Cardiac Manifestations of Lyme Disease.

Lyme carditis is an uncommon manifestation of Lyme disease. Most cases present with heart block of varying degrees, but the spectrum of disease includes other transient arrhythmias and structural manifestations, such as myopericarditis or cardiomyopathy. Antibiotics hasten the resolution of Lyme carditis, and cardiac pacing can be an adjunctive therapy. Outcomes are generally good, but there are rare fatalities associated with Lyme carditis. The latter underscores the continued need for improved modes of prevention of Lyme disease and the importance of its early recognition and treatment.

Lyme Carditis in Hospitalized Children and Adults, a Case Series.

BACKGROUND: Lyme carditis is an uncommon manifestation of Lyme disease. This report compares Lyme carditis presentation, management, and outcomes in pediatric and adult populations. METHODS: Charts of pediatric and adult patients with heart block (PR interval >300 ms) and positive Lyme serologies hospitalized in Portland, Maine, between January 2010 and December 2018 were analyzed. Data on medical history, presentation, treatment, and outcomes are described. RESULTS: Ten children and 20 adults were admitted for Lyme carditis between June and October. Ninety percent were male, and 87% had no prior cardiac history. Seventeen had outpatient evaluation before admission. Of these, a minority (41%) had Lyme disease suspected in the outpatient setting, and fewer (12%) were initiated on Lyme disease treatment. The most common alternate diagnoses were viral illness and erythema multiforme. More children than adults had disseminated erythema migrans and fever. First-degree heart block was more prevalent in children, and Mobitz type 2 heart block was more prevalent in adults. Ten patients presented with syncope. Proportionately more adults needed temporary pacing. Children had shorter antibiotic durations compared with adults. Of the 30 cases, 27 had improved heart block, while 3 adults required a pacemaker at discharge. Nine children and 14 adults were discharged with a PR 200-300 ms. There was a single death in this series. CONCLUSIONS: Cases tended to be younger males. Most patients had some heart block on discharge. Of patients evaluated as outpatients, Lyme disease was suspected in 41%. Improved early recognition and treatment of Lyme disease may decrease Lyme carditis.

Bedaquiline for multidrug-resistant tuberculosis and QTc prolongation in California.

BACKGROUND: Bedaquiline (BDQ) is recommended for the treatment of multidrug-resistant tuberculosis (MDR TB), however, it has the potential to prolong QTc interval. We assessed the frequency and severity of QTc prolongation in patients receiving BDQ in California. METHODS: Based on chart review for patients receiving BDQ as part of MDR TB therapy from January 2013-May 2019, we analyzed QTc values at six pre-specified time points during BDQ therapy (baseline, 2, 4, 8, 12, and 24 weeks), as well as peak QTc, time to peak QTc, and the clinical characteristics of patients who had QTc elevation >500 milliseconds (ms) during therapy. RESULTS: A total of 37 patients were treated with BDQ during the analysis period, with a total of 449 QTc measurements available for analysis. Most patients (89%) received at least one QTc-prolonging drug in addition to BDQ. Median QTc values at all pre-specified time points were <450 ms. Median peak QTc was 455 ms (interquartile range [IQR]: 437-486) and median time to peak was 57 days (IQR: 19-156). Four patients (11%) had a non-transient elevation in QTc to >500 ms, including one patient with profound hypokalemia and one receiving concurrent chemotherapy, but none had cardiac arrhythmia. Less than 10% of patient in our cohort had ECGs performed at all six pre-specified time points. DISCUSSION: BDQ was generally well-tolerated in a cohort of patients treated for MDR TB in California, with 11% of patients experiencing a non-transient QTc elevation >500 ms, and no episodes of arrhythmia. Frequent ECG monitoring during BDQ therapy presents a challenge for TB clinicians, even in well-resourced countries.

Development and Validation of an Ultradeep Next-Generation Sequencing Assay for Testing of Plasma Cell-Free DNA from Patients with Advanced Cancer.

Purpose: Tumor-derived cell-free DNA (cfDNA) in plasma can be used for molecular testing and provide an attractive alternative to tumor tissue. Commonly used PCR-based technologies can test for limited number of alterations at the time. Therefore, novel ultrasensitive technologies capable of testing for a broad spectrum of molecular alterations are needed to further personalized cancer therapy.Experimental Design: We developed a highly sensitive ultradeep next-generation sequencing (NGS) assay using reagents from TruSeqNano library preparation and NexteraRapid Capture target enrichment kits to generate plasma cfDNA sequencing libraries for mutational analysis in 61 cancer-related genes using common bioinformatics tools. The results were retrospectively compared with molecular testing of archival primary or metastatic tumor tissue obtained at different points of clinical care.Results: In a study of 55 patients with advanced cancer, the ultradeep NGS assay detected 82% (complete detection) to 87% (complete and partial detection) of the aberrations identified in discordantly collected corresponding archival tumor tissue. Patients with a low variant allele frequency (VAF) of mutant cfDNA survived longer than those with a high VAF did (P = 0.018). In patients undergoing systemic therapy, radiological response was positively associated with changes in cfDNA VAF (P = 0.02), and compared with unchanged/increased mutant cfDNA VAF, decreased cfDNA VAF was associated with longer time to treatment failure (TTF; P = 0.03).Conclusions: Ultradeep NGS assay has good sensitivity compared with conventional clinical mutation testing of archival specimens. A high VAF in mutant cfDNA corresponded with shorter survival. Changes in VAF of mutated cfDNA were associated with TTF. Clin Cancer Res; 23(18); 5648-56. ©2017 AACR.

Neuronal subtypes and diversity revealed by single-nucleus RNA sequencing of the human brain.

The human brain has enormously complex cellular diversity and connectivities fundamental to our neural functions, yet difficulties in interrogating individual neurons has impeded understanding of the underlying transcriptional landscape. We developed a scalable approach to sequence and quantify RNA molecules in isolated neuronal nuclei from a postmortem brain, generating 3227 sets of single-neuron data from six distinct regions of the cerebral cortex. Using an iterative clustering and classification approach, we identified 16 neuronal subtypes that were further annotated on the basis of known markers and cortical cytoarchitecture. These data demonstrate a robust and scalable method for identifying and categorizing single nuclear transcriptomes, revealing shared genes sufficient to distinguish previously unknown and orthologous neuronal subtypes as well as regional identity and transcriptomic heterogeneity within the human brain.

High density DNA methylation array with single CpG site resolution.

We have developed a new generation of genome-wide DNA methylation BeadChip which allows high-throughput methylation profiling of the human genome. The new high density BeadChip can assay over 480K CpG sites and analyze twelve samples in parallel. The innovative content includes coverage of 99% of RefSeq genes with multiple probes per gene, 96% of CpG islands from the UCSC database, CpG island shores and additional content selected from whole-genome bisulfite sequencing data and input from DNA methylation experts. The well-characterized Infinium® Assay is used for analysis of CpG methylation using bisulfite-converted genomic DNA. We applied this technology to analyze DNA methylation in normal and tumor DNA samples and compared results with whole-genome bisulfite sequencing (WGBS) data obtained for the same samples. Highly comparable DNA methylation profiles were generated by the array and sequencing methods (average R2 of 0.95). The ability to determine genome-wide methylation patterns will rapidly advance methylation research.

Strengthening and neuromuscular reeducation of the gluteus maximus in a triathlete with exercise-associated cramping of the hamstrings.

STUDY DESIGN: Case report. OBJECTIVE: To highlight the effects of an intervention program consisting of strengthening and neuromuscular reeducation of the gluteus maximus in an elite triathlete with exercise-associated muscle cramping (EAMC). BACKGROUND: Researchers have described 2 theories concerning the etiology of EAMC: (1) muscle fatigue and (2) electrolyte deficit. As such, interventions for EAMC typically consist of stretching/strengthening of the involved muscle and/or supplements to restore electrolyte imbalances. CASE DESCRIPTION: The patient was a 42-year-old male triathlete with a primary complaint of recurrent cramping of his right hamstring muscle, which prevented him from completing races at his desired pace. Strength testing revealed gluteus maximus muscle weakness bilaterally. Electromyographic (EMG) analysis (surface electrodes, 1560 Hz) revealed that the right hamstrings were being activated excessively during terminal swing and the first half of the stance phase (48.1% maximum voluntary isometric contraction [MVIC]). OUTCOMES: Following the intervention, the patient was able to complete 3 triathlons without hamstring cramping. Strength testing revealed that the right hip extension strength improved from 35.6 to 54.7 kg, and activation of the hamstrings during terminal swing and the first half of the stance phase decreased to 36.4% of MVIC. DISCUSSION: A program of gluteus maximus strengthening and neuromuscular training eliminated EAMC of the hamstrings in this patient. Given that the hamstrings and gluteus maximus work as agonists to decelerate the thigh during terminal swing phase and control hip flexion during loading response of running, we postulate that strengthening of the gluteus maximus decreased the relative effort required by the hamstrings, thus reducing EAMC. The results of the EMG evaluation that was performed as part of this case report provides support for this hypothesis. LEVEL OF EVIDENCE: Therapy, level 4.

Medium- to high-throughput SNP genotyping using VeraCode microbeads.

Recent breakthroughs in multiplexed SNP (single nucleotide polymorphism) genotyping technology have enabled global mapping of the relationships between genetic variation and disease. Discoveries made by such whole-genome association studies often spur further interest in surveying more focused subsets of SNPs for validation or research purposes. Here we describe a new SNP genotyping platform that is flexible in assay content and multiplexing (up to 384 analytes), and can serve medium- to high-throughput applications. The Illumina BeadXpress platform supports the GoldenGate Genotyping Assay on digitally inscribed VeraCode microbeads to allow streamlined workflow, rapid detection, unparalleled data reproducibility and consistency. Thus, it is a highly valuable tool for biomarker research and validation, pharmaceutical development, as well as the development of molecular diagnostic tests.

Genome-wide DNA methylation profiling using Infinium® assay.

AIMS: Bisulfite sequence analysis of individual CpG sites within genomic DNA is a powerful approach for methylation analysis in the genome. The major limitation of bisulfite-based methods is parallelization. Both array and next-generation sequencing technology are capable of addressing this bottleneck. In this report, we describe the application of Infinium® genotyping technology to analyze bisulfite-converted DNA to simultaneously query the methylation state of over 27,000 CpG sites from promoters of consensus coding sequences (CCDS) genes. MATERIALS & METHODS: We adapted the Infinium genotyping assay to readout an array of over 27,000 pairs of CpG methylation-specific query probes complementary to bisulfite-converted DNA. Two probes were designed to each CpG site: a 'methylated' and an 'unmethylated' query probe. The probe design assumed that all underlying CpG sites were 'in phase' with the queried CpG site due to their close proximity. Bisulfite conversion was performed with a modified version of the Zymo EZ DNA Methylation™ kit. RESULTS: We applied this technology to measuring methylation levels across a panel of 14 different human tissues, four Coriell cell lines and six cancer cell lines. We observed that CpG sites within CpG islands (CGIs) were largely unmethylated across all tissues (~80% sites unmethylated, ß < 0.2), whereas CpG sites in non-CGIs were moderately to highly methylated (only ~12% sites unmethylated, ß < 0.2). Within CGIs, only approximately 3-6% of the loci were highly methylated; in contrast, outside of CGIs approximately 25-40% of loci were highly methylated. Moreover, tissue-specific methylation (variation in methylation across tissues) was much more prevalent in non-CGIs than within CGIs. CONCLUSION: Our results demonstrate a genome-wide scalable array-based methylation readout platform that is both highly reproducible and quantitative. In the near future, this platform should enable the analysis of hundreds of thousands to millions of CpG sites per sample.

Power to detect risk alleles using genome-wide tag SNP panels.

Advances in high-throughput genotyping and the International HapMap Project have enabled association studies at the whole-genome level. We have constructed whole-genome genotyping panels of over 550,000 (HumanHap550) and 650,000 (HumanHap650Y) SNP loci by choosing tag SNPs from all populations genotyped by the International HapMap Project. These panels also contain additional SNP content in regions that have historically been overrepresented in diseases, such as nonsynonymous sites, the MHC region, copy number variant regions and mitochondrial DNA. We estimate that the tag SNP loci in these panels cover the majority of all common variation in the genome as measured by coverage of both all common HapMap SNPs and an independent set of SNPs derived from complete resequencing of genes obtained from SeattleSNPs. We also estimate that, given a sample size of 1,000 cases and 1,000 controls, these panels have the power to detect single disease loci of moderate risk (lambda approximately 1.8-2.0). Relative risks as low as lambda approximately 1.1-1.3 can be detected using 10,000 cases and 10,000 controls depending on the sample population and disease model. If multiple loci are involved, the power increases significantly to detect at least one locus such that relative risks 20%-35% lower can be detected with 80% power if between two and four independent loci are involved. Although our SNP selection was based on HapMap data, which is a subset of all common SNPs, these panels effectively capture the majority of all common variation and provide high power to detect risk alleles that are not represented in the HapMap data.

High-resolution genomic profiling of chromosomal aberrations using Infinium whole-genome genotyping.

Array-CGH is a powerful tool for the detection of chromosomal aberrations. The introduction of high-density SNP genotyping technology to genomic profiling, termed SNP-CGH, represents a further advance, since simultaneous measurement of both signal intensity variations and changes in allelic composition makes it possible to detect both copy number changes and copy-neutral loss-of-heterozygosity (LOH) events. We demonstrate the utility of SNP-CGH with two Infinium whole-genome genotyping BeadChips, assaying 109,000 and 317,000 SNP loci, to detect chromosomal aberrations in samples bearing constitutional aberrations as well tumor samples at sub-100 kb effective resolution. Detected aberrations include homozygous deletions, hemizygous deletions, copy-neutral LOH, duplications, and amplifications. The statistical ability to detect common aberrations was modeled by analysis of an X chromosome titration model system, and sensitivity was modeled by titration of gDNA from a tumor cell with that of its paired normal cell line. Analysis was facilitated by using a genome browser that plots log ratios of normalized intensities and allelic ratios along the chromosomes. We developed two modes of SNP-CGH analysis, a single sample and a paired sample mode. The single sample mode computes log intensity ratios and allelic ratios by referencing to canonical genotype clusters generated from approximately 120 reference samples, whereas the paired sample mode uses a paired normal reference sample from the same individual. Finally, the two analysis modes are compared and contrasted for their utility in analyzing different types of input gDNA: low input amounts, fragmented gDNA, and Phi29 whole-genome pre-amplified DNA.

Illumina universal bead arrays.

This chapter describes an accurate, scalable, and flexible microarray technology. It includes a miniaturized array platform where each individual feature is quality controlled and a versatile assay that can be adapted for various genetic analyses, such as single nucleotide polymorphism genotyping, DNA methylation detection, and gene expression profiling. This chapter describes the concept of the BeadArray technology, two different Array of Arrays formats, the assay scheme and protocol, the performance of the system, and its use in large-scale genetic, epigenetic, and expression studies.

Whole-genome genotyping.

We have developed an array-based whole-genome genotyping (WGG) assay (Infinium) using our BeadChip platform that effectively enables unlimited multiplexing and unconstrained single nucleotide polymorphism (SNP) selection. A single tube whole-genome amplification reaction is used to amplify the genome, and loci of interest are captured by specific hybridization of amplified gDNA to 50-mer probe arrays. After target capture, SNPs are genotyped on the array by a primer extension reaction in the presence of hapten-labeled nucleotides. The resultant signal is amplified during staining and the array is read out on a high-resolution confocal scanner. We have employed our high-density BeadChips supporting up to 288,000 bead types to create an array that can query over 100,000 SNPs using the Infinium assay. In addition, we have developed an automated BeadChip processing platform using Tecan's GenePaint slide processing system. Hybridization, washing, array-based primer extension, and staining are performed directly in Tecan's capillary gap Te-Flow chambers. This automation process increases assay robustness and throughput greatly while enabling laboratory information management system control of sample tracking.

Whole-genome genotyping of haplotype tag single nucleotide polymorphisms.

The International HapMap Consortium recently completed genotyping over 3.8 million single nucleotide polymorphisms (SNPs) in three major populations, and the results of studying patterns of linkage disequilibrium indicate that characterization of 300,000-500,000 tag SNPs is sufficient to provide good genomic coverage for linkage-disequilibrium-based association studies in many populations. These whole-genome association studies will be used to dissect the genetics of complex diseases and pharmacogenomic drug responses. As such, the development of a cost-effective genotyping platform that can assay hundred of thousands of SNPs across thousands of samples is essential. In this review, we describe the development of a whole-genome genotyping (WGG) assay that enables unconstrained SNP selection and effectively unlimited multiplexing from a single sample preparation. The development of WGG in concert with high-density BeadChips has enabled the creation of three different high-density SNP genotyping BeadChips: the Sentrix Human-1 Genotyping BeadChip containing over 109,000 exon-centric SNPs; the HumanHap300 BeadChip containing over 317,000 tag SNPs, and the HumanHap550 Beadchip containing over 550,000 tag SNPs.

Whole-genome genotyping with the single-base extension assay.

We describe an efficient, accurate and robust whole-genome genotyping (WGG) assay based on a two-color, single-base extension (SBE), single-nucleotide polymorphism (SNP)-scoring step. We report genotyping results for biallelic International HapMap quality control (QC) SNPs using a single probe per locus. We show scalability, throughput and accuracy of the system by resequencing homozygous loci from our 100k Human-1 Genotyping BeadChip.

High-throughput SNP genotyping on universal bead arrays.

We have developed a flexible, accurate and highly multiplexed SNP genotyping assay for high-throughput genetic analysis of large populations on a bead array platform. The novel genotyping system combines high assay conversion rate and data quality with >1500 multiplexing, and Array of Arrays formats. Genotyping assay oligos corresponding to specific SNP sequences are each linked to a unique sequence (address) that can hybridize to its complementary strand on universal arrays. The arrays are made of beads located in microwells of optical fiber bundles (Sentrix Array Matrix) or silicon slides (Sentrix BeadChip). The optical fiber bundles are further organized into a matrix that matches a 96-well microtiter plate. The arrays on the silicon slides are multi-channel pipette compatible for loading multiple samples onto a single silicon slide. These formats allow many samples to be processed in parallel. This genotyping system enables investigators to generate approximately 300,000 genotypes per day with minimal equipment requirements and greater than 1.6 million genotypes per day in a robotics-assisted process. With a streamlined and comprehensive assay, this system brings a new level of flexibility, throughput, and affordability to genetic research.

A versatile assay for high-throughput gene expression profiling on universal array matrices.

We report a flexible, sensitive, and quantitative gene-expression profiling system for assaying more than 400 genes, with three probes per gene, for 96 samples in parallel. The cDNA-mediated annealing, selection, extension and ligation (DASL) assay targets specific transcripts, using oligonucleotides containing unique address sequences that can hybridize to universal arrays. Cell-specific gene expression profiles were obtained using this assay for hormone-treated cell lines and laser-capture microdissected cancer tissues. Gene expression profiles derived from this assay were consistent with those determined by qRT-PCR. The DASL assay has been automated for use with a bead-based 96-array matrix system. The combined high-throughput assay and readout system is accurate and efficient, and can cost-effectively profile the expression of hundreds of genes in thousands of samples.

Two methods of whole-genome amplification enable accurate genotyping across a 2320-SNP linkage panel.

Comprehensive genome scans involving many thousands of SNP assays will require significant amounts of genomic DNA from each sample. We report two successful methods for amplifying whole-genomic DNA prior to SNP analysis, multiple displacement amplification, and OmniPlex technology. We determined the coverage of amplification by analyzing a SNP linkage marker set that contained 2320 SNP markers spread across the genome at an average distance of 2.5 cM. We observed a concordance of >99.8% in genotyping results from genomic DNA and amplified DNA, strongly indicating the ability of both methods used to amplify genomic DNA in a highly representative manner. Furthermore, we were able to achieve a SNP call rate of >98% in both genomic and amplified DNA. The combination of whole-genome amplification and comprehensive SNP linkage analysis offers new opportunities for genetic analysis in clinical trials, disease association studies, and archiving of DNA samples.

A highly informative SNP linkage panel for human genetic studies.

We have developed a highly informative set of single-nucleotide polymorphism (SNP) assays designed for linkage mapping of the human genome. These assays were developed on a robust multiplexed assay system to provide a combination of very high accuracy and data completeness with high throughput for linkage studies. The linkage panel is comprised of approximately 4,700 SNPs with 0.39 average minor allele frequency and 624-kb average spacing. Based on almost 2 million genotypes, data quality was shown to be extremely high, with a 99.94% call rate, >99.99% reproducibility and 99.995% genotypes consistent with mendelian inheritance. We constructed a genetic map with an average 1.5-cM resolution using series of 28 CEPH pedigrees. The relative information content of this panel was higher than those of commonly used STR marker panels. The potent combination of this SNP linkage panel with the multiplexed assay system provides a previously unattainable level of performance for linkage studies.

A draft sequence of the rice genome (Oryza sativa L. ssp. japonica).

The genome of the japonica subspecies of rice, an important cereal and model monocot, was sequenced and assembled by whole-genome shotgun sequencing. The assembled sequence covers 93% of the 420-megabase genome. Gene predictions on the assembled sequence suggest that the genome contains 32,000 to 50,000 genes. Homologs of 98% of the known maize, wheat, and barley proteins are found in rice. Synteny and gene homology between rice and the other cereal genomes are extensive, whereas synteny with Arabidopsis is limited. Assignment of candidate rice orthologs to Arabidopsis genes is possible in many cases. The rice genome sequence provides a foundation for the improvement of cereals, our most important crops.