Exome-based case-control association study using extreme phenotype design reveals novel candidates with protective effect in diabetic retinopathy.

UNLABELLED: Diabetic retinopathy (DR) is a common clinical expression of diabetes mellitus-induced vasculopathy and is a major cause of vision loss. Significant gaps remain in our understanding of the molecular pathoetiology of DR, and it is hoped that human genetic approaches can reveal novel targets especially since DR is a heritable trait. Previous studies have focused on genetic risk factors of DR but their results have been mixed. In this study, we hypothesized that the use of the extreme phenotype design will increase the power of a genomewide search for "protective" genetic variants. We enrolled a small yet atypical cohort of 43 diabetics who did not develop DR a decade or more after diagnosis (cases), and 64 diabetics with DR (controls), all of similar ethnic background (Saudi). Whole-exome sequencing of the entire cohort was followed by statistical analysis employing combined multivariate and collapsing methods at the gene level, to identify genes that are enriched for rare variants in cases vs. CONTROLS: Three genes (NME3, LOC728699, and FASTK) reached gene-based genome-wide significance at the 10(-08) threshold (p value = 1.55 × 10(-10), 6.23 × 10(-10), 3.21 × 10(-08), respectively). Our results reveal novel candidate genes whose increased rare variant burden appears to protect against DR, thus highlighting them as attractive candidate targets, if replicated by future studies, for the treatment and prevention of DR. Extreme phenotype design when implemented in sequencing-based genome-wide case-control studies has the potential to reveal novel candidates with a smaller cohort size compared to standard study designs.

Detection and correction of artefacts in estimation of rare copy number variants and analysis of rare deletions in type 1 diabetes.

Copy number variants (CNVs) have been proposed as a possible source of 'missing heritability' in complex human diseases. Two studies of type 1 diabetes (T1D) found null associations with common copy number polymorphisms, but CNVs of low frequency and high penetrance could still play a role. We used the Log-R-ratio intensity data from a dense single nucleotide polymorphism (SNP) array, ImmunoChip, to detect rare CNV deletions (rDELs) and duplications (rDUPs) in 6808 T1D cases, 9954 controls and 2206 families with T1D-affected offspring. Initial analyses detected CNV associations. However, these were shown to be false-positive findings, failing replication with polymerase chain reaction. We developed a pipeline of quality control (QC) tests that were calibrated using systematic testing of sensitivity and specificity. The case-control odds ratios (OR) of CNV burden on T1D risk resulting from this QC pipeline converged on unity, suggesting no global frequency difference in rDELs or rDUPs. There was evidence that deletions could impact T1D risk for a small minority of cases, with enrichment for rDELs longer than 400 kb (OR = 1.57, P = 0.005). There were also 18 de novo rDELs detected in affected offspring but none for unaffected siblings (P = 0.03). No specific CNV regions showed robust evidence for association with T1D, although frequencies were lower than expected (most less than 0.1%), substantially reducing statistical power, which was examined in detail. We present an R-package, plumbCNV, which provides an automated approach for QC and detection of rare CNVs that can facilitate equivalent analyses of large-scale SNP array datasets.

A genome-wide assessment of the role of untagged copy number variants in type 1 diabetes.

Genome-wide association studies (GWAS) for type 1 diabetes (T1D) have successfully identified more than 40 independent T1D associated tagging single nucleotide polymorphisms (SNPs). However, owing to technical limitations of copy number variants (CNVs) genotyping assays, the assessment of the role of CNVs has been limited to the subset of these in high linkage disequilibrium with tag SNPs. The contribution of untagged CNVs, often multi-allelic and difficult to genotype using existing assays, to the heritability of T1D remains an open question. To investigate this issue, we designed a custom comparative genetic hybridization array (aCGH) specifically designed to assay untagged CNV loci identified from a variety of sources. To overcome the technical limitations of the case control design for this class of CNVs, we genotyped the Type 1 Diabetes Genetics Consortium (T1DGC) family resource (representing 3,903 transmissions from parents to affected offspring) and used an association testing strategy that does not necessitate obtaining discrete genotypes. Our design targeted 4,309 CNVs, of which 3,410 passed stringent quality control filters. As a positive control, the scan confirmed the known T1D association at the INS locus by direct typing of the 5' variable number of tandem repeat (VNTR) locus. Our results clarify the fact that the disease association is indistinguishable from the two main polymorphic allele classes of the INS VNTR, class I-and class III. We also identified novel technical artifacts resulting into spurious associations at the somatically rearranging loci, T cell receptor, TCRA/TCRD and TCRB, and Immunoglobulin heavy chain, IGH, loci on chromosomes 14q11.2, 7q34 and 14q32.33, respectively. However, our data did not identify novel T1D loci. Our results do not support a major role of untagged CNVs in T1D heritability.

Genome-wide association study of intelligence: additive effects of novel brain expressed genes.

OBJECTIVE: The purpose of the present study was to identify common genetic variants that are associated with human intelligence or general cognitive ability. METHOD: We performed a genome-wide association analysis with a dense set of 1 million single-nucleotide polymorphisms (SNPs) and quantitative intelligence scores within an ancestrally homogeneous family sample of 656 individuals with at least one child affected by attention-deficit/hyperactivity disorder (ADHD). RESULTS: Haplotype trend regression analysis with sliding four-SNP windows identified haplotypes of genome-wide significance in genes involved in synaptic signaling (KIF16B; p = 1.27E-08) and neurodevelopment (PAX5; p = 3.58E-08), and highlight findings from a recent genetic study of cognitive ability (RXRA; p = 7.7E-08; GYPC; p = 2.5E-07). Further interrogation of SNPs within top haplotypes reveals that the minor alleles are associated with higher intelligence, whereas others are associated with relatively lower (but still average range) intelligence. Effects of the eight genes are additive, as a greater number of the associated genotypes in a given individual predict higher intelligence (p = 5.36E-08) and account for 8% of variance in intelligence. CONCLUSIONS: Analyses that examine additive genetic effects may be useful in identifying regions where the additive effects of SNPs have a significant effect on phenotype. These results describe novel variants and additive effects of genes involved in brain development on variability in intelligence within an ADHD sample. The precise mechanisms of these loci in relation to determining individual differences in general cognitive ability require further investigation.

Genome-wide analysis of copy number variants in attention deficit hyperactivity disorder: the role of rare variants and duplications at 15q13.3.

OBJECTIVE: Attention deficit hyperactivity disorder (ADHD) is a common, highly heritable psychiatric disorder. Because of its multifactorial etiology, however, identifying the genes involved has been difficult. The authors followed up on recent findings suggesting that rare copy number variants (CNVs) may be important for ADHD etiology. METHOD: The authors performed a genome-wide analysis of large, rare CNVs (<1% population frequency) in children with ADHD (N=896) and comparison subjects (N=2,455) from the IMAGE II Consortium. RESULTS: The authors observed 1,562 individually rare CNVs >100 kb in size, which segregated into 912 independent loci. Overall, the rate of rare CNVs >100 kb was 1.15 times higher in ADHD case subjects relative to comparison subjects, with duplications spanning known genes showing a 1.2-fold enrichment. In accordance with a previous study, rare CNVs >500 kb showed the greatest enrichment (1.28-fold). CNVs identified in ADHD case subjects were significantly enriched for loci implicated in autism and in schizophrenia. Duplications spanning the CHRNA7 gene at chromosome 15q13.3 were associated with ADHD in single-locus analysis. This finding was consistently replicated in an additional 2,242 ADHD case subjects and 8,552 comparison subjects from four independent cohorts from the United Kingdom, the United States, and Canada. Presence of the duplication at 15q13.3 appeared to be associated with comorbid conduct disorder. CONCLUSIONS: These findings support the enrichment of large, rare CNVs in ADHD and implicate duplications at 15q13.3 as a novel risk factor for ADHD. With a frequency of 0.6% in the populations investigated and a relatively large effect size (odds ratio=2.22, 95% confidence interval=1.5­3.6), this locus could be an important contributor to ADHD etiology.

Genome-wide copy number variation study associates metabotropic glutamate receptor gene networks with attention deficit hyperactivity disorder.

Attention deficit hyperactivity disorder (ADHD) is a common, heritable neuropsychiatric disorder of unknown etiology. We performed a whole-genome copy number variation (CNV) study on 1,013 cases with ADHD and 4,105 healthy children of European ancestry using 550,000 SNPs. We evaluated statistically significant findings in multiple independent cohorts, with a total of 2,493 cases with ADHD and 9,222 controls of European ancestry, using matched platforms. CNVs affecting metabotropic glutamate receptor genes were enriched across all cohorts (P = 2.1 × 10(-9)). We saw GRM5 (encoding glutamate receptor, metabotropic 5) deletions in ten cases and one control (P = 1.36 × 10(-6)). We saw GRM7 deletions in six cases, and we saw GRM8 deletions in eight cases and no controls. GRM1 was duplicated in eight cases. We experimentally validated the observed variants using quantitative RT-PCR. A gene network analysis showed that genes interacting with the genes in the GRM family are enriched for CNVs in ∼10% of the cases (P = 4.38 × 10(-10)) after correction for occurrence in the controls. We identified rare recurrent CNVs affecting glutamatergic neurotransmission genes that were overrepresented in multiple ADHD cohorts.

Severity of diabetic retinopathy and health-related quality of life: the Los Angeles Latino Eye Study.

OBJECTIVE: To assess the impact of diabetic retinopathy (DR) and its severity on health-related quality of life (HRQOL) in a population-based sample of Latinos with type 2 diabetes mellitus (DM). DESIGN: Cross-sectional population-based study, the Los Angeles Latino Eye Study (LALES). PARTICIPANTS: We included 1064 LALES participants with DM. METHODS: We measured HRQOL by the 25-item National Eye Institute Visual Function Questionnaire (NEI-VFQ-25) and the Medical Outcomes Study 12-item Short Form Health Survey (SF-12). We assessed DR by masked standardized grading of stereoscopic photographs from 7 standard fields. Severity of DR in eyes was graded using a modified Airlie House classification. The severity scores from each eye were then concatenated to create a single per person grade ranging from 1 (no DR in either eye) to 15 (bilateral PDR). Multiple linear regression analyses were performed to determine the independent relationship between severity of DR and HRQOL after adjusting for covariates. MAIN OUTCOME MEASURES: Scores on the NEI-VFQ-25 and SF-12. RESULTS: More severe DR was associated with worse HRQOL scores on all of the NEI-VFQ-25 and SF-12 subscales (P<0.05). Individuals with DR from grade 2 (minimum nonproliferative diabetic retinopathy [NPDR]) through grade 8 (unilateral moderate NPDR) show a modest decline in HRQOL. However, the decline becomes significantly steeper between steps 8 (unilateral moderate NPDR) and 9-15 (bilateral moderate NPDR to bilateral PDR). The domains with the most significant impact were for vision-related daily activities, dependency, and mental health. CONCLUSIONS: Greater severity of DR was associated with lower general and vision-specific HRQOL. Persons with bilateral moderate NPDR had the most substantial decrease in quality of life compared with those with less severe DR. The prevention of incident DR and, more important, its progression from unilateral to bilateral NPDR is likely to have a positive impact on a person's HRQOL and should be considered an important goal in management of individuals with DM.

Copy number variation in the Framingham Heart Study.

In this paper we test for association between copy number variation and diabetes in a subset of individuals from the Framingham Heart Study. We used the 500 k SNP data and called copy number variation using two algorithms: the genome alteration detection algorithm of Pique-Regi et al. and the software Golden Helix. We then tested for association between copy number and diabetes using a gene-based analysis. Our results show little evidence of association between copy number and diabetes status. Furthermore, our results indicate a relatively poor level of agreement between copy number calls resulting from the two programs. We then examined potential causes for this difference in results and the implications for future studies.

Variation in genetic admixture and population structure among Latinos: the Los Angeles Latino eye study (LALES).

BACKGROUND: Population structure and admixture have strong confounding effects on genetic association studies. Discordant frequencies for age-related macular degeneration (AMD) risk alleles and for AMD incidence and prevalence rates are reported across different ethnic groups. We examined the genomic ancestry characterizing 538 Latinos drawn from the Los Angeles Latino Eye Study [LALES] as part of an ongoing AMD-association study. To help assess the degree of Native American ancestry inherited by Latino populations we sampled 25 Mayans and 5 Mexican Indians collected through Coriell's Institute. Levels of European, Asian, and African descent in Latinos were inferred through the USC Multiethnic Panel (USC MEP), formed from a sample from the Multiethnic Cohort (MEC) study, the Yoruba African samples from HapMap II, the Singapore Chinese Health Study, and a prospective cohort from Shanghai, China. A total of 233 ancestry informative markers were genotyped for 538 LALES Latinos, 30 Native Americans, and 355 USC MEP individuals (African Americans, Japanese, Chinese, European Americans, Latinos, and Native Hawaiians). Sensitivity of ancestry estimates to relative sample size was considered. RESULTS: We detected strong evidence for recent population admixture in LALES Latinos. Gradients of increasing Native American background and of correspondingly decreasing European ancestry were observed as a function of birth origin from North to South. The strongest excess of homozygosity, a reflection of recent population admixture, was observed in non-US born Latinos that recently populated the US. A set of 42 SNPs especially informative for distinguishing between Native Americans and Europeans were identified. CONCLUSION: These findings reflect the historic migration patterns of Native Americans and suggest that while the 'Latino' label is used to categorize the entire population, there exists a strong degree of heterogeneity within that population, and that it will be important to assess this heterogeneity within future association studies on Latino populations. Our study raises awareness of the diversity within "Latinos" and the necessity to assess appropriate risk and treatment management.

Intraocular pressure, central corneal thickness, and prevalence of open-angle glaucoma: the Los Angeles Latino Eye Study.

PURPOSE: To examine the relationship between the prevalence of open-angle glaucoma (OAG) and intraocular pressure (IOP) and the impact of central corneal thickness (CCT) on this relationship. DESIGN: Population-based cross-sectional study. METHODS: The study cohort consisted of 5,970 participants from the Los Angeles Latino Eye Study (LALES) with no history of glaucoma treatment and with complete ophthalmic examination data. The relationship between the prevalence of OAG and IOP was contrasted across persons with CCT designated as thin, normal, or thick. RESULTS: Prevalence of OAG was exponentially related to IOP. When stratified by CCT, persons with thin CCT had a significantly higher prevalence of OAG than did those with normal or thick CCTs at all levels of IOP. Adjusting each IOP individually for CCT did not impact significantly the relationship between the prevalence of OAG and IOP. CONCLUSIONS: These findings suggest that adjusting for the impact of CCT on IOP by correction algorithms is not necessary in a population based assessment of glaucoma prevalence; CCT, however, is an important independent risk factor for the prevalence of OAG.

Subsets of Finns with high HDL to total cholesterol ratio show evidence for linkage to type 2 diabetes on chromosome 6q.

OBJECTIVES: The purpose of this study was to examine carefully heterogeneity underlying evidence for linkage to type 2 diabetes (T2DM) on chromosome 6q from two sets of FUSION families. METHODS: Ordered subsets analysis (OSA) was performed on two sets of FUSION families. For OSA results showing significant improvement in evidence for linkage, T2DM-related phenotypes were compared between individuals with T2DM within the subset versus the complement. RESULTS: OSA analysis revealed 105 families with the highest average HDL to total cholesterol ratio (HDL ratio) that had strongly increased evidence for linkage (MLS = 7.91 at 78.0 cM; uncorrected p = 0.00002). Subjects with T2DM within this subset were significantly leaner, had lower fasting glucose, insulin, and C-peptide, and more favorable cardiovascular risk profile compared to the complement set of subjects with T2DM. OSA also revealed 33 families with the lowest average fasting insulin that had increased evidence for linkage at a second locus (MLS = 3.45 at 128 cM; uncorrected p = 0.017) coincident with quantitative trait locus linkage analysis results for fasting and 2-hour insulin in subjects without T2DM. CONCLUSIONS: These results suggest two diabetes susceptibility loci on chromosome 6q that may affect subsets of individuals with a milder form of T2DM.

The Trager approach in the treatment of chronic headache: a pilot study.

CONTEXT: Although the traditional treatment of headache has been pharmacological, there have been many attempts to treat headaches with other methods with mixed levels of success. OBJECTIVE: To obtain preliminary data on the efficacy of the Trager approach in the treatment of chronic headache. DESIGN: Small-scale randomized controlled clinical trial. SETTING: University-based clinic. PATIENTS: Thirty-three volunteers with a self-reported history of chronic headache with at least one headache per week for at least 6 months. INTERVENTIONS: Medication only control group, medication and attention control group, and medication and Trager treatment group. MAIN OUTCOME MEASURES: Self-reported frequency, duration, and intensity of headache, medication usage and headache quality of life (HQOL) obtained at baseline and after a 6-week treatment period. RESULTS: Analyses of variance demonstrated significant improvement in HQOL for the Trager and attention control groups, and reduction in medication usage for the Trager group (P < 0.05). Within-group analyses revealed that participants randomized to Trager demonstrated a significant decrease in the frequency of headaches (P = 0.045), improvement in HQOL (P = 0.045), and a 44% decrease in medication usage (P = 0.03). Participants randomized to the attention control group demonstrated a significant improvement in HQOL (P = 0.035) and a 19% decrease in medication usage (P = 0.15). Participants randomized to the no-treatment control group revealed a significant increase in headache duration (P = 0.025) and intensity (P = 0.025), and a declination in HQOL (P = 0.035). CONCLUSIONS: The Trager approach decreased headache frequency and medication usage. Trager and physician attention improved HQOL. A larger, multi-site study is recommended.