GATA2 deficiency phenotype associated with tandem duplication GATA2 and over-expression of GATA2-AS1

A 3-year old girl of non-consanguineous healthy parents presented with cervical and mediastinal lymphadenopathy due to Mycobacterium fortuitum infection. Routine blood analysis showed normal hemoglobin, neutrophils and platelets but profound mononuclear cell deficiency (monocytes <0.1x109/L; B cells 78/µL; NK cells 48/µL). A 548,902bp region containing GATA2 was sequenced by targeted capture and deep sequencing. This revealed a de novo 187Kb duplication of the entire GATA2 locus, containing a maternally inherited copy number variation deletion of 25Kb (GRCh37: esv2725896 and nsv513733). Many GATA2-associated phenotypes have been attributed to amino acid substitution, frameshift/deletion, loss of intronic enhancer function or aberrant splicing. Gene deletion has been described but other structural variation has not been reported in the germline configuration. In this case, duplication of the GATA2 locus was paradoxically associated with skewed, diminished expression of GATA2 mRNA and loss of GATA2 protein. Chimeric RNA fusion transcripts were not detected. A possible mechanism involves increased transcription of the anti-sense long-non-coding (lnc)RNA GATA2-AS1 (RP11-472.220) which was increased several-fold. This case further highlights that evaluation of the allele count is essential in any case of suspected GATA2-related syndrome.

The EGFRvIII transcriptome in glioblastoma, a meta-omics analysis

Background EGFR is among the genes most frequently altered in glioblastoma, with exons 2-7 deletions (EGFRvIII) being amongst its most common genomic mutations. There are conflicting reports about its prognostic role and it remains unclear whether and how it differs in signalling compared with wildtype EGFR. Methods To better understand the oncogenic role of EGFRvIII, we leveraged four large datasets into one large glioblastoma transcriptome dataset (n=741) alongside 81 whole-genome samples from two datasets. Results The EGFRvIII/EGFR expression ratios differ strongly between tumours and ranges from 1% to 95%. Interestingly, the slope of relative EGFRvIII expression is near-linear, which argues against a more positive selection pressure than EGFR wildtype. An absence of selection pressure is also suggested by the similar survival between EGFRvIII positive and negative glioblastoma patients. EGFRvIII levels are inversely correlated with pan-EGFR (all wildtype and mutant variants) expression, which indicates that EGFRvIII has a higher potency in downstream pathway activation. EGFRvIII-positive glioblastomas have a lower CDK4 or MDM2 amplification incidence than EGFRvIII-negative (p=0.007), which may point towards crosstalk between these pathways. EGFRvIII-expressing tumours have an upregulation of ‘classical’ subtype genes compared to those with EGFR-amplification only (p=3.873e-6). Genomic breakpoints of the EGFRvIII deletions have a preference towards the 3’ end of the large intron-1. These preferred breakpoints preserve a cryptic exon resulting in a novel EGFRvIII variant and preserve an intronic enhancer. Conclusions These data provide deeper insights into the complex EGFRvIII biology and provide new insights for targeting EGFRvIII mutated tumours.

Variant Intronic Enhancer Controls SCN10A-Short Expression and Heart Conduction

Background: Genetic variants in SCN10A , encoding the neural voltage-gated sodium channel NaV1.8, are strongly associated with atrial fibrillation, Brugada syndrome, cardiac conduction velocities and heart rate. The cardiac function of SCN10A has not been resolved, however, and diverging mechanisms have been proposed. Here, we investigated the cardiac expression of SCN10A and the function of a variant-sensitive intronic enhancer previously linked to the regulation of SCN5A , encoding the major essential cardiac sodium channel NaV1.5. Methods: The expression of SCN10A was investigated in mouse and human hearts. Using CRISPR/Cas9 genome editing, the mouse intronic enhancer was disrupted, and mutant mice were characterized by transcriptomic and electrophysiological analyses. The association of genetic variants at SCN5A-SCN10A enhancer regions and gene expression were evaluated by GWAS SNP mapping and expression QTL analysis. Results: We found that cardiomyocytes of the atria, sinoatrial node and ventricular conduction system express a short transcript comprising the last 7 exons of the gene ( Scn10a-short ). Transcription occurs from an intronic enhancer-promoter complex, while full length Scn10a transcript was undetectable in the human and mouse heart. Expression QTL analysis revealed that the genetic variants in linkage disequilibrium with genetic variant rs6801957 in the intronic enhancer associate with SCN10A transcript levels in the heart. Genetic modification of the enhancer in the mouse genome led to reduced cardiac Scn10a-short expression in atria and ventricles, reduced cardiac sodium current in atrial cardiomyocytes, atrial conduction slowing and arrhythmia, while expression of Scn5a , the presumed enhancer target gene, remained unaffected. In patch-clamp transfection experiments, expression of Scn10a-short -encoded NaV1.8-short increased NaV1.5-mediated sodium current. We propose that non-coding genetic variation modulates transcriptional regulation of Scn10a-short in cardiomyocytes that impacts on NaV1.5-mediated sodium current and heart rhythm. Conclusions: Genetic variants in and around SCN10A modulate enhancer function and expression of a cardiac-specific SCN10A-short transcript. We propose that non-coding genetic variation modulates transcriptional regulation of a functional C-terminal portion of NaV1.8 in cardiomyocytes that impacts on NaV1.5 function, cardiac conduction velocities and arrhythmia susceptibility.

Intronic enhancer region governs transcript-specific Bdnf expression in rodent neurons

Brain-derived neurotrophic factor (BDNF) controls the survival, growth, and function of neurons both during the development and in the adult nervous system. Bdnf is transcribed from several distinct promoters generating transcripts with alternative 5' exons. Bdnf transcripts initiated at the first cluster of exons have been associated with the regulation of body weight and various aspects of social behavior, but the mechanisms driving the expression of these transcripts have remained poorly understood. Here, we identify an evolutionarily conserved intronic enhancer region inside the Bdnf gene that regulates both basal and stimulus-dependent expression of the Bdnf transcripts starting from the first cluster of 5' exons in mouse and rat neurons. We further uncover a functional E-box element in the enhancer region, linking the expression of Bdnf and various pro-neural basic helix–loop–helix transcription factors. Collectively, our results shed new light on the cell-type- and stimulus-specific regulation of the important neurotrophic factor BDNF.

Intronic enhancer region governs transcript-specific BDNF expression in neurons

ABSTRACTBrain-derived neurotrophic factor (BDNF) controls the survival, growth, and function of neurons both during the development and in the adult nervous system. BDNF gene is transcribed from several distinct promoters generating transcripts with alternative 5’ exons. BDNF transcripts initiated at the first cluster of exons have been associated with the regulation of body weight and various aspects of social behavior, but the mechanisms driving the expression of these transcripts have remained poorly understood. Here, we identify an evolutionarily conserved intronic enhancer region inside the BDNF gene that regulates both basal and stimulus-dependent expression of the BDNF transcripts starting from the first cluster of 5’ exons in neurons. We further uncover a functional E-box element in the enhancer region, linking the expression of BDNF and various pro-neural basic helix-loop-helix transcription factors. Collectively, our results shed new light on the cell type- and stimulus-specific regulation of the important neurotrophic factor BDNF.