© 2012 Nature America, Inc. All rights reserved. to to this work. Illinois, USA. Street Hospital for Children, London, UK. Seville, Spain. Perelman School of Medicine, USA. Pennsylvania, Philadelphia, Informatics, Children’s Hospital of USA. Pennsylvania, Philadelphia, Philadelphia, 13 INSERM–Direction de l’Hospitalisation et de l’Organisation des Soins (DHOS) Centre d’Investigation Clinique 503, Paris, France. la Vision, Paris, France. et de la Recherche Médicale (INSERM) U968, Paris, France. Kallam Anji Reddy Campus, LV Prasad Marg, Hyderabad, India. of Pennsylvania Perelman School of Medicine, USA. Pennsylvania, Philadelphia, Ophthalmology, Eye Massachusetts and Ear Infirmary, Harvard Medical School, Boston, USA. Massachusetts, Eye and Ear Infirmary, Harvard Medical School, Boston, USA. Massachusetts, University of Pennsylvania Perelman School of Medicine, USA. Pennsylvania, Philadelphia, USA. Pennsylvania, Philadelphia, 1 cells and of death the rod and phot sive cone dysfunction retinal progres by characterized are that disorders blindness early-onset of Inherited retinal diseases, such as LCA, represent a heterogeneous group mutations isoform individuals. identified Sequencing p.Val9Met (NAD a isoform previously in This of NMNAT1 a Network in vision form Leber Qin Liu Mark Consugar Subhadra Jalali Magdalena Staniszewska Chakarova Christina Marni J Falk NMNAT1 Nature Ge Nature Received 12 February; accepted 29 June; published online 29 July 2012; Department Department of Pediatrics, Division of Human Genetics, The Children’s Hospital of Philadelphia and University of Pennsylvania Perelman School of Medicine,

Srimati Srimati Kanuri Santhamma Centre for Diseases, Vitreoretinal LVPEI, Kallam Anji Reddy Campus, LV Prasad Marg, Hyderabad, India. rate-limiting homozygous 1

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, Xiaowu , Gai Xiaowu 2 These These authors jointly directed this work. should Correspondence be addressed to E.A.P. ([email protected]). 17 These etics NMNAT1

Center Center for Biomedical Informatics, Loyola University Chicago Health Sciences Division, Maywood, Illinois, USA. .

disease-associated

rare Fondation Ophtalmologique Fondation Adolphe Ophtalmologique de Rothschild, Paris, France. Two-thirds

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Université Université Pierre et Marie Curie (UPMC Paris 06), Unité Mixte de Recherche (UMR)_S 968, Institut de identified

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activity. 16 Institute Institute of Ophthalmology, University College of London, London, UK.

, Lakshmi Palavalli , Lakshmi

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Kallam Kallam Anji Reddy Molecular Genetics Laboratory, LV Prasad Eye Institute (LVPEI), 5 15 , , Chitra Kannabiran 18 Department Department of and Biostatistics Epidemiology, University of Pennsylvania etrs osset ih uoiioi ( mucolipidosis with consistent clinical exhibited features but development a normal had and who exam (IV-2), eye brother normal 8-year-old her in and (IV-1) 11-year-old proband the in present was loss Severe-to-profound hearing facies. sensorineural bilateral dysmorphic and hypermobility joint no delay, develop mental global had also children Both details). clinical additional (see infancy early in testing (ERG) phy (<10 detectable Clinic no and LCA nystagmus of congenital impairment, evaluation vision severe of for Ophthalmology-Genetics because Philadelphia of the Hospital to Children’s the at came initially tively), ( brother genes. LCA-causing known in mutations have not did who LCA with affected children five including family, we used whole-exome sequencing in a large consanguineous Pakistani inherited retinal disease–associated genes, the genetic etiology etiology retinal disease inherited genetic with individuals the of 40–50% in genes, uncertain remains disease–associated retinal inherited cess of clinical gene therapy trials for for trials therapy gene clinical of cess the guide to suc by recent the as highlighted essential therapies, of potential is development diseases retinal inherited of basis genetic (ref. 1p36 the chromosome to as mapped such locus LCA9 identified, been yet not have genes disease-associated Académie Académie des de Sciences–Institut France, Paris, France. 7 8 , Christina , Zeitz Christina – Two Pakistani siblings, an 11-year-old girl and her 3-year-old 3-year-old her and girl 11-year-old an siblings, Pakistani Two 11 3 , 17 Department Department of Ophthalmology, Ocular Genomics Institute, Massachusetts 6 7 , Saddek , Mohand-SaidSaddek (RetNet). Additional loci have been identified at which which at identified been have loci Additional (RetNet). , 1 18 i. 1 Fig. , , Anthony T Moore 11 1 , , Rui Xiao Centre Centre Hospitalier National des d’Ophtalmologie Quinze-Vingts, µ a n V) retinal function by full-field electroretinogra full-field by function retinal V) fml 07 sbet I- ad V3 respec IV-3, and IV-1 subjects 047, family , verbal autism with stereotypies, hypotonia with with hypotonia stereotypies, with autism verbal 5 Department Department of Biochemistry and Biophysics, University 8 – 6 10 , Zoe , Fonseca-Kelly Zoe 15 , Arundhati Dev , Borman Arundhati Dev , , Shomi S Bhattacharya 7 12 8 22 , 12 – Moorfields Eye Hospital, London, UK. These These authors contributed equally 11 RPE65 , 19 , , Naushin H Waseem 14 , , Eliot L Berson Supplementary Note Supplementary 8 Center Center for Biomedical Institut Institut National de la Santé upeetr Note Supplementary -related LCA -related 3 4). Identifying the the Identifying 4). 19 , 4 s r e t t e l Great Great Ormond ,

7 7 8– , , 16 12 1 2 4 , , . Here, Here, .

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© 2012 Nature America, Inc. All rights reserved. 132, 1000 Genomes Project data and National Heart, Lung, and and Lung, Heart, National and data Project Genomes 1000 132, 1 Fig. ( criteria these met that genes 86 in variants mous nonsynony 113 of total a Weidentified inheritance. LCA of likely mode most the be to postulated was acuity visual normal with ling sib their by not but children affected both by shared was that ance inherit of mutation a consanguinity, known biparental homozygous 11-year-old ( the proband of family nuclear the of sequencing whole-exome ( kindred larger the in phenotype loss hearing the segregated with GJB2 in p.Trp24* encoding mutation homozygous the genes LCA-causing known ( the of any in no but IV-1, identified was subject in mutation loss hearing sensorineural of cause the as in mutation homozygous a testing identified diagnostic genetic Clinical (IV-6). LCA isolated with child nonverbal IV-7 one IV-8) and and and (subjects proband the as vision phenotypes autism similar with children two had together and ( another one married had siblings ( normal tally developmen and visually were who cousins first were parents Their in shown are traces sequence Sample type. wild +, (p.Glu257Lys). of sequencing Sanger by shown was LCA with proband of sequencing ( lane). last the in shown products mutant and wild-type digested of (sizes sequences 2 of exon of amplification PCR by genotyped were Members ( individuals. deceased indicate Slashes number. unknown of individuals multiple N indicates and gender, unknown of individuals represent Diamonds gender. a given of offspring multiple indicate symbols within Numbers individuals. affected indicate symbols M1, sequencing. Sanger by validation with sequencing whole-exome by LCA with children five in identified were NMNAT1 1 Figure s r e t t e l  data (ESP) Project Sequencing Exome (NHLBI) Institute Blood Supplementary Note Supplementary b III a II IV I To search for the genetic cause of LCA in this family, we performed NMNAT1 Family 047 ). Four of these variants were rare or novel according to were rare dbSNP or ). novel variants Four according of these

IV-2 . . ( Pedigrees of three unrelated kindreds with LCA in which mutations were identified in identified were mutations which in LCA with kindreds unrelated three of Pedigrees Fig. 1 Fig. a ) Family 047. Consanguineous Pakistani kindred in which homozygous homozygous which in kindred Pakistani Consanguineous 047. ) Family 3 mutation (c.25G>A, p.Val9Met); M2, M2, p.Val9Met); (c.25G>A, mutation NMNAT1 IV-3 Fig. 1 Fig. M2/M M1/M M1/+ III-3 IV-1 Mucolipidosis typeIII LCA, congenitaldeafness,globaldevelopmentaldelay,autism a , subjects IV-1, IV-2, IV-3, III-4 and III-5). Given Given III-5). and III-4 IV-3, IV-2, IV-1, subjects , M 2 1 III-4 a , subjects III-4 and III-5). Notably, the parents’ parents’ the Notably, III-5). and III-4 , subjects M2/+ M1/+ III-4 to harbor compound heterozygous mutations c.196C>T (p.Arg66Trp) and c.709C>T (p.Arg237Cys). ( (p.Arg237Cys). c.709C>T and (p.Arg66Trp) c.196C>T mutations heterozygous compound harbor to IV-2 ). Additional sequencing analyses verified that verified analyses ). sequencing Additional +/+ 2/M2 IV-1 III-5 M IV-3 +/+ 1/M1 III-6 M2/+ M1/+ III-5 Deafness M2/M2 M1/+ Fig. 1 Fig. IV-4 III-3 GJB2 a M1/+ IV-5 +/+ , subjects III-3 and III-6) III-6) and III-3 subjects , IV-4 (c.71G>A, p.Trp24*) p.Trp24*) (c.71G>A, LCA, globaldevelopmentaldelay,autism M1/M1 GJB2 IV-5 IV-6 +/+ NMNAT1 Supplementary Supplementary mutation (c.71G>A, p.Trp24*). A representative sequence trace for M1 is shown. Colored Colored shown. is M1 for trace sequence A representative p.Trp24*). (c.71G>A, mutation IV-6 M1/M M2/M2 IV-7 NMNAT1 Fig. 1 Fig. M1/+ III-6 M 1 to harbor compound heterozygous mutations c.205A>G (p.Met69Val) and c.769G>A c.769G>A and (p.Met69Val) c.205A>G mutations heterozygous compound harbor to IV-7 M2/M2 IV-8 c 1/M1 a and and N ). , and PCR products were digested with AcuI to distinguish wild-type and mutant mutant and wild-type distinguish to AcuI with digested were products PCR , and 13 IV-8 , 1 LCA 4 d - - - - . . N WT M1: c.25G>A(p.Val9Met) G as described below. described as LCA, non-syndromic with individuals in identified were mutations additional and alone deafness has IV-2 individual alone, from etiology LCA has IV-6 individual as family, genetic this in deafness and LCA separate of that a have likely presentations These IV-1, IV-3, members IV-7in family and facies IV-8. and dysmorphic hypotonia autism, delay, nonverbal of developmental cause the to be databases any public in allele ( wild-type the only harbor vision normal with children three in generation III carry both the mutant and wild-type alleles, and their children these of parents unaffected IV,four the generation whereas in LCA with children five the in detected was NMNAT1p.Val9Met ( one LCA the isolated with including cousins, affected similarly proband’s the in and kindred nuclear original the in phenotype LCA the with segregation in variant c.25G>A the of sequencing Sanger in (p.Val9Met) c.25G>A programs: other and SIFT,PolyPhen-2 by function protein damage sequencing RNA analyses retina (RNA-seq) mouse from determined was which sion, expres retinal known had variants four these harboring genes The Fig. 1 Fig. A 5 N No clearly pathogenic mutations were identified that were likely likely were that identified were mutations pathogenic clearly No A NMNAT1 c b A ) Family 007. A single proband with LCA was shown by Sanger Sanger by shown was LCA with proband A single 007. ) Family ). The p.Val9Met variant was not present in 501 controls or or controls 501 in present not was p.Val9Metvariant The ). Normal Mutant T G mutations mutations G a DVANCE ONLINE PUBLICATION ONLINE DVANCE Fig. 1a Fig. 1 c 5 d . Only one of these variants was predicted to to predicted was variants these of one Only . 13

Family 007 , II II c.196C>T (p.Arg66Trp) I 1 c.205A>G (p.Met69Val) I Family 053 C C A p.Arg66Trp/+ 4 , NMNAT1 .

b p.Met69Val/+ A T ). Only the mutant M1 allele encoding encoding allele M1 mutant the Only ). I-1 C p.Arg66Trp/+ I-1 C A / /G T b p.Met69Val/p.Glu257Lys II-1 ) Genotyping of family 047. 047. family of ) Genotyping G G T (refs. (refs. G G G p.Arg66Trp/p.Arg237Cys II-1 d ) Family 053. A single A single 053. ) Family 62) ( 16–20) NMNAT1 T T c.769G>A (p.Glu257Lys) c.709C>T (p.Arg237Cys) A

Nature Ge Nature T T C p.Glu257Lys/+ II-2 p.Arg237Cys/+ N I-2 G/ C validated its its validated M / I-2 A A T _ NMNAT1 G 0 2 n G C 2 etics 7 8 T A 7 ). ). -

© 2012 Nature America, Inc. All rights reserved. the Massachusetts Eye and Ear Infirmary (MEEI). We found rare rare found We (MEEI). Infirmary Ear and Eye Massachusetts and the (CHOP) Philadelphia of Hospital Children’s The at evaluated we families, sequenced a UCL, University College London. CHOP, Children’s Hospital of Philadelphia; LVPEI, LV Prasad Eye Hospital; MEEI, Massachusetts Eye and Ear Infirmary; LCA-7 LCA-6 LCA-5 LCA-4 LCA-3 LCA-2 LCA-1 UC LCA-128 LCA-100 LCA-79 LCA-73 L LCA-053 LCA-007 LCA-047 CHOP/M T Nature Ge Nature consistent withahighlikelihoodthatthisvariantispathogenic. (0/1,002 chromosomes=0%; p.Glu257Lys variantwassignificantlyhigherintheLCAcases(6/568chromosomes =1.056%)comparedtobothourcontrols estimate theprobabilitythatp.Glu257Lys variantcausesdisease.Thisanalysis showedthattheallelefrequencyfor positive andnegativeratesfrequentlydonotagreewithoneanother to damageproteinfunctionbyPolyPhen-2orSIFT. However, itisknownthatthesepredictionprogramshavesignificantfalse associated withLCA(estimatedprevalenceof1in30,000) d score: PrD,probablydamaging;PoD,possiblyB,benign. Data fromExomeVariant Server, numberoftimesvariantdetected/numberexomesanalyzed. The p.Glu257Lys variantinNMNAT1 wasdetectedinatotalofsixfamilies.Althoughthisvariantissufficientlyraretobe VP able able 1 To determine whether whether determine To L E I EE

Identified Identified I n (European descent) British (European descent) British Polish Caribbean, Caribbean Caribbean, European Indian Indian Indian Indian African American Asian American Pakistani etics Irish Sri Lankan descent nationality Ancestry or

NMNAT1 ADVANCE ONLINE PUBLICATION ONLINE ADVANCE

P NMNAT1 =0.002)andESPsamples(13/10,758chromosomes0.121%;

NMNAT1 mutations c.769G>A c.466G>C c.769G>A c.552A>G c.769G>A c.59T>A c.769G>A c.723delA c.293T>G c.37G>A c.293T>G c.161C>T c.769G>A c.205A>G c.215T>A c.565delG c.709C>T c.98A>G c.25G>A c.769G>A c.205A>G c.709C>T c.196C>T c.25G>A DNA in 56 unrelated probands with LCA mutations cause LCA in other other in LCA cause mutations

Alterations

3 p.Glu257Lys p.Gly156Arg p.Glu257Lys p.Ile184Met p.Glu257Lys p.Ile20Asn p.Glu257Lys p.Pro241Profs*45 p.Val98Gly p.Ala13Thr p.Val98Gly p.Ala54Val p.Glu257Lys p.Met69Val p.Leu72His (homo) p.Ala189Leufs*25 p.Arg237Cys p.Asp33Gly (homo) p.Val9Met (homo) p.Glu257Lys p.Met69Val p.Arg237Cys p.Arg66Trp p.Val9Met (homo) 3 accordingtoESPdata(13/10,745=0.12%),itisnotpredicted Protein c SIFT: D,damaging;T, tolerated;N/A,notapplicable. 34

,

3

d

5 . We therefore

13/10,745 Novel 11/10,747 1/10,757 Novel 1/10,757 13/10,745 Novel Novel 1/7,019 Novel Novel Novel 13/10,745 Novel 1/7,019 Novel Novel 13/10,745 Novel 13/10,745 Novel 13/10,745 Novel

EVS employed Fisher’s exacttestto

a b PolyPhen-2 Hum-Var

photoreceptors leads to photoreceptor cell degeneration cell photoreceptor to leads photoreceptors disc is ~1.75 mm in diameter. in mm ~1.75 is disc optic The retina. peripheral the in clumping pigment scattered and (arrow) macula the in changes atrophic disruption, pigment vessels, blood retinal the of attenuation disc, optic the of pallor showing LCA-100, LVPEI family NMNAT1 2 Figure development normal embryonic lethal, whereas heterozygous neur have to shown been has and factor, multiubiquitination Ube4b the (Wld component cipal of the mouse Wallerian protein fusion degeneration DNA and metabolism cell signaling NAD nuclear the in involved NAD regenerates mitochondria, respectively NMNAT1, with NMNAT2 compartments, and NMNAT3 localizing to cellular the nucleus, Golgi complex and distinct within identified NMNAT mammalian by genes have different encoded isoforms been (NMN) ( (NaMN) and in a salvage pathway from nicotinamide mononucle P =0.0002),whichis PolyPhen-2 o s protective activity protective ), which also includes a 70-residue N-terminal sequence from from sequence N-terminal 70-residue a includes also which ), PoD PoD PoD N/A PrD PrD PrD PrD PrD PrD PrD PrD PrD PrD PrD PrD PrD

B B B B B B . Composite fundus image of the right eye of subject II-1 from from II-1 subject of eye right the of image fundus . Composite Retinal image from individual with LCA due to mutations in mutations to due LCA with individual from image Retinal Supplementary Fig. 3 Supplementary

b

SIFT N/A

D D D D D D D D D D D D D D T T T T T T T T +

for cellular energetics, whereas NMNAT1 is is NMNAT1 whereas energetics, cellular for c

2 4 . Loss of of Loss . and and ( LCA with families additional 11 in com in or mutations heterozygous pound homozygous identified analyses at and University College London (UCL). These ascertained India LVPEI in Paris, in Vision la de Institut probands additional 228 included analysis This groups. ancestry ing populations of affected additional individuals from vary screened we LCA, in mutations samples. control 501 in identified not were subjects these in identified variants ( the proband was a 20-year-old man with LCA which in 053, family in disease with regated in variants heterozygous and ( disease the of history family no was a was there and LCA proband isolated with girl the 5-year-old which in 007, in family mutations NMNAT1 heterozygous compound pathway from nicotinic acid mon NAD generates that ( lesions cated that the majority have atrophic macular ( LCA of cause NMNAT1 in whom for individuals information clinical available of Review samples. control 501 in subjects were identified by Sanger sequencing the of ( SIFT and/or PolyPhen-2 by damaging be to predicted were variant, the with p.Glu257Lys the all, encoding one of and exception rare, are detected tions 2 i. 1 Fig. 3 . Homozygous . Homozygous 5 To investigate the frequency of of frequency the Toinvestigate NMNAT1 , 2 Supplementary Fig. 2 Fig. Supplementary 2 Table + . The mitochondrial isoform, NMNAT3, homeostasis that is necessary for both for both necessary is that homeostasis NMNAT1 d ) 2 Fig. Fig. and and 1 nmnat mutations were identified as the the as identified were mutations that segregated with disease in in disease with segregated that . Three functionally nonredundant . Three functionally 1 5 ). We also identified compound compound identified Wealso ). encodes a rate-limiting enzyme enzyme rate-limiting a encodes . . Of interest, Nmnat1 is the prin 2 ). Table Supplementary Note Supplementary in in Nmnat1 variants identified in these these in identified variants Nmnat1 Drosophila melanogaster Drosophila + 1 , both in a biosynthetic biosynthetic a in both , ). The four four The ). -knockout mice have -knockout mice are mice -knockout NMNAT1 ). All of the muta of the All ). s r e t t e l Table 2 o 5 . nucleotide nucleotide

NMNAT1 NMNAT1 NMNAT1 1 that seg that ). None None ). Table Fig. 1 Fig. ) indi ) o tide tide  1 c - - - - - ­

© 2012 Nature America, Inc. All rights reserved. tified in family 007. We observed notably lower enzyme activity activity enzyme lower notably for iden observed We were 007. that constructs family in variants with tified p.Arg237Cys and cotransfected p.Arg66Trp the cells both from purified recombinant protein in activity NMNAT1 measured we protein in involved multimerization, is that NMNAT1 of related region be a in could location its alteration to p.Arg237Cys the of effect pathogenic interactions protein 234–238 these in acids participate amino that and homo-oligomers NMNAT1 that functional forms observed been has It disease. causes mutation this Wilcoxon 41.1–90.1; pro range test wild-type rank-sum interquartile of that reduction, than (18.9% lower tein marginally only was mutant cells mIMCD3 and CHO in ( localization nuclear and expression ( mutant this of version Flag-tagged the of purification effective achieve to able not range test rank-sum Wilcoxon interquartile 0.01–0.11; reduction, median also (99.5% was lower mutant p.Arg66Trp significantly the of activity test enzyme rank-sum The Wilcoxon 0.0015). 31.4–88.7; reduc range median interquartile (63.4% day tion, same the on protein than lower wild-type of that significantly and NMNAT1 reproducibly the was of variant activity p.Val9Met protein the days, experimental between ( proteins NMNAT1 recombinant purified mutant and of wild-type activity NAD biosynthetic the measured therefore We function. enzymatic NMNAT1 of p.Val9Met,be on the might variants p.Arg66Trp and p.Arg237Cys effect deleterious the that we postulated NMNAT1 mutant proteins, ( 047 of a fibroblast cell line obtained from the proband with LCA in family nucleus the to localized p.Val9Met correctly the mutant addition, In ( cells NMNAT1heterologous in proteins recombinant of expression following levels expression normal and localization nuclear correct showed proteins mutant these of three protein structure and stability by several prediction programs protein ( protein. These altered residues are located in regions conserved of the NMNAT1 the on p.Arg237Cys and p.Arg66Trpp.Val9Met, variants and acid, nicotinic to exposed NAD on effect no had but cells control in content ( cells wild-type in that to relative 16% by decreased was LCA with proband the from in as fibroblasts mutant and control same the in (two-tailed NMNAT NAD cellular total the of 27% had cells mutant The NMNAT1 variant. p.Val9Met the for homozygous was who 047) family IV-1 from (subject LCA with a proband and control a healthy from fibroblasts of extracts whole-cell in measured was activity NMNAT enzyme ( activity NMNAT1no had enzyme ( NMNAT1 activity wild-type ( activity) NMNAT1 wild-type of (37% activity enzyme lower significantly had mutant ( activity NMNAT1no had enzyme mutant p.Trp169Ala The variable. analysis the of values maximum and minimum the to extend box the from issuing lines vertical and mean, the represents cross interior the median, the 25 the represents box the of length The measured. and generated independently were that preparations protein replicate from measurements activity show plots Box measured. were NMNAT1 proteins recombinant purified ( 3 Figure s r e t t e l  combined the in shown) not data rate; control wild-type of (18% a Supplementary Fig. 5 Fig. Supplementary ) The NAD ) The Given the normal nuclear localization and expression of the the of expression and localization nuclear normal the Given missense novel the of effects deleterious potential We the assessed Fig. 1 Fig. th to 75 to

n Supplementary Fig. 4 n NMNAT1 enzyme activity and cellular NAD cellular and activity NMNAT1 enzyme = 6; = 6; = 7; = 7; t a test test + , subject IV-1, , and subject biosynthetic activities of wild-type (WT) and mutant mutant and (WT) wild-type of activities biosynthetic th P interquartile range, the interior horizontal line represents represents line horizontal interior the range, interquartile P = 0.0014), as was previously reported previously was as = 0.0014), P P = 0.0015). The p.Arg237Cys mutant had 81% of 81% had mutant p.Arg237Cys The = 0.0015). Supplementary Fig. 6 Fig. Supplementary = 0.016; = 0.016; 004 ( 0.034) = Fig. 3 Fig. ). The enzyme activity of the p.Arg237Cys p.Arg237Cys the of activity enzyme The ). n n = 6; = 6; = 6 for wild-type, wild-type, = 6 for a n i. 3 Fig. ). Despite variability in enzyme rates rates enzyme in variability Despite ). = 4 for mutant cells exposed to nicotinic acid. In In acid. nicotinic to exposed cells mutant = 4 for ) and are predicted to damage NMNAT1 Supplementary Fig. 5c Fig. Supplementary n P = 6; = 6; = 0.034). The p.Arg66Trp mutant mutant p.Arg66Trp The = 0.034). a ), raising the question of how how of question the raising ), P P = 0.0014), although we were were we although 0.0014), = = 0.0014). * = 0.0014). ), despite its clearly normal normal clearly its despite ), 5 Supplementary Fig. 5a Fig. Supplementary . To evaluate whether the the whether Toevaluate . b n + at baseline and after exposure to 10 mM nicotinic acid (NA) for 24 h. NAD h. 24 for (NA) acid nicotinic mM 10 to exposure after and baseline at = 5 for mutant cells). ( cells). mutant = 5 for content in the mutant cells. cells. mutant the in content + content. content. 3 P 2 < 0.05; ** < 0.05; . The p.Val9Met p.Val9Met . The ) 2 2 .

26 , 2

P 7 < 0.01; determined by non-parametric Wilcoxon rank-sum test. ( test. rank-sum Wilcoxon non-parametric by determined < 0.01; . . All P ,

b = ). ). + c - - -

) Cellular NAD ) Cellular blast cells of the proband with the p.Val9Met alteration ( alteration NMNATacti enzyme cellular total lower p.Val9Met IV-1) 73% showed subject the with proband the of cells blast protein from preparation. Additional studies using the extracts fibro unrelated probands with LCA, we identified mutations of in 14 unrelated sequencing Sanger subsequent and LCA with kindred Pakistani in a consanguineous sequencing exome Through communication). personal Inglehearn, Toomes C. and (C. origi was study this in disease analysis for available not was which locus to this in linked nally family the although locus, LCA9 to the linked previously disease for basis pathogenic likely the are and isoform NMNAT an with ciation activity. NMNAT enzymatic cellular fibroblasts in mutant deficiency substantial a have p.Val9Met they that evidence NMNAT1 further provides in content NAD+ the ( LCA with proband the two-tailed from fibroblasts the in content (two-tailed acid cells control NAD nicotinic cellular whereas total the Notably, increased h. 24 significantly for acid nicotinic mM to 10 exposure by altered was IV-1) subject (from mutant p.Val9Met lular NAD to conversion its for NAD activity NMNAT requires which acid, tinic NAD cellular total affect to sufficient be may tion in by NMNAT1caused the p.Val9Met activity enzyme alteration (two-tailed controls, type was this significant difference although not statistically NAD less 16% had LCA with proband the from total NADcellular affected protein significantly NMNAT1 nuclear-localized the in p.Val9Met the alteration whether LCA ( with proband the from line determinants buting the of LCA. of function phenotype the investigate retinal to isolated the given cells, retinal in interest NMNAT1 proteins mutant of be will It activity. enzyme NMNAT1 reduced significantly to part, in least at related, are mutations of these effects pathogenic the that suggest data These vity (two-tailed a n = 7 for both cell lines without treatment, treatment, without lines cell both = 7 for In summary, we report here the first instance of disease asso disease of instance first the here report we summary, In NAD Cellular NAD total The NMNAT activity (% of WT

b average on same sample day) and and 100 + 20 40 60 80 P ( 0 = 0.067). Nicotinic acid exposure significantly increased NAD increased significantly exposure acid Nicotinic = 0.067). Supplementary Fig. 3 Fig. Supplementary c

+ WT + + , data are shown as the mean with standard error; * error; standard with mean the as shown are , data t levels. Total cellular NAD Total cellular levels. t

concentration in the expressing fibroblasts the NMNAT1 W169A test test test ** + t

a V9M test P ** P + + DVANCE ONLINE PUBLICATION ONLINE DVANCE concentrations can be directly increased by nico by increased directly be can concentrations > 0.05). The inability of nicotinic acid to increase increase to acid nicotinic of inability The 0.05). > = 0.067; + R66W concentration of human cells has many contri many has cells human of concentration ** + P 21 R237C = 0.016) relative to control wild-type ( , 2 + * 8 . We measured NAD Wemeasured . t test test Fig. 3 b Total cellular NMNAT P )

2 activity (nmol per minute = 0.021), it had no effect on NAD on effect no had it 0.021), = c 9 ). ). These data suggest that the reduc 5 Fig. 1 Fig. . We therefore asked whether cel whether asked Wetherefore . per milligram protein) . . + 0. 0. 0. 0. content was quantified by HPLC HPLC by quantified was content NMNAT1 2 4 6 8 0 + synthetic activity of control cells cells control of activity synthetic WT a NMNAT1

, subject IV-1) to determine IV-1) to , determine subject V9M n * = 6 for control cells cells control = 6 for + content in fibroblasts fibroblasts in content + c mutations cause LCA LCA cause mutations + content. + in the fibroblast cell cell fibroblast the in + + Cellular NAD content

+ content. Fibroblasts content by 53% in in 53% by content

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© 2012 Nature America, Inc. All rights reserved. Hyderabad Eye Research Foundation (C.K.); a senior research fellowship from Government of India and the Champalimaud Foundation, Portugal (C.K.); the (UL1-RR-024134) (M.J.F. and E.A.P.); the Department of Biotechnology, the and Translational Research Center at the Children’s Hospital of Philadelphia Metabolic atDisease the Children’s Hospital of Philadelphia (M.J.F.); the Clinical the Angelina Foundation Fund from the Division of DevelopmentChild and for Excellence from the Children’s Hospital of Philadelphia (M.J.F. and X.G.); the Loyola University Stritch School of Medicine (X.G.); the Foerderer Award and X.G.); the Institutional Fund to the Center for Biomedical Informatics by Silbermann Foundation (E.A.P.); the Penn Genome Frontiers Institute (E.A.P. E.L.B., S.S.B., Q.L., A.T.M., D.S.M., E.A.P., J.-A.S., S.M.-S. and A.R.W.); the Rosanne coreInfirmary support)); the Foundation Fighting Blindness USA (I.A., A.D.B., (M.J.F.P30HD026979 and andR.X.) P30EY014104 (Massachusetts Eye and Ear (RO1-EY12910 (E.A.P.), R03-DK082446 (M.J.F.), (E.N.-O.),R01-GM097409 this study. grateful to the individuals with LCA and their relatives for their participation in tissue culture and J. Baur for his helpful discussions on NAD Laboratory for assistance with the establishment of the fibroblast cell lines and technical assistance, The Children’s Hospital of Philadelphia CytoGenomics We thank M. Sousa, D. Harnley, M.-E. Lancelot and A. Antonio for their excellent Note: Supplementary information is available in the the NCBI Read Sequence Archive (SRA) under accession codes. Accession the pape the of in version available are references associated any and Methods Me g h Network, (ESP), URLs. for strategy therapeutic a offer may cells NAD cellular restoring at directed therapies diseases retinal inherited of heterogeneity genetic the underscoring further LCA, of case, NAD of neuroprotective effect of NMNAT1 or a previously unappreciated role in mutations by caused degeneration retinal whether determined be to NAD its of independent as the of protein chimeric portion NMNAT1 active enzymatically an and component neur of Wld the studies from ings NAD with individuals in observed phenotype degeneration data retinal the The that suggest presented phenotype. retina-specific a to lead protein biosynthetic NAD expressed widely a in mutations how of question intriguing data. these by overestimated be to likely is genes, the proportion of all LCA caused bycases LCA-causing known in mutations without subjects for enriched are studies these for used individuals of cohorts the because However, suggests work This cases). in mutations unrelated that of 4.9% = (14/285 families Nature Ge Nature A e t cknowledgments t n This This work was supported by grants from the US National Institutes of Health The identification of of identification The Drosophila p NMNAT1 t o : o / m ho + NMNAT1 / protective effect of the Wld the of effect protective biosynthetic activity. This hypothesis is consistent with find with consistent is hypothesis This activity. biosynthetic Exome Variant Server, NHLBI Exome Sequencing Project Project Sequencing Exome NHLBI Server, Variant Exome http w e . + w u ds -mediated signaling in retinal health and disease. In either either In disease. and health retinal in signaling -mediated c w h : s / n . t c / 1 t e etics results primarily from the loss of a potentially novel novel potentially a of loss the from primarily results . p 0 e , , v s d 0 s nmnat mutations represent a new pathophysiological cause cause pathophysiological new a represent mutations 3 : . u 0 / , g 6 / 3 Exome sequence data for family 047 is available at available is 047 family for data sequence Exome g / s . We postulate that pharmacologic and/or genetic genetic and/or pharmacologic that Wepostulate . s NMNAT1 0 .

. e p . However, it seems that, in some systems, such such systems, some in that, seems it However, . w r n . h ADVANCE ONLINE PUBLICATION ONLINE ADVANCE a o . NMNAT1 alone has a neuroprotective role that may be be may that role neuroprotective a has alone s u m h t NMNAT1 i h + e n biosynthetic activity biosynthetic s . g t . o m t o are a relatively common cause of LCA of cause common relatively a are s r protein in mice, which showed that the that showed which in mice, protein c n g . . / e e ; UCSC Genome Browser, Browser, Genome UCSC ; mutations results from decreased decreased from results mutations d d u u as an LCA-causing gene raises the the raises gene LCA-causing an as s protein required both the Ube4b Ube4b the both required protein / / r E e V t n S e / ; RetNet Retinal Information t NMNAT1 / online version of the pape ; 1000 Genomes Project, Project, Genomes 1000 ; + homeostasis in retinal retinal in homeostasis 25 NMNAT1 , 3 + 1 metabolism. We are -related LCA. -related . Thus, it remains remains it Thus, .

S R mutations P 0 h 1 online online r .

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Q.Z., E.N.-O., J.O., Q.L. and M.S. The manuscript was written by M.J.F., Q.Z., E.N.-O., M.J.F., E.A.P. and R.X. assay development was performed by E.N.-O., with data analysis performed by chromatography NMNAT(HPLC)-based enzyme activity assay and NAD M.C., Z.F.-K., D.S.M., L.P., N.H.W.,R.S., C.Z. and Q.Z. High-performance liquid NMNAT1 M.C. Exome data analyses were performed by J.C.P., X.G., Z.F.-K. and E.A.P. Bioinformatics pipeline development was performed by X.G., M.J.F., E.A.P. and evaluated clinically by the authors). Exome sequencing was performed by M.C. evaluated by M.J.F. and E.A.P. (individuals III-3, III-6 and IV-4 to IV-7 were not C.K., S.J. and E.P. Individuals III-4, III-5, IV-1, IV-2 and IV-3 were clinically E.P., S.M.-S., J.-A.S., A.R.W. and E.A.P. Pedigrees were compiled by A.D.B., C.C., and controls were provided by I.A., A.D.B., E.L.B., S.S.B., C.K., M.J.F., S.J., A.T.M., Experiments were bydesigned Q.Z., M.J.F., X.G. and E.A.P. LCA case samples interpretations or conclusions. Department of Health, which disclaims responsibility for any analyses, by the Penn Genome Frontiers Institute under a grant with the Pennsylvania organizations or the National Institutes of Health. This project is funded, in part, authors and does not necessarily represent ofviews the official the funding A.T.M., D.S.M. and A.R.W.). The content is solely the responsibility of the and A.R.W.); and the TrusteesSpecial of Moorfields Eye Hospital (A.D.B., S.S.B., Research Council (BRC) for (A.D.B.,Ophthalmology S.S.B., A.T.M., D.S.M. Moorfields Eye Hospital National Institute of Health Research (NIHR) British (UK)(A.R.W.); Fight For Sight (UK) (A.D.B., S.S.B., A.T.M., D.S.M. and A.R.W.); Entendre, Ville de Paris and Région Ille de France (C.Z.); RP Fighting Blindness the Council for and Scientific Industrial Research the (R.S.); Foundation Voir et 17. 16. 15. 14. 13. 12. 11. 10. 9. 8. 7. 6. 5. 4. 3. 2. 1. reprints/index.html. http://www.nature.com/ at online available is information permissions and Reprints Published online at The authors declare no competing interests.financial X.G. and E.A.P. AUTHOR CONTRIBUTIONS COMPETING FINANCIAL INTERESTS FINANCIAL COMPETING

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27 ,

© 2012 Nature America, Inc. All rights reserved. liquid nitrogen for HPLC analysis. HPLC for nitrogen liquid in frozen flash and (Gibco) solution salt Hank’s with balanced twice washed for 24 h and were trypsinized, (Sigma) acid to 10 mM nicotinic were exposed grown to confluence before undergoing functional analyses. Cells in T75 flasks l CO per standard protocol, in DMEM supplemented with 20% (Gibco) FBS, 2 °C mM 37 a in flasks T75 in maintained subsequently were and Pennsylvania of Children’sHospital the at Laboratory CytoGenomics the in Hospital of Philadelphia (08-6177, M.J.F.). Fibroblast cell lines accordance were established in obtained was of board the Children’s review approved the protocol with by the institutional consent informed after III-5) and III-4 IV-3, on with two LCA siblings performed and parents ( their Human fibroblast culture and exposure to acid. nicotinic sequence. mutant the not but type wild- the cuts which AcuI, with digested was product PCR relevant the 047, or 3100 ABI an 3730 DNA analyzer on (Applied Biosystems). To kit genotype the members of family Reaction Ready V2.0 Sequencing Cycle Terminator 1 of Table boundaries intron-exon amplify and to exons primers four using all analysis sequence Sanger for selected were LCA tion. NMNAT1 models. genetic as such criteria, ing filter different fit that variants candidate to identify and used developed also scores conservation phastCons and predictions, and SIFT PolyPhen frequencies, allele include annotations These URLs). (see Browser Genome UCSC the Exome and NHLBI Server Variant Exome the Project website, Sequencing Project Genomes 1000 sets the data from using provided downloaded were call variant each of annotations any. if Additional frames, translation and acids amino encoded codons, genes, scripts, tran corresponding its to annotations gene 65 Release on Ensembl of genome basis the reference human the in database. position base codon each maps base-pair database This human custom our variant using Resulting annotated were homozygous. calls called be to 0.75 above and called be to 0.75 heterozygous and 0.25 between be to had base variant a of fraction The bases all of values quality using performed was correction Benjamini-Hochberg after position program custom The at a candidate counts base raw calls. adjust to approach rate indel discovery and a false used SNP the refine further to used and deletion and/or command) pileup the (with calls insertion (indel) and SNP initial make to and the (with command) duplicates rmdup potential remove to used was r859) or 0.1.12 (version URLs) (see website Project from the Genomes 1000 downloaded was used to the align reads sequence to the human reference genome GRCh37 analyses. data Exome exome. targeted the of 92–95% for depth read 10× of minimum a obtain to densities of ~800,000/mm Sequencing system using v2.5 SBS chemistry, with average Next-Generation flow-cell 2000 lane HiSeq cluster Illumina an on sequencing paired-end 101-bp manual kit the with accordance in Technologies Agilent from kit Enrichment Targeted Exon All Exome sequencing. Children’s Philadelphia. at of the Hospital Clinic Ophthalmology-Genetics IV-1, (subjects 047 family of IV-1 and IV-3), family members 007 II-2) (subject and family 053 were at performed of the evaluations genetic clinical and ophthalmic Complete participants. the from obtained was consent Informed Helsinki. of Declaration the of tenets the to conformed and London, College V, France de University and Ile LVEye Hospital (CDP) the Prasad Personnes des Protection de Comité Philadelphia, of Children’s the Hospital Infirmary, evaluations. approved clinical by boards of review the institutional The Eye Massachusetts and Ear and recruitment Subject O doi:10.1038/ng.2361 -glutamine, 1 mM pyruvate and 50 50 and pyruvate mM 1 -glutamine, NLIN Subjects in families 007 and 047 and probands from other families with with families other from probands and 047 and 007 families in Subjects ). PCR products were sequenced with the ABI PRISM BigDye BigDye PRISM ABI the with sequenced were products PCR ). E E PCR amplification, Sanger sequencing and genotype confirma genotype and sequencing Sanger amplification, PCR M E T HO 3 Exome capture was performed using the SureSelect 50 Mb 3 9 6 DS . A coverage depth cutoff of 10× was then applied. applied. then was 10× of cutoff depth coverage A . . The resulting exome-capture libraries underwent 2 × 2 underwent libraries exome-capture resulting The . Burrows-Wheeler Aligner (BWA, version 0.5.9-r16) 0.5.9-r16) (BWA,version Aligner Burrows-Wheeler 2 . . One exome sample was analyzed per flow-cell lane µ g/ml uridine (Calbiochem). Cells were were Cells (Calbiochem). uridine g/ml 3 8 . A custom program was developed developed was . program A custom NMNAT1 16 The clinical study was was study clinical The , 1 7 . Custom scripts were were scripts Custom . Fig. Fig. 1 Skin biopsies were ( Supplementary Supplementary a , subjects IV-1,, subjects 3 7 2 . . SAMtools incubator, incubator, - - -

dilution)or 1:5,000 dilution), human NMNAT1 (Novus Biologicals, H00064802-B01P; 1:500 described Bis-Tris Gel (Invitrogen) and were transferred V5- to a PVDF membrane, as pr blotting. Protein microscope. fluorescent dilution) 1:1,000 Fluor Alexa with then A21127; and IgG (Invitrogen, to mouse antibody secondary goat dilution) 555–conjugated 1:1,000 46-0705; (Invitrogen, V5 to in described X-100 Triton previously 0.2% as and BSA PBS, 1% with blocked then and permeabilized analyses. Immunofluorescence immun going under before confluence to grown were and supplement growth low-serum with (Invitrogen) 106 Medium in maintained were and III-V) III-IV subjects two from immunocytochemistry obtained LCA with ( siblings cells for fibroblast skin Human processed transfection. were after h 48–72 cells and (Invitrogen), 2000 and FBS 10% supplemented with 10% FBS. Transfection was medium with performed Lipofectamine with F12 in performed was culture supplemented cell CHO pyruvate. sodium mM medium 0.5 DMEM:F12 in were cells maintained mIMCD3 (ATCC). Collection Culture Type American the from culture. Cell −80 °C and were subjected to HPLC analysis. HPLC to subjected were and °C −80 at stored were Samples estimation. protein for used was extraction PCA the from pellet The material. insoluble remove to centrifuged was and carbonate 16,000 at centrifuged then and min 15 for ice on placed was suspension the vortexing, After metabisulfite. sodium EDTA 0.15% mM and 20 containing PCA M 1.2 ice-cold argon-bubbled, of volumes NAD four with including extracted was suspension dinucleotides, cell oxidized the of analysis for Tris-HCl 7.4) mM 20 (pH argon-bubbled in resuspended was pellet cell The min. 5 2,150 at centrifuged were and twice solution salt Hank’sbalanced with Sample preparation for HPLC analyses of NAD analysis. HPLC until °C −80 K 20 of addition the by neutralized further was supernatant 16,000 at min 10 for centrifuged was mixture the °C, 4 at incubation 15-min a After reaction. the 20 to mM containing EDTAstop (PCA) acid metabisulfite and sodium 0.15% 40- a for mixture 10, 30 or of 120 min at reaction the 37 incubation °C, After strate. sub NMN of addition by started was reaction The ml. 0.2 of volume final a 40 or 20 (typically sample enzyme of amount appropriate the 1.5 mM ATP, 1 mM NAD NMN and 10 mM MgCl product, reaction the of quantitation assay. activity enzyme NMNAT assay. activity enzyme subsequent for (Sigma) in was CHO using expressed a and cells kit FLAG was purified M Purification NMNAT1 Recombinant tag) Flag kit (with maxi (Qiagen). plasmid EndoFree IRES- (pCAG-V5-IRES- V5 N-terminal for sequence a encode to modified vector expression destination Gateway-compatible a to recombination by moved was sequence coding The fully sequencing. was by construct verified the (Invitrogen); vector entry pENTR/D-TOPO a into cloned and (OpenBiosystem) clone cDNA a from RT-PCR by amplified was Recombinant protein production and purification. Antibody binding was detected with an dilutions).Odyssey infrared imager 1:15,000 (LI-COR). 926-68071; 680RD, anti-rabbit goat 926-32210; 800CW, was then incubated with IRDye secondary antibodies (LI-COR, goat anti-mouse 2 CO NMNAT1 µ EGFP 3 l aliquot was removed and added to 20 20 to added and removed was aliquot l and was centrifuged again. The supernatant was isolated and was stored atisolated The supernatant again. and was centrifuged g 4 1 for 10 min. The supernatant was neutralized with 1 M potassium potassium M 1 with neutralized was supernatant The min. 10 for . The membrane was probed with antibody to V5 (Invitrogen, 46-0705; ) epitope tags in frame. Plasmid DNA was purified using the the using purified was DNA Plasmid frame. in tags epitope ) β -IRES- -actin (Santa Cruz Biotechnology,(SantaCruz -actindilution) 1:1,000andsc-1615; CHO-K1 and wild-type mIMCD3 cell lines were purchased purchased were lines cell mIMCD3 wild-type and CHO-K1 o fluorescence and immunoblot analyses. immunoblot and fluorescence 4 Total cell lysates from CHO cells transfected with pCAG- with transfected cells CHO from lysates cell Total 0 . Fluorescent signals were visualized using a Nikon Eclipse a Nikon Eclipse using visualized were signals . Fluorescent Fig. Fig. 1 EGFP g in a Beckman microcentrifuge. A 55- A microcentrifuge. Beckman a in a plasmid were separated on a precast NuPAGE 4–12% , subjects IV-1 , subjects and IV-3) and parents ( their 4 0 Cells were fixed in 4% paraformaldehyde, paraformaldehyde, 4% in fixed were Cells . Cells were then stained with antibody antibody with stained then were Cells . NMNAT activity was measured by HPLC HPLC by measured was activity NMNAT 2 in 25 mM Tris-HCl (pH 7.4) and + . The assay mixture contained contained mixture assay The . µ l of ice-cold 1.2 M perchloric perchloric M 1.2 ice-cold of l + EGFP . Harvested cells were rinsed Human ) or Flag (pCAG-Flag- Flag or ) Nature Ge Nature µ l of ice-cold 1 M 1 ice-cold of l µ NMNAT1 l aliquot of the the of aliquot l µ l) to achieve achieve to l) n e Fig. Fig. 1 cDNA viously + etics . The The . g for for a ­ - ,

© 2012 Nature America, Inc. All rights reserved. dates. The significance of differences between groups was evaluated using using evaluated was groups between differences of significance The dates. to day account for same variation in the the absolute on enzyme activity rates on different analyzed analysis protein wild-type the of rate mean the by ized normal were rates protein, wild-type of that with NMNAT1 proteins binant analyses. Statistical curves. standard using quantified were and Shimadzu from software with Shimadzu nm SPD-M20A. Pertinent 340 peak areas and were integrated260 using at LabSolution monitored was absorbance UV min. 5 for 100% to B phase mobile increasing by separation each after washed was column The phate buffer (pH 6.0) containing 30% methanol), increasing to 50% over 15 min. concentration was linearly increased with mobile phase B (0.1 M sodium phos at M sodium phosphate (0.1 set buffer (pH A 6.0) containing 3.75% phase methanol). was The methanol mobile of rate 100% Flow initially was °C. phase 40 mobile The at ml/min. 0.5 column guard a by preceded 90Å) C18 XL (5 column C18 a on out carried was tides HPLC conditions for of analyses NAD Nature Ge Nature n etics For comparison of the activity rates of purified recom purified of rates activity the of comparison For + . Separation of the oxidized dinucleo µ m, 4.6 × 250 mm, Adsorbosphere Adsorbosphere mm, 250 × 4.6 m, - - - ­

41. 40. 39. 38. 37. 36. Student’s using two-tailed performed were groups cellular of measurements For NADand size. NMNAT activity sample small and data skewness of the in because 4.3 observed SAS in test rank-sum Wilcoxon nonparametric a

Davis, E.E. Davis, photoreceptor a is protein 1 pigmentosa retinitis The E.A. Pierce, & J. Zuo, Q., Liu, and practical a rate: discovery false the Controlling Y. Hochberg, & Y. Benjamini, H. Li, Burrows-Wheeler with alignment read short accurate and Fast R. Durbin, & H. Li, A. Gnirke, ciliopathy spectrum. ciliopathy protein. microtubule-associated (1995). 289–300 testing. multiple to approach powerful 25 transform. sequencing. targeted parallel massively , 2078–2079 (2009). 2078–2079 , t al. et Bioinformatics et al. et t al. et h Sqec AinetMp omt n SAMtools. and format Alignment/Map Sequence The

TTC21B ouin yrd eeto wt utaln oiouloie for oligonucleotides ultra-long with selection hybrid Solution Nat. Genet. Nat.

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