Biallelic Mutations in the Autophagy Regulator DRAM2 Cause Retinal Dystrophy with Early Macular Involvement
2015; Elsevier BV; Volume: 96; Issue: 6 Linguagem: Inglês
10.1016/j.ajhg.2015.04.006
ISSN1537-6605
AutoresMohammed E. El‐Asrag, Panagiotis I. Sergouniotis, Martin McKibbin, Vincent Plagnol, Eamonn Sheridan, Naushin Waseem, Zakia Abdelhamed, Declan J. McKeefry, Kristof Van Schil, James A. Poulter, Colin A. Johnson, Ian Carr, Bart P. Leroy, Elfride De Baere, Chris F. Inglehearn, Andrew R. Webster, Carmel Toomes, Manir Ali, Graeme Black, Georgina Hall, Stuart Ingram, Rachel Gillespie, Simon Ramsden, Forbes D.C. Manson, Alison J. Hardcastle, Michel Michaelides, Michael E. Cheetham, Gavin Arno, Niclas Thomas, Shomi S. Bhattacharya, Tony Moore, Andrea H. Németh, Susan M. Downes, Stefano Lise, Emma Lord,
Tópico(s)Retinal Development and Disorders
ResumoRetinal dystrophies are an overlapping group of genetically heterogeneous conditions resulting from mutations in more than 250 genes. Here we describe five families affected by an adult-onset retinal dystrophy with early macular involvement and associated central visual loss in the third or fourth decade of life. Affected individuals were found to harbor disease-causing variants in DRAM2 (DNA-damage regulated autophagy modulator protein 2). Homozygosity mapping and exome sequencing in a large, consanguineous British family of Pakistani origin revealed a homozygous frameshift variant (c.140delG [p.Gly47Valfs∗3]) in nine affected family members. Sanger sequencing of DRAM2 in 322 unrelated probands with retinal dystrophy revealed one European subject with compound heterozygous DRAM2 changes (c.494G>A [p.Trp165∗] and c.131G>A [p.Ser44Asn]). Inspection of previously generated exome sequencing data in unsolved retinal dystrophy cases identified a homozygous variant in an individual of Indian origin (c.64_66del [p.Ala22del]). Independently, a gene-based case-control association study was conducted via an exome sequencing dataset of 18 phenotypically similar case subjects and 1,917 control subjects. Using a recessive model and a binomial test for rare, presumed biallelic, variants, we found DRAM2 to be the most statistically enriched gene; one subject was a homozygote (c.362A>T [p.His121Leu]) and another a compound heterozygote (c.79T>C [p.Tyr27His] and c.217_225del [p.Val73_Tyr75del]). DRAM2 encodes a transmembrane lysosomal protein thought to play a role in the initiation of autophagy. Immunohistochemical analysis showed DRAM2 localization to photoreceptor inner segments and to the apical surface of retinal pigment epithelial cells where it might be involved in the process of photoreceptor renewal and recycling to preserve visual function. Retinal dystrophies are an overlapping group of genetically heterogeneous conditions resulting from mutations in more than 250 genes. Here we describe five families affected by an adult-onset retinal dystrophy with early macular involvement and associated central visual loss in the third or fourth decade of life. Affected individuals were found to harbor disease-causing variants in DRAM2 (DNA-damage regulated autophagy modulator protein 2). Homozygosity mapping and exome sequencing in a large, consanguineous British family of Pakistani origin revealed a homozygous frameshift variant (c.140delG [p.Gly47Valfs∗3]) in nine affected family members. Sanger sequencing of DRAM2 in 322 unrelated probands with retinal dystrophy revealed one European subject with compound heterozygous DRAM2 changes (c.494G>A [p.Trp165∗] and c.131G>A [p.Ser44Asn]). Inspection of previously generated exome sequencing data in unsolved retinal dystrophy cases identified a homozygous variant in an individual of Indian origin (c.64_66del [p.Ala22del]). Independently, a gene-based case-control association study was conducted via an exome sequencing dataset of 18 phenotypically similar case subjects and 1,917 control subjects. Using a recessive model and a binomial test for rare, presumed biallelic, variants, we found DRAM2 to be the most statistically enriched gene; one subject was a homozygote (c.362A>T [p.His121Leu]) and another a compound heterozygote (c.79T>C [p.Tyr27His] and c.217_225del [p.Val73_Tyr75del]). DRAM2 encodes a transmembrane lysosomal protein thought to play a role in the initiation of autophagy. Immunohistochemical analysis showed DRAM2 localization to photoreceptor inner segments and to the apical surface of retinal pigment epithelial cells where it might be involved in the process of photoreceptor renewal and recycling to preserve visual function. Retinal dystrophies are a clinically and genetically heterogeneous group of disorders characterized by progressive photoreceptor degeneration.1Wright A.F. Chakarova C.F. Abd El-Aziz M.M. Bhattacharya S.S. Photoreceptor degeneration: genetic and mechanistic dissection of a complex trait.Nat. Rev. Genet. 2010; 11: 273-284Crossref PubMed Scopus (453) Google Scholar The pattern of visual loss and retinal appearance varies and is related to the degree to which cone and rod photoreceptors are affected. In subjects with retinitis pigmentosa (RP), for example, the rods are affected more severely and earlier than the cones, and the presenting symptoms are typically night blindness and/or visual field loss. Disorders in which the cones are more severely affected than the rods include macular dystrophies (MDs; localized loss of central/macular cones as a primary or secondary event) and cone-rod dystrophies (CRDs; central and peripheral cone involvement). MDs and CRDs show clinical overlap and loss of central vision is often the common presenting symptom. Frequently, subjects with CRDs also report light sensitivity, a symptom that can suggest generalized cone system dysfunction. Assigning a disease category can sometimes be challenging, with confounding factors being inter- and intra-familial phenotypic variability and the presence of age-dependent phenotypic transitions. RP, MDs, and CRDs can be transmitted in a dominant, recessive, or X-linked manner and, to date, variants in 70, 14, and 30 genes, respectively, have been shown to give rise to these conditions (RetNet, accessed February 2015). The initial aim of this study was to identify the genetic basis of an adult-onset retinal dystrophy with early macular involvement (Figure 1) in a consanguineous Pakistani family with multiple affected members living in the UK (family ES1; Figure 2). Affected individuals became symptomatic early in the third decade of life, describing increasing difficulty with close visual tasks. Neither light sensitivity nor night blindness were significant early symptoms. There was progressive loss of visual acuity in all symptomatic individuals; light sensitivity and difficulty seeing in dim illumination were inconsistent features of advanced disease. Fundus examination revealed maculopathy in all symptomatic individuals tested, with peripheral retinal degeneration being a frequent finding in older subjects. Notably, optical coherence tomography (OCT) imaging in the pre-symptomatic second decade of life (subject IV.9, family ES1; Figure 2) suggested early central photoreceptor cell loss.Figure 2Pedigrees of Families and Case Subjects Reported in This Study and DRAM2 Mutation Segregation DataShow full captionAffected individuals are shaded black. The maternal grandmother of individual gc17004 has age-related macular degeneration in her 90s (shaded gray). The genotypes for all tested family members are shown below each individual, with M representing the mutant allele and + representing the wild-type allele.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Affected individuals are shaded black. The maternal grandmother of individual gc17004 has age-related macular degeneration in her 90s (shaded gray). The genotypes for all tested family members are shown below each individual, with M representing the mutant allele and + representing the wild-type allele. This study was approved by the Leeds East (Project number 03/362), Moorfields Eye Hospital, and Ghent University Hospital (PA2015/012) Research Ethics Committees and adhered to the tenets of the Declaration of Helsinki. Individuals participated in the study after giving their informed consent. Peripheral blood was collected from affected individuals, parents, and unaffected relatives where these were available. Genomic DNA was extracted from blood leukocytes according to standard procedures. Homozygosity mapping was performed with Affymetrix 250K SNP arrays on genomic DNA from seven affected individuals from family ES1. Data were analyzed with the AgileMultiIdeogram software. Two homozygous regions were shared among all seven affected individuals: a 10.1 Mb interval on chromosome 1 (between rs6677953 and rs814987; containing 160 genes) and a 2.9 Mb region on chromosome 7 (between rs17140297 and rs12706292; containing 5 genes) (Figure S1). Given the absence of genes previously reported to be associated with retinal dystrophy within these intervals, a whole exome sequencing (WES) strategy was utilized to identify the molecular pathology in the family. DNA from one affected family member (subject IV.6, family ES1; Figure 2) was analyzed with a HiSeq2000 system (Illumina). After aligning the sequencing data output against the reference genome (hg19/GRCh37) as well as variant calling and filtration steps, a list of 33 homozygous variants was generated (Table S1). Only one of these sequence alterations mapped within the shared regions of homozygosity identified in family ES1. This was a homozygous single-base deletion in DRAM2 (DNA-damage regulated autophagy modulator protein 2 [MIM: 613360; GenBank: NM_178454.4]) that creates a frameshift and is predicted to lead to premature truncation of the protein (c.140delG [p.Gly47Valfs∗3]). Segregation of this variant with the disease in the family (Figure 2) was confirmed by Sanger sequencing of DRAM2 exon 4 (Figure 3A; primer pairs are shown in Table S2). This change was excluded from 159 ethnically matched control individuals and was not present in the dbSNP and EVS databases. It was found once in heterozygous state in WES data from 61,486 unrelated individuals sequenced as part of various disease-specific and population genetic studies (accessed via the Exome Aggregation Consortium [ExAC] browser, v.0.2). Notably, no homozygous presumed loss-of-function variant in DRAM2 was present in the ExAC dataset. A maximum two-point LOD score of 2.4 was obtained between c.140delG and the disease in nine genotyped family members using Superlink.2Silberstein M. Tzemach A. Dovgolevsky N. Fishelson M. Schuster A. Geiger D. Online system for faster multipoint linkage analysis via parallel execution on thousands of personal computers.Am. J. Hum. Genet. 2006; 78: 922-935Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar For this analysis the c.140delG change was treated as a genetic marker with a MAF of 0.01%, and the disease was assumed to segregate in the family in a recessive fashion with full penetrance. In an attempt to identify additional families with DRAM2-associated retinopathy, the seven coding DRAM2 exons and flanking splice sites were PCR amplified and Sanger sequenced in 74 individuals diagnosed with RP, 154 with CRD or MD, and 94 with infantile-onset retinal dystrophy (Leber congenital amaurosis) (primer pairs are shown in Table S2). This screen identified an isolated female case (subject 1325) of European ancestry in the CRD/MD panel who was compound heterozygous for a nonsense variant in exon 6 (c.494G>A [p.Trp165∗]) and for a missense change in exon 3 (c.131G>A [p.Ser44Asn]). The latter affects a serine residue that is conserved from human to nematodes (Figures 3 and S2). This missense change was predicted to be pathogenic by a number of bioinformatics prediction tools (Table S3) and was not present in the dbSNP, EVS, or ExAC databases. The c.494G>A change is an annotated variant in dbSNP (rs201422368) with a MAF of 0.008% (1/13,003) in EVS and 0.003% (3/118,572) in ExAC; it is reported only in heterozygous state in these databases. Subject 1325 experienced blurred vision at age 29 and was soon after found to have maculopathy on fundus examination. At age 35, she also complained of night vision problems and sensitivity to light; fundus examination revealed mild peripheral retinal degeneration in addition to the maculopathy. At the age of 47, she had acuity of 1.0 logMAR in each eye and electrophysiology revealed severely attenuated or absent full-field electroretinograms (ERGs) and pattern ERGs (Figure S3). Meanwhile, interrogation of previously generated WES data from unsolved cases with retinal dystrophy led to the identification of a homozygous variant in exon 3 of DRAM2 (c.64_66del [p.Ala22del]) in a subject of Indian origin. This change, which removes an alanine residue from the first transmembrane domain of the molecule, was predicted to be pathogenic by bioinformatics prediction tools (Table S3) and was absent in the dbSNP, EVS, and ExAC databases. The variant was confirmed by Sanger sequencing (Figure 3) and segregated in family BL1 with the disease as expected in a recessive manner (Figure 2). The affected subject, a woman in her early forties, has maculopathy with a normal full-field ERG indicating absence of a generalized retinal dysfunction. She also suffers from iron-deficiency anemia, which might have been brought on by a vegetarian diet or might be the result of a genetic factor resulting from parental consanguinity. Independently, in a study designed to identify novel genes associated with retinal disease, 28 families from the inherited retinal disease clinics at Moorfields Eye Hospital (London) were ascertained. Details on this cohort have been previously reported.3Sergouniotis P.I. Chakarova C. Murphy C. Becker M. Lenassi E. Arno G. Lek M. MacArthur D.G. Bhattacharya S.S. Moore A.T. et al.UCL-Exomes ConsortiumBiallelic variants in TTLL5, encoding a tubulin glutamylase, cause retinal dystrophy.Am. J. Hum. Genet. 2014; 94: 760-769Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar The main inclusion criteria included a CRD or MD phenotype and an absence of retinal imaging findings suggestive of ABCA4 retinopathy. Genomic DNA from the probands was analyzed by WES and variant filtering was performed as previously described.3Sergouniotis P.I. Chakarova C. Murphy C. Becker M. Lenassi E. Arno G. Lek M. MacArthur D.G. Bhattacharya S.S. Moore A.T. et al.UCL-Exomes ConsortiumBiallelic variants in TTLL5, encoding a tubulin glutamylase, cause retinal dystrophy.Am. J. Hum. Genet. 2014; 94: 760-769Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar The molecular diagnosis was identified in 10 of 28 families.3Sergouniotis P.I. Chakarova C. Murphy C. Becker M. Lenassi E. Arno G. Lek M. MacArthur D.G. Bhattacharya S.S. Moore A.T. et al.UCL-Exomes ConsortiumBiallelic variants in TTLL5, encoding a tubulin glutamylase, cause retinal dystrophy.Am. J. Hum. Genet. 2014; 94: 760-769Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar On the 18 unsolved case subjects, a gene-based case-control association analysis was performed utilizing WES data generated by a consortium of UK-based researchers ("UCL-exomes," Table S4). Aiming to minimize bias,4Pearson R.D. Bias due to selection of rare variants using frequency in controls.Nat. Genet. 2011; 43 (author reply 394–395): 392-393Crossref PubMed Scopus (13) Google Scholar UCL-exome control subjects were initially split into two sets. The first set of 500 randomly selected samples was used in conjunction with EVS to determine variant frequency for inclusion in case-control tests. In that context, "rare" variants are those with MAF < 0.5% in EVS and no more than two occurrences in this first set of 500 UCL-exome control samples. The second set of 1,917 unrelated UCL-exome control subjects was used to directly compute gene-based association p values, using a recessive disease mode, i.e., samples were labeled as potential carriers only if they carried at least two rare (using the definition stated above) and potentially functional (presumed loss-of-function, non-synonymous, or splice site altering) variants. A binomial test was used for excess of such potential biallelic variants in the 18 case subjects compared to the 1,917 control subjects (Table S4). The most significant gene-based p value was obtained for DRAM2 (Table S4). Two of the 18 case subjects were found to harbor likely disease-associated variants in this gene. A 37-year-old female proband (family gc17004, Figure 2) of European ancestry was a compound heterozygote for a missense variant (c.79T>C [p.Tyr27His]) and an in-frame deletion (c.217_225del [p.Val73_Tyr75del]). Furthermore, a 47-year-old male proband of South Asian origin (family gc4728, Figure 2; parents not knowingly related) was homozygous for a missense change (c.362A>T [p.His121Leu]). None of these three changes which are reported to be pathogenic by a number of prediction tools (Table S3) exist in dbSNP, EVS, or ExAC. Both missense variants, p.Tyr27His and p.His121Leu, affect a tyrosine and histidine residue, respectively, that are evolutionarily conserved from human to nematodes (Figure S2). All changes were confirmed by Sanger sequencing (Figure 3) and segregated with the disease phenotype in the family as expected for a recessive condition (Figure 2). Both probands presented with central visual loss (at age 29 for the proband of family gc17004 and at age 34 for the proband of family gc4728). At presentation, fundus examination and retinal imaging revealed macular photoreceptor loss with an apparently normal peripheral retina. These observations were consistent with electrophysiological findings. Notably, 8 years after presentation, the central areas of atrophy have expanded and peripheral changes were observed. The phenotype was notably similar to the affected members of families ES1 and BL1 and to subject 1325 described above. Given that affected members of family ES1 are homozygous for a DRAM2 variant that is likely to lead either to nonsense-mediated decay of the encoded mRNA or to a truncated protein of only 47 amino acids, the molecular pathology of the disease is likely to be loss of DRAM2 function. This speculation is further supported by the biallelic state and predicted severity of the additional six likely disease-causing variants identified as well as by the similar phenotype in all five families. DRAM2, also known as TMEM77 (transmembrane protein 77), encodes a 266-amino-acid protein containing six putative transmembrane domains (Figure 3B). Previous overexpression studies in HEK293 cells localized it to lysosomal membranes5Park S.M. Kim K. Lee E.J. Kim B.K. Lee T.J. Seo T. Jang I.S. Lee S.H. Kim S. Lee J.H. Park J. Reduced expression of DRAM2/TMEM77 in tumor cells interferes with cell death.Biochem. Biophys. Res. Commun. 2009; 390: 1340-1344Crossref PubMed Scopus (25) Google Scholar, 6O'Prey J. Skommer J. Wilkinson S. Ryan K.M. Analysis of DRAM-related proteins reveals evolutionarily conserved and divergent roles in the control of autophagy.Cell Cycle. 2009; 8: 2260-2265Crossref PubMed Scopus (56) Google Scholar where it initiates the conversion of endogenous LC3-I (microtubule-associated protein light chain 3) to the general autophagosome marker protein, LC3-II (LC3-1/phosphatidylethanolamine conjugate). This suggests that DRAM2 induces the autophagy process.5Park S.M. Kim K. Lee E.J. Kim B.K. Lee T.J. Seo T. Jang I.S. Lee S.H. Kim S. Lee J.H. Park J. Reduced expression of DRAM2/TMEM77 in tumor cells interferes with cell death.Biochem. Biophys. Res. Commun. 2009; 390: 1340-1344Crossref PubMed Scopus (25) Google Scholar Autophagy is a natural cell survival mechanism triggered in response to stress stimuli such as nutrient starvation or the accumulation of damaged organelles. It is responsible for degrading and recycling cytoplasmic proteins and lipids as well as organelles within the cell.7Levine B. Klionsky D.J. Development by self-digestion: molecular mechanisms and biological functions of autophagy.Dev. Cell. 2004; 6: 463-477Abstract Full Text Full Text PDF PubMed Scopus (3187) Google Scholar This usually begins with isolation of the macromolecules and organelles within the cytoplasm into single membrane vesicles, which fuse together to produce an autophagosome. These autophagosomes subsequently fuse with lysosomes containing acid hydrolases and form a double-membrane autolysosome.8Yoshimori T. Autophagy: a regulated bulk degradation process inside cells.Biochem. Biophys. Res. Commun. 2004; 313: 453-458Crossref PubMed Scopus (462) Google Scholar, 9Kroemer G. Jäättelä M. Lysosomes and autophagy in cell death control.Nat. Rev. Cancer. 2005; 5: 886-897Crossref PubMed Scopus (1037) Google Scholar Although the aim of autophagy is to relieve cellular stress, its excessive induction can in some cases lead to apoptosis rather than protection from cell death.10Yuan J. Lipinski M. Degterev A. Diversity in the mechanisms of neuronal cell death.Neuron. 2003; 40: 401-413Abstract Full Text Full Text PDF PubMed Scopus (404) Google Scholar There is also some evidence to suggest that DRAM2 might have tumor-suppressor capability. DRAM2 transcript and protein expression are reduced in ovarian tumors compared to normal matched tissues.5Park S.M. Kim K. Lee E.J. Kim B.K. Lee T.J. Seo T. Jang I.S. Lee S.H. Kim S. Lee J.H. Park J. Reduced expression of DRAM2/TMEM77 in tumor cells interferes with cell death.Biochem. Biophys. Res. Commun. 2009; 390: 1340-1344Crossref PubMed Scopus (25) Google Scholar Also, siRNA knockdown of endogenous DRAM2 results in reduced conversion to LC3-II in cells subject to starvation-induced autophagy11Yoon J.H. Her S. Kim M. Jang I.S. Park J. The expression of damage-regulated autophagy modulator 2 (DRAM2) contributes to autophagy induction.Mol. Biol. Rep. 2012; 39: 1087-1093Crossref PubMed Scopus (43) Google Scholar and increased survival in deoxyrubicin-treated cells that would normally undergo p53-mediated apoptosis.5Park S.M. Kim K. Lee E.J. Kim B.K. Lee T.J. Seo T. Jang I.S. Lee S.H. Kim S. Lee J.H. Park J. Reduced expression of DRAM2/TMEM77 in tumor cells interferes with cell death.Biochem. Biophys. Res. Commun. 2009; 390: 1340-1344Crossref PubMed Scopus (25) Google Scholar We note that examination of medical histories in the reported subjects with DRAM2-associated retinal dystrophy provided no evidence of increased susceptibility to cancer. Although DRAM2 is transcribed ubiquitously (Figure S4),6O'Prey J. Skommer J. Wilkinson S. Ryan K.M. Analysis of DRAM-related proteins reveals evolutionarily conserved and divergent roles in the control of autophagy.Cell Cycle. 2009; 8: 2260-2265Crossref PubMed Scopus (56) Google Scholar in light of the finding that human DRAM2 variants cause retinal dystrophy, we investigated the precise distribution of the normal protein in the mouse retina. Serial sections were taken from mouse eyes at postnatal day 30 and were stained with a goat polyclonal antiserum against DRAM2 (Figure 4). Confocal immunofluorescence microscopy showed that DRAM2 localized to the inner segment of the photoreceptor layer and the apical surface of the retinal pigment epithelium (RPE), which are located at the basal and distal ends of the outer segment, respectively. This coincides with the primary pathology observed on pre-symptomatic OCT analysis in which the photoreceptor layer appeared specifically affected. This localization is consistent with a role for DRAM2 in photoreceptor autophagy. Photoreceptor outer segments are in a constant state of renewal by ciliogenesis in response to light-induced damage. Recent studies have suggested that there is interplay between ciliogenesis and autophagy. In one study it was shown that disruption of ciliogenesis partially inhibited autophagy, whereas blocking autophagy enhanced primary cilia growth and cilia-associated signaling during normal nutritional conditions. The authors therefore proposed that basal autophagy regulated ciliary growth through the degradation of proteins required for intraflagellar transport.12Pampliega O. Orhon I. Patel B. Sridhar S. Díaz-Carretero A. Beau I. Codogno P. Satir B.H. Satir P. Cuervo A.M. Functional interaction between autophagy and ciliogenesis.Nature. 2013; 502: 194-200Crossref PubMed Scopus (302) Google Scholar In another study, the protein OFD1 (oral facial digital syndrome 1), which accumulated at centriolar satellites located close to the base of the cilium, was rapidly degraded by serum starvation-induced autophagy. This led to ciliary growth, suggesting that OFD1 normally inhibited ciliogenesis.13Tang Z. Lin M.G. Stowe T.R. Chen S. Zhu M. Stearns T. Franco B. Zhong Q. Autophagy promotes primary ciliogenesis by removing OFD1 from centriolar satellites.Nature. 2013; 502: 254-257Crossref PubMed Scopus (279) Google Scholar A high level of autophagy is also expected to take place in the RPE. These cells have a key role in processing shed photoreceptor outer segment discs and consequently in removing toxic metabolites and recycling phototransduction components. This process, which involves RPE phagocytosis, causes up to 10% photoreceptor volume loss each day and is entrained to the circadian rhythm.14Young R.W. Bok D. Participation of the retinal pigment epithelium in the rod outer segment renewal process.J. Cell Biol. 1969; 42: 392-403Crossref PubMed Scopus (912) Google Scholar, 15Nguyen-Legros J. Hicks D. Renewal of photoreceptor outer segments and their phagocytosis by the retinal pigment epithelium.Int. Rev. Cytol. 2000; 196: 245-313Crossref PubMed Google Scholar Indeed there is increasing interest in the role of autophagy in preserving photoreceptor function in connection with the circadian cycle,16Yao J. Jia L. Shelby S.J. Ganios A.M. Feathers K. Thompson D.A. Zacks D.N. Circadian and noncircadian modulation of autophagy in photoreceptors and retinal pigment epithelium.Invest. Ophthalmol. Vis. Sci. 2014; 55: 3237-3246Crossref PubMed Scopus (55) Google Scholar the aging process,17Rodríguez-Muela N. Koga H. García-Ledo L. de la Villa P. de la Rosa E.J. Cuervo A.M. Boya P. Balance between autophagic pathways preserves retinal homeostasis.Aging Cell. 2013; 12: 478-488Crossref PubMed Scopus (125) Google Scholar and retinal disease pathology.18Métrailler S. Schorderet D.F. Cottet S. Early apoptosis of rod photoreceptors in Rpe65(-/-) mice is associated with the upregulated expression of lysosomal-mediated autophagic genes.Exp. Eye Res. 2012; 96: 70-81Crossref PubMed Scopus (22) Google Scholar It is therefore likely that the absence of DRAM2 in the retina reduces the efficiency of autophagy in recycling cell components, which in turn reduces photoreceptor renewal, leading to the thin photoreceptor layer observed on OCT, which is the first presenting feature in pre-symptomatic patients. To summarize, we have shown that biallelic missense, nonsense, and frameshift variants in DRAM2 cause retinal dystrophy with early macular cone photoreceptor involvement. The clinical features and course of retinal degeneration were highly similar among affected individuals from the five reported families. Our findings suggest that DRAM2 is essential for photoreceptor survival and further studies are expected to provide important insights into its precise role in the retina. The UK Inherited Retinal Disease Consortium includes Graeme Black, Georgina Hall, Stuart Ingram, Rachel Gillespie, Simon Ramsden, Forbes Manson, Panagiotis Sergouniotis, Andrew Webster, Alison Hardcastle, Michel Michaelides, Vincent Plagnol, Michael Cheetham, Gavin Arno, Niclas Thomas, Shomi Bhattacharya, Tony Moore, Chris Inglehearn, Carmel Toomes, Manir Ali, Martin McKibbin, James Poulter, Emma Lord, Andrea Nemeth, Susan Downes, Stefano Lise, and Veronica van Heyningen. We would like to thank the subjects and their families who participated in this study. We thank our colleagues at University College London (UK) for kindly contributing to the UCL-exomes control panel. This work was supported by grants from NERC (Yorkshire branch), Macular Society UK, the UK National Institute for Health Research (Biomedical Research Centre, Moorfields Eye Hospital and Institute of Ophthalmology), RP Fighting Blindness and Fight For Sight (RP Genome Project GR586), the Belspo IAP project P7/43, the Belgium Medical Genomics Initiative (BeMGI), the Ghent University Special Research Fund (BOF15/GOA/011), and the Funds for Research in Ophthalmology (FRO). C.A.J. and C.F.I. were supported by a grant from the Jules Thorn Charitable Trust. M.E.E. is funded by an Egyptian Government Scholarship and K.V.S. is a doctoral fellow from the Institute for Innovation by Science and Technology (IWT). E.D.B. and B.P.L. are senior clinical investigators of the Research Foundation Flanders (FWO). The variants reported in this paper have been deposited into the ClinVar database at the National Centre for Biotechnology Information under accession numbers ClinVar: SCV000222243-49. 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Figures S1–S4 and Tables S1–S4 The URLs for data presented herein are as follows:AgileMultiIdeogram, http://dna.leeds.ac.uk/agile/AgileMultiIdeogram/ANNOVAR, http://www.openbioinformatics.org/annovar/CADD, http://cadd.gs.washington.edu/ClustalW, http://www.ebi.ac.uk/Tools/msa/clustalw2/dbSNP, http://www.ncbi.nlm.nih.gov/projects/SNP/ExAC Browser, http://exac.broadinstitute.org/Galaxy, https://usegalaxy.org/GATK, http://www.broadinstitute.org/gatk/IGV, http://www.broadinstitute.org/igv/Macular Society, http://www.macularsociety.org/MutationTaster, http://www.mutationtaster.org/MutPred, http://mutpred.mutdb.org/NHLBI Exome Sequencing Project (ESP) Exome Variant Server, http://evs.gs.washington.edu/EVS/OMIM, http://www.omim.org/Picard, http://picard.sourceforge.net/PolyPhen-2, http://www.genetics.bwh.harvard.edu/pph2/Primer3, http://bioinfo.ut.ee/primer3PROVEAN, http://provean.jcvi.org/index.phpRefSeq, http://www.ncbi.nlm.nih.gov/RefSeqRetNet – Retinal Information Network, https://sph.uth.edu/retnet/home.htmSIFT, http://sift.bii.a-star.edu.sg/Superlink Online SNP, http://cbl-hap.cs.technion.ac.il/superlink-snp/
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