The IFIH1-A946T risk variant promotes diabetes in a sex-dependent manner.

Type 1 diabetes (T1D) is an autoimmune disease in which pancreatic islet ß-cells are attacked by the immune system, resulting in insulin deficiency and hyperglycemia. One of the top non-synonymous single-nucleotide polymorphisms (SNP) associated with T1D is in the interferon-induced helicase C domain-containing protein 1 (IFIH1), which encodes an anti-viral cytosolic RNA sensor. This SNP results in an alanine to threonine substitution at amino acid 946 (IFIH1A946T) and confers an increased risk for several autoimmune diseases, including T1D. We hypothesized that the IFIH1A946T risk variant, (IFIH1R) would promote T1D pathogenesis by stimulating type I interferon (IFN I) signaling leading to immune cell alterations. To test this, we developed Ifih1R knock-in mice on the non-obese diabetic (NOD) mouse background, a spontaneous T1D model. Our results revealed a modest increase in diabetes incidence and insulitis in Ifih1R compared to non-risk Ifih1 (Ifih1NR) mice and a significant acceleration of diabetes onset in Ifih1R females. Ifih1R mice exhibited a significantly enhanced interferon stimulated gene (ISG) signature compared to Ifih1NR, indicative of increased IFN I signaling. Ifih1R mice exhibited an increased frequency of plasma cells as well as tissue-dependent changes in the frequency and activation of CD8+ T cells. Our results indicate that IFIH1R may contribute to T1D pathogenesis by altering the frequency and activation of immune cells. These findings advance our knowledge on the connection between the rs1990760 variant and T1D. Further, these data are the first to demonstrate effects of Ifih1R in NOD mice, which will be important to consider for the development of therapeutics for T1D.

The IFIH1-A946T risk variant promotes diabetes in a sex-dependent manner.

Type 1 diabetes (T1D) is an autoimmune disease in which pancreatic islet ß-cells are attacked by the immune system, resulting in insulin deficiency and hyperglycemia. One of the top non-synonymous single-nucleotide polymorphisms (SNP) associated with T1D is in the interferon-induced helicase C domain-containing protein 1 ( IFIH1 ), which encodes an anti-viral cytosolic RNA sensor. This SNP results in an alanine to threonine substitution at amino acid 946 (IFIH1 A946T ) and confers an increased risk for several autoimmune diseases, including T1D. We hypothesized that the IFIH1 A946T risk variant, ( IFIH1 R ) would promote T1D pathogenesis by stimulating type I interferon (IFN I) signaling leading to immune cell alterations. To test this, we developed Ifih1 R knock-in mice on the non-obese diabetic (NOD) mouse background, a spontaneous T1D model. Our results revealed a modest increase in diabetes incidence and insulitis in Ifih1 R compared to non-risk Ifih1 ( Ifih1 NR ) mice and a significant acceleration of diabetes onset in Ifih1 R females. Ifih1 R mice exhibited a significantly enhanced interferon stimulated gene (ISG) signature compared to Ifih1 NR , indicative of increased IFN I signaling. Ifih1 R mice exhibited an increased frequency of plasma cells as well as tissue-dependent changes in the frequency and activation of CD8 + T cells. Our results indicate that IFIH1 R may contribute to T1D pathogenesis by altering the frequency and activation of immune cells. These findings advance our knowledge on the connection between the rs1990760 variant and T1D. Further, these data are the first to demonstrate effects of Ifih1 R in NOD mice, which will be important to consider for the development of therapeutics for T1D.

Genome Report: chromosome-scale genome assembly of the African spiny mouse (Acomys cahirinus).

There is increasing interest in the African spiny mouse (Acomys cahirinus) as a model organism because of its ability for regeneration of tissue after injury in skin, muscle, and internal organs such as the kidneys. A high-quality reference genome is needed to better understand these regenerative properties at the molecular level. Here, we present an improved reference genome for A. cahirinus generated from long Nanopore sequencing reads. We confirm the quality of our annotations using RNA sequencing data from 4 different tissues. Our genome is of higher contiguity and quality than previously reported genomes from this species and will facilitate ongoing efforts to better understand the regenerative properties of this organism.

GENOME REPORT: Chromosome-scale genome assembly of the African spiny mouse ( Acomys cahirinus ).

There is increasing interest in the African spiny mouse ( Acomys cahirinus ) as a model organism because of its ability for regeneration of tissue after injury in skin, muscle, and internal organs such as the kidneys. A high-quality reference genome is needed to better understand these regenerative properties at the molecular level. Here, we present an improved reference genome for A. cahirinus generated from long Nanopore sequencing reads. We confirm the quality of our annotations using RNA sequencing data from four different tissues. Our genome is of higher contiguity and quality than previously reported genomes from this species and will facilitate ongoing efforts to better understand the regenerative properties of this organism.

Human KCNQ5 de novo mutations underlie epilepsy and intellectual disability.

We identified six novel de novo human KCNQ5 variants in children with motor/language delay, intellectual disability (ID), and/or epilepsy by whole exome sequencing. These variants, comprising two nonsense and four missense alterations, were functionally characterized by electrophysiology in HEK293/CHO cells, together with four previously reported KCNQ5 missense variants (Lehman A, Thouta S, Mancini GM, Naidu S, van Slegtenhorst M, McWalter K, Person R, Mwenifumbo J, Salvarinova R; CAUSES Study; EPGEN Study; Guella I, McKenzie MB, Datta A, Connolly MB, Kalkhoran SM, Poburko D, Friedman JM, Farrer MJ, Demos M, Desai S, Claydon T. Am J Hum Genet 101: 65-74, 2017). Surprisingly, all eight missense variants resulted in gain of function (GOF) due to hyperpolarized voltage dependence of activation or slowed deactivation kinetics, whereas the two nonsense variants were confirmed to be loss of function (LOF). One severe GOF allele (P369T) was tested and found to extend a dominant GOF effect to heteromeric KCNQ5/3 channels. Clinical presentations were associated with altered KCNQ5 channel gating: milder presentations with LOF or smaller GOF shifts in voltage dependence [change in voltage at half-maximal conduction (ΔV50) = ∼-15 mV] and severe presentations with larger GOF shifts in voltage dependence (ΔV50 = ∼-30 mV). To examine LOF pathogenicity, two Kcnq5 LOF mouse lines were created with CRISPR/Cas9. Both lines exhibited handling- and thermal-induced seizures and abnormal cortical EEGs consistent with epileptiform activity. Our study thus provides evidence for in vivo KCNQ5 LOF pathogenicity and strengthens the contribution of both LOF and GOF mutations to global pediatric neurological impairment, including ID/epilepsy.NEW & NOTEWORTHY Six novel de novo human KCNQ5 variants were identified from children with neurodevelopmental delay, intellectual disability, and/or epilepsy. Expression of these variants along with four previously reported KCNQ5 variants from a similar cohort revealed GOF potassium channels, negatively shifted in V50 of activation and/or delayed deactivation kinetics. GOF is extended to KCNQ5/3 heteromeric channels, making these the predominant channels affected in heterozygous de novo patients. Kcnq5 LOF mice exhibited seizures, consistent with in vivo pathogenicity.

Spiny mice activate unique transcriptional programs after severe kidney injury regenerating organ function without fibrosis.

Fibrosis-driven solid organ failure is an enormous burden on global health. Spiny mice (Acomys) are terrestrial mammals that can regenerate severe skin wounds without scars to avoid predation. Whether spiny mice also regenerate internal organ injuries is unknown. Here, we show that despite equivalent acute obstructive or ischemic kidney injury, spiny mice fully regenerate nephron structure and organ function without fibrosis, whereas C57Bl/6 or CD1 mice progress to complete organ failure with extensive renal fibrosis. Two mechanisms for vertebrate regeneration have been proposed that emphasize either extrinsic (pro-regenerative macrophages) or intrinsic (surviving cells of the organ itself) controls. Comparative transcriptome analysis revealed that the Acomys genome appears poised at the time of injury to initiate regeneration by surviving kidney cells, whereas macrophage accumulation was not detected until about day 7. Thus, we provide evidence for rapid activation of a gene expression signature for regenerative wound healing in the spiny mouse kidney.

Adaptations in Hippo-Yap signaling and myofibroblast fate underlie scar-free ear appendage wound healing in spiny mice.

Spiny mice (Acomys cahirinus) are terrestrial mammals that evolved unique scar-free regenerative wound-healing properties. Myofibroblasts (MFs) are the major scar-forming cell type in skin. We found that following traumatic injury to ear pinnae, MFs appeared rapidly in both Acomys and mouse yet persisted only in mouse. The timing of MF loss in Acomys correlated with wound closure, blastema differentiation, and nuclear localization of the Hippo pathway target protein Yap. Experiments in vitro revealed an accelerated PP2A-dependent dephosphorylation activity that maintained nuclear Yap in Acomys dermal fibroblasts (DFs) and was not detected in mouse or human DFs. Treatment of Acomys in vivo with the nuclear Yap-TEAD inhibitor verteporfin prolonged MF persistence and converted tissue regeneration to fibrosis. Forced Yap activity prevented and rescued TGF-ß1-induced human MF formation in vitro. These results suggest that Acomys evolved modifications of Yap activity and MF fate important for scar-free regenerative wound healing in vivo.

A novel intergenic ETnII-ß insertion mutation causes multiple malformations in polypodia mice.

Mouse early transposon insertions are responsible for ~10% of spontaneous mutant phenotypes. We previously reported the phenotypes and genetic mapping of Polypodia, (Ppd), a spontaneous, X-linked dominant mutation with profound effects on body plan morphogenesis. Our new data shows that mutant mice are not born in expected Mendelian ratios secondary to loss after E9.5. In addition, we refined the Ppd genetic interval and discovered a novel ETnII-ß early transposon insertion between the genes for Dusp9 and Pnck. The ETn inserted 1.6 kb downstream and antisense to Dusp9 and does not disrupt polyadenylation or splicing of either gene. Knock-in mice engineered to carry the ETn display Ppd characteristic ectopic caudal limb phenotypes, showing that the ETn insertion is the Ppd molecular lesion. Early transposons are actively expressed in the early blastocyst. To explore the consequences of the ETn on the genomic landscape at an early stage of development, we compared interval gene expression between wild-type and mutant ES cells. Mutant ES cell expression analysis revealed marked upregulation of Dusp9 mRNA and protein expression. Evaluation of the 5' LTR CpG methylation state in adult mice revealed no correlation with the occurrence or severity of Ppd phenotypes at birth. Thus, the broad range of phenotypes observed in this mutant is secondary to a novel intergenic ETn insertion whose effects include dysregulation of nearby interval gene expression at early stages of development.

Phenotypic and genetic analysis of the cerebellar mutant tmgc26, a new ENU-induced ROR-alpha allele.

ROR-alpha is an orphan nuclear receptor, inactivation of which cell-autonomously blocks differentiation of cerebellar Purkinje cells with a secondary loss of granule neurons. As part of our ENU mutagenesis screen we isolated the recessive tmgc26 mouse mutant, characterized by early-onset progressive ataxia, cerebellar degeneration and juvenile lethality. Detailed analysis of the tmgc26-/- cerebella revealed Purkinje cell and granule cell abnormalities, and defects in molecular layer interneurons and radial glia. Chimera studies suggested a cell-autonomous effect of the tmgc26 mutation in Purkinje cells and molecular layer interneurons, and a non-cell-autonomous effect in granule cells. The mutation was mapped to a 13-Mb interval on chromosome 9, a region that contains the ROR-alpha gene. Sequencing of genomic DNA revealed a T-to-A transition in exon 5 of the ROR-alpha gene, resulting in a nonsense mutation C257X and severe truncation of the ROR-alpha protein. Together, our data identify new roles for ROR-alpha in molecular layer interneurons and radial glia development and suggest tmgc26 as a novel ROR-alpha allele that may be used to further delineate the molecular mechanisms of ROR-alpha action.

Floxin, a resource for genetically engineering mouse ESCs.

We describe a method for the highly efficient and precise targeted modification of gene trap loci in mouse embryonic stem cells (ESCs). Through the Floxin method, gene trap mutations were reverted and new DNA sequences inserted using Cre recombinase and a shuttle vector, pFloxin. Floxin technology is applicable to the existing collection of 24,149 compatible gene trap cell lines, which should enable high-throughput modification of many genes in mouse ESCs.