SCIP: software for efficient clinical interpretation of copy number variants detected by whole-genome sequencing.

Copy number variants (CNVs) represent major etiologic factors in rare genetic diseases. Current clinical CNV interpretation workflows require extensive back-and-forth with multiple tools and databases. This increases complexity and time burden, potentially resulting in missed genetic diagnoses. We present the Suite for CNV Interpretation and Prioritization (SCIP), a software package for the clinical interpretation of CNVs detected by whole-genome sequencing (WGS). The SCIP Visualization Module near-instantaneously displays all information necessary for CNV interpretation (variant quality, population frequency, inheritance pattern, and clinical relevance) on a single page-supported by modules providing variant filtration and prioritization. SCIP was comprehensively evaluated using WGS data from 1027 families with congenital cardiac disease and/or autism spectrum disorder, containing 187 pathogenic or likely pathogenic (P/LP) CNVs identified in previous curations. SCIP was efficient in filtration and prioritization: a median of just two CNVs per case were selected for review, yet it captured all P/LP findings (92.5% of which ranked 1st). SCIP was also able to identify one pathogenic CNV previously missed. SCIP was benchmarked against AnnotSV and a spreadsheet-based manual workflow and performed superiorly than both. In conclusion, SCIP is a novel software package for efficient clinical CNV interpretation, substantially faster and more accurate than previous tools (available at https://github.com/qd29/SCIP , a video tutorial series is available at https://bit.ly/SCIPVideos ).

The Cardiac Genome Clinic: implementing genome sequencing in pediatric heart disease.

PURPOSE: This study investigated the diagnostic utility of nontargeted genomic testing in patients with pediatric heart disease. METHODS: We analyzed genome sequencing data of 111 families with cardiac lesions for rare, disease-associated variation. RESULTS: In 14 families (12.6%), we identified causative variants: seven were de novo (ANKRD11, KMT2D, NR2F2, POGZ, PTPN11, PURA, SALL1) and six were inherited from parents with no or subclinical heart phenotypes (FLT4, DNAH9, MYH11, NEXMIF, NIPBL, PTPN11). Outcome of the testing was associated with the presence of extracardiac features (p = 0.02), but not a positive family history for cardiac lesions (p = 0.67). We also report novel plausible gene-disease associations for tetralogy of Fallot/pulmonary stenosis (CDC42BPA, FGD5), hypoplastic left or right heart (SMARCC1, TLN2, TRPM4, VASP), congenitally corrected transposition of the great arteries (UBXN10), and early-onset cardiomyopathy (TPCN1). The identified candidate genes have critical functions in heart development, such as angiogenesis, mechanotransduction, regulation of heart size, chromatin remodeling, or ciliogenesis. CONCLUSION: This data set demonstrates the diagnostic and scientific value of genome sequencing in pediatric heart disease, anticipating its role as a first-tier diagnostic test. The genetic heterogeneity will necessitate large-scale genomic initiatives for delineating novel gene-disease associations.

A three-gene DNA methylation biomarker accurately classifies early stage prostate cancer.

BACKGROUND: We identify and validate accurate diagnostic biomarkers for prostate cancer through a systematic evaluation of DNA methylation alterations. MATERIALS AND METHODS: We assembled three early prostate cancer cohorts (total patients = 699) from which we collected and processed over 1300 prostatectomy tissue samples for DNA extraction. Using real-time methylation-specific PCR, we measured normalized methylation levels at 15 frequently methylated loci. After partitioning sample sets into independent training and validation cohorts, classifiers were developed using logistic regression, analyzed, and validated. RESULTS: In the training dataset, DNA methylation levels at 7 of 15 genomic loci (glutathione S-transferase Pi 1 [GSTP1], CCDC181, hyaluronan, and proteoglycan link protein 3 [HAPLN3], GSTM2, growth arrest-specific 6 [GAS6], RASSF1, and APC) showed large differences between cancer and benign samples. The best binary classifier was the GAS6/GSTP1/HAPLN3 logistic regression model, with an area under these curves of 0.97, which showed a sensitivity of 94%, and a specificity of 93% after external validation. CONCLUSION: We created and validated a multigene model for the classification of benign and malignant prostate tissue. With false positive and negative rates below 7%, this three-gene biomarker represents a promising basis for more accurate prostate cancer diagnosis.

Genetic consequences of population expansions and contractions in the common hippopotamus (Hippopotamus amphibius) since the Late Pleistocene.

Over the past two decades, an increasing amount of phylogeographic work has substantially improved our understanding of African biogeography, in particular the role played by Pleistocene pluvial-drought cycles on terrestrial vertebrates. However, still little is known on the evolutionary history of semi-aquatic animals, which faced tremendous challenges imposed by unpredictable availability of water resources. In this study, we investigate the Late Pleistocene history of the common hippopotamus (Hippopotamus amphibius), using mitochondrial and nuclear DNA sequence variation and range-wide sampling. We documented a global demographic and spatial expansion approximately 0.1-0.3 Myr ago, most likely associated with an episode of massive drainage overflow. These events presumably enabled a historical continent-wide gene flow among hippopotamus populations, and hence, no clear continental-scale genetic structuring remains. Nevertheless, present-day hippopotamus populations are genetically disconnected, probably as a result of the mid-Holocene aridification and contemporary anthropogenic pressures. This unique pattern contrasts with the biogeographic paradigms established for savannah-adapted ungulate mammals and should be further investigated in other water-associated taxa. Our study has important consequences for the conservation of the hippo, an emblematic but threatened species that requires specific protection to curtail its long-term decline.

GeneTerpret: a customizable multilayer approach to genomic variant prioritization and interpretation.

BACKGROUND: Variant interpretation is the main bottleneck in medical genomic sequencing efforts. This usually involves genome analysts manually searching through a multitude of independent databases, often with the aid of several, mostly independent, computational tools. To streamline variant interpretation, we developed the GeneTerpret platform which collates data from current interpretation tools and databases, and applies a phenotype-driven query to categorize the variants identified in the genome(s). The platform assigns quantitative validity scores to genes by query and assembly of the genotype-phenotype data, sequence homology, molecular interactions, expression data, and animal models. It also uses the American College of Medical Genetics and Genomics (ACMG) criteria to categorize variants into five tiers of pathogenicity. The final output is a prioritized list of potentially causal variants/genes. RESULTS: We tested GeneTerpret by comparing its performance to expert-curated genes (ClinGen's gene-validity database) and variant pathogenicity reports (DECIPHER database). Output from GeneTerpret was 97.2% and 83.5% concordant with the expert-curated sources, respectively. Additionally, similar concordance was observed when GeneTerpret's performance was compared with our internal expert-interpreted clinical datasets. CONCLUSIONS: GeneTerpret is a flexible platform designed to streamline the genome interpretation process, through a unique interface, with improved ease, speed and accuracy. This modular and customizable system allows the user to tailor the component-programs in the analysis process to their preference. GeneTerpret is available online at https://geneterpret.com .

Assessment of the Implementation of Pharmacogenomic Testing in a Pediatric Tertiary Care Setting.

Importance: Pharmacogenomic (PGx) testing provides preemptive pharmacotherapeutic guidance regarding the lack of therapeutic benefit or adverse drug reactions of PGx targeted drugs. Pharmacogenomic information is of particular value among children with complex medical conditions who receive multiple medications and are at higher risk of developing adverse drug reactions. Objectives: To assess the implementation outcomes of a PGx testing program comprising both a point-of-care model that examined targeted drugs and a preemptive model informed by whole-genome sequencing that evaluated a broad range of drugs for potential therapy among children in a pediatric tertiary care setting. Design, Setting, and Participants: This cohort study was conducted at The Hospital for Sick Children in Toronto, Ontario, from January 2017 to September 2020. Pharmacogenomic analyses were performed among 172 children who were categorized into 2 groups: a point-of-care cohort and a preemptive cohort. The point-of-care cohort comprised 57 patients referred to the consultation clinic for planned therapy with PGx targeted drugs and/or for adverse drug reactions, including lack of therapeutic benefit, after the receipt of current or past medications. The preemptive cohort comprised 115 patients who received exploratory whole-genome sequencing-guided PGx testing for their heart conditions from the cardiac genome clinic at the Ted Rogers Centre for Heart Research. Exposures: Patients received PGx analysis of whole-genome sequencing data and/or multiplex genotyping of 6 pharmacogenes (CYP2C19, CYP2C9, CYP2D6, CYP3A5, VKORC1, and TPMT) that have established PGx clinical guidelines. Main Outcomes and Measures: The number of patients for whom PGx test results warranted deviation from standard dosing regimens. Results: A total of 172 children (mean [SD] age, 8.5 [5.6] years; 108 boys [62.8%]) were enrolled in the study. In the point-of-care cohort, a median of 2 target genes (range, 1-5 genes) were investigated per individual, with CYP2C19 being the most frequently examined; genotypes in 21 of 57 children (36.8%) were incompatible with standard treatment regimens. As expected from population allelic frequencies, among the 115 children in the whole-genome sequencing-guided preemptive cohort, 92 children (80.0%) were recommended to receive nonstandard treatment regimens for potential drug therapies based on their 6-gene pharmacogenetic profile. Conclusions and Relevance: In this cohort study, among both the point-of-care and preemptive cohorts, the multiplex PGx testing program provided dosing recommendations that deviated from standard regimens at an overall rate that was similar to the population frequencies of relevant variants.

Comparison of methods in the recovery of nucleic acids from archival formalin-fixed paraffin-embedded autopsy tissues.

Archival formalin-fixed paraffin-embedded (FFPE) human tissue collections are typically in poor states of storage across the developing world. With advances in biomolecular techniques, these extraordinary and virtually untapped resources have become an essential part of retrospective epidemiological studies. To successfully use such tissues in genomic studies, scientists require high nucleic acid yields and purity. In spite of the increasing number of FFPE tissue kits available, few studies have analyzed their applicability in recovering high-quality nucleic acids from archived human autopsy samples. Here we provide a study involving 10 major extraction methods used to isolate total nucleic acid from FFPE tissues ranging in age from 3 to 13years. Although all 10 methods recovered quantifiable amounts of DNA, only 6 recovered quantifiable RNA, varying considerably and generally yielding lower DNA concentrations. Overall, we show quantitatively that TrimGen's WaxFree method and our in-house phenol-chloroform extraction method recovered the highest yields of amplifiable DNA, with considerable polymerase chain reaction (PCR) inhibition, whereas Ambion's RecoverAll method recovered the most amplifiable RNA.

Genes and Pathways Implicated in Tetralogy of Fallot Revealed by Ultra-Rare Variant Burden Analysis in 231 Genome Sequences.

Recent genome-wide studies of rare genetic variants have begun to implicate novel mechanisms for tetralogy of Fallot (TOF), a severe congenital heart defect (CHD). To provide statistical support for case-only data without parental genomes, we re-analyzed genome sequences of 231 individuals with TOF (n = 175) or related CHD. We adapted a burden test originally developed for de novo variants to assess ultra-rare variant burden in individual genes, and in gene-sets corresponding to functional pathways and mouse phenotypes, accounting for highly correlated gene-sets and for multiple testing. For truncating variants, the gene burden test confirmed significant burden in FLT4 (Bonferroni corrected p-value < 0.01). For missense variants, burden in NOTCH1 achieved genome-wide significance only when restricted to constrained genes (i.e., under negative selection, Bonferroni corrected p-value = 0.004), and showed enrichment for variants affecting the extracellular domain, especially those disrupting cysteine residues forming disulfide bonds (OR = 39.8 vs. gnomAD). Individuals with NOTCH1 ultra-rare missense variants, all with TOF, were enriched for positive family history of CHD. Other genes not previously implicated in CHD had more modest statistical support in gene burden tests. Gene-set burden tests for truncating variants identified a cluster of pathways corresponding to VEGF signaling (FDR = 0%), and of mouse phenotypes corresponding to abnormal vasculature (FDR = 0.8%); these suggested additional candidate genes not previously identified (e.g., WNT5A and ZFAND5). Results for the most promising genes were driven by the TOF subset of the cohort. The findings support the importance of ultra-rare variants disrupting genes involved in VEGF and NOTCH signaling in the genetic architecture of TOF, accounting for 11-14% of individuals in the TOF cohort. These proof-of-principle data indicate that this statistical methodology could assist in analyzing case-only sequencing data in which ultra-rare variants, whether de novo or inherited, contribute to the genetic etiopathogenesis of a complex disorder.

Where sociality and relatedness diverge: the genetic basis for hierarchical social organization in African elephants.

Hierarchical properties characterize elephant fission-fusion social organization whereby stable groups of individuals coalesce into higher order groups or split in a predictable manner. This hierarchical complexity is rare among animals and, as such, an examination of the factors driving its emergence offers unique insight into the evolution of social behaviour. Investigation of the genetic basis for such social affiliation demonstrates that while the majority of core social groups (second-tier affiliates) are significantly related, this is not exclusively the case. As such, direct benefits received through membership of these groups appear to be salient to their formation and maintenance. Further analysis revealed that the majority of groups in the two higher social echelons (third and fourth tiers) are typically not significantly related. The majority of third-tier members are matrilocal, carrying the same mtDNA control region haplotype, while matrilocality among fourth-tier groups was slightly less than expected at random. Comparison of results to those from a less disturbed population suggests that human depredation, leading to social disruption, altered the genetic underpinning of social relations in the study population. These results suggest that inclusive fitness benefits may crystallize elephant hierarchical social structuring along genetic lines when populations are undisturbed. However, indirect benefits are not critical to the formation and maintenance of second-, third- or fourth-tier level bonds, indicating the importance of direct benefits in the emergence of complex, hierarchical social relations among elephants. Future directions and conservation implications are discussed.

Population genetic structure of savannah elephants in Kenya: conservation and management implications.

We investigated population genetic structure and regional differentiation among African savannah elephants in Kenya using mitochondrial and microsatellite markers. We observed mitochondrial DNA (mtDNA) nucleotide diversity of 1.68% and microsatellite variation in terms of average number of alleles, expected and observed heterozygosities in the total study population of 10.20, 0.75, and 0.69, respectively. Hierarchical analysis of molecular variance of mtDNA variation revealed significant differentiation among the 3 geographical regions studied (F(CT) = 0.264; P < 0.05) and a relatively lower differentiation among populations within regions (F(SC) = 0.218; P < 0.0001). Microsatellite variation significantly differentiated among populations within regions (F(SC) = 0.019; P < 0.0001) but not at the regional levels (F(CT) = 0.000; P > 0.500). We attribute the high differentiation at the mitochondrial genome to the matrilineal social structure of elephant populations, female natal philopatry, and probably ancient vicariance. Lack of significant regional differentiation at the nuclear loci vis-a-vis strong differences at mtDNA loci between regions is likely the effect of subsequent homogenization through male-mediated gene flow. Our results depicting 3 broad regional mtDNA groups and the observed population genetic differentiation as well as connectivity patterns should be incorporated in the planning of future management activities such as translocations.

Reliability and performance of commercial RNA and DNA extraction kits for FFPE tissue cores.

Cancer biomarker studies often require nucleic acid extraction from limited amounts of formalin-fixed, paraffin-embedded (FFPE) tissues, such as histologic sections or needle cores. A major challenge is low quantity and quality of extracted nucleic acids, which can limit our ability to perform genetic analyses, and have a significant influence on overall study design. This study was aimed at identifying the most reliable and reproducible method of obtaining sufficient high-quality nucleic acids from FFPE tissues. We compared the yield and quality of nucleic acids from 0.6-mm FFPE prostate tissue cores across 16 DNA and RNA extraction protocols, using 14 commercially available kits. Nucleic acid yield was determined by fluorometry, and quality was determined by spectrophotometry. All protocols yielded nucleic acids in quantities that are compatible with downstream molecular applications. However, the protocols varied widely in the quality of the extracted RNA and DNA. Four RNA and five DNA extraction protocols, including protocols from two kits for dual-extraction of RNA and DNA from the same tissue source, were prioritized for further quality assessment based on the yield and purity of their products. Specifically, their compatibility with downstream reactions was assessed using both NanoString nCounter gene expression assays and reverse-transcriptase real-time PCR for RNA, and methylation-specific PCR assays for DNA. The kit deemed most suitable for FFPE tissue was the AllPrep kit by Qiagen because of its yield, quality, and ability to purify both RNA and DNA from the same sample, which would be advantageous in biomarker studies.

Preparation of Formalin-fixed Paraffin-embedded Tissue Cores for both RNA and DNA Extraction.

Formalin-fixed paraffin embedded tissue (FFPET) represents a valuable, well-annotated substrate for molecular investigations. The utility of FFPET in molecular analysis is complicated both by heterogeneous tissue composition and low yields when extracting nucleic acids. A literature search revealed a paucity of protocols addressing these issues, and none that showed a validated method for simultaneous extraction of RNA and DNA from regions of interest in FFPET. This method addresses both issues. Tissue specificity was achieved by mapping cancer areas of interest on microscope slides and transferring annotations onto FFPET blocks. Tissue cores were harvested from areas of interest using 0.6 mm microarray punches. Nucleic acid extraction was performed using a commercial FFPET extraction system, with modifications to homogenization, deparaffinization, and Proteinase K digestion steps to improve tissue digestion and increase nucleic acid yields. The modified protocol yields sufficient quantity and quality of nucleic acids for use in a number of downstream analyses, including a multi-analyte gene expression platform, as well as reverse transcriptase coupled real time PCR analysis of mRNA expression, and methylation-specific PCR (MSP) analysis of DNA methylation.

Quantitative assessment of the sensitivity of various commercial reverse transcriptases based on armored HIV RNA.

BACKGROUND: The in-vitro reverse transcription of RNA to its complementary DNA, catalyzed by the enzyme reverse transcriptase, is the most fundamental step in the quantitative RNA detection in genomic studies. As such, this step should be as analytically sensitive, efficient and reproducible as possible, especially when dealing with degraded or low copy RNA samples. While there are many reverse transcriptases in the market, all claiming to be highly sensitive, there is need for a systematic independent comparison of their applicability in quantification of rare RNA transcripts or low copy RNA, such as those obtained from archival tissues. METHODOLOGY/PRINCIPAL FINDINGS: We performed RT-qPCR to assess the sensitivity and reproducibility of 11 commercially available reverse transcriptases in cDNA synthesis from low copy number RNA levels. As target RNA, we used a serially known number of Armored HIV RNA molecules, and observed that 9 enzymes we tested were consistently sensitive to ∼1,000 copies, seven of which were sensitive to ∼100 copies, while only 5 were sensitive to ∼10 RNA template copies across all replicates tested. Despite their demonstrated sensitivity, these five best performing enzymes (Accuscript, HIV-RT, M-MLV, Superscript III and Thermoscript) showed considerable variation in their reproducibility as well as their overall amplification efficiency. Accuscript and Superscript III were the most sensitive and consistent within runs, with Accuscript and Superscript II ranking as the most reproducible enzymes between assays. CONCLUSIONS/SIGNIFICANCE: We therefore recommend the use of Accuscript or Superscript III when dealing with low copy number RNA levels, and suggest purification of the RT reactions prior to downstream applications (eg qPCR) to augment detection. Although the results presented in this study were based on a viral RNA surrogate, and applied to nucleic acid lysates derived from archival formalin-fixed paraffin embedded tissue, their relative performance on RNA obtained from other tissue types may vary, and needs future evaluation.