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Impulsive behavior and impulsivity are heritable phenotypes that are strongly associated with risk for substance use disorders. Identifying the neurogenetic mechanisms that influence impulsivity may also reveal novel biological insights into addiction vulnerability. Our past studies using the BXD and Collaborative Cross (CC) recombinant inbred mouse panels have revealed that behavioral indicators of impulsivity measured in a reversal-learning task are heritable and are genetically correlated with aspects of intravenous cocaine self-administration. Genome-wide linkage studies in the BXD panel revealed a quantitative trait locus (QTL) on chromosome 10, but we expect to identify additional QTL by testing in a population with more genetic diversity. To this end, we turned to Diversity Outbred (DO) mice; 392 DO mice (156 males, 236 females) were phenotyped using the same reversal learning test utilized previously. Our primary indicator of impulsive responding, a measure that isolates the relative difficulty mice have with reaching performance criteria under reversal conditions, revealed a genome-wide significant QTL on chromosome 7 (max LOD score = 8.73, genome-wide corrected p<0.05). A measure of premature responding akin to that implemented in the 5-choice serial reaction time task yielded a suggestive QTL on chromosome 17 (max LOD score = 9.14, genome-wide corrected <0.1). Candidate genes were prioritized ( 2900076A07Rik, Wdr73 and Zscan2) based upon expression QTL data we collected in DO and CC mice and analyses using publicly available gene expression and phenotype databases. These findings may advance understanding of the genetics that drive impulsive behavior and enhance risk for substance use disorders., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Background: The exposure risks to front-line health care workers caring for patients with SARS-CoV-2 infection undergoing surgery or obstetric delivery are unclear, and an understanding of sample types that may harbour virus is important for evaluating risk. We sought to determine whether SARS-CoV-2 viral RNA from patients with SARS-CoV-2 infection undergoing surgery or obstetric delivery was present in the peritoneal cavity of male and female patients, in the female reproductive tract, in the environment of the surgery or delivery suite (surgical instruments or equipment used, air or floors), and inside the masks of the attending health care workers., Methods: We conducted a cross-sectional study from November 2020 to May 2021 at 2 tertiary academic Toronto hospitals, during urgent surgeries or obstetric deliveries for patients with SARS-CoV-2 infection. The presence of SARS-CoV-2 viral RNA in patient, environmental and air samples was identified by real-time reverse transcription polymerase chain reaction (RT-PCR). Air samples were collected using both active and passive sampling techniques. The primary outcome was the proportion of health care workers' masks positive for SARS-CoV-2 RNA. We included adult patients with positive RT-PCR nasal swab undergoing obstetric delivery or urgent surgery (from across all surgical specialties)., Results: A total of 32 patients (age 20-88 yr) were included. Nine patients had obstetric deliveries (6 cesarean deliveries), and 23 patients (14 male) required urgent surgery from the orthopedic or trauma, general surgery, burn, plastic surgery, cardiac surgery, neurosurgery, vascular surgery, gastroenterology and gynecologic oncology divisions. SARS-CoV-2 RNA was detected in 20 of 332 (6%) patient and environmental samples collected: 4 of 24 (17%) patient samples, 5 of 60 (8%) floor samples, 1 of 54 (2%) air samples, 10 of 23 (43%) surgical instrument or equipment samples, 0 of 24 cautery filter samples and 0 of 143 (95% confidence interval 0-0.026) inner surface of mask samples., Interpretation: During the study period of November 2020 to May 2021, we found evidence of SARS-CoV-2 RNA in a small but important number of samples obtained in the surgical and obstetric operative environment. The finding of no detectable virus inside the masks worn by the health care teams would suggest a low risk of infection for health care workers using appropriate personal protective equipment., Competing Interests: Competing interests: Robert Kozak reports a grant from the Ontario Together Fund. Grace Liu reports a speaker payment from Hologic and reports an unpaid position as a board member for the American Association of Gynecologic Laparoscopists, 2019–2021. No other competing interests were declared., (© 2022 CMA Impact Inc. or its licensors.)

Circadian variability is driven by genetics and Diversity Outbred (DO) mice is a powerful tool for examining the genetics of complex traits because their high genetic and phenotypic diversity compared to conventional mouse crosses. The DO population combines the genetic diversity of eight founder strains including five common inbred and three wild-derived strains. In DO mice and their founders, we established a high-throughput system to measure cellular rhythms using in vitro preparations of skin fibroblasts. Among the founders, we observed strong heritability for rhythm period, robustness, phase and amplitude. We also found significant sex and strain differences for these rhythms. Extreme differences in period for molecular and behavioral rhythms were found between the inbred A/J strain and the wild-derived CAST/EiJ strain, where A/J had the longest period and CAST/EiJ had the shortest. In addition, we measured cellular rhythms in 329 DO mice, which displayed far greater phenotypic variability than the founders-80% of founders compared to only 25% of DO mice had periods of ~ 24 h. Collectively, our findings demonstrate that genetic diversity contributes to phenotypic variability in circadian rhythms, and high-throughput characterization of fibroblast rhythms in DO mice is a tractable system for examining the genetics of circadian traits.

Distal enhancers are thought to play important roles in the spatiotemporal regulation of gene expression during embryonic development, but few predicted enhancer elements have been shown to affect transcription of their endogenous genes or to alter phenotypes when disrupted. Here, we demonstrate that a 123.6-kb deletion within the mouse Slc25a13 gene is associated with reduced transcription of Dlx5 , a gene located 660 kb away. Mice homozygous for the Slc25a13 deletion mutation [named hyperspin ( hspn )] have malformed inner ears and are deaf with balance defects, whereas previously reported Slc25a13 knockout mice showed no phenotypic abnormalities. Inner ears of Slc25a13 hspn/hspn mice have malformations similar to those of Dlx5 - / - embryos, and Dlx5 expression is severely reduced in the otocyst but not the branchial arches of Slc25a13 hspn/hspn embryos, indicating that the Slc25a13 hspn deletion affects otic-specific enhancers of Dlx5 In addition, transheterozygous Slc25a13 +/hspn Dlx5 +/ - mice exhibit noncomplementation with inner ear dysmorphologies similar to those of Slc25a13 hspn/hspn and Dlx5 - / - embryos, verifying a cis -acting effect of the Slc25a13 hspn deletion on Dlx5 expression. CRISPR/Cas9-mediated deletions of putative enhancer elements located within the Slc25a13 hspn deleted region failed to phenocopy the defects of Slc25a13 hspn/hspn mice, suggesting the possibility of multiple enhancers with redundant functions. Our findings in mice suggest that analogous enhancer elements in the human SLC25A13 gene may regulate DLX5 expression and underlie the hearing loss that is associated with split-hand/-foot malformation 1 syndrome. Slc25a13 hspn/hspn mice provide a new animal model for studying long-range enhancer effects on Dlx5 expression in the developing inner ear., (Copyright © 2018 by the Genetics Society of America.)

Mutations of the human ATP6V1B1 gene cause distal renal tubular acidosis (dRTA; OMIM #267300) often associated with sensorineural hearing impairment; however, mice with a knockout mutation of Atp6v1b1 were reported to exhibit a compensated acidosis and normal hearing. We discovered a new spontaneous mutation (vortex, symbol vtx) of Atp6v1b1 in an MRL/MpJ (MRL) colony of mice. In contrast to the reported phenotype of the knockout mouse, which was developed on a primarily C57BL/6 (B6) strain background, MRL-Atp6v1b1vtx/vtx mutant mice exhibit profound hearing impairment, which is associated with enlarged endolymphatic compartments of the inner ear. Mutant mice have alkaline urine but do not exhibit overt metabolic acidosis, a renal phenotype similar to that of the Atpbv1b1 knockout mouse. The abnormal inner ear phenotype of MRL- Atp6v1b1vtx/vtx mice was lost when the mutation was transferred onto the C57BL/6J (B6) background, indicating the influence of strain-specific genetic modifiers. To genetically map modifier loci in Atp6v1b1vtx/vtx mice, we analysed ABR thresholds of progeny from a backcross segregating MRL and B6 alleles. We found statistically significant linkage with a locus on Chr 13 that accounts for about 20% of the hearing threshold variation in the backcross mice. The important effect that genetic background has on the inner ear phenotype of Atp6v1b1 mutant mice provides insight into the hearing loss variability associated with dRTA caused by ATP6V1B1 mutations. Because MRL-Atp6v1b1vxt/vtx mice do not recapitulate the metabolic acidosis of dRTA patients, they provide a new genetic model for nonsyndromic deafness with enlarged vestibular aqueduct (EVA; OMIM #600791)., (© The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

A single nucleotide variant (SNV) of the cadherin 23 gene (Cdh23 c.753A ), common to many inbred mouse strains, accelerates age-related hearing loss (AHL) and can worsen auditory phenotypes of other mutations. We used homologous recombination in C57BL/6 NJ (B6N) and 129S1/SvImJ (129S1) embryonic stem cells to engineer mouse strains with reciprocal single base pair substitutions (B6-Cdh23 c.753A>G and 129S1-Cdh23 c.753G>A ). We compared ABR thresholds and cochlear pathologies of these SNV mice with those of congenic (B6.129S1-Cdh23 Ahl+ and 129S1.B6-Cdh23 ahl ) and parental (B6N and 129S1) strain mice. Results verified the protective effect of the Cdh23 c.753G allele, which prevented high frequency hearing loss in B6 mice to at least 18 months of age, and the AHL-inducing effect of the Cdh23 c.753A allele, which worsened hearing loss in 129S1 mice. ABR thresholds differed between 129S-Cdh23 c.753A SNV and 129S1.B6-Cdh23 ahl congenic mice, and a linkage backcross involving these strains localized a Chr 10 QTL contributing to the difference. These results illustrate the large effects that strain background and congenic regions have on the hearing loss associated with Cdh23 c.753 alleles. Importantly, the B6-Cdh23 c.753G strain can be used to eliminate the confounding influence of the Cdh23 c.753A variant in hearing studies of B6 mice and mutant mice on the B6 background.

Thyroid hormone (TH) is essential for proper cochlear development and function, and TH deficiencies cause variable hearing impairment in humans and mice. Thyroid peroxidase (TPO) catalyzes key reactions in TH synthesis, and TPO mutations have been found to underlie many cases of congenital hypothyroidism in human patients. In contrast, only a single mutation of the mouse TPO gene has been reported previously (Tpo(R479C)) but was not evaluated for auditory function. Here, we describe and characterize two new mouse mutations of Tpo with an emphasis on their associated auditory deficits. Mice homozygous for these recessive mutations have dysplastic thyroid glands and lack detectable levels of TH. Because of the small size of mutant mice, the mutations were named teeny (symbol Tpo(tee)) and teeny-2 Jackson (Tpo(tee-2J)). Tpo(tee) is a single base-pair missense mutation that was induced by ENU, and Tpo(tee-2J) is a 64 bp intragenic deletion that arose spontaneously. The Tpo(tee) mutation changes the codon for a highly conserved tyrosine to asparagine (p.Y614N), and the Tpo(tee-2J) mutation deletes a splice donor site, which results in exon skipping and aberrant transcripts. Mutant mice are profoundly hearing impaired with auditory brainstem response (ABR) thresholds about 60 dB above those of non-mutant controls. The maturation of cochlear structures is delayed in mutant mice and tectorial membranes are abnormally thick. To evaluate the effect of genetic background on auditory phenotype, we produced a C3.B6-Tpo(tee-2J) congenic strain and found that ABR thresholds of mutant mice on the C3H/HeJ strain background are 10-12 dB lower than those of mutant mice on the C57BL/6 J background. The Tpo mutant strains described here provide new heritable mouse models of congenital hypothyroidism that will be valuable for future studies of thyroid hormones' role in auditory development and function.

Stereocilia, the modified microvilli projecting from the apical surfaces of the sensory hair cells of the inner ear, are essential to the mechanoelectrical transduction process underlying hearing and balance. The actin-filled stereocilia on each hair cell are tethered together by fibrous links to form a highly patterned hair bundle. Although many structural components of hair bundles have been identified, little is known about the signaling mechanisms that regulate their development, morphology, and maintenance. Here, we describe two naturally occurring, allelic mutations that result in hearing and balance deficits in mice, named roundabout (rda) and roundabout-2J (rda(2J)). Positional cloning identified both as mutations of the mouse ELMO domain containing 1 gene (Elmod1), a poorly characterized gene with no previously reported mutant phenotypes. The rda mutation is a 138 kb deletion that includes exons 1-5 of Elmod1, and rda(2J) is an intragenic duplication of exons 3-8 of Elmod1. The deafness associated with these mutations is caused by cochlear hair cell dysfunction, as indicated by conspicuous elongations and fusions of inner hair cell stereocilia and progressive degeneration of outer hair cell stereocilia. Mammalian ELMO-family proteins are known to be involved in complexes that activate small GTPases to regulate the actin cytoskeleton during phagocytosis and cell migration. ELMOD1 and ELMOD2 recently were shown to function as GTPase-activating proteins (GAPs) for the Arf family of small G proteins. Our finding connecting ELMOD1 deficiencies with stereocilia dysmorphologies thus establishes a link between the Ras superfamily of small regulatory GTPases and the actin cytoskeleton dynamics of hair cell stereocilia.

Thyroid hormone has pleiotropic effects on cochlear development, and genomic variation influences the severity of associated hearing deficits. DW/J-Pou1f1dw/dw mutant mice lack pituitary thyrotropin, which causes severe thyroid hormone deficiency and profound hearing impairment. To assess the genetic complexity of protective effects on hypothyroidism-induced hearing impairment, an F1 intercross was generated between DW/J-Pou1f1dw/+ carriers and an inbred strain with excellent hearing derived from Mus castaneus, CAST/EiJ. Approximately 24% of the (DW/J×CAST/EiJ) Pou1f1dw/dw F2 progeny had normal hearing. A genome scan revealed a locus on chromosome 2, named modifier of dw hearing, or Mdwh, that rescues hearing despite persistent hypothyroidism. This chromosomal region contains the modifier of tubby hearing 1 (Moth1) locus that encodes a protective allele of the microtubule-associated protein MTAP1A. DW/J-Pou1f1dw/+ carriers were crossed with the AKR strain, which also carries a protective allele of Mtap1a, and we found that AKR is not protective for hearing in the (DW/J×AKR) Pou1f1dw/dw F2 progeny. Thus, protective alleles of Mtap1a are not sufficient to rescue DW/J-Pou1f1dw/dw hearing. We expect that identification of protective modifiers will enhance our understanding of the mechanisms of hypothyroidism-induced hearing impairment.

The quantitative trait locus ahl8 is a key contributor to the early-onset, age-related hearing loss of DBA/2J mice. A nonsynonymous nucleotide substitution in the mouse fascin-2 gene (Fscn2) is responsible for this phenotype, confirmed by wild-type BAC transgene rescue of hearing loss in DBA/2J mice. In chickens and mice, FSCN2 protein is abundant in hair-cell stereocilia, the actin-rich structures comprising the mechanically sensitive hair bundle, and is concentrated toward stereocilia tips of the bundle's longest stereocilia. FSCN2 expression increases when these stereocilia differentially elongate, suggesting that FSCN2 controls filament growth, stiffens exposed stereocilia, or both. Because ahl8 accelerates hearing loss only in the presence of mutant cadherin 23, a component of hair-cell tip links, mechanotransduction and actin crosslinking must be functionally interrelated.

Recessive mutations at the mouse pirouette (pi) locus result in hearing loss and vestibular dysfunction due to neuroepithelial defects in the inner ear. Using a positional cloning strategy, we have identified mutations in the gene Grxcr1 (glutaredoxin cysteine-rich 1) in five independent allelic strains of pirouette mice. We also provide sequence data of GRXCR1 from humans with profound hearing loss suggesting that pirouette is a model for studying the mechanism of nonsyndromic deafness DFNB25. Grxcr1 encodes a 290 amino acid protein that contains a region of similarity to glutaredoxin proteins and a cysteine-rich region at its C terminus. Grxcr1 is expressed in sensory epithelia of the inner ear, and its encoded protein is localized along the length of stereocilia, the actin-filament-rich mechanosensory structures at the apical surface of auditory and vestibular hair cells. The precise architecture of hair cell stereocilia is essential for normal hearing. Loss of function of Grxcr1 in homozygous pirouette mice results in abnormally thin and slightly shortened stereocilia. When overexpressed in transfected cells, GRXCR1 localizes along the length of actin-filament-rich structures at the dorsal-apical surface and induces structures with greater actin filament content and/or increased lengths in a subset of cells. Our results suggest that deafness in pirouette mutants is associated with loss of GRXCR1 function in modulating actin cytoskeletal architecture in the developing stereocilia of sensory hair cells., (Copyright (c) 2010 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

The DBA/2J inbred strain of mice is used extensively in hearing research, yet little is known about the genetic basis for its early onset, progressive hearing loss. To map underlying genetic factors we analyzed recombinant inbred strains and linkage backcrosses. Analysis of 213 mice from 31 BXD recombinant inbred strains detected linkage of auditory brain-stem response thresholds with a locus on distal chromosome 11, which we designate ahl8. Analysis of 225 N2 mice from a backcross of (C57BL/6JxDBA/2J) F1 hybrids to DBA/2J mice confirmed this linkage (LOD>50) and refined the ahl8 candidate gene interval. Analysis of 214 mice from a backcross of (B6.CAST-Cdh23 Ahl+ xDBA/2J) F1 hybrids to DBA/2J mice demonstrated a genetic interaction of Cdh23 with ahl8. We conclude that ahl8 is a major contributor to the hearing loss of DBA/2J mice and that its effects are dependent on the predisposing Cdh23 ahl genotype of this strain.

Dual oxidases generate the hydrogen peroxide needed by thyroid peroxidase for the incorporation of iodine into thyroglobulin, an essential step in thyroid hormone synthesis. Mutations in the human dual oxidase 2 gene, DUOX2, have been shown to underlie several cases of congenital hypothyroidism. We report here the first mouse Duox2 mutation, which provides a new genetic model for studying the specific function of DUOX2 in the thyroid gland and in other organ systems where it is hypothesized to play a role. We mapped the new spontaneous mouse mutation to chromosome 2 and identified it as a T>G base pair change in exon 16 of Duox2. The mutation changes a highly conserved valine to glycine at amino acid position 674 (V674G) and was named "thyroid dyshormonogenesis" (symbol thyd) to signify a defect in thyroid hormone synthesis. Thyroid glands of mutant mice are goitrous and contain few normal follicles, and anterior pituitaries are dysplastic. Serum T(4) in homozygotes is about one-tenth the level of controls and is accompanied by a more than 100-fold increase in TSH. The weight of adult mutant mice is approximately half that of littermate controls, and serum IGF-I is reduced. The cochleae of mutant mice exhibit abnormalities characteristic of hypothyroidism, including a delayed formation of the inner sulcus and tunnel of Corti and an abnormally thickened tectorial membrane. Hearing thresholds of adult mutant mice are on average 50-60 decibels (dB) above those of controls.

Although CLIC5 is a member of the chloride intracellular channel protein family, its association with actin-based cytoskeletal structures suggests that it may play an important role in their assembly or maintenance. Mice homozygous for a new spontaneous recessive mutation of the Clic5 gene, named jitterbug (jbg), exhibit impaired hearing and vestibular dysfunction. The jbg mutation is a 97 bp intragenic deletion that causes skipping of exon 5, which creates a translational frame shift and premature stop codon. Western blot and immunohistochemistry results confirmed the predicted absence of CLIC5 protein in tissues of jbg/jbg mutant mice. Histological analysis of mutant inner ears revealed dysmorphic stereocilia and progressive hair cell degeneration. In wild-type mice, CLIC5-specific immunofluorescence was detected in stereocilia of both cochlear and vestibular hair cells and also along the apical surface of Kolliker's organ during cochlear development. Refined immunolocalization in rat and chicken vestibular hair cells showed that CLIC5 is limited to the basal region of the hair bundle, similar to the known location of radixin. Radixin immunostaining appeared reduced in hair bundles of jbg mutant mice. By mass spectrometry and immunoblotting, CLIC5 was shown to be expressed at high levels in stereocilia of the chicken utricle, in an approximate 1:1 molar ratio with radixin. These results suggest that CLIC5 associates with radixin in hair cell stereocilia and may help form or stabilize connections between the plasma membrane and the filamentous actin core.

Mouse deafness mutations provide valuable models of human hearing disorders and entry points into molecular pathways important to the hearing process. A newly discovered mouse mutation named hurry-scurry (hscy) causes deafness and vestibular dysfunction. Scanning electron microscopy of cochleae from 8-day-old mutants revealed disorganized hair bundles, and by 50 days of age, many hair cells are missing. To positionally clone hscy, 1,160 F(2) mice were produced from an intercross of (C57BL/6-hscy x CAST/EiJ) F(1) hybrids, and the mutation was localized to a 182-kb region of chromosome 17. A missense mutation causing a critical cysteine to phenylalanine codon change was discovered in a previously undescribed gene within this candidate interval. The gene is predicted to encode an integral membrane protein with four transmembrane helices. A synthetic peptide designed from the predicted protein was used to produce specific polyclonal antibodies, and strong immunoreactivity was observed on hair bundles of both inner and outer hair cells in cochleae of newborn +/+ controls and +/hscy heterozygotes but was absent in hscy/hscy mutants. Accordingly, the gene was given the name "tetraspan membrane protein of hair cell stereocilia," symbol Tmhs. Two related proteins (>60% amino acid identity) are encoded by genes on mouse chromosomes 5 and 6 and, together with the Tmhs-encoded protein (TMHS), comprise a distinct tetraspan subfamily. Our localization of TMHS to the apical membrane of inner ear hair cells during the period of stereocilia formation suggests a function in hair bundle morphogenesis.

Mutations in the mouse dreher (dr) gene cause skeletal defects, hyperactivity, abnormal gait, deafness, white belly spotting, and hypoplasia of Müllerian duct derivatives. To map dr to high resolution, we utilized two crosses. Initially, we analyzed an intersubspecific intercross to construct a detailed genetic map of simple sequence length polymorphism markers within a 6.3-cM region surrounding the dr locus. Subsequently, we analyzed a second intersubspecific intercross segregating for the dr(6J) allele, which positioned dr within a 0.13-cM region between Rxrg and D1Mit370. A physical contig of BAC clones spanning the dr critical region was constructed, and eight potential dr candidate genes were excluded by genetic or physical mapping. Together these results lay the foundation for positional cloning of the dr gene., (Copyright 1999 Academic Press.)