ENU Mutagenesis Reveals a Novel Phenotype of Reduced Limb Strength in Mice Lacking Fibrillin 2

Background: Fibrillins 1 (FBN1) and 2 (FBN2) are components of microfibrils, microfilaments that are present in many connective tissues, either alone or in association with elastin. Marfan's syndrome and congenital contractural arachnodactyly (CCA) result from dominant mutations in the genes FBN1 and FBN2 respectively. Patients with both conditions often present with specific muscle atrophy or weakness, yet this has not been reported in the mouse models. In the case of Fbn1, this is due to perinatal lethality of the homozygous null mice making measurements of strength difficult. In the case of Fbn2, four different mutant alleles have been described in the mouse and in all cases syndactyly was reported as the defining phenotypic feature of homozygotes.Methodology/Principal Findings: As part of a large-scale N-ethyl-N-nitrosourea (ENU) mutagenesis screen, we identified a mouse mutant, Mariusz, which exhibited muscle weakness along with hindlimb syndactyly. We identified an amber nonsense mutation in Fbn2 in this mouse mutant. Examination of a previously characterised Fbn2-null mutant, Fbn2(fp), identified a similar muscle weakness phenotype. The two Fbn2 mutant alleles complement each other confirming that the weakness is the result of a lack of Fbn2 activity. Skeletal muscle from mutants proved to be abnormal with higher than average numbers of fibres with centrally placed nuclei, an indicator that there are some regenerating muscle fibres. Physiological tests indicated that the mutant muscle produces significantly less maximal force, possibly as a result of the muscles being relatively smaller in Mariusz mice.Conclusions: These findings indicate that Fbn2 is involved in integrity of structures required for strength in limb movement. As human patients with mutations in the fibrillin genes FBN1 and FBN2 often present with muscle weakness and atrophy as a symptom, Fbn2-null mice will be a useful model for examining this aspect of the disease process further

A mutation in the mitochondrial fission gene Dnm1l leads to cardiomyopathy

Mutations in a number of genes have been linked to inherited dilated cardiomyopathy (DCM). However, such mutations account for only a small proportion of the clinical cases emphasising the need for alternative discovery approaches to uncovering novel pathogenic mutations in hitherto unidentified pathways. Accordingly, as part of a large-scale N-ethyl-N-nitrosourea mutagenesis screen, we identified a mouse mutant, Python, which develops DCM. We demonstrate that the Python phenotype is attributable to a dominant fully penetrant mutation in the dynamin-1-like (Dnm1l) gene, which has been shown to be critical for mitochondrial fission. The C452F mutation is in a highly conserved region of the M domain of Dnm1l that alters protein interactions in a yeast two-hybrid system, suggesting that the mutation might alter intramolecular interactions within the Dnm1l monomer. Heterozygous Python fibroblasts exhibit abnormal mitochondria and peroxisomes. Homozygosity for the mutation results in the death of embryos midway though gestation. Heterozygous Python hearts show reduced levels of mitochondria enzyme complexes and suffer from cardiac ATP depletion. The resulting energy deficiency may contribute to cardiomyopathy. This is the first demonstration that a defect in a gene involved in mitochondrial remodelling can result in cardiomyopathy, showing that the function of this gene is needed for the maintenance of normal cellular function in a relatively tissue-specific manner. This disease model attests to the importance of mitochondrial remodelling in the heart; similar defects might underlie human heart muscle disease

The muscle weakness phenotype of the Mariusz mutant.

<p>(<b>A</b>) The original pedigree in which the Mariusz mutant was identified. (<b>B</b>) Grip-strength comparison using 3-month-old Mariusz homozygotes (n = 30) and wild type (n = 26) littermate controls. The average force generated by wild type mice has been set as 100%. Mean and SD are shown. The differences are highly significant; P-values (Student's t test): *** <i>P</i><0.0001. (<b>C</b>) Close-up photos showing the different grasp of Mariusz homozygotes (<i>Mz/Mz</i>) and wild type littermate controls. (<b>D</b>) Example of clasping phenotype observed in a Mariusz homozygote and littermate control. (<b>E</b>) A wire manoeuvre test of Mariusz mice and littermate controls. Scores are as follows: 0 = mouse swings hindlegs on to bar and makes way to edge rapidly; 1 = mouse swings hindlegs on to bar after a delay and makes way to edge rapidly; 2 = mouse is suspended by using underarms to straddle bar and does not lift hind limbs, eventually falls; 3 = mouse falls after short delay; 4 = mouse falls immediately.</p

Muscle force measurements in Mariusz mice.

<p>(<b>A</b>) Morphometric measurement of cross-sectional area of Mariusz muscles in histological sections normalized to the area of the muscle in wild type controls (n = 3 mice each genotype at 5 weeks of age; n = 50 fibres counted per sample). Muscle abbreviations are: FDS, Flexor Digitorum Sublimis; FCR, Flexor Carpi Radialis; ECU, Extensor Carpi Ulnaris; ECR, Extensor Carpi Radialis. ‘Area forelimb’ refers to the entire area of the forelimb to emphasize the size difference between Mariusz mice and littermates controls. (<b>B</b>) Example trace of a tetanic contraction in a Mariusz soleus muscle and a wild type control. (<b>C</b>) Mean peak tetanic force for control (n = 5) and Mariusz (n = 6) mice with SEM shown by error bars. (<b>D</b>) Mean isometric stress of the soleus muscle for wild type (n = 5) and Mariusz (n = 6) mice with SEM shown by error bars. (<b>E</b>) The mean ratio of the mass of the soleus muscle to the mass of the mouse for control (n = 5) and Mariusz (n = 6) mice with SEM shown by error bars. (<b>F</b>) The mean maximum contraction velocity of the soleus muscle in wild type control (n = 4) and Mariusz (n = 5) mice with SEM shown by error bars. P-values: * <i>P</i><0.05; ** <i>P</i><0.01.</p

The digit fusion phenotype of the Mariusz mutant.

<p>(<b>A</b>) Examples of different fusion phenotypes observed - four toes (#1), four toes with two toes fused (#2), four toes with skewed digit (#3), mixture of two digit phenotypes in a single mouse (#4). (<b>B</b>) Hard and soft tissue fusion seen in Mariusz mice. Alcian Blue/Alizarin Red-stained left hindlimbs of P7 mice showing a littermate heterozygous control (left), soft tissue syndactyly of toes 3 and 4 (centre), and syndactyly of toes 2,3 and 4 with fusion of the distal phalanges (right). (<b>C</b>) X-ray analysis of a hindlimb of a Mariusz mutant demonstrating fusion of the soft tissue as well as the distal phalanx bones. (<b>D</b>) Morag test on Mariusz mice and wild type littermates. The proportion of trials in which the mice were able to grasp the food pellets and retrieve the pellets to their mouths are shown. (<b>E</b>) Mariusz mice have a reduced body weight. Growth curves of Mariusz male mice and littermate controls. Picture shows a typical example of the size difference between an adult Mariusz female mouse and a littermate control at 3 months of age.</p

Skeletal muscle histology in Mariusz mice.

<p>(<b>A</b>) Morphometric analysis of muscle fibre cross-sectional area in the soleus and quadriceps muscles in Mariusz (n = 3) and wild type controls (n = 3). Two-way ANOVA was not significant. (<b>B</b>) Typical H&E sections of Mariusz quadriceps muscle from mice at 4 and 9 weeks of age. Scale bar = 100 um. (<b>C</b>) Quantification of central nucleation. The number of muscle fibres with central nuclei is represented as percentage of the total number of muscle fibres analyzed in >100 fibres. Arrows indicate centrally displaced nuclei. Mice were aged 4 weeks and 9 weeks (n = 3 <i>Mz/Mz</i>; n = 3<i>+/+</i>). P-values (Student's t test): * <i>P</i><0.05, ** <i>P</i><0.01.</p

Genetic linkage analysis and positional cloning of the <i>Mz</i> mutation.

<p>(<b>A</b>) Genotypes of seven informative recombinants exhibiting the Mariusz phenotype. The mutation was localized to a 2.5 Mb interval (indicated by the bracketed region). (<b>B</b>) Sequence of part of exon 14 of the <i>Fbn2</i> gene reveals a T-A substitution in the Mariusz allele. The reading frame of part of exon 14 is shown with the amino acid substitution that results. (<b>C</b>) Schematic of the Fbn2 wild type protein and the truncation that results from the Mariusz mutation.</p