Identification and characterisation of spontaneous mutations causing deafness from a targeted knockout programme.

BACKGROUND: Mice carrying targeted mutations are important for investigating gene function and the role of genes in disease, but off-target mutagenic effects associated with the processes of generating targeted alleles, for instance using Crispr, and culturing embryonic stem cells, offer opportunities for spontaneous mutations to arise. Identifying spontaneous mutations relies on the detection of phenotypes segregating independently of targeted alleles, and having a broad estimate of the level of mutations generated by intensive breeding programmes is difficult given that many phenotypes are easy to miss if not specifically looked for. Here we present data from a large, targeted knockout programme in which mice were analysed through a phenotyping pipeline. Such spontaneous mutations segregating within mutant lines may confound phenotypic analyses, highlighting the importance of record-keeping and maintaining correct pedigrees. RESULTS: Twenty-five lines out of 1311 displayed different deafness phenotypes that did not segregate with the targeted allele. We observed a variety of phenotypes by Auditory Brainstem Response (ABR) and behavioural assessment and isolated eight lines showing early-onset severe progressive hearing loss, later-onset progressive hearing loss, low frequency hearing loss, or complete deafness, with vestibular dysfunction. The causative mutations identified include deletions, insertions, and point mutations, some of which involve new genes not previously associated with deafness while others are new alleles of genes known to underlie hearing loss. Two of the latter show a phenotype much reduced in severity compared to other mutant alleles of the same gene. We investigated the ES cells from which these lines were derived and determined that only one of the 8 mutations could have arisen in the ES cell, and in that case, only after targeting. Instead, most of the non-segregating mutations appear to have occurred during breeding of mutant mice. In one case, the mutation arose within the wildtype colony used for expanding mutant lines. CONCLUSIONS: Our data show that spontaneous mutations with observable effects on phenotype are a common side effect of intensive breeding programmes, including those underlying targeted mutation programmes. Such spontaneous mutations segregating within mutant lines may confound phenotypic analyses, highlighting the importance of record-keeping and maintaining correct pedigrees.

Training welfare caseworkers in service excellence: increasing children's Medicaid coverage.

Many low-income children who are eligible for public sector health insurance remain uninsured. There are many barriers to enrolling these children, but one key issue is parents' reluctance to use the services of the local enrollment agency, which is usually the welfare office. The Eastside Access Partnership, a community-academic coalition on the Eastside of Detroit, addressed the problem of uninsured-but-eligible children through a variety of interventions focused on (1) enhancing community members' understanding of the enrollment process and (2) reducing institutional barriers to enrollment. One of these interventions addressed the institutional barriers by developing a customer service excellence training program for welfare caseworkers. The training program curriculum, which was developed following the principles of community-based participatory research, included extensive input from community residents, welfare agency staff, and academic researchers. The training sessions received positive evaluations from participants and agency executives. A more thorough evaluation of the project is under way.