Exome_sequencing_of_the_mouse_Iso_mutant

Mouse mutant line "Iso" was identified in ENU mutagenesis-based recessive screen. We analyzed 3rd generation embryos using magnetic resonace imaging (MRI) for any cardiac and other developmental anomalies. Analysis was done at 15.5dpc (days post coitum) when cardiac development shall be completed. Iso embryos showed anomalies in cardio-pulmonary left-right patterning (atrial and pulmonary isomerism, abnormal ventricular topology), heart development (atrial and ventricular septal defects) and neural tube defects. Underlying mutation was mapped to chromosome 14 (~20Mb), but identified interval does not contain any gene currently known to be involved in cardiac development or left-right patterning. Paralogs of genes involved in cardiac development in this interval are Gpc5 and Gpc6. Sequencing identified a mutation in Gpc5 exon 2 that results in a nonsynonymous change (E74G) in the protein; it is likely that the GPC5-E74G mutation is causative, as GPCs through NODAL, WNT, or HH signaling could affect left-right patterning. Supporting this hypothesis, GPC5 is closely related to GPC3, which is known to have a role in heart development. However, not only Gpc5 expression in the heart has never been reported but also with ENU mutagenesis, there is always a concern about a second, closely linked mutation. To exclude such a possibility, the interval should be narrowed as much as possible or/and all coding sequences within it shall be checked. If such a putative mutation was close to the one identified in Gpc5, it would be very difficult to separate one from another by breeding more animals and counting on more recombination. Actual sequencing of coding regions is required but it would prove to be too expensive and time and labor consuming, to sequence over 20Mb in a traditional way. Exome sequencing provided by Sanger Institute (Dr David Adams) would be an answer and would give as unbiased, detailed information about any possible additional mutation in the interval or it would prove Gpc5 mutation to be a causative one. Either way, the project will result in the confirmation (Gpc5) or identification (any other gene in the interval) of new left-right patterning gene.