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Mutations in NMNAT1 cause Leber congenital amaurosis with early-onset severe macular and optic atrophy Isabelle Perrault1,13, Sylvain Hanein1,13, Xavier Zanlonghi2, Valérie Serre1, Michael Nicouleau1, Sabine Defoort-Delhemmes3, Nathalie Delphin1, Lucas Fares-Taie1, Sylvie Gerber1, Olivia Xerri1, Catherine Edelson4, Alice Goldenberg5, Alice Duncombe5, Gylène Le Meur6, Christian Hamel7, Eduardo Silva8, Patrick Nitschke9, Patrick Calvas10, Arnold Munnich1, Olivier Roche11, Hélène Dollfus12, Josseline Kaplan1 & Jean-Michel Rozet1 In addition to its activity in nicotinamide adenine dinucleotide (NAD+) synthesis, the nuclear nicotinamide mononucleotide adenyltransferase NMNAT1 acts as a chaperone that protects against neuronal activity–induced degeneration. Here we report that compound heterozygous and homozygous NMNAT1 mutations cause severe neonatal neurodegeneration of the central retina and early-onset optic atrophy in 22 unrelated individuals. Their clinical presentation is consistent with Leber congenital amaurosis and suggests that the mutations affect neuroprotection of photoreceptor cells. Leber congenital amaurosis (LCA; MIM 204000) is a group of severe congenital neurodegenerative diseases of the retina that is the most common cause of incurable childhood blindness (10–18%)1. LCA is typically an autosomal recessive condition, but rare dominant cases have been reported1. Mutations in 16 genes involved in various retinal functions have been identified as the cause of 70% of LCA cases1–3. To provide further insights into the molecular bases of the disease, we subjected the DNA of five French index LCA cases without mutations in known genes to whole-exome resequencing. In two of the five cases, exome sequencing analyses identified NMNAT1 as a potential candidate gene, which encodes nuclear nicotinamide mononucleotide adenyltransferase 1 (MIM 608700; NM_022787.3; Supplementary Tables 1 and 2). Subject P15 carried the c.634G>T (p.Glu215*) variant and the rare c.769G>A (p.Glu257Lys) variant (rs150726175; dbSNP allele frequency of T
(p.Glu107*) and c.709C>T (p.Arg237Cys) variants (Fig. 1a, Table 1 and Supplementary Table 3a). Sanger sequencing showed cosegregation of these changes with the disease (Supplementary Fig. 1) and their absence in 200 control individuals (100/200 ancestry-matched individuals; Supplementary Methods). Subsequently, we screened NMNAT1 coding exons and intron-exon boundaries by Sanger sequencing in 256 additional index cases with no mutations in known LCA-causing genes (Supplementary Table 4 and Supplementary Methods). Another 20 cases were found to carry homozygous (1/20) or compound heterozygous (19/20) NMNAT1 mutations that were absent in our cohort of controls. We confirmed cosegregation of mutations with the disease by sequencing DNA from the affected individuals’ relatives (for subjects P1–P11.1, P15 and P17; Supplementary Fig. 1) or by allele-specific PCR amplification (for subjects P12–P14 and P16; Supplementary Fig. 2, Supplementary Table 4 and Supplementary Methods). A sporadic case born to consanguineous Algerian parents (P21) carried a homozygous nonsense mutation c.507G>A encoding p.Trp169*. In addition to P15 and P22.1 who were analyzed by exome sequencing, another four cases were identified by Sanger sequencing who carried a convincingly pathogenic mutation in combination with a missense change in NMNAT1. P20 had the c.362delA (p.Glu121Glufs*20) and the unreported c.532G>A (p.Val178Met) variants. P1, P3 and P5 had the c.769G>A (p.Glu257Lys) variant with the c.1A>G (p.Met1?), c.362delA (p.Glu121Glufs*20) or c.439+1G>C variants. The remaining 15 cases carried two missense variants. P18 had the rare c.37G>A (p.Ala13Thr) variant (rs138613460; dbSNP allele frequency of T (p.Leu153Pro) variant. P19 harbored the unreported c.205A>G (p.Met69Val) and c.650T>A (p.Ile217Asn) variants. Of the remaining 15 cases, 13 had the c.769G>A (p.Glu257Lys) variant with the c.205A>G (p.Met69Val), c.439G>C (p.Ala147Pro), c.518A>G (p.Asp173Gly), c.542A>G (p.Tyr181Cys), c.752A>C (p.His251Pro) or c.716T>C (p.Leu239Ser) variant (P2, P4, P6, P7.2, P16 and P17, respectively) or in combination with the rare c.619C>T (p.Arg207Trp) variant (dbSNP allele frequency of A (p.Trp169*) variant (P24) and 6 of whom had the c.769G>A (p.Glu257Lys) variant (P23.1, P25, P26.1, P27, P28.1 and P29; Supplementary Fig. 1 and Supplementary Table 3b). We assessed whether the recurrent mutations c.769G>A (p.Glu257Lys) and c.619C>T (p.Arg207Trp) represented founder effects through the
1Department
of Genetics, Institut National de la Santé et de la Recherche Médicale (INSERM) U781, Université Paris Descartes–Sorbonne Paris Cité, Imagine Institute, Paris, France. 2Department of Vision Functional Exploration, Clinique Sourdille, Nantes, France. 3Department of Exploration of Vision and NeuroOphthalmology, Hôpital Roger Salengro, Centre Hospitalier Universitaire Régional, Lille, France. 4Department of Ophthalmology, Fondation Ophtalmologique Adolphe de Rothschild, Paris, France. 5Department of Genetics, Centre Hospitalier Universitaire, Rouen, France. 6Department of Ophthalmology, Centre Hospitalier Universitaire, Nantes, France. 7Neurosciences Institute, Hôpital Saint Eloi, Montpellier, France. 8Department of Ophthalmology, Coimbra University Hospital, Coimbra, Portugal. 9Bioinformatics Platform, Université Paris Descartes–Sorbonne Paris Cité, Paris, France. 10Department of Medical Genetics, Purpan Hospital, Centre Hospitalier Universitaire, Toulouse, France. 11Department of Ophthalmology, Université Paris Descartes–Sorbonne Paris Cité, Paris, France. 12Department of Clinical Genetics, Strasbourg University Hospital, Strasbourg, France. 13These authors contributed equally to this work. Correspondence should be addressed to J.K. (
[email protected]) or J.-M.R. (
[email protected]). Received 9 April; accepted 27 June; published online 29 July 2012; doi:10.1038/ng.2357
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b p.Leu153Pro p.Trp169* p.Asp173Gly p.Val178Met p.Tyr181Cys p.Arg207Trp p.Glu215* p.Ile217Asn p.Glu107* p.Arg237Cys p.Glu121Glufs*20 p.Leu239Ser p.Ala147Pro p.Met1? p.His251Pro p.Ala13Thr c.439+1g>c p.Glu257Lys p.Met69Val ATG
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© 2012 Nature America, Inc. All rights reserved.
1
Right eye
Left eye
c
d
e
f
g
h
4 months
6 months
N/A
14 months
TAG 2
3
4
24 months
5
Figure 1 NMNAT1 variants identified in individuals with LCA and funduscopic evidence of early-onset and fast-progressing macular atrophy. (a) Structures of the NMNAT1 gene and NMNAT1 protein and the position and nature of the encoded alterations identified in homozygous and compound heterozygous individuals. (b) Progression of the macular lesions in the right and left eyes of subject P4. Pictures taken at age 4 months already show marked macular changes that progressed rapidly, as shown in photographs taken 2 and 10 months later. N/A, not available. (c–g) The fundi of subjects P10 (c), P15 (d), P9 (e), P11.1 (f) and P11.2 (g) (age range of 4.5–20 years) show a large macular atrophy with pigmented edges, optic atrophy and attenuated retinal vessels. (h) The fundus of subject P14 at 49 years of age shows marked macular atrophy, with the appearance of congenital coloboma, optic atrophy, extremely thin retinal vessels and peripheral pigmentary clumping. Scale bars, 5 mm.
analysis of microsatellite markers flanking NMNAT1 (Supplementary Methods). We found that, in 12 of 18 families (66.7%), the c.769G>A mutation encoding the p.Glu257Lys variant segregated with a very rare allele at the D1S244 locus 0.5 Mb away, suggesting that it might have occurred on an ancestral allele (Supplementary Fig. 3). The small number of available samples with the c.619C>T (p.Arg207Trp) alteration precluded the search for linkage disequilibrium (LD) for this allele. All of the missense variants identified except for the c.769G>A (p.Glu257Lys) and c.439G>C (p.Ala147Pro) variants were predicted to be deleterious by bioinformatics analysis using Alamut Mutation Interpretation Software (Table 1). NMNAT1 encodes a homohexameric nuclear enzyme that catalyzes the formation of NAD+ from nicotinamide mononucleotide and ATP4. We used the 2.20-Å coordinate set for human NMNAT1 complexed with NAD (Protein Data Bank (PDB) 1KQN) to assess the structural and functional effects of the missense variants (Supplementary Methods). The Glu257 residue is located at the solvent-accessible surface of the protein
(Supplementary Fig. 4). The replacement of this negatively charged glutamic acid with a positively charged lysine might be tolerated. Taking into consideration that the frequency of the c.769G>A allele encoding p.Glu257Lys is significantly higher in cases (17 compound heterozygotes (Supplementary Table 3a) and 6 single heterozygotes (Supplementary Table 3b) for a total of 23 of 261 LCA cases; 23/522 disease alleles = 0.044) than in ancestry-matched controls (0), it is possible that this variant is not causative but rather is in LD with an undetected mutation. Conversely, introducing a proline instead of an alanine at position 147 (p.Ala147Pro) is likely to alter the protein function by destabilizing the native conformation of the nuclear localization signal (Supplementary Fig. 4). The other 11 missense variants are likely to alter the catalytic activity (p.Ala13Thr, p.Leu153Pro, p.Asp173Gly and p.Val178Met), the hexamerization (p.Arg207Trp, p.Ile217Asn, p.Arg237Cys and p.Leu239Ser) and the hydrophobic interactions or stability (p.Met69Val, p.Tyr181Cys and p.His251Pro) of the enzyme (Supplementary Fig. 4 and Supplementary Tables 4 and 5).
Table 1 NMNAT1 variants identified in 22 individuals with LCA Allele 1 Family
Mutation
1 2 3 4 5 6 7 8–14 15 16 17 18 19 20 21 22
c.1A>G c.205A>G c.362delA c.439G>C c.439+1G>C c.518A>G c.542A>G c.619C>T c.643G>T c.752A>C c.716T>C c.37G>A c.205A>G c.362delA c.507G>A c.319G>T
Allele 2
Predicted effecta p.Met1? p.Met69Val p.Glu121Glufs*20 p.Ala147Pro Splice p.Asp173Gly p.Tyr181Cys p.Arg207Trp p.Glu215* p.His251Pro p.Leu239Ser p.Ala13Thr p.Met69Val p.Glu121Glufs*20 p.Trp169* p.Glu107*
Deleterious Deleterious Deleterious Tolerated Deleterious Deleterious Deleterious Deleterious Deleterious Deleterious Deleterious Deleterious Deleterious Deleterious Deleterious Deleterious
Mutation
rs142968179c
rs138613460d
c.769G>A c.769G>A c.769G>A c.769G>A c.769G>A c.769G>A c.769G>A c.769G>A c.769G>A c.769G>A c.769G>A c.468T>C c.650T>A c.532G>A c.507G>A c.709C>T
Predicted effecta p.Glu257Lys p.Glu257Lys p.Glu257Lys p.Glu257Lys p.Glu257Lys p.Glu257Lys p.Glu257Lys p.Glu257Lys p.Glu257Lys p.Glu257Lys p.Glu257Lys p.Leu153Pro p.Ile217Asn p.Val178Met p.Trp169* p.Arg237Cys
Tolerated Tolerated Tolerated Tolerated Tolerated Tolerated Tolerated Tolerated Tolerated Tolerated Tolerated Deleterious Deleterious Deleterious Deleterious Deleterious
rs150726175b rs150726175b rs150726175b rs150726175b rs150726175b rs150726175b rs150726175b rs150726175b rs150726175b rs150726175b rs150726175b
aVariant
pathogenicity according to Align DGVD, Polyphen-2, SIFT, SpliceSiteFinder-like, MaxEntScan, NNSPLICE and Human Splicing Finder, available through Alamut Interpretation Software 2.0. bMinor allele frequency (MAF) of 0.044 in LCA (based on the screening of 261 unrelated cases). cMAF of 0.013 in LCA. dMAF of 0.002 in LCA. The rs numbers of NMNAT1 variants reported in the dbSNP database are indicated (MAFs in the general population were G mutation that accounts for 60% of CEP290 disease-related alleles in LCA5, these cases who share the same ancestry (French) could carry a common NMNAT1 mutation lying in an unscreened region. NMNAT1 encodes one isoform of the nuclear NAD+-synthesizing enzyme nicotinamide mononucleotide adenylyltransferase 6. The other two isoforms, NMNAT2 and NMNAT3, localize to the Golgi apparatus and to cytoplasm and mitochondria, respectively7. In mice, Nmnat1 knockout results in embryonic lethality, indicating that the other isoforms cannot compensate for its function8. In Drosophila melanogaster, the complete inactivation of the unique nmnat isoform is also lethal but causes a severe early-onset degeneration of normally developed photoreceptors when the mutation is restricted to the retina9. In that study, light exposure was shown to trigger the loss of photoreceptor cells, suggesting that degeneration was worsened by neuronal activity9. Notably, the human retinal phenotype associated with NMNAT1 mutations is consistent with that observed in Drosophila. Pseudo-coloboma and optic atrophy are not present at birth (Fig. 1b). Rather, they arise through the progressive yet rapid degeneration of central photoreceptors and retinal ganglion cells. Considering that the central retina receives most of the photons entering the eye, it is likely that light exposure triggers this degeneration. Nmnat1 constitutes most of the Wallerian degeneration slow (WldS) fusion protein that delays axon degeneration after injury in rodents and flies10,11. Many studies have shown that catalytically active Nmnat1 is necessary but not sufficient for axonal protection8,12–14. In Drosophila, enzymatically inactive Nmnat can prevent photoreceptor degeneration9, suggesting that the enzyme functions as a chaperone that protects against neuronal activity–induced degeneration in mature photoreceptors, independent of its enzymatic activity, which is required for viability9. We could not assess the chaperone activity of the enzyme, but both the genetic data and structural analyses are consistent with a residual activity of the enzyme in LCA. A single case carried a homozygous NMNAT1 nonsense mutation (P21, c.507G>A, encoding p.Trp169*; Table 1). Structural analyses indicate that the protein lacking the last 110 amino acids retains the domain from Glu107–Lys146 that mediates the nuclear localization of the enzyme15. In addition, despite the alteration of the protein’s hexamerization interfaces, the mutant protein is predicted to preserve the NAD-binding pocket region (Supplementary Fig. 5). This suggests that, as in other LCA cases with NMNAT1 mutations, P21 may harbor an enzyme that retains residual catalytic activity. This would be consistent with data reported in Drosophila suggesting that homozygosity for null NMNAT1 alleles would result in embryonic lethality. Additionally, milder NMNAT1 mutations could cause late-onset retinal dystrophies, as reported for several genes involved in LCA1. Together, these data suggest that retinal delivery of exogenous NAD+ might not be sufficient to prevent the degeneration of photoreceptors Nature Genetics VOLUME 44 | NUMBER 9 | SEPTEMBER 2012
in individuals with LCA with NMNAT1 mutations. Gene replacement therapy could be a preferable option. Furthermore, considering that protection against light prevents degeneration of Nmnat-mutant photoreceptors in Drosophila and that the degradation of the central retina is progressive, it is worth considering whether strict protection against light at birth might slow the retinal lesions. In summary, we report the identification of convincingly pathogenic or recurrent NMNAT1 mutations whose frequency in LCA cases versus controls provides robust evidence that they are disease causing. The specific and well-characterized phenotype further supports the notion that NMNAT1 should be regarded as a new LCA-causing gene, while adding inability to protect against neuronal degeneration to the list of mechanisms underlying LCA. URLs. Align DGVD, Polyphen-2, SIFT, SpliceSiteFinder-like, MaxEntScan, NNSPLICE and Human Splicing Finder are available through Alamut Interpretation Software 2.0, http://www.interactivebiosoftware.com/; UCSC Genome Browser, http://genome.ucsc.edu/; Online Mendelian Inheritance in Man (OMIM), http://www.omim. org/; RCSB Protein Data Bank (PDB), http://www.rcsb.org/pdb/home/ home.do/; Swiss-PdbViewer 3.7, http://www.expasy.org/spdbv/. Note: Supplementary information is available in the online version of the paper. Acknowledgments We are grateful to the individuals with Leber congenital amaurosis and their families for their participation in this study. We thank P. Debruyne for making available the fundus pictures of subject P4. This research was supported by the Association Retina France (grant to J.-M.R.) and the Assistance Publique–Hôpitaux de Paris (AP-HP; grant PHRC National-2009 AOM 09058/P081257 to J.K.). We also thank the Association Retina France for supporting the 100 Exomes project of the French Research Network aiming to speed the genetic analysis of inherited retinal diseases through whole-exome resequencing. Written consent was obtained from participants or legally authorized representatives. The study was conducted in strict adherence to the tenets of the Declaration of Helsinki and was approved by the Comité de Protection des Personnes Ile-de-France II. AUTHOR CONTRIBUTIONS J.-M.R., J.K., H.D. and A.M. designed the study. I.P., S.H. and P.N. analyzed data from exome sequencing. I.P. and S.H. performed and analyzed data from Sanger sequencing of affected individuals and controls. M.N., N.D., L.F.-T. and S.G. performed linkage analyses at the LCA loci and Sanger sequencing of candidates to select the subjects to be screened for NMNAT1 mutations. O.X. performed and analyzed the LD analyses at the NMNAT1 locus. V.S. performed the threedimensional structural analysis of NMNAT1 missense variants. J.K., H.D., X.Z., S.D.-D., C.E., A.G., A.D., G.L.M., C.H., E.S., P.C. and O.R. provided and analyzed clinical material from subjects. All authors contributed to the manuscript written by J.-M.R. and J.K. COMPETING FINANCIAL INTERESTS The authors declare no competing financial interests. Published online at http://www.nature.com/doifinder/10.1038/ng.2357. Reprints and permissions information is available online at http://www.nature.com/ reprints/index.html. 1. den Hollander, A.I. et al. Prog. Retin. Eye Res. 27, 391–419 (2008). 2. Sergouniotis, P.I. et al. Am. J. Hum. Genet. 89, 183–190 (2011). 3. Estrada-Cuzcano, A. et al. Invest. Ophthalmol. Vis. Sci. 52, 834–839 (2011). 4. Zhou, T. et al. J. Biol. Chem. 277, 13148–13154 (2002). 5. Perrault, I. et al. Hum. Mutat. 28, 416 (2007). 6. Raffaelli, N. et al. Biochem. Biophys. Res. Commun. 297, 835–840 (2002). 7. Berger, F. et al. J. Biol. Chem. 280, 36334–36341 (2005). 8. Conforti, L. et al. FEBS J. 278, 2666–2679 (2011). 9. Zhai, R.G. et al. PLoS Biol. 4, e416 (2006). 10. Mack, T.G. et al. Nat. Neurosci. 4, 1199–1206 (2001). 11. Coleman, M.P. et al. Annu. Rev. Neurosci. 33, 245–267 (2010). 12. Avery, M.A. et al. J. Cell Biol. 184, 501–513 (2009). 13. Feng, Y. et al. Protein Cell 1, 237–245 (2010). 14. Sasaki, Y. et al. J. Neurosci. 29, 5525–5535 (2009). 15. Lau, C. et al. J. Biol. Chem. 285, 18868–18876 (2010).
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