Evaluating Human Mutation Databases for 'Treatability' Using Personalized Antisense Oligonucleotides

Genome sequencing in the clinic often allows patients to receive a molecular diagnosis. However, variants are most often evaluated for pathogenicity, neglecting potential "treatability", and thus often yielding limited clinical benefit. Several collaborative efforts now aim to provide a therapy based upon the genetic variants, even if the drug will benefit only a single patient. Antisense oligonucleotide (ASO) therapies, among others, offer attractive "programmable" and relatively safe platforms for individualized therapy. The landscape of "ASO-treatable" variants is largely uncharted, with new developments emerging for loss-of-function (LOF), haploinsufficient, and gain-of-function (GOF) variants. ASOs can access the genome to target splice-gain variants, poison exons, untranslated/regulatory regions, and naturally-occurring antisense transcripts. Many of these approaches have yet to be proven clinically beneficial, and it is unclear if disease in some patients has progressed past the point where benefit could reasonably be expected. Here we mine public variant databases to identify potential future therapeutic targets. We found that the majority of human pathogenic genetic variants have one or more approaches that could be targeted therapeutically, advantaging the many ways that ASOs can regulate gene expression. The future might see medical teams considering "treatability" when interpreting genome sequencing results, to fully realize benefits for patients.

Overactive STAT3 drives accumulation of disease-associated CD21low B cells

Dysregulated STAT3 signalling is correlated with antibody-mediated autoimmunity and B- cell neoplasia, but its effect on B cells is underexplored. Here we address this in children with STAT3 gain-of-function (GOF) syndrome and in mice with STAT3T716M, the most common STAT3 GOF syndrome human mutation, or STAT3K658N, a dimerization interface mutation responsible for STAT3 GOF syndrome in two children. The main B cell consequence of overactive STAT3 was accumulation of CD19high CD21low atypical memory B cells in humans and of CD21low CD23low B cells in mice resembling age-associated B cells expressing T-bet, CD11c and plasma cell differentiation genes. Overactive STAT3 within B cells increased expression of many genes in the B cell receptor and T cell help pathways, increased the tolerogenic receptor CD22, but opposed B cell tolerance checkpoints and increased formation of T-bet+ B cells upon BCR and CD40 stimulation. These results reveal overactive STAT3 as a central driver of a key class of disease-associated B-lymphocytes in humans and mice.

CREBRF p.Arg457Gln Promotes Obesity and Improves Insulin Sensitivity by Promoting Preadipocyte Differentiation and Reducing Oxidative Metabolism in Gene-modified Pig Models

Obesity is one of the most important risk factors for type 2 diabetes (T2DM). The CREBRF missense allele of rs373863828 (p.Arg457Gln) is associated with increased body mass index (BMI), yet reduced risk of T2DM in people with Pacific ancestry. To investigate the functional consequences of the CREBRF variant, we introduced the corresponding human mutation p.Arg457Gln into porcine genome by using a CRISPR/Cas9-mediated homologous recombination (HR)-dependent approach. The CREBRF p.Arg457Gln pig models displayed dramatically increased fat deposition, yet improved sensitivity to insulin. Transcriptome and metabolome analyses of subcutaneous white adipose tissues showed that the CREBRF p.Arg457Gln mutation promoted preadipocyte differentiation, which indicated that obesity was caused by increased number (hyperplasia) rather than size (hypertrophy) of adipocytes. In addition, the oxidative capacity decreased in the adipose tissue of pigs with CREBRF p.Arg457Gln variant. The pre-oxidative metabolite content (4-HNE and MDA) significantly decreased, while activity of antioxidant enzymes (GPX, SOD, and CAT) increased, thereby repressing oxidative metabolism of adipose tissue and reducing level of reactive oxygen species (ROS). The low reactive oxygen species could prevent insulin resistance and reduce risk of obesity-induced type 2 diabetes. This study provides further mechanistic insights into favourable adiposity resulting from CREBRF p.Arg457Gln.

Abstract P439: Harnessing Multiscale Models Of A Dilated Cardiomyopathy Mutation For Precision Medicine

Familial dilated cardiomyopathy (DCM) is a leading cause of both adult and pediatric heart failure. Currently, there is no cure for DCM, and the 5-year transplant free survival rate is <50%. There is therefore an outstanding need to develop new therapeutics. Prior studies have established a strong genetic basis for DCM and identified causative genetic mutations. These observations provide unique opportunities to apply precision medicine approaches that target and circumvent the effects of deleterious mutations. Here, we used a multiscale approach to study the consequences of a human mutation in troponin T that causes DCM, ΔK210. We found that at the molecular scale ΔK210 changes the positioning of tropomyosin along the thin filament, leading to molecular hypocontractility. Using genome edited human stem cell derived cardiomyocytes heterozygous for the mutation, we show reduced cellular contractility at the single cell and tissue levels. Importantly, we demonstrate that mutant tissues show a reduced Frank-Starling response, increased stiffness, and misaligned myocytes. Based on our molecular mechanism, we hypothesized that treatment of ΔK210 with Omecamtiv Mecarbil (OM), a thin filament activator in clinical trials for heart failure, would improve the function of mutant tissues. We found that treatment of ΔK210 molecular complexes and tissues with OM causes a dose-dependent increase in cardiac function, reversing the mutation-induced contractile defect. Taken together, our study demonstrates how mechanistic molecular studies can be harnessed to identify precision medicine therapeutics.

Pathophysiological Roles of Abnormal Axon Initial Segments in Neurodevelopmental Disorders

The 20–60 μm axon initial segment (AIS) is proximally located at the interface between the axon and cell body. AIS has characteristic molecular and structural properties regulated by the crucial protein, ankyrin-G. The AIS contains a high density of Na+ channels relative to the cell body, which allows low thresholds for the initiation of action potential (AP). Molecular and physiological studies have shown that the AIS is also a key domain for the control of neuronal excitability by homeostatic mechanisms. The AIS has high plasticity in normal developmental processes and pathological activities, such as injury, neurodegeneration, and neurodevelopmental disorders (NDDs). In the first half of this review, we provide an overview of the molecular, structural, and ion-channel characteristics of AIS, AIS regulation through axo-axonic synapses, and axo−glial interactions. In the second half, to understand the relationship between NDDs and AIS, we discuss the activity-dependent plasticity of AIS, the human mutation of AIS regulatory genes, and the pathophysiological role of an abnormal AIS in NDD model animals and patients. We propose that the AIS may provide a potentially valuable structural biomarker in response to abnormal network activity in vivo as well as a new treatment concept at the neural circuit level.

Nucleoporin107 mediates female sexual differentiation via Dsx

We recently identified a missense mutation in Nucleoporin107 (Nup107; D447N) underlying XX-ovarian-dysgenesis, a rare disorder characterized by underdeveloped and dysfunctional ovaries. Modelling of the human mutation in Drosophila or specific knockdown of Nup107 in the gonadal soma resulted in ovarian-dysgenesis-like phenotypes. Transcriptomic analysis identified the somatic sex-determination gene doublesex (dsx) as a target of Nup107. Establishing Dsx as a primary relevant target of Nup107, either loss or gain of Dsx in the gonadal soma is sufficient to mimic or rescue the phenotypes induced by Nup107 loss. Importantly, the aberrant phenotypes induced by compromising either Nup107 or dsx are reminiscent of BMP signaling hyperactivation. Remarkably, in this context, the metalloprotease AdamTS-A, a transcriptional target of both Dsx and Nup107, is necessary for the calibration of BMP signaling. As modulation of BMP signaling is a conserved critical determinant of soma-germline interaction, the sex and tissue specific deployment of Dsx-F by Nup107 seems crucial for the maintenance of the homeostatic balance between the germ cells and somatic gonadal cells.

Pathophysiological Roles of Abnormal Axon Initial Segments in Neurodevelopmental Disorders

The 20&ndash;60-&mu;m axon initial segment (AIS) is proximally located at the interface between the axon and cell body. AIS has characteristic molecular and structural properties regulated by the crucial protein, ankyrin-G. The AIS contains a high density of Na+ channels relative to the cell body, which allows low thresholds for initiation of action potential (AP). Molecular and physiological studies have shown that the AIS is also a key domain for the control of neuronal excitability by homeostatic mechanisms. The AIS has high plasticity in normal developmental processes and pathological activities such as injury, neurodegeneration, and neurodevelopmental disorders (NDDs). In the first half of this review, we provide an overview of the molecular, structural, and ion-channel characteristics of AIS, AIS regulation through axo-axonic synapses, and axo-glial interactions. In the second half, to understand the relationship between NDDs and AIS, we discuss the activity-dependent plasticity of AIS, the human mutation of AIS regulatory genes, and the pathophysiological role of the abnormal AIS in the NDD model animals and patients. We propose that AIS may provide a potentially valuable structural biomarker in response to abnormal network activity in vivo as well as a new treatment concept at the neural circuit level.