Functional Characterization of Genetic Variant Effects on Expression.

Thousands of common genetic variants in the human population have been associated with disease risk and phenotypic variation by genome-wide association studies (GWAS). However, the majority of GWAS variants fall into noncoding regions of the genome, complicating our understanding of their regulatory functions, and few molecular mechanisms of GWAS variant effects have been clearly elucidated. Here, we set out to review genetic variant effects, focusing on expression quantitative trait loci (eQTLs), including their utility in interpreting GWAS variant mechanisms. We discuss the interrelated challenges and opportunities for eQTL analysis, covering determining causal variants, elucidating molecular mechanisms of action, and understanding context variability. Addressing these questions can enable better functional characterization of disease-associated loci and provide insights into fundamental biological questions of the noncoding genetic regulatory code and its control of gene expression.

Transcription factor regulation of eQTL activity across individuals and tissues.

Tens of thousands of genetic variants associated with gene expression (cis-eQTLs) have been discovered in the human population. These eQTLs are active in various tissues and contexts, but the molecular mechanisms of eQTL variability are poorly understood, hindering our understanding of genetic regulation across biological contexts. Since many eQTLs are believed to act by altering transcription factor (TF) binding affinity, we hypothesized that analyzing eQTL effect size as a function of TF level may allow discovery of mechanisms of eQTL variability. Using GTEx Consortium eQTL data from 49 tissues, we analyzed the interaction between eQTL effect size and TF level across tissues and across individuals within specific tissues and generated a list of 10,098 TF-eQTL interactions across 2,136 genes that are supported by at least two lines of evidence. These TF-eQTLs were enriched for various TF binding measures, supporting with orthogonal evidence that these eQTLs are regulated by the implicated TFs. We also found that our TF-eQTLs tend to overlap genes with gene-by-environment regulatory effects and to colocalize with GWAS loci, implying that our approach can help to elucidate mechanisms of context-specificity and trait associations. Finally, we highlight an interesting example of IKZF1 TF regulation of an APBB1IP gene eQTL that colocalizes with a GWAS signal for blood cell traits. Together, our findings provide candidate TF mechanisms for a large number of eQTLs and offer a generalizable approach for researchers to discover TF regulators of genetic variant effects in additional QTL datasets.

Modeling RNA-Binding Protein Specificity In Vivo by Precisely Registering Protein-RNA Crosslink Sites.

RNA-binding proteins (RBPs) regulate post-transcriptional gene expression by recognizing short and degenerate sequence motifs in their target transcripts, but precisely defining their binding specificity remains challenging. Crosslinking and immunoprecipitation (CLIP) allows for mapping of the exact protein-RNA crosslink sites, which frequently reside at specific positions in RBP motifs at single-nucleotide resolution. Here, we have developed a computational method, named mCross, to jointly model RBP binding specificity while precisely registering the crosslinking position in motif sites. We applied mCross to 112 RBPs using ENCODE eCLIP data and validated the reliability of the discovered motifs by genome-wide analysis of allelic binding sites. Our analyses revealed that the prototypical SR protein SRSF1 recognizes clusters of GGA half-sites in addition to its canonical GGAGGA motif. Therefore, SRSF1 regulates splicing of a much larger repertoire of transcripts than previously appreciated, including HNRNPD and HNRNPDL, which are involved in multivalent protein assemblies and phase separation.

Antithrombotic management and thrombosis rates in children post-liver transplantation: A case series and literature review.

Thrombosis is a major postoperative complication in pediatric liver transplantation. There is marked heterogeneity in prophylactic antithrombotic therapies used, without established guidelines. This review summarizes current worldwide incidence of thrombotic events and compares antithrombotic therapies in children post-liver transplant, with comparison to our institution's experience. Of the twenty-three articles with sufficient detail to compare antithrombotic regimens, the overall incidence of thrombosis ranged from 2.4% to 17.3%. Incidence of HAT ranged from 0% to 28.1%, of HVT from 0% to 4.7%, of PVT from 1.5% to 11.2%, and of IVC thrombosis from 0% to 2.8%. Re-transplantation due to thrombosis ranged from 0% to 4.8%. Prophylactic antithrombotic therapies varied between studies, and bleeding complications were infrequently reported. Since 2010, 96 children underwent 100 liver transplants at our institution with thrombosis incidence comparable to international literature (HAT 6%, PVT 5%, IVC 1%, and HVT 0%). Re-transplantation due to thrombosis occurred in 2% and major bleeding occurred in 10%. The prophylactic antithrombotic therapies used post-liver transplantation in children remain varied. Low rates of thrombosis have been reported with antiplatelet use both with and without anticoagulation. Standard definitions and consistent reporting of bleeding complications are required, in addition to thrombosis rates, so that true risk-benefit assessment of reported regimes can be understood.

Distinct epigenomic patterns are associated with haploinsufficiency and predict risk genes of developmental disorders.

Haploinsufficiency is a major mechanism of genetic risk in developmental disorders. Accurate prediction of haploinsufficient genes is essential for prioritizing and interpreting deleterious variants in genetic studies. Current methods based on mutation intolerance in population data suffer from inadequate power for genes with short transcripts. Here we show haploinsufficiency is strongly associated with epigenomic patterns, and develop a computational method (Episcore) to predict haploinsufficiency leveraging epigenomic data from a broad range of tissue and cell types by machine learning methods. Based on data from recent exome sequencing studies on developmental disorders, Episcore achieves better performance in prioritizing likely-gene-disrupting (LGD) de novo variants than current methods. We further show that Episcore is less-biased by gene size, and complementary to mutation intolerance metrics for prioritizing LGD variants. Our approach enables new applications of epigenomic data and facilitates discovery and interpretation of novel risk variants implicated in developmental disorders.

Defining Disease, Diagnosis, and Translational Medicine within a Homeostatic Perturbation Paradigm: The National Institutes of Health Undiagnosed Diseases Program Experience.

Traditionally, the use of genomic information for personalized medical decisions relies on prior discovery and validation of genotype-phenotype associations. This approach constrains care for patients presenting with undescribed problems. The National Institutes of Health (NIH) Undiagnosed Diseases Program (UDP) hypothesized that defining disease as maladaptation to an ecological niche allows delineation of a logical framework to diagnose and evaluate such patients. Herein, we present the philosophical bases, methodologies, and processes implemented by the NIH UDP. The NIH UDP incorporated use of the Human Phenotype Ontology, developed a genomic alignment strategy cognizant of parental genotypes, pursued agnostic biochemical analyses, implemented functional validation, and established virtual villages of global experts. This systematic approach provided a foundation for the diagnostic or non-diagnostic answers provided to patients and serves as a paradigm for scalable translational research.

Phenotypic evolution of UNC80 loss of function.

Failure to thrive arises as a complication of a heterogeneous group of disorders. We describe two female siblings with spastic paraplegia and global developmental delay but also, atypically for the HSPs, poor weight gain classified as failure to thrive. After extensive clinical and biochemical investigations failed to identify the etiology, we used exome sequencing to identify biallelic UNC80 mutations (NM_032504.1:c.[3983-3_3994delinsA];[2431C>T]. The paternally inherited NM_032504.1:c.3983-3_3994delinsA is predicted to encode p.Ser1328Argfs*19 and the maternally inherited NM_032504.1:c.2431C>T is predicted to encode p.Arg811*. No UNC80 mRNA was detectable in patient cultured skin fibroblasts, suggesting UNC80 loss of function by nonsense mediated mRNA decay. Further supporting the UNC80 mutations as causative of these siblings' disorder, biallelic mutations in UNC80 have recently been described among individuals with an overlapping phenotype. This report expands the disease spectrum associated with UNC80 mutations. © 2016 Wiley Periodicals, Inc.

Pharmacogenomic incidental findings in 308 families: The NIH Undiagnosed Diseases Program experience.

PURPOSE: Using single-nucleotide polymorphism (SNP) chip and exome sequence data from individuals participating in the National Institutes of Health (NIH) Undiagnosed Diseases Program (UDP), we evaluated the number and therapeutic informativeness of incidental pharmacogenetic variants. METHODS: Pharmacogenomics Knowledgebase (PharmGKB) annotated sequence variants were identified in 1,101 individuals. Medication records of participants were used to identify individuals prescribed medications with a genetic variant that might alter efficacy. RESULTS: A total of 395 sequence variants, including 19 PharmGKB 1A and 1B variants, were identified in SNP chip sequence data, and 388 variants, including 21 PharmGKB 1A and 1B variants, were identified in the exome sequence data. Nine participants had incidental pharmacogenetic variants associated with altered efficacy of a prescribed medication. CONCLUSIONS: Despite the small size of the NIH UDP patient cohort, we identified pharmacogenetic incidental findings potentially useful for guiding therapy. Consequently, groups conducting clinical genomic studies might consider reporting of pharmacogenetic incidental findings.Genet Med 18 12, 1303-1307.

Computational evaluation of exome sequence data using human and model organism phenotypes improves diagnostic efficiency.

PURPOSE: Medical diagnosis and molecular or biochemical confirmation typically rely on the knowledge of the clinician. Although this is very difficult in extremely rare diseases, we hypothesized that the recording of patient phenotypes in Human Phenotype Ontology (HPO) terms and computationally ranking putative disease-associated sequence variants improves diagnosis, particularly for patients with atypical clinical profiles. METHODS: Using simulated exomes and the National Institutes of Health Undiagnosed Diseases Program (UDP) patient cohort and associated exome sequence, we tested our hypothesis using Exomiser. Exomiser ranks candidate variants based on patient phenotype similarity to (i) known disease-gene phenotypes, (ii) model organism phenotypes of candidate orthologs, and (iii) phenotypes of protein-protein association neighbors. RESULTS: Benchmarking showed Exomiser ranked the causal variant as the top hit in 97% of known disease-gene associations and ranked the correct seeded variant in up to 87% when detectable disease-gene associations were unavailable. Using UDP data, Exomiser ranked the causative variant(s) within the top 10 variants for 11 previously diagnosed variants and achieved a diagnosis for 4 of 23 cases undiagnosed by clinical evaluation. CONCLUSION: Structured phenotyping of patients and computational analysis are effective adjuncts for diagnosing patients with genetic disorders.Genet Med 18 6, 608-617.

Bowel perforation complicating an ACTH-secreting phaeochromocytoma.

ACTH-secreting phaeochromocytoma (ASP) is a rare cause of ACTH-dependent Cushing's syndrome (CS). We report the case of a 63-year-old female presenting with CS secondary to an ASP complicated by bowel perforation. This case report highlights ASP as an uncommon but important cause of ectopic ACTH secretion (EAS). There have been 29 cases of ASP, all of which were unilateral and benign, but associated with significant complications. Patients presenting with ASP have the potential for cure with unilateral adrenalectomy. Given this promising prognosis if recognised, ASP should be considered in the diagnostic workup of ACTH-dependent CS. As this case demonstrates, gastrointestinal complications can arise from severe hypercortisolaemia associated with CS. Early medical and surgical intervention is imperative as mortality approaches 50% once bowel perforation occurs. LEARNING POINTS: Consider phaeochromocytoma in the diagnostic workup of ACTH-dependent CS; screen with plasma metanephrines or urinary catecholamines.Serial screening may be required if ACTH-secreting phaeochromocytoma is suspected, as absolute levels can be misleading.Early catecholamine receptor blockade and adrenal synthesis blockade may avoid the need for rescue bilateral adrenalectomy in ACTH-secreting phaeochromocytoma.Consider early medical or surgical management when gastrointestinal features are present in patients with CS, as bowel perforation due to severe hypercortisolaemia can occur and is associated with significant mortality.