MVB-12, a Fourth Subunit of Metazoan ESCRT-I, Functions in Receptor Downregulation

After ligand binding and endocytosis, cell surface receptors can continue to signal from endosomal compartments until sequestered from the cytoplasm. An important mechanism for receptor downregulation in vivo is via the inward budding of receptors into intralumenal vesicles to form specialized endosomes called multivesicular bodies (MVBs) that subsequently fuse with lysosomes, degrading their cargo. This process requires four heterooligomeric protein complexes collectively termed the ESCRT machinery. In yeast, ESCRT-I is a heterotetrameric complex comprised of three conserved subunits and a fourth subunit for which identifiable metazoan homologs were lacking. Using C. elegans, we identify MVB-12, a fourth metazoan ESCRT-I subunit. Depletion of MVB-12 slows the kinetics of receptor downregulation in vivo, but to a lesser extent than inhibition of other ESCRT-I subunits. Consistent with these findings, targeting of MVB-12 to membranes requires the other ESCRT-I subunits, but MVB-12 is not required to target the remaining ESCRT-I components. Both endogenous and recombinant ESCRT-I are stable complexes with a 1:1:1:1 subunit stoichiometry. MVB-12 has two human homologs that co-localize and co-immunoprecipitate with the ESCRT-I component TSG101. Thus, MVB-12 is a conserved core component of metazoan ESCRT-I that regulates its activity during MVB biogenesis

Genomic analyses in Cornelia de Lange Syndrome and related diagnoses: Novel candidate genes, <scp>genotype–phenotype</scp> correlations and common mechanisms

Cornelia de Lange Syndrome (CdLS) is a rare, dominantly inherited multisystem developmental disorder characterized by highly variable manifestations of growth and developmental delays, upper limb involvement, hypertrichosis, cardiac, gastrointestinal, craniofacial, and other systemic features. Pathogenic variants in genes encoding cohesin complex structural subunits and regulatory proteins (NIPBL, SMC1A, SMC3, HDAC8, and RAD21) are the major pathogenic contributors to CdLS. Heterozygous or hemizygous variants in the genes encoding these five proteins have been found to be contributory to CdLS, with variants in NIPBL accounting for the majority (&gt;60%) of cases, and the only gene identified to date that results in the severe or classic form of CdLS when mutated. Pathogenic variants in cohesin genes other than NIPBL tend to result in a less severe phenotype. Causative variants in additional genes, such as ANKRD11, EP300, AFF4, TAF1, and BRD4, can cause a CdLS‐like phenotype. The common role that these genes, and others, play as critical regulators of developmental transcriptional control has led to the conditions they cause being referred to as disorders of transcriptional regulation (or “DTRs”). Here, we report the results of a comprehensive molecular analysis in a cohort of 716 probands with typical and atypical CdLS in order to delineate the genetic contribution of causative variants in cohesin complex genes as well as novel candidate genes, genotype–phenotype correlations, and the utility of genome sequencing in understanding the mutational landscape in this population

Kif18a is specifically required for mitotic progression during germ line development.

Genome integrity in the developing germ line is strictly required for fecundity. In proliferating somatic cells and in germ cells, there are mitotic checkpoint mechanisms that ensure accurate chromosome segregation and euploidy. There is growing evidence of mitotic cell cycle components that are uniquely required in the germ line to ensure genome integrity. We previously showed that the primary phenotype of germ cell deficient 2 (gcd2) mutant mice is infertility due to germ cell depletion during embryogenesis. Here we show that the underlying mutation is a mis-sense mutation, R308K, in the motor domain of the kinesin-8 family member, KIF18A, a protein that is expressed in a variety of proliferative tissues and is a key regulator of chromosome alignment during mitosis. Despite the conservative nature of the mutation, we show that its functional consequences are equivalent to KIF18A deficiency in HeLa cells. We also show that somatic cells progress through mitosis, despite having chromosome alignment defects, while germ cells with similar chromosome alignment defects undergo mitotic arrest and apoptosis. Our data provide evidence for differential requirements for chromosome alignment in germ and somatic cells and show that Kif18a is one of a growing number of genes that are specifically required for cell cycle progression in proliferating germ cells. Dev Biol 2015 Jun 15; 402(2):253-62

Discovery of transgene insertion sites by high throughput sequencing of mate pair libraries.

BACKGROUND: Transgenesis by random integration of a transgene into the genome of a zygote has become a reliable and powerful method for the creation of new mouse strains that express exogenous genes, including human disease genes, tissue specific reporter genes or genes that allow for tissue specific recombination. Nearly 6,500 transgenic alleles have been created by random integration in embryos over the last 30 years, but for the vast majority of these strains, the transgene insertion sites remain uncharacterized. RESULTS: To obtain a complete understanding of how insertion sites might contribute to phenotypic outcomes, to more cost effectively manage transgenic strains, and to fully understand mechanisms of instability in transgene expression, we\u27ve developed methodology and a scoring scheme for transgene insertion site discovery using high throughput sequencing data. CONCLUSIONS: Similar to other molecular approaches to transgene insertion site discovery, high-throughput sequencing of standard paired-end libraries is hindered by low signal to noise ratios. This problem is exacerbated when the transgene consists of sequences that are also present in the host genome. We\u27ve found that high throughput sequencing data from mate-pair libraries are more informative when compared to data from standard paired end libraries. We also show examples of the genomic regions that harbor transgenes, which have in common a preponderance of repetitive sequences. BMC Genomics 2014 May 14; 15(1):367

Derivation and characterization of mouse embryonic stem cells from permissive and nonpermissive strains.

Mouse embryonic stem cells (mESCs) are key tools for genetic engineering, development of stem cell-based therapies and basic research on pluripotency and early lineage commitment. However, successful derivation of germline-competent embryonic stem cell lines has, until recently, been limited to a small number of inbred mouse strains. Recently, there have been considerable advances in the field of embryonic stem cell biology, particularly in the area of pluripotency maintenance in the epiblast from which the mESCs are derived. Here we describe a protocol for efficient derivation of germline-competent mESCs from any mouse strain, including strains previously deemed nonpermissive. We provide a protocol that is generally applicable to most inbred strains, as well as a variant for nonpermissive strains. By using this protocol, mESCs can be derived in 3 weeks and fully characterized after an additional 12 weeks, at efficiencies as high as 90% and in any strain background. Nat Protoc 2014 Mar; 9(3):559-74

Systemic and local antimicrobial use in periodontal therapy in England and Wales.

BACKGROUND/AIMS: The aim of this study was to investigate antimicrobial use during periodontal therapy in dental practice in England & Wales. METHOD: This was a postal questionnaire survey of 800 dentists, 400 general dental practitioners (GDP) in National Health Service practice and 400 members of the British Society of Periodontology (Periodontal Society) primarily in dental practice. We designed and piloted a questionnaire to evaluate both systemic and local antibiotic use with periodontal therapy as well as factors affecting their prescription. In addition, we also investigated the potential use of antibiotic sensitivity testing, since this has been recommended prior to prescribing antibiotics. Two follow-up mailings were used to encourage non-responders. RESULTS: The useable return rate for the questionnaires was 587/800 (73%). Systemic antibiotics were used by 7.4% Periodontal Society members and 18.4% GDP for untreated adult periodontitis patients (p<0.001). Antimicrobials were prescribed more frequently by Periodontal Society members in early onset (52.7%) and refractory periodontitis patients (49.6%), and this was highly statistically significantly greater usage than GDP (p<0.001). Regarding local antimicrobials, usage for untreated adult periodontitis was Periodontal Society 8.9% and GDP 5.4%. Higher usage of local antimicrobials was found both for the treatment of recurrent pocketing in adult periodontitis (Periodontal Society 26.3%, GDP 14.8%, p<0.014) and refractory periodontitis (Periodontal Society 30.8%, GDP 15.2%, p<0.001). As reasons for using local antimicrobials, more than 80% of all respondents stated superiority over root debridement alone. Barriers to use included cost, no perceived need and lack of supporting research data. The percentage of responders considering diagnostic microbiology either theoretically or at a cost of pound 60 were by group, Periodontal Society 83% & 70.4% and GDP 76% & 51.2%. 33% of Periodontal Society members and 3.8% of GDP spent at least 45 min per quadrant on root planing and Periodontal Society members had a greater exposure to lectures on both systemic and local drug therapy compared with GDP (p<0.001). CONCLUSIONS: Systemic antimicrobial use was infrequent for adult periodontitis and generally in line with current recommendations for other disease types. Whilst local antimicrobial therapy for periodontitis was not widespread, a substantial minority of dentists use this form of therapy and most believe that it is more effective than root debridement alone

Dsp(rul): A spontaneous mouse mutation in desmoplakin as a model of Carvajal-Huerta syndrome.

Studies of spontaneous mutations in mice have provided valuable disease models and important insights into the mechanisms of human disease. Ruffled (rul) is a new autosomal recessive mutation causing abnormal hair coat in mice. The rul allele arose spontaneously in the RB156Bnr/EiJ inbred mouse strain. In addition to an abnormal coat texture, we found diffuse epidermal blistering, abnormal electrocardiograms (ECGs), and ventricular fibrosis in mutant animals. Using high-throughput sequencing (HTS) we found a frameshift mutation at 38,288,978bp of chromosome 13 in the desmoplakin gene (Dsp). The predicted mutant protein is truncated at the c-terminus and missing the majority of the plakin repeat domain. The phenotypes found in Dsp(rul) mice closely model a rare human disorder, Carvajal-Huerta syndrome. Carvajal-Huerta syndrome (CHS) is a rare cardiocutaneous disorder that presents in humans with wooly hair, palmoplantar keratoderma and ventricular cardiomyopathy. CHS results from an autosomal recessive mutation on the 3\u27 end of desmoplakin (DSP) truncating the full length protein. The Dsp(rul) mouse provides a new model to investigate the pathogenesis of CHS, as well as the underlying basic biology of the adhesion molecules coded by the desmosomal genes. Exp Mol Pathol 2015 Feb 7; 98(2):164-17