shRNAs Targeting a Common KCNQ1 Variant Could Alleviate Long-QT1 Disease Severity by Inhibiting a Mutant Allele

Long-QT syndrome type 1 (LQT1) is caused by mutations in KCNQ1. Patients heterozygous for such a mutation co-assemble both mutant and wild-type KCNQ1-encoded subunits into tetrameric Kv7.1 potassium channels. Here, we investigated whether allele-specific inhibition of mutant KCNQ1 by targeting a common variant can shift the balance towards increased incorporation of the wildtype allele to alleviate the disease in human-induced pluripotent stem-cell-derived cardiomyocytes (hiPSC-CMs). We identified the single nucleotide polymorphisms (SNP) rs1057128 (G/A) in KCNQ1, with a heterozygosity of 27% in the European population. Next, we determined allele-specificity of short-hairpin RNAs (shRNAs) targeting either allele of this SNP in hiPSC-CMs that carry an LQT1 mutation. Our shRNAs downregulated 60% of the A allele and 40% of the G allele without affecting the non-targeted allele. Suppression of the mutant KCNQ1 allele by 60% decreased the occurrence of arrhythmic events in hiPSC-CMs measured by a voltage-sensitive reporter, while suppression of the wild-type allele increased the occurrence of arrhythmic events. Furthermore, computer simulations based on another LQT1 mutation revealed that 60% suppression of the mutant KCNQ1 allele shortens the prolonged action potential in an adult cardiomyocyte model. We conclude that allele-specific inhibition of a mutant KCNQ1 allele by targeting a common variant may alleviate the disease. This novel approach avoids the need to design shRNAs to target every single mutation and opens up the exciting possibility of treating multiple LQT1-causing mutations with only two shRNAs

shRNAs Targeting a Common KCNQ1 Variant Could Alleviate Long-QT1 Disease Severity by Inhibiting a Mutant Allele

Long-QT syndrome type 1 (LQT1) is caused by mutations in KCNQ1. Patients heterozygous for such a mutation co-assemble both mutant and wild-type KCNQ1-encoded subunits into tetrameric Kv7.1 potassium channels. Here, we investigated whether allele-specific inhibition of mutant KCNQ1 by targeting a common variant can shift the balance towards increased incorporation of the wild-type allele to alleviate the disease in human-induced pluripotent stem-cell-derived cardiomyocytes (hiPSC-CMs). We identified the single nucleotide polymorphisms (SNP) rs1057128 (G/A) in KCNQ1, with a heterozygosity of 27% in the European population. Next, we determined allele-specificity of short-hairpin RNAs (shRNAs) targeting either allele of this SNP in hiPSC-CMs that carry an LQT1 mutation. Our shRNAs downregulated 60% of the A allele and 40% of the G allele without affecting the non-targeted allele. Suppression of the mutant KCNQ1 allele by 60% decreased the occurrence of arrhythmic events in hiPSC-CMs measured by a voltage-sensitive reporter, while suppression of the wild-type allele increased the occurrence of arrhythmic events. Furthermore, computer simulations based on another LQT1 mutation revealed that 60% suppression of the mutant KCNQ1 allele shortens the prolonged action potential in an adult cardiomyocyte model. We conclude that allele-specific inhibition of a mutant KCNQ1 allele by targeting a common variant may alleviate the disease. This novel approach avoids the need to design shRNAs to target every single mutation and opens up the exciting possibility of treating multiple LQT1-causing mutations with only two shRNAs

Identification of a differentiation stall in epithelial mesenchymal transition in histone H3-mutant diffuse midline glioma.

BackgroundDiffuse midline gliomas with histone H3 K27M (H3K27M) mutations occur in early childhood and are marked by an invasive phenotype and global decrease in H3K27me3, an epigenetic mark that regulates differentiation and development. H3K27M mutation timing and effect on early embryonic brain development are not fully characterized.ResultsWe analyzed multiple publicly available RNA sequencing datasets to identify differentially expressed genes between H3K27M and non-K27M pediatric gliomas. We found that genes involved in the epithelial-mesenchymal transition (EMT) were significantly overrepresented among differentially expressed genes. Overall, the expression of pre-EMT genes was increased in the H3K27M tumors as compared to non-K27M tumors, while the expression of post-EMT genes was decreased. We hypothesized that H3K27M may contribute to gliomagenesis by stalling an EMT required for early brain development, and evaluated this hypothesis by using another publicly available dataset of single-cell and bulk RNA sequencing data from developing cerebral organoids. This analysis revealed similarities between H3K27M tumors and pre-EMT normal brain cells. Finally, a previously published single-cell RNA sequencing dataset of H3K27M and non-K27M gliomas revealed subgroups of cells at different stages of EMT. In particular, H3.1K27M tumors resemble a later EMT stage compared to H3.3K27M tumors.ConclusionsOur data analyses indicate that this mutation may be associated with a differentiation stall evident from the failure to proceed through the EMT-like developmental processes, and that H3K27M cells preferentially exist in a pre-EMT cell phenotype. This study demonstrates how novel biological insights could be derived from combined analysis of several previously published datasets, highlighting the importance of making genomic data available to the community in a timely manner

Identification of a differentiation stall in epithelial mesenchymal transition in histone H3-mutant diffuse midline glioma.

Diffuse midline gliomas with histone H3 K27M (H3K27M) mutations occur in early childhood and are marked by an invasive phenotype and global decrease in H3K27me3, an epigenetic mark that regulates differentiation and development. H3K27M mutation timing and effect on early embryonic brain development are not fully characterized. We analyzed multiple publicly available RNA sequencing datasets to identify differentially expressed genes between H3K27M and non-K27M pediatric gliomas. We found that genes involved in the epithelial-mesenchymal transition (EMT) were significantly overrepresented among differentially expressed genes. Overall, the expression of pre-EMT genes was increased in the H3K27M tumors as compared to non-K27M tumors, while the expression of post-EMT genes was decreased. We hypothesized that H3K27M may contribute to gliomagenesis by stalling an EMT required for early brain development, and evaluated this hypothesis by using another publicly available dataset of single-cell and bulk RNA sequencing data from developing cerebral organoids. This analysis revealed similarities between H3K27M tumors and pre-EMT normal brain cells. Finally, a previously published single-cell RNA sequencing dataset of H3K27M and non-K27M gliomas revealed subgroups of cells at different stages of EMT. In particular, H3.1K27M tumors resemble a later EMT stage compared to H3.3K27M tumors. Our data analyses indicate that this mutation may be associated with a differentiation stall evident from the failure to proceed through the EMT-like developmental processes, and that H3K27M cells preferentially exist in a pre-EMT cell phenotype. This study demonstrates how novel biological insights could be derived from combined analysis of several previously published datasets, highlighting the importance of making genomic data available to the community in a timely manner

Titin Circular RNAs Create a Back-Splice Motif Essential for SRSF10 Splicing

BACKGROUND: TTN (Titin), the largest protein in humans, forms the molecular spring that spans half of the sarcomere to provide passive elasticity to the cardiomyocyte. Mutations that disrupt the TTN transcript are the most frequent cause of hereditary heart failure. We showed before that TTN produces a class of circular RNAs (circRNAs) that depend on RBM20 to be formed. In this study, we show that the back-splice junction formed by this class of circRNAs creates a unique motif that binds SRSF10 to enable it to regulate splicing. Furthermore, we show that one of these circRNAs (cTTN1) distorts both localization of and splicing by RBM20. METHODS: We calculated genetic constraint of the identified motif in 125 748 exomes collected from the gnomAD database. Furthermore, we focused on the highest expressed RBM20-dependent circRNA in the human heart, which we named cTTN1. We used shRNAs directed to the back-splice junction to induce selective loss of cTTN1 in human induced pluripotent stem cell-derived cardiomyocytes. RESULTS: Human genetics suggests reduced genetic tolerance of the generated motif, indicating that mutations in this motif might lead to disease. RNA immunoprecipitation confirmed binding of circRNAs with this motif to SRSF10. Selective loss of cTTN1 in human induced pluripotent stem cell-derived cardiomyocytes induced structural abnormalities, apoptosis, and reduced contractile force in engineered heart tissue. In line with its SRSF10 binding, loss of cTTN1 caused abnormal splicing of important cardiomyocyte SRSF10 targets such as MEF2A and CASQ2. Strikingly, loss of cTTN1 also caused abnormal splicing of TTN itself. Mechanistically, we show that loss of cTTN1 distorts both localization of and splicing by RBM20. CONCLUSIONS: We demonstrate that circRNAs formed from the TTN transcript are essential for normal splicing of key muscle genes by enabling splice regulators RBM20 and SRSF10. This shows that the TTN transcript also has regulatory roles, besides its well-known signaling and structural function. In addition, we demonstrate that the specific sequence created by the back-splice junction of these circRNAs has important functions. This highlights the existence of functionally important sequences that cannot be recognized as such in the human genome but provides an as-yet unrecognized source for functional sequence variation

A method for campus-wide SARS-CoV-2 surveillance at a large public university

The systematic screening of asymptomatic and pre-symptomatic individuals is a powerful tool for controlling community transmission of infectious disease on college campuses. Faced with a paucity of testing in the beginning of the COVID-19 pandemic, many universities developed molecular diagnostic laboratories focused on SARS-CoV-2 diagnostic testing on campus and in their broader communities. We established the UC Santa Cruz Molecular Diagnostic Lab in early April 2020 and began testing clinical samples just five weeks later. Using a clinically-validated laboratory developed test (LDT) that avoided supply chain constraints, an automated sample pooling and processing workflow, and a custom laboratory information management system (LIMS), we expanded testing from a handful of clinical samples per day to thousands per day with the testing capacity to screen our entire campus population twice per week. In this report we describe the technical, logistical, and regulatory processes that enabled our pop-up lab to scale testing and reporting capacity to thousands of tests per day

Human-specific NOTCH2NL genes affect notch signaling and cortical neurogenesis

Genetic changes causing brain size expansion in human evolution have remained elusive. Notch signaling is essential for radial glia stem cell proliferation and is a determinant of neuronal number in the mammalian cortex. We find that three paralogs of human-specific NOTCH2NL are highly expressed in radial glia. Functional analysis reveals that different alleles of NOTCH2NL have varying potencies to enhance Notch signaling by interacting directly with NOTCH receptors. Consistent with a role in Notch signaling, NOTCH2NL ectopic expression delays differentiation of neuronal progenitors, while deletion accelerates differentiation into cortical neurons. Furthermore, NOTCH2NL genes provide the breakpoints in 1q21.1 distal deletion/duplication syndrome, where duplications are associated with macrocephaly and autism and deletions with microcephaly and schizophrenia. Thus, the emergence of human-specific NOTCH2NL genes may have contributed to the rapid evolution of the larger human neocortex, accompanied by loss of genomic stability at the 1q21.1 locus and resulting recurrent neurodevelopmental disorders