An ENU-induced mutation of miR-96 associated with progressive hearing loss in mice.

Progressive hearing loss is common in the human population, but little is known about the molecular basis. We report a new N-ethyl-N-nitrosurea (ENU)-induced mouse mutant, diminuendo, with a single base change in the seed region of Mirn96. Heterozygotes show progressive loss of hearing and hair cell anomalies, whereas homozygotes have no cochlear responses. Most microRNAs are believed to downregulate target genes by binding to specific sites on their mRNAs, so mutation of the seed should lead to target gene upregulation. Microarray analysis revealed 96 transcripts with significantly altered expression in homozygotes; notably, Slc26a5, Ocm, Gfi1, Ptprq and Pitpnm1 were downregulated. Hypergeometric P-value analysis showed that hundreds of genes were upregulated in mutants. Different genes, with target sites complementary to the mutant seed, were downregulated. This is the first microRNA found associated with deafness, and diminuendo represents a model for understanding and potentially moderating progressive hair cell degeneration in hearing loss more generally

FLT4/VEGFR3 and Milroy Disease: Novel Mutations, a Review of Published Variants and Database Update

Milroy disease (MD) is an autosomal dominantly inherited primary lymphedema. In 1998, the gene locus for MD was mapped to 5q35.3 and variants in the VEGFR3 (FLT4) gene, encoding vascular endothelial growth factor receptor 3 (VEGFR3), were identified as being responsible for the majority of MD cases. Several reports have since been published detailing pathogenic FLT4 mutations. To date, a total of 58 different variants in FLT4, 20 of which are unpublished, have been observed in 95 families with MD. A review of published mutations is presented in this update. Furthermore, the unpublished variants are presented including clinical data. Comparison of clinical features in patients and their families with the same mutations reveals incomplete penetrance and variable expression, making genotype–phenotype correlations difficult. Most mutations are missense, but a few deletions and one splicing variant have also been reported. Several animal models have confirmed the role of VEGFR3 in lymphangiogenesis and studies show mutant VEGFR3 receptors are not phosphorylated. Here, an MD patient with the same p.Ile1053Phe change as seen in the Chy mouse is presented for the first time. This finding confirms that this mouse lineage is an excellent model for MD. All the data reviewed here has been submitted to a database based on the Leiden Open (source) Variation Database (LOVD) and is accessible online at www.lovd.nl/flt4

CA2+ IMAGING PRINCIPLES OF ANALYSIS AND ENHANCEMENT

In this chapter, we review the theoretical and experimental foundations underling a quantitative approach to Ca2+ imaging, discuss equilibrium conditions and their violations and present a computational framework that can be used to estimate the spatial and temporal dynamics of Ca2+ signals based of fluorescence measurements with Ca2+ indicators

Structure of the human plasma membrane Ca2+-ATPase 1 in complex with its obligatory subunit neuroplastin

The plasma membrane Ca2+ ATPase (PMCA) is essential for maintaining Ca2+ homeostasis in eukaryotic cells, and neuroplastin (NPTN) was recently identified as an obligatory subunit of PMCA. Here the authors present the cryo-EM structure of NPTN bound to human PMCA1, which reveals that the NPTN transmembrane (TM) helix interacts with TM10 and the TM8-9-linker of PMCA1

CRISPR: a versatile tool for both forward and reverse genetics research

Human genetics research employs the two opposing approaches of forward and reverse genetics. While forward genetics identifies and links a mutation to an observed disease etiology, reverse genetics induces mutations in model organisms to study their role in disease. In most cases, causality for mutations identified by forward genetics is confirmed by reverse genetics through the development of genetically engineered animal models and an assessment of whether the model can recapitulate the disease. While many technological advances have helped improve these approaches, some gaps still remain. CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated) system, which has emerged as a revolutionary genetic engineering tool, holds great promise for closing such gaps. By combining the benefits of forward and reverse genetics, it has dramatically expedited human genetics research. We provide a perspective on the power of CRISPR-based forward and reverse genetics tools in human genetics and discuss its applications using some disease examples

PMCA2w/a Splice Variant: A Key Regulator of Hair Cell Mechano-transduction Machinery

Sensory hair cells of the inner ear detect sound stimuli, inertial or gravi- tational forces. These mechanical inputs cause de\ufb02ection of the cell stereociliary bundle and activate a small number of cation-selective mechano-transduction (MET) channels that admit K+ and Ca2+ ions into the cytoplasm. Stereociliary Ca2+ levels are homeostatically regulated by an unusual splicing isoform (w/a) of plasma membrane calcium-pump isoform 2 (PMCA2w/a), ablation or missense mutations of which cause deafness and loss of balance in humans and mice. At variance with other PMCA2 isoforms, PMCA2w/a expressed in CHO transfectants increases only marginally its activity in response to a rapid increase of the cytoplasmic free Ca2+ concentration ([Ca2+]c). In this expression system, deafness-related mutations of PMCA2w/a decrease the pump ability to extrude Ca2+ both at steady state and in response to a [Ca2+]c rise. Consistent with these \ufb01ndings, mouse strains in which the pump is genetically ablated or mutated show hearing impairment correlated with defects in homeostatic regulation of stereociliary Ca2+, decreased sensitivity of the MET channels to hair bundle displacement, and morphological abnormalities in the organ of Corti. These results highlight a critical role played by PMCA2w/a in the control of hair cell function and survival and provide mechanistic insight into the etiology of deafness and vestibular disorders