ABSTRACT
Advanced genomic technologies such as whole exome or whole genome sequencing have improved diagnoses and disease outcomes for individuals with genetic diseases. Yet, variants of unknown significance (VUS) require rigorous validation to establish disease causality or modification, or to exclude them from further analysis. Here, we describe a young individual of Polynesian ancestry who in the first 13 mo of life presented with SARS-CoV-2 pneumonia, severe enterovirus meningitis and adenovirus gastroenteritis, and severe adverse reaction to MMR vaccination. Genomic analysis identified a previously reported pathogenic homozygous variant in IFNAR1 (c.1156G > T, p.Glu386* LOF), which is common in Western Polynesia. Moreover, a new and putatively deleterious canonical splice site variant in DOCK8 was also found in homozygosity (c.3234 + 2T > C). This DOCK8 variant is common in Polynesians and other under-represented ancestries in large genomic databases. Despite in silico bioinformatic predictions, extensive in vitro and ex vivo analysis revealed the DOCK8 variant likely be neutral. Thus, our study reports a novel case of IFNAR1 deficiency, but also highlights the importance of functional validation of VUS, including those predicted to be deleterious, and the pressing need to expand our knowledge of the genomic architecture and landscape of under-represented populations and ancestries.
Subject(s)
COVID-19 , Guanine Nucleotide Exchange Factors , Receptor, Interferon alpha-beta , SARS-CoV-2 , Humans , Guanine Nucleotide Exchange Factors/genetics , Guanine Nucleotide Exchange Factors/deficiency , Receptor, Interferon alpha-beta/genetics , Receptor, Interferon alpha-beta/deficiency , COVID-19/genetics , SARS-CoV-2/genetics , Infant , RNA Splice Sites/genetics , Male , Female , Mutation/genetics , HomozygoteABSTRACT
BACKGROUND: Many families and individuals do not meet criteria for a known hereditary cancer syndrome but display unusual clusters of cancers. These families may carry pathogenic variants in cancer predisposition genes and be at higher risk for developing cancer. METHODS: This multi-centre prospective study recruited 195 cancer-affected participants suspected to have a hereditary cancer syndrome for whom previous clinical targeted genetic testing was either not informative or not available. To identify pathogenic disease-causing variants explaining participant presentation, germline whole-genome sequencing (WGS) and a comprehensive cancer virtual gene panel analysis were undertaken. RESULTS: Pathogenic variants consistent with the presenting cancer(s) were identified in 5.1% (10/195) of participants and pathogenic variants considered secondary findings with potential risk management implications were identified in another 9.7% (19/195) of participants. Health economic analysis estimated the marginal cost per case with an actionable variant was significantly lower for upfront WGS with virtual panel ($8744AUD) compared to standard testing followed by WGS ($24,894AUD). Financial analysis suggests that national adoption of diagnostic WGS testing would require a ninefold increase in government annual expenditure compared to conventional testing. CONCLUSIONS: These findings make a case for replacing conventional testing with WGS to deliver clinically important benefits for cancer patients and families. The uptake of such an approach will depend on the perspectives of different payers on affordability.
Subject(s)
Neoplastic Syndromes, Hereditary , Humans , Prospective Studies , Oncogenes , Genetic Testing , Germ CellsABSTRACT
Molecular diagnosis of paediatric inborn errors of immunity (IEI) influences management decisions and alters clinical outcomes, through early use of targeted and curative therapies. The increasing demand for genetic services has resulted in growing waitlists and delayed access to vital genomic testing. To address this issue, the Queensland Paediatric Immunology and Allergy Service, Australia, developed and evaluated a mainstreaming model of care to support point-of-care genomic testing for paediatric IEI. Key features of the model of care included a genetic counsellor embedded in the department, state-wide multidisciplinary team meetings, and variant prioritisation meetings to review whole exome sequencing (WES) data. Of the 62 children presented at the MDT, 43 proceeded to WES, of which nine (21%) received a confirmed molecular diagnosis. Changes to treatment and management were reported for all children with a positive result, including curative hematopoietic stem cell transplantation (n = 4). Four children were also referred for further investigations of variants of uncertain significance or additional testing due to ongoing suspicion of genetic cause after negative result. Demonstrating engagement with the model of care, 45% of the patients were from regional areas and on average, 14 healthcare providers attended the state-wide multidisciplinary team meetings. Parents demonstrated understanding of the implications of testing, reported minimal decisional regret post-test, and identified benefits to genomic testing. Overall, our program demonstrated the feasibility of a mainstreaming model of care for paediatric IEI, improved access to genomic testing, facilitated treatment decision-making, and was acceptable to parents and clinicians alike.
Subject(s)
Genomics , Parents , Humans , Child , Exome Sequencing , Australia , Genetic TestingSubject(s)
Breast Neoplasms , Neoplasms , Humans , Female , Breast Neoplasms/therapy , Pilot Projects , Queensland , Precision Medicine , BreastABSTRACT
Sharing genomic variant interpretations across laboratories promotes consistency in variant assertions. A landscape analysis of Australian clinical genetic-testing laboratories in 2017 identified that, despite the national-accreditation-body recommendations encouraging laboratories to submit genotypic data to clinical databases, fewer than 300 variants had been shared to the ClinVar public database. Consultations with Australian laboratories identified resource constraints limiting routine application of manual processes, consent issues, and differences in interpretation systems as barriers to sharing. This information was used to define key needs and solutions required to enable national sharing of variant interpretations. The Shariant platform, using both the GRCh37 and GRCh38 genome builds, was developed to enable ongoing sharing of variant interpretations and associated evidence between Australian clinical genetic-testing laboratories. Where possible, two-way automated sharing was implemented so that disruption to laboratory workflows would be minimized. Terms of use were developed through consultation and currently restrict access to Australian clinical genetic-testing laboratories. Shariant was designed to store and compare structured evidence, to promote and record resolution of inter-laboratory classification discrepancies, and to streamline the submission of variant assertions to ClinVar. As of December 2021, more than 14,000 largely prospectively curated variant records from 11 participating laboratories have been shared. Discrepant classifications have been identified for 11% (28/260) of variants submitted by more than one laboratory. We have demonstrated that co-design with clinical laboratories is vital to developing and implementing a national variant-interpretation sharing effort. This approach has improved inter-laboratory concordance and enabled opportunities to standardize interpretation practices.
Subject(s)
Databases, Genetic , Laboratories , Humans , Genetic Variation , Australia , Genetic TestingABSTRACT
OBJECTIVE: Existing gene panels were developed to understand the etiology of epilepsy, and further benefits will arise from an effective pharmacogenomics panel for personalizing therapy and achieving seizure control. Our study assessed the cost-effectiveness of a pharmacogenomics panel for patients with drug-resistant epilepsy, compared with usual care. METHODS: A cost-utility analysis was employed using a discrete event simulation model. The microsimulation model aggregated the costs and benefits of genetically guided treatment versus usual care for 5000 simulated patients. The 10-year model combined data from various sources including genomic databases on prevalence of variants, population-level pharmaceutical claims on antiseizure medications, published long-term therapy retention rates, patient-level cost data, and systematic reviews. Incremental cost per quality-adjusted life-year (QALY) gained was computed. Deterministic and probabilistic sensitivity analyses were undertaken to address uncertainty in model parameters. RESULTS: The mean cost of the genetically guided treatment option was AU$98 199 compared with AU$95 386 for usual care. Corresponding mean QALYs were 4.67 compared with 4.28 for genetically guided and usual care strategies, respectively. The incremental cost per QALY gained was AU$7381. In probabilistic sensitivity analyses, the incremental cost per QALY gained was AU$6321 (95% uncertainty interval = AU$3604-AU$9621), with a 100% likelihood of being cost-effective in the Australian health care system. The most influential drivers of the findings were the monthly health care costs associated with reduced seizures, costs when seizures continued, and the quality-of-life estimates under genetically guided and usual care strategies. SIGNIFICANCE: This early economic evaluation of a pharmacogenomics panel to guide treatment for drug-resistant epilepsy could potentially be cost-effective in the Australian health care system. Clinical trial evidence is necessary to confirm these findings.
Subject(s)
Epilepsy , Quality of Life , Humans , Cost-Benefit Analysis , Australia , SeizuresABSTRACT
The clinical classification of variants may change with new information, however, there is limited guidance on how often significant changes in variant classification occur. We used ClinVar to examine how variant classification changes over time. We developed a custom parser and accessed variant data from ClinVar between January 2015 and July 2021. The ClinVar-assigned "aggregate" classification of variants in 121 hereditary cancer genes was harmonized across releases to align to the American College of Medical Genetics and Genomics and the Association for Molecular Pathology terms. Aggregate classification categories were grouped as: benign/likely benign (B/LB); likely pathogenic/pathogenic (LP/P); variant of uncertain significance (VUS); conflicting interpretations of pathogenicity (Conflicting); or Other. We profiled changes in aggregate variant classification between consecutive semi-annual ClinVar releases. The proportion of variants that changed aggregate classification between semi-annual ClinVar releases ranged from 0.6% to 6.4%. The most frequent changes were "VUS to conflicting," "other to LP/P," and "B/LB to Conflicting." A limited number of variants changed aggregate classification from "LP/P to B/LB," or vice versa. Our analysis indicates need for regular reassessment of clinical variant interpretations. The parser developed for this project will facilitate extraction of relevant interpretation data from ClinVar.
Subject(s)
Genetic Testing , Neoplasms , Humans , United States , Genetic Variation , Genetic Predisposition to Disease , Genomics , Software , Neoplasms/diagnosis , Neoplasms/geneticsABSTRACT
PURPOSE: Deficiency of adenosine deaminase type 2 (ADA2) (DADA2) is a rare inborn error of immunity caused by deleterious biallelic mutations in ADA2. Clinical manifestations are diverse, ranging from severe vasculopathy with lacunar strokes to immunodeficiency with viral infections, hypogammaglobulinemia and bone marrow failure. Limited data are available on the phenotype and function of leukocytes from DADA2 patients. The aim of this study was to perform in-depth immunophenotyping and functional analysis of the impact of DADA2 on human lymphocytes. METHODS: In-depth immunophenotyping and functional analyses were performed on ten patients with confirmed DADA2 and compared to heterozygous carriers of pathogenic ADA2 mutations and normal healthy controls. RESULTS: The median age of the patients was 10 years (mean 20.7 years, range 1-44 years). Four out of ten patients were on treatment with steroids and/or etanercept or other immunosuppressives. We confirmed a defect in terminal B cell differentiation in DADA2 and reveal a block in B cell development in the bone marrow at the pro-B to pre-B cell stage. We also show impaired differentiation of CD4+ and CD8+ memory T cells, accelerated exhaustion/senescence, and impaired survival and granzyme production by ADA2 deficient CD8+ T cells. Unconventional T cells (i.e. iNKT, MAIT, Vδ2+ γδT) were diminished whereas pro-inflammatory monocytes and CD56bright immature NK cells were increased. Expression of the IFN-induced lectin SIGLEC1 was increased on all monocyte subsets in DADA2 patients compared to healthy donors. Interestingly, the phenotype and function of lymphocytes from healthy heterozygous carriers were often intermediate to that of healthy donors and ADA2-deficient patients. CONCLUSION: Extended immunophenotyping in DADA2 patients shows a complex immunophenotype. Our findings provide insight into the cellular mechanisms underlying some of the complex and heterogenous clinical features of DADA2. More research is needed to design targeted therapy to prevent viral infections in these patients with excessive inflammation as the overarching phenotype.
Subject(s)
Agammaglobulinemia/immunology , B-Lymphocytes/immunology , Severe Combined Immunodeficiency/immunology , T-Lymphocytes/immunology , Adenosine Deaminase/blood , Adenosine Deaminase/deficiency , Adenosine Deaminase/genetics , Adolescent , Adult , Agammaglobulinemia/blood , Agammaglobulinemia/genetics , Aged , Cell Differentiation , Child , Child, Preschool , Dendritic Cells/immunology , Humans , Infant , Intercellular Signaling Peptides and Proteins/blood , Intercellular Signaling Peptides and Proteins/deficiency , Intercellular Signaling Peptides and Proteins/genetics , Killer Cells, Natural/immunology , Middle Aged , Severe Combined Immunodeficiency/blood , Severe Combined Immunodeficiency/genetics , Young AdultABSTRACT
Evaluating genes involved in the pharmacodynamics and pharmacokinetics of epilepsy drugs is critical to better understand pharmacoresistant epilepsy. We reviewed the pharmacogenetics literature on six antiseizure medicines (carbamazepine, perampanel, lamotrigine, levetiracetam, sodium valproate and zonisamide) and compared the genes found with those present on epilepsy gene panels using a functional annotation pathway analysis. Little overlap was found between the two gene lists; pharmacogenetic genes are mainly involved in detoxification processes, while epilepsy panel genes are involved in cell signaling and gene expression. Our work provides support for a specific pharmacoresistant epilepsy gene panel to assist antiseizure medicine selection, enabling personalized approaches to treatment. Future efforts will seek to include this panel in genomic analyses of pharmacoresistant patients, to determine clinical utility and patient treatment responses.
Subject(s)
Drug Resistant Epilepsy/drug therapy , Drug Resistant Epilepsy/genetics , Pharmacogenetics , Anticonvulsants/therapeutic use , HumansABSTRACT
Aggregate population genomics data from large cohorts are vital for assessing germline variant pathogenicity. However, there are no specifications on how sequencing quality metrics should be considered, and whether exome-derived and genome-derived allele frequencies should be considered in isolation. Germline genome sequence data were simulated for nine read-depths to identify a minimum acceptable read-depth for detecting variants. gnomAD exome-derived and genome-derived datasets were assessed for read-depth, for six key cancer genes selected for variant curation by ClinGen expert panels. Non-Finnish European allele frequency (AF) or filter AF of coding variants in these genes, assigned into frequency bins using modified ACMG-AMP criteria, was compared between exome-derived and genome-derived datasets. A 30X read-depth achieved acceptable precision and recall for detection of substitutions, but poor recall for small insertions/deletions. Exome-derived and genome-derived datasets exhibited low read-depth for different gene exons. Individual variants were mostly assigned to non-divergent AF bins (>95%) or filter AF bins (>97%). Two major bin divergences were resolved by applying the minimal acceptable read-depth threshold. These findings show the importance of assessing read-depth separately for population datasets sourced from different short-read sequencing technologies before assigning a frequency-based ACMG-AMP classification code for variant interpretation.
Subject(s)
Genome, Human , Neoplasms , Gene Frequency , Genetic Testing , Genetic Variation , Genomics , Germ Cells , Humans , Neoplasms/geneticsABSTRACT
Autosomal Dominant Polycystic Kidney Disease (ADPKD) is common, with a prevalence of 1/1000 and predominantly caused by disease-causing variants in PKD1 or PKD2. Clinical diagnosis is usually by age-dependent imaging criteria, which is challenging in patients with atypical clinical features, without family history, or younger age. However, there is increasing need for definitive diagnosis of ADPKD with new treatments available. Sequencing is complicated by six pseudogenes that share 97% homology to PKD1 and by recently identified phenocopy genes. Whole-genome sequencing can definitively diagnose ADPKD, but requires validation for clinical use. We initially performed a validation study, in which 42 ADPKD patients underwent sequencing of PKD1 and PKD2 by both whole-genome and Sanger sequencing, using a blinded, cross-over method. Whole-genome sequencing identified all PKD1 and PKD2 germline pathogenic variants in the validation study (sensitivity and specificity 100%). Two mosaic variants outside pipeline thresholds were not detected. We then examined the first 144 samples referred to a clinically-accredited diagnostic laboratory for clinical whole-genome sequencing, with targeted-analysis to a polycystic kidney disease gene-panel. In this unselected, diagnostic cohort (71 males :73 females), the diagnostic rate was 70%, including a diagnostic rate of 81% in patients with typical ADPKD (98% with PKD1/PKD2 variants) and 60% in those with atypical features (56% PKD1/PKD2; 44% PKHD1/HNF1B/GANAB/ DNAJB11/PRKCSH/TSC2). Most patients with atypical disease did not have clinical features that predicted likelihood of a genetic diagnosis. These results suggest clinicians should consider diagnostic genomics as part of their assessment in polycystic kidney disease, particularly in atypical disease.
Subject(s)
Gene Frequency , Genetic Testing/methods , Polycystic Kidney Diseases/genetics , Whole Genome Sequencing/methods , Adolescent , Adult , Aged , Child , Child, Preschool , Female , Genetic Testing/standards , Glucosidases/genetics , HSP40 Heat-Shock Proteins/genetics , Hepatocyte Nuclear Factor 1-alpha/genetics , Humans , Infant , Male , Middle Aged , Polycystic Kidney Diseases/diagnosis , Receptors, Cell Surface/genetics , Sensitivity and Specificity , TRPP Cation Channels/genetics , Tuberous Sclerosis Complex 2 Protein/genetics , Whole Genome Sequencing/standardsABSTRACT
Radiotherapy is essential to the treatment of most solid tumors and acquired or innate resistance to this therapeutic modality is a major clinical problem. Here we show that miR-139-5p is a potent modulator of radiotherapy response in breast cancer via its regulation of genes involved in multiple DNA repair and reactive oxygen species defense pathways. Treatment of breast cancer cells with a miR-139-5p mimic strongly synergized with radiation both in vitro and in vivo, resulting in significantly increased oxidative stress, accumulation of unrepaired DNA damage, and induction of apoptosis. Several miR-139-5p target genes were also strongly predictive of outcome in radiotherapy-treated patients across multiple independent breast cancer cohorts. These prognostically relevant miR-139-5p target genes were used as companion biomarkers to identify radioresistant breast cancer xenografts highly amenable to sensitization by cotreatment with a miR-139-5p mimetic.Significance: The microRNA described in this study offers a potentially useful predictive biomarker of radiosensitivity in solid tumors and a generally applicable druggable target for tumor radiosensitization. Cancer Res; 78(2); 501-15. ©2017 AACR.
Subject(s)
Biomarkers, Tumor/genetics , Breast Neoplasms/radiotherapy , DNA Repair/radiation effects , Gene Regulatory Networks/radiation effects , MicroRNAs/genetics , Radiation Tolerance/genetics , Reactive Oxygen Species/metabolism , Animals , Apoptosis , Breast Neoplasms/genetics , Breast Neoplasms/pathology , Case-Control Studies , Cell Proliferation , DNA Damage/radiation effects , Female , Follow-Up Studies , Gene Expression Profiling , Gene Expression Regulation, Neoplastic/radiation effects , Humans , Mice , Mice, Inbred BALB C , Mice, Nude , Neoplasm Recurrence, Local/genetics , Neoplasm Recurrence, Local/pathology , Neoplasm Recurrence, Local/radiotherapy , Prognosis , Survival Rate , Tumor Cells, Cultured , Xenograft Model Antitumor AssaysABSTRACT
Haematopoietic stem cells (HSCs) are self-renewing stem cells capable of replenishing all blood lineages. In all vertebrate embryos that have been studied, definitive HSCs are generated initially within the dorsal aorta (DA) of the embryonic vasculature by a series of poorly understood inductive events. Previous studies have identified that signalling relayed from adjacent somites coordinates HSC induction, but the nature of this signal has remained elusive. Here we reveal that somite specification of HSCs occurs via the deployment of a specific endothelial precursor population, which arises within a sub-compartment of the zebrafish somite that we have defined as the endotome. Endothelial cells of the endotome are specified within the nascent somite by the activity of the homeobox gene meox1. Specified endotomal cells consequently migrate and colonize the DA, where they induce HSC formation through the deployment of chemokine signalling activated in these cells during endotome formation. Loss of meox1 activity expands the endotome at the expense of a second somitic cell type, the muscle precursors of the dermomyotomal equivalent in zebrafish, the external cell layer. The resulting increase in endotome-derived cells that migrate to colonize the DA generates a dramatic increase in chemokine-dependent HSC induction. This study reveals the molecular basis for a novel somite lineage restriction mechanism and defines a new paradigm in induction of definitive HSCs.
Subject(s)
Endothelial Cells/cytology , Hematopoietic Stem Cells/cytology , Homeodomain Proteins/metabolism , Somites/cytology , Transcription Factors/metabolism , Zebrafish Proteins/metabolism , Animals , Aorta/cytology , Aorta/embryology , Biomarkers/analysis , Cell Movement , Chemokine CXCL12/analysis , Chemokine CXCL12/metabolism , Chick Embryo , Endothelial Cells/metabolism , Hematopoietic Stem Cells/metabolism , Homeodomain Proteins/analysis , Homeodomain Proteins/genetics , Humans , Mice , Muscles/cytology , Muscles/metabolism , Mutation/genetics , Somites/metabolism , Transcription Factors/analysis , Transcription Factors/genetics , Wnt Proteins/analysis , Wnt Proteins/metabolism , Zebrafish/embryology , Zebrafish/metabolism , Zebrafish Proteins/analysis , Zebrafish Proteins/geneticsABSTRACT
Stac3 was identified as a nutritionally regulated gene from an Atlantic salmon subtractive hybridization library with highest expression in skeletal muscle. Salmon Stac3 mRNA was highly correlated with myogenin and myoD1a expression during differentiation of a salmon primary myogenic culture and was regulated by amino acid availability. In zebrafish embryos, stac3 was initially expressed in myotomal adaxial cells and in fast muscle fibers post-segmentation. Morpholino knockdown resulted in defects in myofibrillar protein assembly, particularly in slow muscle fibers, and decreased levels of the hedgehog receptor patched. The function of Stac3 was further characterized in vitro using the mammalian C2C12 myogenic cell line. Stac3 mRNA expression increased during the differentiation of the C2C12 myogenic cell line. Knockdown of Stac3 by RNAi inhibited myotube formation, and microarray analysis revealed that transcripts involved in cell cycle, focal adhesion, cytoskeleton, and the pro-myogenic factors Igfbp-5 and Igf2 were down-regulated. RNAi-treated cells had suppressed Akt signaling and exogenous insulin-like growth factor (Igf) 2 was unable to rescue the phenotype, however, Igf/Akt signaling was not blocked. Overexpression of Stac3, which results in increased levels of Igfbp-5 mRNA, did not lead to increased differentiation. In synchronized cells, Stac3 mRNA was most abundant during the G(1) phase of the cell cycle. RNAi-treated cells were smaller, had higher proliferation rates and a decreased proportion of cells in G(1) phase when compared with controls, suggesting a role in the G(1) phase checkpoint. These results identify Stac3 as a new gene required for myogenic differentiation and myofibrillar protein assembly in vertebrates.
Subject(s)
Cell Differentiation/physiology , Fish Proteins/biosynthesis , Gene Expression Regulation/physiology , Muscle Fibers, Skeletal/metabolism , Muscle Proteins/biosynthesis , Salmo salar/metabolism , Animals , Cell Line , Fish Proteins/genetics , G1 Phase Cell Cycle Checkpoints/physiology , Gene Expression Profiling , Muscle Fibers, Skeletal/cytology , Muscle Proteins/genetics , Salmo salar/genetics , Signal Transduction/physiology , ZebrafishABSTRACT
The Hedgehog (HH) signaling pathway is a central regulator of embryonic development, controlling the pattern and proliferation of a wide variety of organs. Previous studies have implicated the secreted protein, Scube2, in HH signal transduction in the zebrafish embryo (Hollway et al., 2006; Kawakami et al., 2005; Woods and Talbot, 2005) although the nature of the molecular function of Scube2 in this process has remained undefined. This analysis has been compounded by the fact that removal of Scube2 activity in the zebrafish embryo leads to only subtle defects in HH signal transduction in vivo (Barresi et al., 2000; Hollway et al., 2006; Ochi and Westerfield, 2007; van Eeden et al., 1996; Wolff et al., 2003). Here we present the discovery of two additional scube genes in zebrafish, scube1 and scube3, and demonstrate their roles in facilitating HH signal transduction. Knocking down the function of all three scube genes simultaneously phenocopies a complete loss of HH signal transduction in the embryo, revealing that Scube signaling is essential for HH signal transduction in vivo. We further define the molecular role of scube2 in HH signaling.
Subject(s)
Calcium-Binding Proteins/genetics , Embryo, Nonmammalian/metabolism , Extracellular Matrix Proteins/genetics , Hedgehog Proteins/genetics , Signal Transduction/genetics , Zebrafish Proteins/genetics , Animals , Blotting, Western , COS Cells , Calcium-Binding Proteins/metabolism , Chlorocebus aethiops , DNA, Complementary/chemistry , DNA, Complementary/genetics , Embryo, Nonmammalian/embryology , Extracellular Matrix Proteins/metabolism , Gene Expression Profiling , Gene Expression Regulation, Developmental , Gene Knockdown Techniques , Hedgehog Proteins/metabolism , In Situ Hybridization , Molecular Sequence Data , Multigene Family , Mutation , Phenotype , Sequence Analysis, DNA , Zebrafish/embryology , Zebrafish/genetics , Zebrafish/metabolism , Zebrafish Proteins/metabolismABSTRACT
Mutations in the human laminin alpha2 (LAMA2) gene result in the most common form of congenital muscular dystrophy (MDC1A). There are currently three models for the molecular basis of cellular pathology in MDC1A: (i) lack of LAMA2 leads to sarcolemmal weakness and failure, followed by cellular necrosis, as is the case in Duchenne muscular dystrophy (DMD); (ii) loss of LAMA2-mediated signaling during the development and maintenance of muscle tissue results in myoblast proliferation and fusion defects; (iii) loss of LAMA2 from the basement membrane of the Schwann cells surrounding the peripheral nerves results in a lack of motor stimulation, leading to effective denervation atrophy. Here we show that the degenerative muscle phenotype in the zebrafish dystrophic mutant, candyfloss (caf) results from mutations in the laminin alpha2 (lama2) gene. In vivo time-lapse analysis of mechanically loaded fibers and membrane permeability assays suggest that, unlike DMD, fiber detachment is not initially associated with sarcolemmal rupture. Early muscle formation and myoblast fusion are normal, indicating that any deficiency in early Lama2 signaling does not lead to muscle pathology. In addition, innervation by the primary motor neurons is unaffected, and fiber detachment stems from muscle contraction, demonstrating that muscle atrophy through lack of motor neuron activity does not contribute to pathology in this system. Using these and other analyses, we present a model of lama2 function where fiber detachment external to the sarcolemma is mechanically induced, and retracted fibers with uncompromised membranes undergo subsequent apoptosis.
Subject(s)
Extracellular Matrix/metabolism , Laminin/deficiency , Muscular Dystrophy, Animal/congenital , Mutant Proteins/metabolism , Zebrafish Proteins/metabolism , Zebrafish/abnormalities , Adhesiveness/drug effects , Alleles , Amino Acid Sequence , Animals , Base Sequence , Cell Death/drug effects , Codon, Nonsense/genetics , Embryo, Nonmammalian/cytology , Embryo, Nonmammalian/drug effects , Embryo, Nonmammalian/innervation , Embryo, Nonmammalian/ultrastructure , Extracellular Matrix/drug effects , Gene Expression Regulation/drug effects , Intercellular Junctions/drug effects , Intercellular Junctions/ultrastructure , Laminin/chemistry , Laminin/genetics , Laminin/metabolism , Molecular Sequence Data , Motor Activity/drug effects , Muscle Fibers, Skeletal/drug effects , Muscle Fibers, Skeletal/pathology , Oligonucleotides, Antisense/pharmacology , Open Reading Frames/genetics , Sarcolemma/drug effects , Sarcolemma/pathology , Sequence Homology, Amino Acid , Zebrafish/embryologyABSTRACT
Pigment pattern formation in zebrafish presents a tractable model system for studying the morphogenesis of neural crest derivatives. Embryos mutant for choker manifest a unique pigment pattern phenotype that combines a loss of lateral stripe melanophores with an ectopic melanophore ;collar' at the head-trunk border. We find that defects in neural crest migration are largely restricted to the lateral migration pathway, affecting both xanthophores (lost) and melanophores (gained) in choker mutants. Double mutant and timelapse analyses demonstrate that these defects are likely to be driven independently, the collar being formed by invasion of melanophores from the dorsal and ventral stripes. Using tissue transplantation, we show that melanophore patterning depends upon the underlying somitic cells, the myotomal derivatives of which--both slow--and fast-twitch muscle fibres--are themselves significantly disorganised in the region of the ectopic collar. In addition, we uncover an aberrant pattern of expression of the gene encoding the chemokine Sdf1a in choker mutant homozygotes that correlates with each aspect of the melanophore pattern defect. Using morpholino knock-down and ectopic expression experiments, we provide evidence to suggest that Sdf1a drives melanophore invasion in the choker mutant collar and normally plays an essential role in patterning the lateral stripe. We thus identify Sdf1 as a key molecule in pigment pattern formation, adding to the growing inventory of its roles in embryonic development.
Subject(s)
Chemokines, CXC/physiology , Melanophores/physiology , Neural Crest/physiology , Somites/physiology , Zebrafish/physiology , Animals , Body Patterning , Cell Movement , Chemokine CXCL12 , Chemokines, CXC/genetics , Chemokines, CXC/metabolism , Melanophores/cytology , Muscle Fibers, Fast-Twitch/physiology , Muscle Fibers, Slow-Twitch/physiology , Mutation , Pigmentation , Somites/cytology , Zebrafish/embryology , Zebrafish/geneticsABSTRACT
Somites are transient, mesodermally derived structures that give rise to a number of different cell types within the vertebrate embryo. To achieve this, somitic cells are partitioned into lineage-restricted domains, whose fates are determined by signals secreted from adjacent tissues. While the molecular nature of many of the inductive signals that trigger formation of different cell fates within the nascent somite has been identified, less is known about the processes that coordinate the formation of the subsomitic compartments from which these cells arise. Utilizing a combination of vital dye-staining and lineage-tracking techniques, we describe a previously uncharacterized, lineage-restricted compartment of the zebrafish somite that generates muscle progenitor cells for the growth of appendicular, hypaxial, and axial muscles during development. We also show that formation of this compartment occurs via whole-somite rotation, a process that requires the action of the Sdf family of secreted cytokines.
Subject(s)
Body Patterning/physiology , Cell Compartmentation , Embryo, Nonmammalian/cytology , Muscle Cells/cytology , Somites/physiology , Stem Cells/cytology , Zebrafish/embryology , Animals , Cell Lineage , Embryo, Nonmammalian/embryology , Gene Expression Regulation, Developmental , Muscle Cells/metabolism , PAX7 Transcription Factor/metabolism , Rotation , Signal Transduction , Somites/cytology , Stem Cells/metabolism , Zebrafish Proteins/genetics , Zebrafish Proteins/metabolismABSTRACT
The Hedgehog family of secreted morphogens specifies the fate of a large number of different cell types within invertebrate and vertebrate embryos, including the muscle cell precursors of the embryonic myotome of zebrafish. Formation of Hedgehog-sensitive muscle fates is disrupted within homozygous zebrafish mutants of the "you"-type class, the majority of which disrupt components of the Hedgehog (HH) signal transduction pathway. We have undertaken a phenotypic and molecular characterisation of one of these mutants, you, which we show results from mutations within the zebrafish orthologue of the mammalian gene scube2. This gene encodes a member of the Scube family of proteins, which is characterised by several protein motifs including EGF and CUB domains. Epistatic and molecular analyses position Scube2 function upstream of Smoothened (Smoh), the signalling component of the HH receptor complex, suggesting that Scube2 may act during HH signal transduction prior to, or during, receipt of the HH signal at the plasma membrane. In support of this model we show that scube2 has homology to cubilin, which encodes an endocytic receptor involved in protein trafficking suggesting a possible mode of function for Scube2 during HH signal transduction.
Subject(s)
Extracellular Matrix Proteins/physiology , Trans-Activators/physiology , Zebrafish Proteins/physiology , Animals , Embryo, Nonmammalian , Hedgehog Proteins , Mutation , Protein Transport , Receptors, Cell Surface , Receptors, G-Protein-Coupled/physiology , Signal Transduction , Smoothened Receptor , ZebrafishABSTRACT
The embryonic myotome generates both the axial musculature and the appendicular muscle of the fins and limbs. Early in embryo development the mesoderm is segmented into somites, and within these the primary myotome forms by a complex series of cellular movements and migrations. A new model of primary myotome formation in amniotes has emerged recently. The myotome also includes the muscle progenitor cells that are known to contribute to the secondary formation of the myotome. The adult myotome contains satellite cells that play an important role in adult muscle regeneration. Recent studies have shed light on how the growth and patterning of the myotome occurs.