A novel knockout mouse for the small EDRK-rich factor 2 (Serf2) showing developmental and other deficits

The small EDRK-rich factor 2 (SERF2) is a highly conserved protein that modifies amyloid fibre assembly in vitro and promotes protein misfolding. However, the role of SERF2 in regulating age-related proteotoxicity remains largely unexplored due to a lack of in vivo models. Here, we report the generation of Serf2 knockout mice using an ES cell targeting approach, with Serf2 knockout alleles being bred onto different defined genetic backgrounds. We highlight phenotyping data from heterozygous Serf2^{+/-} mice, including unexpected male-specific phenotypes in startle response and pre-pulse inhibition. We report embryonic lethality in Serf2^{-/-} null animals when bred onto a C57BL/6 N background. However, homozygous null animals were viable on a mixed genetic background and, remarkably, developed without obvious abnormalities. The Serf2 knockout mice provide a powerful tool to further investigate the role of SERF2 protein in previously unexplored pathophysiological pathways in the context of a whole organism

Whole genome sequencing in paediatric channelopathy and cardiomyopathy

BackgroundPrecision medicine in paediatric cardiac channelopathy and cardiomyopathy has a rapid advancement over the past years. Compared to conventional gene panel and exome-based testing, whole genome sequencing (WGS) offers additional coverage at the promoter, intronic regions and the mitochondrial genome. However, the data on use of WGS to evaluate the genetic cause of these cardiovascular conditions in children and adolescents are limited.MethodsIn a tertiary paediatric cardiology center, we recruited all patients diagnosed with cardiac channelopathy and cardiomyopathy between the ages of 0 and 18 years old, who had negative genetic findings with prior gene panel or exome-based testing. After genetic counselling, blood samples were collected from the subjects and both their parents for WGS analysis.ResultsA total of 31 patients (11 cardiac channelopathy and 20 cardiomyopathy) were recruited. Four intronic splice-site variants were identified in three cardiomyopathy patients, which were not identified in previous whole exome sequencing. These included a pathogenic variant in TAFAZZIN:c.284+5G>A (Barth syndrome), a variant of unknown significance (VUS) in MYBPC3:c.1224-80G>A and 2 compound heterozygous LP variants in LZTR1 (LZTR1:c.1943-256C>T and LZTR1:c1261-3C>G) in a patient with clinical features of RASopathy. There was an additional diagnostic yield of 1.94% using WGS for identification of intronic variants, on top of conventional gene testing.ConclusionWGS plays a role in identifying additional intronic splice-site variants in paediatric patients with isolated cardiomyopathy. With the demonstrated low extra yield of WGS albeit its ability to provide potential clinically important information, WGS should be considered in selected paediatric cases of cardiac channelopathy and cardiomyopathy in a cost-effective manner

Mice with endogenous TDP-43 mutations exhibit gain of splicing function and characteristics of amyotrophic lateral sclerosis

TDP-43 (encoded by the gene TARDBP) is an RNA binding protein central to the pathogenesis of amyotrophic lateral sclerosis (ALS). However, how TARDBP mutations trigger pathogenesis remains unknown. Here, we use novel mouse mutants carrying point mutations in endogenous Tardbp to dissect TDP-43 function at physiological levels both in vitro and in vivo Interestingly, we find that mutations within the C-terminal domain of TDP-43 lead to a gain of splicing function. Using two different strains, we are able to separate TDP-43 loss- and gain-of-function effects. TDP-43 gain-of-function effects in these mice reveal a novel category of splicing events controlled by TDP-43, referred to as "skiptic" exons, in which skipping of constitutive exons causes changes in gene expression. In vivo, this gain-of-function mutation in endogenous Tardbp causes an adult-onset neuromuscular phenotype accompanied by motor neuron loss and neurodegenerative changes. Furthermore, we have validated the splicing gain-of-function and skiptic exons in ALS patient-derived cells. Our findings provide a novel pathogenic mechanism and highlight how TDP-43 gain of function and loss of function affect RNA processing differently, suggesting they may act at different disease stages

Linking the FTO obesity rs1421085 variant circuitry to cellular, metabolic, and organismal phenotypes in vivo

Variants in FTO have the strongest association with obesity; however, it is still unclear how those noncoding variants mechanistically affect whole-body physiology. We engineered a deletion of the rs1421085 conserved cis-regulatory module (CRM) in mice and confirmed in vivo that the CRM modulates Irx3 and Irx5 gene expression and mitochondrial function in adipocytes. The CRM affects molecular and cellular phenotypes in an adipose depot–dependent manner and affects organismal phenotypes that are relevant for obesity, including decreased high-fat diet–induced weight gain, decreased whole-body fat mass, and decreased skin fat thickness. Last, we connected the CRM to a genetically determined effect on steroid patterns in males that was dependent on nutritional challenge and conserved across mice and humans. Together, our data establish cross-species conservation of the rs1421085 regulatory circuitry at the molecular, cellular, metabolic, and organismal level, revealing previously unknown contextual dependence of the variant’s action

Supporting data for "Unrevealing Parental Mosaicism: The hidden answer to the recurrence of apparent de novo variants"

In this study, I investigated the diagnostic yield of parental mosaicism in a cohort of 21 families with apparent de novo variants (DNVs) associated with neurodevelopmental disorders, and determined the usefulness of the detection of parental mosaicism to the management and genetic counselling of patients. Buccal and sperm samples were collected from the parents to detect potential parental mosaicism by digital PCR (dPCR)/droplet digital PCR (ddPCR), with blocker displacement amplification (BDA) whenever possible. This dataset consisted of 2 parts. First part includes sensitive clinical data including but not limited to patients’ rare diseases and clinical records, parental age of conception/childbirth, parental health status, family history and clinical records, all of which were strictly confidential. Second part includes experimental primer/probe sequence/condition for dPCR/ddPCR/BDA that can be shared.</p

In silico assessment of moringa oleifera as an antiviral agent against the N501Y mutation in the receptor-binding domain of the spike glycoprotein of SARS-Cov-2 South African variant (B.1.351)

An in-silico method was employed to predict which of the fifteen Moringa oleifera phytocompounds would exhibit inhibitory action towards the spike glycoprotein with N501Y mutation of the SARS-CoV-2 B.1.351 variant. The phytocompounds were screened for their drug-likeness and Absorption, Distribution, Metabolism, Excretion and Toxicity (ADMET) properties. Molecular docking results from AutoDock Vina reveal that chlorogenic acid and rutin had the greatest affinity for the spike glycoprotein. (-5.2 kcal/mol), even stronger than the positive control, arbidol (-4.2 kcal/mol). The conformations were visualized and analyzed using BIOVIA Discovery Studio Visualizer. Hydrogen bonds and electrostatic and hydrophobic interactions dominated the complex. Furthermore, CABS-flex 2.0 showed that chlorogenic acid and rutin can stably bind to the spike glycoprotein based on the minimal root mean square fluctuations between the unbound and bound structures of the protein and paired t-test (p \u3e 0.05). Results showed that chlorogenic acid and rutin compounds have promising antiviral properties that could potentially block the N501Y spike glycoprotein from binding into target human cells