Scalable approaches for generating, validating and incorporating data from high-throughput functional assays to improve clinical variant classification.

As the adoption and scope of genetic testing continue to expand, interpreting the clinical significance of DNA sequence variants at scale remains a formidable challenge, with a high proportion classified as variants of uncertain significance (VUSs). Genetic testing laboratories have historically relied, in part, on functional data from academic literature to support variant classification. High-throughput functional assays or multiplex assays of variant effect (MAVEs), designed to assess the effects of DNA variants on protein stability and function, represent an important and increasingly available source of evidence for variant classification, but their potential is just beginning to be realized in clinical lab settings. Here, we describe a framework for generating, validating and incorporating data from MAVEs into a semi-quantitative variant classification method applied to clinical genetic testing. Using single-cell gene expression measurements, cellular evidence models were built to assess the effects of DNA variation in 44 genes of clinical interest. This framework was also applied to models for an additional 22 genes with previously published MAVE datasets. In total, modeling data was incorporated from 24 genes into our variant classification method. These data contributed evidence for classifying 4043 observed variants in over 57,000 individuals. Genetic testing laboratories are uniquely positioned to generate, analyze, validate, and incorporate evidence from high-throughput functional data and ultimately enable the use of these data to provide definitive clinical variant classifications for more patients.

Prioritizing genes for systematic variant effect mapping.

MOTIVATION: When rare missense variants are clinically interpreted as to their pathogenicity, most are classified as variants of uncertain significance (VUS). Although functional assays can provide strong evidence for variant classification, such results are generally unavailable. Multiplexed assays of variant effect can generate experimental 'variant effect maps' that score nearly all possible missense variants in selected protein targets for their impact on protein function. However, these efforts have not always prioritized proteins for which variant effect maps would have the greatest impact on clinical variant interpretation. RESULTS: Here, we mined databases of clinically interpreted variants and applied three strategies, each building on the previous, to prioritize genes for systematic functional testing of missense variation. The strategies ranked genes (i) by the number of unique missense VUS that had been reported to ClinVar; (ii) by movability- and reappearance-weighted impact scores, to give extra weight to reappearing, movable VUS and (iii) by difficulty-adjusted impact scores, to account for the more resource-intensive nature of generating variant effect maps for longer genes. Our results could be used to guide systematic functional testing of missense variation toward greater impact on clinical variant interpretation. AVAILABILITY AND IMPLEMENTATION: Source code available at: https://github.com/rothlab/mave-gene-prioritization. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Regulatory analysis of the C. elegans genome with spatiotemporal resolution.

Discovering the structure and dynamics of transcriptional regulatory events in the genome with cellular and temporal resolution is crucial to understanding the regulatory underpinnings of development and disease. We determined the genomic distribution of binding sites for 92 transcription factors and regulatory proteins across multiple stages of Caenorhabditis elegans development by performing 241 ChIP-seq (chromatin immunoprecipitation followed by sequencing) experiments. Integration of regulatory binding and cellular-resolution expression data produced a spatiotemporally resolved metazoan transcription factor binding map. Using this map, we explore developmental regulatory circuits that encode combinatorial logic at the levels of co-binding and co-expression of transcription factors, characterizing the genomic coverage and clustering of regulatory binding, the binding preferences of, and biological processes regulated by, transcription factors, the global transcription factor co-associations and genomic subdomains that suggest shared patterns of regulation, and identifying key transcription factors and transcription factor co-associations for fate specification of individual lineages and cell types.

Comparative analysis of regulatory information and circuits across distant species.

Despite the large evolutionary distances between metazoan species, they can show remarkable commonalities in their biology, and this has helped to establish fly and worm as model organisms for human biology. Although studies of individual elements and factors have explored similarities in gene regulation, a large-scale comparative analysis of basic principles of transcriptional regulatory features is lacking. Here we map the genome-wide binding locations of 165 human, 93 worm and 52 fly transcription regulatory factors, generating a total of 1,019 data sets from diverse cell types, developmental stages, or conditions in the three species, of which 498 (48.9%) are presented here for the first time. We find that structural properties of regulatory networks are remarkably conserved and that orthologous regulatory factor families recognize similar binding motifs in vivo and show some similar co-associations. Our results suggest that gene-regulatory properties previously observed for individual factors are general principles of metazoan regulation that are remarkably well-preserved despite extensive functional divergence of individual network connections. The comparative maps of regulatory circuitry provided here will drive an improved understanding of the regulatory underpinnings of model organism biology and how these relate to human biology, development and disease.

Integrative analysis of RNA, translation, and protein levels reveals distinct regulatory variation across humans.

Elucidating the consequences of genetic differences between humans is essential for understanding phenotypic diversity and personalized medicine. Although variation in RNA levels, transcription factor binding, and chromatin have been explored, little is known about global variation in translation and its genetic determinants. We used ribosome profiling, RNA sequencing, and mass spectrometry to perform an integrated analysis in lymphoblastoid cell lines from a diverse group of individuals. We find significant differences in RNA, translation, and protein levels suggesting diverse mechanisms of personalized gene expression control. Combined analysis of RNA expression and ribosome occupancy improves the identification of individual protein level differences. Finally, we identify genetic differences that specifically modulate ribosome occupancy--many of these differences lie close to start codons and upstream ORFs. Our results reveal a new level of gene expression variation among humans and indicate that genetic variants can cause changes in protein levels through effects on translation.

Sushi.R: flexible, quantitative and integrative genomic visualizations for publication-quality multi-panel figures.

MOTIVATION: Interpretation and communication of genomic data require flexible and quantitative tools to analyze and visualize diverse data types, and yet, a comprehensive tool to display all common genomic data types in publication quality figures does not exist to date. To address this shortcoming, we present Sushi.R, an R/Bioconductor package that allows flexible integration of genomic visualizations into highly customizable, publication-ready, multi-panel figures from common genomic data formats including Browser Extensible Data (BED), bedGraph and Browser Extensible Data Paired-End (BEDPE). Sushi.R is open source and made publicly available through GitHub (https://github.com/dphansti/Sushi) and Bioconductor (http://bioconductor.org/packages/release/bioc/html/Sushi.html).

A fundamental protein property, thermodynamic stability, revealed solely from large-scale measurements of protein function.

The ability of a protein to carry out a given function results from fundamental physicochemical properties that include the protein's structure, mechanism of action, and thermodynamic stability. Traditional approaches to study these properties have typically required the direct measurement of the property of interest, oftentimes a laborious undertaking. Although protein properties can be probed by mutagenesis, this approach has been limited by its low throughput. Recent technological developments have enabled the rapid quantification of a protein's function, such as binding to a ligand, for numerous variants of that protein. Here, we measure the ability of 47,000 variants of a WW domain to bind to a peptide ligand and use these functional measurements to identify stabilizing mutations without directly assaying stability. Our approach is rooted in the well-established concept that protein function is closely related to stability. Protein function is generally reduced by destabilizing mutations, but this decrease can be rescued by stabilizing mutations. Based on this observation, we introduce partner potentiation, a metric that uses this rescue ability to identify stabilizing mutations, and identify 15 candidate stabilizing mutations in the WW domain. We tested six candidates by thermal denaturation and found two highly stabilizing mutations, one more stabilizing than any previously known mutation. Thus, physicochemical properties such as stability are latent within these large-scale protein functional data and can be revealed by systematic analysis. This approach should allow other protein properties to be discovered.

First reported magnesium pyrophosphate kidney stone prompts diagnosis of hypophosphatasia.

Hypophosphatasia (HPP) is a rare genetic condition associated with poor bone mineralization, low serum alkaline phosphatase, high urinary pyrophosphate excretion, and nephrocalcinosis. Nephrocalcinosis is thought to develop due to the increased filtered loads associated with hypercalcemia and hyperphosphatemia, but the composition of these calcifications is incompletely understood. We report the first ever magnesium pyrophosphate (MgPPi) urinary stone, which prompted the new diagnosis of HPP in a 12-year-old boy. Stone analysis labs should include infrared spectra of PPi salts in their reference libraries to facilitate identification of these rare but clinically important stones.

High-resolution mapping of protein sequence-function relationships.

We present a large-scale approach to investigate the functional consequences of sequence variation in a protein. The approach entails the display of hundreds of thousands of protein variants, moderate selection for activity and high-throughput DNA sequencing to quantify the performance of each variant. Using this strategy, we tracked the performance of >600,000 variants of a human WW domain after three and six rounds of selection by phage display for binding to its peptide ligand. Binding properties of these variants defined a high-resolution map of mutational preference across the WW domain; each position had unique features that could not be captured by a few representative mutations. Our approach could be applied to many in vitro or in vivo protein assays, providing a general means for understanding how protein function relates to sequence.

Missed viral surveillance testing visits associate with full blown viral diseases in children receiving kidney transplants.

Surveillance testing for major viral infections such as CMV, EBV, and BKV early in their natural history course may allow for early intervention and prevention of FBVD, but the testing is expensive and optimal interval/frequency are uncertain. At our center we initiated routine monthly viral surveillance for CMV, EBV, and BKV in July 2008 for the first 12 months post-transplant. Here, we retrospectively analyzed for outcome of the patients who missed three or more surveillance tests in the first 12 months post-transplant vs. those who did not. Of 21 patients, five missed three or more surveillance tests. Two of those five developed FBVD (one BKV nephropathy and one EBV-PTLD). None of the 16 patients with more regular surveillance testing developed FBVD. The incidence of viral replication was similar in both groups. The odds ratio for FBVD if viral surveillance tests were missed was 23.57 (p-value of 0.047). In this small group of contemporaneous patients on identical immunosuppression, those patients who missed regular viral surveillance were more likely to develop FBVD. Prospective randomized trials to confirm the benefit of regular viral testing are recommended.

Serum from minimal change patients in relapse increases CD80 expression in cultured podocytes.

BACKGROUND: Minimal change disease (MCD) is the most common cause of nephrotic syndrome in children and is associated with the expression of CD80 in podocytes and the increased excretion of CD80 in urine. We hypothesized that serum from patients with MCD might stimulate CD80 expression in cultured podocytes. METHODS: Sera and peripheral blood mononuclear cells (PBMCs) were collected from subjects with MCD in relapse and remission and from normal controls. Immortalized human podocytes were incubated with culture media containing patient sera or supernatants from patient and control PBMC cultures. CD80 expression was measured by quantitative PCR and western blot analysis. RESULTS: Sera collected from patients with MCD in relapse, but not in remission, significantly increased CD80 expression (mean ± standard deviation: 1.8 ± 0.7 vs. 0.8 ± 0.2; p < 0.004) and CD80 protein secretion by podocytes (p < 0.05 between relapse and normal controls). No such CD80 increase was observed when podocytes were incubated with supernatants of PBMC cultures from patients in relapse. CONCLUSIONS: Sera from MCD patients in relapse, but not in remission, stimulated CD80 expression in cultured podocytes. Identifying this factor in sera could provide insights into the pathogenesis of this disorder. No role in CD80 expression by podocytes was found for cytokines released by PBMCs.

Minimal change disease in graft versus host disease: a podocyte response to the graft?

Nephrotic syndrome is a rare complication of hematopoietic cell transplantation. It has been suggested that nephrotic syndrome may represent a limited form of graft-versus-host disease although the pathological link between these two entities remains unclear. In this paper, we report a case of a 61-year-old female who underwent nonmyeloablative allogenic stem cell transplantation for T-cell prolymphocytic leukemia and subsequently developed biopsy proven minimal change disease shortly after cessation of her immunosuppression therapy. Urinary CD80 was markedly elevated during active disease and disappeared following corticosteroid-induced remission. We hypothesize that alloreactive donor T cells target the kidney and induce podocyte expression of CD80 that results in proteinuria from limited 'graft versus host' disease.

Effectiveness of an intervention to reduce stigma towards people with a mental disorder diagnosis in university students of healthcare careers.

Students in healthcare careers present stigma towards people with psychiatric diagnoses, so the development of interventions to reduce it is essential. This study aimed to evaluate the effectiveness of an intervention to reduce stigma towards people diagnosed with mental disorders in healthcare students in Chile. A randomized clinical trial with a before and after measurement was carried out. The intervention was part of a compulsory course and combined educational and contact strategies. A total of 244 fourth-semester students of medicine, nursing, dentistry, obstetrics, psychology, and social work participated. The intervention was effective in reducing stigmatizing attitudes and the desire for social distance. For almost all variables, the magnitude of the stigma reduction depended on the initial level of stigma, not on the profession. The intervention had positive effects on all careers. In conclusion, incorporating a stigma reduction intervention into mandatory professional training, with the active participation of the teacher in charge and experts by experience, can be a valuable tool to promote humanized and non-stigmatizing treatment.

Enrich: software for analysis of protein function by enrichment and depletion of variants.

SUMMARY: Measuring the consequences of mutation in proteins is critical to understanding their function. These measurements are essential in such applications as protein engineering, drug development, protein design and genome sequence analysis. Recently, high-throughput sequencing has been coupled to assays of protein activity, enabling the analysis of large numbers of mutations in parallel. We present Enrich, a tool for analyzing such deep mutational scanning data. Enrich identifies all unique variants (mutants) of a protein in high-throughput sequencing datasets and can correct for sequencing errors using overlapping paired-end reads. Enrich uses the frequency of each variant before and after selection to calculate an enrichment ratio, which is used to estimate fitness. Enrich provides an interactive interface to guide users. It generates user-accessible output for downstream analyses as well as several visualizations of the effects of mutation on function, thereby allowing the user to rapidly quantify and comprehend sequence-function relationships. AVAILABILITY AND IMPLEMENTATION: Enrich is implemented in Python and is available under a FreeBSD license at http://depts.washington.edu/sfields/software/enrich/. Enrich includes detailed documentation as well as a small example dataset. CONTACT: dfowler@uw.edu; fields@uw.edu SUPPLEMENTARY INFORMATION: Supplementary data is available at Bioinformatics online.

The BK virus in renal transplant recipients-review of pathogenesis, diagnosis, and treatment.

The BK virus, a DNA virus from the Polyomavirus group, represents an opportunistic infection of immunosuppressed transplant recipients. Though the virus was discovered approximately 40 years ago, the emergence of BK virus nephropathy since 1995 onwards, with associated high graft loss rates, has revolutionized renal transplantation medicine. Kidney transplant professionals realized that the consequences of over-immunosuppression were as severe as the consequences of under-immunosuppression and we entered the era of immunosuppressive minimization. Despite this recognition, the optimal testing type for BK virus infections and frequency of testing are hotly debated. Similarly, optimal treatment strategies remain sources of intense controversy. The authors review the current strategies of screening, diagnosis, and possible treatment, and also review the amount and quality of evidence in favor or against. Similarities and differences between cytomegalovirus, Epstein-Barr virus, and BV virus, the three major viral infections in kidney transplantation, are highlighted.

Minimal change disease: a "two-hit" podocyte immune disorder?

Minimal change disease (MCD) is the most common nephrotic syndrome in children and is commonly thought to be a T-cell disorder mediated by a circulating factor that alters podocyte function resulting in massive proteinuria. We suggest that MCD is a "two-hit" disorder. As originally hypothesized by Reiser et al. in 2004, we propose that the initial hit is the induction of CD80 (also known as B7.1) on the podocyte, and that this results in an alteration in shape with actin rearrangement that alters glomerular permeability and causes proteinuria. We propose that CD80 expression may result from either direct binding of the podocyte by cytokines from activated T cells or by activation of podocyte toll-like receptors (TLR) by viral products or allergens. We further hypothesize that under normal circumstances, CD80 expression is only transiently expressed and proteinuria is minimal due to rapid autoregulatory response by circulating T regulatory cells or by the podocyte itself, probably due to the expression of factors [cytotoxic T-lymphocyte-associated (CTLA)-4, interleukin (IL)-10, and possibly transforming growth factor (TGF)-ß] that downregulate the podocyte CD80 response. In MCD, however, there is a defect in CD80 podocyte autoregulation. This results in persistent CD80 expression and persistent proteinuria. If correct, this hypothesis may lead to both new diagnostic tests and potential therapeutics for this important renal disease.

Whole-genome sequencing of a laboratory-evolved yeast strain.

BACKGROUND: Experimental evolution of microbial populations provides a unique opportunity to study evolutionary adaptation in response to controlled selective pressures. However, until recently it has been difficult to identify the precise genetic changes underlying adaptation at a genome-wide scale. New DNA sequencing technologies now allow the genome of parental and evolved strains of microorganisms to be rapidly determined. RESULTS: We sequenced >93.5% of the genome of a laboratory-evolved strain of the yeast Saccharomyces cerevisiae and its ancestor at >28x depth. Both single nucleotide polymorphisms and copy number amplifications were found, with specific gains over array-based methodologies previously used to analyze these genomes. Applying a segmentation algorithm to quantify structural changes, we determined the approximate genomic boundaries of a 5x gene amplification. These boundaries guided the recovery of breakpoint sequences, which provide insights into the nature of a complex genomic rearrangement. CONCLUSIONS: This study suggests that whole-genome sequencing can provide a rapid approach to uncover the genetic basis of evolutionary adaptations, with further applications in the study of laboratory selections and mutagenesis screens. In addition, we show how single-end, short read sequencing data can provide detailed information about structural rearrangements, and generate predictions about the genomic features and processes that underlie genome plasticity.

Urinary CD80 is elevated in minimal change disease but not in focal segmental glomerulosclerosis.

Controversy exists as to whether minimal change disease (MCD) and focal segmental glomerulosclerosis (FSGS) represent different diseases or are manifestations within the same disease spectrum. Urinary excretion of CD80 (also known as B7.1) is elevated in patients with MCD and hence we tested whether urinary CD80 excretion might distinguish between patients with MCD from those with FSGS. Urinary CD80 was measured in 17 patients with biopsy-proven MCD and 22 with proven FSGS using a commercially available enzyme-linked immunosorbent assay and its molecular size determined by western blot analysis. A significant increase in urinary CD80, normalized to urinary creatinine, was found in patients with MCD in relapse compared to those in remission or those with FSGS. No significant differences were seen when CD80 urinary excretion from MCD patients in remission were compared to those with FSGS. In seven of eight MCD patients in relapse, CD80 was found in glomeruli by immunohistochemical analysis of their biopsy specimen. No CD80 was found in glomeruli of two patients with FSGS and another MCD patient in remission. Thus, our study supports the hypothesis that MCD and FSGS represent two different diseases rather than a continuum of one disease. Urinary CD80 excretion may be a useful marker to differentiate between MCD and FSGS.

Comparison of generic tacrolimus and Prograf drug levels in a pediatric kidney transplant program: brief communication.

A generic version of tacrolimus was approved for use in the USA in August 2009. These narrow therapeutic index generics are tested for bioequivalence only in adults. No data are available on generic tacrolimus levels in children with allografts. Four patients with stable renal allografts in our pediatric program were inadvertently switched to generic tacrolimus. We retrospectively analyzed pre- and post-switch trough tacrolimus and serum creatinine levels. Twelve-h trough tacrolimus levels (mean ± s.e.) were (i) patient 1 (12-yr-old girl): 7.0 ± 0.69 and 9.7 ± 3.5 (p =NS); (ii) patient 2 (eight-yr-old boy): 4.7 ± 0.68 and 3.4 ± 0.84 (p = 0.04); (iii) patient 3 (22-yr-old woman): 6.8 ± 0.17 and 6.6 ± 0.4 (p = NS); (iv) patient 4 (20-yr-old woman): 5.4 ± 0.25 and 4.9 ± 1.4 (p = NS). Creatinine levels were similar pre- and post-switch (eGFR > 75 mL/min/1.73 m²) in the first three. Patient 4 experienced a biopsy proven acute rejection immediately after switching. Mean creatinine rose from 1.15 ± 0.05 to 2.168 ± 0.07 after switch (p < 0.001). Given our mixed picture with the early data, we suggest careful monitoring of pediatric patients who get switched to generic tacrolimus.