Cigarette Smoking Strongly Modifies the Association of LOC387715 and Age-Related Macular Degeneration
2006; Elsevier BV; Volume: 78; Issue: 5 Linguagem: Inglês
10.1086/503822
ISSN1537-6605
AutoresSilke Schmidt, Michael A. Hauser, William K. Scott, Eric A. Postel, Anita Agarwal, Paul J. Gallins, Frank Wong, Yu Sarah Chen, Kylee L. Spencer, Nathalie Schnetz‐Boutaud, Jonathan L. Haines, Margaret A. Pericak‐Vance,
Tópico(s)Antioxidant Activity and Oxidative Stress
ResumoWe used iterative association mapping to identify a susceptibility gene for age-related macular degeneration (AMD) on chromosome 10q26, which is one of the most consistently implicated linkage regions for this disorder. We employed linkage analysis methods, followed by family-based and case-control association analyses, using two independent data sets. To identify statistically the most likely AMD-susceptibility allele, we used the Genotype-IBD Sharing Test (GIST) and conditional haplotype analysis. To incorporate the two most important known AMD risk factors—smoking and the Y402H variant of the complement factor H gene (CFH)—we used logistic regression modeling to test for gene-gene and gene-environment interactions in the case-control data set and used the ordered-subset analysis to account for genetic linkage heterogeneity in the family-based data set. Our results strongly implicate a coding change (Ala69Ser) in the LOC387715 gene as the second major identified AMD-susceptibility allele, confirming earlier suggestions. This variant's effect on AMD is statistically independent of CFH and is of similar magnitude to the effect of Y402H. The overall effect is driven primarily by a strong association in smokers, since we observed significant evidence for a statistical interaction between the LOC387715 variant and a history of cigarette smoking. This gene-environment interaction is supported by statistically independent family-based and case-control analysis methods. We estimate that CFH, LOC387715, and cigarette smoking together explain 61% of the population-attributable risk (PAR) of AMD. The adjusted PAR percentage estimates are 20% for smoking, 36% for LOC387715, and 43% for CFH. We demonstrate, for the first time, that a genetic susceptibility coupled with a modifiable lifestyle factor such as cigarette smoking confers a significantly higher risk of AMD than either factor alone. We used iterative association mapping to identify a susceptibility gene for age-related macular degeneration (AMD) on chromosome 10q26, which is one of the most consistently implicated linkage regions for this disorder. We employed linkage analysis methods, followed by family-based and case-control association analyses, using two independent data sets. To identify statistically the most likely AMD-susceptibility allele, we used the Genotype-IBD Sharing Test (GIST) and conditional haplotype analysis. To incorporate the two most important known AMD risk factors—smoking and the Y402H variant of the complement factor H gene (CFH)—we used logistic regression modeling to test for gene-gene and gene-environment interactions in the case-control data set and used the ordered-subset analysis to account for genetic linkage heterogeneity in the family-based data set. Our results strongly implicate a coding change (Ala69Ser) in the LOC387715 gene as the second major identified AMD-susceptibility allele, confirming earlier suggestions. This variant's effect on AMD is statistically independent of CFH and is of similar magnitude to the effect of Y402H. The overall effect is driven primarily by a strong association in smokers, since we observed significant evidence for a statistical interaction between the LOC387715 variant and a history of cigarette smoking. This gene-environment interaction is supported by statistically independent family-based and case-control analysis methods. We estimate that CFH, LOC387715, and cigarette smoking together explain 61% of the population-attributable risk (PAR) of AMD. The adjusted PAR percentage estimates are 20% for smoking, 36% for LOC387715, and 43% for CFH. We demonstrate, for the first time, that a genetic susceptibility coupled with a modifiable lifestyle factor such as cigarette smoking confers a significantly higher risk of AMD than either factor alone. Age-related macular degeneration (AMD) is a common complex disorder that affects the central region of the retina (macula) and is the leading cause of legal blindness in white Americans aged >65 years. The prevalence of AMD and its significant morbidity will rise sharply as the population ages. AMD is a clinically heterogeneous disorder with a poorly understood etiology. Population-based longitudinal studies1Klaver CCW Assink JJM van Leeuwen R Wolfs RCW Vingerling JR Stijnen T Hofman A de Jong PTVM Incidence and progression rates of age-related maculopathy: the Rotterdam Study.Invest Ophthalmol Vis Sci. 2001; 42: 2237-2241PubMed Google Scholar, 2van Leeuwen R Klaver CC Vingerling JR Hofman A de Jong PT The risk and natural course of age-related maculopathy: follow-up at 6 1/2 years in the Rotterdam Study.Arch Ophthalmol. 2003; 121: 519-526Crossref PubMed Scopus (279) Google Scholar, 3Klein R Klein BE Tomany SC Cruickshanks KJ The association of cardiovascular disease with the long-term incidence of age-related maculopathy: the Beaver Dam Eye Study.Ophthalmology. 2003; 110: 1273-1280Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar have established that the presence of extracellular protein and/or lipid deposits (drusen) between the basal lamina of the retinal pigment epithelium (RPE) and the inner layer of the Bruch's membrane is associated with an increased risk of progressing to an advanced form of AMD, either geographic atrophy (dry AMD) or choroidal neovascularization (wet AMD). The presence of large and indistinct (soft) drusen, coupled with RPE abnormalities, is considered to be an early form of the disorder and is often referred to as “age-related maculopathy.” Epidemiologically, AMD is a complex disorder, with contributions of environmental factors and genetic susceptibility.4Klein R Peto T Bird A Vannewkirk MR The epidemiology of age-related macular degeneration.Am J Ophthalmol. 2004; 137: 486-495Abstract Full Text Full Text PDF PubMed Scopus (759) Google Scholar Many environmental and lifestyle factors have been postulated, but the strongest nongenetic risk factor for AMD is clearly cigarette smoking.5Smith W Assink J Klein R Mitchell P Klaver CC Klein BE Hofman A Jensen S Wang JJ de Jong PT Risk factors for age-related macular degeneration: pooled findings from three continents.Ophthalmology. 2001; 108: 697-704Abstract Full Text Full Text PDF PubMed Scopus (826) Google Scholar, 6Khan JC Thurlby DA Shahid H Clayton DG Yates JR Bradley M Moore AT Bird AC Smoking and age related macular degeneration: the number of pack years of cigarette smoking is a major determinant of risk for both geographic atrophy and choroidal neovascularisation.Br J Ophthalmol. 2006; 90: 75-80Crossref PubMed Scopus (298) Google Scholar Much progress has been made recently in identifying and characterizing the genetic basis of AMD. In a remarkable example of the convergence of methods for disease-gene discovery, multiple independent research efforts identified the Y402H variant in CFH (the complement factor H [MIM 134370]) on chromosome 1q32 as the first major AMD-susceptibility allele.7Haines JL Hauser MA Schmidt S Scott WK Olson LM Gallins P Spencer KL Kwan SY Noureddine M Gilbert JR Schnetz-Boutaud N Agarwal A Postel EA Pericak-Vance MA Complement factor H variant increases the risk of age-related macular degeneration.Science. 2005; 308: 419-421Crossref PubMed Scopus (2069) Google Scholar, 8Hageman GS Anderson DH Johnson LV Hancox LS Taiber AJ Hardisty LI Hageman JL et al.A common haplotype in the complement regulatory gene factor H (HF1/CFH) predisposes individuals to age-related macular degeneration.Proc Natl Acad Sci USA. 2005; 102: 7227-7232Crossref PubMed Scopus (1692) Google Scholar, 9Klein RJ Zeiss C Chew EY Tsai JY Sackler RS Haynes C Henning AK Sangiovanni JP Mane SM Mayne ST Bracken MB Ferris FL Ott J Barnstable C Hoh J Complement factor H polymorphism in age-related macular degeneration.Science. 2005; 308: 385-389Crossref PubMed Scopus (3515) Google Scholar, 10Edwards AO Ritter III, R Abel KJ Manning A Panhuysen C Farrer LA Complement factor H polymorphism and age-related macular degeneration.Science. 2005; 308: 421-424Crossref PubMed Scopus (2078) Google Scholar, 11Zareparsi S Branham KE Li M Shah S Klein RJ Ott J Hoh J Abecasis GR Swaroop A Strong association of the Y402H variant in complement factor H at 1q32 with susceptibility to age-related macular degeneration.Am J Hum Genet. 2005; 77: 149-153Abstract Full Text Full Text PDF PubMed Scopus (311) Google Scholar, 12Conley YP Thalamuthu A Jakobsdottir J Weeks DE Mah T Ferrell RE Gorin MB Candidate gene analysis suggests a role for fatty acid biosynthesis and regulation of the complement system in the etiology of age-related maculopathy.Hum Mol Genet. 2005; 14: 1991-2002Crossref PubMed Scopus (139) Google Scholar Whereas one of the studies was able to pinpoint CFH on the basis of a whole-genome association study,9Klein RJ Zeiss C Chew EY Tsai JY Sackler RS Haynes C Henning AK Sangiovanni JP Mane SM Mayne ST Bracken MB Ferris FL Ott J Barnstable C Hoh J Complement factor H polymorphism in age-related macular degeneration.Science. 2005; 308: 385-389Crossref PubMed Scopus (3515) Google Scholar most focused on the 1q32 region because it had been implicated consistently by several whole-genome linkage scans. A second genomic region with similarly consistent linkage evidence is chromosome 10q26, which was identified as the single most-promising region by a recent meta-analysis of published linkage screens.13Fisher SA Abecasis GR Yashar BM Zareparsi S Swaroop A Iyengar SK Klein BE Klein R Lee KE Majewski J Schultz DW Klein ML Seddon JM Santangelo SL Weeks DE Conley YP Mah TS Schmidt S Haines JL Pericak-Vance MA Gorin MB Schulz HL Pardi F Lewis CM Weber BH Meta-analysis of genome scans of age-related macular degeneration.Hum Mol Genet. 2005; 14: 2257-2264Crossref PubMed Scopus (194) Google Scholar Two recent studies have suggested specific AMD-susceptibility genes that are located on chromosome 10q26. One study used a combination of family-based and case-control analyses to implicate PLEKHA1 (pleckstrin homology domain–containing, family A, member 1 [MIM 607772]) and the predicted LOC387715 gene.14Jakobsdottir J Conley YP Weeks DE Mah TS Ferrell RE Gorin MB Susceptibility genes for age-related maculopathy on chromosome 10q26.Am J Hum Genet. 2005; 77: 389-407Abstract Full Text Full Text PDF PubMed Scopus (476) Google Scholar However, the association signals for SNPs in these two genes were statistically indistinguishable. The other study used two independent case-control data sets and concluded that the T allele of SNP rs10490924 in LOC387715, a coding change (Ala69Ser) in exon 1 of this poorly characterized gene, was the most likely AMD-susceptibility allele.15Rivera A Fisher SA Fritsche LG Keilhauer CN Lichtner P Meitinger T Weber BH Hypothetical LOC387715 is a second major susceptibility gene for age-related macular degeneration, contributing independently of complement factor H to disease risk.Hum Mol Genet. 2005; 14: 3227-3236Crossref PubMed Scopus (661) Google Scholar Both studies reported that the chromosome 10q26 variant confers an AMD risk similar in magnitude to that conferred by the Y402H variant in CFH. Here, we describe highly significant association of SNPs in LOC387715 with AMD. In our data, only SNPs in this gene, including rs10490924, explained the strong linkage and association signal in this region. Given a previous report of an effect of cigarette smoking on the linkage findings in the 10q26 region,16Weeks DE Conley YP Tsai HJ Mah TS Schmidt S Postel EA Agarwal A Haines JL Pericak-Vance MA Rosenfeld PJ Paul TO Eller AW Morse LS Dailey JP Ferrell RE Gorin MB Age-related maculopathy: a genomewide scan with continued evidence of susceptibility loci within the 1q31, 10q26, and 17q25 regions.Am J Hum Genet. 2004; 75: 174-189Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar we tested whether smoking modifies this association. Our data suggest that variant genotypes at rs10490924 confer a substantially larger AMD risk to cigarette smokers than to nonsmokers. This observation is supported by traditional case-control modeling; by ordered-subset linkage analysis (OSA), incorporating pack-years of cigarette smoking as a covariate; and by family-based association analysis, using a more homogeneous set of families as defined by OSA. As part of an ongoing, large-scale study of genetic and environmental risk factors for AMD, we ascertained patients with AMD, their affected and unaffected family members, and a group of unrelated controls of similar age and ethnic background at two sites in the southeastern United States: Duke University Eye Center (DUEC) and Vanderbilt University Medical Center (VUMC). By use of stereoscopic color fundus photographs, all enrolled individuals were assigned (by E.A.P. and A.A.) one of five different grades of macular findings, as described elsewhere17Schmidt S Saunders AM De La Paz MA Postel EA Heinis RM Agarwal A Scott WK Gilbert JR McDowell JG Bazyk A Gass JD Haines JL Pericak-Vance MA Association of the apolipoprotein E gene with age-related macular degeneration: possible effect modification by family history, age, and gender.Mol Vis. 2000; 6: 287-293PubMed Google Scholar, 18Seddon JM Ajani UA Mitchell BD Familial aggregation of age-related maculopathy.Am J Ophthalmol. 1997; 123: 199-206Abstract Full Text PDF PubMed Scopus (330) Google Scholar and summarized in table 1. Our AMD classification is a modification of the Age-Related Eye Disease Study grading system, with the use of Wisconsin grading-system example slides19Klein R Davis MD Magli YL Segal P Klein BE Hubbard L The Wisconsin age-related maculopathy grading system.Ophthalmology. 1991; 98: 1128-1134Abstract Full Text PDF PubMed Scopus (960) Google Scholar and the International Classification System20Bird AC Bressler NM Bressler SB Chisholm IH Coscas G Davis MD de Jong PT Klaver CC Klein BE Klein R An international classification and grading system for age-related maculopathy and age-related macular degeneration. The International ARM Epidemiological Study Group.Surv Ophthalmol. 1995; 39: 367-374Abstract Full Text PDF PubMed Scopus (1648) Google Scholar as guides. The more severely affected eye was used to classify each individual. Unrelated controls were enrolled via (i) study advertisement in DUEC- and VUMC-specific newsletters; (ii) recruitment presentations by study coordinators at local retirement communities, the residents of which were likely to obtain health care at DUEC or VUMC; and (iii) AMD-related seminars for the general public sponsored by DUEC or VUMC ophthalmology clinics. Spouses of patients with AMD were also asked to participate as controls. All cases and controls included in this study were white and at least 55 years old. The study protocol was approved by the institutional review boards of the Duke University Medical Center and VUMC, the research adhered to the tenets of the Declaration of Helsinki, and informed consent was obtained from all study participants. Blood samples were collected, and genomic DNA was extracted from whole blood by using the PureGene system (Gentra Systems) on an Autopure LS.Table 1Demographic and Clinical Characteristics of the Study PopulationIndependent Case-Control Data SetCharacteristicFamily Data SetCasesControlsNo. of multiplex families140……No. of affected sibling pairsaIn multiplex families.169……No. of other affected relative pairsaIn multiplex families.37……No. of singleton families60……No. of discordant sibling pairsbIn multiplex and singleton families.158……No. of individuals526610259No. (%) with macular findings: Grade 1cNo drusen or nonextensive small (<63 μm) drusen without RPE abnormalities.85 (16.2)…193 (74.5) Grade 2dExtensive small drusen or nonextensive intermediate (≥63 μm and <125 μm) drusen and/or RPE hyper- or hypopigmentation.50 (9.5)…66 (25.5) Grade 3eExtensive intermediate drusen or any large (≥125 μm), soft drusen, including drusenoid RPE detachment.109 (20.7)140 (23.0)… Grade 4fGeographic atrophy (area of RPE atrophy with sharp margins, usually visible choroidal vessels, at least 175 μm in diameter).61 (11.6)77 (12.6)… Grade 5gExudative AMD, including nondrusenoid RPE detachment, choroidal neovascularization, subretinal hemorrhage or fibrosis, or photocoagulation scar consistent with treatment of AMD.221 (42.0)393 (64.4)…No. (%) female348 (66.2)396 (64.9)148 (57.1)Mean (±SD) age at examination (years)72.6 ± 9.976.8 ± 7.766.7 ± 8.1a In multiplex families.b In multiplex and singleton families.c No drusen or nonextensive small (<63 μm) drusen without RPE abnormalities.d Extensive small drusen or nonextensive intermediate (≥63 μm and <125 μm) drusen and/or RPE hyper- or hypopigmentation.e Extensive intermediate drusen or any large (≥125 μm), soft drusen, including drusenoid RPE detachment.f Geographic atrophy (area of RPE atrophy with sharp margins, usually visible choroidal vessels, at least 175 μm in diameter).g Exudative AMD, including nondrusenoid RPE detachment, choroidal neovascularization, subretinal hemorrhage or fibrosis, or photocoagulation scar consistent with treatment of AMD. Open table in a new tab Information about the smoking history of study participants was obtained from a self-administered questionnaire that was formatted to maximize readability for individuals with low vision. However, if participants indicated that they could not complete the form, a project coordinator offered to assist the participants in filling out the questionnaire. Regular cigarette smoking was assessed by two questions: (1) “Have you smoked at least 100 cigarettes in your lifetime?” and (2) “Did you ever smoke cigarettes at least once per week?”. Individuals answering “yes” to both questions were asked the average number of cigarettes they smoked per day, the year that they started smoking, whether they had quit smoking, and, if so, what year. This information was used to calculate pack-years of smoking as (cigarettes per day × years smoked)/20 cigarettes per pack. The most general measurement of smoking history was constructed as a binary “ever or never” variable based on a participant's response to question (1) above. The study population for the analysis presented here included 810 unrelated patients with early (grade 3) or advanced (grades 4 and 5) AMD. Of these, 200 had at least one sampled (affected or unaffected) relative and thus contributed to the family-based association analysis. The remaining 610 patients with AMD who did not have sampled relatives and 259 unrelated controls without AMD (grades 1 and 2) made up an independent case-control data set. Demographic and clinical information for these individuals and their relatives (1,395 individuals total) is shown in table 1. Previous work by our group21Kenealy SJ Schmidt S Agarwal A Postel EA De La Paz MA Pericak-Vance MA Haines JL Linkage analysis for age-related macular degeneration supports a gene on chromosome 10q26.Mol Vis. 2004; 10: 57-61PubMed Google Scholar and by others16Weeks DE Conley YP Tsai HJ Mah TS Schmidt S Postel EA Agarwal A Haines JL Pericak-Vance MA Rosenfeld PJ Paul TO Eller AW Morse LS Dailey JP Ferrell RE Gorin MB Age-related maculopathy: a genomewide scan with continued evidence of susceptibility loci within the 1q31, 10q26, and 17q25 regions.Am J Hum Genet. 2004; 75: 174-189Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar, 22Majewski J Schultz DW Weleber RG Schain MB Edwards AO Matise TC Acott TS Ott J Klein ML Age-related macular degeneration—a genome scan in extended families.Am J Hum Genet. 2003; 73: 540-550Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar, 23Seddon JM Santangelo SL Book K Chong S Cote J A genomewide scan for age-related macular degeneration provides evidence for linkage to several chromosomal regions.Am J Hum Genet. 2003; 73: 780-790Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar, 24Iyengar SK Song D Klein BE Klein R Schick JH Humphrey J Millard C Liptak R Russo K Jun G Lee KE Fijal B Elston RC Dissection of genomewide-scan data in extended families reveals a major locus and oligogenic susceptibility for age-related macular degeneration.Am J Hum Genet. 2004; 74: 20-39Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar suggested the presence of an AMD-susceptibility locus on chromosome 10q26, with the linkage peak centered at ∼122 Mb. To narrow the region most likely to harbor an AMD-susceptibility allele, we genotyped SNPs in the 112–132-Mb interval, which extended 10 Mb on either side of the reported linkage peak. We started with a density of ∼1 SNP per 1 Mb and filled in the 117–127-Mb region immediately surrounding the 122-Mb peak with a higher density of 1 SNP per 140 kb on average. All SNPs were selected using SNPSelector software25Xu H Gregory SG Hauser ER Stenger JE Pericak-Vance MA Vance JM Zuchner S Hauser MA SNPselector: a web tool for selecting SNPs for genetic association studies.Bioinformatics. 2005; 21: 4181-4186Crossref PubMed Scopus (92) Google Scholar to have approximately equal spacing and minor-allele frequency (MAF) ≥5%. Genotyping was performed with the TaqMan allelic discrimination assay, by use of either Assays-On-Demand or Assays-By-Design products (Applied Biosystems). For quality-control purposes, two CEPH standards were included in each 96-well plate, and samples from six individuals were duplicated across all plates, with the laboratory technicians blinded to their identities. Analysis required matching quality-controlled genotypes within and across plates and at least 95% genotyping efficiency. The Y402H variant in CFH was genotyped by sequencing, as described elsewhere.7Haines JL Hauser MA Schmidt S Scott WK Olson LM Gallins P Spencer KL Kwan SY Noureddine M Gilbert JR Schnetz-Boutaud N Agarwal A Postel EA Pericak-Vance MA Complement factor H variant increases the risk of age-related macular degeneration.Science. 2005; 308: 419-421Crossref PubMed Scopus (2069) Google Scholar After the first round of genotyping and statistical analysis, we applied iterative association mapping26Oliveira SA Li YJ Noureddine MA Zuchner S Qin X Pericak-Vance MA Vance JM Identification of risk and age-at-onset genes on chromosome 1p in Parkinson disease.Am J Hum Genet. 2005; 77: 252-264Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar to select another set of SNPs in the peak region, defined approximately as the 1-LOD support interval surrounding the peak multipoint LOD score (123.8–126.6 Mb). Our final SNP density was an average of 1 SNP per 33 kb, for a total of 84 SNPs in the 2.8-Mb support interval. Outside this interval, 101 SNPs were genotyped in the 112–132-Mb region, for a total of 185 SNPs. In addition to using SNPSelector,25Xu H Gregory SG Hauser ER Stenger JE Pericak-Vance MA Vance JM Zuchner S Hauser MA SNPselector: a web tool for selecting SNPs for genetic association studies.Bioinformatics. 2005; 21: 4181-4186Crossref PubMed Scopus (92) Google Scholar SNPs were identified through resequencing of LOC387715 and CUZD1 (CUB and zona pellucida–like domains 1 [HGNC accession number 17937]) in 48–72 unrelated, affected and unaffected individuals. We chose to sequence only LOC387715 and CUZD1, because they were the only genes that harbored SNPs with statistically significant association signals when the false-discovery rate (FDR) was used to correct for multiple testing and controlled at a level of 5%. Individuals were selected for resequencing on the basis of homozygosity at SNP rs10490924 in LOC387715 and SNP rs1891110 in CUZD1. The reason for this selection was that individuals homozygous for the risk-associated variant would be most likely to carry the risk allele at a different intragenic sequence variant. Thus, if the observed association were due to linkage disequilibrium (LD) with an untyped causal variant, this should maximize the probability of identifying this variant. The genotype data were analyzed with MERLIN27Abecasis GR Cherny SS Cookson WO Cardon LR Merlin—rapid analysis of dense genetic maps using sparse gene flow trees.Nat Genet. 2002; 30: 97-101Crossref PubMed Scopus (2779) Google Scholar (for MERLIN software, see Center for Statistical Genetics Web site) to calculate nonparametric two-point and multipoint LOD scores under the exponential model, denoted as LOD*.28Kong A Cox NJ Allele-sharing models: LOD scores and accurate linkage tests.Am J Hum Genet. 1997; 61: 1179-1188Abstract Full Text Full Text PDF PubMed Scopus (836) Google Scholar Allele frequencies were estimated from all genotyped individuals. Parametric affecteds-only heterogeneity LOD scores (HLODs) under a dominant (disease-allele frequency 0.01) or recessive (disease-allele frequency 0.2) model were also computed with MERLIN. To avoid an inflation of linkage evidence due to intermarker LD,29Boyles AL Scott WK Martin ER Schmidt S Li YJ Ashley-Koch A Bass MP Schmidt M Pericak-Vance MA Speer MC Hauser ER Linkage disequilibrium inflates type I error rates in multipoint linkage analysis when parental genotypes are missing.Hum Hered. 2005; 59: 220-227Crossref PubMed Scopus (69) Google Scholar we used recently described methods for estimating haplotype frequencies of SNP clusters in high pairwise LD, using a threshold of r2=0.16 to define these clusters.30Abecasis GR Wigginton JE Handling marker-marker linkage disequilibrium: pedigree analysis with clustered markers.Am J Hum Genet. 2005; 77: 754-767Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar The LD pattern in the region of interest was analyzed with the Haploview program,31Barrett JC Fry B Maller J Daly MJ Haploview: analysis and visualization of LD and haplotype maps.Bioinformatics. 2005; 21: 263-265Crossref PubMed Scopus (12020) Google Scholar with the generated genotypes from unrelated patients with AMD as the input. Association analysis was applied to all 185 SNPs, by use of the family-based association in the presence of linkage (APL) test32Martin ER Bass MP Hauser ER Kaplan NL Accounting for linkage in family-based tests of association with missing parental genotypes.Am J Hum Genet. 2003; 73: 1016-1026Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar (for APL software, see Center for Human Genetics Web site) and standard logistic regression analysis for case-control comparisons with adjustment for age and sex (SAS version 8.02 [SAS Institute]). An additive coding scheme was used, with the SNP model covariate taking on the values −1, 0, and 1 for genotypes 1/1, 1/2, and 2/2, respectively, where 2 is the minor allele in controls. As described above, we divided our total sample into cases contributing to the APL analysis (affected individuals with at least one sampled relative; n=200 families) and an independent sample of cases without sampled relatives (n=610) who were compared with 259 unrelated controls. As mentioned above, we used the FDR to correct for multiple testing.33Benjamini Y Hochberg Y Controlling the false discovery rate: a practical and powerful approach to multiple testing.J R Stat Soc. 1995; 57: 289-300Google Scholar Only SNPs that were significant with an FDR of 5% were analyzed with the Genotype-IBD Sharing Test (GIST) method34Li C Scott LJ Boehnke M Assessing whether an allele can account in part for a linkage signal: the Genotype-IBD Sharing Test (GIST).Am J Hum Genet. 2004; 74: 418-431Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar (for GIST software, see Center for Human Genetics Research Web site) to examine which of the most strongly associated SNPs best explained the linkage evidence in the region. We also used the COCAPHASE module of the UNPHASED software package35Dudbridge F Pedigree disequilibrium tests for multilocus haplotypes.Genet Epidemiol. 2003; 25: 115-121Crossref PubMed Scopus (1048) Google Scholar (see UNPHASED page at the Medical Research Council Biostatisics Unit Web site) to perform conditional haplotype analysis. This analysis tested whether conditioning on the risk allele at a particular SNP accounted for the association signal in the region. If the association signal in the region was driven by a single SNP, conditioning on its effect was expected to remove all evidence of association for the remaining SNPs. We conducted additional analyses to incorporate effects of the two most important known AMD risk factors—smoking and the CFH gene. First, we fit a series of logistic regression models to the combined case-control data set (including probands from the family data set) to identify the model that best described (1) the joint effects of CFH and LOC387715 and (2) the joint effects of smoking and LOC387715. We followed a recently proposed modeling strategy36North BV Curtis D Sham PC Application of logistic regression to case-control association studies involving two causative loci.Hum Hered. 2005; 59: 79-87Crossref PubMed Scopus (41) Google Scholar in which the best-fitting model was derived on the basis of Akaike's information criterion (AIC). The AIC compares different models with a log-likelihood ratio test that is penalized for the number of model parameters to identify the most parsimonious model that adequately fits the data. For each genotype, two model terms were tested: one coding for additive effects at the first, second, or both loci (ADD1, ADD2, and ADDBOTH), using the coding described above, and the other coding for dominance effects (DOM1, DOM2, and DOMBOTH). For example, the ADD1 model contained only model term x1, coded as −1 for genotype GG at rs10490924, as 0 for genotype GT, and as 1 for genotype TT. The ADD2 model included only model term x2, coded as −1 for genotype TT at Y402H, as 0 for genotype TC, and as 1 for genotype CC. Both models were nested within the larger ADDBOTH model, which included both x1 and x2 model terms. This model was again nested within the larger DOMBOTH model, which included x1, x2, and two ad
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