Exome sequencing identifies NMNAT1 mutations as a cause of Leber congenital amaurosis

 

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Leber congenital amaurosis (LCA) is an autosomal recessive retinal dystrophy that manifests with genetic heterogeneity. We sequenced the exome of an individual with LCA and identified nonsense (c.507G>A, p.Trp169*) and missense (c.769G>A, p.Glu257Lys) mut

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b riefcommunic at ions exome sequencing identifies nmnat1 mutations as a cause of leber congenital amaurosis sequence analysis supplementary tables 1 and 2 and supplementary methods we identified 2,460 previously unreported variants we prioritized ten genes each with a previously unreported nonsense variant predicted to truncate the encoded protein for further analysis genecards indicates that five of these ten genes are expressed in the retina including nmnat1 which maps to the 1p36 region lca9 pei-wen chiang1,12 juan wang2,12 yang chen2 quan fu3 which is the only known lca locus that has not yet been defined jing zhong2 yanhua chen2 xin yi2 renhua wu2 molecularly was previously mapped to chromosome 1p36 in a conhaixue gan1 yong shi1 yanling chen4 christopher barnett5 sanguineous pakistani family5 dianna wheaton6 megan day7 joanne sutherland7 elise heon7 we identified a nonsense variant c.507g>a p.trp169 and a richard g weleber8 luis alexandre rassi gabriel9 missense variant c.769g>a p.glu257lys in nmnat1 by exome peikuan cong4 kuanghsiang chuang10 sheng ye3 sequencing and confirmed these alterations by sanger sequencing juliana maria ferraz sallum11 ming qi2,4,10 pcr primer sequences are provided in supplementary table 3 parental testing determined that the nonsense variant was inherited lebercongenitalamaurosislcaisanautosomalrecessive paternally and the missense variant maternally analysis showed that retinaldystrophythatmanifestswithgeneticheterogeneity.we the unaffected sibling harbored neither variant taken together these sequencedtheexomeofanindividualwithlcaandidentified findings supported the classification of nmnat1 as a candidate gene nonsensec.507g>a,p.trp69 andmissensec.769g>a for lca p.glu257lysmutationsinnmnat1,whichencodesanenzyme to further evaluate the role of nmnat1 in lca we analyzed by inthenicotinamideadeninedinucleotidenadbiosynthesis sanger sequencing 50 unrelated lca cases with no previously identipathwayimplicatedinprotectionagainstaxonaldegeneration fied mutations in known lca-linked genes we identified ten addiwealsofoundnmnat1mutationsintenotherindividualswith tional cases with compound heterozygous mutations in nmnat1 lca,allofwhomcarrythep.glu257lysvariant fig 1 and table 1 notably all 11 cases carried the common missense variant c.769g>a p.glu257lys as 1 of the 2 variant alleles leber congenital amaurosis the most common cause of inherited the identified mutations all cosegregated with the lca phenotype blindness in childhood1 is a severe retinal dystrophy that manifests as as shown by pedigree analysis supplementary fig 2 low vision and nystagmus at birth or during infancy lca is associated nmnat1 encoding nicotinamide mononucleotide adenylylwith mutations in at least 17 genes an identifiable molecular etiology transferase 1 contains four coding exons and encodes a 279-residue is absent in 20­30 of the cases screened suggesting that there are protein with an important role in nad biosynthesis in which it causative genes that are yet to be discovered2­4 current clinical trials catalyzes the formation of nad from nicotinamide mononucleotide address the molecular defect by gene replacement or bypass the block nmn and atp nmnat1 has not previously been linked to lca in metabolism by oral administration of 9-cis-retinal for deficiency but is associated with axonal degeneration6 homozygous nmnat1of rpe65 and lrat knockout mice do not survive to birth and a homozygous nmnat we sequenced the coding regions and all exon-intron boundaries mutation in drosophila melanogaster is also lethal these observations to at least 50 intronic nucleotides from the intron-exon boundary support the idea that nmnat1 has an essential function in certain for the 17 known genes associated with lca namely aipl1 cabp4 species7 nmnat1 is located at 1p36.22 near the previously mapped cep290 crb1 crx gucy2d iqcb1 lca5 lrat rd3 rdh12 lca9 locus the affected individuals in the lca9 family all had conrpe65 rpgrip1 spata7 tulp1 impdh1 and otx2 among 220 genital non-progressive severe visual impairment 5 similarly the individuals with lca screened we identified mutant alleles on both 11 lca cases reported here all have very early­onset severe vision chromosomes in 160 cases and a single mutant allele in 10 cases loss supplementary table 4 and supplementary note because we found no mutations in known lca-linked genes in this study provides evidence that nmnat1 is a new lca-causing subject 1 for clinical phenotype information see supplementary gene through the discovery of disease-associated mutations in 9 unrefig 1 and the supplementary note we performed whole-exome lated families and 11 total cases from 4 different countries table 1 1casey © 2012 nature america inc all rights reserved eye institute molecular diagnostic laboratory portland oregon usa 2beijing genomics institute bgi shenzhen shenzhen china 3center for structural biology life sciences institute zhejiang university hangzhou china 4center for genetic and genomic medicine zhejiang university school of medicine first affiliated hospital and james d watson institute of genome sciences hangzhou china 5sa clinical genetics service women s and children s hospital north adelaide south australia australia 6retina foundation of the southwest dallas texas usa 7department of ophthalmology sickkids hospital toronto ontario canada 8casey eye institute oregon health science university portland oregon usa 9department of ocular genetics centro de referência em oftalmologia cerof federal university of goias goiania brazil 10department of pathology university of rochester medical center rochester new york usa 11department of ophthalmology federal university of sao paulo sao paulo brazil 12these authors contributed equally to this work correspondence should be addressed to m.q ming_qi@urmc.rochester.edu received 5 december 2011 accepted 9 july 2012 published online 29 july 2012 doi:10.1038/ng.2370 nature genetics advance online publication

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b riefcommunic at ions coding utr heterozygous in every subject with lca studied in cis e257k m35t v98g w85 v151f l153v w169 n273d figure 1 schematic of the nmnat1 gene depicting nmnat1 variants identified in individuals with lca clinically all 11 cases have severe presentations supplementary table 4 and supplementary note although the data are sparse disease severity may correlate with the nature of the mutations the concept of `allele clamping 8 involves a process whereby the phenotypes of individuals with a common variant such as p.glu257lys in the presence of a variable second allele are examined to infer the effect of the second allele the four individuals carrying the nonsense variant p.trp169 were all blind at birth with light perception still present to various extents for the five individuals carrying only missense variants vision decreased within a few years after birth the retinas of all affected individuals had a mottled aspect in the periphery and atrophic macular coloboma­like lesions supplementary fig 1 described in subject 8 as macular scarring enlargement of the macular lesion was recorded in follow-up examinations in subject 2 between 6 and 9 months of age and in subject 4 between 5 and 7 years of age subject 4 who had two variant alleles on the second chromosome had a visual acuity of 20/200 od right eye and 20/400 os left eye at 8 years of age and was the only case in whom the electroretinogram erg showed primarily cone dysfunction rather than profound loss of all responses this individual s diagnosis was revised to a cone-rod dystrophy subject 5 presented with coats disease in the left eye at 3 years of age subject 6 the only individual harboring the c.451g>t p.val151phe allele sustained major loss of vision with respect to light perception between the ages of 18 and 26 years the allele frequency of c.769g>a p.glu257lys is estimated to be 0.001 dbsnp rs150726175 in this study we found no individuals who were homozygous for the c.769g>a p.glu257lys allele the rest of the variants we identified have not been reported in any public table 1 nmnat1 variants in individuals with lca variant 1 subject 1 2 3 4 5 6 7,8 9,10 11 dna c.507g>a paternal c.507g>a paternal c.507g>a maternal c.457c>g paternal c.817a>g paternal c.293t>g paternal c.451g>t parental samples not available c.104t>c paternal c.255g>a maternal c.507g>a maternal proteina p.trp169 p.trp169 p.trp169 p.leu153val 0.987 p.asn273asp 0.915 p.val98gly 0.909 p.val151phe 0.986 p.met35thr 0.601 p.trp85 p.trp169 dna c.769g>a maternal c.769g>a maternal c.769g>a maternal c.769g>a maternal c.769g>a maternal database the p.trp169 variant was found in four of the nine families the reason for this high incidence rate is unknown numerous studies involving several different organisms have linked the function of nmnat1 to axonal degeneration6 in the wlds mouse model a hybrid protein formed by fusing a ube4b sequence to nmnat1 acts to protect severed axons9,10 also loss of nmnat function in drosophila eyes causes a severe neurodegenerative phenotype although catalytic function is not required for photoreceptor differentiation or development rather nmnat is involved in the maintenance and integrity of mature neurons loss of nmnat function in drosophila photoreceptors resulted in the degeneration of developed neuronal cells a phenotype that can be regarded as a retinal dystrophy11 nmnat1 has not been associated with human disease before this study therefore we have identified the eye as a biologically relevant organ for studying nmnat1 function in vivo nmnat1 is ubiquitously expressed and shows the highest specific activity in catalyzing the final step of nad biosynthesis human nmnat1 is a homohexamer and each protomer contains a central six-stranded parallel sheet flanked by several helices12,13 supplementary fig 3 in this study all 11 individuals with lca carried the p.glu257lys variant according to the crystal structure of human nmnat1 glu257 is located on the external surface within a short c-terminal helix notably the adjacent residue ser256 was predicted to be a phosphorylation site and might be involved in physical interactions with other associated proteins such as polyadp-ribose polymerase or protein kinases12,13 substitution of the negatively charged glutamic acid residue with positively charged lysine might change the electrostatic properties of the surface thereby affecting the proposed physical interactions asn273 is located in the last short helix residues 267­274 and is involved in the coordination of an active site water molecule12,13 substitution of this residue with an acidic residue aspartic acid would likely affect enzymatic activity met35 and val151 are located in the hydrophobic core of the protein whereas val98 and leu153 are very close to the site of ligand binding approximately 6­7 Å away the four variants p.met35thr p.val98gly p.val151phe and p.leu153val most likely disturb local interactions which in consequence alter the active site thereby affecting the enzymatic activity © 2012 nature america inc all rights reserved variant 2 proteina p.glu257lys 0.089 p.glu257lys 0.089 p.glu257lys 0.089 p.glu257lys 0.089 p.glu257lys 0.089 country ancestry paternal united states chinese hispanic caucasian maternal united states cherokee hispanic caucasian paternal brazil turkish german italian portuguese maternal brazil polish portuguese lebanese italian paternal brazil brazilian maternal brazil brazilian paternal canada western european maternal canada western european paternal brazil african and portuguese maternal brazil hispanic caucasian paternal canada greek maternal canada greek paternal united states european maternal united states european paternal canada british and dutch maternal canada british and dutch paternal australia australian maternal australia english c.769g>a parental p.glu257lys 0.089 samples not available c.769g>a maternal p.glu257lys 0.089 c.769g>a paternal c.769g>a paternal p.glu257lys 0.089 p.glu257lys 0.089 apolyphen-2 predictions of the identified missense variants are shown in parentheses the highest score for a predicted damaging variant is 1.0 these predictions should be treated with caution the p.glu257lys variant was originally predicted to be possibly damaging score of 0.903 but the latest prediction suggests that the variant is benign score of 0.089 conversely the p.met35thr variant was originally predicted to be benign score of 0.059 but the latest prediction is possibly damaging score of 0.601 2 advance online publication nature genetics

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b riefcommunic at ions identifying nmnat1 as the causative gene for a subset of individuals with lca provides a new direction for the study of its biological function due to its small size nmnat1 is an attractive target for gene replacement therapy because nmnat1 has a role in neuroprotection and is an enzyme pharmacological intervention may also be possible restriction of clinical presentation to the retina may facilitate these therapeutic approaches recent progress in gene therapy­mediated recovery of retinal function14,15 underscores the importance of finding the underlying genetic cause for all individuals with lca urls leiden open variation database lovd for nmnat1 http www.genomed.org/lovd/lca genecards http www.genecards org polyphen-2 http genetics.bwh.harvard.edu/pph2 accession codes exome sequencing data are available at the ncbi sequence read archive sra under accession sra055680 note supplementary information is available in the online version of the paper author contributions p w.c and m.q conceived the project and planned the experiments j.s m.d d.w c.b e.h l.a.r.g and j.m.f.s clinically characterized the lca cases and collected blood samples j.w yang chen j.z yanhua chen x.y r.w yanling chen and m.q performed next-generation sequencing experiments h.g and y.s performed validation experiments p w.c analyzed and interpreted the data r.g.w interpreted the clinical data q.f and s.y analyzed and interpreted the structural data p.c and k.c built the public lovd database for the nmnat1 gene and curated the clinical and mutation data all authors contributed to the final manuscript competing financial interests the authors declare no competing financial interests published online at http www.nature.com/doifinder/10.1038/ng.2370 reprints and permissions information is available online at http www.nature.com reprints/index.html © 2012 nature america inc all rights reserved acknowledgments we thank the subjects and their parents for participation in this study the primary appointment of m.q is at the zhejiang university school of medicine p w.c and r.g.w are grateful for the support of the foundation fighting blindness ffb p.w.c is especially thankful to the parents of subject 1 without their persistence and commitment this work would not have been possible this project was partially supported by a 985 project grant from the ministry of education of china to m.q and by the qiangjiang research talent grant 2006r10018 to m.q from the science and technology department of zhejiang province q.f and s.y are supported by funds from the natural science foundation of zhejiang province r2100439 informed consent was obtained through the casey eye institute molecular diagnostic laboratory for the purpose of clinical testing and subsequently research testing if no mutations could be identified this study was approved by the oregon health science university research integrity office irb00008083 1 weleber r.g francis p.j trzupek k.m genereviews eds pagon r.a bird t.d dolan c.r stephens k university of washington seattle 1993 2 stone e.m am j ophthalmol 144 791­811 2007 3 berger w kloecknner-gruissem b neidhardt j prog retin eye res 29 335­375 2010 4 den hollander a.i black a bennett j cremers f.p j clin invest 120 3042­3053 2010 5 keen t.j et al eur j hum genet 11 420­423 2003 6 coleman m.p freeman m.r annu rev neurosci 33 245­267 2010 7 conforti l et al febs j 278 2666­2679 2011 8 schindler e.i et al hum mol genet 19 3693­3701 2010 9 conforti l et al proc natl acad sci usa 97 11377­11382 2000 10 mack t.g et al nat neurosci 4 1199­1206 2001 11 zhai r.g et al plos biol 4 e416 2006 12 garavaglia s et al j biol chem 277 8524­8530 2002 13 zhou t et al j biol chem 277 13148­13154 2002 14 maguire a.m et al n engl j med 358 2240­2248 2008 15 maguire a.m et al lancet 374 1597­1605 2009 nature genetics advance online publication

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