in human female carrier patients. Heterozygous Cacna1f carrier mice have a retinal mosaic consistent with differential X-chromosomal inactivation, characterized by adjacent vertical columns of affected and non-affected wild-type-like retinal network. Vertical columns in heterozygous mice are well comparable to either the wild-type retinal network of normal mice or to the retina of homozygous mice. Affected retinal columns display pronounced rod and cone photoreceptor synaptopathy and cone degeneration. These changes lead to vastly impaired vision-guided navigation under dark and normal light conditions and reduced retinal electroretinography (ERG) responses in Cacna1f carrier mice. Similar abnormal ERG responses were found in five human CACNA1F carriers, four of which had novel mutations. In conclusion, our data on Cav1.4 deficient mice and human female carriers of mutations in CACNA1F are consistent with a phenotype of mosaic CSNB2.

Enrichment of LOVD-USHbases with 152 USH2A Genotypes Defines an Extensive Mutational Spectrum and Highlights Missense Hotspots

Alterations of USH2A, encoding usherin, are responsible for more than 70% of cases of Usher syndrome type II (USH2), a recessive disorder that combines moderate to severe hearing loss and retinal degeneration. The longest USH2A transcript encodes usherin isoform b, a 5,202‐amino‐acid transmembrane protein with an exceptionally large extracellular domain consisting notably of a Laminin N‐terminal domain and numerous Laminin EGF‐like (LE) and Fibronectin type III (FN3) repeats. Mutations of USH2A are scattered throughout the gene and mostly private. Annotating these variants is therefore of major importance to correctly assign pathogenicity. We have extensively genotyped a novel cohort of 152 Usher patients and identified 158 different mutations, of which 93 are newly described. Pooling this new data with the existing pathogenic variants already incorporated in USHbases reveals several previously unappreciated features of the mutational spectrum. We show that parts of the protein are more likely to tolerate single amino acid variations, whereas others constitute pathogenic missense hotspots. We have found, in repeated LE and FN3 domains, a nonequal distribution of the missense mutations that highlights some crucial positions in usherin with possible consequences for the assessment of the pathogenicity of the numerous missense variants identified in USH2A.

Recessive Mutations in RTN4IP1 Cause Isolated and Syndromic Optic Neuropathies

Autosomal-recessive optic neuropathies are rare blinding conditions related to retinal ganglion cell (RGC) and optic-nerve degeneration, for which only mutations in TMEM126A and ACO2 are known. In four families with early-onset recessive optic neuropathy, we identified mutations in RTN4IP1, which encodes a mitochondrial ubiquinol oxydo-reductase. RTN4IP1 is a partner of RTN4 (also known as NOGO), and its ortholog Rad8 in C. elegans is involved in UV light response. Analysis of fibroblasts from affected individuals with a RTN4IP1 mutation showed loss of the altered protein, a deficit of mitochondrial respiratory complex I and IV activities, and increased susceptibility to UV light. Silencing of RTN4IP1 altered the number and morphogenesis of mouse RGC dendrites in vitro and the eye size, neuro-retinal development, and swimming behavior in zebrafish in vivo. Altogether, these data point to a pathophysiological mechanism responsible for RGC early degeneration and optic neuropathy and linking RTN4IP1 functions to mitochondrial physiology, response to UV light, and dendrite growth during eye maturation.

Combining gene mapping and phenotype assessment for fast mutation finding in non-consanguineous autosomal recessive retinitis pigmentosa families.

Among inherited retinal dystrophies, autosomal recessive retinitis pigmentosa (arRP) is the most genetically heterogenous condition with 32 genes currently known that account for ∼60 % of patients. Molecular diagnosis thus requires the tedious systematic sequencing of 506 exons. To rapidly identify the causative mutations, we devised a strategy that combines gene mapping and phenotype assessment in small non-consanguineous families. Two unrelated sibships with arRP had whole-genome scan using SNP microchips. Chromosomal regions were selected by calculating a score based on SNP coverage and genotype identity of affected patients. Candidate genes from the regions with the highest scores were then selected based on phenotype concordance of affected patients with previously described phenotype for each candidate gene. For families RP127 and RP1459, 33 and 40 chromosomal regions showed possible linkage, respectively. By comparing the scores with the phenotypes, we ended with one best candidate gene for each family, namely tubby-like protein 1 (TULP1) and C2ORF71 for RP127 and RP1459, respectively. We found that RP127 patients were compound heterozygous for two novel TULP1 mutations, p.Arg311Gln and p.Arg342Gln, and that RP1459 patients were compound heterozygous for two novel C2ORF71 mutations, p.Leu777PhefsX34 and p.Leu777AsnfsX28. Phenotype assessment showed that TULP1 patients had severe early onset arRP and that C2ORF71 patients had a cone rod dystrophy type of arRP. Only two affected individuals in each sibship were sufficient to lead to mutation identification by screening the best candidate gene selected by a combination of gene mapping and phenotype characterization.

Frequency and Clinical Pattern of Vitelliform Macular Dystrophy Caused by Mutations of Interphotoreceptor Matrix IMPG1 and IMPG2 Genes

PURPOSE To assess the frequency of and to characterize the clinical spectrum and optical coherence tomography findings of vitelliform macular dystrophy linked to IMPG1 and IMPG2, 2 new causal genes expressed in the interphotoreceptor matrix. DESIGN Retrospective epidemiologic, clinical, electrophysiologic, and molecular genetic study. PARTICIPANTS The database of a national referral center specialized in genetic sensory diseases was screened for patients with a macular vitelliform dystrophy without identified mutation or small deletion or large rearrangement in BEST1 and PRPH2 genes. Forty-nine families were included. METHODS Clinical, imaging, and electro-oculogram findings were reviewed. Mutation screening of IMPG1 and IMPG2 genes were performed systematically. MAIN OUTCOME MEASURES Frequency, inheritance, and clinical pattern of vitelliform dystrophy associated with IMPG1 and IMPG2 mutations were characterized. RESULTS IMPG1 was the causal gene in 3 families (IMPG1 1-3, 11 patients) and IMPG2 in a fourth family (2 patients). With an autosomal dominant transmission, families 1 and 2 had the c.713T→G (p.Leu238Arg) mutation in IMPG1 and family 4 had the c.3230G→T (p.Cys1077Phe) mutation in IMPG2. Patients with IMPG1 or IMPG2 mutations had a late onset and moderate visual impairment (mean visual acuity, 20/40; mean age of onset, 42 years), even in the sporadic case of family 3 with a presumed recessive transmission (age at onset, 38 years; mean visual acuity, 20/50). Drusen-like lesions adjacent to the vitelliform deposits were observed in 9 of 13 patients. The vitelliform material was above the retinal pigment epithelium (RPE) at any stage of the macular dystrophy, and this epithelium was well preserved and maintained its classical reflectivity on spectral-domain optical coherence tomography (SD-OCT). Electro-oculogram results were normal or borderline in 9 cases. CONCLUSIONS IMPG1 and IMPG2 are new causal genes in 8% of families negative for BEST1 and PRPH2 mutations. These genes should be screened in adult-onset vitelliform dystrophy with (1) moderate visual impairment, (2) drusen-like lesions, (3) normal reflectivity of the RPE line on SD-OCT, and (4) vitelliform deposits located between ellipsoid and interdigitation lines on SD-OCT. These clinical characteristics are not observed in the classical forms of BEST1 or PRPH2 vitelliform dystrophies.

Isolated and Syndromic Retinal Dystrophy Caused by Biallelic Mutations in RCBTB1, a Gene Implicated in Ubiquitination

Inherited retinal dystrophies (iRDs) are a group of genetically and clinically heterogeneous conditions resulting from mutations in over 250 genes. Here, homozygosity mapping and whole-exome sequencing (WES) in a consanguineous family revealed a homozygous missense mutation, c.973C>T (p.His325Tyr), in RCBTB1. In affected individuals, it was found to segregate with retinitis pigmentosa (RP), goiter, primary ovarian insufficiency, and mild intellectual disability. Subsequent analysis of WES data in different cohorts uncovered four additional homozygous missense mutations in five unrelated families in whom iRD segregates with or without syndromic features. Ocular phenotypes ranged from typical RP starting in the second decade to chorioretinal dystrophy with a later age of onset. The five missense mutations affect highly conserved residues either in the sixth repeat of the RCC1 domain or in the BTB1 domain. A founder haplotype was identified for mutation c.919G>A (p.Val307Met), occurring in two families of Mediterranean origin. We showed ubiquitous mRNA expression of RCBTB1 and demonstrated predominant RCBTB1 localization in human inner retina. RCBTB1 was very recently shown to be involved in ubiquitination, more specifically as a CUL3 substrate adaptor. Therefore, the effect on different components of the CUL3 and NFE2L2 (NRF2) pathway was assessed in affected individuals’ lymphocytes, revealing decreased mRNA expression of NFE2L2 and several NFE2L2 target genes. In conclusion, our study puts forward mutations in RCBTB1 as a cause of autosomal-recessive non-syndromic and syndromic iRD. Finally, our data support a role for impaired ubiquitination in the pathogenetic mechanism of RCBTB1 mutations.