© 2017 Nature America, Inc., part of Springer Nature. All rights reserved. Received Received 30 September 2016; accepted 26 April 2017; published online 29 May 2017; A full list of authors and affiliations appears at the end of the paper. gene in 5,570 XFS and XFG cases and 6,279 controls from LOXL1nine countries (p.Leu141Arg) rs3825942[G>A] (p.Gly153Asp) and, to a lesser extent, rs1048661[T>G] of the allele reversals seen for common polymorphisms at at further understanding the genetic basis of the disorder. First, because genesis are essential in providing new biological leads in complex disease XFS patho underlying XFS disease biology is complex and worthy of further study. tions inconsistentare ‘flipped’allelesbeingriskowingtocertainpopula in genetic association seen at polymorphisms mappingassociated towith this disease oxidasegeneticfirstlysylreporteding be thelocushomologtowas 1, genetic contribution to disease pathology globally. blindness of cause major a represent XFG and intervention and surgical laser requiring often more treatment, medical intraocular-pressure-lowering to resistant often is it and glaucoma, of types major other than prognosis worse affected individuals 60–70 million estimated an with populations, many in common is disease also a prognostic factor for progression of open-angle glaucomaworldwide glaucoma secondary of cause common most the tissues in various material extracellular abnormal an of accumulation and progressive production excessive the by characterized is It (ECM). matrix extracellular the involving disorder systemic age-related an is XFS pathology 11 1 countries. at inconsistent genetic worldwide. Exfoliation five new susceptibility loci identifies a protective rare variant at association Genetic study of exfoliation syndrome Nature Ge Nature 8

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© 2017 Nature America, Inc., part of Springer Nature. All rights reserved. Japan Collection T ( SNP sentinel rs3825942[G>A] the at cosegregation for accounting after even function significant LOXL1 remained affect to predicted conservatively alleles of effect variant burden (OR = 0.18, by rare the conferred effect protective larger a substantially observed 2–HumDiv, LRT score, MutationTaster and Condel) (SIFT, used PolyPhen- algorithms prediction effect functional of the five all by deleterious be to predicted conservatively variants mous to the only analysis aggregate rare nonsynony test restricting second effects functional exert not did variants mous 0.46, variants in the normal controls in comparison to the XFS (OR cases = nonsynonymous rare for enrichment risk. broad a XFS to Weobserved contribute collectively could 1%) < (MAF) frequency within that rare alleles hypothesis the evaluated common reversed the from available 4 Fig. ( studied onymous across the nine variants countries sample. worldwide our across XFS with association sistent con shows that variant common ‘unflipped’ an missed have we that with locus, no exceptions ( windows. All analyzed haplotypes showed reversal of across effect the sequenced entire the SNPs across populations, we followed up our search by phasing the haplotypes of 57 consistent a have missed could analysis single-variant that Recognizing 98.3%). ( upper 95%confidencelimit. No additionalfiltersbyfunctionaleffectpredictionalgorithmwereapplied.Frequenciesforrarevariantcarriersaregivenaspercentages.L95,lower95%confidencelimit;U95, Stratified meta-analysisforallsequencedcollections Pakistan India South Africa Mexico United States Russia Italy Greece Japan Collection T s e l c i t r A  a ers aregivenaspercentages. NA,notpossibletoestimatebecause ofzerocountsincases. Variants weretaggedasdeleterious byfivefunctionalpredictionalgorithms(SIFT, PolyPhen-2–HumDiv, LRT score,MutationTaster andCondel).Frequenciesforrarevariant carri Meta-analysis excludingRussia,Mexico, SouthAfricaandPakistan Stratified meta-analysisforallsequenced collections Pakistan India South Africa Mexico United States Russia Italy Greece Excluding collectionswhere norare,deleteriousnonsynonymoussubstitutions werefoundineithercasesorcontrols. P able able 2 able 1 = 0.53) conditioning on rs3825942[G>A] ( rs3825942[G>A] on conditioning 0.53) = The The resequencing of P and and = × 4.2 10

Association Association of all rare, deleterious variants nonsynonymous at Association Association of rare variants nonsynonymous at LOXL1 Supplementary Table7 Supplementary n n 2,827 2,827 cases cases −7 383 648 116 212 476 454 355 haplotypic association that is not reversed across across reversed not is that association haplotypic 383 648 116 212 476 454 355 95 ; ; 95 Table LOXL1 Supplementary Data 1 Supplementary 1 P ). As the vast majority of nonsynony the ). As majority vast the n n = 4.23 × 10 controls controls 1,075 3,013 1,075 3,013 identified a identified total of 63 unique nonsyn 186 263 250 205 161 859 267 186 263 250 205 161 859 267 Supplementary Table 8 Supplementary LOXL1 ). Because of the limited insights insights limited the of Because ). Allele burden, Allele burden, LOXL1 −11 locus using 20-SNP sliding sliding 20-SNP using locus cases cases ; ; 11 12 10 34 Table 0 5 0 0 1 0 1 1 7 1 2 2 2 3 31– haplotypes, we next next we haplotypes, P LOXL1 het 3 ). ). It is thus unlikely 2 4 < 1 × 10 × 1 < 3 , we performed a performed we , Allele burden, Allele burden, ). ). This protective 3 Supplementary Supplementary . . In so doing, we controls controls 100 (minor allele allele (minor 85 21 17 LOXL1 ). a 4 8 9 2 5 3 3 7 2 0 1 4 3 6 −10 LOXL1 with risk of exfoliation syndrome ; ; I Carrier freq., Carrier freq., 2 = - - - - 0.39 0 0.77 0 0 0.47 0 0.22 0.28 cases cases 1.83 1.85 1.05 1.72 0.94 0.42 2.20 0.85 1.20

rs201011613[T] (LOXL1 p.407Phe) allele was observed in only 2 of the 3 ( population Japanese the in exclusively found LOXL1is and affect function to algorithms prediction function protein five all by predicted conservatively was variant This analysis. single-variant in ing LOXL1 p.Tyr407Phe, showed genome-wide significant association major axes of population stratification ( structure but found no evidence that these carriers clustered along the undergone genome-wide genotyping for the presence of populationWe subexamined individuals carrying the rare p.Phe407 allele who had (Fisher’sXFS alsoresistance25-foldto test,ferring exact hospital-matched controls with no eye disorder (0.64%) ( 3,909 XFS cases (0.026%) but was observed in 68 of the 5,338 age- and positions 141, 153 and 407) on LOXL1 function. LOXL1 on 407) and 153 141, positions acid amino (at variants nonsynonymous three all of the impact relative assess to needed be would testing biological functional phism, polymor p.Arg141Leu common the with segregate not does allele 3 Table Supplementary underwent that of collections all in the meta-analysis significant thus only nominally was common and Africans black in XFS to this susceptibility conferred but Japan discussed, in XFS from previously protection with associated was haplotype rs3825942[A] As haplotype. (p.Asp153) rs3825942[A] common the with segregated allele p.Phe407 the als, for the sequencing underwent who allele p.407Phe the carrying individuals LOXL1 , , One of the rare nonsynonymous variants, rs201011613[A>T], encod We next examined the haplotype background for all 37 Japanese Japanese 37 all for background haplotype the examined Wenext Supplementary Fig. 4a,b Fig. Supplementary Carrier freq., Carrier freq., LOXL1 with risk of exfoliation syndrome controls controls 1.61 2.66 0.80 0 0.62 0.47 1.12 0.56 2.82 2.15 3.04 8.40 4.39 1.24 0.58 1.12 1.58 3.32 locus ( a LOXL1 DVANCE ONLINE PUBLICATION ONLINE DVANCE Table Allele 0.14 0.19 0.18 0 0.29 0 0 0.76 0 0.20 0.50 0.46 0.85 0.61 0.12 0.39 0.76 0.72 1.97 0.53 0.36 OR OR resequencing (random-effects (random-effects resequencing ). Although the rare, protective p.Phe407 p.Phe407 protective rare, the Although ).

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Nature Ge Nature P = 2.9 × 10 × 2.9 = Table 1.41 ×10 4.23 ×10 0.035 0.047 0.38 1 1 0.56 0.15 1 3.49 ×10 4.2 ×10 0.76 0.32 0.014 0.34 1 1 0.39 0.44 8.03 ×10 P ). The rare The ). = 0.0039; 0.0039; = ) 3 n 35 P P ), con etics Table , 3 −7 −14 6 −10 −11 −13 . −8 - ). - - - - - © 2017 Nature America, Inc., part of Springer Nature. All rights reserved. are shown from four independent experiments. ** experiments. independent four from shown are Data values. maximum and minimum the to extend whiskers the and range, interquartile the shows box the median, the represents line middle the plot, tested four the overexpressing HLECs of nucleofection h 35 following over measured strength) adhesion cellular for surrogate ( expression). (decreased purple to expression) (increased red from staining fibrillin-1 of intensity the indicates fibrillin-1 for map heat The blue. in stained are nuclei Cell (green). fibrillin-1 to antibody and (red) LOXL1 of forms overexpressed of detection ( expression). (decreased purple to expression) (increased red from staining IV collagen of intensity the indicates IV collagen for map heat The blue. in stained are nuclei Cell (green). IV collagen to antibody and (red) LOXL1 of forms overexpressed of detection for HA to antibody with labeled HLECs in overexpressed variants LOXL1 tagged ( expression). (decreased purple to expression) (increased red from staining elastin of intensity the indicates elastin for map heat The blue. in stained are nuclei Cell (green). elastin to antibody and (red) LOXL1 of forms overexpressed of detection for HA to antibody study. this in ( evaluated variants the for positions 1 Figure burden variant rare additional an harbor GWASloci variant common some only as surprising, not is CACNA1A rare any between variant of burden consistent atevidence association and In Asians. South to contrast Africans of mous amino in acid substitutions were the sequence coding observed Japan replication Japan sequencing Collection T Nature Ge Nature were tested haplotypes Europe sequencing Japan combined South Asiasequencing South Africasequencing Fisher’s exacttests. The p.Tyr407Phe rarevariantwasfoundexclusivelyintheJapanesepopulation andwasnotpolymorphicinEurope,AfricaorSouthAsia. c a able able 3 (1–25 aa) T- G-G-A- G-A-A- G-A-T G-A- At the the At SP CACNA1A -L LO LO OX LO XL1-HA XL1-HA L1-H XL1-HA (26–94 aa) Propetide H1: rs1048661[G>T] A

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thus performed experiments assaying the effects of of effects the assaying experiments performed thus tissues connective in collagen and elastin cross-linking by lial cells to tin, the localized of surface various cell including types lens epithe containing ECM components such as and elastin, fibrillin-1 fibronec material, exfoliation termed material, fibrillar abnormal an of lation XFS is characterized by excessive production and progressive accumu Biological 25 aa API API API API cases (%) 10 10 10 10 Freq., 0.018 0.0 0.026 0.046 0.0 0.0 µ µ µ µ m m m m Fibrillin-1 heat ma Fibrillin-1 heat ma Fibrillin-1 heat ma Fibrillin-1 heat ma 1 , 38

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P < 40– variants variants T-G-A ** P – – – 0.002 4 2 . We . −14 −10 −5  - - - © 2017 Nature America, Inc., part of Springer Nature. All rights reserved. lower cell–cell adhesion, also irrespective of the p.Arg141Leu and and ( p.Arg141Leu alleles p.Gly153Asp the of irrespective also significantly adhesion, had cell–cell lower allele (rs201011613[A]) p.Tyr407 wild- baseline the type, carrying p.Gly153Asp haplotypes and the contrast, In p.Arg141Leu alleles. the background of irrespective p.Phe407 adhesion cell– protective cell physiological rare, increased the significantly of allele (rs201011613[T]) introduction the that observe ( differences adhesion for together cell–cell cloned. in constructs were haplotype six all haplotypes retested We (Leu141–Gly153–Phe407) T-G-T and (Arg141–Gly153–Phe407) G-G-T the covering constructs haplotype additional two allele, (rs201011613[T]) p.Phe407 protective rare, the to unique was adhesion cell–cell of strength the in increase the that To assay. ensure this in effect adhesion cellular of significant strength the on a have not do polymorphisms p.Gly153Asp and ( another one to binations of the p.Arg141Leu and p.Gly153Asp alleles) were compared three remaining haplotypes carrying p.Tyr407 (but with different com the when adhesion cellular of strength relative the in difference cant ( allele p.Tyr407 wild-type the carrying haplotypes three remaining the to significant increase in the strength of cellular adhesion in comparison G-A-T p.Phe407-carrying protective tional to the quality of cell attachment propor directly is readout impedance cellular in change the ology, In system. method this analysis cell real-time xCelligence the Roche of microelectrodes the using strength adhesion cellular relative for HLEC 3D spheroids overexpressing the four haplotypes were analyzed end, To this adhesion. cell–cell affecting outcome physiological a to rare the of LOXL1 effects biochemical functional observed these whether fibrillin. and elastin as such components ECM on effect This allele. result p.Tyr407 suggests that the rare p.Phe407 wild-type allele has an the overall upregulating included that haplotypes three ( levels in fibrillin-1 an increase ( levels IV collagen in decrease a in resulted also lotype cultures ( spheroid 3D of analysis immunofluorescence in recapitulated levels on levels elastin ( in immunoblots increase dose-dependent a observed we tures, (G-A-T) Arg141–Asp153–Phe407 haplotypes four the of any ( between observed be could secretion LOXL1 in difference significant no and levels, detectable at secreted that LOXL1 protein was we observed the four haplotypes, expressing transiently cultures cell 3D (HLEC) cell epithelial lens Using human allele. p.Asp153 the with segregates allele p.Phe407 protective rare, as the important is of particularly for p.Gly153Asp account the effect p.Gly153Asp haplotype combinations. The ability to p.Arg141Leu– condition on all and of effects the on conditioning of while p.Tyr407Phe effect the of ( measurement for allowed haplotypes also design occurring experimental naturally in variants three these carrying constructs four using variant p.Tyr407Phe rare, protective the as well as polymorphisms p.Gly153Asp and p.Arg141Leu readout. physiological cellular a as adhesion cell relative overall and readouts biochemical cellular as fibronectin and IV type collagen fibrillin-1, elastin, using metabolism, ECM on s e l c i t r A  7 Fig. Supplementary Supplementary Fig. 6a Fig. Supplementary As the ECM is important for cellular adhesion, we next asked asked next we adhesion, cellular for important is ECM the As p.Phe407-carrying rare the overexpressed we when contrast, In flipped common the of effects functional the assessed We P p.Phe407 allele on ECM components on components ECM allele p.Phe407 < 0.01 for all comparisons; comparisons; for < all 0.01 Fig. Fig. 1 b Supplementary Fig. 6b Fig. Supplementary ). ). Overexpression of this rare p.Phe407-carrying hap Fig. 1 Fig. P < 1 × 10 e ). ), suggesting that the common p.Arg141Leu p.Arg141Leu common the that suggesting ), upeetr Fg 7 Fig. Supplementary ). −4 Fig. 1 Fig. for all comparisons, unpaired unpaired for comparisons, all LOXL1 Fig. 1 Fig. 43– d LOXL1 ) when compared to other the ) compared when ), with the increase in elastin elastin in increase the with ), 4 5 e . . We that observed the rare, ). We observed no ). Wesignifi observed haplotype in HLEC cul HLEC in haplotype in in vitro haplotype conferred a conferred haplotype ) and continued to to continued and ) would translate translate would i. 1 Fig. Fig. 1 Fig. a LOXL1 ). This This ). c t ) and and ) test; ------

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© 2017 Nature America, Inc., part of Springer Nature. All rights reserved. coma and (vi) genes highlighted from unbiased genome-wide molec coma from genome-wide and unbiased (vi) genes highlighted glau of forms other with pleiotropy showing genes (v) PubMed, in (eQTLs) loci trait titative mice, (iii) genes whose expression is regulated by knockout in phenotypes eye-related relevant with associated genes and ciliary body,iris as determined the using publicly as available databases such tissues segment anterior in expressed genes (i) the 33 genes by determining whether these genes showed overlap with variant; index the of (inclusive variant by proxy formed the SNPs region the showing within and bp) (<100,000 regions genomic narrow relatively within were located loci significant genome-wide for seven the sets credible ( genes with SNPs proxy containing r region the as or variant index the of ‘associated region’locus as the either generically region within 150 kb to have long-range complex LD patterns, we observed that defining an complex patibility (MHC) on locus chromosome 6 that is well known the credible set analysis ( SNP sentinel the with 0.5 showing markers SNP and SNP sentinel the on centered region seven genome-widethe to significant loci (identifiedclosest using the located 150,000-bp or genomic to mapping genes 33 annotated We Biological ( regions ratio of the risk allele highest in polar regions and lowest in equatorial 13 Chr. T Nature Ge Nature information additional this summarize We analysis. pathway ular a Chr., chromosome. 11 5 3 6 This summaryincludes7,113casesand95,863controlsfromNorthAmericanorthern,southern,easterncentral–westernEurope,doesnotincludeLatinSouth America. 2 able able 4 > 0.5 with the index variant resulted in identification of the same same the of identification in resulted variant index the with 0.5 > We next assessed the potential biological contribution for each of each for contribution biological potential the Weassessed next AGPAT Supplementary Fig. 10 Fig. Supplementary s Supplementary Table 13 Supplementary rs7329408 (A/G) SNP (effect/reference) rs11827818 (G/A) rs10072088 (G/A) rs12490863 (A/G) rs3130283 (A/C) ummary ummary of genetic associations for the five newly identified loci

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) or defined by defined or ) GWAS discovery European summary All datasummary Replication summary Asian summary European summary All datasummary Replication summary European summary All datasummary Replication summary European summary All datasummary Replication summary GWAS discovery Asian summary GWAS discovery Asian summary GWAS discovery Asian summary European summary All datasummary Replication summary GWAS discovery Asian summary 50– ). Stage 5 ). All All ). 4 , , (ii) ). r 2 > - - - -

a a a a a Expression levels in these tissues did not significantly correlate correlate significantly not did tissues these in levels (retina). Expression pathophysiology glaucoma for and body) ciliary and (iris material exfoliation of synthesis the for relevant tissues in observed ( lyzed ana of tissues eye panel in the levels at moderate detected was genes six of all mRNA expression used. were male) 10 female, 11 80.1 years; 7.9 age, (mean XFS with eyes 21 and male) 21 female, 20 years; 41 normal eyes with no known ocular disease (mean age, 77.1 77.1 age, (mean disease ocular known no with eyes normal 41 appropriate with For analyses, mRNA expression Methods). (Online consent research eyes donor human from obtained tissues ocular ( 6p21 ( were 13q12 genes) (and loci These loci. associated significantly most ( loci significant significant biological pathways highlighted by the seven genome-wide regions relevant within biologically gene sets, did not anyidentify statistically genomic LD-independent for signals association enriched package software tool) analysis enrichment 12 Fig. from harbors (which for except RNAs noncoding long any harbor loci significant UCSC Genome Browser showed that none of the genome-wide seven pathways. A disease biological search broader, of the yet-undescribed implicating be could loci associated significantly the that suggesting ( loci significant genome-wide seven the between the proteins encoded by several of the 33 genes located within databases available in POMP We next studied the expression of genes associated with the three three the with associated genes of expression the Westudied next Supplementary Table 15 Table Supplementary 1.17 1.22 1.17 1.18 1.19 1.14 1.14 1.14 1.18 1.10 1.13 0.88 0.90 0.89 1.12 1.12 1.15 1.24 1.13 1.17 1.15 1.19 1.15 0.88 0.89 LOXL1 OR AGPAT1 Supplementary Fig. 13 Fig. Supplementary ). Further interrogation using the INRICH (interval-based (interval-based INRICH the using interrogation Further ). , , FLT1 ) ) and the 1.13 1.11 1.11 1.09 1.11 1.05 1.07 1.15 0.81 0.85 1.05 1.11 1.09 1.04 1.09 1.14 1.06 1.11 1.07 1.11 1.08 1.08 0.85 0.83 0.85 , , L95 Association tests ). Expression for these six genes was tested in fresh fresh in tested was genes six these for Expression ). SLC46A3 Supplementary Data 3 Data Supplementary LOXL1 5 7 FLT1 identified potential molecular interactions interactions molecular potential identified 1.29 1.22 1.25 1.22 1.27 1.19 1.20 1.18 1.25 1.16 1.19 0.96 0.94 0.93 1.20 1.20 1.22 1.34 1.22 1.23 1.24 1.27 1.22 0.94 0.94 U95 - AS1 , 12. ( 11q23.3 ), – POMP . A genome-wide search using publicly publicly using search genome-wide A . , transcribed in the opposite direction direction opposite the in transcribed , 2.97 ×10 7.82 ×10 1.56 ×10 9.63 ×10 1.64 ×10 7 ×10 2.09 ×10 5.86 ×10 1.96 ×10 0.0001 1.61 ×10 0.0024 2.83 ×10 1.5 ×10 0.00053 0.002 4.9 ×10 2.27 ×10 0.00034 7.62 ×10 0.00013 1.29 ×10 4.35 ×10 0.00017 2.3 ×10 ). The highest expression levels were were levels expression highest The ). – P −9 SLC46A3 −5 −7 −8 −12 −16 −8 −10 −11 −8 −5 −5 −6 −7 −10 −6 −6 −6 TMEM136 ). Supplementary Table15 Supplementary locus ( 5 8 P 0.62 0.17 0.9 0.23 0.13 0.69 0.09 0.36 0.10 0.66 0.10 0.85 0.81 0.96 0.38 Heterogeneity tests , designed to detect detect to designed , het , , s e l c i t r A ARHGEF12 Supplementary Supplementary 23.20 36 12.50 26.50 23.10 28.10 I 0.00 0.00 0.00 0.00 0.00 8.00 0.00 0.00 5.60 2 (%) LOXL1 ) and and ) ± 8.1 8.1 ), ), ±  -

© 2017 Nature America, Inc., part of Springer Nature. All rights reserved. (rather than than (rather 15 Fig. XFS eyes in comparison to age-matched control eyes ( to body,such as 41% in tissues, and iris anterior segment from ciliary of sion levels loci three the ( underlying SNPs sentinel the of genotypes the with 20 100 bars, Scale blue. in shown is counterstaining nuclear DAPI meshwork. trabecular ST,TM, canal; stroma; Schlemm’s SC, layer; nuclear outer ONL, epithelium; limbal LE, epithelium; pigment iris IPE, layer; nuclear inner INL, layer; cell ganglion retinal GCL, membrane; descemet DM, epithelium; corneal CoE, epithelium, ciliary CE, vessel; in shown are immunoblots all of * Data are shown as the POMP/ ( eyes control to compared as eyes XFS of lysates tissue body ciliary and iris in expression protein POMP reduced show analysis densitometry ( walls vessel blood iris and ( epithelium pigment iris the of surface the on immunofluorescence) (red accumulations material exfoliation positive LOXL1- with associated is tissues XFS in intensity staining Reduced ( ciliary and ( analysis immunoblot by shown controls age-matched to compared as eyes XFS of tissues body ( ( endothelium ( ( epithelium corneal the of cytoplasm the in fluorescence) (green immunopositivity POMP punctate shows tissues eye ( immunohistochemistry. and immunoblotting by determined as XFS, with eyes donor and eyes donor 3 Figure s e l c i t r A  that and locus 13 chromosome the for gene disease-related likely g b Supplementary Fig. 14 Fig. Supplementary P – ), the limbal epithelium and stromal cells ( cells stromal and epithelium limbal the ), < 0.01, ** < 0.01, Comparing tissues from XFS and control eyes, the mRNA expres the eyes, control and XFS from tissues Comparing

m XFS Control β β µ POM POM g k h d a -acti -acti m in m in ) Reduced POMP protein expression levels in the iris and ciliary ciliary and iris the in levels expression protein POMP ) Reduced P P n n ). These results in XFS-relevant tissues suggest that that suggest tissues XFS-relevant in results These ).

Expression of POMP protein in ocular tissues of normal human human normal of tissues ocular in protein POMP of Expression a Control Control TM , , i ST b , Ciliary body l Co IP , , SC P ) epithelia, as well as vascular endothelia in the iris ( iris the in endothelia vascular as well as ) epithelia, d POMP FLT1 E e < 0.005, unpaired two-sided two-sided unpaired < 0.005, ), the ciliary epithelium ( epithelium ciliary the ), E Iris and and IP E XFS XFS and and h and g – ) and immunofluorescence labeling of iridal ( iridal of labeling immunofluorescence ) and m . - 42kDa - 16kDa - 42kDa - 16kDa SLC46A3 ). TMEM136 b e m β l i a ). Immunoblots (cropped images) and and images) (cropped Immunoblots ). -actin ratio ( – f ) Immunofluorescence labeling of normal normal of labeling ) Immunofluorescence S

upplementary Figure 16 Figure upplementary POMP protein (% of control) 12 16 10 14 ST 20 40 60 80 , which are located nearby) is the the is nearby) located are which , DM ST 0 0 0 CE 0 0 ST were significantly reduced by reduced up were significantly ST e CE ) and the retinal cell layers ( layers cell retinal the ) and n CE = 6 for each group; mean a Iris c ), the corneal endothelium endothelium corneal the ), t XF Control ), the trabecular meshwork meshwork trabecular the ), test); uncropped versions versions uncropped test); k –45 ), ciliary epithelium ( epithelium ciliary ), ** S c % µ f m in m in m j POMP protein (% of control) 100 120 140 160 c LE Supplementary Supplementary 20 40 60 80 , , . . BV, blood 0 d and and BV BV ST Ciliary body f and and k POMP g XF Control , –33% h m ± m ). ). ON GC * f , s.d.; IN S ) k ). ). ). ). L L L

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levels were significantly reduced in iris (45% decrease) and ciliary ciliary and decrease) (45% iris in reduced significantly were levels ( analysis immunofluorescence in types cell ocular most in expressed was protein, maturation teasome immunoblotting and immunofluorescence microscopy. POMP, a pro locus. 11 chromosome the for gene disease-related TMEM136 b 200 bars, Scale blue. in is counterstaining nuclear DAPI meshwork. trabecular ST,TM, canal; stroma; Schlemm’s SC, layer; nuclear outer ONL, epithelium; limbal LE, epithelium; pigment iris IPE, layer; nuclear inner INL, layer; cell ganglion retinal GCL, muscle; dilator 17 Figure in shown are immunoblots all of versions uncropped n TMEM136/ the as shown are Data ( eyes control to compared as eyes XFS of lysates tissue body ciliary and iris in expression KDa) 31 3 at isoform and KDa 28 1 at (isoform protein TMEM136 reduced show analysis densitometry and images) (cropped ( epithelium pigment iris the of surface the on immunofluorescence) (red accumulations material exfoliation LOXL1-positive with associated is ( iris the in endothelia ( iridal of labeling ( analysis immunoblot by shown controls age-matched to compared as eyes XFS of tissues body ciliary and iris in ( layers cell and ( body ciliary the of epithelia and vessels ( (arrows) veins aqueous ( endothelium canal Schlemm’s and meshwork trabecular ( vessels blood limbal in fluorescence) (green immunopositivity TMEM136 cytoplasmic shows tissues eye normal of labeling ( immunohistochemistry. and immunoblotting by determined as XFS, with eyes donor and eyes donor human normal 4 Figure k – * group; each = 6 for TMEM1 TMEM1 ), ciliary epithelium ( epithelium ciliary ), f g POMP and TMEM136 protein expression was further analyzed by analyzed was further protein POMP expression and TMEM136 XFS Control and 20 20 and h k β β d a -actin -actin 36 36

Expression of TMEM136 protein in the ocular tissues of tissues ocular the in protein TMEM136 of Expression . AV, aqueous vein; BV, blood vessel; CE, ciliary epithelium; DIL, DIL, epithelium; ciliary . AV,CE, BV, vein; vessel; blood aqueous C C µ LE (rather than the neighboring neighboring the than (rather ontr ontr IPE IPE m in m in BV Cilia DIL ST DIL ol ol IPE f ). ( ). Ir ry a is h h body DVANCE ONLINE PUBLICATION ONLINE DVANCE , – g k XFS XFS m – ) and ciliary ( ciliary ) and k ST j . , P ) Reduced TMEM136 protein expression levels levels expression protein TMEM136 ) Reduced l m ) and iris blood vessel walls ( walls vessel blood iris ) and c < 0.01, ** < 0.01, ), blood vessels of the iris (arrows) ( (arrows) iris the of vessels blood ), ). Reduced staining intensity in XFS tissues tissues XFS in intensity staining Reduced ). - 42kD - 28kD - 31kD - 42kD - 28kD - 31kD l i e a a a a a a b CE β CE i BV ST , CE -actin ratio (mean (mean ratio -actin TMEM136 P l ST ) epithelia, as well as vascular vascular as well as ) epithelia,

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© 2017 Nature America, Inc., part of Springer Nature. All rights reserved. We the that rare, speculate carrying protective p.Tyr407 variant could of tissues in patients impaired the with XFS cell adhesion, ocular tions of decreased elastic fiber formation observa and tissue stiffness, as well histopathological as by supported is notion This fibrillin-1 deposition. and elastin increased to due ECM the of stabilization of cal experiments suggest that the protective effect may be a consequence arily conserved catalytic domain of LOXL1 (ref. clear. less are polymorphisms and of roles p.Leu141Arg p.Gly153Asp common the but the allele, role for rare p.Phe407 the and physiological functional clear a confirm experiments Our disease. complex a being XFS with the in allele risk rs11827818[G] the of the as a copy carried absolute, also in cases two Both XFS cases. variant was observed not is provided protection the strong, is effect the Although on own. its significance genome-wide to surpass OR = 25) p.Tyr407Phe variant had a protective strong effect enough (resistance ( architecture ( broad the along haplotypes phased all across occur als revers the as populations, different across structures LD different to due be to unlikely also are They reversals. these confirming tions replica multiple of light in population same the within differences at reversals allele common of effect reversal the genetic significant had also genome-wide of activity promoter the influencing variants associated moter that influences 1 of intron in region a describing study recent a Even doubt. in remain lotypes hap these of consequences functional the effect, genetic of reversal com all As pathogenesis. mon disease in role central a have to esized hypoth LOXL1 with ECM, the involving aging of disorder a is XFS DISCUSSION ( ( magnification segment anterior ocular low both using eyes, to control comparison in eyes XFS in important structures in TMEM136 and POMP of expression reduced Weconfirmed TMEM136. and POMP LOXL1, for co-staining antibodies, of batch independent an using controls in tissues from a further three donor eyes with XFS and three matched obtained using microscopy also immunofluorescence ( were eyes control to comparison in eyes XFS in cells endothelial and 15 ferential mRNA expression analysis shown in eyes ( control in observed levels the to comparison in decrease) (32% body significantly reduced showed expression eyes levels XFS in from iris (26% tissues decrease) in and ciliary immunoblotting by levels protein lial cells of vessels blood in eye tissues ( endothe vascular the to immunofluorescence by localized primarily in shown expression mRNA differential ( microscopy cence ( analysis immunoblot by investigated was expression protein when eyes trol body (33% from specimens decrease) XFS eyes in comparison to con Nature Ge Nature maintain and integrity cellular render morecells resistant to environ Supplementary Figs. 19 19 Figs. Supplementary Supplementary Data 1 Data Supplementary . Similar findings of reduced TMEM136 protein staining in epithelial The The protective p.Tyr407Phe substitution is located in the evolution of resequencing Deep findings microscopy immunofluorescence the We replicated then was function, unknown of protein transmembrane a TMEM136, LOXL1 Fig. 4 Fig. 3 Fig. g LOXL1 n and haplotypes detected by the resequencing effort showed showed effort resequencing the by detected haplotypes etics Supplementary Fig. 21 Fig. Supplementary g Supplementary Fig. 18 Fig. Supplementary and and Supplementary Fig. 17 and 5 and

Fig. 3h– Fig. ADVANCE ONLINE PUBLICATION ONLINE ADVANCE LOXL1 Supplementary Fig. 16 Fig. Supplementary ′ ) in the absence of gross differences in LD LD in differences gross of absence the in ) upstream of upstream LOXL1 and LOXL1 m - AS1 20 ). These results are consistent with the the with consistent are results These ). enabled us to observe that the rare rare the that observe to us enabled ). expression showed that all strongly are unlikely to due be to sampling are unlikely ). LOXL1 Fig. Fig. TMEM136 ), also consistent with the dif ) and higher magnification magnification higher and ) Supplementary Figure 15 Figure Supplementary 4 ). ). Analysis of TMEM136 Supplementary Figure Supplementary - ) and immunofluores and ) 5 AS1 9 ). Follow-up biologi containing a pro containing locus, in keeping keeping in locus,

Fig. Fig. 4h– LOXL1 LOXL1 2 1 locus locus m . . The - 60 AS1 ). , 6 1 . ------.

as a susceptibility locus for omega-6 (n-6) polyunsaturated fatty fatty polyunsaturated (n-6) omega-6 for locus susceptibility a as consortium, CHARGE the In (5q23). to (6p21), region MHC III class to addition pathophysiology XFS in material XFS ( of vessels blood deposition ocular around involving partly and vasculopathy, pronounced a early Interestingly, endothelia. vascular to localized in XFS implicated signaling been ubiquitin–proteasome involves also that process a tissues pathway, autophagy XFS related in closely the in enzymes Abnormalities ubiquitin-conjugating of reduction a suggests tissues XFS in protein, maturation proteasome expressed ( POMP of downregulation marked exam the For ple, genetic types. from tissue different arise and can pathways that multiple in aging lesions of is disease XFS systemic that complex hypothesis a the supporting pathway, pathogenesis gle that p.Tyr407Phe RNApossibility affect could stability were not tested at the RNA might level, which be relevant as there is a p.Tyr407Phe for mechanisms biological the that is approach this of limitation One ECM. the disrupt or destabilize that stressors mental for Inherited Research Disease (CIDR). CIDR is fully funded through a federal were providedservices through a grant to J.L.W. (HG008597) by the Center R01 EY015473 to J.L.W. For XFS cases in the US GWAS data set, genotyping CA49449, R01 AR056291, R01 CA131332, P01 CA055075, R01 CA134958 and and by grants from the US National Institutes of Health: UM1 CA186107, R01 für ForschungZentrum Klinische (IZKF-E23) from Germany to F.P. and U.S.-S., to T.A. and NMRC/CBRG/0032/2013 to E.N.V.), by the Interdisziplinäres Council, Singapore NMRC/TCR/008-SERI/2013 and NMRC/STAR/0023/2014 Foundation of New York (to C.C.K.), by grants from the National Medical Research TechnologyScience, and Research, Singapore (to C.C.K.), by the Glaucoma This research is supported by the Biomedical Research Council, Agency for online version of the pape Note: Any Supplementary Information and Source Data files are available in the pa the the in available are references, and codes accession statements of including Methods, and data availability any associated M software, Primer3 lator for genetic studies, association https://ma cal program package, URLs. disease for important pathogenesis. be could that pathways biological new cate impli that loci XFS-associated new five we identified have addition, purposes therapeutic for LOXL1 of targeting macological strongly protects against XFS, raising the possibility of potential phar biology. disease XFS into research for avenues further up opens loci these at and ciation asso RBMS3 of evidence AGPAT1, consistent the of understood, roles well not are SEMA6A biological the Although ditions. disease Parkinson’s and Alzheimer’s as such conditions in involved be to reported been also populations in aging risk cardiovascular to related be may which levels, (PUFA) acid Acknowledgments et n hs td, e dniid he XS ucpiiiy oi in loci susceptibility XFS three identified we study, this In sin a implicate GWASnot do the from emerging loci seven The In summary, we now show that a rare h pe PLINK software, ods r . thgen.stats.ox.ac.uk POMP 7 http: 1 and and r . The MHC locus (where (where locus MHC The . . https://www.r-pro 6 //primer3.ut.e http://zzz. 8 72 69 . TMEM136 expression was predominantly was predominantly expression . TMEM136 TMEM136 , 7 , 7 3 0 , which, like XFS, are age-related con age-related are XFS, like which, , . /impute/impute_v2.ht Fig. Fig. RBMS3 bwh.harvard.edu/plin 4 http://zzz.bwh.ha , which map to to map which , ), appears to have a major role role major a have to appears ), e . AGPAT1 ject.org LOXL1 (3p24) and near near and (3p24) Fig. Fig. variant, p.Tyr407Phe, / AGPAT1 ; ; IMPUTE2 software, has been identified identified been has s e l c i t r A 3 onli ), a ubiquitously ubiquitously a ), m AGPAT1 rvard.edu/gpc 66 l 62– ; ; power calcu , 6 ne version of version ne resides) has has resides) k 7 6 / , have also also have , 4 ; ; R statisti 29 . SEMA6A , 74– in the the in 7 6 . In . 6 5 /  ------. ;

© 2017 Nature America, Inc., part of Springer Nature. All rights reserved. 12. 11. 10. 9. 8. 7. 6. 5. 4. 3. 2. 1. claims jurisdictional in published maps and affiliations. institutional reprints/index. at online available is information permissions and Reprints The authors declare no competing interests.financial for publication. from T.A., T.K., U.T., J.L.W., L.R.P. and F.P. All co-authors approved the manuscript analysis of deCODE data. The manuscript was written by C.C.K., with inputcritical and processing. U.T., G.T. and K. Stefanssonconducted and supervised, provided P. Sundaresan, M.D. and K.T. were involved in sample phenotypingcollection, T.R.R., Kubota, S. Micheal, F.T., J.E.C., K.A.-A., M.H., J.H.K., S. Nelson, D.M., B.J.S., N. Wang, D.C., R.Q., T. Kivela, A. Reis, F.E.K., R.N.W., L.R.P., F.J., R.R.A., Crandall, L.M.Z., T.Y.W., M.N., S. Kinoshita, A.I.d.H., E.V., J.H.F., A.J.S.,R.K.L., D.A., B.K., M.R.W., A.L.C., Y.L., P.C., L. Herndon, R.W.K., J.K., K.C., C.J.C., A. A.Z., T.R.C., L.A., M.R., M.G., H.G.-I., P.P.R.-C., L.F.-V.C., C.O., N.T., E.A., B.B., A.B., D.S.K., M.L.H., S.D., S. Herms, S. Heegaard, M.M.N., S. Moebus, R.M.R., B. Wanichwecharugruang, N.K., A. Sakuntabhai, H.X.N., G.T.T.N., T.V.N., W.Z., S.-L.H., F.A.E.-D.,R.H., F.M.-T.,R.G.-S., A. Salas, K.P., L. Hansapinyo, M.C., J.G.C., S.Y.A., E.L.A., A.E., V.V., G.K., R.F., S.A.A.-O., O.O., L.A.A., B.C., P. Fornero, O.C., D.S., T. Zompa, E. R.A.M., Souzeau, P. Mitchell, J.J.W., A.W.H., F.A., E.K.-J., U.L., I.L., V.C., R.P.G., G.S.M., S. Roy, E.D., E. Silke, A. Rao, P. Sahay, M.I.K., O.O.O., A.O.A., I.U., A.O., N.K.-A., C.T., Y.S., W.S., S.O., N.J.U., I.A., H.A., M.U., C. Sotozono, J.W.J., M.S., K.H.P., J.A., M.C.-A., S.M.E., A. Rafei, V.H.K.Y., T.O., T. Sakurai, T. Sugimoto, H.C., M.A., M. Inatani, M.M., N.G., F.M., N.Y., Y.I., P. Frezzotti, D.P., E.S., P. Manunta, Y.M., K. Miyata, T.H., E.C., S.I., A.Y., M.Y., Y.K., N. Kobakhidze, A.N.B., M.R.K., S.Y., A.I., H.N., N. Khatibi, A.F., C.L., L.D., T.R., P. Founti, A. T.P.,Chatzikyriakidou, E.A., A.L., N.P.,R.S., V.S., R.V., C. Shivkumar, Y.-X.W., L.X., S.L., P.R., G.C., S.T., G.M., N. Weisschuh, U.H., U.-C.W.-L., C.M., B. Wirostko, S.T., D.G., K.B., W.L.G., X.C., J.S., H.J., L.J., C.Q., H.Z., X.L., B.Z., T.D., D.P.E., L.d.J.M., M.P., S. Moghimi, D.B.-H.,R.I., P. Kappelgaard, Y.N., M.B., K.H.P., S.C.C., K.Y., J.C.Z., J.B.J., S.A.P.,R.S.K., N. Kalpana, L.V.,R.G., T. Zarnowski, K.I., M. Irkec, M.C.-P., K. Sugiyama, P. Schlottmann, S.F.L., H.L., S.E.W., Y.S.A., A.C.O., S. Nakano, K. Mori, A.P.C., K.H., S. Manabe, S. Kazama, conducted genotyping and sequencing experiments. T.A., M.O., T.M., A.H., inputcritical on statistical analysis. Z.X., S.Q.M., H.M.S., X.Y.C., S.Q.P. and K.K.H. E.N.V., C.-Y.C. and J.L.H. conducted statistical analysis. S. Raychaudhuri provided S.U., Z.Y., L. Huang, J.N.F., R.Q.S., K.S.S., P. Kraft, I.J., A.G., M.A.P.-V., A.M.H., biological functional experiments. G.T., R.P.I., K.P.B., Z.L., G.P., S.S., J.N.C.B., M.Z., D.B., Y.F.C., X.Y.N., A.W.O.C., E.N.V., A.S.Y.C.S.R.G., and Y.C. conducted C.C.K., F.P., J.L.W., T.A. and M.O. jointly conceived the project. M.C.L., U.S.-S., all of the exfoliation syndrome cases from Finland. the ophthalmologist Eva Forsman from Finland who awaypassed after diagnosing the Health System of Beijing to (2009-1-05) N. Wang. We dedicate this article to Development and (2011ZX09302-007-05) Talents–High-LevelLeading Talents of Major and Scientific Technological Project Special for Significant New Drugs (81030016 and the 81570837), Program of Beijing Scholars (2013), the National supported by funding from the National Natural FoundationScience of China University, contract The HHSN268201200008I. Beijing, China, was collection contract from the US National Institutes of Health to The Johns Hopkins s e l c i t r A  COMPETING FINANCIAL INTERESTS COMPETING AU

T Williams, S.E. Williams, G. Thorleifsson, A.C. Orr, R.R. Allingham, open-angle of cause identifiable common most syndrome—the Exfoliation R. Ritch, syndrome. exfoliation of manifestations systemic and Ocular R. Ritch, 2. act span: life and syndrome pseudoexfoliation Ocular T. Kivelä, Olawoye, O.O. associated glaucoma is Why A.G. Konstas, & U. Schlötzer-Schrehardt, R., Ritch, M.C. Leske, syndrome. Exfoliation U. Schlötzer-Schrehardt, & R. Ritch, glaucoma. exfoliation and syndrome Exfoliation T. Kivelä, & E. Vesti, a black South African population. African South black a glaucoma. exfoliation to susceptibility Genet. Ophthalmol. glaucoma. 23 2 19 syndrome? exfoliation with trial. glaucoma manifest early the (2001). 265–315 Res. Eye , 640–641 (2015). 640–641 , H (Suppl. 1), S1–S8 (2014). S1–S8 1), (Suppl. , 402–405 (2012). 402–405 , O R R

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M D 71 , Robert , Robert 16 C haiwat , 32 1 16 123 187 31 146 , , 14 K ilek ilek Aktas , , raig Zangwill , , Paul Department Department of Ophthalmology, Flinders University, , , Ignacio , , 24 Shinjo Shinjo Eye Clinic, Miyazaki, Japan. 35 S D Department Department of Ophthalmology, Dalhousie University, ubota , , E , , Fridbert Jonasson S huang Ru 144 , , 48 , , , 126 34 S 135 36 aniele aniele 148 ija Vesti armiento D S Department Department of Ophthalmology, Otsu Red Cross 41 114 ylvain ylvain Roy 16 Department Department of Ophthalmology, Hacettepe 141 , hi hi Qi , 50 aniela aniela 145 , , Fundación Fundación para el Estudio del Glaucoma, 23 154 , , Axel 131 T L , , 193 Biochemistry Biochemistry Department, Faculty of M 151 O , Department Department of Medicine, Duke University N uis Fernández-Vega eekhasaenee 116 , , 13 18

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© 2017 Nature America, Inc., part of Springer Nature. All rights reserved. Denmark. Denmark. Institutet and Karolinska University Hospital Solna, Stockholm, Sweden. USA. Massachusetts, for Personalized Genetic Medicine, Boston, USA. Massachusetts, Genetics and Rheumatology, Department of Medicine, Brigham and Women’s Hospital and Harvard Medical School, Boston, USA. Massachusetts, USA. Massachusetts, Nafees Medical College and Hospital, Isra University, Islamabad, Pakistan. CNR), Italy.Segrate-Milano, Minnesota, USA. 169 University of Iowa, Iowa City, Iowa, USA. Nijmegen, the Netherlands. Technology for Ophthalmology, Kyoto Prefectural University of Medicine, Kyoto, Japan. USA. Tennessee, USA. James and Jean Culver Discovery Institute, Augusta University, Augusta, Georgia, USA. Geffen School of Medicine at UCLA, Los Angeles, California, USA. Ankara, Turkey. Osmangazi University, Meselik, Eskisehir, Turkey. Institute for Molecular Bioscience, Faculty of Health Sciences, University of the Witwatersrand, South Johannesburg, Africa. Essen, Duisburg-Essen, Germany. Human Genetics, University of Bonn, Bonn, Germany. 149 Disease, London School of Hygiene and Tropical Medicine, London, UK. Recherche Associée 3012, Paris, France. Functional Genetics of Infectious Diseases Unit, Department of Genomes and Genetics, Paris, France. Hospital, Bangkok, Thailand. Compostela, Spain. Compostela, Santiago de Compostela, Spain. Xenética, Departamento de Anatomía Patolóxica e Ciencias Forenses, Instituto de Ciencias Forenses, Facultade de Medicina, Universidade de Santiago de Santiago de Compostela, Spain. Department of Medicine, University of Salamanca, Salamanca, Spain. Medicine, Nanyang Technological University, Singapore. Pharmacology Laboratory, National Cancer Centre, Singapore. Hospital, Melbourne, Victoria, Australia. University of Sydney, Sydney, New South Wales, Australia. 127 University Misericordiae Hospital, Dublin, Ireland. 122 Shenzhen, China. Pakistan. Pakistan. Sciences, COMSATS Institute of Information Technology, Abbottabad, Pakistan. 117 Hospital, Mahidol University, Bangkok, Thailand. Ophthalmology, ESUT Teaching Hospital Parklane, Enugu, Nigeria. 111 University of Ibadan, Ibadan, Nigeria. Morocco. Ophthalmology, Seoul Metropolitan Government Seoul National University Boramae Medical Center, Seoul, Republic of Korea. Hospital, Osaka, Japan. Genomic Medicine, INSERM U852, Kyoto University Graduate School of Medicine, Kyoto, Japan. Ophthalmology, University of Tokyo, Tokyo, Japan. Sciences, Hiroshima University, Hiroshima, Japan. University, Asahikawa, Japan. Hospital, Miyazaki, Japan. 91 Department of Medicine, Surgery and University Neuroscience, of Siena, Siena, Italy. Shahid Beheshti University of Medical Sciences, Tehran, Iran. Hospital, Tirunelveli, India. Foundation, Bangalore, India. 80 University of Tübingen, Tübingen, Germany. Nürnberg, Erlangen, Germany. Angers, Universitaire, France. Beijing Tongren Hospital, Capital Medical University, Beijing, China. People’s Hospital, Chengdu, China. Urumchi, China. University of Electronic Science and Technology of China, Chengdu, China. Human Disease Gene Study, Sichuan Provincial People’s Hospital, University of Electronic Science and Technology of China, Chengdu, China. Glostrup, Denmark. Eye Research Center, Tehran University of Medical Sciences, Tehran, Iran. (IBSAL), Salamanca, Spain. Chicago, Illinois, USA. Riyadh, Saudi Arabia. Glaucoma, Medical Research Foundation, Chennai, India. China. Germany. Medicine, UNAM, Mexico City, Mexico. Nature Ge Nature Department Department of Health Sciences, University of Milan, Milan, Italy. Laboratory of General Biology, School of Medicine, Aristotle University of Greece. Thessaloniki, Thessaloniki, Bascom Bascom Palmer Eye Institute, University of Miami Miller School of Medicine, Miami, Florida, USA. Department Department of Biomedicine, University of Basel, Basel, Switzerland. Centro Charles, Oftalmologico Buenos Aires, Argentina. Instituto Instituto de Glaucoma y Catarata, Lima, Peru. Department of Ophthalmology, College of Medicine, University of Nigeria, Nsukka, Ituku Ozalla Campus, Enugu, Nigeria. Department of Ophthalmology, University of Lagos, Lagos, Nigeria. 163 53 Department Department of Genomic Medical Sciences, Kyoto Prefectural University of Medicine, Kyoto, Japan. Narayana Narayana Nethralaya Eye Hospital, Bangalore, India. 107 120 52 183 Beijing Beijing Tongren Eye Center, Beijing Tongren Hospital, Capital Medical University, Beijing and Ophthalmology Visual Science Key Laboratory, Beijing, Department Department of Human Genetics, Radboud University Medical Centre, Nijmegen, the Netherlands. Centro Oftalmologico Centro Lischinsky,Oftalmologico Tucumán, Argentina. n Department Department of Pathology, Eye Rigshospitalet, Pathology Section, University of Copenhagen, Copenhagen, Denmark. etics 158 69 162 171 73 School School of Medicine, Wayne State University, Detroit, Michigan, USA. Center Center for Human Molecular Biology and Genetics, Institute of Laboratory Medicine, Sichuan Academy of Medical Sciences and Sichuan Provincial 141 65 Département d’Ophtalmologie, Centre Département d’Ophtalmologie, Hospitalier Angers, Universitaire, France. Hamilton Hamilton Glaucoma Center, Department of and Ophthalmology Shiley Eye Institute, University of California, San Diego, San Diego, California, Ophthalmic Ophthalmic Consultants of Boston, Boston, USA. Massachusetts, 180 176

John John A. Moran Eye Center, Department of Ophthalmology, University of Utah, Salt Lake City, Utah, USA. 58 59 Department Department of Ophthalmology, Faculty of Medicine, Chiang Mai University, Chiang Mai, Thailand. 104 ADVANCE ONLINE PUBLICATION ONLINE ADVANCE Department Department of and Ophthalmology Visual Sciences, Illinois Eye and Ear Infirmary, College of Medicine, University of Illinois at Chicago, Institute Institute of Inflammation and Repair, University of Manchester, Manchester, UK. John P. Hussman Institute for Human Genomics, University of Miami Miller School of Medicine, Miami, Florida, USA. Department Department of Ophthalmology, University Hospital of Salamanca, Salamanca, Spain. 94 Department Department of Ophthalmology, Seoul National University Bundang Hospital, Gyeonggi, Republic of Korea. 85 166 173 61 Sensho-kai Sensho-kai Eye Institute, Kyoto, Japan. 143 Aravind Aravind Eye Hospital, Coimbatore, India. 96 75 82 Ophthalmic Ophthalmic Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran. 77 Department Department of Ophthalmology, University of Turku and Turku University Hospital, Turku, Finland. Department Department of Biosciences, COMSATS Institute of Information Technology, Islamabad, Pakistan. Department Department of Ophthalmology, Asahikawa Medical University, Asahikawa, Japan. David David Tvildiani Medical University, Tbilisi, Georgia. 138 Santa Santa Lucia Eye Hospital from Buenos Aires, Buenos Aires, Argentina. Department Department of Ophthalmology, Faculty of Medicine, Siriraj Hospital, Mahidol University, Bangkok, Thailand. Department Department of Ophthalmology, Medical University Graz, Graz, Austria. 153 GENVIP GENVIP Research Group, Instituto de Sanitaria Investigación de Santiago, Santiago de Compostela, Spain. 70 Division Division of Ophthalmology, Stellenbosch University and Tygerberg Hospital, Cape Town, South Africa. Sichuan Sichuan Translational Research Hospital, Chinese Academy of Sciences, Chengdu, China. 109 51 130 168 Department Department of Ophthalmology, University College Hospital, Ibadan, Nigeria. Department Department of Ophthalmology, Medical Faculty Mannheim of the Ruprecht Karls University of Heidelberg, Mannheim, 146 St. St. Petersburg Academic University, St. Petersburg, Russia. Department Department of and Ophthalmology Visual Sciences, Carver College of Medicine, University of Iowa, Iowa City, Iowa, USA. 79 Department Department of General Pediatrics, Medical University of Graz, Graz, Austria. 140 Department Department of Ophthalmology, School of Medicine, Aristotle University of Greece. Thessaloniki, Thessaloniki, Instituto Instituto de Ciencias Forenses, Facultade de Medicina, Universidade de Santiago de Compostela, Santiago de 116 123 156 101 125 98 Department Department of Ophthalmology, University of Nigeria Teaching Hospital, Enugu, Ituku-Ozalla, Nigeria. Clinical Clinical Research Centre Adolphe de Rothschild, Société Médicale de Beaulieu, Geneva, Switzerland. Istanbul Istanbul University Cerrahpasa Faculty of Medicine, Istanbul, Turkey. Ohashi Ohashi Eye Center, Sapporo, Japan. Department Department of Ophthalmology, Faculty of Medical Science, University of Fukui, Fukui, Japan. Shri Shri Mithu Tulsi, LV Prasad Eye Institute, Bhubaneswar, India. 152 135 Institute Institute for Medical Informatics, Biometry and Epidemiology, University Hospital of Essen, University 56 129 128 Department Department of Ophthalmology, Tan Tock Seng Hospital, Singapore. Vietnam Vietnam National Institute of Ophthalmology, Hanoi, Vietnam. Centre Centre for Eye Research Australia (CERA), University of Melbourne, Royal Victorian Eye and Ear Centre Centre for Vision Research, Department of and Ophthalmology Westmead Institute for Medical Research, 133 54

88 Chichua Chichua Medical Center Mzera, LLC, Tbilisi, Georgia. Dipartimento Dipartimento di Scienze Chirurgiche, Università di Torino, Turin, Italy. Office Office of Clinical Sciences, Duke–NUS Medical School, Singapore. 179 160 114 92 121 72 Program Program in Medical and Population Genetics, Broad Institute of MIT and Harvard, Cambridge, 95 Department Department of Nephrology, University Vita-Salute San Raffaele, Milan, Italy. Department Department of Cellular Biology and Anatomy, Center for and Biotechnology Genomic Medicine, 112 Eye Eye Specialists Hospital, Enugu, Nigeria. 137 Shenzhen Shenzhen Key Laboratory of Ophthalmology, Shenzhen Eye Hospital, Jinan University, Universidad Universidad Peruana Cayetano Heredia, Hospital Nacional Arzobispo Loayza, Lima, Peru. Department Department of Medicine and Engineering Combined Research Institute, Asahikawa Medical 86 150 148 182 Vision Vision Research Foundation, Chennai, India. Guinness Guinness Eye Centre, Lagos University Teaching Hospital, Lagos, Nigeria. Translational Pediatrics and Infectious Diseases, Hospital Clínico de Universitario Santiago, 63 Division Division of Medical Genetics, University Hospital Basel, Basel, Switzerland. 175 68 Department Department of Genomics, Life & Brain Center, University of Bonn, Bonn, Germany. Department Department of Ophthalmology, University Rigshospitalet, of Copenhagen, Copenhagen, Ideta Ideta Eye Hospital, Kumamoto City, Japan. Department Department of Ophthalmology, First Affiliated Hospital of Xinjiang Medical University, Department Department of Epidemiology, Harvard T.H. Chan School of Public Health, Boston, 119 Pakistan Pakistan Institute of Ophthalmology, Al-Shifa Trust Eye Hospital, Rawalpindi, 76 90 Institute Institute of Human Genetics, Friedrich-Alexander-Universität Erlangen- 172 165 161 Department Department of Ophthalmology, Monfalcone Hospital, Gorizia, Italy. 159 Institute Institute of Biomedical Technologies, Italian National Research Centre (ITB- 99 Department Department of Ophthalmology, Radboud University Medical Centre, Vanderbilt Eye Institute, Vanderbilt University Medical Center, Nashville, Center Center for Community Outreach and Policy, Stein Eye Institute, David Tane Memorial Eye Hospital, Osaka, Japan. 103 74 Tazuke Kofukai Foundation, Medical Research Institute, Kitano 170 145 Département Département de Biochimie et Génétique, Centre Hospitalier 83 78 131 Department Department of Ophthalmology, Mayo Clinic, Rochester, Aravind Aravind Eye Hospital, Pondicherry, India. Institute Institute for Ophthalmic Research, Centre for Ophthalmology, Centre Centre National de la Recherche Unité Scientifique, de 181 Clinical Clinical Pharmacology, SingHealth, Singapore. 164 Rheumatology Rheumatology Unit, Department of Medicine, Karolinska 60 108 81 Department Department of Frontier Medical Science and 115 Institute Institute for Biomedical Research of Salamanca GROW GROW Research Laboratory, Narayana Nethralaya 97 Department Department of Ophthalmology, College of Medicine, Department Department of Ophthalmology, Ramathibodi Department Department of and Ophthalmology Visual 126 110 55 87 Hospital Hospital Córdoba, Córdoba, Argentina. 64 Jadhavbhai Jadhavbhai Nathamal Singhvi Department of Ocular Ocular Tissue Engineering Research Center, ECWA Eye Hospital, Kano, Nigeria. 142 147 Eye Clinic, Rigshospitalet–Glostrup, Eye Clinic, Rigshospitalet–Glostrup, 157 57 62 118 Department Department of Ophthalmology, Rajavithi 155 Faculty Faculty of Infectious and Tropical King King Khaled Eye Specialist Hospital, DAMAGEN DAMAGEN Genetic Diagnostic Center, Farabi Farabi Eye Hospital, Tehran University 106 66 136 Department Department of Environmental Department Department of Genetics, Eskisehir 71 167 174 Sichuan Sichuan Provincial Key Laboratory for 184 Laboratoires Laboratoires RAFEI, Mohammedia, Molecular Molecular Medicine Unit, Beijing Beijing Institute of Ophthalmology, Institute Institute for Vision Research, Department Department of Biochemistry, Al- Department Department of Ophthalmology, 134 105 89 100 Lee Lee Kong Chian School of Department Department of Ophthalmology Unit, Ophthalmology Department Department of 154 144 Sydney Sydney Brenner 67 139 177 Institut Institut Pasteur, s e l c i t r A School School of Medicine, 93 Unidade Unidade de 178 Divisions Divisions of 84 113 102 Miyata Miyata Eye Aravind Aravind Eye Partners Partners Center Department Department of Center Center for 151 Institute Institute of 132 124 Clinical Clinical Mater Mater 11 © 2017 Nature America, Inc., part of Springer Nature. All rights reserved. F.P. ( contributed equally to this work. Aravind Eye Hospital, Madurai, India. New York Eye and Ear Infirmary of Mount Sinai, New York, New York, USA. University of Iceland, Reykjavik, Iceland. Infirmary, Boston, USA. Massachusetts, University of Helsinki and Helsinki University Hospital, Helsinki, Finland. s e l c i t r A 1 2

[email protected] 193 These These authors jointly directed this work. should Correspondence be addressed to T.A. ( 191 ), ), J.L.W. ( 186 Department Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, Singapore. 188 Institute Institute of Biology,Computational Case Western Reserve University, Cleveland, Ohio, USA. Department Department of Ophthalmology, Landspitali University Hospital, Reykjavik, Iceland. [email protected] 185 190 Department Department of Ophthalmology, Harvard Medical School, Eye Massachusetts and Ear Dr. G.Venkataswamy Eye Research Institute, Aravind Medical Research Foundation, ) ) or C.C.K. ( [email protected] a DVANCE ONLINE PUBLICATION ONLINE DVANCE ). [email protected] 189 Einhorn Einhorn Clinical Research Center, 187 Faculty Faculty of Medicine,

Nature Ge Nature 192 ), ),

These These authors n etics © 2017 Nature America, Inc., part of Springer Nature. All rights reserved. ( in illustrated manner a in distributed is which statistic, test regression median the using SNPs, genomic inflation estimate ( all SNP markers from the GWAS are appended as of statistics summary association The genome-wide stratification. population residual minimize to collections sample for stratification genetic of ponents association analysis was additionally adjusted for the first three principal com elsewhere as described was conducted, before meta-analysis strata for country separate regression each logistic using URLs). (see package software statistical R the using generated to adjust for residual population stratification. Principal-component plots were r pairwise by (defined markers unlinked of set pruned a from calculated than with samples outlying ancestry from remove further analysis. to Principal-component scores were separately country/site each for performed was analysis described previously underwent as that genotyping, samples genome-wide all for substructure population and stratification analysis. further from removed was rate completeness > (IBD 0.1875) relatedness of cryptic status for each sample pair comparison. For each sample pair identify-by-state for showing evidence information derive to URLs) (see software PLINK used ther exclusion of samples using the principle of variability in allele sharing. We analysis principal-component by outlying (defined genetic ancestry being more than 6 s.d. from the mean on as excess heterozygosity (defined being >3 s.d. from the mean genotyping success rate as (defined genotyping completeness < 95%), showing a with poor and those checked, similarly was sample Each analysis. discovery GWAS further from removed also were of <1% frequency allele a minor with ( equilibrium Hardy–Weinberg from tion devia significant showing SNPs as well as <95%, of rates completion typing geno showed that markers SNP performing OmniExpress poorly We removed Illumina BeadChip. the by genotyped markers SNP genotyped directly included only stage GWAS the for discovery protocol analysis statistical Our sample. individual each and marker SNP each for performed were checks control for quality GWASthe Stringent analysis stage Statistical discovery the in appended are stage lication ( minimized were groups ancestry different the across frequencies allele minor varying SNPs with imputed for content information and insufficient discovery uncertainty to imputation relating issues that means analysis primary the in imputation of absence genotyped The controls. uniformly and markers cases in SNP genotyped directly only used analysis discovery primary the that ensuring array, OmniExpress Illumina the using genotyped were stage GWAS for the controls discovery 17,008 and cases XFS To bias different genotyping minimize arrays between and platforms, all 9,035 described previously as BeadChip, OmniExpress Illumina the using taken of Republic the Bashkortostan; from one and Petersburg St. from one collections, distinct to be two from 25 owing across to strata (considered Russia contributing tries Genotyping of samples. the in appended are review board or ethics committee. Details for each XFS case–control collection institutional respective their from approval obtained sites study All Helsinki. of Declaration the of tenets the to adherence strict in obtained human were samples All participant. each from consent written informed after were obtained XFS without controls normal with together glaucoma exfoliation and collections. Patient ONLINE doi: correction, double-GC underwent loci significant for genome-wide the seven Supplementary Table 2 Supplementary Supplementary Table 2 Supplementary 2 < 0.1) for each country/site separately. These scores were used as covariates as were covariates used scores separately. These < for 0.1) country/site each Association between SNP genotypes and XFS disease status was measured measured was status disease XFS and genotypes SNP between Association We analysis principal-component to performed assess the degree of genetic fur after remaining samples all of relationships biological the We verified countries 18 from collections XFS included stage replication The 10.1038/ng.3875 8 3 . .

λ MET GC Supplementary Fig. 22 Fig. Supplementary is listed for GWASeach individual stratum (and discovery also Supplementary Supplementary Table 2 H ODS Supplementary Note Supplementary DNA and tissue samples from all patients with XFS XFS with patients all from samples tissue and DNA For the GWAS discovery stage performed in 24 coun ). Details on the genotyping and analysis for on and the analysis rep ). the genotyping Details ). In line with well-described methodologies, results results methodologies, ). In well-described with line λ GC 8 0 ) ) was using calculated only directly genotyped ) were excluded from further analysis. further from excluded were ) Supplementary Note Supplementary 7 ) as well as for the GWAS meta-analysis GWAS the for as well meta-analysis as ) 8 , the sample with the lower genotyping , the lower genotyping with the sample 15 ), genome-wide genotyping was under , 81 . P , 8 < 1 × 10 × 1 < 2 . . For GWASthe stage, discovery Supplementary Data Supplementary 2 8 0 7 −6 . Principal-component Principal-component . 7 . for deviation). SNPs SNPs deviation). for . 78 , 7 9 ) ) and having . . The 1 χ 4 2 ------.

2014) release, as described elsewhere. To minimize the effect of imputation imputation of effect the Tominimize elsewhere. (June 3 described as Phase release, Project 2014) Genomes 1000 the of part are data These world. the around populations from 26 distinct individuals from 2,535 on obtained data based haplotypes population from cosmopolitan constructed panel reference the with URLs) (see software IMPUTE2 using out carried were genotypes of sets underlying each of the five newly identified loci of credible the delineation for better allow also would This ity checks. control via imputation using samples fine-mapping and SNP qual markers strict passing arrays GWAS standard-content on included currently SNPs genotyped directly the by provided resolution genetic the on improve to sought we loci, imputation. Genotype ( stage ward to the replication stage and analyzed in a manner similar to the discovery surpassing XFS stage. for replication the analysis Statistical ( at reversal strong population validated for method meta-analysis account The stratification. appropriately not would measure this as analysis, association for groups continental across or within pooled samples were analy sis the in point no At country/site. separate each from errors) standard and ratios odds (adjusted results summary control–corrected genomic using meta-analysis during again once then and which forcorrects genomic first inflation at the population individual stratum Roche NimbleGen SeqCap Easy probe kit. Enrichment and amplification of of amplification and Enrichment kit. probe the Easy SeqCap using NimbleGen captured Roche were 13,745,000 to chr19:13,307,000 and 74,260,000 to 5 introns, ( countries nine from controls 6,279 and cases XFS 5,570 of total a on formed of sequencing Deep in footnotes, Table 15 as Supplementary appended models mouse the detailing references relevant avail publicly Informatics database Genome able Mouse the in ( lookups loci performed distinct we seven in SNPs significant wide phenotypes. Mouse model latitude. graphical geo of increments 10° increasing with increased disease for ratios odds the whether assess to test trend a GWASWe the conducted during analysis. then and analysis adjustment for genomic control principal-component correction ( drawn were controls and cases XFS which from more precise) was whichever zone, (or country the for band latitude a assigned were tested SNP each for ciation with XFS. The odds ratio and standard error for the odds ratio estimate taken for the five loci showing asso newly identified significant genome-wide under were latitude geographical and markers genetic between interaction latitude. test forStatistical with geographical interaction uncertainty. imputation across average to software SNPTEST with analyses for were used the imputed data association dosages step, tation allele of imputation on quality Forthe set analysis. credible the impu fine-mapping frequency >1% and imputation information content >0.95 to reduce the impact ping imputation variants were included using similar thresholds of minor allele with variants genotyped rates >1%. Fine-map of success genotyping >95% and frequency all minor allele included we sets, credible of construction For signal association for locus-specific each accounted driving of probability that the of >95% variants genetic of number minimum the as described, analysis. set Credible uncertainty. imputation across average to software SNPTEST with of score information an with genotypes imputed included only we uncertainty, Supplementary Supplementary Table 1 Supplementary Fig. 23 Fig. Supplementary Supplementary Table 2 Supplementary ≥ Meta-analysis was performed via the inverse-variance, fixed-effects model model fixed-effects the inverse-variance, via was performed Meta-analysis The The odds ratios and standard errors used in this test had already undergone 0.95. Allele dosages were used for the imputed data association analyses analyses association data imputed the for used were dosages Allele 0.95. Supplementary Note Supplementary ′ and 3 and 8 6 . The output was manually checked and curated, with the the with curated, and checked manually was output The . P ′

flanking regions) spanning coordinates chr15:74,200,000 chr15:74,200,000 coordinates spanning regions) flanking ≤ 1 × 10 × 1 LOXL1 For the five newly identified genome-wide significant significant genome-wide For identified the five newly Credible sets of SNPs were defined, as previously previously as defined, were SNPs of sets Credible ). ). Both the ). ). . −4 For the 33 by genes implicated genome- the seven ). in the GWAS discovery stage were brought for brought were stage GWAS the discovery in and and LOXL1 CACNA1A 84 and , 8 5 SNPs showing association with with association SNPs showing . CACNA1A . ep eunig a per was sequencing Deep upeetr Tbe 15 Table Supplementary 4 8 . . Imputation and phasing Nature Ge Nature genetic loci (exons, Statistical tests forStatistical n LOXL1 etics 48 , 4 ), ), 9 ------.

© 2017 Nature America, Inc., part of Springer Nature. All rights reserved. gene expression levels, mRNA ratios relative to the housekeeping gene in appended are conditions and PCR URLs), (see software Primer3 using designed Genomics), (Eurofins for The sequences EvaGreen primers the (Bio-Rad). Supermix exon-spanning cDNA, 0.4 strand (25 reactions PCR Rad). PCR was performed using the CFX Connect thermal cycler and software (Bio- 20 a in (Invitrogen) transcriptase from 0.5 synthesis cDNA an DNase step. I includes on-column This First-strand tions. digestion with together kit manufacturer’s to the instruc according DNA/RNA (Qiagen) kit AllPrep the lysing and homogenizer 24 Precellys the tissues. using eye extracted human of PCR real-time Quantitative study. this approved (4218-CH), Tübingen and the University of Würzburg, as well as the local ethics committee of University the of Erlangen-Nuremberg, University of the faculties medical by Sanger sequencing ( liquid in nitrogen. DNA samples frozen obtained from tissues ocular and cells and were genotyped compound temperature cutting optimal in embedded normal human donor eyes (mean age, 72.3 78.7 age, (mean XFS with eyes donor sectors. tissue small of analysis microscopy tron macroscopic of inspection anterior segment and structures by by confirmed elec assessed was deposits material exfoliation characteristic of presence The and were snap in nitrogen. liquid frozen microscope a under pared dissecting pre were tissues Ocular used. were disease ocular known any without male) 77.1 age, (mean eyes control age-matched healthy, age, XFS with (mean eyes 80.1 15 h 21 For donor of RNAwithin death. and DNA and processed extractions, obtained were consent research appropriate with transplantation corneal for GWASloci. the of analysis for specimens Tissue PLINK GWAS experiments. rate and of completeness call with a from 100% the sequencing both genotype individuals we only included ably and common capture rare both haplotypes, for the phasing Haplotype studies ciation asso genetic in reported as is well controls, and of cases number asymmetric 10 in Table shown are Supplementary and URLs) (see methods well-described using model genetic additive an with performed were stages replication and GWAS the discovery studies. association genetic for calculations Power analysis. statistical further for forward brought were >95% completeness calling genotype variant with individuals and recalibration score quality variant by ‘PASS’ scores assigned variants ity were otypes using the Only called high-qual GATKguidelines. best-practices using Burrows–Wheeler aligner software, which is (hg19) well described genome reference human the to aligned were individual each annotation. and detection variant mapping, Read entire the of sequencing deep undergo not did and 2016 December and 2015 February between were collected samples These stage. for replication the were enrolled 2007 to January 2015. A 1,082 further XFS cases and 2,325 controls from Japan from December were enrolled resequencing from trols Japan underwent who 60×. be to observed was be covered by at 10× least samples the of >95% that We required platform. 2500 HiSeq Illumina the on techniques laboratory routine well-described, using out carried then were libraries the Nature Ge Nature comparative the by calculated were For immunostaining experiments, ocular tissue samples obtained from 10 from obtained samples tissue ocular experiments, For immunostaining For the analysis of 9 0 software packages, as previously described previously as packages, software LOXL1 3 6 n . Sequencing was performed using 2 × 101-bp paired-end reads reads paired-end 101-bp × 2 using performed was Sequencing . etics 82 , 8 locus. 8 µ . µ M of each upstream and downstream primer, and SsoFast primer, and SsoFast and downstream M upstream of each g of total RNA was performed with Superscript II reverse II reverse g Superscript with of RNA total was performed LOXL1 LOXL1 Supplementary Supplementary Note µ 8 7 . These power calculations take into account the the account into take calculations power These . l) were run in duplicate and contained 2 contained and duplicate in run were l) . . Mean coverage for across the samples sequencing p.Tyr407Phe, the initial 2,827 cases and 3,013 con LOXL1 haplotypes were phased using the BEAGLE Supplementary Table 16 Table Supplementary ± 7.9 years; 11 female, 10 male) and 10 11 7.9 years; male) 41 female, normal, µ locus. l reaction volume. Quantitative real-time real-time Quantitative volume. reaction l C ± T 9.7 years; 6 female, 4 male) and 10 10 and male) 4 female, 6 years; 9.7 ehd ( method To ensure accurate phasing to reli Tophasing accurate ensure ± ). ). The ethical review boards of the 11.6 years; 5 female, 5 male) were Human donor eyes used used eyes donor Human ± 3 2 8.1 years; 20 female, 21 21 female, 20 years; 8.1 5 Power calculations for for calculations Power All sequence reads in in reads sequence All −∆ . . For normalization of of normalization For . Ocular tissues were were tissues Ocular C T 3 6 ). Amplification Amplification ). . . Consensus gen µ l l of first- GAPDH 8 9 and ------

enzyme sites EcoRI and SalI were added in a second amplification using using amplification second a in added were SalI and EcoRI sites enzyme in shown primers the using fied constructs. LOXL1 the in described are which procedures, densitometry. by computerized analyzed was intensity band and Scientific), (Thermo kit ECL West Femto Signal Super the and PBST in T20 SuperBlock 10% in antibody secondary (HRP)-conjugated peroxidase radish horse a with performed was PBST. by Immunodetection replaced was body anti primary the experiments, PBST. in negative-control T20 In SuperBlock to with mouse antibody human verified was loading PBST. in Equal T20 SuperBlock 10% in diluted Abcam) °C 4 with antibodies at against POMP (ab170865, Abcam) and overnight TMEM136 (ab182495, or temperature room at h 1 for incubated and min 30 for system (Bio-Rad). Membranes transfer were Turboblocked with SuperBlock Trans-Blot T20 (Thermo Scientific) the with membranes nitrocellulose onto transferred and DTT) (6% conditions reducing SDS–PAGE under 4–15% by (10 Proteins Scientific). (Thermo kit assay tein 0.1% SDS). Protein concentrations were determined with the Micro-BCA pro RIPA mM (50 Tris-HCl, mM 150 1% NaCl, DOC, pH NP-40, 8.0, buffer 0.5% of tissues six XFS with eyes and using body eyes six normal and the iris ciliary Immunoblot analysis of human eye tissues. Biosystems). (Applied DNA sequencer 3100 Prism on the analyses sequence and curve melt using checked was specificity tion; sc-17581, Santa Cruz Biotechnology), mouse antibody to fibrillin-1 fibrillin-1 to antibody mouse Biotechnology), Cruz Santa sc-17581, tion; dilu (1:100 elastin to antibody goat sc-7392 Biotechnology), Cruz Santa dilution; sc-805, and (1:100 HA to antibody rabbit or mouse antibodies: mary pri following the with °C 4 at temperature. overnight room incubated at h subsequently were 1 They for PBS) in BSA (3% and buffer PBS) 1× blocking in in Tween-20 blocked (0.1% PBST with washed were °C. 4 spheroids at stored Fixed and temperature room at min with 10 fixed for were paraformaldehyde spheroids 4% The h. 72 at to collected left were were and Cells spheroids well. form per cells 300,000 at Bio-One) (Greiner surfaces hydropho bic with plates six-well low-attachment in medium growth their in negative. be to found was and mycoplasma for tested was line CO 5% with °C 37 at and 2 mM GLUTAMAX (Invitrogen) (Sigma-Aldrich) FBS 20% with mented supple DMEM in maintained were Cells (ATCC). Collection Culture Type Spheroid cultures. the in appended are which procedures, laboratory routine followed IV collagen and fibronectin elastin, the in are appended assay assay.for this More details and (G-A-A) Arg141–Asp153–Phe407 (G-A-T) was performed Arg141–Asp153–Tyr407 using the NanoLuc luciferase (T-G-A), Leu141–Gly153–Tyr407 (G-G-A), Arg141–Gly153–Tyr407 haplotypes tested four the to respect with LOXL1. for assay secretion luciferase Nano in appended are constructs LOXL1 ing clone. of primer All pairs oligonucleotide used to the full-length create the by sequenc Sanger was confirmed constructed of the haplotypes all accuracy 17 Table ( pairs primer oligonucleotide respective with (Clontech) kit mutagenesis site-directed Transformer the using strategy PCR-based a using mutagenesis. site-directed subsequent for plasmid plate tem the as served this and (G-A-A), Arg141–Asp153–Tyr407 was generated haplotype first The rs1048661[G>T]–rs3825942[G>A]–rs201011613[A>T]. order: following the in variants genetic contained and generated were A-T), (T-G-A), Arg141–Asp153–Tyr407 (G-A-A) (G- and Arg141–Asp153–Phe407 tag. HA an encoding Leu141–Gly153–Tyr407 (G-G-A), vector Arg141–Gly153–Tyr407 Four haplotypes, pcipuro a into subcloned then was kb) (~1.7 ( primers of set second a Immunohistochemistry of human eye tissues followed routine laboratory laboratory routine followed tissues eye human of Immunohistochemistry Nucleofected HLECs were trypsinized 48 h after nucleofection and seeded and seeded 48 h nucleofection after were trypsinized HLECs Nucleofected on haplotypes LOXL1 HA-tagged different the of analysis Immunoblot mutagenesis site-directed by achieved was substitution base Targeted ) on the the on ) LOXL1 The HLEC (B-3) cell line was obtained from the American h fl-egh DA noig OL ws ampli was LOXL1 encoding cDNA full-length The 2 and were passaged every 2–3 d in a 1:4 ratio. The cell cell The ratio. 1:4 a in d 2–3 every passaged were and Arg141–Asp153–Tyr407 (G-A-A) haplotype. The The haplotype. (G-A-A) Arg141–Asp153–Tyr407 Supplementary Table 17 Table Supplementary Supplementary Note Supplementary β -actin (clone AC-15, Sigma-Aldrich) in 10% (clone AC-15, Sigma-Aldrich) -actin Supplementary Table 16 Table Supplementary Supplementary Table 17 Supplementary Supplementary Note Supplementary Total protein was from extracted The secretion assay for LOXL1 LOXL1 for assay secretion The µ . g per lane) were separated separated were lane) per g ). The LOXL1 fragment fragment LOXL1 The ). Supplementary Note Supplementary doi: . The restriction restriction The . Supplementary Supplementary 10.1038/ng.3875 . . . ------

© 2017 Nature America, Inc., part of Springer Nature. All rights reserved. software (IBM) with an unpaired two-tailed two-tailed unpaired an with (IBM) software patients and between sion controls differences using was SPSS performed v.20 Statistical procedures for analyses. biological times. independent seven follow-up independent experiment (shown in in shown assay The haplotype. (G-A-T) Arg141–Asp153–Phe407 the for those against compared readings at the first time point, and the normalized readings were subsequently initial respective their against normalized were variant for each readings The each haplotype to allow for robust evaluation statistical of the results obtained. LOXL1 different the between adhesion cell–cell in differences for analyzed and ware RTCA the from soft were extracted data 48 h. Impedance for subsequent the intervals 24 h and at for first the 30-min intervals RTCA at SP 15-min system CO 5% with °C 37 at incubated were plates 96-well The well. per cells 120,000 of density a at Biosciences) (ACEA RTCA for plated SP into were the plates xCELLigence designed instrument 96-well constructs haplotype (G-A-T) Arg141–Asp153–Phe407 and (G-A-A) (T-G-A), Arg141–Asp153–Tyr407 Leu141–Gly153–Tyr407 Tyr407 (G-G-A), assay. adhesion Cell–cell times. three independently acquired also images with times, three independently repeated was experiment Each figures. the in indicated as range, color by the defined scale same for the values intensity fluorescence maximum and minimum the to relative generated were signals ofluorescent five image of on of Analysis steps. a spheroids was performed maximal-projection platform. laser-scanning confocal SP8 Leica a TCS with Services, Health Singapore Academia, the at Core Bioimaging Advanced the at acquired were images spheroid Immunolabeled Millipore). (Merck Reagent FluorSave and Scientific) Fisher (Thermo cytocentrifuge a (1 DAPI with stained were spheroids The Laboratories). Jackson dilution; (1:300 or 647 Cy3 Fluor Alexa FITC, with conjugated antibody secondary anti-goat or anti-rabbit anti-mouse, were used antibodies secondary The temperature. room at h 1 for antibodies secondary respective their with labeled and PBST with times three antibody, were washed spheroids the primary the with incubation After (1:100 dilution;IV ab6586, Abcam). All antibodies collagen were dilutedto with the blocking buffer.antibody rabbit and Abcam) ab6328, dilution; (1:100 doi: 10.1038/ng.3875 z planes projected onto a single image. Heat maps of respective immun respective of maps Heat image. single a onto projected planes haplotypes. Triplicates were performed for HLECs nucleofected with with nucleofected HLECs for performed were Triplicates haplotypes. Figure 1 Figure µ g/ml) and mounted on glass microscope slides using using slides microscope glass on mounted and g/ml) HLECs nucleofected with nucleofected HLECs e was repeated four independent times, with the the with times, independent four repeated was 2 and monitored on the xCELLigence xCELLigence the on monitored and Supplementary Supplementary Fig. 7 z t Statistical evaluation of expres planes were imaged in 1- in imaged were planes test. test. LOXL1 P < 0.05 was considered considered was 0.05 < Arg141–Gly153– ) ) repeated µ m - - - 83. 82. 81. 80. 79. 78. 77. and analysis) as appended are analysis as appended is plot the in reflected markers SNP all for statistics summary availability. Data We considered calculated. were assays homoscedastic secretion unpaired an and by analyzed adhesion for Data significant. statistically be to 90. 89. 88. 87. 86. 85. 84.

rno, P.G. Bronson, K. Kiryluk, V.J.Verhoeven, Price, A.L. G.F. Mells, Anderson, C.A. J.C. Barrett, Purcell, S. Purcell, and imputation genotype to approach unified A S.R. Browning, & B.L. Browning, T.H.Cheng, J.N. Foo, Mouse The J.E. Richardson, & J.A. Kadin, J.T., Eppig, C.J., Bult, J.A., Blake, J.S. Kooner, Y. Okada, (2016). CLEC16A (2014). pathogens. intestinal against immunity in involved myopia. (2013). and 314–318 error refractive for loci susceptibility new multiple studies. association wide cirrhosis. biliary primary for loci Protoc. Nat. the including (2009). 1330–1334 loci, susceptibility new three based linkage analyses. linkage based individuals. unrelated and Genet. Hum. trios J. Am. of sets data large for inference haplotype-phase analysis. association Genet. populations. Asian East in genes susceptibility and pathogenic related laboratory the about knowledge to mouse. access and of integration Database: Genome loci. susceptibility 2diabetes (2011). 984–989 type new six identifies ancestry discovery.

23 Nucleic Acids Res. Acids Nucleic are associated with selective IgA deficiency.IgA selective with associated are et al. et , 3891–3897 (2014). 3891–3897 , Nature t al. et et al. Supplementary Data 3 Data Supplementary et al. et et al. et t al. et

et al. et 5 t al. et t al. et , 1564–1573 (2010). 1564–1573 , Analysis of non-synonymous-coding variants of Parkinson’sof disease– variants non-synonymous-coding of Analysis et al. et al. et t al. et Principal components analysis corrects for stratification in genome- eeis f huaod rhii cnrbts o ilg ad drug and biology to contributes arthritis rheumatoid of Genetics The primary data set comprising genome-wide association association genome-wide comprising data set The primary PLINK: a tool set for whole-genome association and population- and association whole-genome for set tool a PLINK: Discovery of new risk loci for IgA nephropathy implicates genes implicates nephropathy IgA for loci risk new of Discovery

Genome-wide association study identifies 12 new susceptibility new 12 identifies study association Genome-wide Five endometrial cancer risk loci identified through genome-wide through identified loci risk cancer endometrial Five 506 eoewd ascain td i idvdas f ot Asian South of individuals in study association Genome-wide eoewd ascain td o ucrtv clts identifies colitis ulcerative of study association Genome-wide Supplementary Data 2 Data Supplementary Data quality control in genetic case–control association studies.

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43 81 PVT1 . The data sets from secondary secondary from sets data The . HNF4A , 329–332 (2011). 329–332 , , 559–575 (2007). 559–575 , , ( a. Genet. Nat. ATG13 LOXL1 Nat. Genet. Nat. region. Nature Ge Nature Figure Figure – AMBRA1 phased haplotype haplotype phased

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