Publications by authors named "Matthew A Field"

18 Publications

  • Page 1 of 1

consensusDE: an R package for assessing consensus of multiple RNA-seq algorithms with RUV correction.

PeerJ 2019 13;7:e8206. Epub 2019 Dec 13.

Australian Institute for Tropical Health and Medicine, Centre for Tropical Bioinformatics and Molecular Biology, Centre for Molecular Therapeutics, James Cook University, Smithfield, Australia.

Extensive evaluation of RNA-seq methods have demonstrated that no single algorithm consistently outperforms all others. Removal of unwanted variation (RUV) has also been proposed as a method for stabilizing differential expression (DE) results. Despite this, it remains a challenge to run multiple RNA-seq algorithms to identify significant differences common to multiple algorithms, whilst also integrating and assessing the impact of RUV into all algorithms. consensusDE was developed to automate the process of identifying significant DE by combining the results from multiple algorithms with minimal user input and with the option to automatically integrate RUV. consensusDE only requires a table describing the sample groups, a directory containing BAM files or preprocessed count tables and an optional transcript database for annotation. It supports merging of technical replicates, paired analyses and outputs a compendium of plots to guide the user in subsequent analyses. Herein, we assess the ability of RUV to improve DE stability when combined with multiple algorithms and between algorithms, through application to real and simulated data. We find that, although RUV increased fold change stability between algorithms, it demonstrated improved FDR in a setting of low replication for the intersect, the effect was algorithm specific and diminished with increased replication, reinforcing increased replication for recovery of true DE genes. We finish by offering some rules and considerations for the application of RUV in a consensus-based setting. consensusDE is freely available, implemented in R and available as a Bioconductor package, under the GPL-3 license, along with a comprehensive vignette describing functionality: http://bioconductor.org/packages/consensusDE/.
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http://dx.doi.org/10.7717/peerj.8206DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6913255PMC
December 2019

Recurrent miscalling of missense variation from short-read genome sequence data.

BMC Genomics 2019 Jul 16;20(Suppl 8):546. Epub 2019 Jul 16.

Department of Immunology and Infectious Disease, The John Curtin School of Medical Research, The Australian National University, Canberra, Australian Capital Territory, Australia.

Background: Short-read resequencing of genomes produces abundant information of the genetic variation of individuals. Due to their numerous nature, these variants are rarely exhaustively validated. Furthermore, low levels of undetected variant miscalling will have a systematic and disproportionate impact on the interpretation of individual genome sequence information, especially should these also be carried through into in reference databases of genomic variation.

Results: We find that sequence variation from short-read sequence data is subject to recurrent-yet-intermittent miscalling that occurs in a sequence intrinsic manner and is very sensitive to sequence read length. The miscalls arise from difficulties aligning short reads to redundant genomic regions, where the rate of sequencing error approaches the sequence diversity between redundant regions. We find the resultant miscalled variants to be sensitive to small sequence variations between genomes, and thereby are often intrinsic to an individual, pedigree, strain or human ethnic group. In human exome sequences, we identify 2-300 recurrent false positive variants per individual, almost all of which are present in public databases of human genomic variation. From the exomes of non-reference strains of inbred mice, we identify 3-5000 recurrent false positive variants per mouse - the number of which increasing with greater distance between an individual mouse strain and the reference C57BL6 mouse genome. We show that recurrently miscalled variants may be reproduced for a given genome from repeated simulation rounds of read resampling, realignment and recalling. As such, it is possible to identify more than two-thirds of false positive variation from only ten rounds of simulation.

Conclusion: Identification and removal of recurrent false positive variants from specific individual variant sets will improve overall data quality. Variant miscalls arising are highly sequence intrinsic and are often specific to an individual, pedigree or ethnicity. Further, read length is a strong determinant of whether given false variants will be called for any given genome - which has profound significance for cohort studies that pool datasets collected and sequenced at different points in time.
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http://dx.doi.org/10.1186/s12864-019-5863-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6631443PMC
July 2019

Functional rare and low frequency variants in BLK and BANK1 contribute to human lupus.

Nat Commun 2019 05 17;10(1):2201. Epub 2019 May 17.

Department of Immunology, The Canberra Hospital, Garran, 2601, ACT, Australia.

Systemic lupus erythematosus (SLE) is the prototypic systemic autoimmune disease. It is thought that many common variant gene loci of weak effect act additively to predispose to common autoimmune diseases, while the contribution of rare variants remains unclear. Here we describe that rare coding variants in lupus-risk genes are present in most SLE patients and healthy controls. We demonstrate the functional consequences of rare and low frequency missense variants in the interacting proteins BLK and BANK1, which are present alone, or in combination, in a substantial proportion of lupus patients. The rare variants found in patients, but not those found exclusively in controls, impair suppression of IRF5 and type-I IFN in human B cell lines and increase pathogenic lymphocytes in lupus-prone mice. Thus, rare gene variants are common in SLE and likely contribute to genetic risk.
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http://dx.doi.org/10.1038/s41467-019-10242-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6525203PMC
May 2019

Gain-of-function mutation causes human combined immune deficiency.

J Exp Med 2018 11 18;215(11):2715-2724. Epub 2018 Oct 18.

Centre for Personalised Immunology, Australian National University, Canberra, Australia

Genetic mutations account for many devastating early onset immune deficiencies. In contrast, less severe and later onset immune diseases, including in patients with no prior family history, remain poorly understood. Whole exome sequencing in two cohorts of such patients identified a novel heterozygous de novo missense mutation (c.607G>A) in two separate kindreds in whom probands presented with immune dysregulation, combined T and B cell deficiency, inflammation, and epithelial defects. encodes IKK2, which activates NF-κB signaling. IKK2 results in enhanced NF-κB signaling, as well as T and B cell functional defects. IKK2 is a highly conserved residue, and to prove causation, we generated an accurate mouse model by introducing the precise orthologous codon change in using CRISPR/Cas9. Mice and humans carrying this missense mutation exhibit remarkably similar cellular and biochemical phenotypes. Accurate mouse models engineered by CRISPR/Cas9 can help characterize novel syndromes arising from de novo germline mutations and yield insight into pathogenesis.
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http://dx.doi.org/10.1084/jem.20180639DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6219745PMC
November 2018

Hookworm Secreted Extracellular Vesicles Interact With Host Cells and Prevent Inducible Colitis in Mice.

Front Immunol 2018 30;9:850. Epub 2018 Apr 30.

Centre for Biodiscovery and Molecular Development of Therapeutics, Australian Institute of Tropical Health and Medicine, James Cook University, Cairns, QLD, Australia.

Gastrointestinal (GI) parasites, hookworms in particular, have evolved to cause minimal harm to their hosts, allowing them to establish chronic infections. This is mediated by creating an immunoregulatory environment. Indeed, hookworms are such potent suppressors of inflammation that they have been used in clinical trials to treat inflammatory bowel diseases (IBD) and celiac disease. Since the recent description of helminths (worms) secreting extracellular vesicles (EVs), exosome-like EVs from different helminths have been characterized and their salient roles in parasite-host interactions have been highlighted. Here, we analyze EVs from the rodent parasite , which has been used as a model for human hookworm infection. EVs (-EVs) are actively internalized by mouse gut organoids, indicating a role in driving parasitism. We used proteomics and RNA-Seq to profile the molecular composition of -EVs. We identified 81 proteins, including proteins frequently present in exosomes (like tetraspanin, enolase, 14-3-3 protein, and heat shock proteins), and 27 sperm-coating protein-like extracellular proteins. RNA-Seq analysis revealed 52 miRNA species, many of which putatively map to mouse genes involved in regulation of inflammation. To determine whether GI nematode EVs had immunomodulatory properties, we assessed their potential to suppress GI inflammation in a mouse model of inducible chemical colitis. EVs from but not those from the whipworm or control vesicles from grapes protected against colitic inflammation in the gut of mice that received a single intraperitoneal injection of EVs. Key cytokines associated with colitic pathology (IL-6, IL-1β, IFNγ, and IL-17a) were significantly suppressed in colon tissues from EV-treated mice. By contrast, high levels of the anti-inflammatory cytokine IL-10 were detected in -EV-treated mice. Proteins and miRNAs contained within helminth EVs hold great potential application in development of drugs to treat helminth infections as well as chronic non-infectious diseases resulting from a dysregulated immune system, such as IBD.
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http://dx.doi.org/10.3389/fimmu.2018.00850DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5936971PMC
June 2019

Characterization of secreted proteins and extracellular vesicles provides new insights into host-parasite communication.

J Extracell Vesicles 2018 21;7(1):1428004. Epub 2018 Jan 21.

Centre for Biodiscovery and Molecular Development of Therapeutics, Australian Institute of Tropical Health and Medicine, James Cook University, Cairns, QLD, Australia.

Whipworms are parasitic nematodes that live in the gut of more than 500 million people worldwide. Owing to the difficulty in obtaining parasite material, the mouse whipworm has been extensively used as a model to study human whipworm infections. These nematodes secrete a multitude of compounds that interact with host tissues where they orchestrate a parasitic existence. Herein we provide the first comprehensive characterization of the excretory/secretory products of . We identify 148 proteins secreted by and show for the first time that the mouse whipworm secretes exosome-like extracellular vesicles (EVs) that can interact with host cells. We use an Optiprep® gradient to purify the EVs, highlighting the suitability of this method for purifying EVs secreted by a parasitic nematode. We also characterize the proteomic and genomic content of the EVs, identifying >350 proteins, 56 miRNAs (22 novel) and 475 full-length mRNA transcripts mapping to gene models. Many of the miRNAs putatively mapped to mouse genes are involved in regulation of inflammation, implying a role in parasite-driven immunomodulation. In addition, for the first time to our knowledge, colonic organoids have been used to demonstrate the internalization of parasite EVs by host cells. Understanding how parasites interact with their host is crucial to develop new control measures. This first characterization of the proteins and EVs secreted by provides important information on whipworm-host communication and forms the basis for future studies.
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http://dx.doi.org/10.1080/20013078.2018.1428004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5795766PMC
January 2018

Whole-genome landscapes of major melanoma subtypes.

Nature 2017 05 3;545(7653):175-180. Epub 2017 May 3.

QIMR Berghofer Medical Research Institute, Brisbane, Queensland 4006, Australia.

Melanoma of the skin is a common cancer only in Europeans, whereas it arises in internal body surfaces (mucosal sites) and on the hands and feet (acral sites) in people throughout the world. Here we report analysis of whole-genome sequences from cutaneous, acral and mucosal subtypes of melanoma. The heavily mutated landscape of coding and non-coding mutations in cutaneous melanoma resolved novel signatures of mutagenesis attributable to ultraviolet radiation. However, acral and mucosal melanomas were dominated by structural changes and mutation signatures of unknown aetiology, not previously identified in melanoma. The number of genes affected by recurrent mutations disrupting non-coding sequences was similar to that affected by recurrent mutations to coding sequences. Significantly mutated genes included BRAF, CDKN2A, NRAS and TP53 in cutaneous melanoma, BRAF, NRAS and NF1 in acral melanoma and SF3B1 in mucosal melanoma. Mutations affecting the TERT promoter were the most frequent of all; however, neither they nor ATRX mutations, which correlate with alternative telomere lengthening, were associated with greater telomere length. Most melanomas had potentially actionable mutations, most in components of the mitogen-activated protein kinase and phosphoinositol kinase pathways. The whole-genome mutation landscape of melanoma reveals diverse carcinogenic processes across its subtypes, some unrelated to sun exposure, and extends potential involvement of the non-coding genome in its pathogenesis.
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http://dx.doi.org/10.1038/nature22071DOI Listing
May 2017

DeepSNVMiner: a sequence analysis tool to detect emergent, rare mutations in subsets of cell populations.

PeerJ 2016 24;4:e2074. Epub 2016 May 24.

Department of Immunology, John Curtin School of Medical Research, Australian National University , Canberra ACT , Australia.

Background. Massively parallel sequencing technology is being used to sequence highly diverse populations of DNA such as that derived from heterogeneous cell mixtures containing both wild-type and disease-related states. At the core of such molecule tagging techniques is the tagging and identification of sequence reads derived from individual input DNA molecules, which must be first computationally disambiguated to generate read groups sharing common sequence tags, with each read group representing a single input DNA molecule. This disambiguation typically generates huge numbers of reads groups, each of which requires additional variant detection analysis steps to be run specific to each read group, thus representing a significant computational challenge. While sequencing technologies for producing these data are approaching maturity, the lack of available computational tools for analysing such heterogeneous sequence data represents an obstacle to the widespread adoption of this technology. Results. Using synthetic data we successfully detect unique variants at dilution levels of 1 in a 1,000,000 molecules, and find DeeepSNVMiner obtains significantly lower false positive and false negative rates compared to popular variant callers GATK, SAMTools, FreeBayes and LoFreq, particularly as the variant concentration levels decrease. In a dilution series with genomic DNA from two cells lines, we find DeepSNVMiner identifies a known somatic variant when present at concentrations of only 1 in 1,000 molecules in the input material, the lowest concentration amongst all variant callers tested. Conclusions. Here we present DeepSNVMiner; a tool to disambiguate tagged sequence groups and robustly identify sequence variants specific to subsets of starting DNA molecules that may indicate the presence of a disease. DeepSNVMiner is an automated workflow of custom sequence analysis utilities and open source tools able to differentiate somatic DNA variants from artefactual sequence variants that likely arose during DNA amplification. The workflow remains flexible such that it may be customised to variants of the data production protocol used, and supports reproducible analysis through detailed logging and reporting of results. DeepSNVMiner is available for academic non-commercial research purposes at https://github.com/mattmattmattmatt/DeepSNVMiner.
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http://dx.doi.org/10.7717/peerj.2074DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4888318PMC
June 2016

Reliably Detecting Clinically Important Variants Requires Both Combined Variant Calls and Optimized Filtering Strategies.

PLoS One 2015 23;10(11):e0143199. Epub 2015 Nov 23.

Department of Immunology, John Curtin School of Medical Research, Australian National University, Canberra, ACT, Australia.

A diversity of tools is available for identification of variants from genome sequence data. Given the current complexity of incorporating external software into a genome analysis infrastructure, a tendency exists to rely on the results from a single tool alone. The quality of the output variant calls is highly variable however, depending on factors such as sequence library quality as well as the choice of short-read aligner, variant caller, and variant caller filtering strategy. Here we present a two-part study first using the high quality 'genome in a bottle' reference set to demonstrate the significant impact the choice of aligner, variant caller, and variant caller filtering strategy has on overall variant call quality and further how certain variant callers outperform others with increased sample contamination, an important consideration when analyzing sequenced cancer samples. This analysis confirms previous work showing that combining variant calls of multiple tools results in the best quality resultant variant set, for either specificity or sensitivity, depending on whether the intersection or union, of all variant calls is used respectively. Second, we analyze a melanoma cell line derived from a control lymphocyte sample to determine whether software choices affect the detection of clinically important melanoma risk-factor variants finding that only one of the three such variants is unanimously detected under all conditions. Finally, we describe a cogent strategy for implementing a clinical variant detection pipeline; a strategy that requires careful software selection, variant caller filtering optimizing, and combined variant calls in order to effectively minimize false negative variants. While implementing such features represents an increase in complexity and computation the results offer indisputable improvements in data quality.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0143199PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4658170PMC
June 2016

Tumour procurement, DNA extraction, coverage analysis and optimisation of mutation-detection algorithms for human melanoma genomes.

Pathology 2015 Dec;47(7):683-93

1Melanoma Institute Australia, North Sydney, NSW 2Sydney Medical School, The University of Sydney, Camperdown, NSW 3Immunogenomics Laboratory, Australian National University, Canberra, ACT 4Oncogenomics Laboratory, QIMR Berghofer Medical Research Institute, Herston, Brisbane, Qld 5Centre for Cancer Research, The University of Sydney at Westmead Millennium Institute, Westmead, NSW 6Bioplatforms Australia, Macquarie University, North Ryde, NSW 7Ludwig Institute for Cancer Research, Olivia Newton-John Cancer and Wellness Centre, Austin Health, Heidelberg, Vic 8The Cancer Development and Treatment Laboratory, Peter MacCallum Cancer Centre and Sir Peter MacCallum Department of Oncology, The University of Melbourne, Vic 9Departments of Melanoma and Surgical Oncology 10Tissue Pathology and Diagnostic Oncology, Royal Prince Alfred Hospital, Camperdown, NSW, Australia; these authors contributed equally.

Whole genome sequencing (WGS) of cancer patients' tumours offers the most comprehensive method of identifying both novel and known clinically-actionable genomic targets. However, the practicalities of performing WGS on clinical samples are poorly defined.This study was designed to test sample preparation, sequencing specifications and bioinformatic algorithms for their effect on accuracy and cost-efficiency in a large WGS analysis of human melanoma samples.WGS was performed on melanoma cell lines (n = 15) and melanoma fresh frozen tumours (n = 222). The appropriate level of coverage and the optimal mutation detection algorithm for the project pipeline were determined.An incremental increase in sequencing coverage from 36X to 132X in melanoma tissue samples and 30X to 103X for cell lines only resulted in a small increase (1-2%) in the number of mutations detected, and the quality scores of the additional mutations indicated a low probability that the mutations were real. The results suggest that 60X coverage for melanoma tissue and 40X for melanoma cell lines empower the detection of 98-99% of informative single nucleotide variants (SNVs), a sensitivity level at which clinical decision making or landscape research projects can be carried out with a high degree of confidence in the results. Likewise the bioinformatic mutation analysis methodology strongly influenced the number and quality of SNVs detected. Detecting mutations in the blood genomes separate to the tumour genomes generated 41% more SNVs than if the blood and melanoma tissue genomes were analysed simultaneously. Therefore, simultaneous analysis should be employed on matched melanoma tissue and blood genomes to reduce errors in mutation detection.This study provided valuable insights into the accuracy of SNV with WGS at various coverage levels in human clinical cancer specimens. Additionally, we investigated the accuracy of the publicly available mutation detection algorithms to detect cancer specific SNVs which will aid researchers and clinicians in study design and implementation of WGS for the identification of somatic mutations in other cancers.
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http://dx.doi.org/10.1097/PAT.0000000000000324DOI Listing
December 2015

Comparison of predicted and actual consequences of missense mutations.

Proc Natl Acad Sci U S A 2015 Sep 12;112(37):E5189-98. Epub 2015 Aug 12.

Immunogenomics Laboratory, John Curtin School of Medical Research, Australian National University, Canberra City, ACT 2601, Australia;

Each person's genome sequence has thousands of missense variants. Practical interpretation of their functional significance must rely on computational inferences in the absence of exhaustive experimental measurements. Here we analyzed the efficacy of these inferences in 33 de novo missense mutations revealed by sequencing in first-generation progeny of N-ethyl-N-nitrosourea-treated mice, involving 23 essential immune system genes. PolyPhen2, SIFT, MutationAssessor, Panther, CADD, and Condel were used to predict each mutation's functional importance, whereas the actual effect was measured by breeding and testing homozygotes for the expected in vivo loss-of-function phenotype. Only 20% of mutations predicted to be deleterious by PolyPhen2 (and 15% by CADD) showed a discernible phenotype in individual homozygotes. Half of all possible missense mutations in the same 23 immune genes were predicted to be deleterious, and most of these appear to become subject to purifying selection because few persist between separate mouse substrains, rodents, or primates. Because defects in immune genes could be phenotypically masked in vivo by compensation and environment, we compared inferences by the same tools with the in vitro phenotype of all 2,314 possible missense variants in TP53; 42% of mutations predicted by PolyPhen2 to be deleterious (and 45% by CADD) had little measurable consequence for TP53-promoted transcription. We conclude that for de novo or low-frequency missense mutations found by genome sequencing, half those inferred as deleterious correspond to nearly neutral mutations that have little impact on the clinical phenotype of individual cases but will nevertheless become subject to purifying selection.
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http://dx.doi.org/10.1073/pnas.1511585112DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4577149PMC
September 2015

Reducing the search space for causal genetic variants with VASP.

Bioinformatics 2015 Jul 8;31(14):2377-9. Epub 2015 Mar 8.

Department of Immunology, John Curtin School of Medical Research, Australian National University, Canberra City, ACT 2601, Australia, Immunogenomics Group, Immunology Research Program, Garvan Institute of Medical Research, Darlinghurst, NSW 2010, Australia.

Motivation: Increasingly, cost-effective high-throughput DNA sequencing technologies are being utilized to sequence human pedigrees to elucidate the genetic cause of a wide variety of human diseases. While numerous tools exist for variant prioritization within a single genome, the ability to concurrently analyze variants within pedigrees remains a challenge, especially should there be no prior indication of the underlying genetic cause of the disease. Here, we present a tool, variant analysis of sequenced pedigrees (VASP), a flexible data integration environment capable of producing a summary of pedigree variation, providing relevant information such as compound heterozygosity, genome phasing and disease inheritance patterns. Designed to aggregate data across a sequenced pedigree, VASP allows both powerful filtering and custom prioritization of both single nucleotide variants (SNVs) and small indels. Hence, clinical and research users with prior knowledge of a disease are able to dramatically reduce the variant search space based on a wide variety of custom prioritization criteria.

Availability And Implementation: Source code available for academic non-commercial research purposes at https://github.com/mattmattmattmatt/VASP.
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http://dx.doi.org/10.1093/bioinformatics/btv135DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4495293PMC
July 2015

Identification of a pathogenic variant in TREX1 in early-onset cerebral systemic lupus erythematosus by Whole-exome sequencing.

Arthritis Rheumatol 2014 Dec;66(12):3382-6

Objective. Systemic lupus erythematosus (SLE) isa chronic and heterogeneous autoimmune disease. Both twin and sibling studies indicate a strong genetic contribution to lupus, but in the majority of cases the pathogenic variant remains to be identified. The genetic contribution to disease is likely to be greatest in cases with early onset and severe phenotypes. Whole-exome sequencing now offers the possibility of identifying rare alleles responsible for disease in such cases. This study was undertaken to identify genetic causes of SLE using whole-exome sequencing.Methods. We performed whole-exome sequencing in a 4-year-old girl with early-onset SLE and conducted biochemical analysis of the putative defect.Results. Whole-exome sequencing in a 4-year-old girl with cerebral lupus identified a rare, homozygous mutation in the three prime repair exonuclease 1 gene(TREX1) that was predicted to be highly deleterious.The TREX1 R97H mutant protein had a 20-fold reduction in exonuclease activity and was associated with an elevated interferon-alpha signature in the patient.The discovery and characterization of a pathogenic TREX1 variant in our proband has therapeutic implications.The patient is now a candidate for therapy. Conclusion. Our study is the first to demonstrate that whole-exome sequencing can be used to identify rare or novel deleterious variants as genetic causes of SLE and, through a personalized approach, improve therapeutic options.
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http://dx.doi.org/10.1002/art.38824DOI Listing
December 2014

Zinc-finger protein ZFP318 is essential for expression of IgD, the alternatively spliced Igh product made by mature B lymphocytes.

Proc Natl Acad Sci U S A 2014 Mar 10;111(12):4513-8. Epub 2014 Mar 10.

Department of Immunology and Australian Phenomics Facility, John Curtin School of Medical Research, Australian National University, Canberra, ACT 2601, Australia.

IgD and IgM are produced by alternative splicing of long primary RNA transcripts from the Ig heavy chain (Igh) locus and serve as the receptors for antigen on naïve mature B lymphocytes. IgM is made selectively in immature B cells, whereas IgD is coexpressed with IgM when the cells mature into follicular or marginal zone B cells, but the transacting factors responsible for this regulated change in splicing have remained elusive. Here, we use a genetic screen in mice to identify ZFP318, a nuclear protein with two U1-type zinc fingers found in RNA-binding proteins and no known role in the immune system, as a critical factor for IgD expression. A point mutation in an evolutionarily conserved lysine-rich domain encoded by the alternatively spliced Zfp318 exon 10 abolished IgD expression on marginal zone B cells, decreased IgD on follicular B cells, and increased IgM, but only slightly decreased the percentage of B cells and did not decrease expression of other maturation markers CD21, CD23, or CD62L. A targeted Zfp318 null allele extinguished IgD expression on mature B cells and increased IgM. Zfp318 mRNA is developmentally regulated in parallel with IgD, with little in pro-B cells, moderate amounts in immature B cells, and high levels selectively in mature follicular B cells. These findings identify ZFP318 as a crucial factor regulating the expression of the two major antibody isotypes on the surface of most mature B cells.
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http://dx.doi.org/10.1073/pnas.1402739111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3970522PMC
March 2014

Rasgrp1 mutation increases naive T-cell CD44 expression and drives mTOR-dependent accumulation of Helios⁺ T cells and autoantibodies.

Elife 2013 Dec 12;2:e01020. Epub 2013 Dec 12.

Department of Immunology, John Curtin School of Medical Research, The Australian National University, Canberra, Australia.

Missense variants are a major source of human genetic variation. Here we analyze a new mouse missense variant, Rasgrp1(Anaef), with an ENU-mutated EF hand in the Rasgrp1 Ras guanine nucleotide exchange factor. Rasgrp1(Anaef) mice exhibit anti-nuclear autoantibodies and gradually accumulate a CD44(hi) Helios(+) PD-1(+) CD4(+) T cell population that is dependent on B cells. Despite reduced Rasgrp1-Ras-ERK activation in vitro, thymocyte selection in Rasgrp1(Anaef) is mostly normal in vivo, although CD44 is overexpressed on naïve thymocytes and T cells in a T-cell-autonomous manner. We identify CD44 expression as a sensitive reporter of tonic mTOR-S6 kinase signaling through a novel mouse strain, chino, with a reduction-of-function mutation in Mtor. Elevated tonic mTOR-S6 signaling occurs in Rasgrp1(Anaef) naïve CD4(+) T cells. CD44 expression, CD4(+) T cell subset ratios and serum autoantibodies all returned to normal in Rasgrp1(Anaef)Mtor(chino) double-mutant mice, demonstrating that increased mTOR activity is essential for the Rasgrp1(Anaef) T cell dysregulation. DOI: http://dx.doi.org/10.7554/eLife.01020.001.
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http://dx.doi.org/10.7554/eLife.01020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3858598PMC
December 2013

B cell survival, surface BCR and BAFFR expression, CD74 metabolism, and CD8- dendritic cells require the intramembrane endopeptidase SPPL2A.

J Exp Med 2013 Jan 24;210(1):31-40. Epub 2012 Dec 24.

Ramaciotti Immunization Genomics Laboratory, John Curtin School of Medical Research, The Australian National University, Canberra, Australian Capital Territory 2600, Australia.

Druggable proteins required for B lymphocyte survival and immune responses are an emerging source of new treatments for autoimmunity and lymphoid malignancy. In this study, we show that mice with an inactivating mutation in the intramembrane protease signal peptide peptidase-like 2A (SPPL2A) unexpectedly exhibit profound humoral immunodeficiency and lack mature B cell subsets, mirroring deficiency of the cytokine B cell-activating factor (BAFF). Accumulation of Sppl2a-deficient B cells was rescued by overexpression of the BAFF-induced survival protein B cell lymphoma 2 (BCL2) but not BAFF and was distinguished by low surface BAFF receptor and IgM and IgD B cell receptors. CD8-negative dendritic cells were also greatly decreased. SPPL2A deficiency blocked the proteolytic processing of CD74 MHC II invariant chain in both cell types, causing dramatic build-up of the p8 product of Cathepsin S and interfering with earlier steps in CD74 endosomal retention and processing. The findings illuminate an important role for the final step in the CD74-MHC II pathway and a new target for protease inhibitor treatment of B cell diseases.
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http://dx.doi.org/10.1084/jem.20121076DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3549710PMC
January 2013

Analyses of deep mammalian sequence alignments and constraint predictions for 1% of the human genome.

Genome Res 2007 Jun;17(6):760-74

Genome Technology Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD 20892, USA.

A key component of the ongoing ENCODE project involves rigorous comparative sequence analyses for the initially targeted 1% of the human genome. Here, we present orthologous sequence generation, alignment, and evolutionary constraint analyses of 23 mammalian species for all ENCODE targets. Alignments were generated using four different methods; comparisons of these methods reveal large-scale consistency but substantial differences in terms of small genomic rearrangements, sensitivity (sequence coverage), and specificity (alignment accuracy). We describe the quantitative and qualitative trade-offs concomitant with alignment method choice and the levels of technical error that need to be accounted for in applications that require multisequence alignments. Using the generated alignments, we identified constrained regions using three different methods. While the different constraint-detecting methods are in general agreement, there are important discrepancies relating to both the underlying alignments and the specific algorithms. However, by integrating the results across the alignments and constraint-detecting methods, we produced constraint annotations that were found to be robust based on multiple independent measures. Analyses of these annotations illustrate that most classes of experimentally annotated functional elements are enriched for constrained sequences; however, large portions of each class (with the exception of protein-coding sequences) do not overlap constrained regions. The latter elements might not be under primary sequence constraint, might not be constrained across all mammals, or might have expendable molecular functions. Conversely, 40% of the constrained sequences do not overlap any of the functional elements that have been experimentally identified. Together, these findings demonstrate and quantify how many genomic functional elements await basic molecular characterization.
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http://dx.doi.org/10.1101/gr.6034307DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1891336PMC
June 2007

Plastid-targeting peptides from the chlorarachniophyte Bigelowiella natans.

J Eukaryot Microbiol 2004 Sep-Oct;51(5):529-35

Canadian Institute for Advanced Research, Department of Botany, University of British Columbia, 3529-6270 University Boulevard, Vancouver, BC, V6T 1Z4, Canada.

Chlorarachniophytes are marine amoeboflagellate protists that have acquired their plastid (chloroplast) through secondary endosymbiosis with a green alga. Like other algae, most of the proteins necessary for plastid function are encoded in the nuclear genome of the secondary host. These proteins are targeted to the organelle using a bipartite leader sequence consisting of a signal peptide (allowing entry in to the endomembrane system) and a chloroplast transit peptide (for transport across the chloroplast envelope membranes). We have examined the leader sequences from 45 full-length predicted plastid-targeted proteins from the chlorarachniophyte Bigelowiella natans with the goal of understanding important features of these sequences and possible conserved motifs. The chemical characteristics of these sequences were compared with a set of 10 B. natans endomembrane-targeted proteins and 38 cytosolic or nuclear proteins, which show that the signal peptides are similar to those of most other eukaryotes, while the transit peptides differ from those of other algae in some characteristics. Consistent with this, the leader sequence from one B. natans protein was tested for function in the apicomplexan parasite, Toxoplasma gondii, and shown to direct the secretion of the protein.
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http://dx.doi.org/10.1111/j.1550-7408.2004.tb00288.xDOI Listing
March 2005