Re-analysis of genomic data: An overview of the mechanisms and complexities of clinical adoption.

Re-analyzing genomic information from a patient suspected of having an underlying genetic condition can improve the diagnostic yield of sequencing tests, potentially providing significant benefits to the patient and to the health care system. Although a significant number of studies have shown the clinical potential of re-analysis, less work has been performed to characterize the mechanisms responsible for driving the increases in diagnostic yield. Complexities surrounding re-analysis have also emerged. The terminology itself represents a challenge because "re-analysis" can refer to a range of different concepts. Other challenges include the increased workload that re-analysis demands of curators, adequate reimbursement pathways for clinical and diagnostic services, and the development of systems to handle large volumes of data. Re-analysis also raises ethical implications for patients and families, most notably when re-classification of a variant alters diagnosis, treatment, and prognosis. This review highlights the possibilities and complexities associated with the re-analysis of existing clinical genomic data. We propose a terminology that builds on the foundation presented in a recent statement from the American College of Medical Genetics and Genomics and describes each re-analysis process. We identify mechanisms for increasing diagnostic yield and provide perspectives on the range of challenges that must be addressed by health care systems and individual patients.

Whole genomes redefine the mutational landscape of pancreatic cancer.

Pancreatic cancer remains one of the most lethal of malignancies and a major health burden. We performed whole-genome sequencing and copy number variation (CNV) analysis of 100 pancreatic ductal adenocarcinomas (PDACs). Chromosomal rearrangements leading to gene disruption were prevalent, affecting genes known to be important in pancreatic cancer (TP53, SMAD4, CDKN2A, ARID1A and ROBO2) and new candidate drivers of pancreatic carcinogenesis (KDM6A and PREX2). Patterns of structural variation (variation in chromosomal structure) classified PDACs into 4 subtypes with potential clinical utility: the subtypes were termed stable, locally rearranged, scattered and unstable. A significant proportion harboured focal amplifications, many of which contained druggable oncogenes (ERBB2, MET, FGFR1, CDK6, PIK3R3 and PIK3CA), but at low individual patient prevalence. Genomic instability co-segregated with inactivation of DNA maintenance genes (BRCA1, BRCA2 or PALB2) and a mutational signature of DNA damage repair deficiency. Of 8 patients who received platinum therapy, 4 of 5 individuals with these measures of defective DNA maintenance responded.

sCNAphase: using haplotype resolved read depth to genotype somatic copy number alterations from low cellularity aneuploid tumors.

Accurate identification of copy number alterations is an essential step in understanding the events driving tumor progression. While a variety of algorithms have been developed to use high-throughput sequencing data to profile copy number changes, no tool is able to reliably characterize ploidy and genotype absolute copy number from tumor samples that contain less than 40% tumor cells. To increase our power to resolve the copy number profile from low-cellularity tumor samples, we developed a novel approach that pre-phases heterozygote germline single nucleotide polymorphisms (SNPs) in order to replace the commonly used 'B-allele frequency' with a more powerful 'parental-haplotype frequency'. We apply our tool-sCNAphase-to characterize the copy number and loss-of-heterozygosity profiles of four publicly available breast cancer cell-lines. Comparisons to previous spectral karyotyping and microarray studies revealed that sCNAphase reliably identified overall ploidy as well as the individual copy number mutations from each cell-line. Analysis of artificial cell-line mixtures demonstrated the capacity of this method to determine the level of tumor cellularity, consistently identify sCNAs and characterize ploidy in samples with as little as 10% tumor cells. This novel methodology has the potential to bring sCNA profiling to low-cellularity tumors, a form of cancer unable to be accurately studied by current methods.

It Is in Our DNA: Bringing Electronic Health Records and Genomic Data Together for Precision Medicine.

Health care is at a turning point. We are shifting from protocolized medicine to precision medicine, and digital health systems are facilitating this shift. By providing clinicians with detailed information for each patient and analytic support for decision-making at the point of care, digital health technologies are enabling a new era of precision medicine. Genomic data also provide clinicians with information that can improve the accuracy and timeliness of diagnosis, optimize prescribing, and target risk reduction strategies, all of which are key elements for precision medicine. However, genomic data are predominantly seen as diagnostic information and are not routinely integrated into the clinical workflows of electronic medical records. The use of genomic data holds significant potential for precision medicine; however, as genomic data are fundamentally different from the information collected during routine practice, special considerations are needed to use this information in a digital health setting. This paper outlines the potential of genomic data integration with electronic records, and how these data can enable precision medicine.

Clinically significant changes in genes and variants associated with epilepsy over time: implications for re-analysis.

Despite the significant advances in understanding the genetic architecture of epilepsy, many patients do not receive a molecular diagnosis after genomic testing. Re-analysing existing genomic data has emerged as a potent method to increase diagnostic yields-providing the benefits of genomic-enabled medicine to more individuals afflicted with a range of different conditions. The primary drivers for these new diagnoses are the discovery of novel gene-disease and variants-disease relationships; however, most decisions to trigger re-analysis are based on the passage of time rather than the accumulation of new knowledge. To explore how our understanding of a specific condition changes and how this impacts re-analysis of genomic data from epilepsy patients, we developed Vigelint. This approach combines the information from PanelApp and ClinVar to characterise how the clinically relevant genes and causative variants available to laboratories change over time, and this approach to five clinical-grade epilepsy panels. Applying the Vigelint pipeline to these panels revealed highly variable patterns in new, clinically relevant knowledge becoming publicly available. This variability indicates that a more dynamic approach to re-analysis may benefit the diagnosis and treatment of epilepsy patients. Moreover, this work suggests that Vigelint can provide empirical data to guide more nuanced, condition-specific approaches to re-analysis.

Stem cell transcriptome profiling via massive-scale mRNA sequencing.

We developed a massive-scale RNA sequencing protocol, short quantitative random RNA libraries or SQRL, to survey the complexity, dynamics and sequence content of transcriptomes in a near-complete fashion. This method generates directional, random-primed, linear cDNA libraries that are optimized for next-generation short-tag sequencing. We surveyed the poly(A)(+) transcriptomes of undifferentiated mouse embryonic stem cells (ESCs) and embryoid bodies (EBs) at an unprecedented depth (10 Gb), using the Applied Biosystems SOLiD technology. These libraries capture the genomic landscape of expression, state-specific expression, single-nucleotide polymorphisms (SNPs), the transcriptional activity of repeat elements, and both known and new alternative splicing events. We investigated the impact of transcriptional complexity on current models of key signaling pathways controlling ESC pluripotency and differentiation, highlighting how SQRL can be used to characterize transcriptome content and dynamics in a quantitative and reproducible manner, and suggesting that our understanding of transcriptional complexity is far from complete.

An improved method for undertaking limiting dilution assays for in vitro cloning of Plasmodium falciparum parasites.

BACKGROUND: Obtaining single parasite clones is required for many techniques in malaria research. Cloning by limiting dilution using microscopy-based assessment for parasite growth is an arduous and labor-intensive process. An alternative method for the detection of parasite growth in limiting dilution assays is using a commercial ELISA histidine-rich protein II (HRP2) detection kit. METHODS: Detection of parasite growth was undertaken using HRP2 ELISA and compared to thick film microscopy. An HRP2 protein standard was used to determine the detection threshold of the HRP2 ELISA assay, and a HRP2 release model was used to extrapolate the amount of parasite growth required for a positive result. RESULTS: The HRP2 ELISA was more sensitive than microscopy for detecting parasite growth. The minimum level of HRP2 protein detection of the ELISA was 0.11 ng/ml. Modeling of HRP2 release determined that 2,116 parasites are required to complete a full erythrocytic cycle to produce sufficient HRP2 to be detected by the ELISA. Under standard culture conditions this number of parasites is likely to be reached between 8 to 14 days of culture. CONCLUSIONS: This method provides an accurate and simple way for the detection of parasite growth in limiting dilution assays, reducing time and resources required in traditional methods. Furthermore the method uses spent culture media instead of the parasite-infected red blood cells, enabling culture to continue.

Phosphorinanes as ligands for palladium-catalyzed cross-coupling chemistry.

[structure: see text] Phosphorinanes are presented as a class of phosphine ligand suitable for organopalladium cross-coupling chemistry. Prepared via a direct double Michael addition of a monoalkyl- or arylphosphine to phorone followed by a Wolf-Kishner reduction, phosphorinanes are relatively inexpensive to manufacture and allow modification of one of the alkyl moieties permitting steric and electronic fine-tuning of the ligands. Library screening and applications of these ligands in the Suzuki, Sonogashira, ketone arylation, and aryl amination reactions are presented.

A Randomized Controlled Trial of Allopurinol in Patients With Peripheral Arterial Disease.

BACKGROUND: Patients with peripheral arterial disease (PAD) are limited by intermittent claudication in the distance they can walk. Allopurinol has been shown in coronary arterial disease to prolong exercise before angina occurs, likely by prevention of oxygen wastage in tissues and reduction of harmful oxidative stress. METHODS: In this study we evaluated whether allopurinol could prolong the time to development of leg pain in participants with PAD. In a double-blind, randomized controlled clinical trial participants were randomized to receive either allopurinol 300 mg twice daily or placebo for 6 months. The primary outcome was change in exercise capacity on treadmill testing at 6 months. Secondary outcomes were 6-minute walking distance, Walking Impairment Questionnaire, SF-36 questionnaire, flow-mediated dilatation, and oxidized low-density lipoprotein. Outcome measures were repeated midstudy and at the end of study. The mean age of the 50 participants was 68.4 ± 1.2 years with 39 of 50 (78%) male. RESULTS: Five participants withdrew during the study (2 active, 3 placebo). There was a significant reduction in uric acid levels in those who received active treatment of 52.1% (P < 0.001), but no significant change in either the pain-free or the maximum walking distance. Other measures of exercise capacity, blood vessel function, and the participants' own assessment of their health and walking ability also did not change during the course of the study. CONCLUSIONS: Although allopurinol has been shown to be of benefit in a number of other diseases, in this study there was no evidence of any improvement after treatment in patients with PAD.

Phosphaadamantanes as ligands for palladium catalyzed cross-coupling chemistry: library synthesis, characterization, and screening in the Suzuki coupling of alkyl halides and tosylates containing beta-hydrogens with boronic acids and alkylboranes.

A 15-member library of phosphaadamantane ligands has been prepared via P-arylation of 1,3,5,7-tetramethyl-2,4,8-trioxa-6-phosphaadamantane. Screening of this tertiary phosphine collection has allowed for the rapid determination of the most suitable ligand, specifically 1,3,5,7-tetramethyl-6-(2,4-dimethoxyphenyl)-2,4,8-trioxa-6-phosphaadamantane, for facilitating Suzuki-type couplings of alkyl halides or tosylates containing beta-hydrogens with either boronic acids or alkylboranes.