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1.  Endophenotypes for Age-Related Macular Degeneration: Extending Our Reach into the Preclinical Stages of Disease 
Journal of clinical medicine  2014;3(4):1335-1356.
The key to reducing the individual and societal burden of age-related macular degeneration (AMD)-related vision loss, is to be able to initiate therapies that slow or halt the progression at a point that will yield the maximum benefit while minimizing personal risk and cost. There is a critical need to find clinical markers that, when combined with the specificity of genetic testing, will identify individuals at the earliest stages of AMD who would benefit from preventive therapies. These clinical markers are endophenotypes for AMD, present in those who are likely to develop AMD, as well as in those who have clinical evidence of AMD. Clinical characteristics associated with AMD may also be possible endophenotypes if they can be detected before or at the earliest stages of the condition, but we and others have shown that this may not always be valid. Several studies have suggested that dynamic changes in rhodopsin regeneration (dark adaptation kinetics and/or critical flicker fusion frequencies) may be more subtle indicators of AMD-associated early retinal dysfunction. One can test for the relevance of these measures using genetic risk profiles based on known genetic risk variants. These functional measures may improve the sensitivity and specificity of predictive models for AMD and may also serve to delineate clinical subtypes of AMD that may differ with respect to prognosis and treatment.
PMCID: PMC4284143  PMID: 25568804
age-related macular degeneration; endophenotype; genetic risk; preclinical diagnostics; retinal function; predictive modeling
2.  Genetic insights into age-related macular degeneration: Controversies addressing Risk, Causality, and Therapeutics 
Molecular Aspects of Medicine  2012;33(4):467-486.
Age-related macular degeneration (AMD) is a common condition among the elderly population that leads to the progressive central vision loss and serious compromise of quality of life for its sufferers. It is also one of the few disorders for whom the investigation of its genetics has yielded rich insights into its diversity and causality and holds the promise of enabling clinicians to provide better risk assessments for individuals as well as to develop and selectively deploy new therapeutics to either prevent or slow the development of disease and lessen the threat of vision loss. The genetics of AMD began initially with the appreciation of familial aggregation and increase risk and expanded with the initial association of APOE variants with the disease. The first major breakthroughs came with family-based linkage studies of affected (and discordant) sibs, which identified a number of genetic loci and led to the targeted search of the 1q31 and 10q26 loci for associated variants. Three of the initial four reports for the CFH variant, Y402H, were based on regional candidate searches, as were the two initial reports of the ARMS2/HTRA1 locus variants. Case-control association studies initially also played a role in discovering the major genetic variants for AMD, and the success of those early studies have been used to fuel enthusiasm for the methodology for a number of diseases. Until 2010, all of the subsequent genetic variants associated with AMD came from candidate gene testing based on the complement factor pathway. In 2010, several large-scale genome-wide association studies (GWAS) identified genes that had not been previously identified. Much of this historical information is available in a number of recent reviews.(Chen et al., 2010b; Deangelis et al., 2011; Fafowora and Gorin, 2012b; Francis and Klein, 2011; Kokotas et al., 2011) Large meta analysis of AMD GWAS has added new loci and variants to this collection.(Chen et al., 2010a; Kopplin et al., 2010; Yu et al., 2011) This paper will focus on the ongoing controversies that are confronting AMD genetics at this time, rather than attempting to summarize this field, which has exploded in the past 5 years.
PMCID: PMC3392516  PMID: 22561651
molecular genetics; Age-related macular degeneration; Association studies; Family-based linkage; Risk factors; Genetics-based therapeutics
3.  Comparative Regional Pupillography as a Noninvasive Biosensor Screening Method for Diabetic Retinopathy 
We describe infrared regional pupillometry as an objective comparative assessment of midperipheral to central retinal sensitivity and to correlate with midperipheral retinal ischemia in diabetic subjects.
We tested 12 normal and 17 diabetic subjects using bilateral infrared pupillometry. The diabetic cohort included seven subjects without, five with mild, three with moderate, and two with severe non-proliferative diabetic retinopathy (NPDR). Central and annular stimuli of varying intensity were presented to one eye, and pupillary amplitude and constriction velocity were measured from both eyes. Light stimulus of increasing intensity was presented as 20 consecutive trials (stimulus duration of 300 ms with 3000 ms intervals). The ratio of central to peripheral responses (Q values) was calculated for each stimulus configuration. Average responses with respect to the stimulus strength were regressed with Gompertz sigmoid function.
Control and moderate/severe NPDR cases comparison showed statistically significant differences in amplitude (QA) and constriction velocity (QCV) (Wilcoxon rank sum test P = 0.002, respectively). Age difference for these groups was not statistically significant (Wilcoxon rank sum test P = 0.15). The comparison of control and diabetic subjects without NPDR/mild NPDR was statistically significant for QA and QCV (Wilcoxon rank sum test P = 0.0002 and P = 0.001, respectively). QA and QCV differences were statistically significant between moderate/severe NPDR cases and subjects without or mild NPDR cases (Wilcoxon rank sum test P = 0.013).
QA and QCV values correlated highly with the severity of diabetic retinopathy, but not with the duration of diabetes. ( number, NCT01546766.)
We describe a novel methodology based on the pupillary response to evaluate midperipheral and central retinal sensitivity. In diabetic subjects, the ratios of peripheral to central amplitude and constriction velocity of the pupillary response are reduced, correlating with severity of retinopathy.
PMCID: PMC3541948  PMID: 23154459
4.  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
5.  Whole exome sequencing detects homozygosity for ABCA4 p.Arg602Trp missense mutation in a pediatric patient with rapidly progressive retinal dystrophy 
BMC Medical Genetics  2014;15:11.
A pediatric patient presented with rapidly progressive vision loss, nyctalopia and retinal dystrophy. This is the first report of homozygosity for the p.Arg602Trp mutation in the ABCA4 gene. The child became legally blind within a period of 2 years.
Case presentation
An eight year-old Hispanic female presented with bilateral decreased vision following a febrile gastrointestinal illness with nausea and vomiting. Extensive workup involved pediatric infectious disease and rheumatology consultations.
Initial visual acuity was 20/60 at distance and 20/30 at near in both eyes. Rapidly progressive vision loss occurred during a 2-year period resulting in visual acuities of 20/200 at distance in both eyes. Fundus exam disclosed attenuated vessels and multiple subretinal blister-like elevations. Optical coherence tomography showed far more lesions than were clinically evident with different levels of elevation. Autofluorescence imagery showed dramatic and widespread geographic areas of atrophy. The deposits that appeared drusen-like on clinical exam were hyperfluorescent, consistent with lipofuscin deposits containing A2e (N-retinylidene-N-retinylethanolamine) indicative of RPE cell dysfunction. Electroretinography was consistent with cone dystrophy, with relative preservation of rod function. Blood analysis and rheumatology evaluation found no evidence of a diffuse post-infectious/inflammatory process. The unique and rapid progression of her subretinal blister-like lesions was documented by fluorescein angiography, optical coherence tomography, autofluorescence imagery, and fundus photography. Family pedigree history disclosed consanguinity, her parents being first cousins. DNA analysis by whole exomic sequencing revealed homozygosity of p.Arg602Trp in the ABCA4 gene.
The pediatric patient presented with a striking clinical appearance and dramatic rate of progression that was clinically more characteristic of an infectious or inflammatory process. This case expands the diverse range of phenotypes attributed to ABCA4 mutations and further supports the role of whole exome sequencing as a powerful new tool available to aid clinicians in establishing diagnosis for challenging cases.
PMCID: PMC3905103  PMID: 24444108
ABCA4 retinopathy; Pediatric; Homozygosity; Consanguinity
6.  Deep intronic mutation in OFD1, identified by targeted genomic next-generation sequencing, causes a severe form of X-linked retinitis pigmentosa (RP23) 
Human Molecular Genetics  2012;21(16):3647-3654.
X-linked retinitis pigmentosa (XLRP) is genetically heterogeneous with two causative genes identified, RPGR and RP2. We previously mapped a locus for a severe form of XLRP, RP23, to a 10.71 Mb interval on Xp22.31-22.13 containing 62 genes. Candidate gene screening failed to identify a causative mutation, so we adopted targeted genomic next-generation sequencing of the disease interval to determine the molecular cause of RP23. No coding variants or variants within or near splice sites were identified. In contrast, a variant deep within intron 9 of OFD1 increased the splice site prediction score 4 bp upstream of the variant. Mutations in OFD1 cause the syndromic ciliopathies orofaciodigital syndrome-1, which is male lethal, Simpson–Golabi–Behmel syndrome type 2 and Joubert syndrome. We tested the effect of the IVS9+706A>G variant on OFD1 splicing in vivo. In RP23 patient-derived RNA, we detected an OFD1 transcript with the insertion of a cryptic exon spliced between exons 9 and 10 causing a frameshift, p.N313fs.X330. Correctly spliced OFD1 was also detected in patient-derived RNA, although at reduced levels (39%), hence the mutation is not male lethal. Our data suggest that photoreceptors are uniquely susceptible to reduced expression of OFD1 and that an alternative disease mechanism can cause XLRP. This disease mechanism of reduced expression for a syndromic ciliopathy gene causing isolated retinal degeneration is reminiscent of CEP290 intronic mutations that cause Leber congenital amaurosis, and we speculate that reduced dosage of correctly spliced ciliopathy genes may be a common disease mechanism in retinal degenerations.
PMCID: PMC3406759  PMID: 22619378
7.  Complement factor H genetic variant and age-related macular degeneration: effect size, modifiers and relationship to disease subtype 
Background Variation in the complement factor H gene (CFH) is associated with risk of late age-related macular degeneration (AMD). Previous studies have been case–control studies in populations of European ancestry with little differentiation in AMD subtype, and insufficient power to confirm or refute effect modification by smoking.
Methods To precisely quantify the association of the single nucleotide polymorphism (SNP rs1061170, ‘Y402H’) with risk of AMD among studies with differing study designs, participant ancestry and AMD grade and to investigate effect modification by smoking, we report two unpublished genetic association studies (n = 2759) combined with data from 24 published studies (26 studies, 26 494 individuals, including 14 174 cases of AMD) of European ancestry, 10 of which provided individual-level data used to test gene–smoking interaction; and 16 published studies from non-European ancestry.
Results In individuals of European ancestry, there was a significant association between Y402H and late-AMD with a per-allele odds ratio (OR) of 2.27 [95% confidence interval (CI) 2.10–2.45; P = 1.1 x 10−161]. There was no evidence of effect modification by smoking (P = 0.75). The frequency of Y402H varied by ancestral origin and the association with AMD in non-Europeans was less clear, limited by paucity of studies.
Conclusion The Y402H variant confers a 2-fold higher risk of late-AMD per copy in individuals of European descent. This was stable to stratification by study design and AMD classification and not modified by smoking. The lack of association in non-Europeans requires further verification. These findings are of direct relevance for disease prediction. New research is needed to ascertain if differences in circulating levels, expression or activity of factor H protein explain the genetic association.
PMCID: PMC3304526  PMID: 22253316
Age-related macular degeneration (AMD); Complement factor H gene; meta-ananlysis
8.  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
9.  Molecular diagnosis of putative Stargardt disease probands by exome sequencing 
BMC Medical Genetics  2012;13:67.
The commonest genetic form of juvenile or early adult onset macular degeneration is Stargardt Disease (STGD) caused by recessive mutations in the gene ABCA4. However, high phenotypic and allelic heterogeneity and a small but non-trivial amount of locus heterogeneity currently impede conclusive molecular diagnosis in a significant proportion of cases.
We performed whole exome sequencing (WES) of nine putative Stargardt Disease probands and searched for potentially disease-causing genetic variants in previously identified retinal or macular dystrophy genes. Follow-up dideoxy sequencing was performed for confirmation and to screen for mutations in an additional set of affected individuals lacking a definitive molecular diagnosis.
Whole exome sequencing revealed seven likely disease-causing variants across four genes, providing a confident genetic diagnosis in six previously uncharacterized participants. We identified four previously missed mutations in ABCA4 across three individuals. Likely disease-causing mutations in RDS/PRPH2, ELOVL, and CRB1 were also identified.
Our findings highlight the enormous potential of whole exome sequencing in Stargardt Disease molecular diagnosis and research. WES adequately assayed all coding sequences and canonical splice sites of ABCA4 in this study. Additionally, WES enables the identification of disease-related alleles in other genes. This work highlights the importance of collecting parental genetic material for WES testing as the current knowledge of human genome variation limits the determination of causality between identified variants and disease. While larger sample sizes are required to establish the precision and accuracy of this type of testing, this study supports WES for inherited early onset macular degeneration disorders as an alternative to standard mutation screening techniques.
PMCID: PMC3459799  PMID: 22863181
Stargardt Disease; Macular Degeneration; Exome; Mutation Screening; Molecular Diagnostics; ABCA4; PRPH2
10.  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
11.  Variants of the Adenosine A2A Receptor Gene Are Protective against Proliferative Diabetic Retinopathy in Patients with Type 1 Diabetes 
Ophthalmic Research  2010;46(1):1-8.
The adenosine A2A receptor (ADORA2A) may ameliorate deleterious physiologic effects associated with tissue injury in individuals with diabetes. We explored associations between variants of the ADORA2A gene and proliferative diabetic retinopathy (PDR) in a cohort of patients with type 1 diabetes (T1D).
The participants were from the Pittsburgh Epidemiology of Diabetes Complications prospective study of childhood-onset T1D. Stereoscopic photographs of the retinal fundus taken at baseline, then biennially, for 10 years were used to define PDR according to the modified Airlie House system. Two tagging single nucleotide polymorphisms (tSNPs; rs2236624-C/T and rs4822489-G/T) in the ADORA2A gene were selected using the HapMap (haplotype map) reference database.
A significant association was observed between SNP rs2236624 and PDR in the recessive genetic model. Participants homozygous for the T allele displayed a decreased risk of developing prevalent PDR (odds ratio, OR = 0.36; p = 0.04) and incident PDR (hazard ratio = 0.156; p = 0.009), and for all cases of PDR combined (OR = 0.23; p = 0.001). The protective effect of T allele homozygosity remained after adjusting for covariates. Similarly, for SNP rs4822489, an association between PDR and T allele homozygosity was observed following covariate adjustment (OR = 0.55; 95% CI: 0.31–0.92; p = 0.04).
Genetic variants of ADORA2A offer statistically significant protection against PDR development in patients with T1D.
PMCID: PMC2997447  PMID: 21088442
Diabetes; Diabetic retinopathy; Single nucleotide polymorphism; Adenosine receptor
12.  Exclusion of Positional Candidate Gene Coding Region Mutations in the Common Posterior Polymorphous Corneal Dystrophy 1 Candidate Gene Interval 
Cornea  2009;28(7):801-807.
Posterior polymorphous corneal dystrophy (PPCD) is an autosomal dominant disorder of the corneal endothelium associated with visually significant corneal edema and glaucoma. Statistical genetic analysis of four families with PPCD has demonstrated linkage to a 2.4 cM common support interval on chromosome 20 bordered by the markers D20S182 and D20S139. We sought to identify the genetic basis of PPCD linked to chromosome 20 (PPCD1) by screening the 26 positional candidate genes between these markers in a family previously mapped to the PPCD1 region.
The coding regions of the 26 positional candidate genes mapped to the common PPCD1 support interval were amplified and sequenced in affected and unaffected individuals from a family previously linked to the PPCD1 locus. Nine other genes positioned just outside of the common PPCD1 support interval but within the autosomal dominant congenital hereditary endothelial dystrophy (CHED1) interval were also screened.
Four DNA sequence variants in three of the positional candidate genes demonstrated complete segregation with the affected phenotype: p.Thr109Thr (rs6111803) in OVOL2, p.Arg56Gln (novel variant - RPSnovel) in RPS19P1, and p.Thr85Thr (rs1053834) and p.Pro99Ser (rs1053839) in C20orf79. Each of these four sequence variants demonstrated significant linkage with the affected phenotype in this family (p-value = 2.5 × 10−7 for RPSnovel, rs1053834 and rs1053839; p-value = 8.6 × 10−7 for rs6111803). However, we also identified each of these four sequence variants in ≥ 9% of unaffected control individuals. The haplotype upon which the disease causing mutation is segregating was found to have a population frequency of 4.2% in the CEPH HapMap trios. While a number of other previously described and novel SNPs were identified in the 35 positional candidate genes located within the PPCD1 and CHED1 intervals, none segregated with the affected phenotype.
We report the absence of a presumed pathogenic coding region mutation in the common PPCD1 support interval. Although minor alleles of four SNPs were identified that segregated with the affected phenotype, the relatively high frequency of each minor allele in the general population indicates that none is a candidate for the causal variant for PPCD. Instead, the causal variant is most likely a coding region deletion or a variant in a non-coding region of the PPCD1 common support interval.
PMCID: PMC2714875  PMID: 19574904
Posterior polymorphous corneal dystrophy; Candidate gene screening; Linkage; Congenital hereditary endothelial dystrophy
13.  Associations between Genetic Polymorphisms of Insulin-like Growth Factor Axis Genes and Risk for Age-Related Macular Degeneration 
This paper provides the first epidemiological evidence that IGF axis genes may be involved in the pathogenesis of advanced AMD and that IGF1R may predispose obese people to higher risk.
To investigate whether insulin-like growth factor (IGF) axis genes, together with a novel dietary risk factor, the dietary glycemic index (dGI), and body mass index (BMI) affect the risk for age-related macular degeneration (AMD).
This case–control study involved 962 subjects originally recruited through the Age-Related Eye Disease Study (AREDS) Genetic Repository. After those with missing covariates or invalid calorie intake (n = 23), diabetes (n = 59), and non-Caucasian race (n = 16) were excluded, 864 participants were used, including 209 AREDS category 1 participants (control group), 354 category 2 or 3 participants (drusen group), and 301 category 4 participants (advanced AMD group). A total of 25 single-nucleotide polymorphisms (SNPs) selected from IGF-1 (n = 9), IGF-2 (n = 1), IGF binding protein 1 (IGFBP1; n = 3), IGFBP3 (n = 3), acid-labile subunit of IGFBP (IGFALS; n = 2), IGF1 receptor (IGF1R; n = 4), and IGF2R (n = 3) were genotyped. SNP-AMD associations were measured with genotype, allele χ2 tests and Armitage's trend test. Odds ratios (OR), 95% confidence intervals (CIs), and SNP-exposure interactions were evaluated by multivariate logistic regression.
One SNP (rs2872060) in IGF1R revealed a significant association with advanced AMD (P-allele = 0.0009, P-trend = 0.0008; the significance level was set at 0.05/25 = 0.002 for multiple comparisons). The risk allele (G) in the heterozygous and homozygous states (OR, 1.67 and 2.93; 95% CI, 1.03–2.71 and 1.60–5.36, respectively) suggests susceptibility and an additive effect on AMD risk. Further stratification analysis remained significant for both neovascularization (OR, 1.49 and 2.61; 95% CI, 0.90–2.48 and 1.39–4.90, respectively) and geographic atrophy (OR, 2.57 and 4.52; 95% CI, 0.99–6.71 and 1.49–13.74, respectively). The G allele interaction analysis with BMI was significant for neovascularization (P = 0.042) but not for geographic atrophy (P = 0.47). No significant interaction was found with dGI.
These data suggest a role of IGF1R on the risk for advanced AMD in this group of subjects.
PMCID: PMC3231967  PMID: 22058336
14.  Dissection of Chromosome 16p12 Linkage Peak Suggests a Possible Role for CACNG3 Variants in Age-Related Macular Degeneration Susceptibility 
Through extensive linkage and association analyses in multiple independent datasets, this study identified CACNG3 as the most likely AMD susceptibility gene on 16p12.
Age-related macular degeneration (AMD) is a complex disorder of the retina, characterized by drusen, geographic atrophy, and choroidal neovascularization. Cigarette smoking and the genetic variants CFH Y402H, ARMS2 A69S, CFB R32Q, and C3 R102G have been strongly and consistently associated with AMD. Multiple linkage studies have found evidence suggestive of another AMD locus on chromosome 16p12 but the gene responsible has yet to be identified.
In the initial phase of the study, single-nucleotide polymorphisms (SNPs) across chromosome 16 were examined for linkage and/or association in 575 Caucasian individuals from 148 multiplex and 77 singleton families. Additional variants were tested in an independent dataset of unrelated cases and controls. According to these results, in combination with gene expression data and biological knowledge, five genes were selected for further study: CACNG3, HS3ST4, IL4R, Q7Z6F8, and ITGAM.
After genotyping additional tagging SNPs across each gene, the strongest evidence for linkage and association was found within CACNG3 (rs757200 nonparametric LOD* = 3.3, APL (association in the presence of linkage) P = 0.06, and rs2238498 MQLS (modified quasi-likelihood score) P = 0.006 in the families; rs2283550 P = 1.3 × 10−6, and rs4787924 P = 0.002 in the case–control dataset). After adjusting for known AMD risk factors, rs2283550 remained strongly associated (P = 2.4 × 10−4). Furthermore, the association signal at rs4787924 was replicated in an independent dataset (P = 0.035) and in a joint analysis of all the data (P = 0.001).
These results suggest that CACNG3 is the best candidate for an AMD risk gene within the 16p12 linkage peak. More studies are needed to confirm this association and clarify the role of the gene in AMD pathogenesis.
PMCID: PMC3101690  PMID: 21169531
15.  Linkage of Posterior Amorphous Corneal Dystrophy to Chromosome 12q21.33 and Exclusion of Coding Region Mutations in KERA, LUM, DCN, and EPYC 
Genomewide linkage analysis has identified a chromosomal locus for posterior amorphous corneal dystrophy at 12q21.33, though no mutations were identified in the positional and functional candidate genes KERA, LUM, DCN, and EPYC.
To identify the genetic basis of posterior amorphous corneal dystrophy (PACD) segregating in a large pedigree.
The authors performed clinical evaluation of a previously unreported pedigree with PACD, light and electron microscopic examination of an excised corneal button, genomewide linkage analysis, fine mapping linkage and haplotype analysis, and screening of four candidate genes (KERA, LUM, DCN, and EPYC).
Twenty-one participants were determined to be affected based on the presence of characteristic clinical features of PACD; 15 affected and 39 unaffected individuals from a single pedigree enrolled in the study and provided DNA for analysis. Histopathologic examination of an excised corneal specimen from an affected individual demonstrated disorganized stromal lamellae and stromal staining with colloidal iron. Genomewide analysis demonstrated significant evidence of linkage to chromosome region 12q21.33 and evidence suggestive of linkage to chromosome region 8q22.3. Fine mapping of the chromosome 12 locus confirmed significant linkage; the largest multipoint log odds ratio score was 5.6 at D12S351. The linkage support interval was approximately 3.5 Mb (3.5 cM) in length between flanking markers D12S1812 and D12S95, roughly the entire chromosome band 12q21.33. No coding region mutations were identified in four candidate genes—KERA, LUM, DCN, EPYC—located in the chromosome 12 linkage support interval.
Linkage and haplotype analyses identified 12q21.33 as a locus for PACD. However, no mutations were identified in the candidate genes (KERA, LUM, DCN, EPYC) within this region.
PMCID: PMC2910638  PMID: 20357198
16.  Novel KRIT1/CCM1 mutation in a patient with retinal cavernous hemangioma and cerebral cavernous malformation 
Retinal cavernous hemangiomas are rare vascular anomalies, and can be associated with cerebral cavernous malformations (CCM). Distinct mutations have been reported in patients who have both CCMs and retinal cavernous hemangiomas.
Fluorescein angiography, spectral domain optical coherence tomography, and genetic testing were performed on a patient with a retinal cavernous hemangioma and a CCM.
Our patient was heterozygous in the KRIT1/CCM1 gene for a frameshift mutation, c.1088delC. This would be predicted to result in premature protein termination.
We have identified a novel mutation in the KRIT1/CCM1 gene in a patient with both CCM and retinal cavernous hemangioma. We hypothesize that the occurrence of retinal cavernous hemangiomas and CCMs is underlaid by a common mechanism present in the KRIT1/CCM1 gene.
PMCID: PMC2910301  PMID: 20306072
Cerebral cavernous malformation; KRIT1; Mutation; Retinal cavernous hemangioma
17.  Interpretation of Genetic Association Studies: Markers with Replicated Highly Significant Odds Ratios May Be Poor Classifiers 
PLoS Genetics  2009;5(2):e1000337.
Recent successful discoveries of potentially causal single nucleotide polymorphisms (SNPs) for complex diseases hold great promise, and commercialization of genomics in personalized medicine has already begun. The hope is that genetic testing will benefit patients and their families, and encourage positive lifestyle changes and guide clinical decisions. However, for many complex diseases, it is arguable whether the era of genomics in personalized medicine is here yet. We focus on the clinical validity of genetic testing with an emphasis on two popular statistical methods for evaluating markers. The two methods, logistic regression and receiver operating characteristic (ROC) curve analysis, are applied to our age-related macular degeneration dataset. By using an additive model of the CFH, LOC387715, and C2 variants, the odds ratios are 2.9, 3.4, and 0.4, with p-values of 10−13, 10−13, and 10−3, respectively. The area under the ROC curve (AUC) is 0.79, but assuming prevalences of 15%, 5.5%, and 1.5% (which are realistic for age groups 80 y, 65 y, and 40 y and older, respectively), only 30%, 12%, and 3% of the group classified as high risk are cases. Additionally, we present examples for four other diseases for which strongly associated variants have been discovered. In type 2 diabetes, our classification model of 12 SNPs has an AUC of only 0.64, and two SNPs achieve an AUC of only 0.56 for prostate cancer. Nine SNPs were not sufficient to improve the discrimination power over that of nongenetic predictors for risk of cardiovascular events. Finally, in Crohn's disease, a model of five SNPs, one with a quite low odds ratio of 0.26, has an AUC of only 0.66. Our analyses and examples show that strong association, although very valuable for establishing etiological hypotheses, does not guarantee effective discrimination between cases and controls. The scientific community should be cautious to avoid overstating the value of association findings in terms of personalized medicine before their time.
PMCID: PMC2629574  PMID: 19197355
18.  C2 and CFB Genes in Age-Related Maculopathy and Joint Action with CFH and LOC387715 Genes 
PLoS ONE  2008;3(5):e2199.
Age-related maculopathy (ARM) is a common cause of visual impairment in the elderly populations of industrialized countries and significantly affects the quality of life of those suffering from the disease. Variants within two genes, the complement factor H (CFH) and the poorly characterized LOC387715 (ARMS2), are widely recognized as ARM risk factors. CFH is important in regulation of the alternative complement pathway suggesting this pathway is involved in ARM pathogenesis. Two other complement pathway genes, the closely linked complement component receptor (C2) and complement factor B (CFB), were recently shown to harbor variants associated with ARM.
Methods/Principal Findings
We investigated two SNPs in C2 and two in CFB in independent case-control and family cohorts of white subjects and found rs547154, an intronic SNP in C2, to be significantly associated with ARM in both our case-control (P-value 0.00007) and family data (P-value 0.00001). Logistic regression analysis suggested that accounting for the effect at this locus significantly (P-value 0.002) improves the fit of a genetic risk model of CFH and LOC387715 effects only. Modeling with the generalized multifactor dimensionality reduction method showed that adding C2 to the two-factor model of CFH and LOC387715 increases the sensitivity (from 63% to 73%). However, the balanced accuracy increases only from 71% to 72%, and the specificity decreases from 80% to 72%.
C2/CFB significantly influences AMD susceptibility and although accounting for effects at this locus does not dramatically increase the overall accuracy of the genetic risk model, the improvement over the CFH-LOC387715 model is statistically significant.
PMCID: PMC2374901  PMID: 18493315
19.  Ordered subset linkage analysis supports a susceptibility locus for age-related macular degeneration on chromosome 16p12 
BMC Genetics  2004;5:18.
Age-related macular degeneration (AMD) is a complex disorder that is responsible for the majority of central vision loss in older adults living in developed countries. Phenotypic and genetic heterogeneity complicate the analysis of genome-wide scans for AMD susceptibility loci. The ordered subset analysis (OSA) method is an approach for reducing heterogeneity, increasing statistical power for detecting linkage, and helping to define the most informative data set for follow-up analysis. OSA assesses the linkage evidence in subsets of potentially more homogeneous families by rank-ordering family-specific lod scores with respect to trait-associated covariates or phenotypic features. Here, we present results of incorporating five continuous covariates into our genome-wide linkage analysis of 389 microsatellite markers in 62 multiplex families: Body mass index (BMI), systolic (SBP) and diastolic (DBP) blood pressure, intraocular pressure (IOP), and pack-years of cigarette smoking. Chromosome-wide significance of increases in nonparametric multipoint lod scores in covariate-defined subsets relative to the overall sample was assessed by permutation.
Using a correction for testing multiple covariates, statistically significant lod score increases were observed for two chromosomal regions: 14q13 with a lod score of 3.2 in 28 families with average IOP ≤ 15.5 (p = 0.002), and 6q14 with a lod score of 1.6 in eight families with average BMI ≥ 30.1 (p = 0.0004). On chromosome 16p12, nominally significant lod score increases (p ≤ 0.05), up to a lod score of 2.9 in 32 families, were observed with several covariate orderings. While less significant, this was the only region where linkage evidence was associated with multiple clinically meaningful covariates and the only nominally significant finding when analysis was restricted to advanced forms of AMD. Families with linkage to 16p12 had higher averages of SBP, IOP and BMI and were primarily affected with neovascular AMD. For all three regions, linkage signals at or very near the peak marker have previously been reported.
Our results suggest that a susceptibility gene on chromosome 16p12 may predispose to AMD, particularly to the neovascular form, and that further research into the previously suggested association of neovascular AMD and systemic hypertension is warranted.
PMCID: PMC481059  PMID: 15238159

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