Novel mutations in CACNA1F and NYX in Dutch families with X-linked congenital stationary night blindness

PURPOSE: To describe the clinical features and genetic analysis of eight X-linked congenital stationary night blindness (XLCSNB) Dutch patients. METHODS: Electroretinogram (ERG) measurements were assessed in Dutch patients. Molecular genetic testing by denaturing high performance liquid chromatography (DHPLC), single stranded conformation polymorphism (SSCP) analysis, and direct sequencing of the CACNA1F and NYX genes were performed in the patients possessing a negative Schubert Bornschein ERG. RESULTS: Molecular genetic testing of CACNA1F and NYX revealed three novel and two known CACNA1F sequence variants as well as two novel sequence alterations in the NYX gene. While one of the CACNA1F sequence variants (5756G>A, R1919H) has been previously described as a common polymorphism in Japanese families, we did not found this transition in 100 European control alleles. CONCLUSIONS: In a pool of eight diagnosed XLCSNB patients, five showed a sequence variation in the CACNA1F and two in the NYX gene. In only one of the eight patients no sequence alteration could be detected. This might be explained by a mutation in other, as yet unidentified coding or regulatory sequences of NYX or CACNA1F or additional genes

Prevalence and novelty of PRPF31 mutations in French autosomal dominant rod-cone dystrophy patients and a review of published reports

Background: Rod-cone dystrophies are heterogeneous group of inherited retinal disorders both clinically and genetically characterized by photoreceptor degeneration. The mode of inheritance can be autosomal dominant, autosomal recessive or X-linked. The purpose of this study was to identify mutations in one of the genes, PRPF31, in French patients with autosomal dominant RP, to perform genotype-phenotype correlations of those patients, to determine the prevalence of PRPF31 mutations in this cohort and to review previously identified PRPF31 mutations from other cohorts.Methods: Detailed phenotypic characterization was performed including precise family history, best corrected visual acuity using the ETDRS chart, slit lamp examination, kinetic and static perimetry, full field and multifocal ERG, fundus autofluorescence imaging and optic coherence tomography. For genetic diagnosis, genomic DNA of ninety families was isolated by standard methods. The coding exons and flanking intronic regions of PRPF31 were PCR amplified, purified and sequenced in the index patient.Results: We showed for the first time that 6.7% cases of a French adRP cohort have a PRPF31 mutation. We identified in total six mutations, which were all novel and not detected in ethnically matched controls. The mutation spectrum from our cohort comprises frameshift and splice site mutations. Co-segregation analysis in available family members revealed that each index patient and all affected family members showed a heterozygous mutation. In five families incomplete penetrance was observed. Most patients showed classical signs of RP with relatively preserved central vision and visual field.Conclusion: Our studies extended the mutation spectrum of PRPF31 and as previously reported in other populations, it is a major cause of adRP in France

Development and application of a next-generation-sequencing (NGS) approach to detect known and novel gene defects underlying retinal diseases

<p>Abstract</p> <p>Background</p> <p>Inherited retinal disorders are clinically and genetically heterogeneous with more than 150 gene defects accounting for the diversity of disease phenotypes. So far, mutation detection was mainly performed by APEX technology and direct Sanger sequencing of known genes. However, these methods are time consuming, expensive and unable to provide a result if the patient carries a new gene mutation. In addition, multiplicity of phenotypes associated with the same gene defect may be overlooked.</p> <p>Methods</p> <p>To overcome these challenges, we designed an exon sequencing array to target 254 known and candidate genes using Agilent capture. Subsequently, 20 DNA samples from 17 different families, including four patients with known mutations were sequenced using Illumina Genome Analyzer IIx next-generation-sequencing (NGS) platform. Different filtering approaches were applied to identify the genetic defect. The most likely disease causing variants were analyzed by Sanger sequencing. Co-segregation and sequencing analysis of control samples validated the pathogenicity of the observed variants.</p> <p>Results</p> <p>The phenotype of the patients included retinitis pigmentosa, congenital stationary night blindness, Best disease, early-onset cone dystrophy and Stargardt disease. In three of four control samples with known genotypes NGS detected the expected mutations. Three known and five novel mutations were identified in <it>NR2E3, PRPF3, EYS, PRPF8, CRB1, TRPM1 </it>and <it>CACNA1F</it>. One of the control samples with a known genotype belongs to a family with two clinical phenotypes (Best and CSNB), where a novel mutation was identified for CSNB. In six families the disease associated mutations were not found, indicating that novel gene defects remain to be identified.</p> <p>Conclusions</p> <p>In summary, this unbiased and time-efficient NGS approach allowed mutation detection in 75% of control cases and in 57% of test cases. Furthermore, it has the possibility of associating known gene defects with novel phenotypes and mode of inheritance.</p

Disease-causing mutations in BEST1 gene are associated with altered sorting of bestrophin-1 protein

Doumanov, Jordan A. et al.Mutations in BEST1 gene, encoding the bestrophin-1 (Best1) protein are associated with macular dystrophies. Best1 is predominantly expressed in the retinal pigment epithelium (RPE), and is inserted in its basolateral membrane. We investigated the cellular localization in polarized MDCKII cells of disease-associated Best1 mutant proteins to study specific sorting motifs of Best1. Real-time PCR and western blots for endogenous expression of BEST1 in MDCK cells were performed. Best1 mutant constructs were generated using site-directed mutagenesis and transfected in MDCK cells. For protein sorting, confocal microscopy studies, biotinylation assays and statistical methods for quantification of mislocalization were used. Analysis of endogenous expression of BEST1 in MDCK cells revealed the presence of BEST1 transcript but no protein. Confocal microscopy and quantitative analyses indicate that transfected normal human Best1 displays a basolateral localization in MDCK cells, while cell sorting of several Best1 mutants (Y85H, Q96R, L100R, Y227N, Y227E) was altered. In contrast to constitutively active Y227E, constitutively inactive Y227F Best1 mutant localized basolaterally similar to the normal Best1 protein. Our data suggest that at least three basolateral sorting motifs might be implicated in proper Best1 basolateral localization. In addition, non-phosphorylated tyrosine 227 could play a role for basolateral delivery. © 2013 by the authors; licensee MDPI, Basel, Switzerland.This work was supported by Agence Nationale de la Recherche (Chaire d’Excellence to Shomi S. Bhattacharya), Fondation Voir et Entendre (Young Investigator Grants to Christina Zeitz and Emeline F. Nandrot), Centre National de la Recherche Scientifique (CNRS) and Fondation Bettencourt Schueller (to Emeline F. Nandrot), Université Pierre et Marie Curie-Paris6 (Bonus Qualité Recherche to Christina Zeitz), Foundation Fighting Blindness (grant number CD-CL-0808-0466-CHNO to Isabelle Audo), Centre d’Investigation Clinique 503 recognized as a Foundation Fighting Blindness Center (grant number C-CMM-0907-0428-INSERM04), Fundacion Progreso y Salud (to Shomi S. Bhattacharya, Maria Luz Bellido Diaz, Abhay Krishna and Paloma Dominguez Gimenez), Instituto de Salud Carlos III (grant number CM06/00183 to Maria Luz Bellido Diaz) and Bulgarian National Science Fund (grant number DDVU 02/10). Additionally, the Institut de la Vision is funded by Institut National de la Santé et de la Recherche Médicale, Université Pierre et Marie Curie-Paris 6, Centre National de la Recherche Scientifique and Départment de Paris.Peer Reviewe

Detailed investigations of proximal tubular function in Imerslund-Grasbeck syndrome

BACKGROUND: Imerslund-Gräsbeck Syndrome (IGS) is a rare genetic disorder characterised by juvenile megaloblastic anaemia. IGS is caused by mutations in either of the genes encoding the intestinal intrinsic factor-vitamin B(12) receptor complex, cubam. The cubam receptor proteins cubilin and amnionless are both expressed in the small intestine as well as the proximal tubules of the kidney and exhibit an interdependent relationship for post-translational processing and trafficking. In the proximal tubules cubilin is involved in the reabsorption of several filtered plasma proteins including vitamin carriers and lipoproteins. Consistent with this, low-molecular-weight proteinuria has been observed in most patients with IGS. The aim of this study was to characterise novel disease-causing mutations and correlate novel and previously reported mutations with the presence of low-molecular-weight proteinuria. METHODS: Genetic screening was performed by direct sequencing of the CUBN and AMN genes and novel identified mutations were characterised by in silico and/or in vitro investigations. Urinary protein excretion was analysed by immunoblotting and high-resolution gel electrophoresis of collected urines from patients and healthy controls to determine renal phenotype. RESULTS: Genetic characterisation of nine IGS patients identified two novel AMN frameshift mutations alongside a frequently reported AMN splice site mutation and two CUBN missense mutations; one novel and one previously reported in Finnish patients. The novel AMN mutations were predicted to result in functionally null AMN alleles with no cell-surface expression of cubilin. Also, the novel CUBN missense mutation was predicted to affect structural integrity of the IF-B(12) binding site of cubilin and hereby most likely cubilin cell-surface expression. Analysis of urinary protein excretion in the patients and 20 healthy controls revealed increased urinary excretion of cubilin ligands including apolipoprotein A-I, transferrin, vitamin D-binding protein, and albumin. This was, however, only observed in patients where plasma membrane expression of cubilin was predicted to be perturbed. CONCLUSIONS: In the present study, mutational characterisation of nine IGS patients coupled with analyses of urinary protein excretion provide additional evidence for a correlation between mutation type and presence of the characteristic low-molecular-weight proteinuria

NMNAT1 Mutations Cause Leber Congenital Amaurosis

Leber congenital amaurosis (LCA) is an infantile-onset form of inherited retinal degeneration characterized by severe vision loss. Two-thirds of LCA cases are caused by mutations in 17 known disease genes (RetNet Retinal Information Network). Using exome sequencing, we identified a homozygous missense mutation (c.25G>A, p.Val9Met) in NMNAT1 as likely disease-causing in two siblings of a consanguineous Pakistani kindred affected by LCA. This mutation segregated with disease in their kindred, including in three other children with LCA. NMNAT1 resides in the previously identified LCA9 locus and encodes the nuclear isoform of nicotinamide mononucleotide adenylyltransferase, a rate-limiting enzyme in nicotinamide adenine dinucleotide $(NAD^+)$ biosynthesis. Functional studies showed the p.Val9Met mutation decreased NMNAT1 enzyme activity. Sequencing NMNAT1 in 284 unrelated LCA families identified 14 rare mutations in 13 additional affected individuals. These results are the first to link an NMNAT isoform to disease and indicate that NMNAT1 mutations cause LCA

WDR34, a candidate gene for non-syndromic rod-cone dystrophy

Rod-cone dystrophy (RCD), also called retinitis pigmentosa, is characterized by rod followed by cone photoreceptor degeneration, leading to gradual visual loss. Mutations in over 65 genes have been associated with non-syndromic RCD explaining 60% to 70% of cases, with novel gene defects possibly accounting for the unsolved cases. Homozygosity mapping and whole-exome sequencing applied to a case of autosomal recessive non-syndromic RCD from a consanguineous union identified a homozygous variant in WDR34. Mutations in WDR34 have been previously associated with severe ciliopathy syndromes possibly associated with a retinal dystrophy. This is the first report of a homozygous mutation in WDR34 associated with non-syndromic RCD.Doctoral funding from the Ministère de l'Enseignement Supérieur et de la Recherche; Europe exchange 2018 Erasmus; European Reintegration Grant, Grant/Award Number: PERG04-GA-2008-231125; Fondation de France-Berthe Fouassier; Foundation Fighting Blindness, Grant/Award Number: Grant # CD-CL-0808-0466-CHNO CIC503 recogn; Foundation Voir et Entendre; French Agence Nationale de la Recherche, Grant/Award Numbers: IHU FOReSIGHT: ANR-18-IAHU-0001, LIFESENSES: ANR-10-LABX-65; National Eye Institute [R01EY012910 (EAP), R01EY026904 (KMB/EAP) and P30EY014104 (MEEI core support)], the Foundation Fightin

Mutation spectrum of EYS in Spanish patients with autosomal recessive retinitis pigmentosa

29 páginas, 4 figuras, 3 tablas.-- et al.Retinitis pigmentosa (RP) is a heterogeneous group of inherited retinal dystrophies characterised ultimately by the loss of photoreceptor cells. We have recently identified a new gene (EYS) encoding an ortholog of Drosophila spacemaker (spam) as a commonly mutated gene in autosomal recessive RP. In the present study, we report the identification of 73 sequence variations in EYS, of which 28 are novel. Of these, 42.9% (12/28) are very likely pathogenic, 17.9% (5/28) are possibly pathogenic, whereas 39.3% (11/28) are SNPs. In addition, we have detected 3 pathogenic changes previously reported in other populations. We are also presenting the characterisation of EYS homologues in different species, and a detailed analysis of the EYS domains, with the identification of an interesting novel feature: a putative coiled-coil domain. Majority of the mutations in the arRP patients have been found within the domain structures of EYS. The minimum observed prevalence of distinct EYS mutations in our group of patients is of 15.9% (15/94), confirming a major involvement of EYS in the pathogenesis of arRP in the Spanish population. Along with the detection of three recurrent mutations in Caucasian population, our hypothesis of EYS being the first prevalent gene in arRP has been reinforced in the present study.This study was funded by PN de I+D+I 2008- 2011, Instituto de Salud Carlos III (ISCIII) -Subdirección General de Evaluación y Fomento de la Investigación, Fondo de Investigación Sanitaria (PI081131), Spain; Consejería de Salud (PI-0334/2007), Consejería de Innovación, Ciencia y Empresa (PI08-CTS-03687), Junta de Andalucía, Spain; El Centro de Investigación Biomédica en Red de Enfermedades Raras is an initiative of the Instituto de Salud Carlos III. J.I. P. was supported by Fondo de Investigación Sanitaria and M. G-dP by Consejería de Innovación, Ciencia y Empresa, Junta de Andalucía, Spain. The research of K.W.L. was funded by the Stichting Wetenschappelijk Onderzoek Oogziekenhuis Prof. Dr. H.J. Flieringa, Rotterdam. Contract grant sponsor: Fondo de Investigación Sanitaria, Spain; Consejería de Salud; Consejería de Innovación, Ciencia y Empresa; Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER). Contract grant number: PI081131, PI-0334/2007, PI08-CTS-03687Peer reviewe

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