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
and
, 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.
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.
.
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 CD56
brigh
t
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.
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.
Modelling changes in classification of variants in cancer genes over time
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.
Mutations in the human laminin α2 (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 α2 (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.
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.
► We describe the function of the zebrafish scube gene family in Hedgehog signaling. ► Three scube genes contribute to Hedgehog signaling during muscle development. ► Elimination of all scube function abrogates Hedgehog signaling during development. ► Zebrafish scube is a hedgehog binding protein.
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. Keywords: Familial cancer, Genetics, Variants, Whole-genome sequencing, Diagnostic testing, Health economics
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 G1 phase of the cell cycle. RNAi-treated cells were smaller, had higher proliferation rates and a decreased proportion of cells in G1 phase when compared with controls, suggesting a role in the G1 phase checkpoint. These results identify Stac3 as a new gene required for myogenic differentiation and myofibrillar protein assembly in vertebrates.
Stac3, an uncharacterised gene, was identified by its high expression levels in vertebrate muscle.
Knockdown of stac3 inhibited zebrafish myofibrillar protein assembly and differentiation in C2C12 cells by perturbing Akt signaling and cell cycle exit.
Stac3 is a novel regulator of myogenic differentiation.
Identifying new genes controlling myogenic differentiation will allow greater understanding of processes leading to muscular diseases.
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.
Mutations in the human laminin alpha 2 (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 alpha 2 (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.
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