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July 19, 2019

Utility of DNA, RNA, protein, and functional approaches to solve cryptic immunodeficiencies.

We report a female infant identified by newborn screening for severe combined immunodeficiencies (NBS SCID) with T cell lymphopenia (TCL). The patient had persistently elevated alpha-fetoprotein (AFP) with IgA deficiency, and elevated IgM. Gene sequencing for a SCID panel was uninformative. We sought to determine the cause of the immunodeficiency in this infant.We performed whole-exome sequencing (WES) on the patient and parents to identify a genetic diagnosis. Based on the WES result, we developed a novel flow cytometric panel for rapid assessment of DNA repair defects using blood samples. We also performed whole transcriptome sequencing (WTS) on fibroblast RNA from the patient and father for abnormal transcript analysis.WES revealed a pathogenic paternally inherited indel in ATM. We used the flow panel to assess several proteins in the DNA repair pathway in lymphocyte subsets. The patient had absent phosphorylation of ATM, resulting in absent or aberrant phosphorylation of downstream proteins, including ?H2AX. However, ataxia-telangiectasia (AT) is an autosomal recessive condition, and the abnormal functional data did not correspond with a single ATM variant. WTS revealed in-frame reciprocal fusion transcripts involving ATM and SLC35F2 indicating a chromosome 11 inversion within 11q22.3, of maternal origin. Inversion breakpoints were identified within ATM intron 16 and SLC35F2 intron 7.We identified a novel ATM-breaking chromosome 11 inversion in trans with a pathogenic indel (compound heterozygote) resulting in non-functional ATM protein, consistent with a diagnosis of AT. Utilization of several molecular and functional assays allowed successful resolution of this case.

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July 19, 2019

HIV envelope glycoform heterogeneity and localized diversity govern the initiation and maturation of a V2 apex broadly neutralizing antibody lineage.

Understanding how broadly neutralizing antibodies (bnAbs) to HIV envelope (Env) develop during natural infection can help guide the rational design of an HIV vaccine. Here, we described a bnAb lineage targeting the Env V2 apex and the Ab-Env co-evolution that led to development of neutralization breadth. The lineage Abs bore an anionic heavy chain complementarity-determining region 3 (CDRH3) of 25 amino acids, among the shortest known for this class of Abs, and achieved breadth with only 10% nucleotide somatic hypermutation and no insertions or deletions. The data suggested a role for Env glycoform heterogeneity in the activation of the lineage germline B cell. Finally, we showed that localized diversity at key V2 epitope residues drove bnAb maturation toward breadth, mirroring the Env evolution pattern described for another donor who developed V2-apex targeting bnAbs. Overall, these findings suggest potential strategies for vaccine approaches based on germline-targeting and serial immunogen design. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

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July 19, 2019

Discordant inheritance of chromosomal and extrachromosomal DNA elements contributes to dynamic disease evolution in glioblastoma.

To understand how genomic heterogeneity of glioblastoma (GBM) contributes to poor therapy response, we performed DNA and RNA sequencing on GBM samples and the neurospheres and orthotopic xenograft models derived from them. We used the resulting dataset to show that somatic driver alterations including single-nucleotide variants, focal DNA alterations and oncogene amplification on extrachromosomal DNA (ecDNA) elements were in majority propagated from tumor to model systems. In several instances, ecDNAs and chromosomal alterations demonstrated divergent inheritance patterns and clonal selection dynamics during cell culture and xenografting. We infer that ecDNA was unevenly inherited by offspring cells, a characteristic that affects the oncogenic potential of cells with more or fewer ecDNAs. Longitudinal patient tumor profiling found that oncogenic ecDNAs are frequently retained throughout the course of disease. Our analysis shows that extrachromosomal elements allow rapid increase of genomic heterogeneity during GBM evolution, independently of chromosomal DNA alterations.

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July 19, 2019

Unexpected diversity in the mobilome of a Pseudomonas aeruginosa strain isolated from a dental unit waterline revealed by SMRT Sequencing.

The Gram-negative bacterium Pseudomonas aeruginosa is found in several habitats, both natural and human-made, and is particularly known for its recurrent presence as a pathogen in the lungs of patients suffering from cystic fibrosis, a genetic disease. Given its clinical importance, several major studies have investigated the genomic adaptation of P. aeruginosa in lungs and its transition as acute infections become chronic. However, our knowledge about the diversity and adaptation of the P. aeruginosa genome to non-clinical environments is still fragmentary, in part due to the lack of accurate reference genomes of strains from the numerous environments colonized by the bacterium. Here, we used PacBio long-read technology to sequence the genome of PPF-1, a strain of P. aeruginosa isolated from a dental unit waterline. Generating this closed genome was an opportunity to investigate genomic features that are difficult to accurately study in a draft genome (contigs state). It was possible to shed light on putative genomic islands, some shared with other reference genomes, new prophages, and the complete content of insertion sequences. In addition, four different group II introns were also found, including two characterized here and not listed in the specialized group II intron database.

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July 19, 2019

Long read assemblies of geographically dispersed Plasmodium falciparum isolates reveal highly structured subtelomeres.

Background: Although thousands of clinical isolates of Plasmodium falciparum are being sequenced and analysed by short read technology, the data do not resolve the highly variable subtelomeric regions of the genomes that contain polymorphic gene families involved in immune evasion and pathogenesis. There is also no current standard definition of the boundaries of these variable subtelomeric regions. Methods: Using long-read sequence data (Pacific Biosciences SMRT technology), we assembled and annotated the genomes of 15 P. falciparum isolates, ten of which are newly cultured clinical isolates. We performed comparative analysis of the entire genome with particular emphasis on the subtelomeric regions and the internal var genes clusters.   Results: The nearly complete sequence of these 15 isolates has enabled us to define a highly conserved core genome, to delineate the boundaries of the subtelomeric regions, and to compare these across isolates. We found highly structured variable regions in the genome. Some exported gene families purportedly involved in release of merozoites show copy number variation. As an example of ongoing genome evolution, we found a novel CLAG gene in six isolates.  We also found a novel gene that was relatively enriched in the South East Asian isolates compared to those from Africa. Conclusions: These 15 manually curated new reference genome sequences with their nearly complete subtelomeric regions and fully assembled genes are an important new resource for the malaria research community. We report the overall conserved structure and pattern of important gene families and the more clearly defined subtelomeric regions.

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July 19, 2019

Antigenic variation in the lyme spirochete: Insights into recombinational switching with a suggested role for error-prone repair.

The Lyme disease spirochete, Borrelia burgdorferi, uses antigenic variation as a strategy to evade the host’s acquired immune response. New variants of surface-localized VlsE are generated efficiently by unidirectional recombination from 15 unexpressed vls cassettes into the vlsE locus. Using algorithms to analyze switching from vlsE sequencing data, we characterize a population of over 45,000 inferred recombination events generated during mouse infection. We present evidence for clustering of these recombination events within the population and along the vlsE gene, a role for the direct repeats flanking the variable region in vlsE, and the importance of sequence homology in determining the location of recombination, despite RecA’s dispensability. Finally, we report that non-templated sequence variation is strongly associated with recombinational switching and occurs predominantly at the 5′ end of conversion tracts. This likely results from an error-prone repair mechanism operational during recombinational switching that elevates the mutation rate > 5,000-fold in switched regions. Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.

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July 19, 2019

Detailed analysis of HTT repeat elements in human blood using targeted amplification-free long-read sequencing.

Amplification of DNA is required as a mandatory step during library preparation in most targeted sequencing protocols. This can be a critical limitation when targeting regions that are highly repetitive or with extreme guanine-cytosine (GC) content, including repeat expansions associated with human disease. Here, we used an amplification-free protocol for targeted enrichment utilizing the CRISPR/Cas9 system (No-Amp Targeted sequencing) in combination with single molecule, real-time (SMRT) sequencing for studying repeat elements in the huntingtin (HTT) gene, where an expanded CAG repeat is causative for Huntington disease. We also developed a robust data analysis pipeline for repeat element analysis that is independent of alignment of reads to a reference genome. The method was applied to 11 diagnostic blood samples, and for all 22 alleles the resulting CAG repeat count agreed with previous results based on fragment analysis. The amplification-free protocol also allowed for studying somatic variability of repeat elements in our samples, without the interference of PCR stutter. In summary, with No-Amp Targeted sequencing in combination with our analysis pipeline, we could accurately study repeat elements that are difficult to investigate using PCR-based methods.© 2018 The Authors. Human Mutation published by Wiley Periodicals, Inc.

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July 19, 2019

De novo repeat interruptions are associated with reduced somatic instability and mild or absent clinical features in myotonic dystrophy type 1.

Myotonic dystrophy type 1 (DM1) is a multisystem disorder, caused by expansion of a CTG trinucleotide repeat in the 3′-untranslated region of the DMPK gene. The repeat expansion is somatically unstable and tends to increase in length with time, contributing to disease progression. In some individuals, the repeat array is interrupted by variant repeats such as CCG and CGG, stabilising the expansion and often leading to milder symptoms. We have characterised three families, each including one person with variant repeats that had arisen de novo on paternal transmission of the repeat expansion. Two individuals were identified for screening due to an unusual result in the laboratory diagnostic test, and the third due to exceptionally mild symptoms. The presence of variant repeats in all three expanded alleles was confirmed by restriction digestion of small pool PCR products, and allele structures were determined by PacBio sequencing. Each was different, but all contained CCG repeats close to the 3′-end of the repeat expansion. All other family members had inherited pure CTG repeats. The variant repeat-containing alleles were more stable in the blood than pure alleles of similar length, which may in part account for the mild symptoms observed in all three individuals. This emphasises the importance of somatic instability as a disease mechanism in DM1. Further, since patients with variant repeats may have unusually mild symptoms, identification of these individuals has important implications for genetic counselling and for patient stratification in DM1 clinical trials.

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July 19, 2019

A Borrelia burgdorferi mini-vls system that undergoes antigenic switching in mice: investigation of the role of plasmid topology and the long inverted repeat.

Borrelia burgdorferi evades the host immune system by switching the surface antigen. VlsE, in a process known as antigenic variation. The DNA mechanisms and genetic elements present on the vls locus that participate in the switching process remain to be elucidated. Manipulating the vls locus has been difficult due to its instability on Escherichia coli plasmids. In this study, we generated for the first time a mini-vls system composed of a single silent vlsE variable region (silent cassette 2) through the vlsE gene by performing some cloning steps directly in a highly transformable B. burgdorferi strain. Variants of the mini system were constructed with or without the long inverted repeat (IR) located upstream of vlsE and on both circular and linear plasmids to investigate the importance of the IR and plasmid topology on recombinational switching at vlsE. Amplicon sequencing using PacBio long read technology and analysis of the data with our recently reported pipeline and VAST software showed that the system undergoes switching in mice in both linear and circular versions and that the presence of the hairpin does not seem to be crucial in the linear version, however it is required when the topology is circular.© 2018 John Wiley & Sons Ltd.

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July 19, 2019

Adaptation and conservation insights from the koala genome.

The koala, the only extant species of the marsupial family Phascolarctidae, is classified as ‘vulnerable’ due to habitat loss and widespread disease. We sequenced the koala genome, producing a complete and contiguous marsupial reference genome, including centromeres. We reveal that the koala’s ability to detoxify eucalypt foliage may be due to expansions within a cytochrome P450 gene family, and its ability to smell, taste and moderate ingestion of plant secondary metabolites may be due to expansions in the vomeronasal and taste receptors. We characterized novel lactation proteins that protect young in the pouch and annotated immune genes important for response to chlamydial disease. Historical demography showed a substantial population crash coincident with the decline of Australian megafauna, while contemporary populations had biogeographic boundaries and increased inbreeding in populations affected by historic translocations. We identified genetically diverse populations that require habitat corridors and instituting of translocation programs to aid the koala’s survival in the wild.

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July 19, 2019

Population genomics shows no distinction between pathogenic Candida krusei and environmental Pichia kudriavzevii: One species, four names.

We investigated genomic diversity of a yeast species that is both an opportunistic pathogen and an important industrial yeast. Under the name Candida krusei, it is responsible for about 2% of yeast infections caused by Candida species in humans. Bloodstream infections with C. krusei are problematic because most isolates are fluconazole-resistant. Under the names Pichia kudriavzevii, Issatchenkia orientalis and Candida glycerinogenes, the same yeast, including genetically modified strains, is used for industrial-scale production of glycerol and succinate. It is also used to make some fermented foods. Here, we sequenced the type strains of C. krusei (CBS573T) and P. kudriavzevii (CBS5147T), as well as 30 other clinical and environmental isolates. Our results show conclusively that they are the same species, with collinear genomes 99.6% identical in DNA sequence. Phylogenetic analysis of SNPs does not segregate clinical and environmental isolates into separate clades, suggesting that C. krusei infections are frequently acquired from the environment. Reduced resistance of strains to fluconazole correlates with the presence of one gene instead of two at the ABC11-ABC1 tandem locus. Most isolates are diploid, but one-quarter are triploid. Loss of heterozygosity is common, including at the mating-type locus. Our PacBio/Illumina assembly of the 10.8 Mb CBS573T genome is resolved into 5 complete chromosomes, and was annotated using RNAseq support. Each of the 5 centromeres is a 35 kb gene desert containing a large inverted repeat. This species is a member of the genus Pichia and family Pichiaceae (the methylotrophic yeasts clade), and so is only distantly related to other pathogenic Candida species.

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July 19, 2019

Loss of maternal EED results in postnatal overgrowth.

Investigating how epigenetic information is transmitted through the mammalian germline is the key to understanding how this information impacts on health and disease susceptibility in offspring. EED is essential for regulating the repressive histone modification, histone 3 lysine 27 tri-methylation (H3K27me3) at many developmental genes.In this study, we used oocyte-specific Zp3-Cre recombinase (Zp3Cre) to delete Eed specifically in mouse growing oocytes, permitting the study of EED function in oocytes and the impact of depleting EED in oocytes on outcomes in offspring. As EED deletion occurred only in growing oocytes and females were mated to normal wild type males, this model allowed the study of oocyte programming without confounding factors such as altered in utero environment. Loss of EED from growing oocytes resulted in a significant overgrowth phenotype that persisted into adult life. Significantly, this involved increased adiposity (total fat) and bone mineral density in offspring. Similar overgrowth occurs in humans with Cohen-Gibson (OMIM 617561) and Weaver (OMIM 277590) syndromes, that result from de novo germline mutations in EED or its co-factor EZH2, respectively. Consistent with a role for EZH2 in human oocytes, we demonstrate that de novo germline mutations in EZH2 occurred in the maternal germline in some cases of Weaver syndrome. However, deletion of Ezh2 in mouse oocytes resulted in a distinct phenotype compared to that resulting from oocyte-specific deletion of Eed.This study provides novel evidence that altering EED-dependent oocyte programming leads to compromised offspring growth and development in the next generation.

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July 19, 2019

Deep genome annotation of the opportunistic human pathogen Streptococcus pneumoniae D39.

A precise understanding of the genomic organization into transcriptional units and their regulation is essential for our comprehension of opportunistic human pathogens and how they cause disease. Using single-molecule real-time (PacBio) sequencing we unambiguously determined the genome sequence of Streptococcus pneumoniae strain D39 and revealed several inversions previously undetected by short-read sequencing. Significantly, a chromosomal inversion results in antigenic variation of PhtD, an important surface-exposed virulence factor. We generated a new genome annotation using automated tools, followed by manual curation, reflecting the current knowledge in the field. By combining sequence-driven terminator prediction, deep paired-end transcriptome sequencing and enrichment of primary transcripts by Cappable-Seq, we mapped 1015 transcriptional start sites and 748 termination sites. We show that the pneumococcal transcriptional landscape is complex and includes many secondary, antisense and internal promoters. Using this new genomic map, we identified several new small RNAs (sRNAs), RNA switches (including sixteen previously misidentified as sRNAs), and antisense RNAs. In total, we annotated 89 new protein-encoding genes, 34 sRNAs and 165 pseudogenes, bringing the S. pneumoniae D39 repertoire to 2146 genetic elements. We report operon structures and observed that 9% of operons are leaderless. The genome data are accessible in an online resource called PneumoBrowse (https://veeninglab.com/pneumobrowse) providing one of the most complete inventories of a bacterial genome to date. PneumoBrowse will accelerate pneumococcal research and the development of new prevention and treatment strategies.

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July 19, 2019

Long-read sequencing across the C9orf72 ‘GGGGCC’ repeat expansion: implications for clinical use and genetic discovery efforts in human disease.

Many neurodegenerative diseases are caused by nucleotide repeat expansions, but most expansions, like the C9orf72 ‘GGGGCC’ (G4C2) repeat that causes approximately 5-7% of all amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) cases, are too long to sequence using short-read sequencing technologies. It is unclear whether long-read sequencing technologies can traverse these long, challenging repeat expansions. Here, we demonstrate that two long-read sequencing technologies, Pacific Biosciences’ (PacBio) and Oxford Nanopore Technologies’ (ONT), can sequence through disease-causing repeats cloned into plasmids, including the FTD/ALS-causing G4C2 repeat expansion. We also report the first long-read sequencing data characterizing the C9orf72 G4C2 repeat expansion at the nucleotide level in two symptomatic expansion carriers using PacBio whole-genome sequencing and a no-amplification (No-Amp) targeted approach based on CRISPR/Cas9.Both the PacBio and ONT platforms successfully sequenced through the repeat expansions in plasmids. Throughput on the MinION was a challenge for whole-genome sequencing; we were unable to attain reads covering the human C9orf72 repeat expansion using 15 flow cells. We obtained 8× coverage across the C9orf72 locus using the PacBio Sequel, accurately reporting the unexpanded allele at eight repeats, and reading through the entire expansion with 1324 repeats (7941 nucleotides). Using the No-Amp targeted approach, we attained >?800× coverage and were able to identify the unexpanded allele, closely estimate expansion size, and assess nucleotide content in a single experiment. We estimate the individual’s repeat region was >?99% G4C2 content, though we cannot rule out small interruptions.Our findings indicate that long-read sequencing is well suited to characterizing known repeat expansions, and for discovering new disease-causing, disease-modifying, or risk-modifying repeat expansions that have gone undetected with conventional short-read sequencing. The PacBio No-Amp targeted approach may have future potential in clinical and genetic counseling environments. Larger and deeper long-read sequencing studies in C9orf72 expansion carriers will be important to determine heterogeneity and whether the repeats are interrupted by non-G4C2 content, potentially mitigating or modifying disease course or age of onset, as interruptions are known to do in other repeat-expansion disorders. These results have broad implications across all diseases where the genetic etiology remains unclear.

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July 19, 2019

Single copy transgene integration in a transcriptionally active site for recombinant protein synthesis.

For the biomanufacturing of protein biologics, establishing stable cell lines with high transgene transcription is critical for high productivity. Modern genome engineering tools can direct transgene insertion to a specified genomic locus and can potentially become a valuable tool for cell line generation. In this study, the authors survey transgene integration sites and their transcriptional activity to identify characteristics of desirable regions. A lentivirus containing destabilized Green Fluorescent Protein (dGFP) is used to infect Chinese hamster ovary cells at a low multiplicity of infection, and cells with high or low GFP fluorescence are isolated. RNA sequencing and Assay for Transposase Accessible Chromatin using sequencing data shows integration sites with high GFP expression are in larger regions of high transcriptional activity and accessibility, but not necessarily within highly transcribed genes. This method is used to obtain high Immunoglobulin G (IgG) expressing cell lines with a single copy of the transgene integrated into transcriptionally active and accessible genomic regions. Dual recombinase-mediated cassette exchange is then employed to swap the IgG transgene for erythropoietin or tumor necrosis factor receptor-Fc. This work thus highlights a strategy to identify desirable sites for transgene integration and to streamline the development of new product producing cell lines.© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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