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Structural variations such as copy number variants (CNVs) have been associated with multiple autoimmune diseases. In this study, we explored the association of the Fc gamma receptor 3B gene (FCGR3B) copy number variation (CNV) with rheumatoid arthritis (RA) susceptibility and related serological traits in the Pakistani population. We also performed a meta-analysis of four published FCGR3B CNV studies along with the current study. A total of 927 subjects (597 RA cases, 330 healthy controls) were recruited from three rheumatology centers in Pakistan. Anti-cyclic citrullinated peptide (anti-CCP) antibodies and rheumatoid factor (RF) were measured in RA patients. FCGR3B copy number was assayed using the TaqMan® CN assay (Hs04211858_cn, Applied Biosystems, Foster City, CA, USA) and the copy number was estimated by using CopyCaller® software (version 2.1; Applied Biosystems, USA). Logistic regression was applied to calculate the odds ratio (OR) of RA risk associated with FCGR3B CNV using sex and age as covariates in R. Meta-analysis on four previously published studies and the current study was performed using the random-effect model. We observed a significant association between FCGR3B copy number < 2 and RA susceptibility (OR = 1.53; 95% CI: 1.05 to 2.22; p = 0.0259) and anti-CCP seropositivity (OR 2.56; 95% CI: 1.34 to 4.89; p = 0.0045). A non-significant association of FCGR3B copy number < 2 was also observed between increased rheumatoid factor (RF) seropositivity (OR = 1.74; 95% CI:0.93 to 3.26; p = 0.0816). Meta-analysis on 13,915 subjects (7005 RA cases and 6907 controls) also showed significant association of copy number < 2 with the increased risk of RA (OR = 1.30; 95% CI: 1.07 to 1.56; p = 0.00671). FCGR3B copy number < 2 is associated with increased RA risk and anti-CCP seropositivity.

To investigate whether MYD88 L265P mutation, which is frequently present in vitreoretinal lymphoma, can be detected in aqueous humor, a specimen that can be obtained in a clinic setting, potentially mitigating the need for more invasive vitrectomy procedures, and whether this approach can be used to monitor treatment response.Observational case series.Patients who were diagnosed with biopsy-confirmed or clinically diagnosed vitreoretinal lymphoma or biopsy-confirmed vitritis.We evaluated aqueous humor-derived (AHD) MYD88 L265P mutation during vitreous biopsy or at the initial presentation in the clinic if vitreous biopsy was not feasible. Demographic or clinical features of patients were retrospectively reviewed. Aqueous humor-derived MYD88 L265P mutation was re-evaluated after patients completed a course of intravitreal methotrexate and rituximab injection therapy. The NM_002468.4: c.794TC (p.L265P) mutation in the MYD88 gene was evaluated in AHD cellular and cell-free DNA using allele-specific polymerase chain reaction.Detection of AHD MYD88 L265P mutation at the initial diagnosis and to monitor the treatment response.Aqueous humor from 18 eyes of 14 patients with biopsy-confirmed or clinically diagnosed vitreoretinal lymphoma and 3 eyes of 3 patients with biopsy-confirmed vitritis were evaluated. Aqueous humor-derived MYD88 L265P mutation was detected in cell-based and cell-free DNA from 15 (83%) of 18 eyes with biopsy-confirmed or clinically diagnosed vitreoretinal lymphoma but not identified in any of the 3 eyes with vitritis. The mutation was less readily detectable in cellular DNA (10 of 18) compared with cell-free DNA (15 of 18). Furthermore, aqueous sampling after intravitreal methotrexate and rituximab injection therapy revealed absence of this mutation after complete response in 7 eyes. The mutation was detected in 1 eye that developed recurrence in a posttreatment window of 6 months. After a mean of follow-up of 9 months, there was no clinical evidence of vitreoretinal lymphoma recurrence in the 7 eyes with no detectable AHD MYD88 L265P mutation.This investigational study suggests that AHD MYD88 L265P can be detected in eyes with lymphoma and may thus serve as a surrogate, less invasive biopsy in the diagnosis and follow-up of vitreoretinal lymphoma, particularly when cell-free DNA is evaluated.

The genetics of late-onset Alzheimer's disease (AD) is complex due to the heterogeneous nature of the disorder. APOE*4 is the strongest genetic risk factor for AD. Genome-wide association studies have identified more than 30 additional loci, each having relatively small effect size. Known AD loci explain only about 30% of the genetic variance, and thus much of the genetic variance remains unexplained. To identify some of the missing heritability of AD, we analyzed whole-exome sequencing (WES) data focusing on non-APOE*4 carriers from two WES datasets: 720 cases and controls from the University of Pittsburgh and 7,252 cases and controls from the Alzheimer's Disease Sequencing Project. Following separate WES analyses in each dataset, we performed meta-analysis for overlapping markers present in both datasets. Among the four variants reaching the exome-wide significance threshold, three were from known AD loci: APOE/rs7412 (odds ratio (OR) = 0.40; p = 5.46E-24), TOMM40/rs157581 (OR = 1.49; p = 4.04E-07), and TREM2/rs75932628 (OR = 4.00; p = 1.15E-07). The fourth significant variant, rs199533, was from a novel locus on chromosome 17 in the NSF gene (OR = 0.78; p = 2.88E-07). NSF was also significant in the gene-based analysis (p = 1.20E-05). In the GTEx data, NSF/rs199533 is a cis-eQTL for multiple genes in the brain and blood, including NSF that is highly expressed across all brain tissues, including regions that typically show amyloid-β accumulation. Further characterization of genes that are affected by NSF/rs199533 may help to shed light on the roles of these genes in AD etiology.

In the course of our search for the gene responsible for X-linked cone-rod dystrophy (COD1), we constructed a physical map and contig (encompassing the region between DXS556 and DXS228), and identified sequences showing homologies to the expressed sequence tags (ESTs) that matched CRSP2 (EXLM1) transcript. We confirmed the expression of the CRSP2 gene in the retina and refined its exact genomic location between DXS1368 and DXS993. We demonstrated that the entire transcript is encoded within 31 exons. Primers were designed for mutation analysis of the exons by direct sequencing of PCR products from genomic DNA, and revealed no mutations in COD1 families. We subsequently excluded CRSP2 as a candidate for COD1 by demonstrating the causative mutations in the RPGR. However, due to its expression in different tissues and its contribution to transcriptional regulation, CRSP2 may be a candidate for other diseases that map to this region of the X chromosome.

Objective. Copy number variation (CNV) is a structural variation in the human genome that has been associated with multiple clinical phenotypes. B cells are important components of rheumatoid arthritis- (RA-) mediated immune response; hence, CNV in the regulators of B cells (such as VPREB1) can influence RA susceptibility. In this study, we aimed to explore the association of CNV in the VPREB1 gene with RA susceptibility in the Pakistani population. Methods. A total of 1,106 subjects (616 RA cases, 490 healthy controls) were selected from three rheumatology centers in Pakistan. VPREB1 CNV was determined using the TaqMan® CN assay (Hs02879734_cn, Applied Biosystems, Foster City, CA, USA), and CNV was estimated by using CopyCaller® (version 2.1; Applied Biosystems, USA) software. Odds ratio (OR) was calculated by logistic regression with sex and age as covariates in R. Results. A significant association between >2 VPREB1 CNV and RA risk was observed with an OR of 3.92 (95% CI: 1.27 - 12.12; p=0.01746) in the total sample. Whereas p=0.00381). Conclusion. CNV>2 of VPREB1 is a risk factor for RA in the total Pakistani population, while CNV<2 is protective in women.

Genome-wide association studies (GWAS) have identified multiple type 2 diabetes (T2D) loci, mostly among populations of European descent. There is a high prevalence of T2D among Pakistanis. Both genetic and environmental factors may be responsible for this high prevalence. In order to understand the shared genetic basis of T2D among Pakistanis and Europeans, we examined 77 genome-wide significant variants previously implicated among European populations. We genotyped 77 single-nucleotide polymorphisms (SNPs) by iPLEX® Gold or TaqMan® assays in a case-control sample of 1,683 individuals. Association analysis was performed using logistic regression. A total of 16 SNPs (TCF7L2/rs7903146, GLIS3/rs7041847, CHCHD9/rs13292136, PLEKHA1/rs2292626, FTO/rs9936385, CDKAL1/rs7756992, KCNJ11/rs5215, LOC105372155/rs12970134, KCNQ1/rs163182, CTRB1/rs7202877, ST6GAL1/rs16861329, ADAMTS9-AS2/rs6795735, LOC105370275/rs1359790, C5orf67/rs459193, ZBED3-AS1/rs6878122 and UBE2E2/rs7612463) showed statistically significant associations after controlling for the false discovery rate. While KCNQ1/rs163182 and ZBED3-AS1/rs6878122 showed opposite allelic effects, the remaining significant SNPs had the same allelic effects as reported previously. Our data indicate that a selected number of T2D loci previously identified among populations of European descent also affect the risk of T2D in the Pakistani population.

Immune checkpoint inhibitors were effective and well tolerated in advanced conjunctival squamous cell carcinoma with orbital extension. Of five tumors, four with high mutation burden showed complete and durable response after a mean follow-up of 6 months.

Genome-wide association studies (GWAS) of coronary artery disease (CAD) have revealed multiple genetic risk loci. We assessed the association of 47 genome-wide significant single-nucleotide polymorphisms (SNPs) at 43 CAD loci with coronary stenosis in a Pakistani sample comprising 663 clinically ascertained and angiographically confirmed cases. Genotypes were determined using the iPLEX Gold technology. All statistical analyses were performed using R software. Linkage disequilibrium (LD) between significant SNPs was determined using SNAP web portal, and functional annotation of SNPs was performed using the RegulomeDB and Genotype-Tissue Expression (GTEx) databases. Genotyping comparison was made between cases with severe stenosis (≥70%) and mild/minimal stenosis (PLG/rs4252120 (p=0.0078), KIAA1462/rs2505083 (p=0.005), and SLC22A3/rs2048327 (p=0.045) and two with recessive models SORT1/rs602633 (p=0.005) and UBE2Z/rs46522 (p=0.03). PLG/rs4252120 was in LD with two functional PLG variants (rs4252126 and rs4252135), each with a RegulomeDB score of 1f. Likewise, KIAA1462/rs2505083 was in LD with a functional SNP, KIAA1462/rs3739998, having a RegulomeDB score of 2b. In the GTEx database, KIAA1462/rs2505083, SLC22A3/rs2048327, SORT1/rs602633, and UBE2Z/rs46522 SNPs were found to be expression quantitative trait loci (eQTLs) in CAD-associated tissues. In conclusion, five genome-wide significant SNPs previously reported in European GWAS were replicated in the Pakistani sample. Further association studies on larger non-European populations are needed to understand the worldwide genetic architecture of CAD.

Rheumatoid arthritis (RA) is a multifactorial autoimmune disease. The interaction of genetic and environmental factors is likely necessary for RA. Among potential genetic factors, many major histocompatibility complex (MHC) and non-MHC variants may be involved in RA susceptibility. CTLA4 is involved in the regulation of T-cell response during an immune reaction, and multiple CTLA4 single nucleotide polymorphisms (SNPs) have been associated with numerous autoimmune diseases, including RA. To our knowledge, the genetic association of CTLA4 with RA risk has not been examined previously in the Pakistani population. In this study, we sequenced the entire CTLA4 gene and flanking regions in 95 Pakistani RA cases followed the screening of identified variants in Study 1 sample consisting of 350 RA cases and controls. Four common significant variants identified in Study 1 sample were further examined in a larger Study 2 replication sample comprising 1,678 independent RA cases and controls. We report significant associations of three variants from the combined analysis: rs3087243 (OR = 1.26, p = 4.47E-03), rs5742909 (OR = 1.78, p = 4.60E-03), and rs11571319 (OR = 1.48, p = 6.64E-03); the latter is a novel association in the Pakistani sample.

Rheumatoid arthritis (RA) is a complex and multifactorial autoimmune disorder with the involvement of multiple genetic and environmental factors. Genome-wide association studies (GWAS) have identified more than 50 RA genetic loci in European populations. Given the anticipated overlap of RA-relevant genes and pathways across different ethnic groups, we sought to replicate 58 GWAS-implicated SNPs reported in Europeans in Pakistani subjects. 1,959 unrelated subjects comprising 1,222 RA cases and 737 controls were collected from three rheumatology facilities in Pakistan. Genotyping was performed using iPLEX or TaqMan® methods. A total of 50 SNPs were included in the final association analysis after excluding those that failed assay design/run or postrun QC analysis. Fourteen SNPs (LINC00824/rs1516971, PADI4/rs2240336, CEP57/rs4409785, CTLA4/rs3087243, STAT4/rs13426947, HLA-B/MICA/rs2596565, C5orf30/rs26232, CCL21/rs951005, GATA3/rs2275806, VPS37C/rs595158, HLA-DRB1/rs660895, EOMES/rs3806624, SPRED2/rs934734, and RUNX1/rs9979383) were replicated in our Pakistani sample at false discovery rate (FDR) of p values ranging from 4.73E-06 to 3.48E-02. Our results indicate that several RA susceptibility loci are shared between Pakistani and European populations, supporting the role of common genes/pathways.

Deposition of amyloid plaques in the brain is one of the two main pathological hallmarks of Alzheimer’s disease (AD). Amyloid positron emission tomography (PET) is a neuroimaging tool that selectively detects in vivo amyloid deposition in the brain and is a reliable endophenotype for AD that complements cerebrospinal fluid biomarkers with regional information. We measured in vivo amyloid deposition in the brains of ~1000 subjects from three collaborative AD centers and ADNI using 11C-labeled Pittsburgh Compound-B (PiB)-PET imaging followed by meta-analysis of genome-wide association studies, first to our knowledge for PiB-PET, to identify novel genetic loci for this endophenotype. The APOE region showed the most significant association where several SNPs surpassed the genome-wide significant threshold, with APOE*4 being most significant (P-meta = 9.09E-30; β = 0.18). Interestingly, after conditioning on APOE*4, 14 SNPs remained significant at P APOE region that were not in linkage disequilibrium with APOE*4. Outside the APOE region, the meta-analysis revealed 15 non-APOE loci with P P-meta = 4.87E-07) and 3 (P-meta = 9.69E-07). Functional analyses of these SNPs indicate their potential relevance with AD pathogenesis. Top 15 non-APOE SNPs along with APOE*4 explained 25–35% of the amyloid variance in different datasets, of which 14–17% was explained by APOE*4 alone. In conclusion, we have identified novel signals in APOE and non-APOE regions that affect amyloid deposition in the brain. Our data also highlights the presence of yet to be discovered variants that may be responsible for the unexplained genetic variance of amyloid deposition.

BackgroundA major systemic lupus erythematosus (SLE) susceptibility locus lies within a common inversion polymorphism region (encompassing 3.8 – 4.5 Mb) located at 8p23. Initially implicated genes includedFAM167A-BLKandXKR6, of whichBLKreceived major attention due to its known role in B-cell biology. Recently, additional SLE risk carried in non-inverted background was also reported.Objective and methodsIn this case –control study, we further investigated the ‘extended’ 8p23 locus (~ 4 Mb) where we observed multiple SLE signals and assessed these signals for their relation to the inversion affecting this region. The study involved a North American discovery data set (~1200 subjects) and a replication data set (> 10 000 subjects) comprising European-descent individuals.ResultsMeta-analysis of 8p23 SNPs, with p < 0.05 in both data sets, identified 51 genome-wide significant SNPs (p < 5.0 × 10−8). While most of these SNPs were related to previously implicated signals (XKR6-FAM167A-BLKsubregion), our results also revealed two ‘new’ SLE signals, includingSGK223-CLDN23-MFHAS1(6.06 × 10−9≤ meta p ≤ 4.88 × 10−8) andCTSB(meta p = 4.87 × 10−8) subregions that are located > 2 Mb upstream and ~ 0.3 Mb downstream from previously reported signals. Functional assessment of relevant SNPs indicated putativecis-effects on the expression of various genes at 8p23. Additional analyses in discovery sample, where the inversion genotypes were inferred, replicated the association of non-inverted status with SLE risk and suggested that a number of SLE risk alleles are predominantly carried in non-inverted background.ConclusionsOur results implicate multiple (known+novel) SLE signals/genes at the extended 8p23 locus, beyond previously reported signals/genes, and suggest that this broad locus contributes to SLE risk through the effects of multiple genes/pathways.

Objective Type 1 diabetes (T1D) and rheumatoid arthritis (RA) are autoimmune diseases. It is known that certain genetic loci and factors that increase the overall autoimmunity risk can be shared among different autoimmune diseases. We sought to replicate seven T1D-related SNPs that have been previously reported to be associated with RA susceptibility in a small set of mixed family-based and case-control Pakistani sample in a relatively large and independent RA case-control sample from the same population. Seven T1D-associted SNPs (GLIS3/rs7020673, BACH2/rs11755527, SKAP2/rs7804356, GDSMB/rs2290400, C6orf173/rs9388489, LOC399716/rs947474 and DLK1-MEG2/rs941576) were genotyped in a large Pakistani RA case-control sample (n=1,959) using TaqMan® SNP genotyping assays. Results None of the tested SNPs showed statistically significant association with RA susceptibility; however, one SNP (GLIS3/rs7020673) showed a trend for association (OR= 0.88, p=7.99E-02). Our study has failed to replicate the previously reported association of seven T1D-associted SNPs with RA risk in a large sample from the same population. Thus, our results do not support a major role of these T1D SNPs in affecting RA susceptibility in the Pakistani population.

Objective Genome-wide association studies (GWAS) in individuals of European ancestry identified a number of systemic lupus erythematosus (SLE) susceptibility loci using earlier versions of high-density genotyping platforms. Followup studies on suggestive GWAS regions using larger samples and more markers identified additional SLE loci in subjects of European descent. This multistage study was undertaken to identify novel SLE loci. Methods In stage 1, we conducted a new GWAS of SLE in a North American case–control sample of subjects of European ancestry (n = 1,166) genotyped on Affymetrix Genome-Wide Human SNP Array 6.0. In stage 2, we further investigated top new suggestive GWAS hits by in silico evaluation and meta-analysis using an additional data set of subjects of European descent (>2,500 individuals), followed by replication of top meta-analysis findings in another data set of subjects of European descent (>10,000 individuals) in stage 3. Results As expected, our GWAS revealed the most significant associations at the major histocompatibility complex locus (6p21), which easily surpassed the genome-wide significance threshold (P < 5 × 10−8). Several other SLE signals/loci previously implicated in Caucasians and/or Asians were also confirmed in the stage 1 discovery sample, and the strongest signals were observed at 2q32/STAT4 (P = 3.6 × 10−7) and at 8p23/BLK (P = 8.1 × 10−6). Stage 2 meta-analyses identified a new genome-wide significant SLE locus at 12q12 (meta P = 3.1 × 10−8), which was replicated in stage 3. Conclusion Our multistage study identified and replicated a new SLE locus that warrants further followup in additional studies. Publicly available databases suggest that this newly identified SLE signal falls within a functionally relevant genomic region and near biologically important genes.

To identify novel loci that affect cognitive decline in older adults free of dementia, we conducted genome-wide and gene-based meta-analyses on longitudinal slopes of 5 cognitive domains (memory, executive function, language, attention/processing speed, and visuospatial ability) derived from 2 population-based cohorts. For decline over time in each cognitive domain, we normalized intraindividual slopes within each cohort, accounting for baseline age, sex, and years of education. Normalized slope for each domain was used in cohort-specific genome-wide analyses after including top principal components as covariates followed by genome-wide and gene-based meta-analyses. Both analyses revealed a novel WDFY2 locus at genome-wide (p = 3.37E-08) and gene-wide (p = 7.10E-07) significance levels for the attention/processing speed domain. In the GTEx eQTL analysis, genome-wide significant single-nucleotide polymorphism was associated with RNA expression levels of WDFY2 in several brain regions: cerebellar hemisphere (p = 1.07E-04), cerebellum (p = 6.92E-04), hippocampus (p = 2.18E-03) and cortex (p = 2.29E-02), and in whole blood (p = 4.41E-05). Our results suggest that WDFY2 genetic variation may affect individual differences in decline over time on tests of attention/processing speed.

IMPORTANCE: Greater understanding of molecular features of conjunctival melanoma (CM) may improve its clinical management. OBJECTIVE: To evaluate molecular features of CM and application of this information into clinical care. DESIGN, SETTING, AND PARTICIPANTS: In a prospective case series of CM with integrative exome and transcriptome analysis, 8 patients at an academic ocular oncology setting were evaluated. The study was conducted from November 2015 to March 2018. INTERVENTIONS/EXPOSURES: Integrative exome and transcriptome analysis of CMs and clinical management of a patient’s care by using this information. MAIN OUTCOMES AND MEASURES: Molecular characterization of CM and its potential clinical application. RESULTS: In the 8 patients (4 men) included in analysis, 4 subgroups of CM were observed, including the BRAF V600E mutation in 1 tumor, NRAS Q61R mutation in 3 tumors, NF1 mutations (Q1188X, R440X, or M1215K+ S15fs) in 3 tumors, and triple-wild type (triple-WT) in 1 tumor. The triple-WT case had CCND1 amplification and mutation in the CIC gene (Q1508X). Five tumors, including the triple-WT, also harbored mutations in MAPK genes. In addition to the genes linked to mitogen-activated protein kinase and phosphoinositol 3-kinase pathways, those involved in cell cycle and/or survival, ubiquitin-mediated protein degradation, and chromatin remodeling/epigenetic regulation (ATRX being the most frequently mutated: noted in 5 tumors) may play an important role. Other frequently mutated genes included PREX2 (n = 3), APOB (n = 4), and RYR1/2 (n = 4), although their relevance remains to be determined. The mutation burden ranged from 1.1 to 15.6 mutations per megabase (Mut/Mb) and was 3.3 Mut/Mb or less in 3 tumors and more than 10 Mut/Mb in 2 tumors. A patient with a large tumor and BRAF V600E mutation was treated with combined systemic BRAF (dabrafenib) and MEK (trametinib) inhibitors. After 3 months of therapy, her CM responded substantially and the residual tumor was removed by local surgical excision. CONCLUSIONS AND RELEVANCE: The NRAS Q61R and NF1 mutations were more common than the BRAF V600E mutation in this series. Although small tumors (where incisional biopsy is not indicated) are treated with surgical excision regardless of mutational profile, in large tumors carrying the BRAF V600E mutation, neoadjuvant therapy with combined systemic BRAF and MEK inhibitors followed by local excision may be used as an alternative to exenteration. Integrative omics analysis of CM may be informative and guide clinical management and treatment in selected cases.

Aim To identify risk alleles contributing towards type 1 diabetes in Pakistani patients. Introduction Type 1 diabetes (T1D) is an autoimmune disease which is caused by destruction of insulin producing β cells by immune system. Genetic predisposition as well as environmental factors contribute to its etiology. To date more than 40 risk loci have been identified for T1D. Methodology A total of 191 family-based and unrelated T1D cases and controls were recruited. DNA was extracted and 32 genome-wide significant single nucleotide polymorphisms (SNPs) previously reported in Europeans were genotyped. Genotyping was performed using TaqMan SNP genotyping assays and the data was analyzed using FamCC software. Results Our results showed significant association of 10 single nucleotide polymorphisms (SNPs) with T1D at p HLA -DQA 1 /rs9272346, ERBB3 /rs2292239, SIRPG /rs2281808, IL2-KIAA1109 /rs4505848, GLIS3 /rs7020673, CD226 /rs763361, PTPN2 /rs478582, IKZF1 /rs10272724, BACH2 /rs11755527, C6orf173 /rs9388489, whereas 5 more SNPs showed their association at 0.01 p Conclusion We have replicated many of the T1D loci established among Europeans in a Pakistani population.

Epigenetic changes including genomic imprinting may affect risk of late-onset Alzheimer's disease (LOAD). There are >100 known imprinted genes and most of them are expressed in human brain. In this study, we examined the association of single nucleotide polymorphisms (SNPs) in 93 imprinted genes with LOAD risk in 1291 LOAD cases and 958 cognitively normal controls. We performed single-site, gene-based, and haplotype analyses. Single-site analysis showed 14 significant associations at p < 0.01. The most significant SNP (rs11770199; p = 0.0003) in single-site analysis was located on chromosome 7 in the GRB10 gene. Gene-based analyses revealed four significant associations in the WT1, ZC3H12C, DLGAP2, and GPR1 genes at p < 0.05. The haplotype analysis also revealed significant associations with three genes (ZC3H12C, DLGAP2, and GPR1). These findings suggest a possible role of imprinted genes in AD pathogenesis that show specific expression in the brain.

Genome-wide association studies have identified several loci associated with plasma lipid levels but those common variants together account only for a small proportion of the genetic variance of lipid traits. It has been hypothesized that the remaining heritability may partly be explained by rare variants with strong effect sizes. Here, we have comprehensively investigated the associations of both common and uncommon/rare variants in the lipoprotein lipase (LPL) gene in relation to plasma lipoprotein–lipid levels in African Blacks (ABs). For variant discovery purposes, the entire LPL gene and flanking regions were resequenced in 95 ABs with extreme high-density lipoprotein cholesterol (HDL-C) levels. A total of 308 variants were identified, of which 64 were novel. Selected common tagSNPs and uncommon/rare variants were genotyped in the entire sample (n=788), and 126 QC-passed variants were evaluated for their associations with lipoprotein–lipid levels by using single-site, haplotype and rare variant (SKAT-O) association analyses. We found eight not highly correlated (r2A, rs8176337:G>C, rs74304285:G>A, rs252:delA, rs316:C>A, rs329:A>G, rs12679834:T>C, and rs4921684:C>T) nominally (P

Associations between whole blood transcriptome and clinical phenotypes in vitamin D-deficient overweight and obese children can provide insight into the biological effects of vitamin D and obesity. We determined differentially expressed genes (DEGs) in relation to body mass index (BMI) in vitamin D-deficient black children with a BMI &ge, 85th percentile and ascertained the cardiometabolic phenotypes associated with the DEGs. We examined whole-blood transcriptome gene expression by RNA sequencing and cardiometabolic profiling in 41, 10- to 18-year-old children. We found 296 DEGs in association with BMI after adjusting for age, race, sex, and pubertal status. Cardiometabolic phenotypes associated with the BMI-related DEGs, after adjusting for age, sex, pubertal status, and %total body fat, were (i) flow-mediated dilation (marker of endothelial function), (ii) c-reactive protein (marker of inflammation), and (iii) leptin (adipocytokine). Canonical pathways of relevance for childhood obesity and its phenotypes that were significantly associated with the BMI-related DEGs affected immune cell function/inflammation, vascular health, metabolic function, and cell survival/death, several immune and inflammatory pathways overlapped across the three phenotypes. We have identified transcriptome-based biomarkers associated with BMI in vitamin D-deficient, overweight and obese black children. Modulating effects of vitamin D supplementation on these biomarkers and their related phenotypes need further exploration.

The apolipoprotein E-C1-C4-C2 gene cluster at 19q13.32 encodes four amphipathic apolipoproteins. The influence of APOE common polymorphisms on plasma lipid/lipoprotein profile, especially on LDL-related traits, is well recognized; however, little is known about the role of other genes/variants in this gene cluster. In this study, we evaluated the role of common and uncommon/rare genetic variation in this gene region on inter-individual variation in plasma lipoprotein levels in non-Hispanic Whites (NHWs) and African blacks (ABs). In the variant discovery step, the APOE, APOC1, APOC4, APOC2 genes were sequenced along with their flanking and hepatic control regions (HCR1 and HCR2) in 190 subjects with extreme HDL-C/TG levels. The next step involved the genotyping of 623 NHWs and 788 ABs for the identified uncommon/rare variants and common tagSNPs along with additional relevant SNPs selected from public resources, followed by association analyses with lipid traits. A total of 230 sequence variants, including 15 indels were identified, of which 65 were novel. A total of 70 QC-passed variants in NHWs and 108 QC-passed variants in ABs were included in the final association analyses. Single-site association analysis of SNPs with MAF>1% revealed 20 variants in NHWs and 24 variants in ABs showing evidence of association with at least one lipid trait, including several variants exhibiting independent associations from the established APOE polymorphism even after multiple-testing correction. Overall, our study has confirmed known associations and also identified novel associations in this genomic region with various lipid traits. Our data also support the contribution of both common and uncommon/rare variation in this gene region in affecting plasma lipid profile in the general population.

Pancreatitis is a complex, progressively destructive inflammatory disorder. Alcohol was long thought to be the primary causative agent, but genetic contributions have been of interest since the discovery that rare PRSS1, CFTR and SPINK1 variants were associated with pancreatitis risk. We now report two associations at genome-wide significance identified and replicated at PRSS1-PRSS2 (P < 1 × 10(-12)) and X-linked CLDN2 (P < 1 × 10(-21)) through a two-stage genome-wide study (stage 1: 676 cases and 4,507 controls; stage 2: 910 cases and 4,170 controls). The PRSS1 variant likely affects disease susceptibility by altering expression of the primary trypsinogen gene. The CLDN2 risk allele is associated with atypical localization of claudin-2 in pancreatic acinar cells. The homozygous (or hemizygous in males) CLDN2 genotype confers the greatest risk, and its alleles interact with alcohol consumption to amplify risk. These results could partially explain the high frequency of alcohol-related pancreatitis in men (male hemizygote frequency is 0.26, whereas female homozygote frequency is 0.07).

The Alzheimer Disease Genetics Consortium (ADGC) performed a genome-wide association study of late-onset Alzheimer disease using a three-stage design consisting of a discovery stage (stage 1) and two replication stages (stages 2 and 3). Both joint analysis and meta-analysis approaches were used. We obtained genome-wide significant results at MS4A4A (rs4938933; stages 1 and 2, meta-analysis P (P(M)) = 1.7 × 10(-9), joint analysis P (P(J)) = 1.7 × 10(-9); stages 1, 2 and 3, P(M) = 8.2 × 10(-12)), CD2AP (rs9349407; stages 1, 2 and 3, P(M) = 8.6 × 10(-9)), EPHA1 (rs11767557; stages 1, 2 and 3, P(M) = 6.0 × 10(-10)) and CD33 (rs3865444; stages 1, 2 and 3, P(M) = 1.6 × 10(-9)). We also replicated previous associations at CR1 (rs6701713; P(M) = 4.6 × 10(-10), P(J) = 5.2 × 10(-11)), CLU (rs1532278; P(M) = 8.3 × 10(-8), P(J) = 1.9 × 10(-8)), BIN1 (rs7561528; P(M) = 4.0 × 10(-14), P(J) = 5.2 × 10(-14)) and PICALM (rs561655; P(M) = 7.0 × 10(-11), P(J) = 1.0 × 10(-10)), but not at EXOC3L2, to late-onset Alzheimer's disease susceptibility.

This study characterized the human apolipoprotein H [APOH; beta(2)-glycoprotein I (beta(2)GPI)] promoter and its variants by in vitro functional experiments and investigated their relationship with human plasma beta(2)GPI levels. We examined the individual effects of 12 APOH promoter single nucleotide polymorphisms in the 5' flanking region of APOH (approximately 1.4 kb) on luciferase activity in COS-1 cells and HepG2 cells and their impact on plasma beta(2)GPI levels in 799 American White people, the DNA binding properties of the APOH promoter using an electrophoretic mobility shift assay in HepG2 cells, the effects of serial deletion analysis of the APOH 5' flanking region in COS-1 and HepG2 cells and cross-species conservation of the APOH promoter sequence. The variant alleles of three single nucleotide polymorphisms (-1219G>A, -643T>C and -32C>A) showed significantly lower luciferase expression (51, 40 and 37%, respectively) as compared with the wild-type allele. The electrophoretic mobility shift assay demonstrated that these three variants specifically bind with protein(s) from HepG2 cell nuclear extracts. Three-site haplotype analysis (-1219G>A, -643T>C and -32C>A) revealed one haplotype carrying -32A (allele frequency = 0.075) to be significantly associated with decreased plasma beta(2)GPI levels (P < 0.001). Deletion analysis localized the core APOH promoter to approximately 160 bp upstream of ATG codon with the presence of critical cis-acting elements between -166 and -65. Cross-species conservation analysis of the APOH promoters of seven species indicated that basic promoter elements are highly conserved across species. In conclusion, we have characterized the functional promoter of APOH and identified functional variants that affect the transcriptional activity of the APOH promoter.

Alzheimer's disease (AD) is a complex and multifactorial disease with the possible involvement of several genes. Complex diseases such as AD have a large affect on the public health. It was estimated in 2007 that over 5 million Americans had AD, and more than $91 billion dollars was spent by medicare on AD and other dementias. Genetics plays a significant role in the etiology of the disease, therefore, it is of public health importance that the genetics of AD be investigated. With the exception of the APOE gene as a susceptibility marker no other genes have been identified for late-onset AD (LOAD). A recent genome wide association study of 17,343 gene-based putative functional single nucleotide polymorphisms (SNPs) found 19 significant variants, including 3 linked to APOE, showing association with LOAD in several population samples. We have set out to replicate the 16 new significant associations in a large case-control cohort of American Whites. Additionally we examined six variants present in positional and/or biological candidate genes for AD. We genotyped the 22 SNPs in up to 1,009 Caucasian Americans with LOAD and up to 1,010 age matched older healthy Caucasian Americans. All variants were genotyped using 5' nuclease assays. We did not observe a statistically significant association between the SNPs with the risk of AD, either individually or stratified by APOE. Our data suggest that the association of the studied variants with LOAD, if it exists, is not statistically significant in our population study.

A polymorphism in the calcium homeostasis modulator 1 gene (CALHM1) has recently been associated with risk of late-onset Alzheimer disease. We examined this variant (rs2986017) in 945 Caucasian Americans with late-onset Alzheimer disease and 875 age-matched Caucasian American controls. No association with risk of late-onset Alzheimer disease (p=0.368 for genotypes; p=0.796 for alleles) was observed in our sample. However, a potential modest association of minor allele homozygosity (TT) with an earlier age-at-onset was seen (p=0.034). © 2009 Wiley-Liss, Inc.

Systemic lupus erythematosus (SLE) is a clinically heterogeneous disease in which the risk of disease is influenced by complex genetic and environmental contributions. Alleles of HLA-DRB1, IRF5, and STAT4 are established susceptibility genes; there is strong evidence for the existence of additional risk loci.We genotyped more than 500,000 single-nucleotide polymorphisms (SNPs) in DNA samples from 1311 case subjects with SLE and 1783 control subjects; all subjects were North Americans of European descent. Genotypes from 1557 additional control subjects were obtained from public data repositories. We measured the association between the SNPs and SLE after applying strict quality-control filters to reduce technical artifacts and to correct for the presence of population stratification. Replication of the top loci was performed in 793 case subjects and 857 control subjects from Sweden.Genetic variation in the region upstream from the transcription initiation site of the gene encoding B lymphoid tyrosine kinase (BLK) and C8orf13 (chromosome 8p23.1) was associated with disease risk in both the U.S. and Swedish case-control series (rs13277113; odds ratio, 1.39; P=1x10(-10)) and also with altered levels of messenger RNA in B-cell lines. In addition, variants on chromosome 16p11.22, near the genes encoding integrin alpha M (ITGAM, or CD11b) and integrin alpha X (ITGAX), were associated with SLE in the combined sample (rs11574637; odds ratio, 1.33; P=3x10(-11)).We identified and then confirmed through replication two new genetic loci for SLE: a promoter-region allele associated with reduced expression of BLK and increased expression of C8orf13 and variants in the ITGAM-ITGAX region.

Importance Mutations in known causal Alzheimer disease (AD) genes account for only 1% to 3% of patients and almost all are dominantly inherited. Recessive inheritance of complex phenotypes can be linked to long (>1-megabase [Mb]) runs of homozygosity (ROHs) detectable by single-nucleotide polymorphism (SNP) arrays. Objective To evaluate the association between ROHs and AD in an African American population known to have a risk for AD up to 3 times higher than white individuals. Design, Setting, and Participants Case-control study of a large African American data set previously genotyped on different genome-wide SNP arrays conducted from December 2013 to January 2015. Global and locus-based ROH measurements were analyzed using raw or imputed genotype data. We studied the raw genotypes from 2 case-control subsets grouped based on SNP array: Alzheimer’s Disease Genetics Consortium data set (871 cases and 1620 control individuals) and Chicago Health and Aging Project–Indianapolis Ibadan Dementia Study data set (279 cases and 1367 control individuals). We then examined the entire data set using imputed genotypes from 1917 cases and 3858 control individuals. Main Outcomes and Measures The ROHs larger than 1 Mb, 2 Mb, or 3 Mb were investigated separately for global burden evaluation, consensus regions, and gene-based analyses. Results The African American cohort had a low degree of inbreeding ( F ~ 0.006). In the Alzheimer’s Disease Genetics Consortium data set, we detected a significantly higher proportion of cases with ROHs greater than 2 Mb ( P = .004) or greater than 3 Mb ( P = .02), as well as a significant 114-kilobase consensus region on chr4q31.3 (empirical P value 2 = .04; ROHs >2 Mb). In the Chicago Health and Aging Project–Indianapolis Ibadan Dementia Study data set, we identified a significant 202-kilobase consensus region on Chr15q24.1 (empirical P value 2 = .02; ROHs >1 Mb) and a cluster of 13 significant genes on Chr3p21.31 (empirical P value 2 = .03; ROHs >3 Mb). A total of 43 of 49 nominally significant genes common for both data sets also mapped to Chr3p21.31. Analyses of imputed SNP data from the entire data set confirmed the association of AD with global ROH measurements (12.38 ROHs >1 Mb in cases vs 12.11 in controls; 2.986 Mb average size of ROHs >2 Mb in cases vs 2.889 Mb in controls; and 22% of cases with ROHs >3 Mb vs 19% of controls) and a gene-cluster on Chr3p21.31 (empirical P value 2 = .006-.04; ROHs >3 Mb). Also, we detected a significant association between AD and CLDN17 (empirical P value 2 = .01; ROHs >1 Mb), encoding a protein from the Claudin family, members of which were previously suggested as AD biomarkers. Conclusions and Relevance To our knowledge, we discovered the first evidence of increased burden of ROHs among patients with AD from an outbred African American population, which could reflect either the cumulative effect of multiple ROHs to AD or the contribution of specific loci harboring recessive mutations and risk haplotypes in a subset of patients. Sequencing is required to uncover AD variants in these individuals.

Late onset Alzheimer's disease (LOAD) is a genetically complex and clinically heterogeneous disease. Recent large-scale genome wide association studies (GWAS) have identified more than twenty loci that modify risk for AD. Despite the identification of these loci, little progress has been made in identifying the functional variants that explain the association with AD risk. Thus, we sought to determine whether the novel LOAD GWAS single nucleotide polymorphisms (SNPs) alter expression of LOAD GWAS genes and whether expression of these genes is altered in AD brains. The majority of LOAD GWAS SNPs occur in gene dense regions under large linkage disequilibrium (LD) blocks, making it unclear which gene(s) are modified by the SNP. Thus, we tested for brain expression quantitative trait loci (eQTLs) between LOAD GWAS SNPs and SNPs in high LD with the LOAD GWAS SNPs in all of the genes within the GWAS loci. We found a significant eQTL between rs1476679 and PILRB and GATS, which occurs within the ZCWPW1 locus. PILRB and GATS expression levels, within the ZCWPW1 locus, were also associated with AD status. Rs7120548 was associated with MTCH2 expression, which occurs within the CELF1 locus. Additionally, expression of several genes within the CELF1 locus, including MTCH2, were highly correlated with one another and were associated with AD status. We further demonstrate that PILRB, as well as other genes within the GWAS loci, are most highly expressed in microglia. These findings together with the function of PILRB as a DAP12 receptor supports the critical role of microglia and neuroinflammation in AD risk.

Background Cholesteryl ester transfer protein (CETP) plays a crucial role in lipid metabolism. Associations of common CETP variants with variation in plasma lipid levels, and/or CETP mass/activity have been extensively studied and well-documented; however, the effects of uncommon/rare CETP variants on plasma lipid profile remain undefined. Hence, resequencing of the gene in extreme phenotypes and follow-up rare-variant association analyses are essential to fill this gap. Objective To identify common and uncommon/rare variants in the CETP gene by resequencing the entire gene and test the effects of both common and uncommon/rare CETP variants on plasma lipid traits in two genetically distinct populations. Methods and results The entire CETP gene plus flanking regions were resequenced in 190 individuals comprising 95 non-Hispanic whites (NHWs) and 95 African blacks with extreme HDL-C levels. A total of 279 sequence variants were identified, of which 25 were novel. Selected variants were genotyped in the entire samples of 623 NHWs and 788 African blacks and 184 QC-passed variants were tested in relation to plasma lipid traits by using gene-based, single-site, haplotype and rare variant association analyses (SKAT-O). Two novel and independent associations of rs1968905 and rs289740 with HDL-C were identified in African blacks. Using SKAT-O analysis, we also identified rare variants with minor allele frequency P = 0.024) and African blacks ( P = 0.009). Conclusions Our results point out that in addition to the common CETP variants, rare genetic variants in the CETP gene also contribute to the phenotypic variation of HDL-C in the general population.

Observational research shows that higher body mass index (BMI) increases Alzheimer's disease (AD) risk, but it is unclear whether this association is causal. We applied genetic variants that predict BMI in Mendelian randomization analyses, an approach that is not biased by reverse causation or confounding, to evaluate whether higher BMI increases AD risk. We evaluated individual-level data from the AD Genetics Consortium (ADGC: 10,079 AD cases and 9613 controls), the Health and Retirement Study (HRS: 8403 participants with algorithm-predicted dementia status), and published associations from the Genetic and Environmental Risk for AD consortium (GERAD1: 3177 AD cases and 7277 controls). No evidence from individual single-nucleotide polymorphisms or polygenic scores indicated BMI increased AD risk. Mendelian randomization effect estimates per BMI point (95% confidence intervals) were as follows: ADGC, odds ratio (OR) = 0.95 (0.90-1.01); HRS, OR = 1.00 (0.75-1.32); GERAD1, OR = 0.96 (0.87-1.07). One subscore (cellular processes not otherwise specified) unexpectedly predicted lower AD risk.

Rheumatoid arthritis (RA) and type 1 diabetes (T1D) are two autoimmune disorders that have been reported to co-occur in the same subjects or in different subjects from the same family. This suggests the sharing of disease susceptibility loci between RA and T1D. This study was aimed to find out such susceptibility loci that are common in both T1D and RA in Pakistani population. A total of 366 Pakistanis comprising related and unrelated RA cases and controls were recruited. Blood samples were collected from all patients followed by DNA isolation. Thirty-one single-nucleotide polymorphisms (SNPs) previously reported to be associated with T1D were genotyped in RA cases and controls using TaqMan SNP genotyping assays. Data was analyzed using FamCC software. We have identified seven SNP associations that survived multiple testing corrections using false discovery rate: SKAP2/rs7804356 (p = 2.47E-04), GLIS3/rs7020673 (p = 2.86E-04), GSDMB/rs2290400 (p = 23.48E-04), BACH2/rs11755527 (p = 9.16E-04), C6orf173/ rs9388489 (p = 3.11E-03), PRKCQ/DKFZp667F0711/ rs947474 (p = 4.53E-03), and DLK1/ rs941576 (p = 9.51E-03). Our results support the presence of overlapping loci between RA and T1D in Pakistani patients.

Genetic variation in lipid regulatory genes, particularlyAPOE, significantly influences the risk of coronary artery disease (CAD). This study aimed to assess the association betweenAPOEpolymorphism and angiographically assessed coronary stenosis in Pakistani population. A total of 695 subjects (22.3% female, mean age =54±11years) presenting with chest pain were enrolled after obtaining written informed consent. CAD stenosis/extent was assessed by angiography. Patients were classified as having severe stenosis (≥70%), moderate stenosis (30–69%), and mild stenosis (n=491) were further categorized based on possessing one, two, or three vessel diseases to assess the disease extent. Genomic DNA from leukocytes was isolated with DNA purification kit (Qiagen) andAPOEpolymorphisms (E2/E3/E4) were determined using TaqMan assays. Six hundred and seventy-two of 695 subjects were successfully genotyped. The frequency ofAPOE∗4carriers (3/4 and 4/4 genotypes) was significantly higher in severe stenosis group (≥70%) as compared to mild group (P=0.01). In multiple regression, the odds ratio forAPOE∗4carriers to develop ≥70% stenosis was 2.16 (95% CI: 1.29–3.79;P<0.005). In conclusion, the presence ofAPOE∗4allele is a significant risk factor to develop severe coronary stenosis (>70%) among Pakistanis.

There is a strong genetic basis for late-onset of Alzheimer's disease (LOAD) and thus far >20 genes/loci have been identified that affect the risk of LOAD. In addition to disease risk, genetic variation at these loci may also affect components of the natural history of AD, such as survival in AD. In this study, we first examined the role of known LOAD genes with survival time in 983 AD patients. We then performed genome-wide single-nucleotide polymorphism (SNP) and gene-based association analyses to identify novel loci that may influence survival of AD. Survival analysis was conducted using Cox proportional hazards regression under an additive genetics model. We found multiple nominally significant associations (P