Thesis

The use of the grip strength meter to measure progressive muscle weakness as a primary phenotype screen in mice who are homozygous for the M712T allele

Hereditary inclusion body myopathy (HIBM) is a genetic neuromuscular disorder characterized by progressive muscle wasting and weakness. HIBM is caused by mutations in the GNE gene which encodes the bifunctional enzyme uridine diphosphospho-N-acetylglucosamine (UDP-GlcNAc) 2-epimerase/N-acetyl-mannosamine (ManNAc) kinase (GNE/MNK). The GNE gene product catalyzes the first two committed, rate-limiting steps in the biosynthesis of 5-N-acetylneuraminic acid (Neu5Ac, also known as sialic acid). One of the most frequent mutations is an exchange of methionine to threonine at position 712 (M712T). There are no effective treatments for HIBM yet. Investigators are working toward finding an effective treatment. In order to make progress toward a cure, it is paramount that an effective animal model be developed. Thus, the purpose of this study was to help determine whether the FVB;B6-GNE M712T/M712T knock-in mice developed a similar phenotype as humans in order to allow future testing of therapeutic approaches. I assessed this through the use of a grip-strength meter to determine the presence of muscle weakness. Grip strength of the mice was measured using the Chatillon DFIS-10 digital force gauge apparatus to determine progressive muscle weakness in the homozygous mutant group. The results of the homozygous mutant group were then compared to results from the mice heterozygous for the mutation and to the wild-type (control) mice. In order to assess the effects of the mutation on muscle strength, it was necessary to determine the genotypes of each mouse in the study. Genotypes were determined by PCR and restriction enzyme digest. Attempts were made to increase the colony size by breeding based on the genotyping results. The average force exerted by the mice revealed that there were no significant differences found between the GNE M712T/M712T and the control mice in grip force of either the forelimb or the combined forelimb and hindlimb. The average force exerted by the mice when using the forelimb was 0.09kg for the wild-type, 0.12 kg for the mutant, and 0.09kg for the heterozygous group. The average force exerted using combined forelimb and hindlimb was 0.24kg for the wild-type, 0.23kg for the mutant, and 0.22kg for the heterozygous group. Unexpectedly, some of the dissected homozygous GneM712T/M712T mice appeared to exhibit signs of abnormal kidneys. Either the grip-strength protocol was not sensitive enough to detect differences in forelimb and combined forelimb and hindlimb strength, or the knock-in mice with the M712T mutation do not show the same muscle-wasting phenotype found in humans at the age in which the mice were tested. The experiment should be repeated with different parameters in the grip-strength protocol and larger sample size. Mouse muscle tissues and necropsies should be arranged for all the mice in poor health to see if their internal organs, especially the kidneys, are healthy. These data could be used to construct a more suitable mouse model to further investigate treatments in alleviating muscle deterioration found in HIBM.

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