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1.  Ancestry of the Timorese: age-related macular degeneration associated genotype and allele sharing among human populations from throughout the world 
Frontiers in Genetics  2015;6:238.
We observed that the third leading cause of blindness in the world, age-related macular degeneration (AMD), occurs at a very low documented frequency in a population-based cohort from Timor-Leste. Thus, we determined a complete catalog of the ancestry of the Timorese by analysis of whole exome chip data and haplogroup analysis of SNP genotypes determined by sequencing the Hypervariable I and II regions of the mitochondrial genome and 17 genotyped YSTR markers obtained from 535 individuals. We genotyped 20 previously reported AMD-associated SNPs in the Timorese to examine their allele frequencies compared to and between previously documented AMD cohorts of varying ethnicities. For those without AMD (average age > 55 years), genotype and allele frequencies were similar for most SNPs with a few exceptions. The major risk allele of HTRA1 rs11200638 (10q26) was at a significantly higher frequency in the Timorese, as well as 3 of the 5 protective CFH (1q32) SNPs (rs800292, rs2284664, and rs12066959). Additionally, the most commonly associated AMD-risk SNP, CFH rs1061170 (Y402H), was also seen at a much lower frequency in the Korean and Timorese populations than in the assessed Caucasian populations (C ~7 vs. ~40%, respectively). The difference in allele frequencies between the Timorese population and the other genotyped populations, along with the haplogroup analysis, also highlight the genetic diversity of the Timorese. Specifically, the most common ancestry groupings were Oceanic (Melanesian and Papuan) and Eastern Asian (specifically Han Chinese). The low prevalence of AMD in the Timorese population (2 of 535 randomly selected participants) may be due to the enrichment of protective alleles in this population at the 1q32 locus.
PMCID: PMC4496576  PMID: 26217379
population genetics; ancestry; age-related macular degeneration; complex disease; and epidemiology
2.  Drusen and Photoreceptor Abnormalities in African-Americans with Intermediate Non-neovascular Age-related Macular Degeneration 
Current eye research  2014;40(4):398-406.
To investigate the relationship of drusen and photoreceptor abnormalities in African-American (AA) patients with intermediate non-neovascular age-related macular degeneration (AMD).
Materials and methods
AA patients with intermediate AMD (n=11; ages 52-77 years) were studied with spectral-domain optical coherence tomography. Macular location and characteristics of large drusen (≥125 μm) were determined. Thickness of photoreceptor laminae was quantified overlying drusen and in other macular regions. A patient with advanced AMD (age 87) was included to illustrate the disease spectrum.
In this AA patient cohort, the spectrum of changes known to occur in AMD, including large drusen, sub-retinal drusenoid deposits and geographic atrophy, were identified. In intermediate AMD eyes (n=17), there were 183 large drusen, the majority of which were pericentral in location. Overlying the drusen there was significant thinning of the photoreceptor outer nuclear layer (termed ONL+) as well as the inner and outer segments (IS+OS). The reductions in IS+OS thickness were directly related to ONL+ thickness. In a fraction (~8%) of paradrusen locations with normal lamination sampled within ~280 μm of peak drusen height, ONL+ was significantly thickened compared to age and retinal-location-matched normal values. Topographical maps of the macula confirmed ONL thickening in regions neighboring and distant to large drusen.
We confirm there is a pericentral distribution of drusen across AA-AMD maculae rather than the central localization in Caucasian AMD. Reductions in the photoreceptor laminae overlying drusen are evident. ONL+ thickening in some macular areas of AA-AMD eyes may be an early phenotypic marker for photoreceptor stress.
PMCID: PMC4489141  PMID: 24912073
drusen; optical coherence tomography; photoreceptor outer nuclear layer; African-American age-related macular degeneration
3.  Association of OCT derived drusen measurements with AMD associated-genotypic SNPs in Amish population 
Journal of clinical medicine  2015;4(2):304-317.
To investigate the association of OCT derived drusen measures in Amish age-related macular degeneration (AMD) patients with known loci for macular degeneration.
Members of the Old Order Amish community in Pennsylvania ages 50 and older were assessed for drusen area, volume and regions of retinal pigment epithelium (RPE) atrophy using a Cirrus High- Definition-OCT. Measurements were obtained in the macula region within a central circle (CC) of 3 mm diameter and a surrounding perifoveal ring (PR) of 3 to 5 mm diameter using the Cirrus OCT RPE analysis software. Other demographic information including age, gender and smoking status were collected. Study subjects were further genotyped to determine their risk for the AMD associated SNPs in SYN3, LIPC, ARMS2, C3, CFB, CETP, CFI and CFH genes using TaqMan genotyping assays. The association of genotypes with OCT measures were assessed using linear trend p-values calculated from univariate and multivariate generalized linear models.
432 eyes were included in the analysis. Multivariate analysis (adjusted by age, gender and smoking status) confirmed the known significant association between AMD and macular drusen with the number of CFH risk alleles for drusen area (area increased 0.12 mm2 for a risk allele increase, p<0.01), drusen volume (volume increased 0.01 mm3 for a risk allele increase, p≤0.05) and area of RPE atrophy (area increased 0.43 mm2 for a risk allele increase, p=0.003). SYN3 risk allele G is significantly associated with larger area PR (area increased 0.09 mm2 for a risk allele increase, p=0.03) and larger drusen volume in central circle (volume increased 0.01 mm3 for a risk allele increase, p=0.04).
Among the genotyped SNPs tested, the CFH risk genotype appears to play a major role in determining the drusen phenotype in the Amish AMD population.
PMCID: PMC4398021  PMID: 25893111
age-related macular degeneration; AMD; Older Order Amish; CFH; SYN3; OCT; drusen area; drusen volume; RPE atrophy Cirrus HD-OCT
4.  Copy number variants encompassing Mendelian disease genes in a large multigenerational family segregating bipolar disorder 
BMC Genetics  2015;16:27.
Bipolar affective disorder (BP) is a common, highly heritable psychiatric disorder characterized by periods of depression and mania. Using dense SNP genotype data, we characterized CNVs in 388 members of an Old Order Amish Pedigree with bipolar disorder. We identified CNV regions arising from common ancestral mutations by utilizing the pedigree information. By combining this analysis with whole genome sequence data in the same individuals, we also explored the role of compound heterozygosity.
Here we describe 541 inherited CNV regions, of which 268 are rare in a control population of European origin but present in a large number of Amish individuals. In addition, we highlight a set of CNVs found at higher frequencies in BP individuals, and within genes known to play a role in human development and disease. As in prior reports, we find no evidence for an increased burden of CNVs in BP individuals, but we report a trend towards a higher burden of CNVs in known Mendelian disease loci in bipolar individuals (BPI and BPII, p = 0.06).
We conclude that CNVs may be contributing factors in the phenotypic presentation of mood disorders and co-morbid medical conditions in this family. These results reinforce the hypothesis of a complex genetic architecture underlying BP disorder, and suggest that the role of CNVs should continue to be investigated in BP data sets.
Electronic supplementary material
The online version of this article (doi:10.1186/s12863-015-0184-1) contains supplementary material, which is available to authorized users.
PMCID: PMC4382929  PMID: 25887117
CNV; Bipolar disorder; Family based studies; Mendelian disease genes; Genetics loci
5.  Association of OCT-Derived Drusen Measurements with AMD-Associated Genotypic SNPs in the Amish Population 
Journal of Clinical Medicine  2015;4(2):304-317.
Purpose: To investigate the association of optical coherence tomography (OCT)-derived drusen measures in Amish age-related macular degeneration (AMD) patients with known loci for macular degeneration. Methods: Members of the Old Order Amish community in Pennsylvania ages 50 and older were assessed for drusen area, volume and regions of retinal pigment epithelium (RPE) atrophy using a Cirrus High-Definition OCT. Measurements were obtained in the macula region within a central circle (CC) of 3 mm in diameter and a surrounding perifoveal ring (PR) of 3 to 5 mm in diameter using the Cirrus OCT RPE analysis software. Other demographic information, including age, gender and smoking status, were collected. Study subjects were further genotyped to determine their risk for the AMD-associated SNPs in the SYN3, LIPC, ARMS2, C3, CFB, CETP, CFI and CFH genes using TaqMan genotyping assays. The association of genotypes with OCT measures were assessed using linear trend p-values calculated from univariate and multivariate generalized linear models. Results: 432 eyes were included in the analysis. Multivariate analysis (adjusted by age, gender and smoking status) confirmed the known significant association between AMD and macular drusen with the number of CFH risk alleles for the drusen area (the area increased 0.12 mm2 for a risk allele increase, p < 0.01), drusen volume (the volume increased 0.01 mm3 for a risk allele increase, p ≤ 0.05) and the area of RPE atrophy (the area increased 0.43 mm2 for a risk allele increase, p = 0.003). SYN3 risk allele G is significantly associated with larger area PR (the area increased 0.09 mm2 for a risk allele increase, p = 0.03) and larger drusen volume in the central circle (the volume increased 0.01 mm3 for a risk allele increase, p = 0.04). Conclusion: Among the genotyped SNPs tested, the CFH risk genotype appears to play a major role in determining the drusen phenotype in the Amish AMD population.
PMCID: PMC4398021  PMID: 25893111
age-related macular degeneration; AMD; Older Order Amish; CFH; SYN3; OCT; drusen area; drusen volume; RPE atrophy; Cirrus HD-OCT
6.  Age-Related Macular Degeneration: Genetics and Biology Coming Together 
Genetic and genomic studies have enhanced our understanding of complex neurodegenerative diseases that exert a devastating impact on individuals and society. One such disease, age-related macular degeneration (AMD), is a major cause of progressive and debilitating visual impairment. Since the pioneering discovery in 2005 of complement factor H (CFH) as a major AMD susceptibility gene, extensive investigations have confirmed 19 additional genetic risk loci, and more are anticipated. In addition to common variants identified by now-conventional genome-wide association studies, targeted genomic sequencing and exome-chip analyses are uncovering rare variant alleles of high impact. Here, we provide a critical review of the ongoing genetic studies and of common and rare risk variants at a total of 20 susceptibility loci, which together explain 40–60% of the disease heritability but provide limited power for diagnostic testing of disease risk. Identification of these susceptibility loci has begun to untangle the complex biological pathways underlying AMD pathophysiology, pointing to new testable paradigms for treatment.
PMCID: PMC4217162  PMID: 24773320
complex disease; genetic susceptibility; neurodegeneration; retina; blindness
7.  Genome-Wide Meta-Analysis of Myopia and Hyperopia Provides Evidence for Replication of 11 Loci 
PLoS ONE  2014;9(9):e107110.
Refractive error (RE) is a complex, multifactorial disorder characterized by a mismatch between the optical power of the eye and its axial length that causes object images to be focused off the retina. The two major subtypes of RE are myopia (nearsightedness) and hyperopia (farsightedness), which represent opposite ends of the distribution of the quantitative measure of spherical refraction. We performed a fixed effects meta-analysis of genome-wide association results of myopia and hyperopia from 9 studies of European-derived populations: AREDS, KORA, FES, OGP-Talana, MESA, RSI, RSII, RSIII and ERF. One genome-wide significant region was observed for myopia, corresponding to a previously identified myopia locus on 8q12 (p = 1.25×10−8), which has been reported by Kiefer et al. as significantly associated with myopia age at onset and Verhoeven et al. as significantly associated to mean spherical-equivalent (MSE) refractive error. We observed two genome-wide significant associations with hyperopia. These regions overlapped with loci on 15q14 (minimum p value = 9.11×10−11) and 8q12 (minimum p value 1.82×10−11) previously reported for MSE and myopia age at onset. We also used an intermarker linkage- disequilibrium-based method for calculating the effective number of tests in targeted regional replication analyses. We analyzed myopia (which represents the closest phenotype in our data to the one used by Kiefer et al.) and showed replication of 10 additional loci associated with myopia previously reported by Kiefer et al. This is the first replication of these loci using myopia as the trait under analysis. “Replication-level” association was also seen between hyperopia and 12 of Kiefer et al.'s published loci. For the loci that show evidence of association to both myopia and hyperopia, the estimated effect of the risk alleles were in opposite directions for the two traits. This suggests that these loci are important contributors to variation of refractive error across the distribution.
PMCID: PMC4169415  PMID: 25233373
8.  Optic Disc and Cup Segmentation from Color Fundus Photograph Using Graph Cut with Priors 
For automatic segmentation of optic disc and cup from color fundus photograph, we describe a fairly general energy function that can naturally fit into a global optimization framework with graph cut. Distinguished from most previous work, our energy function includes priors on the shape & location of disc & cup, the rim thickness and the geometric interaction of “disc contains cup”. These priors together with the effective optimization of graph cut enable our algorithm to generate reliable and robust solutions. Our approach is able to outperform several state-of-the-art segmentation methods, as shown by a set of experimental comparisons with manual delineations and a series of results of correlations with the assessments of a merchant-provided software from Optical Coherence Tomography (OCT) regarding several cup and disc parameters.
PMCID: PMC4165089  PMID: 24579126
9.  Genome-wide Scan of African-American and White Families for Linkage to Myopia 
American journal of ophthalmology  2008;147(3):512-517.e2.
To identify myopia susceptibility genes influencing common myopia in 94 African-American and 36 White families.
A prospective study of families with myopia consisting of a minimum of two individuals affected with myopia.
Extended families consisting of at least two siblings affected with myopia were ascertained. A genome-wide linkage scan using 387 markers was conducted by the Center for Inherited Disease Research. Linkage analyses were conducted with parametric and nonparametric methods. Model-free linkage analysis was performed maximizing over penetrance and over dominance (that is, fitting a wide range of both dominant and recessive models).
Under the model-free analysis, the maximum two point heterogeneity logarithm of the odds score (MALOD) was 2.87 at D6S1009 in the White cohort and the maximum multipoint MALOD was 2.42 at D12S373-D12S1042 in the same cohort. The nonpara-metric linkage (NPL) maximum multipoint at D6S1035 had a P value of .005. An overall multipoint NPL score was obtained by combining NPL scores from both populations. The highest combined NPL score was observed at D20S478 with a significant P value of .008. Suggestive evidence of linkage in the White cohort mapped to a previously mapped locus on chromosome 11 at D11S1981 (NPL = 2.14; P = .02).
Suggestive evidence of linkage to myopia in both African Americans and Whites was seen on chromosome 20 and became more significant when the scores were combined for both groups. The locus on chromosome 11 independently confirms a report by Hammond and associates mapping a myopia quantitative trait locus to this region.
PMCID: PMC4152232  PMID: 19026404
10.  Genetic Susceptibility and Mechanisms for Refractive Error 
Clinical genetics  2013;84(2):102-108.
Refractive errors, myopia and hyperopia, are the most common causes of visual impairment worldwide. Recent advances in genetics have been utilized to identify a wealth of genetic loci believed to contain susceptibility genes for refractive error. The current genetic evidence confirms that refractive error is influenced by both common and rare variants with a significant environmental component. These studies argue that only by combining genetic knowledge with in vivo measurements of biological states will it be possible to understand the underlying biology of refractive error that will lead to novel therapeutic targets and accurate genetic predictions.
PMCID: PMC4136757  PMID: 23647423
Refractive Error; Myopia; Hyperopia; Genetic Variation; Animal Models; Gene-Environment Interaction
11.  Ancestry Estimation and Control of Population Stratification for Sequence-based Association Studies 
Nature genetics  2014;46(4):409-415.
Knowledge of individual ancestry is important for genetic association studies where population structure leads to false positive signals. Estimating individual ancestry with targeted sequence data, which constitutes the bulk of current sequence datasets, is challenging. Here, we propose a new method for accurate estimation of genetic ancestry. Our method skips genotype calling and directly analyzes sequence reads. We validate the method using simulated and empirical data and show that the method can accurately infer worldwide continental ancestry with whole genome shotgun coverage as low as 0.001X. For estimates of fine-scale ancestry within Europe, the method performs well with coverage of 0.1X. At an even finer-scale, the method improves discrimination between exome-sequenced participants originating from different provinces within Finland. Finally, we show that our method can be used to improve case-control matching in genetic association studies and reduce the risk of spurious findings due to population structure.
PMCID: PMC4084909  PMID: 24633160
12.  Meta-analysis of genome-wide association studies in five cohorts reveals common variants in RBFOX1, a regulator of tissue-specific splicing, associated with refractive error 
Human Molecular Genetics  2013;22(13):2754-2764.
Visual refractive errors (REs) are complex genetic traits with a largely unknown etiology. To date, genome-wide association studies (GWASs) of moderate size have identified several novel risk markers for RE, measured here as mean spherical equivalent (MSE). We performed a GWAS using a total of 7280 samples from five cohorts: the Age-Related Eye Disease Study (AREDS); the KORA study (‘Cooperative Health Research in the Region of Augsburg’); the Framingham Eye Study (FES); the Ogliastra Genetic Park-Talana (OGP-Talana) Study and the Multiethnic Study of Atherosclerosis (MESA). Genotyping was performed on Illumina and Affymetrix platforms with additional markers imputed to the HapMap II reference panel. We identified a new genome-wide significant locus on chromosome 16 (rs10500355, P = 3.9 × 10−9) in a combined discovery and replication set (26 953 samples). This single nucleotide polymorphism (SNP) is located within the RBFOX1 gene which is a neuron-specific splicing factor regulating a wide range of alternative splicing events implicated in neuronal development and maturation, including transcription factors, other splicing factors and synaptic proteins.
PMCID: PMC3674806  PMID: 23474815
13.  Identification of a Rare Coding Variant in Complement 3 Associated with Age-related Macular Degeneration 
Nature genetics  2013;45(11):10.1038/ng.2758.
Macular degeneration is a common cause of blindness in the elderly. To identify rare coding variants associated with a large increase in risk of age-related macular degeneration (AMD), we sequenced 2,335 cases and 789 controls in 10 candidate loci (57 genes). To increase power, we augmented our control set with ancestry-matched exome sequenced controls. An analysis of coding variation in 2,268 AMD cases and 2,268 ancestry matched controls revealed two large-effect rare variants; previously described R1210C in the CFH gene (fcase = 0.51%, fcontrol = 0.02%, OR = 23.11), and newly identified K155Q in the C3 gene (fcase = 1.06%, fcontrol = 0.39%, OR = 2.68). The variants suggest decreased inhibition of C3 by Factor H, resulting in increased activation of the alternative complement pathway, as a key component of disease biology.
PMCID: PMC3812337  PMID: 24036949
14.  Matrix metalloproteinases and educational attainment in refractive error: evidence of gene-environment interactions in the AREDS study 
Ophthalmology  2012;120(2):298-305.
A previous study of Old Order Amish families has shown association of ocular refraction with markers proximal to matrix metalloproteinase (MMP) genes MMP1 and MMP10 and intragenic to MMP2. We conducted a candidate gene replication study of association between refraction and single nucleotide polymorphisms (SNPs) within these genomic regions.
Candidate gene genetic association study.
2,000 participants drawn from the Age Related Eye Disease Study (AREDS) were chosen for genotyping. After quality control filtering, 1912 individuals were available for analysis.
Microarray genotyping was performed using the HumanOmni 2.5 bead array. SNPs originally typed in the previous Amish association study were extracted for analysis. In addition, haplotype tagging SNPs were genotyped using TaqMan assays. Quantitative trait association analyses of mean spherical equivalent refraction (MSE) were performed on 30 markers using linear regression models and an additive genetic risk model, while adjusting for age, sex, education, and population substructure. Post-hoc analyses were performed after stratifying on a dichotomous education variable. Pointwise (P-emp) and multiple-test study-wise (P-multi) significance levels were calculated empirically through permutation.
Main outcome measures
MSE was used as a quantitative measure of ocular refraction.
The mean age and ocular refraction were 68 years (SD=4.7) and +0.55 D (SD=2.14), respectively. Pointwise statistical significance was obtained for rs1939008 (P-emp=0.0326). No SNP attained statistical significance after correcting for multiple testing. In stratified analyses, multiple SNPs reached pointwise significance in the lower-education group: 2 of these were statistically significant after multiple testing correction. The two highest-ranking SNPs in Amish families (rs1939008 and rs9928731) showed pointwise P-emp<0.01 in the lower-education stratum of AREDS participants.
We show suggestive evidence of replication of an association signal for ocular refraction to a marker between MMP1 and MMP10. We also provide evidence of a gene-environment interaction between previously-reported markers and education on refractive error. Variants in MMP1- MMP10 and MMP2 regions appear to affect population variation in ocular refraction in environmental conditions less favorable for myopia development.
PMCID: PMC3563738  PMID: 23098370
refraction; refractive error; myopia; association study; gene-environment interaction; matrix metalloproteinase; MMP; genetics
15.  Genome-wide meta-analyses of multi-ethnic cohorts identify multiple new susceptibility loci for refractive error and myopia 
Verhoeven, Virginie J.M. | Hysi, Pirro G. | Wojciechowski, Robert | Fan, Qiao | Guggenheim, Jeremy A. | Höhn, René | MacGregor, Stuart | Hewitt, Alex W. | Nag, Abhishek | Cheng, Ching-Yu | Yonova-Doing, Ekaterina | Zhou, Xin | Ikram, M. Kamran | Buitendijk, Gabriëlle H.S. | McMahon, George | Kemp, John P. | St. Pourcain, Beate | Simpson, Claire L. | Mäkelä, Kari-Matti | Lehtimäki, Terho | Kähönen, Mika | Paterson, Andrew D. | Hosseini, S. Mohsen | Wong, Hoi Suen | Xu, Liang | Jonas, Jost B. | Pärssinen, Olavi | Wedenoja, Juho | Yip, Shea Ping | Ho, Daniel W. H. | Pang, Chi Pui | Chen, Li Jia | Burdon, Kathryn P. | Craig, Jamie E. | Klein, Barbara E. K. | Klein, Ronald | Haller, Toomas | Metspalu, Andres | Khor, Chiea-Chuen | Tai, E-Shyong | Aung, Tin | Vithana, Eranga | Tay, Wan-Ting | Barathi, Veluchamy A. | Chen, Peng | Li, Ruoying | Liao, Jiemin | Zheng, Yingfeng | Ong, Rick T. | Döring, Angela | Evans, David M. | Timpson, Nicholas J. | Verkerk, Annemieke J.M.H. | Meitinger, Thomas | Raitakari, Olli | Hawthorne, Felicia | Spector, Tim D. | Karssen, Lennart C. | Pirastu, Mario | Murgia, Federico | Ang, Wei | Mishra, Aniket | Montgomery, Grant W. | Pennell, Craig E. | Cumberland, Phillippa M. | Cotlarciuc, Ioana | Mitchell, Paul | Wang, Jie Jin | Schache, Maria | Janmahasathian, Sarayut | Igo, Robert P. | Lass, Jonathan H. | Chew, Emily | Iyengar, Sudha K. | Gorgels, Theo G.M.F. | Rudan, Igor | Hayward, Caroline | Wright, Alan F. | Polasek, Ozren | Vatavuk, Zoran | Wilson, James F. | Fleck, Brian | Zeller, Tanja | Mirshahi, Alireza | Müller, Christian | Uitterlinden, Andre’ G. | Rivadeneira, Fernando | Vingerling, Johannes R. | Hofman, Albert | Oostra, Ben A. | Amin, Najaf | Bergen, Arthur A.B. | Teo, Yik-Ying | Rahi, Jugnoo S. | Vitart, Veronique | Williams, Cathy | Baird, Paul N. | Wong, Tien-Yin | Oexle, Konrad | Pfeiffer, Norbert | Mackey, David A. | Young, Terri L. | van Duijn, Cornelia M. | Saw, Seang-Mei | Wilson, Joan E. Bailey | Stambolian, Dwight | Klaver, Caroline C. | Hammond, Christopher J.
Nature genetics  2013;45(3):314-318.
Refractive error is the most common eye disorder worldwide, and a prominent cause of blindness. Myopia affects over 30% of Western populations, and up to 80% of Asians. The CREAM consortium conducted genome-wide meta-analyses including 37,382 individuals from 27 studies of European ancestry, and 8,376 from 5 Asian cohorts. We identified 16 new loci for refractive error in subjects of European ancestry, of which 8 were shared with Asians. Combined analysis revealed 8 additional loci. The new loci include genes with functions in neurotransmission (GRIA4), ion channels (KCNQ5), retinoic acid metabolism (RDH5), extracellular matrix remodeling (LAMA2, BMP2), and eye development (SIX6, PRSS56). We also confirmed previously reported associations with GJD2 and RASGRF1. Risk score analysis using associated SNPs showed a tenfold increased risk of myopia for subjects with the highest genetic load. Our results, accumulated across independent multi-ethnic studies, considerably advance understanding of mechanisms involved in refractive error and myopia.
PMCID: PMC3740568  PMID: 23396134
16.  MATCHCLIP: locate precise breakpoints for copy number variation using CIGAR string by matching soft clipped reads 
Frontiers in Genetics  2013;4:157.
Copy number variations (CNVs) are associated with many complex diseases. Next generation sequencing data enable one to identify precise CNV breakpoints to better under the underlying molecular mechanisms and to design more efficient assays. Using the CIGAR strings of the reads, we develop a method that can identify the exact CNV breakpoints, and in cases when the breakpoints are in a repeated region, the method reports a range where the breakpoints can slide. Our method identifies the breakpoints of a CNV using both the positions and CIGAR strings of the reads that cover breakpoints of a CNV. A read with a long soft clipped part (denoted as S in CIGAR) at its 3′(right) end can be used to identify the 5′(left)-side of the breakpoints, and a read with a long S part at the 5′ end can be used to identify the breakpoint at the 3′-side. To ensure both types of reads cover the same CNV, we require the overlapped common string to include both of the soft clipped parts. When a CNV starts and ends in the same repeated regions, its breakpoints are not unique, in which case our method reports the left most positions for the breakpoints and a range within which the breakpoints can be incremented without changing the variant sequence. We have implemented the methods in a C++ package intended for the current Illumina Miseq and Hiseq platforms for both whole genome and exon-sequencing. Our simulation studies have shown that our method compares favorably with other similar methods in terms of true discovery rate, false positive rate and breakpoint accuracy. Our results from a real application have shown that the detected CNVs are consistent with zygosity and read depth information. The software package is available at
PMCID: PMC3744852  PMID: 23967014
structural variation; breakpoint; duplication; deletion; exon sequencing
17.  Regional replication of association with refractive error on 15q14 and 15q25 in the Age-Related Eye Disease Study cohort 
Molecular Vision  2013;19:2173-2186.
Refractive error is a complex trait with multiple genetic and environmental risk factors, and is the most common cause of preventable blindness worldwide. The common nature of the trait suggests the presence of many genetic factors that individually may have modest effects. To achieve an adequate sample size to detect these common variants, large, international collaborations have formed. These consortia typically use meta-analysis to combine multiple studies from many different populations. This approach is robust to differences between populations; however, it does not compensate for the different haplotypes in each genetic background evidenced by different alleles in linkage disequilibrium with the causative variant. We used the Age-Related Eye Disease Study (AREDS) cohort to replicate published significant associations at two loci on chromosome 15 from two genome-wide association studies (GWASs). The single nucleotide polymorphisms (SNPs) that exhibited association on chromosome 15 in the original studies did not show evidence of association with refractive error in the AREDS cohort. This paper seeks to determine whether the non-replication in this AREDS sample may be due to the limited number of SNPs chosen for replication.
We selected all SNPs genotyped on the Illumina Omni2.5v1_B array or custom TaqMan assays or imputed from the GWAS data, in the region surrounding the SNPs from the Consortium for Refractive Error and Myopia study. We analyzed the SNPs for association with refractive error using standard regression methods in PLINK. The effective number of tests was calculated using the Genetic Type I Error Calculator.
Although use of the same SNPs used in the Consortium for Refractive Error and Myopia study did not show any evidence of association with refractive error in this AREDS sample, other SNPs within the candidate regions demonstrated an association with refractive error. Significant evidence of association was found using the hyperopia categorical trait, with the most significant SNPs rs1357179 on 15q14 (p=1.69×10−3) and rs7164400 on 15q25 (p=8.39×10−4), which passed the replication thresholds.
This study adds to the growing body of evidence that attempting to replicate the most significant SNPs found in one population may not be significant in another population due to differences in the linkage disequilibrium structure and/or allele frequency. This suggests that replication studies should include less significant SNPs in an associated region rather than only a few selected SNPs chosen by a significance threshold.
PMCID: PMC3826323  PMID: 24227913
18.  Evidence of association of APOE with age-related macular degeneration - a pooled analysis of 15 studies 
Human mutation  2011;32(12):1407-1416.
Age-related macular degeneration (AMD) is the most common cause of incurable visual impairment in high-income countries. Previous studies report inconsistent associations between AMD and apolipoprotein E (APOE), a lipid transport protein involved in low-density cholesterol modulation. Potential interaction between APOE and sex, and smoking status, has been reported. We present a pooled analysis (n=21,160) demonstrating associations between late AMD and APOε4 (OR=0.72 per haplotype; CI: 0.65–0.74; P=4.41×10−11) and APOε2 (OR=1.83 for homozygote carriers; CI: 1.04–3.23; P=0.04), following adjustment for age-group and sex within each study and smoking status. No evidence of interaction between APOE and sex or smoking was found. Ever smokers had significant increased risk relative to never smokers for both neovascular (OR=1.54; CI: 1.38–1.72; P=2.8×10−15) and atrophic (OR=1.38; CI: 1.18–1.61; P=3.37×10−5) AMD but not early AMD (OR=0.94; CI: 0.86–1.03; P=0.16), implicating smoking as a major contributing factor to disease progression from early signs to the visually disabling late forms. Extended haplotype analysis incorporating rs405509 did not identify additional risks beyondε2 and ε4 haplotypes. Our expanded analysis substantially improves our understanding of the association between the APOE locus and AMD. It further provides evidence supporting the role of cholesterol modulation, and low-density cholesterol specifically, in AMD disease etiology.
PMCID: PMC3217135  PMID: 21882290
age-related macular degeneration; AMD; apolipoprotein E; APOE; case-control association study
19.  Variations in Apolipoprotein E Frequency With Age in a Pooled Analysis of a Large Group of Older People 
American Journal of Epidemiology  2011;173(12):1357-1364.
Variation in the apolipoprotein E gene (APOE) has been reported to be associated with longevity in humans. The authors assessed the allelic distribution of APOE isoforms ε2, ε3, and ε4 among 10,623 participants from 15 case-control and cohort studies of age-related macular degeneration (AMD) in populations of European ancestry (study dates ranged from 1990 to 2009). The authors included only the 10,623 control subjects from these studies who were classified as having no evidence of AMD, since variation within the APOE gene has previously been associated with AMD. In an analysis stratified by study center, gender, and smoking status, there was a decreasing frequency of the APOE ε4 isoform with increasing age (χ2 for trend = 14.9 (1 df); P = 0.0001), with a concomitant increase in the ε3 isoform (χ2 for trend = 11.3 (1 df); P = 0.001). The association with age was strongest in ε4 homozygotes; the frequency of ε4 homozygosity decreased from 2.7% for participants aged 60 years or less to 0.8% for those over age 85 years, while the proportion of participants with the ε3/ε4 genotype decreased from 26.8% to 17.5% across the same age range. Gender had no significant effect on the isoform frequencies. This study provides strong support for an association of the APOE gene with human longevity.
PMCID: PMC3145394  PMID: 21498624
aged; apolipoprotein E2; apolipoprotein E3; apolipoprotein E4; apolipoproteins E; longevity; meta-analysis; multicenter study
20.  Large scale international replication and meta-analysis study confirms association of the 15q14 locus with myopia. The CREAM consortium 
Verhoeven, Virginie J. M. | Hysi, Pirro G. | Saw, Seang-Mei | Vitart, Veronique | Mirshahi, Alireza | Guggenheim, Jeremy A. | Cotch, Mary Frances | Yamashiro, Kenji | Baird, Paul N. | Mackey, David A. | Wojciechowski, Robert |  Ikram, M. Kamran | Hewitt, Alex W. | Duggal, Priya | Janmahasatian, Sarayut | Khor, Chiea-Chuen | Fan, Qiao | Zhou, Xin | Young, Terri L. | Tai, E-Shyong | Goh, Liang-Kee | Li, Yi-Ju | Aung, Tin | Vithana, Eranga | Teo, Yik-Ying | Tay, Wanting | Sim, Xueling | Rudan, Igor | Hayward, Caroline | Wright, Alan F. | Polasek, Ozren | Campbell, Harry | Wilson, James F. | Fleck, Brian W. | Nakata, Isao | Yoshimura, Nagahisa | Yamada, Ryo | Matsuda, Fumihiko | Ohno-Matsui, Kyoko | Nag, Abhishek | McMahon, George | Pourcain, Beate St. | Lu, Yi | Rahi, Jugnoo S. | Cumberland, Phillippa M. | Bhattacharya, Shomi | Simpson, Claire L. | Atwood, Larry D. | Li, Xiaohui | Raffel, Leslie J. | Murgia, Federico | Portas, Laura | Despriet, Dominiek D. G. | van Koolwijk, Leonieke M. E. | Wolfram, Christian | Lackner, Karl J. | Tönjes, Anke | Mägi, Reedik | Lehtimäki, Terho | Kähönen, Mika | Esko, Tõnu | Metspalu, Andres | Rantanen, Taina | Pärssinen, Olavi | Klein, Barbara E. | Meitinger, Thomas | Spector, Timothy D. | Oostra, Ben A. | Smith, Albert V. | de Jong, Paulus T. V. M. | Hofman, Albert | Amin, Najaf | Karssen, Lennart C. | Rivadeneira, Fernando | Vingerling, Johannes R. | Eiríksdóttir, Guðný | Gudnason, Vilmundur | Döring, Angela | Bettecken, Thomas | Uitterlinden, André G. | Williams, Cathy | Zeller, Tanja | Castagné, Raphaële | Oexle, Konrad | van Duijn, Cornelia M. | Iyengar, Sudha K. | Mitchell, Paul | Wang, Jie Jin | Höhn, René | Pfeiffer, Norbert | Bailey-Wilson, Joan E. | Stambolian, Dwight | Wong, Tien-Yin | Hammond, Christopher J. | Klaver, Caroline C. W.
Human Genetics  2012;131(9):1467-1480.
Myopia is a complex genetic disorder and a common cause of visual impairment among working age adults. Genome-wide association studies have identified susceptibility loci on chromosomes 15q14 and 15q25 in Caucasian populations of European ancestry. Here, we present a confirmation and meta-analysis study in which we assessed whether these two loci are also associated with myopia in other populations. The study population comprised 31 cohorts from the Consortium of Refractive Error and Myopia (CREAM) representing 4 different continents with 55,177 individuals; 42,845 Caucasians and 12,332 Asians. We performed a meta-analysis of 14 single nucleotide polymorphisms (SNPs) on 15q14 and 5 SNPs on 15q25 using linear regression analysis with spherical equivalent as a quantitative outcome, adjusted for age and sex. We calculated the odds ratio (OR) of myopia versus hyperopia for carriers of the top-SNP alleles using a fixed effects meta-analysis. At locus 15q14, all SNPs were significantly replicated, with the lowest P value 3.87 × 10−12 for SNP rs634990 in Caucasians, and 9.65 × 10−4 for rs8032019 in Asians. The overall meta-analysis provided P value 9.20 × 10−23 for the top SNP rs634990. The risk of myopia versus hyperopia was OR 1.88 (95 % CI 1.64, 2.16, P < 0.001) for homozygous carriers of the risk allele at the top SNP rs634990, and OR 1.33 (95 % CI 1.19, 1.49, P < 0.001) for heterozygous carriers. SNPs at locus 15q25 did not replicate significantly (P value 5.81 × 10−2 for top SNP rs939661). We conclude that common variants at chromosome 15q14 influence susceptibility for myopia in Caucasian and Asian populations world-wide.
Electronic supplementary material
The online version of this article (doi:10.1007/s00439-012-1176-0) contains supplementary material, which is available to authorized users.
PMCID: PMC3418496  PMID: 22665138
21.  Complement Factor D in Age-Related Macular Degeneration 
Complement factor D catalyzes a critical step in the alternative complement activation pathway. The authors report a significant elevation in plasma CFD concentrations in age-related macular degeneration (AMD) patients compared with controls and a weak genetic association between CFD gene variants and AMD.
To examine the role of complement factor D (CFD) in age-related macular degeneration (AMD) by analysis of genetic association, copy number variation, and plasma CFD concentrations.
Single nucleotide polymorphisms (SNPs) in the CFD gene were genotyped and the results analyzed by binary logistic regression. CFD gene copy number was analyzed by gene copy number assay. Plasma CFD was measured by an enzyme-linked immunosorbent assay.
Genetic association was found between CFD gene SNP rs3826945 and AMD (odds ratio 1.44; P = 0.028) in a small discovery case-control series (462 cases and 325 controls) and replicated in a combined cohorts meta-analysis of 4765 cases and 2693 controls, with an odds ratio of 1.11 (P = 0.032), with the association almost confined to females. Copy number variation in the CFD gene was identified in 13 out of 640 samples examined but there was no difference in frequency between AMD cases (1.3%) and controls (2.7%). Plasma CFD concentration was measured in 751 AMD cases and 474 controls and found to be elevated in AMD cases (P = 0.00025). The odds ratio for those in the highest versus lowest quartile for plasma CFD was 1.81. The difference in plasma CFD was again almost confined to females.
CFD regulates activation of the alternative complement pathway, which is implicated in AMD pathogenesis. The authors found evidence for genetic association between a CFD gene SNP and AMD and a significant increase in plasma CFD concentration in AMD cases compared with controls, consistent with a role for CFD in AMD pathogenesis.
PMCID: PMC3230905  PMID: 22003108
22.  Quasi-least squares with mixed linear correlation structures 
Statistics and its interface  2010;3(2):223-234.
Quasi-least squares (QLS) is a two-stage computational approach for estimation of the correlation parameters in the framework of generalized estimating equations. We prove two general results for the class of mixed linear correlation structures: namely, that the stage one QLS estimate of the correlation parameter always exists and is feasible (yields a positive definite estimated correlation matrix) for any correlation structure, while the stage two estimator exists and is unique (and therefore consistent) with probability one, for the class of mixed linear correlation structures. Our general results justify the implementation of QLS for particular members of the class of mixed linear correlation structures that are appropriate for analysis of data from families that may vary in size and composition. We describe the familial structures and implement them in an analysis of optical spherical values in the Old Order Amish (OOA). For the OOA analysis, we show that we would suffer a substantial loss in efficiency, if the familial structures were the true structures, but were misspecified as simpler approximate structures. To help bridge the interface between Statistics and Medicine, we also provide R software so that medical researchers can implement the familial structures in a QLS analysis of their own data.
PMCID: PMC3328409  PMID: 22518205
Quasi-least squares; Linear correlation structure; Mixed correlation structure; Familial data
23.  A 32 kb Critical Region Excluding Y402H in CFH Mediates Risk for Age-Related Macular Degeneration 
PLoS ONE  2011;6(10):e25598.
Complement factor H shows very strong association with Age-related Macular Degeneration (AMD), and recent data suggest that multiple causal variants are associated with disease. To refine the location of the disease associated variants, we characterized in detail the structural variation at CFH and its paralogs, including two copy number polymorphisms (CNP), CNP147 and CNP148, and several rare deletions and duplications. Examination of 34 AMD-enriched extended families (N = 293) and AMD cases (White N = 4210 Indian = 134; Malay = 140) and controls (White N = 3229; Indian = 117; Malay = 2390) demonstrated that deletion CNP148 was protective against AMD, independent of SNPs at CFH. Regression analysis of seven common haplotypes showed three haplotypes, H1, H6 and H7, as conferring risk for AMD development. Being the most common haplotype H1 confers the greatest risk by increasing the odds of AMD by 2.75-fold (95% CI = [2.51, 3.01]; p = 8.31×10−109); Caucasian (H6) and Indian-specific (H7) recombinant haplotypes increase the odds of AMD by 1.85-fold (p = 3.52×10−9) and by 15.57-fold (P = 0.007), respectively. We identified a 32-kb region downstream of Y402H (rs1061170), shared by all three risk haplotypes, suggesting that this region may be critical for AMD development. Further analysis showed that two SNPs within the 32 kb block, rs1329428 and rs203687, optimally explain disease association. rs1329428 resides in 20 kb unique sequence block, but rs203687 resides in a 12 kb block that is 89% similar to a noncoding region contained in ΔCNP148. We conclude that causal variation in this region potentially encompasses both regulatory effects at single markers and copy number.
PMCID: PMC3192039  PMID: 22022419
24.  Genomewide scan in Ashkenazi Jewish families demonstrates evidence of linkage of ocular refraction to a QTL on chromosome 1p36 
Human genetics  2006;119(4):389-399.
The development of refractive error is mediated by both environmental and genetic factors. We performed regression-based quantitative trait locus (QTL) linkage analysis on Ashkenazi Jewish families to identify regions in the genome responsible for ocular refraction. We measured refractive error on individuals in 49 multi-generational American families of Ashkenazi Jewish descent. The average family size was 11.1 individuals and was composed of 2.7 generations. Recruitment criteria specified that each family contain at least two myopic members. The mean spherical equivalent refractive error in the sample was −3.46D (SD=3.29) and 87% of individuals were myopic. Microsatellite genotyping with 387 markers was performed on 411 individuals. We performed multipoint regression-based linkage analysis for ocular refraction and a log transformation of the trait using the statistical package Merlin-Regress. Empirical genomewide significance levels were estimated through gene-dropping simulations by generating random genotypes at each of the 387 markers in 200 replicates of our pedigrees. Maximum LOD scores of 9.5 for ocular refraction and 8.7 for log-transformed refraction (LTR) were observed at 49.1 cM on chromosome 1p36 between markers D1S552 and D1S1622. The empirical genomewide significance levels were P=0.065 for ocular refraction and P<0.005 for LTR, providing strong evidence for linkage of refraction to this locus. The inter-marker region containing the peak spans 11 Mb and contains approximately 189 genes. Conclusion: We found genomewide significant evidence for linkage of refractive error to a novel QTL on chromosome 1p36 in an Ashkenazi Jewish population.
PMCID: PMC3123998  PMID: 16501916
25.  Genomewide Scan of Ocular Refraction in African-American Families Shows Significant Linkage to Chromosome 7p15 
Genetic epidemiology  2008;32(5):454-463.
Refractive development is influenced by environmental and genetic factors. Genetic studies have identified several regions of linkage to ocular refraction, but none have been carried out in African-derived populations. We performed quantitative trait locus linkage analyses in African-American (AA) families to identify genomic regions responsible for refraction. We recruited 493 AA individuals in 96 families to participate in the Myopia Family Study. Genotyping of 387 microsatellite markers was performed on 398 participants. The mean refraction among genotyped individuals was −2.87 D (SD = 3.58) and myopia of at least 1 D was present in 267 (68%) participants. Multipoint, regression-based, linkage analyses were carried out on a logarithmic transformation of ocular refraction using the statistical package MERLIN-REGRESS. Empirical significance levels were determined via 4,898 whole-genome gene-dropping simulations. Linkage analyses were repeated after clustering families into two subgroups based on admixture proportions as determined by the software package STRUCTURE. Genomewide significant linkage was seen at 47 cM on chromosome 7 (logarithm of the odds ratio (LOD) = 5.87, P = 0.00005). In addition, three regions on chromosomes 2p, 3p and 10p showed suggestive evidence of linkage (LOD > 2, P < 0.005) for ocular refraction. We mapped the first quantitative trait locus for ocular refraction in an AA population to chr.7p15. Two previous studies in European-derived families reported some evidence of linkage to a nearby region, suggesting that this region may contain polymorphisms that mediate refraction across populations. The genomic region under our linkage peak spans ~17 Mb and contains ~170 genes. Further refinement of this region will be pursued in future studies.
PMCID: PMC3097031  PMID: 18293391
ocular refraction; myopia; linkage; genetics; African-American

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