Search

ABCA4 is the most frequently mutated gene leading to inherited retinal disease (IRD) with over 2200 pathogenic variants reported to date. Of these, ~1% are copy number variants (CNVs) involving the deletion or duplication of genomic regions, typically >50 nucleotides in length. An in-depth assessment of the current literature based on the public database LOVD, regarding the presence of known CNVs and structural variants in ABCA4 , and additional sequencing analysis of ABCA4 using single-molecule Molecular Inversion Probes (smMIPs) for 148 probands highlighted recurrent and novel CNVs associated with ABCA4 -associated retinopathies. An analysis of the coverage depth in the sequencing data led to the identification of eleven deletions (six novel and five recurrent), three duplications (one novel and two recurrent) and one complex CNV. Of particular interest was the identification of a complex defect, i.e., a 15.3 kb duplicated segment encompassing exon 31 through intron 41 that was inserted at the junction of a downstream 2.7 kb deletion encompassing intron 44 through intron 47. In addition, we identified a 7.0 kb tandem duplication of intron 1 in three cases. The identification of CNVs in ABCA4 can provide patients and their families with a genetic diagnosis whilst expanding our understanding of the complexity of diseases caused by ABCA4 variants.

Inherited macular dystrophies (iMDs) are a group of genetic disorders, which affect the central region of the retina. To investigate the genetic basis of iMDs, we used single-molecule Molecular Inversion Probes to sequence 105 maculopathy-associated genes in 1352 patients diagnosed with iMDs. Within this cohort, 39.8% of patients were considered genetically explained by 460 different variants in 49 distinct genes of which 73 were novel variants, with some affecting splicing. The top five most frequent causative genes were ABCA4 (37.2%), PRPH2 (6.7%), CDHR1 (6.1%), PROM1 (4.3%) and RP1L1 (3.1%). Interestingly, variants with incomplete penetrance were revealed in almost one-third of patients considered solved (28.1%), and therefore, a proportion of patients may not be explained solely by the variants reported. This includes eight previously reported variants with incomplete penetrance in addition to CDHR1 :c.783G>A and CNGB3 :c.1208G>A. Notably, segregation analysis was not routinely performed for variant phasing-a limitation, which may also impact the overall diagnostic yield. The relatively high proportion of probands without any putative causal variant (60.2%) highlights the need to explore variants with incomplete penetrance, the potential modifiers of disease and the genetic overlap between iMDs and age-related macular degeneration. Our results provide valuable insights into the genetic landscape of iMDs and warrant future exploration to determine the involvement of other maculopathy genes.

Introduction: Patients with autosomal dominant tubulointerstitial kidney disease (ADTKD) usually present with nonspecific progressive chronic kidney disease (CKD) with mild to negative proteinuria and a family history. ADTKD- MUC1 leads to the formation of a frameshift protein that accumulates in the cytoplasm, leading to tubulointerstitial damage. ADTKD- MUC1 prevalence remains unclear because MUC1 variants are not routinely detected by standard next-generation sequencing (NGS) techniques., Methods: We developed a bioinformatic counting script that can detect specific genetic sequences and count the number of occurrences. We used DNA samples from 27 patients for validation, 11 of them were patients from the Lille University Hospital in France and 16 were from the Wake Forest Hospital, NC. All patients from Lille were tested with an NGS gene panel with our script and all patients from Wake Forest Hospital were tested with the snapshot reference technique. Between January 2018 and February 2023, we collected data on all patients diagnosed with MUC1 variants with this script., Results: A total of 27 samples were tested anonymously by the BROAD Institute reference technique for confirmation and we were able to get a 100% concordance for MUC1 diagnosis. Clinico-biologic characteristics in our cohort were similar to those previously described in ADTKD- MUC1., Conclusion: We describe a new simple and cost-effective method for molecular testing of ADTKD- MUC1. Genetic analyses in our cohort suggest that MUC1 might be the first cause of ADTKD. Increasing the availability of MUC1 diagnosis tools will contribute to a better understanding of the disease and to the development of specific treatments., (© 2024 International Society of Nephrology. Published by Elsevier Inc.)

The ABCA4 gene is the most frequently mutated Mendelian retinopathy-associated gene. Biallelic variants lead to a variety of phenotypes, however, for thousands of cases the underlying variants remain unknown. Here, we aim to shed further light on the missing heritability of ABCA4-associated retinopathy by analyzing a large cohort of macular dystrophy probands. A total of 858 probands were collected from 26 centers, of whom 722 carried no or one pathogenic ABCA4 variant, while 136 cases carried two ABCA4 alleles, one of which was a frequent mild variant, suggesting that deep-intronic variants (DIVs) or other cis-modifiers might have been missed. After single molecule molecular inversion probes (smMIPs)-based sequencing of the complete 128-kb ABCA4 locus, the effect of putative splice variants was assessed in vitro by midigene splice assays in HEK293T cells. The breakpoints of copy number variants (CNVs) were determined by junction PCR and Sanger sequencing. ABCA4 sequence analysis solved 207 of 520 (39.8%) naive or unsolved cases and 70 of 202 (34.7%) monoallelic cases, while additional causal variants were identified in 54 of 136 (39.7%) probands carrying two variants. Seven novel DIVs and six novel non-canonical splice site variants were detected in a total of 35 alleles and characterized, including the c.6283-321C>G variant leading to a complex splicing defect. Additionally, four novel CNVs were identified and characterized in five alleles. These results confirm that smMIPs-based sequencing of the complete ABCA4 gene provides a cost-effective method to genetically solve retinopathy cases and that several rare structural and splice altering defects remain undiscovered in Stargardt disease cases., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: ABCA4, the gene implicated in Stargardt disease (STGD1), contains 50 exons, of which 17 contain multiples of three nucleotides. The impact of in-frame exon skipping is yet to be determined. Antisense oligonucleotides (AONs) have been investigated in Usher syndrome-associated genes to induce skipping of in-frame exons carrying severe variants and mitigate their disease-linked effect. Upon the identification of a STGD1 proband carrying a novel exon 17 canonical splice site variant, the activity of ABCA4 lacking 22 amino acids encoded by exon 17 was examined, followed by design of AONs able to induce exon 17 skipping., Methods: A STGD1 proband was compound heterozygous for the splice variant c.2653+1G>A, that was predicted to result in in-frame skipping of exon 17, and a null variant [c.735T>G, p.(Tyr245*)]. Clinical characteristics of this proband were studied using multi-modal imaging and complete ophthalmological examination. The aberrant splicing of c.2653+1G>A was investigated in vitro in HEK293T cells with wild-type and mutant midigenes. The residual activity of the mutant ABCA4 protein lacking Asp864-Gly885 encoded by exon 17 was analyzed with all-trans-retinal-activated ATPase activity assay, along with its subcellular localization. To induce exon 17 skipping, the effect of 40 AONs was examined in vitro in WT WERI-Rb-1 cells and 3D human retinal organoids., Results: Late onset STGD1 in the proband suggests that c.2653+1G>A does not have a fully deleterious effect. The in vitro splice assay confirmed that this variant leads to ABCA4 transcripts without exon 17. ABCA4 Asp864_Gly863del was stable and retained 58% all-trans-retinal-activated ATPase activity compared to WT ABCA4. This sequence is located in an unstructured linker region between transmembrane domain 6 and nucleotide-binding domain-1 of ABCA4. AONs were designed to possibly reduce pathogenicity of severe variants harbored in exon 17. The best AON achieved 59% of exon 17 skipping in retinal organoids., Conclusions: Exon 17 deletion in ABCA4 does not result in the absence of protein activity and does not cause a severe STGD1 phenotype when in trans with a null allele. By applying AONs, the effect of severe variants in exon 17 can potentially be ameliorated by exon skipping, thus generating partial ABCA4 activity in STGD1 patients., (© 2023. BioMed Central Ltd., part of Springer Nature.)

To suggest a unique missense variant candidate based on long-term ophthalmological changes and associated systemic signs described in five patients from two unrelated families affected by an autosomal dominant multi-systemic disorder including Retinal dystrophy, Optic nerve oedema, Splenomegaly, Anhidrosis and migraine Headaches, called ROSAH syndrome, related to a unique missense variant in ALPK1 gene. Observational longitudinal follow-up study of unrelated families. Clinical analysis of ophthalmological and systemic examinations was performed, followed by genetic analysis, including targeted Next Generation Sequencing (NGS) and Whole-Genome Sequencing (WGS). The ophthalmological phenotype showed extensive optic nerve swelling associated with early macular oedema and vascular leakage. The main associated systemic manifestations were recurrent fever, splenomegaly, anhidrosis, mild cytopenia, anicocytosis and hypersegmented polynuclear cells. WGS, shortened in the second family by the gene candidate suggestion, revealed in all patients the heterozygous missense variant c.710C>T; p.(Thr237Met) in ALPK1. The primary morbidity in ROSAH syndrome in this cohort appeared ophthalmological. Comprehensive, detailed phenotype changes aided by the advancement in genetic testing could allow an early genetic diagnosis of ROSAH syndrome and targeted treatment. The unique missense variant may be suggested as a target of gene correction therapy., (© 2022 The Authors. Clinical Genetics published by John Wiley & Sons Ltd.)

CNGB1 gene mutations are a well-known cause of autosomal recessive retinitis pigmentosa (RP), which was recently associated with olfactory dysfunction. The purpose of this study was to report the molecular spectrum and the ocular and olfactory phenotypes of a multiethnic cohort with CNGB1 -associated RP. A cross-sectional case series was conducted at two ophthalmic genetics referral centers. Consecutive patients with molecularly confirmed CNGB1 -related RP were included. All patients underwent a complete ophthalmological examination complemented by psychophysical olfactory evaluation. Fifteen patients (10 families: 8 Portuguese, 1 French, and 1 Turkish), mean aged 57.13 ± 15.37 years old (yo), were enrolled. Seven disease-causing variants were identified, two of which are reported for the first time: c.2565_2566del and c.2285G > T. Although 11/15 patients reported onset of nyctalopia before age 10, diagnosis was only established after 30 yo in 9/15. Despite widespread retinal degeneration being present in 14/15 probands, a relatively preserved visual acuity was observed throughout follow-up. Olfactory function was preserved in only 4/15 patients, all of whom carried at least one missense variant. Our study supports previous reports of an autosomal recessive RP-olfactory dysfunction syndrome in association with certain disease-causing variants in the CNGB1 gene and expands the mutational spectrum of CNGB1 -related disease by reporting two novel variants.

Background: Patients with mitochondrial diseases are at risk of heart failure (HF) and arrhythmic major adverse cardiac events (MACE)., Objectives: We developed prediction models to estimate the risk of HF and arrhythmic MACE in this population., Methods: We determined the incidence and searched for predictors of HF and arrhythmic MACE using Cox regression in 600 adult patients from a multicenter registry with genetically confirmed mitochondrial diseases., Results: Over a median follow-up time of 6.67 years, 29 patients (4.9%) reached the HF endpoint, including 19 hospitalizations for nonterminal HF, 2 cardiac transplantations, and 8 deaths from HF. Thirty others (5.1%) reached the arrhythmic MACE, including 21 with third-degree or type II second-degree atrioventricular blocks, 4 with sinus node dysfunction, and 5 sudden cardiac deaths. Predictors of HF were the m.3243A>G variant (HR: 4.3; 95% CI: 1.8-10.1), conduction defects (HR: 3.0; 95% CI: 1.3-6.9), left ventricular (LV) hypertrophy (HR: 2.6; 95% CI: 1.1-5.8), LV ejection fraction <50% (HR: 10.2; 95% CI: 4.6-22.3), and premature ventricular beats (HR: 4.1; 95% CI: 1.7-9.9). Independent predictors for arrhythmia were single, large-scale mtDNA deletions (HR: 4.3; 95% CI: 1.7-10.4), conduction defects (HR: 6.8; 95% CI: 3.0-15.4), and LV ejection fraction <50% (HR: 2.7; 95% CI: 1.1-7.1). C-indexes of the Cox regression models were 0.91 (95% CI: 0.88-0.95) and 0.80 (95% CI: 0.70-0.90) for the HF and arrhythmic MACE, respectively., Conclusions: We developed the first prediction models for HF and arrhythmic MACE in patients with mitochondrial diseases using genetic variant type and simple cardiac assessments., Competing Interests: Funding Support and Author Disclosures This study was funded by grants from the Association Française contre les Myopathies (French Alliance against Myopathies), which was not involved in the design and conduct of the study; the collection, management, analysis, and interpretation of the data; the preparation, review or approval of the manuscript; nor in the decision to submit the manuscript for publication. Dr Pitceathly is supported by a Medical Research Council (United Kingdom) Clinician Scientist Fellowship (MR/S002065/1). Drs Pitceathly and Hanna receive funding from a Medical Research Council (United Kingdom) strategic award to establish an International Centre for Genomic Medicine in Neuromuscular Diseases (ICGNMD) (MR/S005021/1). Dr Lopes is supported by a Medical Research Council (United Kingdom) clinical academic partnership (CARP) award. Dr Quinlivan was funded by National Institute for Health and Care Research Biomedical Research Centre, University College Hospitals Foundation Trust. The University College London Hospitals/University College London Queen Square Institute of Neurology sequencing facility receives a proportion of funding from the Department of Health's National Institute for Health and Care Research Biomedical Research Centre’s funding scheme. The clinical and diagnostic “Rare Mitochondrial Disorders” Service in London is funded by the U.K. NHS Highly Specialised Commissioners. Dr Elliott has received consultancy fees from Pfizer, Sanofi, Sarepta, DinaQor, Freeline, Novo Nordisk, and Bristol Myers Squibb. All other authors have reported that they have no relationships relevant to the contents of this paper to disclose., (Copyright © 2022 American College of Cardiology Foundation. Published by Elsevier Inc. All rights reserved.)

Biallelic gene defects in MFSD8 are not only a cause of the late-infantile form of neuronal ceroid lipofuscinosis, but also of rare isolated retinal degeneration. We report clinical and genetic data of seven patients compound heterozygous or homozygous for variants in MFSD8, issued from a French cohort with inherited retinal degeneration, and two additional patients retrieved from a Swiss cohort. Next-generation sequencing of large panels combined with whole-genome sequencing allowed for the identification of twelve variants from which seven were novel. Among them were one deep intronic variant c.998+1669A>G, one large deletion encompassing exon 9 and 10, and a silent change c.750A>G. Transcript analysis performed on patients’ lymphoblastoid cell lines revealed the creation of a donor splice site by c.998+1669A>G, resulting in a 140 bp pseudoexon insertion in intron 10. Variant c.750A>G produced exon 8 skipping. In silico and in cellulo studies of these variants allowed us to assign the pathogenic effect, and showed that the combination of at least one severe variant with a moderate one leads to isolated retinal dystrophy, whereas the combination in trans of two severe variants is responsible for early onset severe retinal dystrophy in the context of late-infantile neuronal ceroid lipofuscinosis.

Purpose: The effect of noncoding variants is often unknown in the absence of functional assays. Here, we characterized an ABCA4 intron 7 variant, c.859-25A>G, identified in Palestinian probands with Stargardt disease (STGD) or cone-rod dystrophy (CRD). We investigated the effect of this variant on the ABCA4 mRNA and retinal phenotype, and its prevalence in Palestine., Methods: The ABCA4 gene was sequenced completely or partially in 1998 cases with STGD or CRD. The effect of c.859-25A>G on splicing was investigated in silico using SpliceAI and in vitro using splice assays. Homozygosity mapping was performed for 16 affected individuals homozygous for c.859-25A>G. The clinical phenotype was assessed using functional and structural analyses including visual acuity, full-field electroretinography, and multimodal imaging., Results: The smMIPs-based ABCA4 sequencing revealed c.859-25A>G in 10 Palestinian probands from Hebron and Jerusalem. SpliceAI predicted a significant effect of this putative branchpoint-inactivating variant on the nearby intron 7 splice acceptor site. Splice assays revealed exon 8 skipping and two partial inclusions of intron 7, each having a deleterious effect. Additional genotyping revealed another 46 affected homozygous or compound heterozygous individuals carrying variant c.859-25A>G. Homozygotes shared a genomic segment of 59.6 to 87.9 kb and showed severe retinal defects on ophthalmoscopic evaluation., Conclusions: The ABCA4 variant c.859-25A>G disrupts a predicted branchpoint, resulting in protein truncation because of different splice defects, and is associated with early-onset STGD1 when present in homozygosity. This variant was found in 25/525 Palestinian inherited retinal dystrophy probands, representing one of the most frequent inherited retinal disease-causing variants in West-Bank Palestine.

Background: Long chain 3-hydroxyacyl-CoA dehydrogenase deficiency (LCHADD) is a rare inherited disease caused by pathogenic variants of HADHA gene. Along with signs common to fatty acid oxidation defects (FAOD), specific retina and heart alterations are observed. Because long-chain fatty acid oxidation is selectively affected, supplementations with short/medium-chain fats represent energetic sources bypassing the enzymatic blockade. Here, we report on an atypical presentation of the disease., Methods: Clinical features were described with medical explorations including ophthalmic and cardiac examination. Biological underlying defects were investigated by measurements of biochemical metabolites and by fluxomic studies of mitochondrial β-oxidation. Whole exome sequencing and molecular validation of variants confirmed the diagnosis., Results: The patient has developed at nine years an unlabeled maculopathy, and at 28 years, an acute cardiac decompensation without any premise. Blood individual acylcarnitine analysis showed a rise in hydroxylated long-chain fatty acids and fluxomic studies validated enzyme blockade consistent with LCHADD. Genetic analysis revealed the common p.(Glu510Gln) variant in HADHA , in trans with a novel variant c.1108G > A, p.(Gly370Arg) located in the NAD binding domain. Patient pathology was responsive to triheptanoin supplementation., Conclusion: This atypical LCHADD form report should encourage the early assessment of biochemical and genetic testing as a specific management is recommended (combination with fast avoidance, low fat-high carbohydrate diet, medium-even-chain triglycerides or triheptanoin supplementation)., Competing Interests: No conflicting relationship exists for any author., (© 2022 The Authors.)

Recurrence risk calculations in autosomal recessive diseases are complicated when the effect of genetic variants and their population frequencies and penetrances are unknown. An example of this is Stargardt disease (STGD1), a frequent recessive retinal disease caused by bi-allelic pathogenic variants in ABCA4. In this cross-sectional study, 1,619 ABCA4 variants from 5,579 individuals with STGD1 were collected and categorized by (1) severity based on statistical comparisons of their frequencies in STGD1-affected individuals versus the general population, (2) their observed versus expected homozygous occurrence in STGD1-affected individuals, (3) their occurrence in combination with established mild alleles in STGD1-affected individuals, and (4) previous functional and clinical studies. We used the sum allele frequencies of these severity categories to estimate recurrence risks for offspring of STGD1-affected individuals and carriers of pathogenic ABCA4 variants. The risk for offspring of an STGD1-affected individual with the "severe|severe" genotype or a "severe|mild with complete penetrance" genotype to develop STGD1 at some moment in life was estimated at 2.8%-3.1% (1 in 36-32 individuals) and 1.6%-1.8% (1 in 62-57 individuals), respectively. The risk to develop STGD1 in childhood was estimated to be 2- to 4-fold lower: 0.68%-0.79% (1 in 148-126) and 0.34%-0.39% (1 in 296-252), respectively. In conclusion, we established personalized recurrence risk calculations for STGD1-affected individuals with different combinations of variants. We thus propose an expanded genotype-based personalized counseling to appreciate the variable recurrence risks for STGD1-affected individuals. This represents a conceptual breakthrough because risk calculations for STGD1 may be exemplary for many other inherited diseases., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

Pathogenic variants in CRB1 lead to diverse recessive retinal disorders from severe Leber congenital amaurosis to isolated macular dystrophy. Until recently, no clear phenotype-genotype correlation and no appropriate mouse models existed. Herein, we reappraise the phenotype-genotype correlation of 50 patients with regards to the recently identified CRB1 isoforms: a canonical long isoform A localized in Müller cells (12 exons) and a short isoform B predominant in photoreceptors (7 exons). Twenty-eight patients with early onset retinal dystrophy (EORD) consistently had a severe Müller impairment, with variable impact on the photoreceptors, regardless of isoform B expression. Among them, two patients expressing wild type isoform B carried one variant in exon 12, which specifically damaged intracellular protein interactions in Müller cells. Thirteen retinitis pigmentosa patients had mainly missense variants in laminin G-like domains and expressed at least 50% of isoform A. Eight patients with the c.498&#95;506del variant had macular dystrophy. In one family homozygous for the c.1562C>T variant, the brother had EORD and the sister macular dystrophy. In contrast with the mouse model, these data highlight the key role of Müller cells in the severity of CRB1 -related dystrophies in humans, which should be taken into consideration for future clinical trials.

Purpose: To identify relevant criteria for gene therapy based on clinical and genetic characteristics of rod-cone dystrophy associated with RLBP1 pathogenic variants in a large cohort comprising children and adults., Design: Retrospective cohort study., Participants: Patients with pathogenic variants in RLBP1 registered in a single French reference center specialized in inherited retinal dystrophies., Methods: Clinical, multimodal imaging, and genetic findings were reviewed., Main Outcome Measures: Age of onset; visual acuity; ellipsoid line length; nasal, temporal, and foveal retinal thickness; and pathogenic variants and related phenotypes, including Newfoundland rod-cone and Bothnia dystrophies (NFRCDs), were reappraised., Results: Twenty-one patients (15 families) were included. The most frequent form was NFRCD with 12 patients (8 families) homozygous for the recurrent deletion of exons 7 through 9 in RLBP1 and 5 patients (4 families) with biallelic protein-truncating variants (2 novel: p.Gln16∗ and p.Tyr251∗). A novel combination of the p.Arg234Trp Bothnia variant with a nonsense variant in trans led to Bothnia dystrophy in 2 sisters. One proband carrying the p.Met266Lys Bothnia variant and in trans p.Arg121Trp and a second, with the p.Arg9Cys and p.Tyr111∗ combination, both demonstrated mild retinitis punctata albescens. Independently of genotype, all patients showed a visual acuity of worse than 20/200, an ellipsoid line width of less than 1000 μm, and a mean foveal thickness of less than 130 to 150 μm, with loss of both the interdigitation and ellipsoid lines., Conclusions: The eligibility for RLBP1 gene therapy first should be determined according to the biallelic variant combination using a robust classification as proposed herein. An ellipsoid line width of more than 1200 μm and a central thickness of more than 130 to 150 μm with detectable ellipsoid and interdigitation lines should be 2 prerequisite imaging indicators for gene therapy., (© 2021 by the American Academy of Ophthalmology.)

Background: Inherited retinal disorders are a clinically and genetically heterogeneous group of conditions and a major cause of visual impairment. Common disease subtypes include vitelliform macular dystrophy (VMD) and retinitis pigmentosa (RP). Despite the identification of over 90 genes associated with RP, conventional genetic testing fails to detect a molecular diagnosis in about one third of patients with RP., Methods: Exome sequencing was carried out for identifying the disease-causing gene in a family with autosomal dominant RP. Gene panel testing and exome sequencing were performed in 596 RP and VMD families to identified additional IMPG1 variants. In vivo analysis in the medaka fish system by knockdown assays was performed to screen IMPG1 possible pathogenic role., Results: Exome sequencing of a family with RP revealed a splice variant in IMPG1 . Subsequently, the same variant was identified in individuals from two families with either RP or VMD. A retrospective study of patients with RP or VMD revealed eight additional families with different missense or nonsense variants in IMPG1 . In addition, the clinical diagnosis of the IMPG1 retinopathy-associated variant, originally described as benign concentric annular macular dystrophy, was also revised to RP with early macular involvement. Using morpholino-mediated ablation of Impg1 and its paralog Impg2 in medaka fish, we confirmed a phenotype consistent with that observed in the families, including a decreased length of rod and cone photoreceptor outer segments., Conclusion: This study discusses a previously unreported association between monoallelic or biallelic IMPG1 variants and RP. Notably, similar observations have been reported for IMPG2 ., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2021. No commercial re-use. See rights and permissions. Published by BMJ.)

Variants of the TTLL5 gene, which encodes tubulin tyrosine ligase-like family member five, are a rare cause of cone dystrophy (COD) or cone-rod dystrophy (CORD). To date, only a few TTLL5 patients have been clinically and genetically described. In this study, we report five patients harbouring biallelic variants of TTLL5. Four adult patients presented either COD or CORD with onset in the late teenage years. The youngest patient had a phenotype of early onset severe retinal dystrophy (EOSRD). Genetic analysis was performed by targeted next generation sequencing of gene panels and assessment of copy number variants (CNV). We identified eight variants, of which six were novel, including two large multiexon deletions in patients with COD or CORD, while the EOSRD patient harboured the novel homozygous p.(Trp640*) variant and three distinct USH2A variants, which might explain the observed rod involvement. Our study highlights the role of TTLL5 in COD/CORD and the importance of large deletions. These findings suggest that COD or CORD patients lacking variants in known genes may harbour CNVs to be discovered in TTLL5, previously undetected by classical sequencing methods. In addition, variable phenotypes in TTLL5 -associated patients might be due to the presence of additional gene defects.

Purpose: Missing heritability in human diseases represents a major challenge, and this is particularly true for ABCA4-associated Stargardt disease (STGD1). We aimed to elucidate the genomic and transcriptomic variation in 1054 unsolved STGD and STGD-like probands., Methods: Sequencing of the complete 128-kb ABCA4 gene was performed using single-molecule molecular inversion probes (smMIPs), based on a semiautomated and cost-effective method. Structural variants (SVs) were identified using relative read coverage analyses and putative splice defects were studied using in vitro assays., Results: In 448 biallelic probands 14 known and 13 novel deep-intronic variants were found, resulting in pseudoexon (PE) insertions or exon elongations in 105 alleles. Intriguingly, intron 13 variants c.1938-621G>A and c.1938-514G>A resulted in dual PE insertions consisting of the same upstream, but different downstream PEs. The intron 44 variant c.6148-84A>T resulted in two PE insertions and flanking exon deletions. Eleven distinct large deletions were found, two of which contained small inverted segments. Uniparental isodisomy of chromosome 1 was identified in one proband., Conclusion: Deep sequencing of ABCA4 and midigene-based splice assays allowed the identification of SVs and causal deep-intronic variants in 25% of biallelic STGD1 cases, which represents a model study that can be applied to other inherited diseases.

Purpose: To evaluate the correlation between the quantification of peripapillary sparing and electroretinogram (ERG) outcomes in autosomal recessive Stargardt disease (STGD1)., Methods: Near infrared fundus autofluorescence (NIR-FAF) images of 101 eyes of 101 patients were retrospectively reviewed. Peripapillary sparing was assessed both qualitatively and quantitatively. The area of spared tissue (AST) was calculated in a 1-mm-wide ring around the optic disc after binarization of the 55° NIR-FAF. These measurements were correlated with the presence of normal ERG (group I), abnormal photopic responses (group II), or abnormal photopic and scotopic responses (group III)., Results: AST showed significant correlations with ERG groups (R = -0.802, P < 0.001). While qualitative assessment of peripapillary sparing (i.e., present or not) also showed a significant correlation with ERG groups (R = -0.435, P < 0.001), it was weaker than by AST quantification. The ordinal regression analysis showed that the increase in AST was associated with a decrease in the odds of belonging to ERG groups II and III, with an odds ratio of 0.82 (95% confidence interval [CI] 0.78-0.87), P < 0.001., Conclusions: The AST around the optic disc in eyes with STGD1 correlates with the impairment of photoreceptors as shown in the ERG. If replicated in future longitudinal studies, the quantification of peripapillary sparing may prove to be a useful parameter for evaluating the visual prognosis of these eyes.

We investigated the prevalence of reported deep-intronic variants in a French cohort of 70 patients with Stargardt disease harboring a monoallelic pathogenic variant on the exonic regions of ABCA4 . Direct Sanger sequencing of selected intronic regions of ABCA4 was conducted. Complete phenotypic analysis and correlation with the genotype was performed in case a known intronic pathogenic variant was identified. All other variants found on the analyzed sequences were queried for minor allele frequency and possible pathogenicity by in silico predictions. The second mutated allele was found in 14 (20%) subjects. The three known deep-intronic variants found were c.5196+1137G>A in intron 36 (6 subjects), c.4539+2064C>T in intron 30 (4 subjects) and c.4253+43G>A in intron 28 (4 subjects). Even though the phenotype depends on the compound effect of the biallelic variants, a genotype-phenotype correlation suggests that the c.5196+1137G>A was mostly associated with a mild phenotype and the c.4539+2064C>T with a more severe one. A variable effect was instead associated with the variant c.4253+43G>A. In addition, two novel variants, c.768+508A>G and c.859-245&#95;859-243delinsTGA never associated with Stargardt disease before, were identified and a possible splice defect was predicted in silico. Our study calls for a larger cohort analysis including targeted locus sequencing and 3D protein modeling to better understand phenotype-genotype correlations associated with deep-intronic changes and patients' selection for clinical trials., Competing Interests: The authors declare no conflict of interest.

Purpose: Using exome sequencing, the underlying variants in many persons with autosomal recessive diseases remain undetected. We explored autosomal recessive Stargardt disease (STGD1) as a model to identify the missing heritability., Methods: Sequencing of ABCA4 was performed in 8 STGD1 cases with one variant and p.Asn1868Ile in trans, 25 cases with one variant, and 3 cases with no ABCA4 variant. The effect of intronic variants was analyzed using in vitro splice assays in HEK293T cells and patient-derived fibroblasts. Antisense oligonucleotides were used to correct splice defects., Results: In 24 of the probands (67%), one known and five novel deep-intronic variants were found. The five novel variants resulted in messenger RNA pseudoexon inclusions, due to strengthening of cryptic splice sites or by disrupting a splicing silencer motif. Variant c.769-784C>T showed partial insertion of a pseudoexon and was found in cis with c.5603A>T (p.Asn1868Ile), so its causal role could not be fully established. Variant c.4253+43G>A resulted in partial skipping of exon 28. Remarkably, antisense oligonucleotides targeting the aberrant splice processes resulted in (partial) correction of all splicing defects., Conclusion: Our data demonstrate the importance of assessing noncoding variants in genetic diseases, and show the great potential of splice modulation therapy for deep-intronic variants.

Background: Myotonic dystrophy (DM1), a neuromuscular disease related to DMPK gene mutations, is associated to endocrine disorders and cancer. A routine endocrine work-up, including thyroid ultrasound (US), was conducted in 115 genetically-proven DM1 patients in a neuromuscular reference center. The aim of this study was to determine the prevalence and the causes of US thyroid abnormalities in DM1., Results: In the whole population (age 45.1 ± 12.2 years, 61.7% female), palpable nodules or goiters were present in 29.2%. The percentage of US goiter (thyroid volume > 18 mL) and US nodules were, respectively, 38.3 and 60.9%. Sixteen of the 115 patients had a thyroidectomy, after 22 fine-needle aspiration cytology guided by thyroid imaging reporting and data system (TIRADS) classification. Six micro- (1/6 pT3) and 3 macro-papillary thyroid carcinoma (PTCs) (2/3 intermediate risk) were diagnosed (7.9% of 115). Thyroid US led to the diagnosis of 4 multifocal and 2 unifocal (including 1 macro-PTC) non-palpable PTCs. Ultrasound thyroid volume was positively correlated to body mass index (BMI) (p = 0.015) and parity (p = 0.036), and was inversely correlated to TSH (p < 0.001) and vitamin D levels (p = 0.023). The BMI, the frequencies of glucose intolerance and PTC were significantly higher in UsGoiter versus non-UsGoiter groups., Conclusion: In this systematically screened DM1 cohort, the frequency of UsGoiter, mainly associated to BMI, was about 40%, US nodules 60%, thyroidectomies 13-14%, and PTCs 8%, two-thirds of them being micro-PTCs with good prognosis. Therefore, a systematic screening remains debatable. A targeted US screening in case of clinical abnormality or high BMI seems more appropriate.

Here we report novel mutations in ABCA4 with the underlying phenotype in a large French cohort with autosomal recessive Stargardt disease. The DNA samples of 397 index subjects were analyzed in exons and flanking intronic regions of ABCA4 (NM&#95;000350.2) by microarray analysis and direct Sanger sequencing. At the end of the screening, at least two likely pathogenic mutations were found in 302 patients (76.1%) while 95 remained unsolved: 40 (10.1%) with no variants identified, 52 (13.1%) with one heterozygous mutation, and 3 (0.7%) with at least one variant of uncertain significance (VUS). Sixty-three novel variants were identified in the cohort. Three of them were variants of uncertain significance. The other 60 mutations were classified as likely pathogenic or pathogenic, and were identified in 61 patients (15.4%). The majority of those were missense (55%) followed by frameshift and nonsense (30%), intronic (11.7%) variants, and in-frame deletions (3.3%). Only patients with variants never reported in literature were further analyzed herein. Recruited subjects underwent complete ophthalmic examination including best corrected visual acuity, kinetic and static perimetry, color vision test, full-field and multifocal electroretinography, color fundus photography, short-wavelength and near-infrared fundus autofluorescence imaging, and spectral domain optical coherence tomography. Clinical evaluation of each subject confirms the tendency that truncating mutations lead to a more severe phenotype with electroretinogram (ERG) impairment ( p = 0.002) and an earlier age of onset ( p = 0.037). Our study further expands the mutation spectrum in the exonic and flanking regions of ABCA4 underlying Stargardt disease.

Alternative promoter usage is an important mechanism for transcriptome diversity and the regulation of gene expression. Indeed, this alternative usage may influence tissue/subcellular specificity, protein translation and function of the proteins. The existence of an alternative promoter for MAPT gene was considered for a long time to explain differential tissue specificity and differential response to transcription and growth factors between mRNA transcripts. The alternative promoter usage could explain partly the different tau proteins expression patterns observed in tauopathies. Here, we report on our discovery of a functional alternative promoter for MAPT, located upstream of the gene's second exon (exon 1). By analyzing genome databases and brain tissue from control individuals and patients with Alzheimer's disease or progressive supranuclear palsy, we identified novel shorter transcripts derived from this alternative promoter. These transcripts are increased in patients' brain tissue as assessed by 5'RACE-PCR and qPCR. We suggest that these new MAPT isoforms can be translated into normal or amino-terminal-truncated tau proteins. We further suggest that activation of MAPT's alternative promoter under pathological conditions leads to the production of truncated proteins, changes in protein localization and function, and thus neurodegeneration.

Purpose: The goal of this analysis was to determine the test-retest variability of functional and structural measures from a cohort of patients with advanced forms of Stargardt Disease (STGD) participating in the SAR422459 (NCT01367444) gene therapy clinical trial., Methods: Twenty-two participants, aged 24 to 66, diagnosed with advanced forms of STGD, with at least one pathogenic ABCA4 mutation on each chromosome participating in the SAR422459 (NCT01367444) gene therapy clinical trial, were screened over three visits within 3 weeks or less. Functional visual evaluations included: best-corrected visual acuity (BCVA) Early Treatment Diabetic Retinopathy Study (ETDRS) letter score, semiautomated kinetic perimetry (SKP) using isopters I4e, III4e, and V4e, hill of vision (HOV) calculated from static visual fields (SVF) by using a 184n point centrally condensed grid with the stimulus size V test target. Retinal structural changes such as central macular thickness and macular volume were assessed by spectral-domain optical coherence tomography (SD-OCT). Repeatability coefficients (RC) and 95% confidential intervals (CI) were calculated for each parameter using a hierarchical mixed-effects model and bootstrapping., Results: Criteria for statistically significant changes for various parameters were found to be the following: BCVA letter score (8 letters), SKP isopters I4e, III4e, and V4e (3478.85; 2488.02 and 2622.46 deg 2 , respectively), SVF full volume HOV (V TOT, 14.62 dB-sr), central macular thickness, and macular volume (4.27 μm and 0.15 mm 3 , respectively)., Conclusions: This analysis provides important information necessary to determine if significant changes are occurring in structural and functional assessments commonly used to measure disease progression in this cohort of patients with STGD. Moreover, this information is useful for future trials assessing safety and efficacy of treatments in STGD., Translational Relevance: Determination of variability of functional and structural measures in participants with advanced stages of the STGD is necessary to assess efficacy and safety in treatment trials involving STGD patients.

To revisit the autosomal dominant Sorsby fundus dystrophy (SFD) as a syndromic condition including late-onset pulmonary disease. We report clinical and imaging data of ten affected individuals from 2 unrelated families with SFD and carrying heterozygous TIMP3 mutations (c.572A > G, p.Y191C, exon 5, in family 1 and c.113C > G, p.S38C, exon 1, in family 2). In family 1, all SFD patients older than 50 (two generations) had also a severe emphysema, despite no history of smoking or asthma. In the preceding generation, the mother died of pulmonary emphysema and she was blind after the age of 50. Her two great-grandsons (<20 years), had abnormal Bruch Membrane thickness, a sign of eye disease. In family 2, eye and lung diseases were also associated in two generations, both occurred later, and lung disease was moderate (bronchiectasis). This is the first report of a syndromic SFD in line with the mouse model uncovering the role of TIMP3 in human lung morphogenesis and functions. The TIMP3 gene should be screened in familial pulmonary diseases with bronchiectasis, associated with a medical history of visual loss. In addition, SFD patients should be advised to avoid tobacco consumption, to practice sports, and to undergo regular pulmonary examinations.

Inherited retinal dystrophies are clinically and genetically heterogeneous with significant number of cases remaining genetically unresolved. We studied a large family from the West Indies islands with a peculiar retinal disease, the Martinique crinkled retinal pigment epitheliopathy that begins around the age of 30 with retinal pigment epithelium (RPE) and Bruch's membrane changes resembling a dry desert land and ends with a retinitis pigmentosa. Whole-exome sequencing identified a heterozygous c.518T>C (p.Leu173Pro) mutation in MAPKAPK3 that segregates with the disease in 14 affected and 28 unaffected siblings from three generations. This unknown variant is predicted to be damaging by bioinformatic predictive tools and the mutated protein to be non-functional by crystal structure analysis. MAPKAPK3 is a serine/threonine protein kinase of the p38 signaling pathway that is activated by a variety of stress stimuli and is implicated in cellular responses and gene regulation. In contrast to other tissues, MAPKAPK3 is highly expressed in the RPE, suggesting a crucial role for retinal physiology. Expression of the mutated allele in HEK cells revealed a mislocalization of the protein in the cytoplasm, leading to cytoskeleton alteration and cytodieresis inhibition. In Mapkapk3-/- mice, Bruch's membrane is irregular with both abnormal thickened and thinned portions. In conclusion, we identified the first pathogenic mutation in MAPKAPK3 associated with a retinal disease. These findings shed new lights on Bruch's membrane/RPE pathophysiology and will open studies of this signaling pathway in diseases with RPE and Bruch's membrane alterations, such as age-related macular degeneration., (© The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Myotonic dystrophy (DM) of type 1 and 2 (DM1 and DM2) are inherited autosomal dominant diseases caused by dynamic and unstable expanded microsatellite sequences (CTG and CCTG, respectively) in the non-coding regions of the genes DMPK and ZNF9, respectively. These mutations result in the intranuclear accumulation of mutated transcripts and the mis-splicing of numerous transcripts. This so-called RNA gain of toxic function is the main feature of an emerging group of pathologies known as RNAopathies. Interestingly, in addition to these RNA inclusions, called foci, the presence of neurofibrillary tangles (NFT) in patient brains also distinguishes DM as a tauopathy. Tauopathies are a group of nearly 30 neurodegenerative diseases that are characterized by intraneuronal protein aggregates of the microtubule-associated protein Tau (MAPT) in patient brains. Furthermore, a number of neurodegenerative diseases involve the dysregulation of splicing regulating factors and have been characterized as spliceopathies. Thus, myotonic dystrophies are pathologies resulting from the interplay among RNAopathy, spliceopathy, and tauopathy. This review will describe how these processes contribute to neurodegeneration. We will first focus on the tauopathy associated with DM1, including clinical symptoms, brain histology, and molecular mechanisms. We will also discuss the features of DM1 that are shared by other tauopathies and, consequently, might participate in the development of a tauopathy. Moreover, we will discuss the determinants common to both RNAopathies and spliceopathies that could interfere with tau-related neurodegeneration.

Background: Mitochondrial DNA (mtDNA) diseases are rare disorders whose prevalence is estimated around 1 in 5000. Patients are usually tested only for deletions and for common mutations of mtDNA which account for 5-40% of cases, depending on the study. However, the prevalence of rare mtDNA mutations is not known., Methods: We analysed the whole mtDNA in a cohort of 743 patients suspected of manifesting a mitochondrial disease, after excluding deletions and common mutations. Both heteroplasmic and homoplasmic variants were identified using two complementary strategies (Surveyor and MitoChip). Multiple correspondence analyses followed by hierarchical ascendant cluster process were used to explore relationships between clinical spectrum, age at onset and localisation of mutations., Results: 7.4% of deleterious mutations and 22.4% of novel putative mutations were identified. Pathogenic heteroplasmic mutations were more frequent than homoplasmic mutations (4.6% vs 2.8%). Patients carrying deleterious mutations showed symptoms before 16 years of age in 67% of cases. Early onset disease (<1 year) was significantly associated with mutations in protein coding genes (mainly in complex I) while late onset disorders (>16 years) were associated with mutations in tRNA genes. MTND5 and MTND6 genes were identified as 'hotspots' of mutations, with Leigh syndrome accounting for the large majority of associated phenotypes., Conclusions: Rare mitochondrial DNA mutations probably account for more than 7.4% of patients with respiratory chain deficiency. This study shows that a comprehensive analysis of mtDNA is essential, and should include young children, for an accurate diagnosis that is now accessible with the development of next generation sequencing technology.

Vitelliform macular dystrophies (VMD) are inherited retinal dystrophies characterized by yellow, round deposits visible upon fundus examination and encountered in individuals with juvenile Best macular dystrophy (BMD) or adult-onset vitelliform macular dystrophy (AVMD). Although many BMD and some AVMD cases harbor mutations in BEST1 or PRPH2, the underlying genetic cause remains unknown for many affected individuals. In a large family with autosomal-dominant VMD, gene mapping and whole-exome sequencing led to the identification of a c.713T>G (p.Leu238Arg) IMPG1 mutation, which was subsequently found in two other families with autosomal-dominant VMD and the same phenotype. IMPG1 encodes the SPACR protein, a component of the rod and cone photoreceptor extracellular matrix domains. Structural modeling indicates that the p.Leu238Arg substitution destabilizes the conserved SEA1 domain of SPACR. Screening of 144 probands who had various forms of macular dystrophy revealed three other IMPG1 mutations. Two individuals from one family affected by autosomal-recessive VMD were homozygous for the splice-site mutation c.807+1G>T, and two from another family were compound heterozygous for the mutations c.461T>C (p.Leu154Pro) and c.1519C>T (p.Arg507(∗)). Most cases had a normal or moderately decreased electrooculogram Arden ratio. We conclude that IMPG1 mutations cause both autosomal-dominant and -recessive forms of VMD, thus indicating that impairment of the interphotoreceptor matrix might be a general cause of VMD., (Copyright © 2013 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

Muscleblind-like-1 (MBNL1) is a splicing regulatory factor controlling the fetal-to-adult alternative splicing transitions during vertebrate muscle development. Its capture by nuclear CUG expansions is one major cause for type 1 myotonic dystrophy (DM1). Alternative splicing produces MBNL1 isoforms that differ by the presence or absence of the exonic regions 3, 5, and 7. To understand better their respective roles and the consequences of the deregulation of their expression in DM1, here we studied the respective roles of MBNL1 alternative and constitutive exons. By combining genetics, molecular and cellular approaches, we found that (i) the exon 5 and 6 regions are both needed to control the nuclear localization of MBNL1; (ii) the exon 3 region strongly enhances the affinity of MBNL1 for its pre-mRNA target sites; (iii) the exon 3 and 6 regions are both required for the splicing regulatory activity, and this function is not enhanced by an exclusive nuclear localization of MBNL1; and finally (iv) the exon 7 region enhances MBNL1-MBNL1 dimerization properties. Consequently, the abnormally high inclusion of the exon 5 and 7 regions in DM1 is expected to enhance the potential of MBNL1 of being sequestered with nuclear CUG expansions, which provides new insight into DM1 pathophysiology.

Among the different mechanisms underlying the etiopathogenesis of myotonic dystrophy type 1 (DM1), a backward reprogramming to a foetal splicing machinery is an interesting hypothesis. To address this possibility, Tau splicing, which is regulated during development and modified in DM1, was analyzed. Indeed, a preferential expression of the foetal Tau isoform, instead of the six normally found, is observed in adult DM1 brains. By using two cell lines, we show here that the cis-regulating elements necessary to generate the unique foetal Tau isoform are dispensable to reproduce the trans-dominant effect induced by DM1 mutation on Tau exon 2 inclusion. Our results suggest that the mis-splicing of Tau in DM1 is resulting from a disease-associated mechanism.

The microtubule-associated protein Tau is mainly expressed in neurons of the CNS and is crucial in axonal maintenance and axonal transport. The rationale for Tau as a biomarker of neurodegenerative diseases is that it is a major component of abnormal intraneuronal aggregates observed in numerous tauopathies, including Alzheimer's disease. The molecular diversity of Tau is very useful when analyzing it in the brain or in the peripheral fluids. Immunohistochemical and biochemical characterization of Tau aggregates in the brain allows the postmortem classification and differential diagnosis of tauopathies. As peripheral biomarkers of Alzheimer's disease in the cerebrospinal fluid, Tau proteins are now validated for diagnosis and predictive purposes. For the future, the detailed characterization of Tau in the brain and in peripheral fluids will lead to novel promising biomarkers for differential diagnosis of dementia and monitoring of therapeutics.