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Background: Plasma proteins are critical mediators of cardiovascular processes and are the targets of many drugs. Previous efforts to characterize the genetic architecture of the plasma proteome have been limited by a focus on individuals of European descent and leveraged genotyping arrays and imputation. Here we describe whole genome sequence analysis of the plasma proteome in individuals with greater African ancestry, increasing our power to identify novel genetic determinants. Methods: Proteomic profiling of 1301 proteins was performed in 1852 Black adults from the Jackson Heart Study using aptamer-based proteomics (SomaScan). Whole genome sequencing association analysis was ascertained for all variants with minor allele count ≥5. Results were validated using an alternative, antibody-based, proteomic platform (Olink) as well as replicated in the Multi-Ethnic Study of Atherosclerosis and the HERITAGE Family Study (Health, Risk Factors, Exercise Training and Genetics). Results: We identify 569 genetic associations between 479 proteins and 438 unique genetic regions at a Bonferroni-adjusted significance level of 3.8×10 -11 . These associations include 114 novel locus-protein relationships and an additional 217 novel sentinel variant-protein relationships. Novel cardiovascular findings include new protein associations at the APOE gene locus including ZAP70 (sentinel single nucleotide polymorphism [SNP] rs7412-T, β=0.61±0.05, P =3.27×10 -30 ) and MMP-3 (β=-0.60±0.05, P =1.67×10 -32 ), as well as a completely novel pleiotropic locus at the HPX gene, associated with 9 proteins. Further, the associations suggest new mechanisms of genetically mediated cardiovascular disease linked to African ancestry; we identify a novel association between variants linked to APOL1-associated chronic kidney and heart disease and the protein CKAP2 (rs73885319-G, β=0.34±0.04, P =1.34×10 -17 ) as well as an association between ATTR amyloidosis and RBP4 levels in community-dwelling individuals without heart failure. Conclusions: Taken together, these results provide evidence for the functional importance of variants in non-European populations, and suggest new biological mechanisms for ancestry-specific determinants of lipids, coagulation, and myocardial function.

BACKGROUND: Asthma is a common respiratory disorder with a highly heterogeneous nature that remains poorly understood. The objective was to use whole genome sequencing (WGS) data to identify regions of common genetic variation contributing to lung function in individuals with a diagnosis of asthma. METHODS: WGS data were generated for 1,053 individuals from trios and extended pedigrees participating in the family-based Genetic Epidemiology of Asthma in Costa Rica study. Asthma affection status was defined through a physician’s diagnosis of asthma, and most participants with asthma also had airway hyperresponsiveness (AHR) to methacholine. Family-based association tests for single variants were performed to assess the associations with lung function phenotypes. RESULTS: A genome-wide significant association was identified between baseline FEV(1)/FVC ratio and a single-nucleotide polymorphism in the top hit cysteine-rich secretory protein LCCL domain-containing 2 (CRISPLD2) (rs12051168; P = 3.6 × 10(−8) in the unadjusted model) that retained suggestive significance in the covariate-adjusted model (P = 5.6 × 10(−6)). Rs12051168 was also nominally associated with other related phenotypes: baseline FEV(1) (P = 3.3 × 10(−3)), postbronchodilator (PB) FEV(1) (7.3 × 10(−3)), and PB FEV(1)/FVC ratio (P = 2.7 × 10(−3)). The identified baseline FEV(1)/FVC ratio and rs12051168 association was meta-analyzed and replicated in three independent cohorts in which most participants with asthma also had confirmed AHR (combined weighted z-score P = .015) but not in cohorts without information about AHR. CONCLUSIONS: These findings suggest that using specific asthma characteristics, such as AHR, can help identify more genetically homogeneous asthma subgroups with genotype-phenotype associations that may not be observed in all children with asthma. CRISPLD2 also may be important for baseline lung function in individuals with asthma who also may have AHR.

Purpose Rh antigens can provoke severe alloimmune reactions, particularly in high-risk transfusion contexts such as, sickle cell disease. Rh antigens are encoded by the paralogs, RHD and RHCE, located in one of the most complex genetic loci. Our goal was to characterize RH genetic variation in multi-ethnic cohorts, with the focus on detecting RH structural variation (SV). Methods We customized analytical methods to estimate paralog-specific copy number from next generation sequencing (NGS) data. We applied these methods to clinically-characterized samples, including four WHO genotyping references and 1135 Asian and Native American blood donors. Subsequently, we surveyed 1715 African American samples from the Jackson Heart Study. Results Most samples in each dataset exhibited SV. SV detection enabled prediction of the immunogenic RhD and RhC antigens in concordance (>99%) with serological phenotyping. RhC antigen expression was associated with exon 2 hybrid alleles (RHCE*CE-D(2)-CE). Clinically-relevant exon 4-7 hybrid alleles (RHD*D-CE(4-7)-D) and exon 9 hybrid alleles (RHCE*CE-D(9)-CE) were prevalent in African Americans. Conclusions This study shows custom NGS methods can accurately detect RH SV, and that SV is important to inform prediction of relevant RH alleles. Additionally, this study provides the first large NGS survey of RH alleles in African Americans.

Lipoprotein(a), Lp(a), is a modified low-density lipoprotein particle that contains apolipoprotein(a), encoded by LPA, and is a highly heritable, causal risk factor for cardiovascular diseases that varies in concentrations across ancestries. Here, we use deep-coverage whole genome sequencing in 8392 individuals of European and African ancestry to discover and interpret both single-nucleotide variants and copy number (CN) variation associated with Lp(a). We observe that genetic determinants between Europeans and Africans have several unique determinants. The common variant rs12740374 associated with Lp(a) cholesterol is an eQTL for SORT1 and independent of LDL cholesterol. Observed associations of aggregates of rare non-coding variants are largely explained by LPA structural variation, namely the LPA kringle IV 2 (KIV2)-CN. Finally, we find that LPA risk genotypes confer greater relative risk for incident atherosclerotic cardiovascular diseases compared to directly measured Lp(a), and are significantly associated with measures of subclinical atherosclerosis in African Americans., Circulating lipoprotein(a) is an important risk factor for cardiovascular disease and shows variability between different ethnic groups. Here, Zekavat et al. perform whole-genome sequencing in individuals of European and African ancestries and find ancestry-specific genetic determinants for lipoprotein(a) levels.

Hemophilia A and B are rare, X-linked bleeding disorders. My Life, Our Future (MLOF) is a collaborative project established to genotype and study hemophilia. Patients were enrolled at US hemophilia treatment centers (HTCs). Genotyping was performed centrally using next-generation sequencing (NGS) with an approach that detected common F8 gene inversions simultaneously with F8 and F9 gene sequencing followed by confirmation using standard genotyping methods. Sixty-nine HTCs enrolled the first 3000 patients in under 3 years. Clinically reportable DNA variants were detected in 98.1% (2357/2401) of hemophilia A and 99.3% (595/599) of hemophilia B patients. Of the 924 unique variants found, 285 were novel. Predicted gene-disrupting variants were common in severe disease; missense variants predominated in mild-moderate disease. Novel DNA variants accounted for ∼30% of variants found and were detected continuously throughout the project, indicating that additional variation likely remains undiscovered. The NGS approach detected >1 reportable variants in 36 patients (10 females), a finding with potential clinical implications. NGS also detected incidental variants unlikely to cause disease, including 11 variants previously reported in hemophilia. Although these genes are thought to be conserved, our findings support caution in interpretation of new variants. In summary, MLOF has contributed significantly toward variant annotation in the F8 and F9 genes. In the near future, investigators will be able to access MLOF data and repository samples for research to advance our understanding of hemophilia.

Background Loss-of-function mutations in the GBA gene are associated with more severe cognitive impairment in PD, but the nature of these deficits is not well understood and whether common GBA polymorphisms influence cognitive performance in PD is not yet known. Methods We screened the GBA coding region for mutations and the E326K polymorphism in 1,369 PD patients enrolled at eight sites from the PD Cognitive Genetics Consortium. Participants underwent assessments of learning and memory (Hopkins Verbal Learning Test–Revised), working memory/executive function (Letter-Number Sequencing Test and Trail Making Test A and B), language processing (semantic and phonemic verbal fluency), visuospatial abilities (Benton Judgment of Line Orientation), and global cognitive function (MoCA). We used linear regression to test for association between genotype and cognitive performance with adjustment for important covariates and accounted for multiple testing using Bonferroni's corrections. Results Mutation carriers (n = 60; 4.4%) and E326K carriers (n = 65; 4.7%) had a higher prevalence of dementia (mutations, odds ratio = 5.1; P = 9.7 × 10−6; E326K, odds ratio = 6.4; P = 5.7 × 10−7) and lower performance on Letter-Number Sequencing (mutations, corrected P[Pc] = 9.0 × 10−4; E326K, Pc = 0.036), Trail Making B-A (mutations, Pc = 0.018; E326K, Pc = 0.018), and Benton Judgment of Line Orientation (mutations, Pc = 0.0045; E326K, Pc = 0.0013). Conclusions Both GBA mutations and E326K are associated with a distinct cognitive profile characterized by greater impairment in working memory/executive function and visuospatial abilities in PD patients. The discovery that E326K negatively impacts cognitive performance approximately doubles the proportion of PD patients we now recognize are at risk for more severe GBA-related cognitive deficits. © 2015 International Parkinson and Movement Disorder Society

Objectives: Mutations in the GBA gene occur in 7% of patients with Parkinson disease (PD) and are a well-established susceptibility factor for PD, which is characterized by Lewy body disease (LBD) neuropathologic changes (LBDNCs). We sought to determine whether GBA influences risk of dementia with LBDNCs, Alzheimer disease (AD) neuropathologic changes (ADNCs), or both. Methods: We screened the entire GBA coding region for mutations in controls and in subjects with dementia and LBDNCs and no or low levels of ADNCs (pure dementia with Lewy bodies [pDLB]), LBDNCs and high-level ADNCs (LBD-AD), and high-level ADNCs but without LBDNCs (AD). Results: Among white subjects, pathogenic GBA mutations were identified in 6 of 79 pDLB cases (7.6%), 8 of 222 LBD-AD cases (3.6%), 2 of 243 AD cases (0.8%), and 3 of 381 controls (0.8%). Subjects with pDLB and LBD-AD were more likely to carry mutations than controls (pDLB: odds ratio [OR] = 7.6; 95% confidence interval [CI] = 1.8–31.9; p = 0.006; LBD-AD: OR = 4.6; CI = 1.2–17.6; p = 0.025), but there was no significant difference in frequencies between the AD and control groups (OR = 1.1; CI = 0.2–6.6; p = 0.92). There was a highly significant trend test across groups (χ 2 (1) = 19.3; p = 1.1 × 10 −5 ), with the likelihood of carrying a GBA mutation increasing in the following direction: control/AD Conclusions: GBA is a susceptibility gene across the LBD spectrum, but not in AD, and appears to convey a higher risk for PD and pDLB than for LBD-AD. PD and pDLB might be more similar to one another in genetic determinants and pathophysiology than either disease is to LBD-AD.

Background The MNS blood group system is second in diversity only to the RH blood group system, with 46 described antigens. MNS system antigens are carried on glycophorins GPA and GPB that are products of the GYPA and GYPB genes, respectively. GYPA and GYPB are homologous paralogs which lie adjacent to each other on chromosome 4 in tandem with a third GYP paralog, GYPE. Current DNA-based testing methods for predicting MNS can be confounded by all types of genetic variation at the GYP locus, particularly in individuals of non-European ancestry. We sought to develop a next generation sequencing (NGS) approach for the systematic characterization of the GYP locus to accurately predict the common M/N and S/s blood group antigens and simultaneously identify other clinically relevant GYP DNA variants. Study Methods A total of 1139 samples were DNA sequenced; 1135 were from a previous study of blood donors self-identified to be of Asian American or Native American descent, and were 4 WHO reference DNAs (NIBSC). Blood donors had been tested for M and N by serology and for M/N and S/s using a single nucleotide variant (SNV) blood group genotyping platform (Bioarray). Samples were selected to enrich for MNS serology-SNV discrepancies or indeterminate results. BloodSeq is a NGS targeted panel that includes capture of 97.4kb over 3 genomic regions including the exons, pseudo-exons, introns, and proximal intergenic regions of GYPA, GYPB, and GYPE. This custom capture was used to generate Illumina, paired-end 100 bp DNA sequence reads. Raw sequence data was aligned to the human reference genome (hg19) and SNVs assessed using standard calling methods (GATK HaplotypeCaller). To predict MNS blood group system antigens, we determined variants which identified ISBT alleles; M/N antigens were defined as codominant alleles with multiple variant sites present in GYPA exon 2, while S/s antigens were defined by GYPB c.143T>C (p.Thr48Met) and 2 known GYPB silencing SNVs. Other DNA variants were cross-referenced with ISBT to predict associated blood group antigens. Results In a preliminary analyses, standard DNA variant calling methods predicted S/s (GYPB) SNVs accurately. However, alleles with M (GYPA) blood group variants exhibited a low call rate. Visualization of aligned reads indicated alleles corresponding to the M blood group sequence align poorly to the reference GYPA sequence. We traced the origin of these poor quality alignments to the presence of a region in GYPE with high sequence homology to the GYPA M allele. Notably, in the reference genome the GYPA gene has DNA variants indicative of theN genotype. With this knowledge, we developed a new approach which considers alignments of all 3 genes (GYPA, GYPB, GYPE) to predict M/N and S/s blood group antigens. Applying this method, BloodSeq predicted M with high concordance with serology (99.2%) and SNV genotype (99.6%), similar to the SNV genotype-serology concordance for M (98.9%). BloodSeq also predicted S/s in high concordance with the SNV predicted genotype (99.4% and 99.8%, respectively for S and s). Prediction of N by both BloodSeq and SNV genotype were similar to each other (99.6%) but exhibited lower accuracy (86.1% and 85.6%, respectively) when compared to serology. Interestingly, most (90%) of the N discrepancies were genetic prediction of absent N antigen but a positive N result by serology. We suspect these discrepancies result from cross-reactivity of reagent antibodies with "N" (an N-like antigen encoded by GYPB), which would require additional DNA sequence curation, or other underlying genetic variation. Additionally, 9 GYPA and GYPB variants indicative of other named ISBT alleles were detected, as well as a novel predicted frameshift variant in GYPA. Conclusion Our results demonstrate that a targeted NGS approach followed by an analysis pipeline customized for the GYP locus can simultaneously predict M/N and S/s blood groups and detect other GYP variants of known clinical significance. We propose that use of GYP locus-specific DNA sequence analysis strategies, such as addition of alternative reference sequences, should allow for automated and reliable classification of the M/N, S/s, and other variants in the MNS blood group system using next generation DNA sequencing. This work provides the foundation for a DNA-based, high resolution blood-typing method for the detection of clinically relevant MNS blood group system genetic variation. Disclosures Johnsen: CSL Behring: Consultancy; Octapharma: Consultancy.

Background Hemophilia A and B are rare X-linked bleeding disorders affecting ~1:5000 male births. Hemophilia genotype is important to inform reproductive planning, pregnancy, and neonatal management, risk of inhibitor formation and bleeding severity, and basic understanding of mechanisms of disease. In 2012, two separate surveys found only ~20% of patients with hemophilia had a genotype determined. MyLifeOurFuture (MLOF) was formed as a multi-sector collaboration between the American Thrombosis and Hemostasis Network (ATHN), the National Hemophilia Foundation (NHF), BloodworksNW (BWNW), and Biogen to provide hemophilia genotype analysis for patients in the U.S. and to create a Research Repository for future scientific discovery. Methods Participating hemophilia treatment centers (HTCs) contract through ATHN, enroll patients, obtain samples, and provide clinical results to patients. ATHN offers HTC provider education, a secure infrastructure for clinical data collection, and access for research proposals. NHF educates the bleeding disorders community about the initiative and supports recruitment. Biogen provides scientific collaboration and financial support. BWNW serves as the central sample processing and genotyping laboratory and houses the research sample repository. Genotyping was performed custom molecular inversion probes (MIPs) targeting the F8 and F9 genes and F8 inversions for simultaneous next generation sequencing (NGS) followed by confirmation of variants using standard genotyping methods.. Clinical results were returned to providers, and new variants were submitted to public databases. Results 69 HTCs enrolled the first 3000 patients in under 3 years. Clinically reportable DNA variants were detected in 98.1% (2357/2401) of hemophilia A and 99.3% (595/599) of hemophilia B patients. 924 unique variants were found; 285 were novel. Predicted gene disrupting variants were common in severe disease, while missense variants predominated in mild-moderate disease. The custom MIP-based NGS inversion screening method successfully detected F8 gene proximal and distal intron 22 inversion and intron 1 inversion variants. Unexpectedly, the NGS approach detected more than one reportable variants in 40 patients (10 females), a finding with potential clinical implications. NGS also detected 108 unique incidental variants unlikely to cause disease; 11 variants were previously reported associated with hemophilia. Interrogation of the ExAC database, which has data from >66,000 individuals without hemophilia, reports DNA variants distributed across the coding regions of both genes. Conclusions MLOF is the largest hemophilia genetics project performed to date, with plans to genotype over 6000 U.S. hemophilia patients. In the first 3000 patients, clinically reportable DNA variants were identifiedin nearly all patients. Our hemophilia NGS approach accurately identified F8 and F9 gene variants and is, to our knowledge, the first NGS method which can detect F8 inversions. The incidence of discovery of novel variation was high (30%) and novel variants were discovered continuously (per patient) over the course of the study, indicating that additional genetic variation in hemophilia likely remains undiscovered. Although both the F8 and F9 genes are thought to be conserved, we identified incidental variation in both genes, supporting caution in the interpretation of new variants. In summary, MLOF is a successful nationwide collaboration to genotype two rare bleeding disorders at scale which has contributed significantly towards DNA variant identification in the F8 and F9 genes in hemophilia. Through a consented research repository, MLOF data and samples, including phenotypic data from the ATHNdataset, will be accessible to providers and research communities for advancing our understanding of hemophilia and other disorders. Disclosures Johnsen: Octapharma: Consultancy; CSL Behring: Consultancy. Meltzer:Biogen: Employment.