A Trans-Acting Protein Effect Causes Severe Eye Malformation in the Mp Mouse

Abstract

Mp is an irradiation-induced mouse mutation associated with microphthalmia, micropinna and hind limb syndactyly. We show that Mp is caused by a 660 kb balanced inversion on chromosome 18 producing reciprocal 3-prime gene fusion events involving Fbn2 and Isoc1. The Isoc1-Fbn2 fusion gene ( Isoc1 ^Mp ) mRNA has a frameshift and early stop codon resulting in nonsense mediated decay. Homozygous deletions of Isoc1 do not support a significant developmental role for this gene. The Fbn2-Isoc1 fusion gene ( Fbn2 ^Mp) predicted protein consists of the N-terminal Fibrillin-2 (amino acids 1–2646, exons 1–62) lacking the C-terminal furin-cleavage site with a short out-of-frame extension encoded by the final exon of Isoc1. The Mp limb phenotype is consistent with that reported in Fbn2 null embryos. However, severe eye malformations, a defining feature of Mp, are not seen in Fbn2 null animals. Fibrillin-2 ^Mp forms large fibrillar structures within the rough endoplasmic reticulum (rER) associated with an unfolded protein response and quantitative mass spectrometry shows a generalised defect in protein secretion in conditioned media from mutant cells. In the embryonic eye Fbn2 is expressed within the peripheral ciliary margin (CM). Mp embryos show reduced canonical Wnt-signalling in the CM – known to be essential for ciliary body development - and show subsequent aplasia of CM-derived structures. We propose that the Mp “worse-than-null” eye phenotype plausibly results from a failure in normal trafficking of proteins that are co-expressed with Fbn2 within the CM. The prediction of similar trans-acting protein effects will be an important challenge in the medical interpretation of human mutations from whole exome sequencing.

Author Summary

With the current increase in large-scale sequencing efforts, correct interpretation of mutation consequences has never been more important. Here, we present evidence for a trans-acting protein effect in a novel mutation of Fbn2, associated with severe developmental eye defects not found in loss of function Fibrillin-2 alleles. The mutant protein is expressed in the developing eye but is unable to exit the cells, instead forming large protein aggregates within the endoplasmic reticulum. We observed ER-stress in mutant eyes, and detected a general reduction to secretion of co-expressed proteins in cell cultures. We propose that similar effects could be caused by mutations to other proteins that are trafficked through the ER, highlighting a disease mechanism that results in different clinical outcomes than observed, or predicted, from loss-off-function alleles.

Contributors

Kathryn S. E. Cheah: Role: Editor

Journal

Journal ID (nlm-ta): PLoS Genet

Journal ID (iso-abbrev): PLoS Genet

Journal ID (publisher-id): plos

Journal ID (pmc): plosgen

Title: PLoS Genetics

Publisher: Public Library of Science (San Francisco, USA )

ISSN (Print): 1553-7390

ISSN (Electronic): 1553-7404

Publication date Collection: December 2013

Publication date (Print): December 2013

Publication date (Electronic): 12 December 2013

Volume: 9

Issue: 12

Affiliations

[1 ]The MRC Human Genetics Unit, MRC Institute of Genetic and Molecular Medicine, University of Edinburgh, Western General Hospital, Edinburgh, United Kingdom

[2 ]Shriners Hospital for Children, Portland, Oregon, United States of America

[3 ]Biomarker and Drug Analysis Core Facility, Medical Research Institute, School of Medicine, University of Dundee, Dundee, United Kingdom

[4 ]Molecular Medicine Centre, MRC Institute of Genetic and Molecular Medicine, University of Edinburgh, Western General Hospital, Edinburgh, United Kingdom

The University of Hong Kong, Hong Kong

Author notes

* E-mail: ian.jackson@ 123456igmm.ed.ac.uk (IJJ); david.fitzpatrick@ 123456igmm.ed.ac.uk (DRF)

The authors have declared that no competing interests exist.

Conceived and designed the experiments: JR DRF. Performed the experiments: JR MK DRK NLC SM JKR JTJH LR RvH. Analyzed the data: JR DRK LYS DRF IJJ. Contributed reagents/materials/analysis tools: MF LYS. Wrote the paper: JR IJJ DRF.

Article

Publisher ID: PGENETICS-D-13-00434

DOI: 10.1371/journal.pgen.1003998

PMC ID: 3861116

License:

This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Page count

Pages: 13

Funding

Funding for this project was provided as an intramural program grant from the Medical Research Council (UK). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Categories

Subject: Research Article