Genome-wide association analyses identify new loci influencing intraocular pressure

Genome-wide association analyses identify new loci influencing intraocular pressure Abstract Elevated intraocular pressure (IOP) is a significant risk factor for glaucoma, the leading cause of irreversible blindness worldwide. While previous studies have identified numerous genetic variants associated with IOP, these loci only explain a fraction of IOP heritability. Recently established of biobank repositories have resulted in large amounts of data, enabling the identification of the remaining heritability for complex traits. Here, we describe the largest genome-wide association study of IOP to date using participants of European ancestry from the UK Biobank. We identified 671 directly genotyped variants that are significantly associated with IOP (P < 5 × 10−8). In addition to 103 novel loci, the top ranked novel IOP genes are LMX1B, NR1H3, MADD and SEPT9. We replicated these findings in an external population and examined the pleiotropic nature of these loci. These discoveries not only further our understanding of the genetic architecture of IOP, but also shed new light on the biological processes underlying glaucoma. Introduction Intraocular pressure (IOP) is the amount of fluid pressure in the eye which is mainly determined by the balance between aqueous humor production by the ciliary body and drainage through the trabecular meshwork (1). Elevated IOP is a major risk factor for glaucoma, a group of progressive optic neuropathies and the leading cause of irreversible blindness worldwide. Currently, IOP is the only modifiable risk factor for glaucoma and lowering IOP helps to prevent the onset and delay the progression of glaucoma (2–4). Identifying factors that contribute to IOP may aid in uncovering the biological mechanisms regulating this trait, provide new management avenues for IOP, and potentially glaucoma. Epidemiological studies have identified numerous factors associated with IOP, including age, body mass index, systolic blood pressure and type 2 diabetes (5–7). In addition to these systemic factors, studies have provided evidence of a genetic component for IOP determination, with heritability estimates ranging from 29% to 67% (8,9). Several genome-wide association studies (GWASs) have been conducted to identify genetic variants associated with IOP. Initial GWASs identified many genetic loci associated with IOP, including GAS7, TMCO1, GLCCI1-ICA1, ADAMTS18-NUDT7, FOXP1, FAM125B and ARHGEF12 (10–13). Meta-analyses consisting of subjects of European and Asian descent identified additional genomic regions, such as CAV1-CAV2, ABO, FNDC3B, RAPSN, PKHD1 and ADAMTS8 (14–16). Moreover, a recent multi-ethnic meta-analysis identified HIVEP3, ANTXR1, AFAP1, ARID5B, FOXO1 and INCA1 as additional IOP-related loci (17). While these studies identified numerous loci associated with IOP, their findings only explain 2.80–3.66% of the variation in this trait (17), indicating that there are additional variants to discover. The recent establishment of large biobank repositories has resulted in a plethora of both clinical and genomic data. The scope and scale of these biobanks will enable researchers to investigate the association between genetic variants and a multitude of complex traits, increasing the statistical power to detect previously unidentified associations and consequently, to further uncover the remaining heritability for various traits. The UK Biobank is one of the largest biobanks containing both ophthalmologic and genetic data, making it possible to identify novel genetic variants associated with ocular traits, including IOP. Due to the large sample size of the UK Biobank, we believe examining genetic variants associated with IOP will yield novel genomic loci associated with this trait. Therefore, the purpose of this study is to perform a GWAS of IOP using data from the UK Biobank. To the best of our knowledge, the current study represents the largest GWAS of IOP. Findings from this study will elucidate the genetic variants regulating this trait, and subsequently, may further our understanding of the underlying biological mechanisms of glaucoma. Results Study sample The characteristics of the study sample are summarized in Table 1. Overall, a total of 115 486 European white study participants were included in this analysis. The mean (standard deviation) age and IOP are 58.1 (7.9) years and 15.9 (3.5) mmHg, respectively. The proportion of females in this study sample is 53.7%. On an average, male subjects exhibited higher IOP compared with females [16.0 (3.6) mmHg versus 15.8 (3.4) mmHg; P < 0.0001]. Table 1. Descriptive statistics of the study sample Sample size  Female, %  Age (years), mean (SD)  IOP (mmHg), mean (SD)  IOP (mmHg) range  115, 486  53.7  58.1 (7.9)  15.9 (3.5)  7.0–39.0  Sample size  Female, %  Age (years), mean (SD)  IOP (mmHg), mean (SD)  IOP (mmHg) range  115, 486  53.7  58.1 (7.9)  15.9 (3.5)  7.0–39.0  Table 1. Descriptive statistics of the study sample Sample size  Female, %  Age (years), mean (SD)  IOP (mmHg), mean (SD)  IOP (mmHg) range  115, 486  53.7  58.1 (7.9)  15.9 (3.5)  7.0–39.0  Sample size  Female, %  Age (years), mean (SD)  IOP (mmHg), mean (SD)  IOP (mmHg) range  115, 486  53.7  58.1 (7.9)  15.9 (3.5)  7.0–39.0  Genome-wide association results For this analysis, the genomic control inflation factor, λ, was 1.16 (Supplementary Material, Fig. S1). Inflation of the genomic control inflation factor may in part be due to the large sample size. The LD score regression intercept, an alternative approach to measure deviation, was 1.004, implying that inflation is primarily due to polygenicity and not confounding. Therefore, our analysis is properly controlled for population structure and cryptic relatedness. Figure 1 presents a Manhattan plot of the genome-wide P values for directly genotyped genetic variants. We identified 671 genome-wide significant genetic variants associated with IOP (P < 5 × 10−8), 66.2% of them are located within functional regions of the genome and 33.8% are located in intergenic regions (Supplementary Material, Table S1). Moreover, these genetic variants map to 149 loci (defined as ±500 kb), including 103 novel loci (Supplementary Material, Table S2). Table 2 presents the summary results for the top genotyped genetic variants for the most significant genes (P < 1 × 10−20). The most significant novel genetic variant is rs10760442 (P = 6.8 × 10−31, GRCh37/hg19 position 129383900), located on chromosome 9q33.3 in an intron of the LMX1B gene. The effect allele G [allele frequency (AF) = 0.63] is associated with an increase in IOP (ß = 0.16). The second most significant novel genetic variant, rs10838681 (P = 9.5 × 10−25, GRCh37/hg19 position 47275064), is located on chromosome 11p11.2 in an intron of NR1H3. The effect allele G (AF = 0.73) is associated with a decrease in IOP (ß = −0.16). The third most significant novel genetic variant is rs326214 (P = 1.0 × 10−23, GRCh37/hg19 position 47298360), located in an exon of MADD on chromosome 11p11.2. The effect allele G (AF = 0.32) is associated with an increase in IOP (ß = 0.15). The fourth most significant genetic variant is rs9038 (P = 1.5 × 10−23, GRCh37/hg19 position 75495397), located on chromosome 17q25.3 in the 3′-UTR of SEPT9. The effect allele T (AF = 0.61) is associated with an increase in IOP (ß = 0.14). Table 2. Lead genotyped SNPs for top-ranking genes associated with IOP             UK Biobank   Replication   SNP  Chr  Pos  Locus  Alleles  AF1  ß  SE  P  ß  SE  P  rs4656461  1  165687205  LOC440700-TMCO1  G/A  0.12  0.29  0.02  9.8 × 10−44  0.25  0.04  1.4 × 10−10  rs7555523  1  165718979  TMCO1  C/A  0.12  0.31  0.02  1.3 × 10−43  0.26  0.04  2.4 × 10−11  rs1700874  1  219182858  MIR548F3  A/G  0.93  0.28  0.03  2.7 × 10−26  0.18  0.05  5.5 × 10−4  rs4380442  3  171930381  FNDC3B  G/A  0.10  −0.26  0.02  1.1 × 10−28  −0.16  0.04  1.4 × 10−4  rs6816389  4  7864457  AFAP1  T/C  0.56  0.17  0.01  1.6 × 10−34  0.10  0.03  2.7 × 10−4  rs1363919  5  52625853  LOC257396-FST  T/G  0.52  0.13  0.01  6.5 × 10−24  0.08  0.03  1.5 × 10−3  rs7739648  6  1540483  FOXF2-FOXCUT  A/G  0.36  −0.14  0.01  3.6 × 10−21  −0.10  0.03  5.3 × 10−4  rs6946058  7  115839253  TFEC-TES  A/C  0.73  0.15  0.02  4.0 × 10−22  0.08  0.03  7.6 × 10−3  rs8940  7  116146074  CAV2  C/G  0.82  −0.23  0.02  3.6 × 10−38  −0.20  0.03  1.5 × 10−9  rs4236601  7  116162729  CAV2-CAV1  G/A  0.73  −0.22  0.02  1.0 × 10−42  −0.19  0.03  2.0 × 10−11  rs13270051  8  108288752  ANGPT1  C/T  0.87  0.23  0.02  9.4 × 10−31  0.09  0.04  1.6 × 10−2  rs1570204  9  4216751  GLIS3  T/C  0.65  −0.17  0.01  7.1 × 10−31  −0.11  0.03  8.2 × 10−5  rs10760442  9  129383900  LMX1B  G/A  0.63  0.16  0.01  6.8 × 10−31  0.11  0.03  1.0 × 10−4  rs12778514  10  63832279  ARID5B  G/A  0.59  0.14  0.01  1.0 × 10−25  0.09  0.03  1.2 × 10−3  rs1222926  10  95039397  CYP26A1-MYOF  C/A  0.36  0.15  0.01  8.5 × 10−27  0.08  0.03  2.7 × 10−3  rs7940065  11  770007  PDDC1  C/T  0.51  0.14  0.01  3.1 × 10−22  0.00  0.03  9.6 × 10−1  rs4963156  11  780827  LOC171391  C/T  0.54  0.14  0.01  3.6 × 10−23  0.00  0.03  9.0 × 10−1  rs10902223  11  817786  RPLP2-PNPLA2  C/T  0.44  0.14  0.01  7.1 × 10−21  0.02  0.03  4.1 × 10−1  rs10838681  11  47275064  NR1H3  G/A  0.73  −0.16  0.02  9.5 × 10−25  −0.11  0.03  1.2 × 10−4  rs326214  11  47298360  MADD  G/A  0.32  0.15  0.01  1.0 × 10−23  0.10  0.03  2.3 × 10−4  rs1052373  11  47354787  MYBPC3  C/T  0.68  −0.15  0.02  1.6 × 10−22  −0.10  0.03  1.7 × 10−4  rs3740689  11  47380593  SPI1  G/A  0.41  0.15  0.01  2.6 × 10−26  0.08  0.03  2.1 × 10−3  rs11824864  11  47411736  SPI1-MIR4487  A/G  0.86  −0.20  0.02  8.5 × 10−23  −0.16  0.04  7.4 × 10−6  rs111228939  11  47456067  PSMC3-RAPSN  T/C  0.87  −0.21  0.02  6.7 × 10−24  −0.21  0.05  3.0 × 10−6  rs4752843  11  47531884  CELF1  T/G  0.86  −0.19  0.02  3.6 × 10−22  −0.18  0.04  1.6 × 10−6  rs4147730  11  47605427  NDUFS3  G/A  0.86  −0.20  0.02  3.6 × 10−23  −0.18  0.04  1.0 × 10−6  rs4752805  11  48018355  PTPRJ  A/G  0.75  −0.15  0.02  1.3 × 10−22  −0.14  0.03  7.1 × 10−6  rs2305013  11  120340060  ARHGEF12  A/T  0.95  −0.32  0.03  4.9 × 10−25  −0.28  0.07  1.8 × 10−5  rs72755233  15  100692953  ADAMTS17  G/A  0.89  −0.31  0.02  4.1 × 10−45  −0.19  0.06  2.3 × 10−3  rs76953588  16  77611387  ADAMTS18-NUDT7  T/C  0.92  −0.26  0.03  9.8 × 10−26  −0.19  0.05  2.1 × 10−4  rs9938149  16  88331640  LOC101928880-ZNF469  C/A  0.37  −0.14  0.01  4.2 × 10−24  −0.07  0.03  7.9 × 10−3  rs12150284  17  10031090  GAS7  C/T  0.63  0.26  0.01  4.6 × 10−77  0.20  0.03  1.7 × 10−12  rs9038  17  75495397  SEPT9  T/C  0.61  0.14  0.01  1.5 × 10−23  0.07  0.03  2.1 × 10−2              UK Biobank   Replication   SNP  Chr  Pos  Locus  Alleles  AF1  ß  SE  P  ß  SE  P  rs4656461  1  165687205  LOC440700-TMCO1  G/A  0.12  0.29  0.02  9.8 × 10−44  0.25  0.04  1.4 × 10−10  rs7555523  1  165718979  TMCO1  C/A  0.12  0.31  0.02  1.3 × 10−43  0.26  0.04  2.4 × 10−11  rs1700874  1  219182858  MIR548F3  A/G  0.93  0.28  0.03  2.7 × 10−26  0.18  0.05  5.5 × 10−4  rs4380442  3  171930381  FNDC3B  G/A  0.10  −0.26  0.02  1.1 × 10−28  −0.16  0.04  1.4 × 10−4  rs6816389  4  7864457  AFAP1  T/C  0.56  0.17  0.01  1.6 × 10−34  0.10  0.03  2.7 × 10−4  rs1363919  5  52625853  LOC257396-FST  T/G  0.52  0.13  0.01  6.5 × 10−24  0.08  0.03  1.5 × 10−3  rs7739648  6  1540483  FOXF2-FOXCUT  A/G  0.36  −0.14  0.01  3.6 × 10−21  −0.10  0.03  5.3 × 10−4  rs6946058  7  115839253  TFEC-TES  A/C  0.73  0.15  0.02  4.0 × 10−22  0.08  0.03  7.6 × 10−3  rs8940  7  116146074  CAV2  C/G  0.82  −0.23  0.02  3.6 × 10−38  −0.20  0.03  1.5 × 10−9  rs4236601  7  116162729  CAV2-CAV1  G/A  0.73  −0.22  0.02  1.0 × 10−42  −0.19  0.03  2.0 × 10−11  rs13270051  8  108288752  ANGPT1  C/T  0.87  0.23  0.02  9.4 × 10−31  0.09  0.04  1.6 × 10−2  rs1570204  9  4216751  GLIS3  T/C  0.65  −0.17  0.01  7.1 × 10−31  −0.11  0.03  8.2 × 10−5  rs10760442  9  129383900  LMX1B  G/A  0.63  0.16  0.01  6.8 × 10−31  0.11  0.03  1.0 × 10−4  rs12778514  10  63832279  ARID5B  G/A  0.59  0.14  0.01  1.0 × 10−25  0.09  0.03  1.2 × 10−3  rs1222926  10  95039397  CYP26A1-MYOF  C/A  0.36  0.15  0.01  8.5 × 10−27  0.08  0.03  2.7 × 10−3  rs7940065  11  770007  PDDC1  C/T  0.51  0.14  0.01  3.1 × 10−22  0.00  0.03  9.6 × 10−1  rs4963156  11  780827  LOC171391  C/T  0.54  0.14  0.01  3.6 × 10−23  0.00  0.03  9.0 × 10−1  rs10902223  11  817786  RPLP2-PNPLA2  C/T  0.44  0.14  0.01  7.1 × 10−21  0.02  0.03  4.1 × 10−1  rs10838681  11  47275064  NR1H3  G/A  0.73  −0.16  0.02  9.5 × 10−25  −0.11  0.03  1.2 × 10−4  rs326214  11  47298360  MADD  G/A  0.32  0.15  0.01  1.0 × 10−23  0.10  0.03  2.3 × 10−4  rs1052373  11  47354787  MYBPC3  C/T  0.68  −0.15  0.02  1.6 × 10−22  −0.10  0.03  1.7 × 10−4  rs3740689  11  47380593  SPI1  G/A  0.41  0.15  0.01  2.6 × 10−26  0.08  0.03  2.1 × 10−3  rs11824864  11  47411736  SPI1-MIR4487  A/G  0.86  −0.20  0.02  8.5 × 10−23  −0.16  0.04  7.4 × 10−6  rs111228939  11  47456067  PSMC3-RAPSN  T/C  0.87  −0.21  0.02  6.7 × 10−24  −0.21  0.05  3.0 × 10−6  rs4752843  11  47531884  CELF1  T/G  0.86  −0.19  0.02  3.6 × 10−22  −0.18  0.04  1.6 × 10−6  rs4147730  11  47605427  NDUFS3  G/A  0.86  −0.20  0.02  3.6 × 10−23  −0.18  0.04  1.0 × 10−6  rs4752805  11  48018355  PTPRJ  A/G  0.75  −0.15  0.02  1.3 × 10−22  −0.14  0.03  7.1 × 10−6  rs2305013  11  120340060  ARHGEF12  A/T  0.95  −0.32  0.03  4.9 × 10−25  −0.28  0.07  1.8 × 10−5  rs72755233  15  100692953  ADAMTS17  G/A  0.89  −0.31  0.02  4.1 × 10−45  −0.19  0.06  2.3 × 10−3  rs76953588  16  77611387  ADAMTS18-NUDT7  T/C  0.92  −0.26  0.03  9.8 × 10−26  −0.19  0.05  2.1 × 10−4  rs9938149  16  88331640  LOC101928880-ZNF469  C/A  0.37  −0.14  0.01  4.2 × 10−24  −0.07  0.03  7.9 × 10−3  rs12150284  17  10031090  GAS7  C/T  0.63  0.26  0.01  4.6 × 10−77  0.20  0.03  1.7 × 10−12  rs9038  17  75495397  SEPT9  T/C  0.61  0.14  0.01  1.5 × 10−23  0.07  0.03  2.1 × 10−2  SNPs with a P < 1.0 × 10−20 are presented. Gene name is in boldface if the genetic variant is located within the gene. Genomic positions are according to GRCh37/hg19. Chr, chromosome; Pos, position; AF1, allele 1 frequency; SE, standard error. Table 2. Lead genotyped SNPs for top-ranking genes associated with IOP             UK Biobank   Replication   SNP  Chr  Pos  Locus  Alleles  AF1  ß  SE  P  ß  SE  P  rs4656461  1  165687205  LOC440700-TMCO1  G/A  0.12  0.29  0.02  9.8 × 10−44  0.25  0.04  1.4 × 10−10  rs7555523  1  165718979  TMCO1  C/A  0.12  0.31  0.02  1.3 × 10−43  0.26  0.04  2.4 × 10−11  rs1700874  1  219182858  MIR548F3  A/G  0.93  0.28  0.03  2.7 × 10−26  0.18  0.05  5.5 × 10−4  rs4380442  3  171930381  FNDC3B  G/A  0.10  −0.26  0.02  1.1 × 10−28  −0.16  0.04  1.4 × 10−4  rs6816389  4  7864457  AFAP1  T/C  0.56  0.17  0.01  1.6 × 10−34  0.10  0.03  2.7 × 10−4  rs1363919  5  52625853  LOC257396-FST  T/G  0.52  0.13  0.01  6.5 × 10−24  0.08  0.03  1.5 × 10−3  rs7739648  6  1540483  FOXF2-FOXCUT  A/G  0.36  −0.14  0.01  3.6 × 10−21  −0.10  0.03  5.3 × 10−4  rs6946058  7  115839253  TFEC-TES  A/C  0.73  0.15  0.02  4.0 × 10−22  0.08  0.03  7.6 × 10−3  rs8940  7  116146074  CAV2  C/G  0.82  −0.23  0.02  3.6 × 10−38  −0.20  0.03  1.5 × 10−9  rs4236601  7  116162729  CAV2-CAV1  G/A  0.73  −0.22  0.02  1.0 × 10−42  −0.19  0.03  2.0 × 10−11  rs13270051  8  108288752  ANGPT1  C/T  0.87  0.23  0.02  9.4 × 10−31  0.09  0.04  1.6 × 10−2  rs1570204  9  4216751  GLIS3  T/C  0.65  −0.17  0.01  7.1 × 10−31  −0.11  0.03  8.2 × 10−5  rs10760442  9  129383900  LMX1B  G/A  0.63  0.16  0.01  6.8 × 10−31  0.11  0.03  1.0 × 10−4  rs12778514  10  63832279  ARID5B  G/A  0.59  0.14  0.01  1.0 × 10−25  0.09  0.03  1.2 × 10−3  rs1222926  10  95039397  CYP26A1-MYOF  C/A  0.36  0.15  0.01  8.5 × 10−27  0.08  0.03  2.7 × 10−3  rs7940065  11  770007  PDDC1  C/T  0.51  0.14  0.01  3.1 × 10−22  0.00  0.03  9.6 × 10−1  rs4963156  11  780827  LOC171391  C/T  0.54  0.14  0.01  3.6 × 10−23  0.00  0.03  9.0 × 10−1  rs10902223  11  817786  RPLP2-PNPLA2  C/T  0.44  0.14  0.01  7.1 × 10−21  0.02  0.03  4.1 × 10−1  rs10838681  11  47275064  NR1H3  G/A  0.73  −0.16  0.02  9.5 × 10−25  −0.11  0.03  1.2 × 10−4  rs326214  11  47298360  MADD  G/A  0.32  0.15  0.01  1.0 × 10−23  0.10  0.03  2.3 × 10−4  rs1052373  11  47354787  MYBPC3  C/T  0.68  −0.15  0.02  1.6 × 10−22  −0.10  0.03  1.7 × 10−4  rs3740689  11  47380593  SPI1  G/A  0.41  0.15  0.01  2.6 × 10−26  0.08  0.03  2.1 × 10−3  rs11824864  11  47411736  SPI1-MIR4487  A/G  0.86  −0.20  0.02  8.5 × 10−23  −0.16  0.04  7.4 × 10−6  rs111228939  11  47456067  PSMC3-RAPSN  T/C  0.87  −0.21  0.02  6.7 × 10−24  −0.21  0.05  3.0 × 10−6  rs4752843  11  47531884  CELF1  T/G  0.86  −0.19  0.02  3.6 × 10−22  −0.18  0.04  1.6 × 10−6  rs4147730  11  47605427  NDUFS3  G/A  0.86  −0.20  0.02  3.6 × 10−23  −0.18  0.04  1.0 × 10−6  rs4752805  11  48018355  PTPRJ  A/G  0.75  −0.15  0.02  1.3 × 10−22  −0.14  0.03  7.1 × 10−6  rs2305013  11  120340060  ARHGEF12  A/T  0.95  −0.32  0.03  4.9 × 10−25  −0.28  0.07  1.8 × 10−5  rs72755233  15  100692953  ADAMTS17  G/A  0.89  −0.31  0.02  4.1 × 10−45  −0.19  0.06  2.3 × 10−3  rs76953588  16  77611387  ADAMTS18-NUDT7  T/C  0.92  −0.26  0.03  9.8 × 10−26  −0.19  0.05  2.1 × 10−4  rs9938149  16  88331640  LOC101928880-ZNF469  C/A  0.37  −0.14  0.01  4.2 × 10−24  −0.07  0.03  7.9 × 10−3  rs12150284  17  10031090  GAS7  C/T  0.63  0.26  0.01  4.6 × 10−77  0.20  0.03  1.7 × 10−12  rs9038  17  75495397  SEPT9  T/C  0.61  0.14  0.01  1.5 × 10−23  0.07  0.03  2.1 × 10−2              UK Biobank   Replication   SNP  Chr  Pos  Locus  Alleles  AF1  ß  SE  P  ß  SE  P  rs4656461  1  165687205  LOC440700-TMCO1  G/A  0.12  0.29  0.02  9.8 × 10−44  0.25  0.04  1.4 × 10−10  rs7555523  1  165718979  TMCO1  C/A  0.12  0.31  0.02  1.3 × 10−43  0.26  0.04  2.4 × 10−11  rs1700874  1  219182858  MIR548F3  A/G  0.93  0.28  0.03  2.7 × 10−26  0.18  0.05  5.5 × 10−4  rs4380442  3  171930381  FNDC3B  G/A  0.10  −0.26  0.02  1.1 × 10−28  −0.16  0.04  1.4 × 10−4  rs6816389  4  7864457  AFAP1  T/C  0.56  0.17  0.01  1.6 × 10−34  0.10  0.03  2.7 × 10−4  rs1363919  5  52625853  LOC257396-FST  T/G  0.52  0.13  0.01  6.5 × 10−24  0.08  0.03  1.5 × 10−3  rs7739648  6  1540483  FOXF2-FOXCUT  A/G  0.36  −0.14  0.01  3.6 × 10−21  −0.10  0.03  5.3 × 10−4  rs6946058  7  115839253  TFEC-TES  A/C  0.73  0.15  0.02  4.0 × 10−22  0.08  0.03  7.6 × 10−3  rs8940  7  116146074  CAV2  C/G  0.82  −0.23  0.02  3.6 × 10−38  −0.20  0.03  1.5 × 10−9  rs4236601  7  116162729  CAV2-CAV1  G/A  0.73  −0.22  0.02  1.0 × 10−42  −0.19  0.03  2.0 × 10−11  rs13270051  8  108288752  ANGPT1  C/T  0.87  0.23  0.02  9.4 × 10−31  0.09  0.04  1.6 × 10−2  rs1570204  9  4216751  GLIS3  T/C  0.65  −0.17  0.01  7.1 × 10−31  −0.11  0.03  8.2 × 10−5  rs10760442  9  129383900  LMX1B  G/A  0.63  0.16  0.01  6.8 × 10−31  0.11  0.03  1.0 × 10−4  rs12778514  10  63832279  ARID5B  G/A  0.59  0.14  0.01  1.0 × 10−25  0.09  0.03  1.2 × 10−3  rs1222926  10  95039397  CYP26A1-MYOF  C/A  0.36  0.15  0.01  8.5 × 10−27  0.08  0.03  2.7 × 10−3  rs7940065  11  770007  PDDC1  C/T  0.51  0.14  0.01  3.1 × 10−22  0.00  0.03  9.6 × 10−1  rs4963156  11  780827  LOC171391  C/T  0.54  0.14  0.01  3.6 × 10−23  0.00  0.03  9.0 × 10−1  rs10902223  11  817786  RPLP2-PNPLA2  C/T  0.44  0.14  0.01  7.1 × 10−21  0.02  0.03  4.1 × 10−1  rs10838681  11  47275064  NR1H3  G/A  0.73  −0.16  0.02  9.5 × 10−25  −0.11  0.03  1.2 × 10−4  rs326214  11  47298360  MADD  G/A  0.32  0.15  0.01  1.0 × 10−23  0.10  0.03  2.3 × 10−4  rs1052373  11  47354787  MYBPC3  C/T  0.68  −0.15  0.02  1.6 × 10−22  −0.10  0.03  1.7 × 10−4  rs3740689  11  47380593  SPI1  G/A  0.41  0.15  0.01  2.6 × 10−26  0.08  0.03  2.1 × 10−3  rs11824864  11  47411736  SPI1-MIR4487  A/G  0.86  −0.20  0.02  8.5 × 10−23  −0.16  0.04  7.4 × 10−6  rs111228939  11  47456067  PSMC3-RAPSN  T/C  0.87  −0.21  0.02  6.7 × 10−24  −0.21  0.05  3.0 × 10−6  rs4752843  11  47531884  CELF1  T/G  0.86  −0.19  0.02  3.6 × 10−22  −0.18  0.04  1.6 × 10−6  rs4147730  11  47605427  NDUFS3  G/A  0.86  −0.20  0.02  3.6 × 10−23  −0.18  0.04  1.0 × 10−6  rs4752805  11  48018355  PTPRJ  A/G  0.75  −0.15  0.02  1.3 × 10−22  −0.14  0.03  7.1 × 10−6  rs2305013  11  120340060  ARHGEF12  A/T  0.95  −0.32  0.03  4.9 × 10−25  −0.28  0.07  1.8 × 10−5  rs72755233  15  100692953  ADAMTS17  G/A  0.89  −0.31  0.02  4.1 × 10−45  −0.19  0.06  2.3 × 10−3  rs76953588  16  77611387  ADAMTS18-NUDT7  T/C  0.92  −0.26  0.03  9.8 × 10−26  −0.19  0.05  2.1 × 10−4  rs9938149  16  88331640  LOC101928880-ZNF469  C/A  0.37  −0.14  0.01  4.2 × 10−24  −0.07  0.03  7.9 × 10−3  rs12150284  17  10031090  GAS7  C/T  0.63  0.26  0.01  4.6 × 10−77  0.20  0.03  1.7 × 10−12  rs9038  17  75495397  SEPT9  T/C  0.61  0.14  0.01  1.5 × 10−23  0.07  0.03  2.1 × 10−2  SNPs with a P < 1.0 × 10−20 are presented. Gene name is in boldface if the genetic variant is located within the gene. Genomic positions are according to GRCh37/hg19. Chr, chromosome; Pos, position; AF1, allele 1 frequency; SE, standard error. Figure 1. View largeDownload slide Manhattan plot displaying the –log10(P values) for the association between IOP and the genotyped genetic variants. The solid and dotted horizontal lines represent genome-wide significant associations (P = 5 × 10−8) and suggestive associations (P = 1.0 × 10−6), respectively. Genetic variants are plotted by genomic position. Figure 1. View largeDownload slide Manhattan plot displaying the –log10(P values) for the association between IOP and the genotyped genetic variants. The solid and dotted horizontal lines represent genome-wide significant associations (P = 5 × 10−8) and suggestive associations (P = 1.0 × 10−6), respectively. Genetic variants are plotted by genomic position. Results from imputed genetic variants Using imputed data, we identified 16 854 genetic variants significantly associated with IOP (P < 5 × 10−8), representing 191 loci (Supplementary Material, Table S3). Among the identified regions, 145 are novel loci. Regional association plots were generated for the top four novel genes identified during the analysis of genotyped genetic variants (Fig. 2). After imputation, several imputed genetic variants were more significant than the genotyped variants in all of the identified regions. Figure 2. View largeDownload slide Regional association plots for the top four novel genes associated with IOP. For (A) LMX1B, (B) NR1H3, (C) MADD and (D) SEPT9, the most significant genotyped SNP is plotted in purple. Squares and circles represent genotyped and imputed genetic variants, respectively. Genes are shown below the SNPs with arrows indicating the strand orientation for each gene. The color-coding in each plot represents the level of linkage disequilibrium with the lead SNP in each plot. Figure 2. View largeDownload slide Regional association plots for the top four novel genes associated with IOP. For (A) LMX1B, (B) NR1H3, (C) MADD and (D) SEPT9, the most significant genotyped SNP is plotted in purple. Squares and circles represent genotyped and imputed genetic variants, respectively. Genes are shown below the SNPs with arrows indicating the strand orientation for each gene. The color-coding in each plot represents the level of linkage disequilibrium with the lead SNP in each plot. Conditional analysis We performed conditional analyses on the top four novel genes associated with IOP to determine whether additional genetic variants contribute to the IOP association. After conditioning on the most significant genetic variant in SEPT9 and MADD respectively, all neighboring SNP associations reduced toward the null, suggesting the identified genetic variants are the lead marker of the IOP associations. For the NR1H3 conditional analysis, we identified an additional independent variant, rs60515486, associated with IOP, which suggests additional evidence of association in this region. For the LMX1B conditional analysis, we identified two additional independent variants, rs11795066 and chr9: 129386031, associated with IOP. After conditioning on the lead and secondary genetic variants, the associations for the neighboring SNPs reduced toward the null (Supplementary Material, Fig. S2). Replication of findings To replicate our significant GWAS findings, we compared our results with the summary statistics provided by Springelkamp et al. (16). We used the simpleM method (18–20) to identify the effective number of independent tests and adjusted for multiple testing. Out of 671 genotype significant variants, 651 overlapped with the Springelkamp et al. (16) data, 632 of which were in same effect direction [409 (65%) with nominal significance P < 0.05]. Using the simpleM method, we estimated the effective number of tests and calculated the adjusted Bonferroni correction threshold as P < 1.0 × 10−4. A total of 89 SNPs were replicated in the Springelkamp data at the P < 1.0 × 10−4 significance level. They mapped to 69 unique genes and 55 of them are novel. Out of all 16 854 imputed significant variants, 15 062 were present in Springelkamp et al. (16) data, 14 587 of which exhibited the same direction of effect [9196 (63%) with nominal significance P < 0.05]. A total of 2464 were significant at the P < 1.0 × 10−4 significance level. A total of 160 unique genes, 131 of which are novel, were replicated in the Springelkamp data. Analysis of previously reported IOP loci We evaluated 95 previously reported IOP genetic variants using this study population. Through simpleM (18–20), we identified 76 independent tests yielding a corrected P of 6.6 × 10−4 (0.05/76). Of the previously reported genetic variants, 71 variants were significant and 64 (67.4%) exhibited consistent direction of effects in the current study (Supplementary Material, Table S4). To replicate genomic regions associated with IOP, we examined genetic variants surrounding the previously reported variants. Using this approach and threshold, we replicated 74 regions. IOP heritability estimates We estimated the heritability of IOP among the study participants using BOLT-REML (21). The estimated heritability of IOP using all genotyped genetic variants was 40.4%. When restricted to only significant genetic variants (n = 671), these variants explained 7.2% of the variance. Pleiotropy among significant loci To assess the pleiotropic nature of significant IOP loci with a replicable significant threshold of 5 × 10−8, we examined these loci in the GWAS Catalog. Of the 671 variants (found in 149 unique loci from significant genotyped variants) examined, 68 SNPs had exact matches in the GWAS Catalog (31 January 2018 version). Figure 3 presents the network of related traits and diseases for these 68 SNPs. Numerous neurological disorders related to eye diseases were directly matched to the included SNPs, such as primary open-angle, primary angle closure and high pressure glaucoma, as well as age-related macular degeneration. Moreover, ocular parameters, including central corneal thickness, axial length, optic cup area and iris characteristics, were also mapped. Additionally, SNPs matched to digestive and immune disorders, cancer, and cardiovascular and hematological measurements, including blood pressure, body mass index and type 2 diabetes (Supplementary Material, Table S5). Figure 3. View largeDownload slide A network of the pleiotropy effects of our genome-wide significant SNPs in the GWAS Catalog. A network of traits and diseases for 68 SNPs that matched directly to other phenotypes through the GWAS Catalog. Yellow, orange and red ovals denote SNPs, individual traits and categories, respectively. Figure 3. View largeDownload slide A network of the pleiotropy effects of our genome-wide significant SNPs in the GWAS Catalog. A network of traits and diseases for 68 SNPs that matched directly to other phenotypes through the GWAS Catalog. Yellow, orange and red ovals denote SNPs, individual traits and categories, respectively. Enrichment analysis Table 3 presents the top five GLAD4U diseases and Reactome pathways from WebGestalt using genes derived directly from genotyped genetic variants. The most significant GLAD4U disease associated with IOP is glaucoma (P = 9.59 × 10−5), open-angle glaucoma (P = 9.59 × 10−5), ocular hypertension (P = 3.56 × 10−4), von Willebrand disease (P = 2.67 × 10−3) and ectopia lentis (P = 6.07 × 10−3). Unsurprisingly, the top three diseases relate to glaucoma and have corresponding risk factors. Moreover, the von Willebrand factor is present in the endothelium of Schlemm’s canal and was upregulated in primary open-angle glaucoma cases (22). Mutations in several genes have also been associated with both glaucoma and ectopic lentis, suggesting similar biological mechanisms underlying these two conditions (23). The most significant Reactome pathway associated with IOP is the olfactory signaling pathway (P = 3.91 × 10−3), followed by defective B3GALTL causes Peters-plus syndrome (P = 5.83 × 10−3), O-glycosylation of TSR domain-containing proteins (P = 5.83 × 10−3), ABC transporters in lipid homeostasis (P = 2.93 × 10−2) and extracellular matrix organization (P = 2.93 × 10−2). Olfactory functioning is associated with several neurological diseases, including glaucoma, suggesting that glaucoma may be associated with multisensory manifestations (24). Peters-plus syndrome, an autosomal recessive disease characterized by ocular abnormalities, short stature and developmental delays, is associated with numerous eye conditions, including glaucoma (25). The thrombospondin type 1 repeat superfamily plays a role in neuronal development and ocular homeostasis (26,27). Several ABC transporters have been associated with ocular traits, including IOP (16,28). The extracellular matrix turnover in the trabecular meshwork has been suggested to regulate IOP (29). En masse, results from the enrichment analysis identified biologically relevant diseases and pathways related to IOP. Table 3. Top enrichment results associated with IOP   Term  Observed/total genes  P-value  Adjusted P-value  Disease    Glaucoma  12/133  7.64 × 10−8  9.59 × 10−5    Glaucoma, open angle  10/86  8.61 × 10−8  9.59 × 10−5    Ocular hypertension  10/103  4.80 × 10−7  3.56 × 10−4    von Willebrand disease  7/56  4.80 × 10−6  2.67 × 10−3    Ectopia lentis  4/13  1.36 × 10−5  6.07 × 10−3  Reactome    Olfactory signaling pathway  19/387  3.00 × 10−6  3.91 × 10−3    Defective B3GALTL causes Peters-plus syndrome  6/35  9.59 × 10−6  5.83 × 10−3    O-glycosylation of TSR domain-containing proteins  6/37  1.34 × 10−5  5.83 × 10−3    ABC transports in lipid homeostasis  4/18  1.10 × 10−4  2.93 × 10−2    Extracellular matrix organization  14/300  1.12 × 10−4  2.93 × 10−2    Term  Observed/total genes  P-value  Adjusted P-value  Disease    Glaucoma  12/133  7.64 × 10−8  9.59 × 10−5    Glaucoma, open angle  10/86  8.61 × 10−8  9.59 × 10−5    Ocular hypertension  10/103  4.80 × 10−7  3.56 × 10−4    von Willebrand disease  7/56  4.80 × 10−6  2.67 × 10−3    Ectopia lentis  4/13  1.36 × 10−5  6.07 × 10−3  Reactome    Olfactory signaling pathway  19/387  3.00 × 10−6  3.91 × 10−3    Defective B3GALTL causes Peters-plus syndrome  6/35  9.59 × 10−6  5.83 × 10−3    O-glycosylation of TSR domain-containing proteins  6/37  1.34 × 10−5  5.83 × 10−3    ABC transports in lipid homeostasis  4/18  1.10 × 10−4  2.93 × 10−2    Extracellular matrix organization  14/300  1.12 × 10−4  2.93 × 10−2  Table 3. Top enrichment results associated with IOP   Term  Observed/total genes  P-value  Adjusted P-value  Disease    Glaucoma  12/133  7.64 × 10−8  9.59 × 10−5    Glaucoma, open angle  10/86  8.61 × 10−8  9.59 × 10−5    Ocular hypertension  10/103  4.80 × 10−7  3.56 × 10−4    von Willebrand disease  7/56  4.80 × 10−6  2.67 × 10−3    Ectopia lentis  4/13  1.36 × 10−5  6.07 × 10−3  Reactome    Olfactory signaling pathway  19/387  3.00 × 10−6  3.91 × 10−3    Defective B3GALTL causes Peters-plus syndrome  6/35  9.59 × 10−6  5.83 × 10−3    O-glycosylation of TSR domain-containing proteins  6/37  1.34 × 10−5  5.83 × 10−3    ABC transports in lipid homeostasis  4/18  1.10 × 10−4  2.93 × 10−2    Extracellular matrix organization  14/300  1.12 × 10−4  2.93 × 10−2    Term  Observed/total genes  P-value  Adjusted P-value  Disease    Glaucoma  12/133  7.64 × 10−8  9.59 × 10−5    Glaucoma, open angle  10/86  8.61 × 10−8  9.59 × 10−5    Ocular hypertension  10/103  4.80 × 10−7  3.56 × 10−4    von Willebrand disease  7/56  4.80 × 10−6  2.67 × 10−3    Ectopia lentis  4/13  1.36 × 10−5  6.07 × 10−3  Reactome    Olfactory signaling pathway  19/387  3.00 × 10−6  3.91 × 10−3    Defective B3GALTL causes Peters-plus syndrome  6/35  9.59 × 10−6  5.83 × 10−3    O-glycosylation of TSR domain-containing proteins  6/37  1.34 × 10−5  5.83 × 10−3    ABC transports in lipid homeostasis  4/18  1.10 × 10−4  2.93 × 10−2    Extracellular matrix organization  14/300  1.12 × 10−4  2.93 × 10−2  Discussion In this study, we conducted the largest GWAS of IOP to date using data from the UK Biobank. Out of the 671 directly genotyped variants associated with IOP we discovered that mapped to 149 loci, 103 loci were novel. This discovery more than doubled the number of known IOP loci. The most significant of these novel genes are LMX1B, NR1H3, MADD and SEPT9. We also replicated 74 previously identified regions associated with IOP. Moreover, results from our enrichment analysis identified several biologically relevant diseases and pathways associated with IOP. Findings from this study demonstrate the polygenic and pleiotrophic nature of IOP loci. Large biobanks have provided an invaluable resource for uncovering the missing heritability of traits and furthering our understanding of the genetic architecture of complex traits. Early GWASs of IOP consisted of several thousand study participants and identified numerous common variants with large effect sizes. These studies, however, were powered to primarily identify the ‘low hanging fruits.’ As such, several strategies hypothesized to identify the ‘mid-hanging fruits’ to uncover the remaining heritability. One strategy included increasing the sample size to increase the statistical power to identify novel variants. By using a sample size nearly twice as large as the most recent IOP GWAS, all genotyped variants were able to account for 40.4% of IOP heritability, while genome-wide significant variants explained 7.2%. Our study illustrates the ability to harvest ‘mid-hanging fruits’ for complex traits by increasing the sample size of a study. The most significant novel genes associated with IOP were LMX1B and NR1H3. LMX1B is a member of the LIM-homeodomain family of transcription factors and plays an essential role in the normal development of serotonergic neurons and the anterior segment of the eye (30,31). Mutations in LMX1B are associated with nail-patella syndrome, of which ocular hypertension and glaucoma are frequently seen in individuals with this syndrome (32,33). Moreover, LMX1B haplotypes were shown to influence glaucoma susceptibility, indicating that alterations in LMX1B may result in glaucomatous damage (34). Transcripts of LMX1B were also detected in numerous ocular tissues, including the ciliary body and the trabecular meshwork, tissues related to the production and drainage of the aqueous humor, respectively (31). NR1H3, also known as the liver x receptor alpha, belongs to the nuclear receptor superfamily that regulates lipid homeostasis and inflammation and may be associated with neuronal degeneration (35). Moreover, activation of the liver x receptors mitigate ocular inflammation and decrease the expression of proinflammatory genes (36). Together, these novel genes represent biologically relevant genes for IOP and may further our understanding of the underlying mechanisms regulating this trait. The third and fourth most significant novel genes were MADD and SEPT9. MADD, from the MAP kinase activating death domain, encodes a protein that interacts with tumor necrosis factor alpha receptor 1 and aids in apoptosis (37). Protein isoforms of MADD were upregulated in glaucomatous retinas (38). Similarly, tumor necrosis factor alpha receptor 1 and the optic nerve head were also upregulated in glaucomatous retinas (38–40). SEPT9 is a member of the septin family, a group of proteins implicated in the formation of the cytoskeleton, cell division and cancer, which interacts with the SEPT2 and SEPT7 proteins. SEPT2, SEPT7 and SEPT9 form a complex in the primary cilium of retinal pigmented epithelial cells (41). Moreover, both the SEPT2 and SEPT7 proteins were detected in the optic nerve head of glaucoma cases and controls (42). These findings suggest that MADD and SEPT9 may relate to pathways associated with neurodegeneration and photoreception. Understanding the pleiotropy of human traits is increasingly important, given the movement toward precision medicine (43). Apropos to the current study, numerous genes associated with IOP also relate to other ocular parameters and systemic factors. Blood pressure is directly associated with IOP and individuals with hypertension have a higher risk of glaucoma (44,45). Such findings suggest that blood pressure treatment may influence IOP and subsequently, an individual’s risk for glaucoma. Similarly, body mass index is positively associated with IOP and interventions to modify body mass index may lead to reductions in IOP (6). Moreover, individuals with diabetes exhibit higher IOP compared with those without diabetes and diabetics are more susceptible to glaucoma (5,46). As such, diabetes interventions may also impact IOP and glaucoma risk. Approved drugs meant for treating a certain disease may possibly be used for IOP management through repositioning based on pleiotropy information. Evidently, elucidating the pleiotropic nature of human traits will uncover the effect of a genetic variant on multiple biological pathways of different traits. This may lead to novel insights for disease prevention and treatment. Despite the aforementioned findings, this study is not without its limitations. Our study did not focus on rare variants with MAF < 0.5%, which may be more appropriate for sequencing data. With the advent of low cost sequencing, however, the ability to discover rare variants will uncover additional heritability. Also, we do not have IOP medication information. Although this is a prospective cohort study and the prevalence of glaucoma is low in our data, the effect of this omission is likely to be insignificant and small. Additionally, the study sample for this analysis consisted of European participants and our findings may not be fully generalizable to other ethnic groups. The nonreplication of some genome-wide significant loci may be due to the limited sample size and different study design of the replication set available. Studies in other ethnic populations are needed to evaluate the transferability of these findings and may lead to the identification of additional novel genetic variants. In conclusion, in the largest GWAS of IOP, we identified 103 novel loci associated with IOP using data from the UK Biobank, substantially increasing the number of loci associated with this trait. In addition, we were able to replicate numerous associations with previously reported IOP genomic regions. We also identified pleiotropic genes that relate to factors associated with IOP, as well as to biologically relevant diseases and pathways through the enrichment analysis. This study identified multiple novel genes associated with IOP, furthering our understanding of the biological mechanisms regulating this trait and potentially, the pathogenesis of glaucoma. Materials and Methods Ethics statement The UK Biobank received approval from the North West Multi-centre Research Ethics Committee and all study participants provided informed consent. Study sample This study was performed using de-identified data from the UK Biobank, as described elsewhere previously (47,48). Briefly, the UK Biobank is a population-based prospective study of 500 000 study participants living in the United Kingdom. Participants between 40 and 69 years of age who were registered with the National Health Service and living less than 25 miles from an assessment center were recruited. Baseline data, collected between 2006 and 2010, consisted of questionnaires regarding lifestyle and medical history, physical measurements, and collection of blood, saliva and urine samples. Additionally, 117 649 study participants at six centers partook in the eye and vision component of the study, where numerous ocular measurements were obtained, including IOP. For the current analysis, we included 115 486 participants who self-reported as white. IOP measurements Initial IOP measurements for each eye were obtained with the Ocular Response Analyzer (Reichert Corp., Philadelphia, PA) between 2009 and 2010, with a repeat assessment between 2012 and 2013. The average Goldmann-corrected IOP of both eyes was used for downstream analysis. If only one IOP measurement was obtained, this measurement was used as a surrogate for the final value. If IOP measurements were available from the repeat assessment, the average of the IOP measurements from the first and second visit was used as the final IOP measurement (Supplementary Material, Fig. S3). The use of a long-term average for quantitative traits has been shown to increase statistical power (49). Study participants who received eye surgery within 4 weeks prior to the ocular assessment or those with possible eye infections did not receive IOP measurements. Furthermore, we excluded study participants with extreme IOP mean values, i.e. IOP < 7 mmHg and IOP > 40 mmHg. Genotyping, imputation and quality control The latest genetic data released on March 2018 was used for this analysis. The genotyping, imputation and quality control protocols are described elsewhere (50). Briefly, this data release includes 488 377 UK Biobank study participants genotyped on either the UK Biobank Axiom Array (825 927 markers; n = 438 427) or the UK BiLEVE Axiom Array (807 411 markers; n = 49 950). Quality control was performed on the genetic markers and individual samples. Individual markers were tested for batch, plate, sex and array effects, as well as departures from the Hardy–Weinberg equilibrium (HWE) and discordance across control replicates and were either excluded or set to missing. Moreover, a small set of duplicate samples were removed. After the above quality control parameters, 805 426 markers from both arrays for 488 377 participants were available in the release. In the current study, autosomal genotyped genetic variants with a minor allele frequency (MAF) ≥ 0.5% and HWE ≥ 1 × 10−12 were retained for downstream analysis, yielding 681 497 variants. We further used imputed genotypes to integrate additional genetic variants not directly genotyped on either array. Phasing of the overlapping genetic variants from the two arrays was performed using SHAPEIT3 (51) with the 1000 Genomes Project (phase 3) dataset. Imputation was conducted using IMPUTE4, a modified version of IMPUTE2, and the 1000 Genomes Project (phase 3), UK10K and Haplotype Reference Consortium reference panels, resulting in 92 693 895 autosomal SNPs, short indels and large structural variants. We applied further quality control parameters to exclude low quality variants (info score < 0.3) and rare variants (MAF < 0.5%), resulting in approximately 11.9 million variants for further analysis. Statistical analysis We performed genetic association analyses using linear mixed models implemented by BOLT-LMM (v2.3) (52), accounting for population structure and cryptic relatedness. Assuming an additive genetic model, we adjusted for age, sex, genotyping array and the first 10 principal components of genetic ancestry (as provided in the data release). Genetic variants with P < 5 × 10−8 were declared genome-wide significant. Independent loci were identified using the PLINK (53) clumping procedure (–clump-r2 0.000001–clump-kb 500). We performed LD score regression using LDSC (v1.0.0) (54). To identify the effective number of independent tests to correct for multiple testing during replication, we used the program simpleM (18–20). SNPs with effects in the same direction as the discovery and P < 0.05 were declared nominally suggestive. Conditional analyses were performed by including the lead genetic variant into the regression model for identified genomic loci. We estimated the heritability of IOP using BOLT-REML (21) and GCTA (55). Enrichment analysis To identify diseases and biological pathways associated with IOP, we performed gene-based association tests and enrichment analysis among unrelated study participants using SKAT-O (56) and WebGestalt (57), respectively. We mapped all genotyped SNPs to genes according to their hg19 assembly genomic position. To capture regulatory elements, we extended gene boundaries to include SNPs ± 50 kb of a gene. For running SKAT-O (56), we adjusted for age, gender and the first 10 principal components of genetic ancestry. Enrichment of GLAD4U, a tool that prioritizes gene lists related to diseases from PubMed, diseases and Reactome pathways associated with IOP were performed using WebGestalt (57). Biological terms were declared significant and reported if P < 0.05 after Benjamin–Hochberg multiple testing correction. Supplementary Material Supplementary Material is available at HMG online. Web Resources The URLs for downloaded data and programs: ANNOVAR, http://annovar.openbioinformatics.org/ BOLT-LMM, https://data.broadinstitute.org/alkesgroup/BOLT-LMM/ GCTA, http://cnsgenomics.com/software/gcta/ GWAS Catalog, https://www.ebi.ac.uk/gwas/ PLINK, https://www.cog-genomics.org/plink2 simpleM, http://simplem.sourceforge.net WebGestalt, http://www.webgestalt.org/LocusZoom, http://locuszoom.org/ Acknowledgements We thank the study participants from the UK Biobank and the staff who aided in data collection and processing. Conflict of Interest statement: None declared. Funding This work was supported by National Institutes of Health (NIH; Bethesda, MD) grants R01EY022651, R01EY027315 and P30EY001792 (departmental core grant). The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH. References 1 Weinreb R.N., Leung C.K., Crowston J.G., Medeiros F.A., Friedman D.S., Wiggs J.L., Martin K.R. ( 2016) Primary open-angle glaucoma. Nat. Rev. Dis. Primers , 2, 16067. Google Scholar CrossRef Search ADS PubMed  2 Collaborative Normal-Tension Glaucoma Study Group. ( 1998) Comparison of glaucomatous progression between untreated patients with normal-tension glaucoma and patients with therapeutically reduced intraocular pressures. Collaborative Normal-Tension Glaucoma Study Group. Am. J. Ophthalmol ., 126, 487– 497. CrossRef Search ADS PubMed  3 Kass M.A., Heuer D.K., Higginbotham E.J., Johnson C.A., Keltner J.L., Miller J.P., Parrish R.K.2nd, Wilson M.R., Gordon M.O. 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Genome-wide association analyses identify new loci influencing intraocular pressure

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Abstract

Abstract Elevated intraocular pressure (IOP) is a significant risk factor for glaucoma, the leading cause of irreversible blindness worldwide. While previous studies have identified numerous genetic variants associated with IOP, these loci only explain a fraction of IOP heritability. Recently established of biobank repositories have resulted in large amounts of data, enabling the identification of the remaining heritability for complex traits. Here, we describe the largest genome-wide association study of IOP to date using participants of European ancestry from the UK Biobank. We identified 671 directly genotyped variants that are significantly associated with IOP (P < 5 × 10−8). In addition to 103 novel loci, the top ranked novel IOP genes are LMX1B, NR1H3, MADD and SEPT9. We replicated these findings in an external population and examined the pleiotropic nature of these loci. These discoveries not only further our understanding of the genetic architecture of IOP, but also shed new light on the biological processes underlying glaucoma. Introduction Intraocular pressure (IOP) is the amount of fluid pressure in the eye which is mainly determined by the balance between aqueous humor production by the ciliary body and drainage through the trabecular meshwork (1). Elevated IOP is a major risk factor for glaucoma, a group of progressive optic neuropathies and the leading cause of irreversible blindness worldwide. Currently, IOP is the only modifiable risk factor for glaucoma and lowering IOP helps to prevent the onset and delay the progression of glaucoma (2–4). Identifying factors that contribute to IOP may aid in uncovering the biological mechanisms regulating this trait, provide new management avenues for IOP, and potentially glaucoma. Epidemiological studies have identified numerous factors associated with IOP, including age, body mass index, systolic blood pressure and type 2 diabetes (5–7). In addition to these systemic factors, studies have provided evidence of a genetic component for IOP determination, with heritability estimates ranging from 29% to 67% (8,9). Several genome-wide association studies (GWASs) have been conducted to identify genetic variants associated with IOP. Initial GWASs identified many genetic loci associated with IOP, including GAS7, TMCO1, GLCCI1-ICA1, ADAMTS18-NUDT7, FOXP1, FAM125B and ARHGEF12 (10–13). Meta-analyses consisting of subjects of European and Asian descent identified additional genomic regions, such as CAV1-CAV2, ABO, FNDC3B, RAPSN, PKHD1 and ADAMTS8 (14–16). Moreover, a recent multi-ethnic meta-analysis identified HIVEP3, ANTXR1, AFAP1, ARID5B, FOXO1 and INCA1 as additional IOP-related loci (17). While these studies identified numerous loci associated with IOP, their findings only explain 2.80–3.66% of the variation in this trait (17), indicating that there are additional variants to discover. The recent establishment of large biobank repositories has resulted in a plethora of both clinical and genomic data. The scope and scale of these biobanks will enable researchers to investigate the association between genetic variants and a multitude of complex traits, increasing the statistical power to detect previously unidentified associations and consequently, to further uncover the remaining heritability for various traits. The UK Biobank is one of the largest biobanks containing both ophthalmologic and genetic data, making it possible to identify novel genetic variants associated with ocular traits, including IOP. Due to the large sample size of the UK Biobank, we believe examining genetic variants associated with IOP will yield novel genomic loci associated with this trait. Therefore, the purpose of this study is to perform a GWAS of IOP using data from the UK Biobank. To the best of our knowledge, the current study represents the largest GWAS of IOP. Findings from this study will elucidate the genetic variants regulating this trait, and subsequently, may further our understanding of the underlying biological mechanisms of glaucoma. Results Study sample The characteristics of the study sample are summarized in Table 1. Overall, a total of 115 486 European white study participants were included in this analysis. The mean (standard deviation) age and IOP are 58.1 (7.9) years and 15.9 (3.5) mmHg, respectively. The proportion of females in this study sample is 53.7%. On an average, male subjects exhibited higher IOP compared with females [16.0 (3.6) mmHg versus 15.8 (3.4) mmHg; P < 0.0001]. Table 1. Descriptive statistics of the study sample Sample size  Female, %  Age (years), mean (SD)  IOP (mmHg), mean (SD)  IOP (mmHg) range  115, 486  53.7  58.1 (7.9)  15.9 (3.5)  7.0–39.0  Sample size  Female, %  Age (years), mean (SD)  IOP (mmHg), mean (SD)  IOP (mmHg) range  115, 486  53.7  58.1 (7.9)  15.9 (3.5)  7.0–39.0  Table 1. Descriptive statistics of the study sample Sample size  Female, %  Age (years), mean (SD)  IOP (mmHg), mean (SD)  IOP (mmHg) range  115, 486  53.7  58.1 (7.9)  15.9 (3.5)  7.0–39.0  Sample size  Female, %  Age (years), mean (SD)  IOP (mmHg), mean (SD)  IOP (mmHg) range  115, 486  53.7  58.1 (7.9)  15.9 (3.5)  7.0–39.0  Genome-wide association results For this analysis, the genomic control inflation factor, λ, was 1.16 (Supplementary Material, Fig. S1). Inflation of the genomic control inflation factor may in part be due to the large sample size. The LD score regression intercept, an alternative approach to measure deviation, was 1.004, implying that inflation is primarily due to polygenicity and not confounding. Therefore, our analysis is properly controlled for population structure and cryptic relatedness. Figure 1 presents a Manhattan plot of the genome-wide P values for directly genotyped genetic variants. We identified 671 genome-wide significant genetic variants associated with IOP (P < 5 × 10−8), 66.2% of them are located within functional regions of the genome and 33.8% are located in intergenic regions (Supplementary Material, Table S1). Moreover, these genetic variants map to 149 loci (defined as ±500 kb), including 103 novel loci (Supplementary Material, Table S2). Table 2 presents the summary results for the top genotyped genetic variants for the most significant genes (P < 1 × 10−20). The most significant novel genetic variant is rs10760442 (P = 6.8 × 10−31, GRCh37/hg19 position 129383900), located on chromosome 9q33.3 in an intron of the LMX1B gene. The effect allele G [allele frequency (AF) = 0.63] is associated with an increase in IOP (ß = 0.16). The second most significant novel genetic variant, rs10838681 (P = 9.5 × 10−25, GRCh37/hg19 position 47275064), is located on chromosome 11p11.2 in an intron of NR1H3. The effect allele G (AF = 0.73) is associated with a decrease in IOP (ß = −0.16). The third most significant novel genetic variant is rs326214 (P = 1.0 × 10−23, GRCh37/hg19 position 47298360), located in an exon of MADD on chromosome 11p11.2. The effect allele G (AF = 0.32) is associated with an increase in IOP (ß = 0.15). The fourth most significant genetic variant is rs9038 (P = 1.5 × 10−23, GRCh37/hg19 position 75495397), located on chromosome 17q25.3 in the 3′-UTR of SEPT9. The effect allele T (AF = 0.61) is associated with an increase in IOP (ß = 0.14). Table 2. Lead genotyped SNPs for top-ranking genes associated with IOP             UK Biobank   Replication   SNP  Chr  Pos  Locus  Alleles  AF1  ß  SE  P  ß  SE  P  rs4656461  1  165687205  LOC440700-TMCO1  G/A  0.12  0.29  0.02  9.8 × 10−44  0.25  0.04  1.4 × 10−10  rs7555523  1  165718979  TMCO1  C/A  0.12  0.31  0.02  1.3 × 10−43  0.26  0.04  2.4 × 10−11  rs1700874  1  219182858  MIR548F3  A/G  0.93  0.28  0.03  2.7 × 10−26  0.18  0.05  5.5 × 10−4  rs4380442  3  171930381  FNDC3B  G/A  0.10  −0.26  0.02  1.1 × 10−28  −0.16  0.04  1.4 × 10−4  rs6816389  4  7864457  AFAP1  T/C  0.56  0.17  0.01  1.6 × 10−34  0.10  0.03  2.7 × 10−4  rs1363919  5  52625853  LOC257396-FST  T/G  0.52  0.13  0.01  6.5 × 10−24  0.08  0.03  1.5 × 10−3  rs7739648  6  1540483  FOXF2-FOXCUT  A/G  0.36  −0.14  0.01  3.6 × 10−21  −0.10  0.03  5.3 × 10−4  rs6946058  7  115839253  TFEC-TES  A/C  0.73  0.15  0.02  4.0 × 10−22  0.08  0.03  7.6 × 10−3  rs8940  7  116146074  CAV2  C/G  0.82  −0.23  0.02  3.6 × 10−38  −0.20  0.03  1.5 × 10−9  rs4236601  7  116162729  CAV2-CAV1  G/A  0.73  −0.22  0.02  1.0 × 10−42  −0.19  0.03  2.0 × 10−11  rs13270051  8  108288752  ANGPT1  C/T  0.87  0.23  0.02  9.4 × 10−31  0.09  0.04  1.6 × 10−2  rs1570204  9  4216751  GLIS3  T/C  0.65  −0.17  0.01  7.1 × 10−31  −0.11  0.03  8.2 × 10−5  rs10760442  9  129383900  LMX1B  G/A  0.63  0.16  0.01  6.8 × 10−31  0.11  0.03  1.0 × 10−4  rs12778514  10  63832279  ARID5B  G/A  0.59  0.14  0.01  1.0 × 10−25  0.09  0.03  1.2 × 10−3  rs1222926  10  95039397  CYP26A1-MYOF  C/A  0.36  0.15  0.01  8.5 × 10−27  0.08  0.03  2.7 × 10−3  rs7940065  11  770007  PDDC1  C/T  0.51  0.14  0.01  3.1 × 10−22  0.00  0.03  9.6 × 10−1  rs4963156  11  780827  LOC171391  C/T  0.54  0.14  0.01  3.6 × 10−23  0.00  0.03  9.0 × 10−1  rs10902223  11  817786  RPLP2-PNPLA2  C/T  0.44  0.14  0.01  7.1 × 10−21  0.02  0.03  4.1 × 10−1  rs10838681  11  47275064  NR1H3  G/A  0.73  −0.16  0.02  9.5 × 10−25  −0.11  0.03  1.2 × 10−4  rs326214  11  47298360  MADD  G/A  0.32  0.15  0.01  1.0 × 10−23  0.10  0.03  2.3 × 10−4  rs1052373  11  47354787  MYBPC3  C/T  0.68  −0.15  0.02  1.6 × 10−22  −0.10  0.03  1.7 × 10−4  rs3740689  11  47380593  SPI1  G/A  0.41  0.15  0.01  2.6 × 10−26  0.08  0.03  2.1 × 10−3  rs11824864  11  47411736  SPI1-MIR4487  A/G  0.86  −0.20  0.02  8.5 × 10−23  −0.16  0.04  7.4 × 10−6  rs111228939  11  47456067  PSMC3-RAPSN  T/C  0.87  −0.21  0.02  6.7 × 10−24  −0.21  0.05  3.0 × 10−6  rs4752843  11  47531884  CELF1  T/G  0.86  −0.19  0.02  3.6 × 10−22  −0.18  0.04  1.6 × 10−6  rs4147730  11  47605427  NDUFS3  G/A  0.86  −0.20  0.02  3.6 × 10−23  −0.18  0.04  1.0 × 10−6  rs4752805  11  48018355  PTPRJ  A/G  0.75  −0.15  0.02  1.3 × 10−22  −0.14  0.03  7.1 × 10−6  rs2305013  11  120340060  ARHGEF12  A/T  0.95  −0.32  0.03  4.9 × 10−25  −0.28  0.07  1.8 × 10−5  rs72755233  15  100692953  ADAMTS17  G/A  0.89  −0.31  0.02  4.1 × 10−45  −0.19  0.06  2.3 × 10−3  rs76953588  16  77611387  ADAMTS18-NUDT7  T/C  0.92  −0.26  0.03  9.8 × 10−26  −0.19  0.05  2.1 × 10−4  rs9938149  16  88331640  LOC101928880-ZNF469  C/A  0.37  −0.14  0.01  4.2 × 10−24  −0.07  0.03  7.9 × 10−3  rs12150284  17  10031090  GAS7  C/T  0.63  0.26  0.01  4.6 × 10−77  0.20  0.03  1.7 × 10−12  rs9038  17  75495397  SEPT9  T/C  0.61  0.14  0.01  1.5 × 10−23  0.07  0.03  2.1 × 10−2              UK Biobank   Replication   SNP  Chr  Pos  Locus  Alleles  AF1  ß  SE  P  ß  SE  P  rs4656461  1  165687205  LOC440700-TMCO1  G/A  0.12  0.29  0.02  9.8 × 10−44  0.25  0.04  1.4 × 10−10  rs7555523  1  165718979  TMCO1  C/A  0.12  0.31  0.02  1.3 × 10−43  0.26  0.04  2.4 × 10−11  rs1700874  1  219182858  MIR548F3  A/G  0.93  0.28  0.03  2.7 × 10−26  0.18  0.05  5.5 × 10−4  rs4380442  3  171930381  FNDC3B  G/A  0.10  −0.26  0.02  1.1 × 10−28  −0.16  0.04  1.4 × 10−4  rs6816389  4  7864457  AFAP1  T/C  0.56  0.17  0.01  1.6 × 10−34  0.10  0.03  2.7 × 10−4  rs1363919  5  52625853  LOC257396-FST  T/G  0.52  0.13  0.01  6.5 × 10−24  0.08  0.03  1.5 × 10−3  rs7739648  6  1540483  FOXF2-FOXCUT  A/G  0.36  −0.14  0.01  3.6 × 10−21  −0.10  0.03  5.3 × 10−4  rs6946058  7  115839253  TFEC-TES  A/C  0.73  0.15  0.02  4.0 × 10−22  0.08  0.03  7.6 × 10−3  rs8940  7  116146074  CAV2  C/G  0.82  −0.23  0.02  3.6 × 10−38  −0.20  0.03  1.5 × 10−9  rs4236601  7  116162729  CAV2-CAV1  G/A  0.73  −0.22  0.02  1.0 × 10−42  −0.19  0.03  2.0 × 10−11  rs13270051  8  108288752  ANGPT1  C/T  0.87  0.23  0.02  9.4 × 10−31  0.09  0.04  1.6 × 10−2  rs1570204  9  4216751  GLIS3  T/C  0.65  −0.17  0.01  7.1 × 10−31  −0.11  0.03  8.2 × 10−5  rs10760442  9  129383900  LMX1B  G/A  0.63  0.16  0.01  6.8 × 10−31  0.11  0.03  1.0 × 10−4  rs12778514  10  63832279  ARID5B  G/A  0.59  0.14  0.01  1.0 × 10−25  0.09  0.03  1.2 × 10−3  rs1222926  10  95039397  CYP26A1-MYOF  C/A  0.36  0.15  0.01  8.5 × 10−27  0.08  0.03  2.7 × 10−3  rs7940065  11  770007  PDDC1  C/T  0.51  0.14  0.01  3.1 × 10−22  0.00  0.03  9.6 × 10−1  rs4963156  11  780827  LOC171391  C/T  0.54  0.14  0.01  3.6 × 10−23  0.00  0.03  9.0 × 10−1  rs10902223  11  817786  RPLP2-PNPLA2  C/T  0.44  0.14  0.01  7.1 × 10−21  0.02  0.03  4.1 × 10−1  rs10838681  11  47275064  NR1H3  G/A  0.73  −0.16  0.02  9.5 × 10−25  −0.11  0.03  1.2 × 10−4  rs326214  11  47298360  MADD  G/A  0.32  0.15  0.01  1.0 × 10−23  0.10  0.03  2.3 × 10−4  rs1052373  11  47354787  MYBPC3  C/T  0.68  −0.15  0.02  1.6 × 10−22  −0.10  0.03  1.7 × 10−4  rs3740689  11  47380593  SPI1  G/A  0.41  0.15  0.01  2.6 × 10−26  0.08  0.03  2.1 × 10−3  rs11824864  11  47411736  SPI1-MIR4487  A/G  0.86  −0.20  0.02  8.5 × 10−23  −0.16  0.04  7.4 × 10−6  rs111228939  11  47456067  PSMC3-RAPSN  T/C  0.87  −0.21  0.02  6.7 × 10−24  −0.21  0.05  3.0 × 10−6  rs4752843  11  47531884  CELF1  T/G  0.86  −0.19  0.02  3.6 × 10−22  −0.18  0.04  1.6 × 10−6  rs4147730  11  47605427  NDUFS3  G/A  0.86  −0.20  0.02  3.6 × 10−23  −0.18  0.04  1.0 × 10−6  rs4752805  11  48018355  PTPRJ  A/G  0.75  −0.15  0.02  1.3 × 10−22  −0.14  0.03  7.1 × 10−6  rs2305013  11  120340060  ARHGEF12  A/T  0.95  −0.32  0.03  4.9 × 10−25  −0.28  0.07  1.8 × 10−5  rs72755233  15  100692953  ADAMTS17  G/A  0.89  −0.31  0.02  4.1 × 10−45  −0.19  0.06  2.3 × 10−3  rs76953588  16  77611387  ADAMTS18-NUDT7  T/C  0.92  −0.26  0.03  9.8 × 10−26  −0.19  0.05  2.1 × 10−4  rs9938149  16  88331640  LOC101928880-ZNF469  C/A  0.37  −0.14  0.01  4.2 × 10−24  −0.07  0.03  7.9 × 10−3  rs12150284  17  10031090  GAS7  C/T  0.63  0.26  0.01  4.6 × 10−77  0.20  0.03  1.7 × 10−12  rs9038  17  75495397  SEPT9  T/C  0.61  0.14  0.01  1.5 × 10−23  0.07  0.03  2.1 × 10−2  SNPs with a P < 1.0 × 10−20 are presented. Gene name is in boldface if the genetic variant is located within the gene. Genomic positions are according to GRCh37/hg19. Chr, chromosome; Pos, position; AF1, allele 1 frequency; SE, standard error. Table 2. Lead genotyped SNPs for top-ranking genes associated with IOP             UK Biobank   Replication   SNP  Chr  Pos  Locus  Alleles  AF1  ß  SE  P  ß  SE  P  rs4656461  1  165687205  LOC440700-TMCO1  G/A  0.12  0.29  0.02  9.8 × 10−44  0.25  0.04  1.4 × 10−10  rs7555523  1  165718979  TMCO1  C/A  0.12  0.31  0.02  1.3 × 10−43  0.26  0.04  2.4 × 10−11  rs1700874  1  219182858  MIR548F3  A/G  0.93  0.28  0.03  2.7 × 10−26  0.18  0.05  5.5 × 10−4  rs4380442  3  171930381  FNDC3B  G/A  0.10  −0.26  0.02  1.1 × 10−28  −0.16  0.04  1.4 × 10−4  rs6816389  4  7864457  AFAP1  T/C  0.56  0.17  0.01  1.6 × 10−34  0.10  0.03  2.7 × 10−4  rs1363919  5  52625853  LOC257396-FST  T/G  0.52  0.13  0.01  6.5 × 10−24  0.08  0.03  1.5 × 10−3  rs7739648  6  1540483  FOXF2-FOXCUT  A/G  0.36  −0.14  0.01  3.6 × 10−21  −0.10  0.03  5.3 × 10−4  rs6946058  7  115839253  TFEC-TES  A/C  0.73  0.15  0.02  4.0 × 10−22  0.08  0.03  7.6 × 10−3  rs8940  7  116146074  CAV2  C/G  0.82  −0.23  0.02  3.6 × 10−38  −0.20  0.03  1.5 × 10−9  rs4236601  7  116162729  CAV2-CAV1  G/A  0.73  −0.22  0.02  1.0 × 10−42  −0.19  0.03  2.0 × 10−11  rs13270051  8  108288752  ANGPT1  C/T  0.87  0.23  0.02  9.4 × 10−31  0.09  0.04  1.6 × 10−2  rs1570204  9  4216751  GLIS3  T/C  0.65  −0.17  0.01  7.1 × 10−31  −0.11  0.03  8.2 × 10−5  rs10760442  9  129383900  LMX1B  G/A  0.63  0.16  0.01  6.8 × 10−31  0.11  0.03  1.0 × 10−4  rs12778514  10  63832279  ARID5B  G/A  0.59  0.14  0.01  1.0 × 10−25  0.09  0.03  1.2 × 10−3  rs1222926  10  95039397  CYP26A1-MYOF  C/A  0.36  0.15  0.01  8.5 × 10−27  0.08  0.03  2.7 × 10−3  rs7940065  11  770007  PDDC1  C/T  0.51  0.14  0.01  3.1 × 10−22  0.00  0.03  9.6 × 10−1  rs4963156  11  780827  LOC171391  C/T  0.54  0.14  0.01  3.6 × 10−23  0.00  0.03  9.0 × 10−1  rs10902223  11  817786  RPLP2-PNPLA2  C/T  0.44  0.14  0.01  7.1 × 10−21  0.02  0.03  4.1 × 10−1  rs10838681  11  47275064  NR1H3  G/A  0.73  −0.16  0.02  9.5 × 10−25  −0.11  0.03  1.2 × 10−4  rs326214  11  47298360  MADD  G/A  0.32  0.15  0.01  1.0 × 10−23  0.10  0.03  2.3 × 10−4  rs1052373  11  47354787  MYBPC3  C/T  0.68  −0.15  0.02  1.6 × 10−22  −0.10  0.03  1.7 × 10−4  rs3740689  11  47380593  SPI1  G/A  0.41  0.15  0.01  2.6 × 10−26  0.08  0.03  2.1 × 10−3  rs11824864  11  47411736  SPI1-MIR4487  A/G  0.86  −0.20  0.02  8.5 × 10−23  −0.16  0.04  7.4 × 10−6  rs111228939  11  47456067  PSMC3-RAPSN  T/C  0.87  −0.21  0.02  6.7 × 10−24  −0.21  0.05  3.0 × 10−6  rs4752843  11  47531884  CELF1  T/G  0.86  −0.19  0.02  3.6 × 10−22  −0.18  0.04  1.6 × 10−6  rs4147730  11  47605427  NDUFS3  G/A  0.86  −0.20  0.02  3.6 × 10−23  −0.18  0.04  1.0 × 10−6  rs4752805  11  48018355  PTPRJ  A/G  0.75  −0.15  0.02  1.3 × 10−22  −0.14  0.03  7.1 × 10−6  rs2305013  11  120340060  ARHGEF12  A/T  0.95  −0.32  0.03  4.9 × 10−25  −0.28  0.07  1.8 × 10−5  rs72755233  15  100692953  ADAMTS17  G/A  0.89  −0.31  0.02  4.1 × 10−45  −0.19  0.06  2.3 × 10−3  rs76953588  16  77611387  ADAMTS18-NUDT7  T/C  0.92  −0.26  0.03  9.8 × 10−26  −0.19  0.05  2.1 × 10−4  rs9938149  16  88331640  LOC101928880-ZNF469  C/A  0.37  −0.14  0.01  4.2 × 10−24  −0.07  0.03  7.9 × 10−3  rs12150284  17  10031090  GAS7  C/T  0.63  0.26  0.01  4.6 × 10−77  0.20  0.03  1.7 × 10−12  rs9038  17  75495397  SEPT9  T/C  0.61  0.14  0.01  1.5 × 10−23  0.07  0.03  2.1 × 10−2              UK Biobank   Replication   SNP  Chr  Pos  Locus  Alleles  AF1  ß  SE  P  ß  SE  P  rs4656461  1  165687205  LOC440700-TMCO1  G/A  0.12  0.29  0.02  9.8 × 10−44  0.25  0.04  1.4 × 10−10  rs7555523  1  165718979  TMCO1  C/A  0.12  0.31  0.02  1.3 × 10−43  0.26  0.04  2.4 × 10−11  rs1700874  1  219182858  MIR548F3  A/G  0.93  0.28  0.03  2.7 × 10−26  0.18  0.05  5.5 × 10−4  rs4380442  3  171930381  FNDC3B  G/A  0.10  −0.26  0.02  1.1 × 10−28  −0.16  0.04  1.4 × 10−4  rs6816389  4  7864457  AFAP1  T/C  0.56  0.17  0.01  1.6 × 10−34  0.10  0.03  2.7 × 10−4  rs1363919  5  52625853  LOC257396-FST  T/G  0.52  0.13  0.01  6.5 × 10−24  0.08  0.03  1.5 × 10−3  rs7739648  6  1540483  FOXF2-FOXCUT  A/G  0.36  −0.14  0.01  3.6 × 10−21  −0.10  0.03  5.3 × 10−4  rs6946058  7  115839253  TFEC-TES  A/C  0.73  0.15  0.02  4.0 × 10−22  0.08  0.03  7.6 × 10−3  rs8940  7  116146074  CAV2  C/G  0.82  −0.23  0.02  3.6 × 10−38  −0.20  0.03  1.5 × 10−9  rs4236601  7  116162729  CAV2-CAV1  G/A  0.73  −0.22  0.02  1.0 × 10−42  −0.19  0.03  2.0 × 10−11  rs13270051  8  108288752  ANGPT1  C/T  0.87  0.23  0.02  9.4 × 10−31  0.09  0.04  1.6 × 10−2  rs1570204  9  4216751  GLIS3  T/C  0.65  −0.17  0.01  7.1 × 10−31  −0.11  0.03  8.2 × 10−5  rs10760442  9  129383900  LMX1B  G/A  0.63  0.16  0.01  6.8 × 10−31  0.11  0.03  1.0 × 10−4  rs12778514  10  63832279  ARID5B  G/A  0.59  0.14  0.01  1.0 × 10−25  0.09  0.03  1.2 × 10−3  rs1222926  10  95039397  CYP26A1-MYOF  C/A  0.36  0.15  0.01  8.5 × 10−27  0.08  0.03  2.7 × 10−3  rs7940065  11  770007  PDDC1  C/T  0.51  0.14  0.01  3.1 × 10−22  0.00  0.03  9.6 × 10−1  rs4963156  11  780827  LOC171391  C/T  0.54  0.14  0.01  3.6 × 10−23  0.00  0.03  9.0 × 10−1  rs10902223  11  817786  RPLP2-PNPLA2  C/T  0.44  0.14  0.01  7.1 × 10−21  0.02  0.03  4.1 × 10−1  rs10838681  11  47275064  NR1H3  G/A  0.73  −0.16  0.02  9.5 × 10−25  −0.11  0.03  1.2 × 10−4  rs326214  11  47298360  MADD  G/A  0.32  0.15  0.01  1.0 × 10−23  0.10  0.03  2.3 × 10−4  rs1052373  11  47354787  MYBPC3  C/T  0.68  −0.15  0.02  1.6 × 10−22  −0.10  0.03  1.7 × 10−4  rs3740689  11  47380593  SPI1  G/A  0.41  0.15  0.01  2.6 × 10−26  0.08  0.03  2.1 × 10−3  rs11824864  11  47411736  SPI1-MIR4487  A/G  0.86  −0.20  0.02  8.5 × 10−23  −0.16  0.04  7.4 × 10−6  rs111228939  11  47456067  PSMC3-RAPSN  T/C  0.87  −0.21  0.02  6.7 × 10−24  −0.21  0.05  3.0 × 10−6  rs4752843  11  47531884  CELF1  T/G  0.86  −0.19  0.02  3.6 × 10−22  −0.18  0.04  1.6 × 10−6  rs4147730  11  47605427  NDUFS3  G/A  0.86  −0.20  0.02  3.6 × 10−23  −0.18  0.04  1.0 × 10−6  rs4752805  11  48018355  PTPRJ  A/G  0.75  −0.15  0.02  1.3 × 10−22  −0.14  0.03  7.1 × 10−6  rs2305013  11  120340060  ARHGEF12  A/T  0.95  −0.32  0.03  4.9 × 10−25  −0.28  0.07  1.8 × 10−5  rs72755233  15  100692953  ADAMTS17  G/A  0.89  −0.31  0.02  4.1 × 10−45  −0.19  0.06  2.3 × 10−3  rs76953588  16  77611387  ADAMTS18-NUDT7  T/C  0.92  −0.26  0.03  9.8 × 10−26  −0.19  0.05  2.1 × 10−4  rs9938149  16  88331640  LOC101928880-ZNF469  C/A  0.37  −0.14  0.01  4.2 × 10−24  −0.07  0.03  7.9 × 10−3  rs12150284  17  10031090  GAS7  C/T  0.63  0.26  0.01  4.6 × 10−77  0.20  0.03  1.7 × 10−12  rs9038  17  75495397  SEPT9  T/C  0.61  0.14  0.01  1.5 × 10−23  0.07  0.03  2.1 × 10−2  SNPs with a P < 1.0 × 10−20 are presented. Gene name is in boldface if the genetic variant is located within the gene. Genomic positions are according to GRCh37/hg19. Chr, chromosome; Pos, position; AF1, allele 1 frequency; SE, standard error. Figure 1. View largeDownload slide Manhattan plot displaying the –log10(P values) for the association between IOP and the genotyped genetic variants. The solid and dotted horizontal lines represent genome-wide significant associations (P = 5 × 10−8) and suggestive associations (P = 1.0 × 10−6), respectively. Genetic variants are plotted by genomic position. Figure 1. View largeDownload slide Manhattan plot displaying the –log10(P values) for the association between IOP and the genotyped genetic variants. The solid and dotted horizontal lines represent genome-wide significant associations (P = 5 × 10−8) and suggestive associations (P = 1.0 × 10−6), respectively. Genetic variants are plotted by genomic position. Results from imputed genetic variants Using imputed data, we identified 16 854 genetic variants significantly associated with IOP (P < 5 × 10−8), representing 191 loci (Supplementary Material, Table S3). Among the identified regions, 145 are novel loci. Regional association plots were generated for the top four novel genes identified during the analysis of genotyped genetic variants (Fig. 2). After imputation, several imputed genetic variants were more significant than the genotyped variants in all of the identified regions. Figure 2. View largeDownload slide Regional association plots for the top four novel genes associated with IOP. For (A) LMX1B, (B) NR1H3, (C) MADD and (D) SEPT9, the most significant genotyped SNP is plotted in purple. Squares and circles represent genotyped and imputed genetic variants, respectively. Genes are shown below the SNPs with arrows indicating the strand orientation for each gene. The color-coding in each plot represents the level of linkage disequilibrium with the lead SNP in each plot. Figure 2. View largeDownload slide Regional association plots for the top four novel genes associated with IOP. For (A) LMX1B, (B) NR1H3, (C) MADD and (D) SEPT9, the most significant genotyped SNP is plotted in purple. Squares and circles represent genotyped and imputed genetic variants, respectively. Genes are shown below the SNPs with arrows indicating the strand orientation for each gene. The color-coding in each plot represents the level of linkage disequilibrium with the lead SNP in each plot. Conditional analysis We performed conditional analyses on the top four novel genes associated with IOP to determine whether additional genetic variants contribute to the IOP association. After conditioning on the most significant genetic variant in SEPT9 and MADD respectively, all neighboring SNP associations reduced toward the null, suggesting the identified genetic variants are the lead marker of the IOP associations. For the NR1H3 conditional analysis, we identified an additional independent variant, rs60515486, associated with IOP, which suggests additional evidence of association in this region. For the LMX1B conditional analysis, we identified two additional independent variants, rs11795066 and chr9: 129386031, associated with IOP. After conditioning on the lead and secondary genetic variants, the associations for the neighboring SNPs reduced toward the null (Supplementary Material, Fig. S2). Replication of findings To replicate our significant GWAS findings, we compared our results with the summary statistics provided by Springelkamp et al. (16). We used the simpleM method (18–20) to identify the effective number of independent tests and adjusted for multiple testing. Out of 671 genotype significant variants, 651 overlapped with the Springelkamp et al. (16) data, 632 of which were in same effect direction [409 (65%) with nominal significance P < 0.05]. Using the simpleM method, we estimated the effective number of tests and calculated the adjusted Bonferroni correction threshold as P < 1.0 × 10−4. A total of 89 SNPs were replicated in the Springelkamp data at the P < 1.0 × 10−4 significance level. They mapped to 69 unique genes and 55 of them are novel. Out of all 16 854 imputed significant variants, 15 062 were present in Springelkamp et al. (16) data, 14 587 of which exhibited the same direction of effect [9196 (63%) with nominal significance P < 0.05]. A total of 2464 were significant at the P < 1.0 × 10−4 significance level. A total of 160 unique genes, 131 of which are novel, were replicated in the Springelkamp data. Analysis of previously reported IOP loci We evaluated 95 previously reported IOP genetic variants using this study population. Through simpleM (18–20), we identified 76 independent tests yielding a corrected P of 6.6 × 10−4 (0.05/76). Of the previously reported genetic variants, 71 variants were significant and 64 (67.4%) exhibited consistent direction of effects in the current study (Supplementary Material, Table S4). To replicate genomic regions associated with IOP, we examined genetic variants surrounding the previously reported variants. Using this approach and threshold, we replicated 74 regions. IOP heritability estimates We estimated the heritability of IOP among the study participants using BOLT-REML (21). The estimated heritability of IOP using all genotyped genetic variants was 40.4%. When restricted to only significant genetic variants (n = 671), these variants explained 7.2% of the variance. Pleiotropy among significant loci To assess the pleiotropic nature of significant IOP loci with a replicable significant threshold of 5 × 10−8, we examined these loci in the GWAS Catalog. Of the 671 variants (found in 149 unique loci from significant genotyped variants) examined, 68 SNPs had exact matches in the GWAS Catalog (31 January 2018 version). Figure 3 presents the network of related traits and diseases for these 68 SNPs. Numerous neurological disorders related to eye diseases were directly matched to the included SNPs, such as primary open-angle, primary angle closure and high pressure glaucoma, as well as age-related macular degeneration. Moreover, ocular parameters, including central corneal thickness, axial length, optic cup area and iris characteristics, were also mapped. Additionally, SNPs matched to digestive and immune disorders, cancer, and cardiovascular and hematological measurements, including blood pressure, body mass index and type 2 diabetes (Supplementary Material, Table S5). Figure 3. View largeDownload slide A network of the pleiotropy effects of our genome-wide significant SNPs in the GWAS Catalog. A network of traits and diseases for 68 SNPs that matched directly to other phenotypes through the GWAS Catalog. Yellow, orange and red ovals denote SNPs, individual traits and categories, respectively. Figure 3. View largeDownload slide A network of the pleiotropy effects of our genome-wide significant SNPs in the GWAS Catalog. A network of traits and diseases for 68 SNPs that matched directly to other phenotypes through the GWAS Catalog. Yellow, orange and red ovals denote SNPs, individual traits and categories, respectively. Enrichment analysis Table 3 presents the top five GLAD4U diseases and Reactome pathways from WebGestalt using genes derived directly from genotyped genetic variants. The most significant GLAD4U disease associated with IOP is glaucoma (P = 9.59 × 10−5), open-angle glaucoma (P = 9.59 × 10−5), ocular hypertension (P = 3.56 × 10−4), von Willebrand disease (P = 2.67 × 10−3) and ectopia lentis (P = 6.07 × 10−3). Unsurprisingly, the top three diseases relate to glaucoma and have corresponding risk factors. Moreover, the von Willebrand factor is present in the endothelium of Schlemm’s canal and was upregulated in primary open-angle glaucoma cases (22). Mutations in several genes have also been associated with both glaucoma and ectopic lentis, suggesting similar biological mechanisms underlying these two conditions (23). The most significant Reactome pathway associated with IOP is the olfactory signaling pathway (P = 3.91 × 10−3), followed by defective B3GALTL causes Peters-plus syndrome (P = 5.83 × 10−3), O-glycosylation of TSR domain-containing proteins (P = 5.83 × 10−3), ABC transporters in lipid homeostasis (P = 2.93 × 10−2) and extracellular matrix organization (P = 2.93 × 10−2). Olfactory functioning is associated with several neurological diseases, including glaucoma, suggesting that glaucoma may be associated with multisensory manifestations (24). Peters-plus syndrome, an autosomal recessive disease characterized by ocular abnormalities, short stature and developmental delays, is associated with numerous eye conditions, including glaucoma (25). The thrombospondin type 1 repeat superfamily plays a role in neuronal development and ocular homeostasis (26,27). Several ABC transporters have been associated with ocular traits, including IOP (16,28). The extracellular matrix turnover in the trabecular meshwork has been suggested to regulate IOP (29). En masse, results from the enrichment analysis identified biologically relevant diseases and pathways related to IOP. Table 3. Top enrichment results associated with IOP   Term  Observed/total genes  P-value  Adjusted P-value  Disease    Glaucoma  12/133  7.64 × 10−8  9.59 × 10−5    Glaucoma, open angle  10/86  8.61 × 10−8  9.59 × 10−5    Ocular hypertension  10/103  4.80 × 10−7  3.56 × 10−4    von Willebrand disease  7/56  4.80 × 10−6  2.67 × 10−3    Ectopia lentis  4/13  1.36 × 10−5  6.07 × 10−3  Reactome    Olfactory signaling pathway  19/387  3.00 × 10−6  3.91 × 10−3    Defective B3GALTL causes Peters-plus syndrome  6/35  9.59 × 10−6  5.83 × 10−3    O-glycosylation of TSR domain-containing proteins  6/37  1.34 × 10−5  5.83 × 10−3    ABC transports in lipid homeostasis  4/18  1.10 × 10−4  2.93 × 10−2    Extracellular matrix organization  14/300  1.12 × 10−4  2.93 × 10−2    Term  Observed/total genes  P-value  Adjusted P-value  Disease    Glaucoma  12/133  7.64 × 10−8  9.59 × 10−5    Glaucoma, open angle  10/86  8.61 × 10−8  9.59 × 10−5    Ocular hypertension  10/103  4.80 × 10−7  3.56 × 10−4    von Willebrand disease  7/56  4.80 × 10−6  2.67 × 10−3    Ectopia lentis  4/13  1.36 × 10−5  6.07 × 10−3  Reactome    Olfactory signaling pathway  19/387  3.00 × 10−6  3.91 × 10−3    Defective B3GALTL causes Peters-plus syndrome  6/35  9.59 × 10−6  5.83 × 10−3    O-glycosylation of TSR domain-containing proteins  6/37  1.34 × 10−5  5.83 × 10−3    ABC transports in lipid homeostasis  4/18  1.10 × 10−4  2.93 × 10−2    Extracellular matrix organization  14/300  1.12 × 10−4  2.93 × 10−2  Table 3. Top enrichment results associated with IOP   Term  Observed/total genes  P-value  Adjusted P-value  Disease    Glaucoma  12/133  7.64 × 10−8  9.59 × 10−5    Glaucoma, open angle  10/86  8.61 × 10−8  9.59 × 10−5    Ocular hypertension  10/103  4.80 × 10−7  3.56 × 10−4    von Willebrand disease  7/56  4.80 × 10−6  2.67 × 10−3    Ectopia lentis  4/13  1.36 × 10−5  6.07 × 10−3  Reactome    Olfactory signaling pathway  19/387  3.00 × 10−6  3.91 × 10−3    Defective B3GALTL causes Peters-plus syndrome  6/35  9.59 × 10−6  5.83 × 10−3    O-glycosylation of TSR domain-containing proteins  6/37  1.34 × 10−5  5.83 × 10−3    ABC transports in lipid homeostasis  4/18  1.10 × 10−4  2.93 × 10−2    Extracellular matrix organization  14/300  1.12 × 10−4  2.93 × 10−2    Term  Observed/total genes  P-value  Adjusted P-value  Disease    Glaucoma  12/133  7.64 × 10−8  9.59 × 10−5    Glaucoma, open angle  10/86  8.61 × 10−8  9.59 × 10−5    Ocular hypertension  10/103  4.80 × 10−7  3.56 × 10−4    von Willebrand disease  7/56  4.80 × 10−6  2.67 × 10−3    Ectopia lentis  4/13  1.36 × 10−5  6.07 × 10−3  Reactome    Olfactory signaling pathway  19/387  3.00 × 10−6  3.91 × 10−3    Defective B3GALTL causes Peters-plus syndrome  6/35  9.59 × 10−6  5.83 × 10−3    O-glycosylation of TSR domain-containing proteins  6/37  1.34 × 10−5  5.83 × 10−3    ABC transports in lipid homeostasis  4/18  1.10 × 10−4  2.93 × 10−2    Extracellular matrix organization  14/300  1.12 × 10−4  2.93 × 10−2  Discussion In this study, we conducted the largest GWAS of IOP to date using data from the UK Biobank. Out of the 671 directly genotyped variants associated with IOP we discovered that mapped to 149 loci, 103 loci were novel. This discovery more than doubled the number of known IOP loci. The most significant of these novel genes are LMX1B, NR1H3, MADD and SEPT9. We also replicated 74 previously identified regions associated with IOP. Moreover, results from our enrichment analysis identified several biologically relevant diseases and pathways associated with IOP. Findings from this study demonstrate the polygenic and pleiotrophic nature of IOP loci. Large biobanks have provided an invaluable resource for uncovering the missing heritability of traits and furthering our understanding of the genetic architecture of complex traits. Early GWASs of IOP consisted of several thousand study participants and identified numerous common variants with large effect sizes. These studies, however, were powered to primarily identify the ‘low hanging fruits.’ As such, several strategies hypothesized to identify the ‘mid-hanging fruits’ to uncover the remaining heritability. One strategy included increasing the sample size to increase the statistical power to identify novel variants. By using a sample size nearly twice as large as the most recent IOP GWAS, all genotyped variants were able to account for 40.4% of IOP heritability, while genome-wide significant variants explained 7.2%. Our study illustrates the ability to harvest ‘mid-hanging fruits’ for complex traits by increasing the sample size of a study. The most significant novel genes associated with IOP were LMX1B and NR1H3. LMX1B is a member of the LIM-homeodomain family of transcription factors and plays an essential role in the normal development of serotonergic neurons and the anterior segment of the eye (30,31). Mutations in LMX1B are associated with nail-patella syndrome, of which ocular hypertension and glaucoma are frequently seen in individuals with this syndrome (32,33). Moreover, LMX1B haplotypes were shown to influence glaucoma susceptibility, indicating that alterations in LMX1B may result in glaucomatous damage (34). Transcripts of LMX1B were also detected in numerous ocular tissues, including the ciliary body and the trabecular meshwork, tissues related to the production and drainage of the aqueous humor, respectively (31). NR1H3, also known as the liver x receptor alpha, belongs to the nuclear receptor superfamily that regulates lipid homeostasis and inflammation and may be associated with neuronal degeneration (35). Moreover, activation of the liver x receptors mitigate ocular inflammation and decrease the expression of proinflammatory genes (36). Together, these novel genes represent biologically relevant genes for IOP and may further our understanding of the underlying mechanisms regulating this trait. The third and fourth most significant novel genes were MADD and SEPT9. MADD, from the MAP kinase activating death domain, encodes a protein that interacts with tumor necrosis factor alpha receptor 1 and aids in apoptosis (37). Protein isoforms of MADD were upregulated in glaucomatous retinas (38). Similarly, tumor necrosis factor alpha receptor 1 and the optic nerve head were also upregulated in glaucomatous retinas (38–40). SEPT9 is a member of the septin family, a group of proteins implicated in the formation of the cytoskeleton, cell division and cancer, which interacts with the SEPT2 and SEPT7 proteins. SEPT2, SEPT7 and SEPT9 form a complex in the primary cilium of retinal pigmented epithelial cells (41). Moreover, both the SEPT2 and SEPT7 proteins were detected in the optic nerve head of glaucoma cases and controls (42). These findings suggest that MADD and SEPT9 may relate to pathways associated with neurodegeneration and photoreception. Understanding the pleiotropy of human traits is increasingly important, given the movement toward precision medicine (43). Apropos to the current study, numerous genes associated with IOP also relate to other ocular parameters and systemic factors. Blood pressure is directly associated with IOP and individuals with hypertension have a higher risk of glaucoma (44,45). Such findings suggest that blood pressure treatment may influence IOP and subsequently, an individual’s risk for glaucoma. Similarly, body mass index is positively associated with IOP and interventions to modify body mass index may lead to reductions in IOP (6). Moreover, individuals with diabetes exhibit higher IOP compared with those without diabetes and diabetics are more susceptible to glaucoma (5,46). As such, diabetes interventions may also impact IOP and glaucoma risk. Approved drugs meant for treating a certain disease may possibly be used for IOP management through repositioning based on pleiotropy information. Evidently, elucidating the pleiotropic nature of human traits will uncover the effect of a genetic variant on multiple biological pathways of different traits. This may lead to novel insights for disease prevention and treatment. Despite the aforementioned findings, this study is not without its limitations. Our study did not focus on rare variants with MAF < 0.5%, which may be more appropriate for sequencing data. With the advent of low cost sequencing, however, the ability to discover rare variants will uncover additional heritability. Also, we do not have IOP medication information. Although this is a prospective cohort study and the prevalence of glaucoma is low in our data, the effect of this omission is likely to be insignificant and small. Additionally, the study sample for this analysis consisted of European participants and our findings may not be fully generalizable to other ethnic groups. The nonreplication of some genome-wide significant loci may be due to the limited sample size and different study design of the replication set available. Studies in other ethnic populations are needed to evaluate the transferability of these findings and may lead to the identification of additional novel genetic variants. In conclusion, in the largest GWAS of IOP, we identified 103 novel loci associated with IOP using data from the UK Biobank, substantially increasing the number of loci associated with this trait. In addition, we were able to replicate numerous associations with previously reported IOP genomic regions. We also identified pleiotropic genes that relate to factors associated with IOP, as well as to biologically relevant diseases and pathways through the enrichment analysis. This study identified multiple novel genes associated with IOP, furthering our understanding of the biological mechanisms regulating this trait and potentially, the pathogenesis of glaucoma. Materials and Methods Ethics statement The UK Biobank received approval from the North West Multi-centre Research Ethics Committee and all study participants provided informed consent. Study sample This study was performed using de-identified data from the UK Biobank, as described elsewhere previously (47,48). Briefly, the UK Biobank is a population-based prospective study of 500 000 study participants living in the United Kingdom. Participants between 40 and 69 years of age who were registered with the National Health Service and living less than 25 miles from an assessment center were recruited. Baseline data, collected between 2006 and 2010, consisted of questionnaires regarding lifestyle and medical history, physical measurements, and collection of blood, saliva and urine samples. Additionally, 117 649 study participants at six centers partook in the eye and vision component of the study, where numerous ocular measurements were obtained, including IOP. For the current analysis, we included 115 486 participants who self-reported as white. IOP measurements Initial IOP measurements for each eye were obtained with the Ocular Response Analyzer (Reichert Corp., Philadelphia, PA) between 2009 and 2010, with a repeat assessment between 2012 and 2013. The average Goldmann-corrected IOP of both eyes was used for downstream analysis. If only one IOP measurement was obtained, this measurement was used as a surrogate for the final value. If IOP measurements were available from the repeat assessment, the average of the IOP measurements from the first and second visit was used as the final IOP measurement (Supplementary Material, Fig. S3). The use of a long-term average for quantitative traits has been shown to increase statistical power (49). Study participants who received eye surgery within 4 weeks prior to the ocular assessment or those with possible eye infections did not receive IOP measurements. Furthermore, we excluded study participants with extreme IOP mean values, i.e. IOP < 7 mmHg and IOP > 40 mmHg. Genotyping, imputation and quality control The latest genetic data released on March 2018 was used for this analysis. The genotyping, imputation and quality control protocols are described elsewhere (50). Briefly, this data release includes 488 377 UK Biobank study participants genotyped on either the UK Biobank Axiom Array (825 927 markers; n = 438 427) or the UK BiLEVE Axiom Array (807 411 markers; n = 49 950). Quality control was performed on the genetic markers and individual samples. Individual markers were tested for batch, plate, sex and array effects, as well as departures from the Hardy–Weinberg equilibrium (HWE) and discordance across control replicates and were either excluded or set to missing. Moreover, a small set of duplicate samples were removed. After the above quality control parameters, 805 426 markers from both arrays for 488 377 participants were available in the release. In the current study, autosomal genotyped genetic variants with a minor allele frequency (MAF) ≥ 0.5% and HWE ≥ 1 × 10−12 were retained for downstream analysis, yielding 681 497 variants. We further used imputed genotypes to integrate additional genetic variants not directly genotyped on either array. Phasing of the overlapping genetic variants from the two arrays was performed using SHAPEIT3 (51) with the 1000 Genomes Project (phase 3) dataset. Imputation was conducted using IMPUTE4, a modified version of IMPUTE2, and the 1000 Genomes Project (phase 3), UK10K and Haplotype Reference Consortium reference panels, resulting in 92 693 895 autosomal SNPs, short indels and large structural variants. We applied further quality control parameters to exclude low quality variants (info score < 0.3) and rare variants (MAF < 0.5%), resulting in approximately 11.9 million variants for further analysis. Statistical analysis We performed genetic association analyses using linear mixed models implemented by BOLT-LMM (v2.3) (52), accounting for population structure and cryptic relatedness. Assuming an additive genetic model, we adjusted for age, sex, genotyping array and the first 10 principal components of genetic ancestry (as provided in the data release). Genetic variants with P < 5 × 10−8 were declared genome-wide significant. Independent loci were identified using the PLINK (53) clumping procedure (–clump-r2 0.000001–clump-kb 500). We performed LD score regression using LDSC (v1.0.0) (54). To identify the effective number of independent tests to correct for multiple testing during replication, we used the program simpleM (18–20). SNPs with effects in the same direction as the discovery and P < 0.05 were declared nominally suggestive. Conditional analyses were performed by including the lead genetic variant into the regression model for identified genomic loci. We estimated the heritability of IOP using BOLT-REML (21) and GCTA (55). Enrichment analysis To identify diseases and biological pathways associated with IOP, we performed gene-based association tests and enrichment analysis among unrelated study participants using SKAT-O (56) and WebGestalt (57), respectively. We mapped all genotyped SNPs to genes according to their hg19 assembly genomic position. To capture regulatory elements, we extended gene boundaries to include SNPs ± 50 kb of a gene. For running SKAT-O (56), we adjusted for age, gender and the first 10 principal components of genetic ancestry. Enrichment of GLAD4U, a tool that prioritizes gene lists related to diseases from PubMed, diseases and Reactome pathways associated with IOP were performed using WebGestalt (57). Biological terms were declared significant and reported if P < 0.05 after Benjamin–Hochberg multiple testing correction. Supplementary Material Supplementary Material is available at HMG online. Web Resources The URLs for downloaded data and programs: ANNOVAR, http://annovar.openbioinformatics.org/ BOLT-LMM, https://data.broadinstitute.org/alkesgroup/BOLT-LMM/ GCTA, http://cnsgenomics.com/software/gcta/ GWAS Catalog, https://www.ebi.ac.uk/gwas/ PLINK, https://www.cog-genomics.org/plink2 simpleM, http://simplem.sourceforge.net WebGestalt, http://www.webgestalt.org/LocusZoom, http://locuszoom.org/ Acknowledgements We thank the study participants from the UK Biobank and the staff who aided in data collection and processing. Conflict of Interest statement: None declared. 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Human Molecular GeneticsOxford University Press

Published: Mar 28, 2018

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