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Context: Congenital hypopituitarism is a genetically heterogeneous condition. Whole exome sequencing (WES) is a promising approach for molecular diagnosis of patients with this condition. Objectives: To conduct WES in a patient with congenital hypopituitarism born to consanguineous parents, CDH2 screening in a cohort of patients with congenital hypopituitarism, and functional testing of a novel CDH2 variant. Design: Genomic DNA from a proband and her consanguineous parents was analyzed by WES. Copy number variants were evaluated. The genetic variants were filtered for population frequency (ExAC, 1000 genomes, gnomAD and ABraOM), in silico prediction of pathogenicity, and gene expression in pituitary and/or hypothalamus. Genomic DNA from 145 patients was screened for CDH2 by Sanger sequencing. Results: One female patient with deficiencies in GH, TSH, ACTH, LH, and FSH and ectopic posterior pituitary gland contained a rare homozygous c.865G>A (p.Val289Ile) variant in CDH2. To determine whether the p.Val289Ile variant in CDH2 affects cell adhesion properties, we stably transfected L1 fibroblast lines, labeled the cells with lipophilic dyes, and quantified aggregation. Large aggregates formed in cells expressing wild type CDH2, but aggregation was impaired in cells transfected with variant CDH2 or non-transfected. Conclusion: A homozygous CDH2 allelic variant was found in 1 hypopituitarism patient, and the variant impaired cell aggregation function in vitro. No disease-causing variants were found in 145 other patients screened for CDH2 variants. Thus, CDH2 is a candidate gene for hypopituitarism that needs to be tested in different populations.

For over a decade, a large research community has relied on a flawed reference assembly of the genome ofRattus norvegicusknown as Rnor_6.0. The seventh assembly of the rat reference genome4mRatBN7.2, based on the inbred Brown Norway rat, corrects numerous misplaced segments, reduces base-level errors by approximately 9-fold, and increases contiguity by 290-fold, despite some remaining regions of potential misassembly. Gene annotations are now more complete, significantly improving the mapping precision of genomic, transcriptomic, and proteomics data sets. SimpleLiftOverfrom Rnor_6.0 to mRatBN7.2 misses ∼12% of variants. To facilitate the transition to mRatBN7.2, we performed a joint analysis of 163 whole genomes representing 120 strains/substrains. We defined 20.0 million sequence variations, of which 18.7 thousand are predicted to potentially impact the function of 6,677 genes. Phylogenetic analysis confirmed historical records and prior results and refined the ancestral relationship of these strains. Sixteen million polymorphisms segregate in the widely studied heterogeneous stock rat population, and 11313 million variants segregate collectively in the HXB/BXH and FXLE/LEXF strain families. Some inbred strains differ by only 132 M variants, and closely related substrains segregate by even fewer variants. We generated a new rat genetic map based on data from 1,893 heterogeneous stock rats and annotated transcription start sites and alternative polyadenylation sites.

Using selective breeding, we have produced two rat lines with highly divergent behaviors: bred Low Responders (bLR) are extremely inhibited and anxious in a novel environment, whereas bred High Responders (bHR) are exploratory and sensation-seeking. These traits map onto temperamental extremes predictive of internalizing vs. externalizing psychiatric disorders in humans and model two divergent paths to drug abuse. To elucidate gene expression underlying these heritable behavioral differences, bHR and bLR rats from the 37thgeneration of selection were used to produce F0, F1cross, and F2intercross generations. We measured exploratory locomotion, anxiety-like behavior, and sensitivity to reward cues (Pavlovian Conditioned Approach) and performed hippocampal RNA sequencing in both F0s (n=24, n=6/phenotype/sex) and F2s (n=250, n=125/sex).Behaviors that diverged during selective breeding remained correlated in F2s, implying a shared genetic basis. F0bHR/bLR differences in gene expression were robust, surpassing differences associated with sex, and predicted expression patterns associated with F2behavior. Among the bHR/bLR differentially expressed genes (DEGs), 17 were associated with exploratory locomotion in the F2s, seven of which were located within related quantitative trait loci we previously identified in F2s. Convergence between our study and others targeting similar behavioral traits revealed an additional 26 genes related to behavioral temperament. Gene set enrichment analysis pointed to growth and proliferation being upregulated with bHR-like behavior, and mitochondrial function, oxidative stress, and microglial activation being upregulated with bLR-like behavior. Our findings implicate bioenergetic regulation of hippocampal function in shaping temperamental differences, thereby modulating vulnerability to psychiatric and addictive disorders.

Common genetic factors likely contribute to multiple psychiatric diseases including mood and substance use disorders. Certain stable, heritable traits reflecting temperament, termed externalizing or internalizing, play a large role in modulating vulnerability to these disorders. To model these heritable tendencies, we selectively bred rats for high and low exploration in a novel environment (bred High Responders (bHR) vs. Low Responders (bLR)). To identify genes underlying the response to selection, we phenotyped and genotyped 558 rats from an F2cross between bHR and bLR. Several behavioral traits show high heritability, including the selection trait: exploratory locomotion (EL) in a novel environment. There were significant phenotypic and genetic correlations between tests that capture facets of EL and anxiety. There were also correlations with Pavlovian conditioned approach (PavCA) behavior despite the lower heritability of that trait.Ten significant and conditionally independent loci for six behavioral traits were identified. Five of the six traits reflect different facets of EL that were captured by three behavioral tests. Distance traveled measures from the open field and the elevated plus maze map onto different loci, thus may represent different aspects of novelty-induced locomotor activity. The sixth behavioral trait, number of fecal boli, is the only anxiety-related trait mapping to a significant locus on chromosome 18 within which thePik3c3gene is located. There were no significant loci for PavCA. We identified a missense variant in thePlekhf1gene on the chromosome 1:95 Mb QTL andFancfandGas2as potential candidate genes that may drive the chromosome 1:107 Mb QTL for EL traits. The identification of a locomotor activity-related QTL on chromosome 7 encompassing thePkhd1l1andTrhrgenes is consistent with our previous finding of these genes being differentially expressed in the hippocampus of bHR vs. bLR rats.The strong heritability coupled with identification of several loci associated with exploratory locomotion and emotionality provide compelling support for this selectively bred rat model in discovering relatively large effect causal variants tied to elements of internalizing and externalizing behaviors inherent to psychiatric and substance use disorders.

People with kidney disease are disproportionately affected by atherosclerosis for unclear reasons. Soluble urokinase plasminogen activator receptor (suPAR) is an immune-derived mediator of kidney disease, levels of which are strongly associated with cardiovascular outcomes. We assessed suPAR's pathogenic involvement in atherosclerosis using epidemiologic, genetic, and experimental approaches. We found serum suPAR levels to be predictive of coronary artery calcification and cardiovascular events in 5,406 participants without known coronary disease. In a genome-wide association meta-analysis including over 25,000 individuals, we identified a missense variant in the plasminogen activator, urokinase receptor (PLAUR) gene (rs4760), confirmed experimentally to lead to higher suPAR levels. Mendelian randomization analysis in the UK Biobank using rs4760 indicated a causal association between genetically predicted suPAR levels and atherosclerotic phenotypes. In an experimental model of atherosclerosis, proprotein convertase subtilisin/kexin-9 (Pcsk9) transfection in mice overexpressing suPAR (suPARTg) led to substantially increased atherosclerotic plaques with necrotic cores and macrophage infiltration compared with those in WT mice, despite similar cholesterol levels. Prior to induction of atherosclerosis, aortas of suPARTg mice excreted higher levels of CCL2 and had higher monocyte counts compared with WT aortas. Aortic and circulating suPARTg monocytes exhibited a proinflammatory profile and enhanced chemotaxis. These findings characterize suPAR as a pathogenic factor for atherosclerosis acting at least partially through modulation of monocyte function.

Loss of function mutations in COQ4 lead to fatal neonatal mitochondrial disorders with CoQ10 deficiency. In two siblings with childhood-onset, slowly progressive ataxia from a consanguineous mating from Turkey, whole exome sequencing identified homozygous missense mutations in COQ4 (Gly55Val). Blood levels of CoQ10 were either below or at the low end of the normal range. The more severely affected of the siblings was given a high dose of CoQ10 (2000 mg/day) for one month, following which significant improvement in neurological signs and symptoms was noted. Our report indicates that COQ4 mutations are a rare cause of ataxia and that CoQ10 supplementation is a personalized treatment for this form of ataxia.

We report four allelic variants (three novel) in three genes previously established as causal for hypopituitarism or related disorders. A novel homozygous variant in the growth hormone gene, GH1 c.171delT (p.Phe 57Leufs*43), was found in a male patient with severe isolated growth hormone deficiency (IGHD) born to consanguineous parents. A hemizygous SOX3 allelic variant (p.Met304Ile) was found in a male patient with IGHD and hypoplastic anterior pituitary. YASARA, a tool to evaluate protein stability, suggests that p.Met304Ile destabilizes the SOX3 protein (ΔΔG = 2.49 kcal/mol). A rare, heterozygous missense variant in the TALE homeobox protein gene, TGIF1 (c.268C&gt, T:p.Arg90Cys) was found in a patient with combined pituitary hormone deficiency (CPHD), diabetes insipidus, and syndromic features of holoprosencephaly (HPE). This variant was previously reported in a patient with severe holoprosencephaly and shown to affect TGIF1 function. A novel heterozygous TGIF1 variant (c.82T&gt, C:p.Ser28Pro) was identified in a patient with CPHD, pituitary aplasia and ectopic posterior lobe. Both TGIF1 variants have an autosomal dominant pattern of inheritance with incomplete penetrance. In conclusion, we have found allelic variants in three genes in hypopituitarism patients. We discuss these variants and associated patient phenotypes in relation to previously reported variants in these genes, expanding our knowledge of the phenotypic spectrum in patient populations.

International audience; Pituitary hormone deficiency occurs in ∼1:4,000 live births. Approximately 3% of the cases are due to mutations in the alpha isoform of POU1F1, a pituitary-specific transcriptional activator. We found four separate heterozygous missense variants in unrelated individuals with hypopituitarism that were predicted to affect a minor isoform, POU1F1 beta, which can act as a transcriptional repressor. These variants retain repressor activity, but they shift splicing to favor the expression of the beta isoform, resulting in dominant-negative loss of function. Using a high-throughput splicing reporter assay, we tested 1,070 single-nucleotide variants in POU1F1. We identified 96 splice-disruptive variants, including 14 synonymous variants. In separate cohorts, we found two additional synonymous variants nominated by this screen that co-segregate with hypopituitarism. This study underlines the importance of evaluating the impact of variants on splicing and provides a catalog for interpretation of variants of unknown significance in POU1F1.

BackgroundUse of targeted exome-arrays with common, rare variants and functionally enriched variation has led to discovery of new genes contributing to population variation in risk factors. Plasminogen activator-inhibitor 1 (PAI-1), tissue plasminogen activator (tPA), and the plasma product D-dimer are important components of the fibrinolytic system. There have been few large-scale genome-wide or exome-wide studies of PAI-1, tPA, and D-dimer.ObjectivesWe sought to discover new genetic loci contributing to variation in these traits using an exome-array approach.MethodsCohort-level analyses and fixed effects meta-analyses of PAI-1 (n=15603), tPA (n=6876,) and D-dimer (n=19306) from 12 cohorts of European ancestry with diverse study design were conducted, including single-variant analyses and gene-based burden testing.ResultsFive variants located in NME7, FGL1, and the fibrinogen locus, all associated with D-dimer levels, achieved genome-wide significance (P&nbsp

Whole Exome Sequencing (WES) has been a useful tool to improve molecular diagnosis in hypopituitarism, leading to the discovery of at least 8 new genes in the last 7 years. However, some genes associated with hypopituitarism show low coverage in this methodology, limiting its use for molecular diagnosis. Our objective is to compare three library prepping kits, NimbleGen (Roche), SureSelect (Agilent) and Nextera (Illumina) examining the best performance related to sequencing quality, exon extension coverage (≥98%) and base depth read (≥20x) of 44 genes associated with hypopituitarism and 32 involved in pituitary development. Three different groups composed of 2 HapMap samples (Group 1), 2 Brazilian patients with hypopituitarism and their respective mothers (Group 2) and 109 random Brazilian samples (Group 3) were sequenced in Illumina platform. Group 1 and 3 were performed using all three library prepping kits, while group 2 was performed with NimbleGen and SureSelect. Although all technologies covered the selected genes with similar efficiency regarding poor (less than 20%) and rich (more than 80%) GC areas, SureSelect has shown to reach the most uniform coverage in the selected region with a lower level of duplicate reads, as well as a higher number of identified pathogenic variants.

Pituitary hormone deficiency occurs in ∼1:4,000 live births. Approximately 3% of the cases are due to mutations in the alpha isoform of POU1F1, a pituitary-specific transcriptional activator. We found four separate heterozygous missense variants in unrelated hypopituitarism patients that were predicted to affect a minor isoform, POU1F1 beta, which can act as a transcriptional repressor. These variants retain repressor activity, but they shift splicing to favor the expression of the beta isoform, resulting in dominant negative loss of function. Using a high throughput splicing reporter assay, we tested 1,080 single nucleotide variants inPOU1F1. We identified 113 splice disruptive variants, including 23 synonymous variants. We evaluated separate cohorts of hypopituitarism patients and found two different synonymous splice disruptive variants that co-segregate with hypopituitarism. This study underlines the importance of evaluating the impact of variants on splicing and provides a catalog for interpretation of variants of unknown significance in thePOU1F1gene.

Objective The transcription factor OTX2is implicated in ocular, craniofacial, and pituitary development. Design We aimed to establish the contribution of OTX2 mutations in congenital hypopituitarism patients with/without eye abnormalities, study functional consequences, and establish OTX2 expression in the human brain, with a view to investigate the mechanism of action. Methods We screened patients from the UK (n = 103), international centres (n = 24), and Brazil (n = 282); 145 were within the septo-optic dysplasia spectrum, and 264 had no eye phenotype. Transactivation ability of OTX2 variants was analysed in murine hypothalamic GT1-7 neurons. In situ hybridization was performed on human embryonic brain sections. Genetically engineered mice were generated with a series of C-terminal OTX2 variants. Results Two chromosomal deletions and six haploinsufficient mutations were identified in individuals with eye abnormalities; an affected relative of one patient harboured the same mutation without an ocular phenotype. OTX2 truncations led to significant transactivation reduction. A missense variant was identified in another patient without eye abnormalities; however, studies revealed it was most likely not causative. In the mouse, truncations proximal to aa219 caused anophthalmia, while distal truncations and the missense variant were tolerated. During human embryogenesis, OTX2 was expressed in the posterior pituitary, retina, ear, thalamus, choroid plexus, and partially in the hypothalamus, but not in the anterior pituitary. Conclusions OTX2 mutations are rarely associated with hypopituitarism in isolation without eye abnormalities, and may be variably penetrant, even within the same pedigree. Our data suggest that the endocrine phenotypes in patients with OTX2 mutations are of hypothalamic origin.

Erythropoietin (EPO) stimulates erythroid differentiation and maturation. Though the transcriptional regulation of EPO has been well studied, the molecular determinants of EPO secretion remain unknown. Here, we generated a HEK293T reporter cell line that provides a quantifiable and selectable readout of intracellular EPO levels and performed a genome-scale CRISPR screen that identified SURF4 as an important mediator of EPO secretion. Targeting SURF4 with multiple independent single guide RNAs (sgRNAs) resulted in intracellular accumulation and extracellular depletion of EPO. Both of these phenotypes were rescued by expression of SURF4 cDNA. Additionally, we found that disruption of SURF4 resulted in accumulation of EPO in the endoplasmic reticulum (ER) compartment and that SURF4 and EPO physically interact. Furthermore, SURF4 disruption in Hep3B cells also caused a defect in the secretion of endogenous EPO under conditions mimicking hypoxia, ruling out an artifact of heterologous overexpression. This work demonstrates that SURF4 functions as an ER cargo receptor that mediates the efficient secretion of EPO. Our findings also suggest that modulating SURF4 may be an effective treatment for disorders of erythropoiesis that are driven by aberrant EPO levels. Finally, we show that SURF4 overexpression results in increased secretion of EPO, suggesting a new strategy for more efficient production of recombinant EPO.

The genetic basis for congenital hypopituitarism and related disorders is beginning to emerge, and over causal 30 genes have been identified, including six in the SHH signaling pathway. Mutations in some of these genes can also cause holoprosencephaly (HPE) or septo-optic dysplasia. SIX3 is a homeodomain protein expressed in the developing brain, pituitary gland, and eye. It activates SHH signaling and represses BMP signaling. Heterozygous mutations in SIX3 cause variable HPE in humans and mice. We identified a rare, heterozygous variant in SIX3 in two children with neonatal GH and TSH deficiency and stalk interruption, p.P74R. Using transient transfection in 3T3 cells, we demonstrated that the variant reduced the ability of SIX3 to transactivate the SHH enhancer and promoter of FOXG1, suggesting that the variant could be deleterious. To understand the role of SIX3 in hypothalamic and pituitary development we used Nkx2.1-cre and Prop1-cre to delete Six3 in mice. The Nkx2.1-cre, Six3flox/flox embryos had no evidence of infundibulum evagination or expression of Fgf10 or Tcf7l2 at e11.5. The oral ectoderm invaginated in mutants, but no definitive Rathke’s pouch formed. There was no evidence of Lhx3 expression and only trace amounts of Pitx1, indicating that pituitary induction failed due to the lack of Six3 in the developing hypothalamus. Similarly, disruption of Six3 expression in Rathke’s pouch using Prop1-cre ablated pituitary development. Together, these data reveal essential roles of Six3 in both the neural and oral ectoderm for hypothalamic and pituitary development, respectively. Heterozygous loss of function variants in SIX3 could be a contributor to multiple pituitary hormone deficiencies in children, especially if there are associated craniofacial abnormalities.

Erythropoietin (EPO), a glycoprotein produced by specialized peritubular fibroblasts in the kidney, is the master regulator of erythropoiesis. EPO is secreted into the plasma in response to tissue hypoxia and stimulates erythroid differentiation and maturation. Though the transcriptional regulation of EPO has been well studied, the molecular determinants of EPO secretion remain unknown. Here, we generated a HEK293T reporter cell line that provides a quantifiable and selectable readout of intracellular EPO levels. Using this cell line, we performed a genome-scale CRISPR screen that identified SURF4 as an important mediator of EPO secretion. TargetingSURF4with multiple independent sgRNAs resulted in intracellular accumulation and extracellular depletion of EPO. Both of these phenotypes were rescued by expression ofSURF4cDNA. Additionally, consistent with a role for SURF4 as an ER cargo receptor of EPO, we found that disruption of SURF4 resulted in accumulation of EPO in the ER compartment, and that SURF4 and EPO physically interact. Furthermore, SURF4 disruption in Hep3B cells also caused a defect in the secretion of endogenous EPO, ruling out an artifact of heterologous overexpression. This work suggests that SURF4 functions as an ER cargo receptor that mediates the efficient secretion of EPO. Our findings also suggest that modulating SURF4 may be an effective treatment for disorders of erythropoeisis that are driven by aberrant EPO levels. Finally, we show that SURF4 overexpression results in increased secretion of EPO, suggesting a new strategy for more efficient production of recombinant EPO.

Deep vein thrombosis and pulmonary embolism, collectively defined as venous thromboembolism (VTE), are the third leading cause of cardiovascular death in the United States. Common genetic variants conferring increased varying degrees of VTE risk have been identified by genome-wide association studies (GWAS). Rare mutations in the anticoagulant genes PROC, PROS1 and SERPINC1 result in perinatal lethal thrombosis in homozygotes and markedly increased VTE risk in heterozygotes. However, currently described VTE variants account for an insufficient portion of risk to be routinely used for clinical decision making. To identify new rare VTE risk variants, we performed a whole-exome study of 393 individuals with unprovoked VTE and 6114 controls. This study identified 4 genes harboring an excess number of rare damaging variants in patients with VTE: PROS1, STAB2, PROC, and SERPINC1. At STAB2, 7.8% of VTE cases and 2.4% of controls had a qualifying rare variant. In cell culture, VTE-associated variants of STAB2 had a reduced surface expression compared with reference STAB2. Common variants in STAB2 have been previously associated with plasma von Willebrand factor and coagulation factor VIII levels in GWAS, suggesting that haploinsufficiency of stabilin-2 may increase VTE risk through elevated levels of these procoagulants. In an independent cohort, we found higher von Willebrand factor levels and equivalent propeptide levels in individuals with rare STAB2 variants compared with controls. Taken together, this study demonstrates the utility of gene-based collapsing analyses to identify loci harboring an excess of rare variants with functional connections to a complex thrombotic disease.

The genetic basis for congenital hypopituitarism and related disorders is beginning to emerge, and over 30 causal genes have been identified. Mutations in some of these genes can also cause holoprosencephaly (HPE) or septo-optic dysplasia. SIX3 is a homeodomain protein expressed in the developing brain, pituitary gland, and eye. Heterozygous mutations in SIX3 cause variable HPE in humans and mice. We identified two children with neonatal GH and TSH deficiency and stalk interruption who were doubly heterozygous for rare, likely deleterious variants in SIX3 and POU1F1. Functional studies demonstrated that both variants are disruptive. We used Six3 and Pou1f1 loss of function mice to assess the genetic interaction between Six3 and Pou1f1. Six3 heterozygotes have variable pituitary gland dysmorphology, while Pou1f1 heterozygotes are normal. A significant portion of the Six3+/-; Pou1f1+/dw doubly heterozygous mice have a more pronounced pituitary phenotype than Six3+/-, supporting the possibility of digenic pituitary disease. To understand the role of SIX3 in pituitary and hypothalamic development, we used Prop1-cre and Nkx2.1-cre to delete Six3. Disruption of Six3 expression in Rathke’s pouch caused poor activation of Lhx3 expression and arrested anterior pituitary development. The Nkx2.1-cre, Six3flox/flox embryos had no evidence of infundibulum evagination and failed to induce FGF and BMP signaling, which normally drive expansion of Rathke’s pouch. By E11.5 cells in Rathke’s pouch underwent apoptosis. The Nkx2.1-cre, Six3flox/flox embryos failed to activate expression of Lhx2 and Tbx3 in the neural ectoderm. These embryos had elevated CCND1, MYCN, and Axin2 expression in the area of the presumptive infundibulum. This indicates that SIX3 is necessary to repress cell proliferation and Wnt/beta-catenin signals to promote formation of the pituitary stalk. Thus, Six3 has essential roles in both the neural and oral ectoderm for hypothalamic and pituitary development, respectively. Heterozygous loss of function variants in SIX3 could be a contributor to multiple pituitary hormone deficiencies in children, especially if there are associated craniofacial abnormalities or PSIS.

Hereditary thrombocytopenias can be subclassified based on mode of inheritance and platelet size. Here we report a family with autosomal dominant (AD) thrombocytopenia with normal platelet size. Linkage analysis and whole exome sequencing identified the R1026W substitution in ITGA2B as the causative defect. The same mutation has been previously reported in 7 Japanese families/patients with AD thrombocytopenia, but all of these patients had macrothrombocytopenia. This is the first report of a family with AD thrombocytopenia with normal platelet size resulting from mutation in ITGA2B. ITGA2B mutations should therefore be included in the differential diagnosis of this latter disorder.

International audience; C-reactive protein (CRP) is a sensitive biomarker of chronic low-grade inflammation and is associated with multiple complex diseases. The genetic determinants of chronic inflammation remain largely unknown, and the causal role of CRP in several clinical outcomes is debated. We performed two genome-wide association studies (GWASs), on HapMap and 1000 Genomes imputed data, of circulating amounts of CRP by using data from 88 studies comprising 204,402 European individuals. Additionally, we performed in silico functional analyses and Mendelian randomization analyses with several clinical outcomes. The GWAS meta-analyses of CRP revealed 58 distinct genetic loci (p < 5 × 10-8). After adjustment for body mass index in the regression analysis, the associations at all except three loci remained. The lead variants at the distinct loci explained up to 7.0% of the variance in circulating amounts of CRP. We identified 66 gene sets that were organized in two substantially correlated clusters, one mainly composed of immune pathways and the other characterized by metabolic pathways in the liver. Mendelian randomization analyses revealed a causal protective effect of CRP on schizophrenia and a risk-increasing effect on bipolar disorder. Our findings provide further insights into the biology of inflammation and could lead to interventions for treating inflammation and its clinical consequences.Copyright © 2018 American Society of Human Genetics. All rights reserved.

Juvenile segmental progeroid syndromes are rare, heterogeneous disorders characterized by signs of premature aging affecting more than one tissue or organ starting in childhood. Hutchinson–Gilford progeria syndrome (HGPS), caused by a recurrent de novo synonymous LMNA mutation resulting in aberrant splicing and generation of a mutant product called progerin, is a prototypical example of such disorders. Here, we performed a joint collaborative study using massively parallel sequencing and targeted Sanger sequencing, aimed at delineating the underlying genetic cause of 14 previously undiagnosed, clinically heterogeneous, non-LMNA-associated juvenile progeroid patients. The molecular diagnosis was achieved in 11 of 14 cases (~ 79%). Furthermore, we firmly establish biallelic mutations in POLR3A as the genetic cause of a recognizable, neonatal, Wiedemann–Rautenstrauch-like progeroid syndrome. Thus, we suggest that POLR3A mutations are causal for a portion of under-diagnosed early-onset segmental progeroid syndromes. We additionally expand the clinical spectrum associated with PYCR1 mutations by showing that they can somewhat resemble HGPS in the first year of life. Moreover, our results lead to clinical reclassification in one single case. Our data emphasize the complex genetic and clinical heterogeneity underlying progeroid disorders.

We performed whole-genome sequencing for eight inbred rat strains commonly used in genetic mapping studies. They are the founders of the NIH heterogeneous stock (HS) outbred colony. We provide their sequences and variant calls to the rat genomics community. When analyzing the variant calls we identified regions with unusually high levels of heterozygosity. These regions are consistent across the eight inbred strains, including Brown Norway, which is the basis of the rat reference genome. These regions show higher read depths than other regions in the genome and contain higher rates of apparent tri-allelic variant sites. The evidence suggests that these regions may correspond to duplicated segments that were incorrectly overlaid as a single segment in the reference genome. We provide masks for these regions of suspected mis-assembly as a resource for the community to flag potentially false interpretations of mapping or functional results.

The comprehensive genetic alterations underlying the pathogenesis of T-cell prolymphocytic leukemia (T-PLL) are unknown. To address this, we performed whole-genome sequencing (WGS), whole-exome sequencing (WES), high-resolution copy-number analysis, and Sanger resequencing of a large cohort of T-PLL. WGS and WES identified novel mutations in recurrently altered genes not previously implicated in T-PLL including EZH2, FBXW10, and CHEK2. Strikingly, WGS and/or WES showed largely mutually exclusive mutations affecting IL2RG, JAK1, JAK3, or STAT5B in 38 of 50 T-PLL genomes (76.0%). Notably, gain-of-function IL2RG mutations are novel and have not been reported in any form of cancer. Further, high-frequency mutations in STAT5B have not been previously reported in T-PLL. Functionally, IL2RG-JAK1-JAK3-STAT5B mutations led to signal transducer and activator of transcription 5 (STAT5) hyperactivation, transformed Ba/F3 cells resulting in cytokine-independent growth, and/or enhanced colony formation in Jurkat T cells. Importantly, primary T-PLL cells exhibited constitutive activation of STAT5, and targeted pharmacologic inhibition of STAT5 with pimozide induced apoptosis in primary T-PLL cells. These results for the first time provide a portrait of the mutational landscape of T-PLL and implicate deregulation of DNA repair and epigenetic modulators as well as high-frequency mutational activation of the IL2RG-JAK1-JAK3-STAT5B axis in the pathogenesis of T-PLL. These findings offer opportunities for novel targeted therapies in this aggressive leukemia.

Nanophthalmos is a rare, potentially devastating eye condition characterized by small eyes with relatively normal anatomy, a high hyperopic refractive error, and frequent association with angle closure glaucoma and vision loss. The condition constitutes the extreme of hyperopia or farsightedness, a common refractive error that is associated with strabismus and amblyopia in children. NNO1 was the first mapped nanophthalmos locus. We used combined pooled exome sequencing and strong linkage data in the large family used to map this locus to identify a canonical splice site alteration upstream of the last exon of the gene encoding myelin regulatory factor (MYRF c.3376-1G>A), a membrane bound transcription factor that undergoes autoproteolytic cleavage for nuclear localization. This variant produced a stable RNA transcript, leading to a frameshift mutation p.Gly1126Valfs*31 in the C-terminus of the protein. In addition, we identified an early truncating MYRF frameshift mutation, c.769dupC (p.S264QfsX74), in a patient with extreme axial hyperopia and syndromic features. Myrf conditional knockout mice (CKO) developed depigmentation of the retinal pigment epithelium (RPE) and retinal degeneration supporting a role of this gene in retinal and RPE development. Furthermore, we demonstrated the reduced expression of Tmem98, another known nanophthalmos gene, in Myrf CKO mice, and the physical interaction of MYRF with TMEM98. Our study establishes MYRF as a nanophthalmos gene and uncovers a new pathway for eye growth and development., Author summary Hyperopia or farsightedness is a common condition that can cause visual impairment especially in children. The extreme of this condition is called nanophthalmos, a small crowded eye in which inappropriate drainage of aqueous humor from the eye can lead to glaucoma and vision loss. We previously described a large family with inherited nanophthalmos, but the genetic defect that segregated in this family was unknown. Here, we have used a new approach combining linkage analysis and pooled sequencing to identify the genetic cause in this family. We identified a splice site mutation that causes the myelin regulatory factor (MYRF) gene to produce an aberrant protein. Additionally, a child with syndromic manifestations and a deleterious MYRF variant shared the same eye condition. Using a mouse model in which MYRF is absent from eye tissue during early development, we established a role for this transcription factor in the development of the retinal pigment epithelium and retina. We showed that MYRF interacts with and regulates expression of another membrane protein, TMEM98, which has been implicated in nanophthalmos. Our study establishes MYRF as a new disease gene for nanophthalmos and a regulator of eye development.

Elevated intraocular pressure (IOP) is an important risk factor in developing glaucoma, and variability in IOP might herald glaucomatous development or progression. We report the results of a genome-wide association study meta-analysis of 18 population cohorts from the International Glaucoma Genetics Consortium (IGGC), comprising 35,296 multi-ancestry participants for IOP. We confirm genetic association of known loci for IOP and primary open-angle glaucoma (POAG) and identify four new IOP-associated loci located on chromosome 3q25.31 within the FNDC3B gene (P = 4.19 x 10(-8) for rs6445055), two on chromosome 9 (P = 2.80 x 10(-11) for rs2472493 near ABCA1 and P = 6.39 x 10(-11) for rs8176693 within ABO) and one on chromosome 11p11.2 (best P = 1.04 x 10(-11) for rs747782). Separate meta-analyses of 4 independent POAG cohorts, totaling 4,284 cases and 95,560 controls, showed that 3 of these loci for IOP were also associated with POAG.

Genome-wide association studies (GWASs) have identified loci for erythrocyte traits in primarily European ancestry populations. We conducted GWAS meta-analyses of six erythrocyte traits in 71,638 individuals from European, East Asian, and African ancestries using a Bayesian approach to account for heterogeneity in allelic effects and variation in the structure of linkage disequilibrium between ethnicities. We identified seven loci for erythrocyte traits including a locus (RBPMS/GTF2E2) associated with mean corpuscular hemoglobin and mean corpuscular volume. Statistical fine-mapping at this locus pointed to RBPMS at this locus and excluded nearby GTF2E2. Using zebrafish morpholino to evaluate loss of function, we observed a strong invivo erythropoietic effect for RBPMS but not for GTF2E2, supporting the statistical fine-mapping at this locus and demonstrating that RBPMS is a regulator of erythropoiesis. Our findings show the utility of trans-ethnic GWASs for discovery and characterization of genetic loci influencing hematologic traits.

The genetic basis for combined pituitary hormone deficiency (CPHD) is complex, involving 30 genes in a variety of syndromic and nonsyndromic presentations. Molecular diagnosis of this disorder is valuable for predicting disease progression, avoiding unnecessary surgery, and family planning. Weexpect that the application of high throughput sequencing will uncover additional contributing genes and eventually become a valuable tool for molecular diagnosis. For example, in the last 3 years, six new genes have been implicated in CPHD using whole-exome sequencing. In this review, we present a historical perspective on gene discovery for CPHD and predict approaches that may facilitate future gene identification projects conducted by clinicians and basic scientists. Guidelines for systematic reporting of genetic variants and assigning causality are emerging. We apply these guidelines retrospectively to reports of the genetic basis of CPHD and summarize modes of inheritance and penetrance for each of the known genes. In recent years, there have been great improvements in databases of genetic information for diverse populations. Some issues remain that make molecular diagnosis challenging in some cases. These include the inherent genetic complexity of this disorder, technical challenges like uneven coverage, differing results from variant calling and interpretation pipelines, the number of tolerated genetic alterations, and imperfect methods for predicting pathogenicity.Wediscuss approaches for future research in the genetics of CPHD. Fil: Fang, Qing. University of Michigan; Estados Unidos Fil: George, Akima S.. University of Michigan; Estados Unidos Fil: Brinkmeier, Michelle L.. University of Michigan; Estados Unidos Fil: Mortensen, Amanda H.. University of Michigan; Estados Unidos Fil: Gergics, Peter. University of Michigan; Estados Unidos Fil: Cheung, Leonard Y.M.. University of Michigan; Estados Unidos Fil: Daly, Alexandre Z.. University of Michigan; Estados Unidos Fil: Ajmal, Adnan. University of Michigan; Estados Unidos Fil: Pérez Millán, María Inés. University of Michigan; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Biomédicas; Argentina Fil: Bilge Ozel, A.. University of Michigan; Estados Unidos Fil: Kitzman, Jacob. University of Michigan; Estados Unidos Fil: Mills, Ryan E.. University of Michigan; Estados Unidos Fil: Li, Jun Z.. University of Michigan; Estados Unidos Fil: Camper, Sally. University of Michigan; Estados Unidos

The design of microarrays is currently based on studies focusing on DNA hybridization reaction in bulk solution. However, the presence of a surface to which the probe strand is attached can make the solution-based approximations invalid, resulting in sub-optimum hybridization conditions. To determine the effect of surfaces on DNA duplex formation, the authors studied the dependence of DNA melting temperature (T(m)) on target concentration. An automated system was developed to capture the melting profiles of a 25-mer perfect-match probe-target pair initially hybridized at 23°C. Target concentrations ranged from 0.0165 to 15 nM with different probe amounts (0.03-0.82 pmol on a surface area of 10(18) Å(2)), a constant probe density (5 × 10(12) molecules/cm(2)) and spacer length (15 dT). The authors found that T(m) for duplexes anchored to a surface is lower than in-solution, and this difference increases with increasing target concentration. In a representative set, a target concentration increase from 0.5 to 15 nM with 0.82 pmol of probe on the surface resulted in a T(m) decrease of 6°C when compared with a 4°C increase in solution. At very low target concentrations, a multi-melting process was observed in low temperature domains of the curves. This was attributed to the presence of truncated or mismatch probes.

UNLABELLED Essentials Variants at ABO, von Willebrand Factor (VWF) and 2q12 contribute to the variation in plasma in VWF. We performed a genome-wide association study of plasma VWF propeptide in 3,238 individuals. ABO, VWF and 2q12 loci had weak or no association or linkage with plasma VWFpp levels. VWF associated variants at ABO, VWF and 2q12 loci primarily affect VWF clearance rates. SUMMARY Background Previous studies identified common variants at the ABO and VWF loci and unknown variants in a chromosome 2q12 linkage interval that contributed to the variation in plasma von Willebrand factor (VWF) levels. Whereas the association with ABO haplotypes can be explained by differential VWF clearance, little is known about the mechanisms underlying the association with VWF single-nucleotide polymorphisms (SNPs) or with variants in the chromosome 2 linkage interval. VWF propeptide (VWFpp) and mature VWF are encoded by the VWF gene and secreted at the same rate, but have different plasma half-lives. Therefore, comparison of VWFpp and VWF association signals can be used to assess whether the variants are primarily affecting synthesis/secretion or clearance. Methods We measured plasma VWFpp levels and performed genome-wide linkage and association studies in 3238 young and healthy individuals for whom VWF levels had been analyzed previously. Results and conclusions Common variants in an intergenic region on chromosome 7q11 were associated with VWFpp levels. We found that ABO serotype-specific SNPs were associated with VWFpp levels in the same direction as for VWF, but with a much lower effect size. Neither the association at VWF nor the linkage on chromosome 2 previously reported for VWF was observed for VWFpp. Taken together, these results suggest that the major genetic factors affecting plasma VWF levels, i.e. variants at ABO, VWF and a locus on chromosome 2, operate primarily through their effects on VWF clearance.

Polydimethyl siloxane (PDMS)-glass microchip has a very strong surface effect on polymerase chain reaction (PCR), leading to a very poor PCR yield. In the work reported here, practical dynamic passivation of surfaces of PDMS-glass microchip using polyethylene glycol (PEG) or polyvinylpyrrolidone (PVP) was achieved using a conventional thermocycler. The passivation procedure was cost-effective and easy to conduct. The effects of polymer molecular weight and polymer concentration on tube PCR efficiency were investigated primarily to prescreen out suitable polymers and polymer concentrations in the PCR mixture. The result from tube PCR indicated that both PEG and PVP could affect the performance of Taq polymerase. A final concentration of 0.025% (w/v) or 0.4% (w/v) polymer in the PCR mixture can enhance the tube PCR, while 1% (w/v) polymer was found to inhibit the reaction. PEG was more effective in tube PCR, although PVP performed better in chip PCR. Instead of employing the polymer directly in the PCR mixture, i.e. the conventional in situ passivation approach, another approach of dynamic passivation by pre-injecting polymers into the microchip achieved better performance. The efficiency of pre-passivation was found to follow the order: PVP10000>PVP55000, PEG8000> PEG10000>PEG400. After pre-passivation with PVP10000, PVP55000 and PEG8000, the PCR efficiency can recover to 93%, 86% and 83%, respectively, of that obtained from tube PCR.  2006 Society of Chemical Industry

Plasminogen is the precursor of the serine protease plasmin, a central enzyme of the fibrinolytic system. Plasma levels of plasminogen vary by almost 2-fold among healthy individuals, yet little is known about its heritability or genetic determinants in the general population. In order to identify genetic factors affecting the natural variation of plasminogen levels, we performed a genome-wide association study and linkage analysis in a sample of 3456 young healthy individuals who participated in the Genes and Blood Clotting Study (GABC) or the Trinity Student Study (TSS). Heritability of plasminogen levels was 48.1% to 60.0%. Tobacco smoking and female sex were associated with higher levels of plasminogen. In the meta-analysis, 11 single-nucleotide polymorphisms (SNPs) in 2 regions reached genome-wide significance (P < 5.0E-8). Of these, 9 SNPs were near the PLG or LPA genes on Chr6q26, whereas 2 were on Chr19q13 and 5′ upstream of SIGLEC14. These 11 SNPs represented 4 independent signals and collectively explained 6.8% of plasminogen level variation in the study populations. The strongest association was observed for a nonsynonymous SNP in the PLG gene (R523W). Individuals bearing an additional copy of this allele had an average decrease of 13.4% in plasma plasminogen level.

Elevated intraocular pressure (IOP) is a major risk factor for glaucoma and is influenced by genetic and environmental factors. Recent genome-wide association studies (GWAS) reported associations with IOP at TMCO1 and GAS7, and with primary open-angle glaucoma (POAG) at CDKN2B-AS1, CAV1/CAV2, and SIX1/SIX6. To identify novel genetic variants and replicate the published findings, we performed GWAS and meta-analysis of IOP in >6,000 subjects of European ancestry collected in three datasets: the NEI Glaucoma Human genetics collaBORation, GLAUcoma Genes and ENvironment study, and a subset of the Age-related Macular Degeneration-Michigan, Mayo, AREDS and Pennsylvania study. While no signal achieved genome-wide significance in individual datasets, a meta-analysis identified significant associations with IOP at TMCO1 (rs7518099-G, p = 8.0 × 10−8). Focused analyses of five loci previously reported for IOP and/or POAG, i.e., TMCO1, CDKN2B-AS1, GAS7, CAV1/CAV2, and SIX1/SIX6, revealed associations with IOP that were largely consistent across our three datasets, and replicated the previously reported associations in both effect size and direction. These results confirm the involvement of common variants in multiple genomic regions in regulating IOP and/or glaucoma risk.

The author manuscript of this article is open access and is freely available online at PubMed Central, Central corneal thickness (CCT) is associated with eye conditions including keratoconus and glaucoma. We performed a meta-analysis on >20,000 individuals in European and Asian populations that identified 16 new loci associated with CCT at genome-wide significance (P < 5 × 10(-8)). We further showed that 2 CCT-associated loci, FOXO1 and FNDC3B, conferred relatively large risks for keratoconus in 2 cohorts with 874 cases and 6,085 controls (rs2721051 near FOXO1 had odds ratio (OR) = 1.62, 95% confidence interval (CI) = 1.4-1.88, P = 2.7 × 10(-10), and rs4894535 in FNDC3B had OR = 1.47, 95% CI = 1.29-1.68, P = 4.9 × 10(-9)). FNDC3B was also associated with primary open-angle glaucoma (P = 5.6 × 10(-4); tested in 3 cohorts with 2,979 cases and 7,399 controls). Further analyses implicate the collagen and extracellular matrix pathways in the regulation of CCT.

Deep vein thrombosis and pulmonary embolism, collectively referred to as venous thromboembolism (VTE), are the third leading cause of cardiovascular death in the United States. Genetic factors account for 50-60% of VTE risk and a recent meta-analysis of genome-wide association studies confirmed that common variants in F5, ABO, and seven other loci are associated with VTE. Rare mutations in the anticoagulant genes PROC, PROS1 and SERPINC1 have been linked to VTE in family studies. In order to identify new genetic variants altering the risk for VTE, we performed whole exome sequencing (WES) in 373 unrelated individuals of European ancestry with unprovoked VTE and compared results to a previously sequenced control cohort of 5784 unrelated Europeans. To avoid variant calling bias, only SNVs from exons with >10X coverage and less than 5% difference in coverage between cases and controls were included, removing 11,813 of 188,689 intervals. We used an emerging framework for a "collapsing" analysis on genes, defining qualifying variants on the basis of annotation and minor allele frequency Disclosures Ginsburg: Shire: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties: recombinant VWF and recombinant ADAMTS13; Portola Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees.

The plasma glycoprotein von Willebrand factor (VWF) exhibits fivefold antigen level variation across the normal human population determined by both genetic and environmental factors. Low levels of VWF are associated with bleeding and elevated levels with increased risk for thrombosis, myocardial infarction, and stroke. To identify additional genetic determinants of VWF antigen levels and to minimize the impact of age and illness-related environmental factors, we performed genome-wide association analysis in two young and healthy cohorts ( n = 1,152 and n = 2,310) and identified signals at ABO ( P < 7.9E-139) and VWF ( P < 5.5E-16), consistent with previous reports. Additionally, linkage analysis based on sibling structure within the cohorts, identified significant signals at chromosome 2q12–2p13 (LOD score 5.3) and at the ABO locus on chromosome 9q34 (LOD score 2.9) that explained 19.2% and 24.5% of the variance in VWF levels, respectively. Given its strong effect, the linkage region on chromosome 2 could harbor a potentially important determinant of bleeding and thrombosis risk. The absence of a chromosome 2 association signal in this or previous association studies suggests a causative gene harboring many genetic variants that are individually rare, but in aggregate common. These results raise the possibility that similar loci could explain a significant portion of the “missing heritability” for other complex genetic traits.

Background: ABO(H) is a carbohydrate blood group system expressed in multiple tissues including red blood cells, blood vessels, and mucosal surfaces. ABO is the largest known genetic modifier of plasma VWF level (VWF:Ag). It has been hypothesized that the effect of ABO on VWF is mediated by H glycan density. FUT2, the gene underlying Secretor phenotype, encodes a glycosyltransferase synthesizing H antigen in mucosal tissues, and variation in the gene has also previously been associated with VWF:Ag, but past studies have been conflicting. To clarify these relationships, we studied the relationship between VWF:Ag, ABH glycans, and FUT2 genotype. Methods: The primary study group was a representative cohort of US blood donors from the Retrovirus Epidemiology Donor Study (REDS, N=499). A validation cohort of unrelated individuals was created from the Genes and Blood Clotting Study (GABC), healthy siblings between ages 14 and 35 years from University of Michigan, Ann Arbor, by choosing a random individual from each family (N=488). VWF:Ag was determined by ELISA in platelet poor plasma. Forensic techniques were adapted to detect ABH glycans in whole blood (REDS) or RBC-rich, frozen buffy coat (GABC). A and B glycans were detected using anti-A or anti-B (Immucor). A biotinylated Ulex europaeus agglutinin (UEA lectin, Vector Labs) was used to detect H. Relative A, B, and H antigen density was quantified on dot blots with ImageQuant (GE). In REDS, functional FUT2 alleles (Secretor) were determined by Sanger sequencing of FUT2 exon 2. FUT2 copy-number was assayed with real-time quantitative PCR. In GABC, FUT2 genotypes were determined through SNP genotyping (Illumina). In REDS, genotype data was phased (BEAGLE) to identify functional haplotypes. In GABC, functional alleles were inferred from the genotypes of two SNPs (rs601338, rs1047781) that determined secretor status in nearly all samples in REDS (see below). Multivariate regression was applied to VWF:Ag as a function H antigen density and O vs. non-O blood group, both separately and within the same model. All models were adjusted for age, gender, and self-reported ethnicity. Results: ABO blood group frequencies in both cohorts were similar to that of the US population. VWF:Ag differed significantly between ABO blood groups with lower values in blood group O versus non-O (REDS: ratio = 0.75, 95% CI [0.71, 0.80], p FUT2 sequencing in the REDS cohort identified 22 distinct FUT2 haplotypes. 99.5% (497/499) of individuals could be accurately assigned FUT2 genotypes based on the common W154* (rs601338) polymorphism alone. In GABC, this decreased to 94% (476/488) due to higher frequency of a hypomorphic allele (rs1047781) more commonly found in Asian individuals. In REDS, quantitative PCR did not identify copy-number variation at FUT2. There was no significant association between FUT2 genotype-predicted function and VWF:Ag or H glycan density in either cohort. Conclusion: H glycan density correlated with VWF:Ag in both cohorts. H glycan density could mediate the association between ABO blood group and VWF:Ag. If so, this suggests a non-linear, complex relationship between H and VWF and a separate ABO mechanism cannot be excluded. In both cohorts, there was no association of FUT2 (Secretor) genotype with VWF. Our data suggest prior focused approaches to FUT2 genotyping are at risk to undercall non-functional alleles, particularly in cohorts with non-European ancestries. Taken together, these indicate blood group H may be a significant modifier of VWF:Ag, and that this effect is not due to the influence of Secretor phenotype. Disclosures Johnsen: Octapharma: Other: Speaker; Biogen: Research Funding; CSL Behring: Other: Speaker.

Homeostatic processing of von Willebrand factor (VWF) in the circulation relies on proteolysis of ultra-large VWF (ULVWF) multimers by the metalloprotease ADAMTS13. Deficiency in ADAMTS13 results in Thrombotic Thrombocytopenic Purpura (TTP) due to the accumulation of thrombogenic ULVWF. ADAMTS13 is secreted into the circulation in its active form and has no known inhibitor. Regulation of ADAMTS13 activity occurs in part through plasma ADAMTS13 levels and by the hemodynamic exposure of the scissile bond in VWF. To identify the genetic determinants of ADAMTS13 levels we performed genome-wide association studies (GWAS) and linkage analysis in two independent cohorts of young healthy individuals. This study recruited 3206 young healthy individuals of European descent who participated in the Genes and Blood Clotting Study (GABC, n=940) and the Trinity Student Study (TSS, n=2266). All subjects provided written informed consent and all genotyping and phenotyping was performed on de-identified samples. Details of the genotyping and data cleaning process for these cohorts were previously described. (Desch KC et al. PNAS 2013, 110:588-593) ADAMTS13 levels were measured by a custom AlphaLISA (Perkin-Elmer, Waltham, MA) assay using polyclonal anti-ADAMTS13 antibodies (a gift from X. Long Zheng, University of Pennsylvania). In GABC, the median ADAMTS13 levels were 115.4 IU/dL and demonstrated a 2.0-fold variation between the 5th and 95th percentiles. Heritability of ADAMTS13 levels was estimated at 77% in GABC and 43% in the TSS. ADAMTS13 levels were associated smoking in both cohorts and was associated with an 8.9% increase in levels of ADAMTS13 (P < 1.5E-11, GABC). GWAS identified 10 SNPs in GABC and 33 SNPs in TSS that were significantly associated with ADAMTS13 levels (P Although ABO mediated glycosylation patterns affect VWF clearance rates, no signal in ABO was detected in our multiple association studies suggesting that the common ABO haplotypes are not associated with ADAMTS13 levels. Although other mutations tagged by the significantly associated SNPs cannot be excluded, a potential candidate mutation (rs2301612, Q448E) was identified in ADAMTS13 and may be responsible for the decrease in ADAMTS13 levels through alteration of synthesis/secretion or plasma clearance rates. Aditionally, linkage analysis confirmed the chromosome 9q34 signal and identified a region on chromosome 5p13 that was not detected by GWAS. This finding suggests that rare variants in this genomic region also contribute to ADAMTS13 levels. The identification of mutations that determine normal ADAMTS13 variation may lead to a better understanding of the genetic risk for TTP and mechanisms of von Willebrand Factor homeostasis. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

The conversion of plasminogen (PLG) to plasmin is a critical step in the activation of the fibrinolytic system. Due to its broad substrate specificity, plasmin plays a pleotropic role in the circulation and extravascular space, controlling the degradation of fibrin clots as well as potential functions in regulating cell migration and angiogenesis. PLG activation is regulated through the interaction of tPA or uPA with plasminogen activator inhibitor type one (PAI-1). Genome-wide association studies (GWAS) have identified polymorphisms associated with the plasma levels of tPA and PAI-1, but to date, no studies have directly examined the control of PLG levels. In order to identify genetic determinants of PLG levels we performed GWAS in a cohort of 3244 young healthy individuals (557 sibships) who participated in the Genes and Blood Clotting Study (GABC) or the Trinity Student Study (TSS).(Desch KC et al. PNAS 2013, 110:588-593) All subjects provided written informed consent. Details of the genotyping and data cleaning process for these cohorts are as previously described. (Desch KC et al. PNAS 2013, 110:588-593) PLG levels were measured in both cohorts by AlphaLISA (Perkin-Elmer, Waltham, MA). Levels were natural log transformed and adjusted for participant age, gender and BMI (TSS only) prior to use in heritability estimates and GWAS, which were performed using the PLINK, EMMAX, MERLIN, GCTA and METAL software packages. The median PLG levels were 101.7 IU/dL and 104.7 IU/dL in the GABC and TSS cohorts respectively and had a 1.7 fold variation between the 5th and 95th percentiles. The narrow-sense heritability using intra-class correlation was 59.7% based on 1277 siblings in GABC and TSS, which was similar to the estimate of 60.2% based on the genotype data and the MERLIN software package. GWAS for PLG in the European only subset of GABC (n=940) did not reveal any genome-wide significant signals. However, GWAS for PLG levels in TSS (n=2304) revealed 9 significant SNPs on chromosome 6q26 at the PLG (plasminogen) and LPA (lipoprotein A) locus that together explained 7.8% of the variance in PLG levels according to the GCTA method, Figure 1. The top SNP was rs4252129 (p = 3.47E-25) which results in a single amino acid substitution (R523W) in PLG. The next strongest associations were with two SNPs near LPA, rs1084651 and rs783149 (p = 1.60E-13 and 1.07E-12 respectively). These SNPs are located between LPA and PLG (Figure 1). To determine if the LPA SNPs were independent from the top PLG SNP, a conditional analysis was performed using rs4252129 as a covariate. Rs1084651 and rs783149 remained significant (p= 7.73E-16 and 6.48E-15 respectively), verifying that they represent a second, independent association signal. Meta-analysis of both cohorts (using 723,715 common SNPs) confirmed the 6q26 signal and revealed another significantly associated region on 19q13.41, Figure 2. Two SNPs, rs10412972 and rs11084102 (p = 2.16E-09 and 1.14E-08, respectively) are intergenic variants 4.8 and 12.6 Kb downstream from SIGLEC14. The significant SNPs in the meta-analysis, 6 on 6q26 and 2 on 19q13.41, explained 5.8% of the variance for PLG. The top SNP in the TSS GWAS, rs4252129, had a minor allele frequency (MAF) of 1.4% in GABC and was not included in the GABC GWAS or meta-analysis. In summary, our data identify two loci (at PLG/LPA and SIGLEC14) that harbor genetic variants contributing to the variation of plasma PLG levels in healthy young adults. The strongest association was observed for a non-synonymous SNP in the PLG gene itself, with a MAF of 1.3% in the HapMap CEU population and 2.2% in TSS. The minor allele (W523) is associated with a 14.1% decrease in mean plasma PLG level. This low frequency, non-synonymous SNP, or another SNP in LD with it, may be the functional mutation leading to decreased PLG levels through unknown mechanisms. Whether the significant SNPs near LPA affect lipoprotein A or PLG is not clear. However, the association with causal variants at LPA is possible and consistent with previous biochemical studies demonstrating competitive inhibition of PLG binding to endothelial surfaces by lipoprotein A, or an effect of lipoprotein A on endothelial PAI-1 expression or the PAI-1 interaction with tPA/uPA. Finally, these studies identified two significant SNPs near SIGLEC14. This gene encodes a sialic acid lectin type receptor protein, closely related to SIGLEC 5 recently implicated in the clearance of von Willebrand factor. Disclosures: Ginsburg: Shire plc: Membership on an entity’s Board of Directors or advisory committees; Portola Pharmaeuticals: Membership on an entity’s Board of Directors or advisory committees; Catalyst Biosciences: Membership on an entity’s Board of Directors or advisory committees; Merck pharmaceuticals: Consultancy; Boston Children's Hospital: From Baxter to Children's Hosp (VWF), From Baxter to Children's Hosp (VWF) Patents & Royalties; University of Michigan: From Baxter to University of Michigan (ADAMTS13) Patents & Royalties.

von Willebrand disease (VWD) type 1 is characterized by incomplete penetrance and variable expressivity. Individuals with very low levels of VWF (< 20 IU/dl) typically have mutations in the VWF gene. However, no such mutations can be identified in a significant subset of type 1 VWD patients, particularly those with VWF levels > 30 IU/dL. ABO is the best-characterized genetic modifier of VWF levels, accounting for up to 30% of the genetic variability and blood type O is overrepresented in patients with VWD type 1. VWF is highly polymorphic with over 1,100 reported single nucleotide variants (SNVs). The impact of many of these changes remains unknown. We hypothesize that common variants in VWF affect VWF levels and may play a role in the diagnosis of VWD type 1. We genotyped 94 tagging SNPs through VWF in a large Amish family (n=445) and study their association with VWF levels. We performed single SNP association tests using age, gender and ABO status as covariates and multiple testing corrections were implemented. We observed significant associations (p value ≤ 0.0005) between VWF levels and twelve tagging SNPs: rs2362479 (telomeric to VWF), rs10849363 and rs7964554 (intron 4); rs216308 (intron 24); rs216310 and rs216311 (exon 28); rs11612370 and rs11612384 (intron 33); rs216330, rs11064003, and rs2239162 (intron 37); and rs216293 (intron 38). To replicate the association of these SNPs with VWF levels in a larger Caucasian population, we conducted association analyses in three population-based cohorts of healthy individuals: 1) the Zimmerman Program for Molecular and Clinical Biology of VWD (ZPMCB-VWD) control cohort (n=150), 2) the Genes and Blood-Clotting Study (GABC) cohort (n=934) and 3) the Trinity Student Study (TSS) cohort (n=2,145). All association analyses were modeled including age, gender, rs687289 (a proxy marker of blood group O) and adjusted for multiple testing. Three SNP associations were replicated in the ZPMCB-VWD control cohort. An additional four SNP associations were replicated in the ZPMCB-VWD control and TSS cohorts. Associations of the intron 37 variants rs11064003 and rs2239162 with VWF levels were replicated in all cohorts and reached genome wide significance with p values of 1.34 x 10 -11 and 7.35 x 10 -15 respectively in the TSS cohort. We then genotyped the 12 candidate SNPs that showed significance in the original family in a cohort of 310 Caucasian VWD type 1 index cases (ZPMCB-VWD). We compared the minor allele frequency (MAF) of the tagging SNPs between the ZPMCB-VWD control cohort (n=150) and the ZPMCB-VWD type 1 index case cohort (n=310). The minor allele of rs7964554 (associated with lower VWF levels) was more common (46.2%) in patients with VWD type 1 than in controls (34.0%), and that difference was statistically significant (p=0.0005). The minor alleles of rs11612370, rs11612384, rs11064003, and rs2239162 (all associated with higher VWF levels) were less common (p=0.018, 0.009, 0.012, and 0.0004, respectively) in VWD type 1 index cases compared to controls. In this population if the subject has VWD type 1, then the odds of being heterozygous for the minor allele of rs7964554 were 1.47 (95% CI: 1.08, 2.00; p=0.014). Similarly, the odds of being heterozygous for the minor allele of rs2239162 were 0.64 (95% CI: 0.45, 0.91; p=0.014). In summary we have identified several common variants in VWF that have a strong effect on VWF levels and more importantly are either under or over represented in patients with VWD type 1 indicating that may influence its diagnosis. Disclosures: Shapiro: Baxter: Consultancy, Global steering committees Other, Membership on an entity’s Board of Directors or advisory committees, Research Funding, Speakers Bureau; Novo Nordisk: Consultancy, Membership on an entity’s Board of Directors or advisory committees, Research Funding, Speakers Bureau; Bayer: Global steering committees, Global steering committees Other, Membership on an entity’s Board of Directors or advisory committees, Research Funding; Inspiration: Consultancy, Membership on an entity’s Board of Directors or advisory committees, Research Funding; CSL Behring: Research Funding; Biogen Idec: Research Funding. Di Paola:CSL Behring: Consultancy; Pfizer: DSMB, DSMB Other.

Abstract 274 The ABO(H) carbohydrate blood group system is highly polymorphic in humans, with >200 ABO alleles described to date. The ABO gene encodes the glycosyltransferase, ABO, which has two main isoforms which generate blood group A or blood group B. Blood group O is the result of a non-functional ABO glycosyltransferase. ABO blood group is commonly imputed in genome-wide association studies where ABO is associated with phenotypes such as von Willebrand Factor level or myocardial infarction. We sought to determine the accuracy of a common ABO genotyping algorithm in a repository of 1092 individuals from a healthy sibling cohort study, the Genes and Blood Clotting Cohort (GABC). We utilized three published single nucleotide variants (SNVs) to assign ABO blood group (A2, B, and O) relative to the A1 reference allele: rs8176704 (G/A) to annotate A2, rs8176749 (C/T) to annotate B, and rs687289 (A/G) to annotate O. We detected ABO glycans in red blood cell-rich buffy coat samples using an adaptation of published ABO forensic dried blood spot techniques. Briefly, samples were diluted and applied to a nitrocellulose membrane, blotted with murine monoclonal anti-A (Immucor) or murine monoclonal anti-B (Immucor) antibodies followed by and streptavidin-conjugated donkey anti-mouse IgG HRP secondary (Jackson ImmunoResearch). Blots were developed, imaged using ImageQuant 350 (GE), and scored semi-quantitatively by two blinded independent observers relative to red blood cell-rich buffy coat reference panels from normal blood donors. Comparison of ABO imputed genotype to detected ABO glycans in red blood cell-rich buffy coat identified discrepancies in the predicted major ABO blood group assignment (i.e. blood group O, A, B, or AB) in 4.4% of individuals. Interestingly, A glycan density was generally low in individuals with a discrepant A phenotype, suggesting that these individuals harbor a less robust blood group A enzyme variant. There was no common pattern for genotype-phenotype discrepancies. Multiple types of errors (both presence and absence of ABO glycans) were observed, and divergent patterns of ABO glycans were observed between homozygotes for ABO SNVs, suggesting multiple ABO alleles or other genetic modifiers underlie the discordant calls. Interrogation of the Exome Variant Server (http://evs.gs.washington.edu/EVS/) found one-third of ABO nonsynonymous polymorphisms in the exome data set are novel, indicating a large proportion of the genetic variation at ABO has not been previously described. Furthermore, the majority of nonsynonymous variants in the EVS database for the genes encoding H antigen (the ABO acceptor structure), FUT1 and FUT2, were also novel. In summary, ABO(H) is more variable genetically than previously suspected. A commonly applied ABO SNV genotyping algorithm resulted in 4.4% frequency of major ABO blood group glycan discrepancy as detected in red blood cell-rich buffy coats, likely due in large part to the underlying genetic diversity at ABO. Further work is needed to deeply characterize the genetic diversity at ABO, FUT1, and FUT2 and correlate the spectrum of allelic variants with ABO(H) carbohydrate phenotypes. Such high resolution ABO and H classification would likely advance our understanding of the complex physiologic processes influenced by ABH blood groups, including disorders of hemostasis and thrombosis, vascular disease, infectious disease susceptibility, complications of pregnancy, and allogeneic exposures. Disclosures: Ginsburg: Shire Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Portola Pharmaceuticals: Consultancy; Catalyst Biosciences: Consultancy; Baxter Pharmaceuticals: benefit from payments to Children's Hosptial, Boston, and the University of Michigan Patents & Royalties; Merck Pharmaceuticals: Consultancy.

Abstract 3313 Plasma VWF levels vary by approximately five-fold in healthy populations and are influenced by both environmental and inherited factors. Low levels of VWF are associated with bleeding and elevated levels with increased risk for thrombosis. Increased levels of VWF occur with age, may rise acutely due to inflammation or infection, and may serve as a biomarker for endothelial dysfunction and atherosclerosis. Genetic factors are thought to account for 60 – 70% of the variance in VWF antigen levels with ∼30% of this effect due to ABO blood type. VWF multimers are assembled in the Golgi apparatus where the VWF propeptide is cleaved to form mature VWF. Because the half life of plasma VWF propeptide (ppVWF) is ∼5X faster than the mature VWF antigen, the ratio of ppVWF/VWF:Ag in plasma primarily reflects clearance rates of mature VWF while alterations in biosynthesis and/or secretion of VWF affect the level of both ppVWF and VWF:Ag. In order to identify additional quantitative trait loci (QTL) for VWF antigen variation and altered clearance of VWF:Ag, we performed genetic analyses in a healthy young sibling cohort, the Genes and Blood Clotting Study (GABC, n=1,152). The cohort was SNP genotyped with an Illumina Omni-1 array resulting in 763,195 SNPs available for analysis after extensive QC. Plasma VWF antigen (VWF:Ag) and VWF propeptide levels were determined by AlphaLISA (Perkin Elmer, Waltham, MA), using polyclonal anti-human VWF antibodies (DAKO, Glostrup, Denmark) or monoclonal anti-ppVWF antibodies (Blood Center of Wisconsin, Milwaukee, WI). The median levels were 108.1 IU/dL, 99.97 IU/dL and 1.0 for VWF:Ag, ppVWF and ppVWF/VWF:Ag respectively. The estimated heritability (h2) was 64.5%, 55.72%, and 64.26% for VWF:Ag, ppVWF, and ppVWF/VWF:Ag respectively. A genome-wide association study (GWAS) for VWF:Ag in this cohort revealed significant signals at the ABO and VWF loci, consistent with previously published reports. However, GWAS for ppVWF levels demonstrated a signal only at 12p13, overlying the VWF gene itself. The top SNP, rs1800378 (beta 4.297, p-value 1.58E–11), encodes a nonsynonymous substitution (His484Arg) in the VWF propeptide. In contrast, GWAS for ppVWF/VWF:Ag showed a strong signal at 9q34, the ABO locus, (top SNP rs687289, beta −0.2131, p-value 1.88E–62) and no significant signal at 12p13. These results suggest that ABO is the major common determinant of variability in VWF clearance rates and a common SNP, rs1800378, is associated with VWF propeptide levels. We next utilized the sibling structure in this cohort to perform linkage analyses. Linkage analysis for VWF:Ag identified a novel locus at 2p12-2q13 in addition to the expected signal at ABO. The effect size of this chromosome 2 locus on VWF variation (19.2% variance explained) was comparable to the effect of the ABO locus (24.5%). Linkage analysis for ppVWF demonstrated novel loci at 7q21 (LOD score 5.03) and at 11q21 (LOD score 4.12), with no significant signal on chromosome 2. In contrast, linkage analysis for ppVWF/VWF:Ag identified signals at 2p12 (LOD score 3.17), 8p22 (LOD score 3.48) and 11q22 (LOD score 3.41). Taken together, these results identify a number of potential candidate loci for genetic regulation of plasma VWF through alteration of synthesis, storage, secretion or clearance mechanisms. We hypothesize that signals present in linkage but not association are due to VWF regulatory genes with high allelic heterogeneity, harboring many genetic variants that are individually rare, but in aggregate present in a large portion of sibships. Segregation of these variants among sibs could then explain the strong signal by linkage, but lack of signal by association in unrelated individuals. Replication of the strong 2p12-2q13 signal in both VWF:Ag and ppVWF/VWF:Ag linkage studies suggests that the responsible variants at this locus affect VWF levels through altered clearance. The novel QTLs for ppVWF/VWF:Ag levels on chromosome 8p22 and 11q22 are also likely to work through altered clearance mechanisms while the novel signals for ppVWF levels on 7q21 and 11q21, not significant in the VWF:Ag linkage study, may affect efficiency of cleavage or clearance of ppVWF. Identification of the underlying genes at these novel loci and their mechanism of action could lead to improved understanding of the severity, penetrance and treatment for von Willebrand Disease as well as the risk for venous thromboembolic disease associated with elevated VWF. Disclosures: Ginsburg: Shire Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Portola Pharmaceuticals: Consultancy; Catalyst Biosciences: Consultancy; Baxter Pharmaceuticals: benefit from payments to Children's Hosptial, Boston, and the University of Michigan Patents & Royalties; Merck Pharmaceuticals: Consultancy.

PURPOSE. To investigate the effects of central corneal thickness (CCT)-associated variants on primary open-angle glaucoma (POAG) risk using single nucleotide polymorphisms (SNP) data from the Glaucoma Genes and Environment (GLAUGEN) and National Eye Institute (NEI) Glaucoma Human Genetics Collaboration (NEIGHBOR) consortia. METHODS. A replication analysis of previously reported CCT SNPs was performed in a CCT dataset (n ¼ 1117) and these SNPs were then tested for association with POAG using a larger POAG dataset (n ¼6470). Then a CCT genome-wide association study (GWAS) was performed. Top SNPs from this analysis were selected and tested for association with POAG. cDNA libraries from fetal and adult brain and ocular tissue samples were generated and used for candidate gene expression analysis. RESULTS. Association with one of 20 previously published CCT SNPs was replicated: rs12447690, near the ZNF469 gene (P ¼ 0.001; b ¼� 5.08 lm/allele). None of these SNPs were significantly associated with POAG. In the CCT GWAS, no SNPs reached genome-wide significance. After testing 50 candidate SNPs for association with POAG, one SNP was identified, rs7481514 within the neurotrimin (NTM) gene, that was significantly associated with POAG in a low-tension subset (P ¼ 0.00099; Odds Ratio [OR] ¼ 1.28). Additionally, SNPs in the CNTNAP4 gene showed suggestive association with POAG (top SNP ¼ rs1428758; P ¼ 0.018; OR ¼ 0.84). NTM and CNTNAP4 were shown to be expressed in ocular tissues. CONCLUSIONS. The results suggest previously reported CCT loci are not significantly associated with POAG susceptibility. By performing a quantitative analysis of CCT and a subsequent analysis of POAG, SNPs in two cell adhesion molecules, NTM and CNTNAP4, were identified and may increase POAG susceptibility in a subset of cases. (Invest Ophthalmol Vis

Abstract 2116 Plasma levels of von Willebrand Factor (VWF) are highly variable among healthy people and ~70% of this variability is heritable. Elevated levels of VWF are associated with thrombosis while low levels of VWF cause the common bleeding disorder, von Willebrand Disease (VWD). The genome-wide association study (GWAS) is a powerful tool for identifying common genetic variants contributing to complex traits, though less common variants are likely to be missed by this approach. We performed combined GWAS and linkage analysis in a cohort of healthy young college students. The study was approved by the University of Michigan IRB (IRBMED #2005-0080). 1189 healthy individuals (14-35 yrs of age) were recruited, comprising 507 sibships. VWF levels were determined by alphalisa assay (mean 109%, range 26 – 410%). VWF level heritability (h2) was 73% among siblings and 82% among monozygotic twins. Samples were de-identified and DNA was extracted from peripheral blood, quantified and genotyped using the Illumina Omni-1 quad SNP chip at the Broad Institute (MIT, Boston). 1182 individuals passed genotyping quality control with >97% call rates for 979,855 SNPs. Genotyping quality control was performed as part of the NIH's GENEVA program. GWAS on log transformed, age and sex adjusted VWF levels identified a major peak over the ABO locus (p Disclosures: No relevant conflicts of interest to declare.