CALIFORNIA STATE UNIVERSITY, NORTHRIDGE
THE USE OF THE GRIP STRENGTH METER TO MEASURE PROGRESSIVE
MUSCLE WEAKNESS AS A PRIMARY PHENOTYPE SCREEN IN MICE THAT
ARE HOMOZYGOUS FOR THE M712T ALLELE
A thesis submitted in partial fulfillment of the requirements
for the degree of Master of Science
in Biology
By
Jessica Mayra Estrada
August 2013
© 2013
Jessica Mayra Estrada
ALL RIGHTS RESERVED
ii
The thesis of Jessica Mayra Estrada is approved:
Dr. Steven B. Oppenheimer
Date
Dr. Stan Metzenberg
Date
Dr. Aida Metzenberg, Chair
Date
California State University, Northridge
iii
Acknowledgements
I would first like to thank my family, especially my parents, Laura Veronica Paso
and Victor Estrada, for supporting my educational career. Watching them diligently work
each day without complaining has made me even more dedicated toward achieving my
goals. I would also like to thank Dr. Aida Metzenberg who tirelessly edited my thesis
darfts. She provided me with extensive guidance and knowledge throughout my graduate
career. I will always be grateful for her support during my journey to become a biologist.
I would also like to thank Dr. Stan Metzenberg for teaching me his recombinant DNA
techniques. He taught me that working with DNA is challenging but rewarding. I am also
grateful for having Dr. Valles supply the lab with the reagents and equipment necessary
to complete this project.
Others who deserve special recognition include Toni Uhlendorf, who helped
answer any mice questions, and Harmanpreet Panesar, who helped me test the mice early
in the mornings. I am grateful to Daniel Thomas who took care of the mice very
diligently even though it was sometimes a bit creepy when he made squeaking sounds to
them in an effort to communicate. Osvaldo Larios also deserves special recognition for
suggesting ideas when I had troubleshooting issues with PCR. Special thanks also go to
Victor Albores who helped me genotype late into the night.
Last but not least, I would like to thank Wheaty, Teddy, and Lizzie for helping me
relieve the stress that came with being a graduate student.
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Table of Contents
Copyright Page
ii
Signature Page
iii
Acknowledgements
iv
List of Figures
viii
List of Tables
x
Abstract
xi
Chapter 1: Introduction
1
HIBM Overview and History
2
Clinical Description of HIBM
3
Diagnosis of HIBM
4
Molecular Basis of HIBM
6
GNE/MNK
7
Sialic Acid
8
Treatments
10
Animal Models of HIBM
12
Grip Strength Test
14
Purpose of Research
14
Chapter 2: Materials and Methods
16
Animal Subjects Approval Form
16
Mice
16
Vivarium
17
Chatillon DFIS-10 Digital Force Gauge Apparatus
18
v
Mouse Handling
19
Breeding
19
Ear Markings and Tailing
20
Grip Strength Procedure
21
Genotyping
24
DNA Isolation and Purification
24
Spectrophotometer (Nanodrop)
25
PCR
26
NlaIII Digestion
27
7.5% Polyacrylamide Gel and Gel Electrophoresis
28
MgCl2 Titration
29
Data Analysis
30
Chapter 3: Results
31
Genotyping
31
Mouse Weights and Grip Strength Force
32
Forelimb and Combined Forelimb and Hind limb Grip Strength Results 36
Chapter 4: Discussion
43
Grip Strength Tests
43
Future Directions
45
Conclusion
48
References
49
Appendix
57
Appendix I: Photograph of the ventral view of the homozygous mouse (7080R)
57
vi
Appendix II: Photograph of the cranial view of the homozygous mouse (7080R)
58
Appendix III: Gross anatomy of the homozygous (7080R) mouse kidney still attached
to body
59
Appendix IV: Photograph of both 7080R mouse’s kidneys
vii
60
List of Figures
Figure 1: Schematic Diagram of the Sialic Acid Biosynthetic Pathway
2
Figure 2: Diagram of Human Chromosome 9
6
Figure 3: Sialic Acid Molecule
9
Figure 4: GNE:p.M712T Mouse Knock-in Locus
17
Figure 5: The Chatillon DFIS-10 Digital Force Gauge Apparatus
19
Figure 6: Photographic Illustration of the Forelimb Grip-Strength Test
23
Figure 7: Photographic Illustration of the Combined Forelimb and Hind limb GripStrength Test
24
Figure 8: PCR Primers
27
Figure 9: Amplification of Segment
27
Figure 10: Phi-X174/Hae III Marker and 1Kb Plus Gene Ruler
29
Figure 11: 7.5 % Polyacrylamide Gel Electrophoresis for the 9000-9004 and 9013 Mice
31
Figure 12: 7.5 % Polyacrylamide Gel Electrophoresis Showing Effects of Using Different
MgCl2 (25mM) on PCR
32
Figure 13: Average Forelimb Measurements of Heterozygous, Homozygous Mutant and
Wild-type mice
37
Figure 14: Average Forelimb and Hind limb Measurements of Heterozygous,
Homozygous Mutant and Wild-Type Mice
37
Figure 15: Average Forelimb Measurements of the Heterozygous and Mutant Mice
38
Figure 16: Average Forelimb Measurements of the Heterozygous and Wild-Type Mice
39
Figure 17: Average Forelimb Measurements of the Mutant and Wild-Type Mice
39
Figure 18: Average Combined Forelimb and Hind limb Measurements of the
Heterozygous and Mutant Mice
40
viii
Figure 19: Average Combined Forelimb and Hind limb Measurements of the
Heterozygous and Wild-Type Mice
41
Figure 20: Average Combined Forelimb and Hind limb Measurements of the Mutant and
Wild-Type Mice.
42
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List of Tables
Table 1: Heterozygous Forelimb and Combined Forelimb and Hindlimb Grip Strength
Measurements
34
Table 2: Wild-Type Forelimb and Combined Forelimb and Hindlimb Grip Strength
Measurements
35
Table 3: Mutant Forelimb and Combined Forelimb and Hindlimb Grip Strength
Measurements
36
Table 4: Correlation of Genotype, Grip Strength Measurements and Weight in Mice
36
x
Abstract
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
By
Jessica Mayra Estrada
Masters of Science
Biology
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-acetylmannosamine (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;B6GNE 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 gripstrength 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
xi
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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Chapter 1: Introduction
Hereditary Inclusion Body Myopathy (HIBM) is a rare genetic disorder that
impacts people of multiple ethnicities. HIBM is a condition that primarily affects skeletal
muscles, which are muscle tissues; this disorder causes muscle weakness, the age of onset
is during late adolescence or early adulthood and it progressively worsens (Inclusion
body myopathy 2, 2008). Although the majority of individuals affected by HIBM are
found among the Japanese and Iranian Jewish communities, HIBM also affects
individuals of other ethnic backgrounds, including individuals of Caucasian, Indian, Thai,
Japanese and African descent (Noshino et al. 2002, Sivakumar K 1996, Liewluck et al.,
2006).
HIBM is also known as UDP-N-acetyl-glucosamine-2-epimerase/N acetylmannosamine kinase gene (GNE) Myopathy, Inclusion Body Myopathy Type 2
(IBM2), Distal Myopathy with Rimmed Vacuoles (DMRV), Quadriceps Sparing
Myopathy (QSM) and Nonaka Myopathy. It is caused by mutations in the UDP-N-acetylglucosamine-2-epimerase/N -acetylmannosamine kinase gene (GNE) (See Figure 1).
GNE encodes the enzyme responsible for making sialic acid. HIBM patients have lower
levels of sialic acid on the surface of certain proteins that are important for muscle
function. Failure of the body to produce enough sialic acid causes muscles to slowly
waste away and can lead to very severe disability within 10 to 20 years of diagnosis, and
many patients are confined to wheelchairs within that time (Jay et al., 2008, Zlotogora
1995).
1
Figure 1: Schematic diagram of the sialic acid biosynthetic pathway. The bifunctional
enzyme UDP-GlcNAc 2-epimerase/ManNAc kinase (GNE/MNK), encoded by the GNE
gene, catalyzes the first two committed, rate-limiting steps in the biosynthesis of Nacetylneuraminic acid (sialic acid). GNE/MNK is feedback inhibited by binding of the
downstream product, CMP-sialic acid in its allosteric site. GNE mutations can result in
two human disorders, hereditary inclusion body myopathy (HIBM) or sialuria. Adapted
from Galeano et al., 2007.
HIBM Overview and History
HIBM was likely first recognized in Japan. In 1981, Nonaka and colleagues
described an autosomal recessive distal myopathy with rimmed vacuoles (DMRV) in
three cases from two families (Nonaka et al., 1981). Nonaka and colleagues gave credit
to Sasaki et al. (1969) and Ideta et al. (1973) for having previously described a distal
myopathy in four other cases.
2
In 1984, Argov and Yarom (1984) published nine cases from four Jewish families
of Iranian descent of autosomal recessive "rimmed vacuole myopathy" sparing the
quadriceps. A larger study of Iranian Jewish individuals with the same disorder was
subsequently published by Sadeh and colleagues (1993). The disorder was characterized
by progressive distal and proximal weakness and wasting beginning in the legs and
sparing the quadriceps, even in the advanced stages. The disorder was further found in
other ethnic groups such as Caucasian American and Asian Indian (Sivakumar and
Dalakas 1996).
Eisenberg et al. (2001) identified mutations in the UDP-N-acetylglucosamine-2epimerase/N-acetylmannosamine kinase (GNE) gene in his research. Eisenberg and his
colleagues described patients from Middle Eastern descent that shared a single
homozygous missense mutation, whereas distinct compound heterozygotes were
identified in affected individuals of families of other ethnic origins. Their findings
indicate for the first time that GNE is the gene responsible for recessive HIBM. In 2002,
it became apparent that distal myopathy with rimmed vacuoles is the same as hereditary
inclusion body myopathy (Nishino et al., 2002).
Clinical Description of HIBM
HIBM starts in young adulthood, usually around the second or third decade,
although there have been studies with patients who experienced symptom onset as young
as 17 and as old as 52 (Hereditary Inclusion Body Myopathy, 2013). The first signs of
HIBM are weakness in distal limb muscles. The initial symptom is difficulty with gait as
a result of foot drop. Weakness in the tibialis anterior muscle also alters the way a person
walks and makes it difficult to run and climb stairs. Other signs are tripping, weakness in
3
the index finger, and frequent loss of balance. The weakness spreads and within several
years involves thigh and hand muscles. In other affected individuals, the hands are
weakened more rapidly than the legs (Hereditary Inclusion Body Myopathy, 2013).
Shoulder girdle muscles also become weak, with relative sparing of the triceps. Neck
flexors are commonly involved as well.
The striking feature of HIBM is quadriceps sparing even at advanced stages of the
disease, which is the reason HIBM is often referred to as Quadriceps Sparing Myopathy
(QSM) (Huizing et al., 2009). However, based on Argov et al., (2003) results of
molecular genetic testing, it has now been shown that quadriceps sparing is not a
common feature found in all individuals with HIBM. In his results, some individuals
without quadriceps sparing have been identified. Affected individuals with HIBM are
usually wheelchair-bound by 20 years after onset of symptoms. If an individual does not
have quadriceps sparing, loss of ambulation tends to occur earlier. Ocular, pharyngeal,
and cardiac muscles are usually spared. Intellectual abilities, internal organs and
sensation are also unaffected. A single affected individual has been described with
respiratory involvement resulting in a reduced forced expiratory volume in 1 second
(FEV1) and vital capacity (VC) (Weihl et al 2011). Occasionally; affected individuals
may have facial weakness (Argov et al 1998).
Diagnosis of HIBM
There are several criteria available with which an individual with HIBM can be
diagnosed. One of the primary indicators of HIBM is weakness in the proximal lowerextremity of the legs with sparing of the quadriceps. Onset in late adolescent or early
adulthood is also observed. An indication of the presence of a recessive condition is
4
supported by the observation of more than one affected individual in a single sibship.
(Argov et al 2003).
A test to determine if an individual has HIBM includes detection of an elevation
of serum creatine kinase (CK) activity. CK is an enzyme present in the cytoplasm of
myocytes and is useful as an indicator for evaluation of a neuromuscular disorder. CK is
elevated as a result of muscle degradation. In muscles, the CK enzyme functions by
making ATP available for contraction. The CK elevation, however, does not distinguish
as to a cause such as trauma, inflammation, or degeneration (Creatine Kinase, 2013).
Normal creatine kinase for adult females is 40-150 units/L and for adult males is 60-400
units/L (Kratz et al., 2004). In HIBM studies conducted by Argov (2003), creatine kinase
levels were between two and four times the normal value.
A muscle biopsy may be used to confirm the presence of rimmed vacuoles in
individuals affected with HIBM. The vacuoles appear empty, contain granular or
amorphous basophilic inclusions or congophilic masses (Sadeh et al., 1993).
Inflammation is not typically observed in an affected muscle. However, in a few studies
conducted by several investigators, a modest inflammatory response has been noted in
some individuals (Argov et al., 2003, Krause et al., 2003, Yabe et al., 2003). For
example, according to Yabe two individuals displayed the presence of inflammatory
changes in the connective tissue between muscle fibers.
Other tests used for diagnosis include computed tomography (CT) or magnetic
resonance imaging (MRI) scans of the muscle showing adipose cell replacement for
muscle. An Electromyogram can also help identify the pattern of muscle involvement in
an individual. If a sequence analysis of GNE is conducted then a muscle biopsy would
5
not be needed. Also, after an individual is diagnosed, neurologic examinations and
medical genetic consultations are recommended.
Molecular Basis of HIBM
HIBM can be inherited either as a heterozygous dominant or as a homozygous
recessive disorder in humans. HIBM is caused by mutations in the GNE gene. The GNE
gene is located on the short arm of chromosome 9 at position 9p13.3 (See Figure 2). The
GNE gene spans about 44 kb of genomic DNA and its major transcript consists of 13
exons. Exons 1 and 13 are non-coding. The GNE mRNA consists of 722 codons. The
most frequent mutation in HIBM patients is the Middle Eastern (Iranian-Jewish) founder
mutation M712T. The M712T mutation results from the change of a T to C in exon 12,
converting the predicted amino acid methionine to threonine at codon 712 (Broccolini et
al., 2011). Also, GNE mutations can result in sialuria (Huizing 2005). Sialuria is the
presence of an increased concentration of sialic acid, instead of the decreased
concentration seen in HIBM. GNE catalyzes the first two steps in the biosynthesis of
sialic acid (SA) (Keppler et al., 1999).The lack of SA production is presumed to cause
decreased sialylation of HIBM muscle glycoproteins, resulting in muscle deterioration
(Nishino et al., 2009 and Saito et al., 2004).
Figure 2: Diagram of human chromosome 9. The GNE gene is located on the short arm
of chromosome 9 at position 9p13.3 marked by a red horizontal line.
More than 70 GNE mutations have been described that are associated with GNE
myopathy (Sim et al., 2013). In a study by Huizing and Krasnewich (2009), out of the 62
GNE mutations that they worked with, 82% were missense mutations and scattered
6
throughout the GNE gene. Also, 11 (18%) out of the 62 GNE mutations were “null”
mutations, nonsense or frame shift mutations.
Other research surrounding GNE mutations suggests that some specific GNE
mutations arose independently in multiple ethnicities. For example, p.A524V in Thailand,
Mexico, and France are believed to have arisen independently (Liewluck et al., 2006,
Darvish et al., 2002, Behin et al., 2008). The process by which the mutations in the
enzyme lead to muscle disease is still not quite understood.
GNE/MNK
One domain of the GNE enzyme carries out epimerase function; the other domain
(MNK) is responsible for kinase activity. The N-terminal portion of GNE/MNK (amino
acids 1–378) has UDP-GlcNAc 2-epimerase catalytic activity (Effertz et al., 1999), which
catalyzes the epimerization of UDP-GlcNAc to ManNAc with the release of UDP. The
C-terminal portion (amino acids 410–722) has ManNAc kinase catalytic activity, which
phosphorylates ManNAc to ManNAc-6-P and phosphoenolpyruvate. The production of
sialic acid on glycoconjugates requires the conversion of N-acetylglucosamine
(conjugated to its carrier nucleotide sugar UDP) to sialic acid. The sialic acid then enters
the nucleus where it is conjugated with its nucleotide sugar carrier cytidine
monophosphate (CMP) to make CMP-sialic acid, which is used as a donor sugar for
glycosylation reactions in the cell. The downstream product, CMP-sialic acid regulates
the activity of GNE by allosteric inhibition (Jay et al., 2009). The exact locations of the
active sites within these domains remain to be determined.
GNE/MNK exists in two major oligomeric states, tetramers and dimmers. The
fully functional tetrameric state of GNE/MNK is stabilized by ligands of the GNE
7
domain, UDP-N-acetylglucosamine and CMP-N-acetylneuraminic acid (Ghaderi et al.,
2007). The GNE/MNK allosteric site appears only in the mammalian enzyme;
prokaryotic 2-epimerases has no allosteric feedback inhibition. In prokaryotes, epimerase
and kinase functions are carried out by two separate enzymes. In mammals, a
bifunctional enzyme may have evolved by fusion of two genes encoding different
enzymes responsible for epimerase and kinase activity. Similarities between mammalian
GNE/MNK N-terminal regions with prokaryotic UDP-GlcNAc 2-epimerases and
mammalian C-terminal regions with members of the sugar kinase superfamily assisted in
identifying several characteristic modifications of the GNE and MNK enzymatic domains
(Effertz et al., 1999).
Sialic Acid
Sialic acids are involved in multiple biological pathways, playing an important
role in many cellular functions, such as adhesion processes, cell migration, inflammation,
wound healing and also in metastasis (Lowe 2003; Varki and Angata 2006). Sialic acids
are also known as N-acetylneuraminic acid (Neu5Ac) and are the most abundant terminal
monosaccharide on glycoproteins and glycolipids of eukaryotic cells. Neu5Ac is a 9carbon acidic sugar and is believed to be one of the metabolic precursors for all other
sialic acids (See Figure 3). In general, they are found at the outermost ends of N-glycans,
O-glycans, and glycosphingolipids. Because of their terminal location and negative
charge, sialic acids have the potential to inhibit many intermolecular and intercellular
interactions. Sialic acids can also be critical components of ligands for recognition by
specific lectins. Unsaturated and dehydro forms of sialic acids are also known to exist.
This structural diversity of sialic acids can determine and/or modify the recognition by
8
antibodies, as well as by a variety of sialic-acid-binding lectins of endogenous and
exogenous origin. Interestingly, they are not typically found in plants, prokaryotes, or
most invertebrates. However, sialic acids have been reported in Drosophila embryos
(Varki et al., 1999).
Figure 3: Sialic acid molecule: 2-keto-5-acetamido-3,5-dideoxy-d-glycero-dgalactononulosonic acid (Neu5Ac).
N-acetylneuraminic acid was discovered by Blix et al. (1957) as a major product
released by mild acid hydrolysis of brain glycolipids or salivary mucins. The name “sialic
acids” comes from the discovery of this product in salivary mucins. Sialic acids are
synthesized in the cytosol from UDP-Nacetylglucosamine by four consecutive reactions.
In 2000, protein kinase C was associated with UDP-GlcNAc 2-epimerase, regulating its
enzymatic activity (Horstkorte et al., 2000). According to Schwarzkopf et al. (2002)
Northern-blot analysis and in situ hybridization revealed the highest expression in liver,
but was also expressed in all other organs investigated. UDP-GlcNAc 2-epimerase is
fully expressed at all stages during mouse development that have been investigated so far
(Horstkorte et al., 2000 ).
The clinical relevance of the UDP-GlcNAc 2-epimerase was demonstrated by the
detection of a binding defect of the feedback inhibitor CMP-sialic acid (Warren L,
9
Felsenfeld H., 1962) leading to sialuria (Seppala et al.,1999). In this sialic acid storage
disease, free sialic acid accumulates in the cytoplasm, which results in severe mental
retardation in the affected individual. The significance of the enzyme is further illustrated
by the observation that, in a variant of HL60 cells, the low expression of sialic acids is
correlated with dramatically reduced enzyme activity (Keppler et al., 1999). Mutations in
the human UDP-GlcNAc 2- epimerase gene, according to Eisenberg and his colleagues
(2001), are responsible for hereditary inclusion body myopathy.
Treatments
The treatment for HIBM is currently palliative only. Examples include
consultation with physiatrists (rehabilitation physician), physiotherapists, and
occupational therapists. Consultation with a pulmonologist to evaluate for nocturnal
hypoventilation or sleep apnea may be beneficial diagnosis of affected individuals.
Mechanical aids such as a brace for the foot may help in preventing further foot drop.
Wheelchairs can improve the mobility of individuals affected with HIBM (O’Ferrall et
al., 2004).
There are several therapies under investigation for HIBM. In one study by
Malicdan et al. (2009), researchers evaluated the efficacy of ManNAc and other sialic
acid metabolites as a treatment for HIBM. In one of their studies the investigators gave
four groups of 10- to 20-week-old DMRV-HIBM mice either ManNAc (n = 6), NeuAc (n
= 5), sialic acid conjugate (sialyllactose, n = 7) or water (as control treatment; n = 10)
continuously until the 54-57 weeks of age. They also treated equal numbers of littermate
controls for each group (n = 18). All groups tolerated a dose of 20 mg per kg body weight
per day. Treatment with any of the three sialic acid compounds improved the survive-
10
ability of the DMRV-HIBM mice versus the control-treated group, from a median
survival at 54-57 weeks of 55.6% to one of 86.9%. The results provided evidence that
hyposialylation is one of the key factors in the pathomechanism of DMRV-HIBM by
showing that muscle atrophy and weakness are prevented in a mouse model of DMRVHIBM after treatment with oral sialic acid metabolites.
In another study, a single trial of intravenous immune globulin (IVIG) in four
molecularly proven HIBM humans, resulted in mild improvement in muscle strength
(Sparks et al., 2007). IVIG is a glycoprotein which can be metabolized by neuraminidase
to provide free sialic acid. These investigators treated four HIBM patients with
intravenous immune globulin, in order to provide sialic acid, because IgG contains 8
μmol of sialic acid/g. IVIG was infused as a loading dose of 1 g/kg on two consecutive
days followed by 3 doses of 400 mg/kg at weekly intervals. For all four patients, mean
quadriceps strength improved from 19.0 kg at baseline to 23.2 kg directly after IVIG
loading to 25.6 kg at the end of the study. Mean shoulder strength improved from 4.1 kg
at baseline to 5.9 kg directly after IVIG loading to 6.0 kg at the end of the study. Also,
the improvement for eight other muscle pairs (right and left), associated with hip flexion,
ankle dorsiflexion, elbow flexion and extension, wrist flexion and extension, grip, and
pinch, was 5% after the initial loading and 19% by the end of the study. It should be
noted that the normal strength levels of these muscle groups were not explained in the
study. Strength of muscle groups of the four HIBM patients were compared at baseline
and after IVIG treatment only.
Recently, a single patient with severe HIBM received four doses of GNE gene
Lipoplex via intramuscular injection (Nemunaitis et al., 2010). The investigators
11
constructed a GNE-wt-DNA vector, using human GNE cDNA and the pUMVC3
expression vector. The same subject was later treated with infusion at the following doses
of the GNE-lipoplex preparation: 0.4, 0.4, 1.0, 4.0, 5.0, 6.0, and 7.0 mg of DNA
(Nemunaitis et al., 2011). GNE transgene expression, downstream induction of sialic
acid, safety, and muscle function were evaluated. Transient low-grade fever, myalgia,
tachycardia, transaminase elevation, hyponatremia, and hypotension were observed after
infusion of each dose of GNE gene lipoplex. Significant durable improvement in skeletal
muscle function was observed in the injected left extensor carpi radialis longus of the
patient in correlation with GNE transgene upregulation and local induction of sialic acid.
Other than transient low grade fever and pain at the injection site, no significant toxicity
was observed. Also, sialic acid-related proteins were increased and stabilization in the
decline of muscle strength was observed. Further studies are required before these
therapies will be available for widespread use.
Animal Models of HIBM
Several mouse models have been generated to further analyze the HIBM disorder.
One mouse model was generated by Malicdan et al. (2007). They knocked out the GNE
gene in mice, by inserting the Neo cassette which replaced 1.4 kb upstream of exon 3,
exon 3 and 1.4 kb downstream of exon 3. With this strategy, only wild-type and GNE
heterozygous mice were generated and no homozygous GNE mice were produced, as
homozygosity evidently resulted in embryonic lethality. They then generated a transgenic
mouse that expressed the human GNE D176V mutation and crossed this with GNE
heterozygous mice to obtain GNE homozygous hGNED176V-Tg. Interestingly, these
mice exhibited hyposialylation in serum, muscle, and other organs. Also, the motor
12
performance of the mice was observed from 30 weeks of age. Their results show that the
GNE homozygous hGNED176V-Tg mouse mimics the clinical, histopathological and
biochemical features of HIBM making it useful for understanding the pathomechanism of
this myopathy and for employing different strategies for therapy. Their findings also
support the idea that hyposialylation plays an important role in the pathophysiology of
HIBM.
Another model of HIBM was generated by creating a gene-targeted knock-in
mouse homozygous for the M712T GNE mutation (Galeano et al., 2007). This
homozygous mutant mouse model died within 72 hours after birth and lacked a muscle
phenotype. According to the investigators, the homozygous mice had severe glomerular
hematuria and podocytopathy, including effacement of the podocyte foot processes and
segmental splitting of the glomerular basement membrane (GBM). They speculate that
the glomerular podocytopathology is likely due to hyposialylation. It is interesting to note
that the podocyte foot processes and GBM integrity was rescued in the homozygous pups
by administering an oral sialic acid precursor (ManNAc) to pregnant mice who were
heterozygous for the GNE gene. This research supports the idea that the biochemical
defect is related to reduced sialylation.
In a study by Valles-Ayoub et al. (2013), heterozygous mice (GNE M712T/+) of
B6 strain were crossed with FVB strain mice. In this study, the homozygous mice showed
glomerular disease and survived longer (mean survival 23.48 ± 13.99 weeks, n=73).
Within the first 2 generations, 26% of the homozygous mice survived past the age of 40
weeks, and within the subsequent 3 generations the frequency of homozygous mice
surviving past age of 40 weeks had increased to 44%. Additionally, the homozygous
13
mice (GNE M712T/M712T) living past the age of 42 weeks began to show muscle
pathology.
Grip Strength Test
The purpose of the grip strength testing is diverse. The grip strength test is a
simple non-invasive method designed to evaluate mouse muscle force in vivo. This test
takes advantage of the animal’s tendency to grasp a horizontal metal bar or grid while
suspended by its tail. Because of its simplicity and economy, the grip strength test is the
most commonly used in vivo test for monitoring impaired limb strength caused by
pathological progression. The method can be used to measure the disease progression as
well as to test the effect of specific therapeutic interventions in mouse models of
neuromuscular disorders. According to a protocol by De Luca et al. (2009), mice older
than 2-3 weeks of age are generally suitable for this test. The grip test was used to
measure the maximal muscle strength of the forelimbs as well as the forelimbs and hind
limbs combined. This test served as a primary phenotypic screen in mice with the M712T
mutation that are past 42 weeks, which is the age when changes were anticipated (VallesAyoub et al.,2013).
Purpose of Research
The purpose of this study was to help determine whether the particular animal
model constructed per Valles-Ayoub et al, reflects the HIBM disorder well enough to
allow testing of different therapeutic approaches. The research study included a gripstrength test to measure progressive muscle weakness as a primary phenotype screen in
mice that are homozygous for the M712T allele. It was my hypothesis that the
homozygous mice would show progressive muscle weakness compared to the wild-type
14
and heterozygous groups. Such a model could be used in future experiments to assess
various treatment options, for the disorder.
15
Chapter 2: Materials and Methods
Materials
Animal Subjects Approval Form
Federal regulations required this study involving the use of animal subjects to
undergo a review by the Institutional Animal Care and Use Committee (IACUC) to
ensure that the mice were handled in an ethical manner and not exposed to unnecessary
risk. The IACUC is regulated by the United States Department of Agriculture (USDA)
and the Department of Health and Human Services (DHHS). All students and faculty
who utilized the animals completed a protocol form and submitted it to Office of
Research and Sponsored Projects (ORSP). The blank protocol forms, as well as, copies of
the federal guidelines for animal related research were downloaded from the California
State University, Northridge Research and Graduate Studies webpage.
Mice
The Gne M712T/M712T knock-in mice given to us by Valles-Ayoub and
colleagues were produced according to a protocol by Galeano et al., (2007). A murine
targeting vector for homologous recombination in C57BL/6J embryonic stem cells was
constructed to include the M712T Gne mutation (See Figure 5). The neomycin
phosphotransferase and thymidine kinase genes were introduced into the vector as
positive and negative selection markers, respectively. The addition of LoxP (flanking
exon 12 and neo) and flippase recombinase target sites (flanking neo) were inserted to
allow for potential future conditional transgenic models. The entire vector was then
sequenced to verify accuracy. Also, since some of the mice in the B6 background
suffered from severe kidney disease and did not survive beyond the first few days of life
16
(Galeano et al., 2007), they back-crossed the heterozygous mouse B6-GneM712T/+ with
the FVB strain, and then crossed the N1 generation to produce mixed inbred homozygous
mice (FVB;B6-GneM712T/M712T).
Figure 4: Gne:p.M712T mouse knock-in locus. Exon 12 is the last exon, which includes
the polyA signal (not shown). LoxP sites allow for tissue specific knockout by mating
with appropriate Cre expressing mice (Galeano et al., 2007).
The original founding colony of mice was delivered on December of 2011 to the
CSUN vivarium. This colony consisted of mice homozygous for the M712T mutation,
heterozygous carriers for the M712T mutation, and wildtype mice. These mice were
identified with an identification number and with an ear-cut (none, right, left, or both).
Vivarium
The mice provided by Valles-Ayoub and their research team lived in the CSUN
vivarium. The mice lived in clear plastic cages. The living area for the mice allowed them
to satisfy their basic physiological and behavioral needs including the ability to eat, drink,
urinate, defecate, forage, explore, hide, climb, play, nest, dig and engage in a range of
social activities. Bedding material in the form of wood shavings was provided for thermal
insulation. The material absorbs fecal and urinary wastes, and in some instances was used
for nest construction. The wood shavings in the cages were sufficient to cover the entire
17
floor. The cage lid incorporated a grid which allowed the animals to climb. Food and
fresh water was provided ad libitum and replaced at least three times per week. A
nutritionally adequate diet, lab block, was provided for the mice from Purina bought at
Red Barn Feed in Tarzana. Occasionally the mice were given sunflower seeds to
stimulate sexual reproduction. The sunflower seeds were dispersed in the bedding and
also encouraged foraging. The temperature in the mouse module ranged from 18°C to
26°C with an average temperature of 22°C. The mouse module was regulated by
automatic timers to provide cycles with 12-14 hours of light and 10-12 hours of dark. The
monitoring of the mice for any behavioral or abnormal physical health was provided by
Toni Uhlendorf, Vivarium Manager, at California State University, Northridge. Her staff
also made sure to clean the cages at least three times per week.
Chatillon DFIS-10 Digital Force Gauge Apparatus
Grip strength of the mouse subjects was measured using the Chatillon DFIS-10
digital force gauge apparatus. The Chatillon DFIS-10 digital force gauge apparatus (See
Figure 4) is straight-forward in its operation. By using a four push button membrane
keypad, users can access peak values, reset to zero, and select units in pounds (lb),
kilograms (kg) or Newton’s (N). A large LCD display shows current values, peak values,
gauge settings and battery status. The temperature for storing the apparatus is 40° to
110°F (5°C to 45°C). The weight of the instrument is 2 lbs (Ametek, 2001). The system
is also supplied with a single metal grid which connects to the sensor. Force gauge data
are recorded manually or automatically. In this study the data were recorded manually.
18
Figure 5: Chatillon DFIS-10 digital force gauge apparatus. This figure is a photograph of
the Chatillon DFIS-10 digital force gauge apparatus used in this study.
Methods
Mouse Handling
When examining mice and transferring them to another cage, the investigator
grasped the middle tail between the thumb and index finger, and lifted the mouse. The
investigator movements were slow and gentle. The one-handed and two-handed methods
for performing minor, non-painful procedures such as ear markings were also techniques
used by the investigator. In the one-handed method, the mouse’s nape was grasped gently
and firmly and the tail was placed between the last two fingers of the hand. In the twohanded method, which was used when two investigators were present, the mouse was
placed on the cage grid while holding the tail firmly. The nape was then gently grasped
with the free hand.
Breeding
19
In order to grow and maintain the colony of HIBM mice, breeding was performed
at the CSUN vivarium. Combinations of heterozygous male and female, homozygous
male and female, or a heterozygous with a homozygous were paired to try and generate
mutant mice. On each breeding cage, the identification of the female and then of the male
was marked on a pink index card. Data were recorded on the data sheet if a pregnancy
was suspected; these included the presence of a yellowish vaginal plug or the physical
observation of abdominal growth. If a female was seen to be pregnant, the male mouse
was removed a few days before her due date in order to ensure a healthy delivery.
Ear Markings and Tailing
Once the pups reached four weeks, they were placed in another cage according to
sex and marked with ear cuts. The identification number and ear marking were recorded
on the appropriate data sheet. It was important to work with one cage at a time to avoid
confusion or incorrect labeling of the mice. The cages were labeled with the following
information on an index card: cage number, mouse colony numbers, dates of birth, and
the total number of mice in that cage. The ear markings were as follows: N (no cut), R
(Right cut), L (Left cut), B (Both cut).
Tail cuts were made from each newborn mouse in order to obtain DNA for
genotyping. To obtain the tail snipping specimen from the N (No cut) mouse, a mouse
was picked up from the cage by its tail and a small portion of the tail was cut. Mouse tail
snippings were approximately 5 mm in length and stored at 20°C in 1.5 mL Eppendorf
tubes. Pressure was applied to the stump of the mouse’s tail until bleeding stopped. Kwik
Stop Styptic Powder was sometimes applied to stop bleeding if bleeding persisted past
two minutes. Tail snips were stored at -20 degrees if DNA was to be isolated later.
20
The first mouse of a series did not need any ear cuts since it was an “N” mouse
(no cut mouse). The second mouse was marked with a right ear cut (“R”). To perform an
ear cut, one hand grasped the nape of the neck of the mouse firmly while the other hand
made a horizontal cut on the right ear, using sterile scissors. The third and fourth mouse
was marked with left ear cut (“L”) and both cut (“B”) following the same procedure.
Grip Strength Procedure
Methods and procedures for the grip strength were obtained from EMPRESS
protocol (Brown et al., 2005). The data in this experiment were collected twice a week in
the morning for a period of three consecutive weeks. In total, there were six data
collection periods. Each mouse was given four trials (two for the forelimb and two for the
forelimbs and hind limbs) on the grip strength meter. The average of the two trials for the
two forelimbs and the average of the two forelimbs and hind limbs were taken during
each session. The experimental groups consisted of six homozygotes, six heterozygotes,
and six wildtype mice. Also, each mouse was given 60 seconds of rest to after each trial,
to recover from pulling on the grip strength meter metal grid.
No eating and no drinking in the Vivarium were allowed. Laboratory coats were
optional and gloves were worn at all times in the work area. The equipment used was the
Chatillon DFIS-10 digital force gauge apparatus that measures the gripping strength of
mice. This system is supplied with a single grid which connects to the sensor. To use the
instrument, it was first ascertained that the connection of the sensor to the grid was tight
to prevent the grid from spinning. Next, the power was connected to the sensor, and the
unit of measurement, kilograms (kg) was selected on the instrument by selecting the units
button. The display was set to zero and a record sheet was prepared which contained the
21
details of the identification of the animal to be studied. To handle mice, the animal was
gripped from the base of the tail between the thumb and the index finger. For each trial, a
mouse was removed from its cage. The mouse was weighed, and the pre-trial weight was
recorded on the data sheet.
The forelimb measurement was tested by gently lowering the mouse over the top
of the grid so that only its front paws could grip the grid (See Figure 6). The torso of the
mouse was kept horizontal during the study. Next, the mouse was pulled back steadily
(without jerking) until its grip was released down the complete length of the grid. The
pulling was at a constant speed and sufficiently slow to permit the mouse to build up
resistance against the grid. When the mouse released the grid, the maximal grip strength
value of the mouse was displayed on the screen. The force in kilograms was then
recorded manually. The mouse rested for 60 seconds and the forelimb procedure was
performed 1 more time to obtain a total of two recordings per mouse.
22
Figure 6: Photographic illustration of the forelimb grip-strength test. Photographic
illustration of the forelimb grip-strength test, demonstrating the position of the animal
subject to be tested.
The combined forelimb and hind limb measurement were tested by gently
lowering the mouse over the top of the grid so that both its front paws and hind paws
could grip the grid (See Figure 7). The torso of the mouse was kept parallel to the grid
and the mouse was pulled back steadily (without jerking) until the grip was released. The
values were recorded manually. The procedure was repeated in order to obtain additional
forelimb and hind limb grip strength measurements. Also, the mouse rested for 60
seconds before the next trial.
23
Figure 7: Photographic illustration of the combined forelimb and hindlimb grip-strength
test. Photographic illustration of the placement of the animal subject during measurement
of the combined forelimb and hindlimb grip-strength test.
Notes were made on the data sheet regarding any special observations that were
made during the test of each subject. For example, if a mouse failed to grip the grid with
any limb. Also, all of the measurements were administered in a blinded fashion so that
the genotype of the mouse was unknown to the examiner. Once the testing was finished,
the grid was cleaned with 50% ethanol and dried with paper towels, before use on the
next mouse subject. At the end of the three week collection period, each mouse was reweighed.
Genotyping
DNA Isolation & Purification
24
In a 1.5 mL Eppendorf tube, snipped mouse tails were placed with 500 uL of
regular alkaline lysis buffer and 5 uL of proteinase K (20mg/uL) (Valles-Ayoub et al.,
2013). The lysis buffer and proteinase K were kindly provided by Valles-Ayoub et al.,
from the HIBM Research Group. The tube was next placed on the vortex (Vortex Genie
2- Fisher Scientific) for 11 seconds. This solution was then incubated in a 55 °C water
bath and left overnight until the sample was completely dissolved. The next day, the
solution was vortexed for 11 additional seconds to mix well. The supernatant was then
aspirated into a fresh 1.5 mL Eppendorf tube. To pellet the debris, the tubes were
balanced in the centrifuge (Centrifuge 5417c- Eppendorf) and centrifuged at 13000 RPM
for 7 minutes. Again, the supernatant was collected in a fresh 1.5 mL Eppendorf tube.
Next, a 1:1 ratio of isopropanol (100%) to supernatant was added. In most cases it was
equal to or less than 500 uL. This solution was vortexed for 11 seconds and centrifuged at
13000 RPM for 20 minutes. Following this step, the supernatant was carefully decanted
without disturbing the pellet which was sometimes not visible. The pellet was washed in
70% ethanol. The sample was re-centrifuged (13000 RPM for 20 minutes), and the
supernatant was removed using a micropipette. The tube was then placed under the fume
hood until the pellet was dry and no ethanol residue was left. Finally, 50 uL of nanopure
water was added to resuspend the DNA pellet. The new solution was vortexed one final
time, to mix thoroughly, for 11 seconds.
Spectrophotometer (Nanodrop)
The concentration of nucleic acid from the mouse tail snips was measured using
The Nanodrop 2000c machine (Thermo Scientific). Before any readings were taken, the
machine was calibrated with 2 uL of nanopure water. This “blanks” the machine and
25
allows for a more accurate measurement. Next, the machine was cleaned with lens paper
and 2 uL of DNA sample was loaded on the nanodrop reader. Each time a new sample
was loaded, a sample ID was typed in the “Sample Id” section to avoid any mix-ups. A
good reading revealed no ethanol residue and had at least 20 ng/uL of nucleic acid. The
samples were measured twice to confirm correct readings. The nanodrop reader was
cleaned once all samples were measured and the program was then closed.
PCR
A region including the location of the M712T mutation was amplified using PCR
of the isolated genomic DNA. A PCR worksheet was used by the investigator to calculate
the quantity of each reagent to be used in the PCR reaction. Primers were ordered from
IDT, and sequences are shown in Figure 9. Sequences are shown in Figure 9, between
primers 1895 F and 2200R are shown. NlaIII cuts wildtype alleles at location 265 and
354. NlaIII cuts mutated alleles at 354 only.
In a 200 uL thin-walled PCR tube (20 uL/reaction) several reagents were mixed:
5.0 uL of nanopure water, 10.0 uL of 2X Buffer A (Epicentre), 2.0 uL of 10 uM Primer
Mix (See Figure 8), 1.0 uL of RedTaq (Sigma), and 2.0 uL of genomic DNA at 20ng/ul
obtained from the specimen. If several samples were tested, a master mix of all the
reagents except for the 2.0 uL of genomic DNA was created. The samples were then
placed in the Thermocycler (Gene Amp PCR System- Perkin Elmer) and proceeded using
the following program: initial denaturation at 95 °C for 60 seconds, 35 cycles of 95 °C
for 10 seconds, 60 °C for 8 seconds, 72 °C for 60 seconds, final extension at 72 °C for 10
minutes. The entire PCR reaction took 3.5 hours.
26
Figure 8: PCR Primers. Forward and Reverse primers were used to create a master mix
to perform PCR.
Figure 9: Amplification of segment. Numbered sequence between primers 1895 F and
2200R are shown. NlaIII cuts the wildtype allele at location 265 and 354. NlaIII cuts the
mutated allele at 354 only.
NlaIII Digestion
Restriction digestion was performed on the amplicon of the PCR product. An
enzyme worksheet was used to display the exact amounts of each reagent. The following
reagents were then mixed in a PCR tube for a 20.0 uL reaction: 11.0 uL H2O, 2.0 uL 10X
Buffer #4 (New England BioLabs), 2.0 uL BSA (100ug/ml, New England BioLabs), 4.0
uL PCR product, 1.0 uL NlaIII (10 units, Thermo Scientific). For more than one sample,
a master mix was created adding all of the reagents except for the 4.0 uL PCR product, so
that a no-DNA control could be run in parallel. The tubes were then placed in the PCR
machine at a constant 37 °C for four hours and then at 65 °C for 20 minutes. The elevated
27
temperature served to inactivate the digestion reaction by denaturing the enzyme. The
tubes were then centrifuged to collect the contents at the bottom. The samples were then
pipetted into polyacrylamide gels and electrophoresed.
7.5 % Polyacrylamide Gel and Gel Electrophoresis
A 7.5 % polyacrylamide gel was chosen to separate the 354- base pair amplicon.
In a Protean III (BioRad) one short glass plate and one long glass plate was placed
together with a grey rubber spacer between the plates on each side. Two glass plates were
set in the green holder. The green holder and the glass plates were then mounted on the
plastic “house” of the apparatus. The plates were then clamped onto the pouring stand.
The gel with the catalysts (TEMED and APS) was then poured between the plates. After
polymerization, the gel was loaded. For the markers, a commercial preparation called
1Kb Plus Gene Ruler (Thermo Scientific) and Phi-X174/Hae III (Agilent Technologies)
were used (See Figure 10). The 1Kb Plus Gene Ruler contains 15 fragments for size
determination of fragments between 75 bp to 20,000bp. PhiX174/Hae II Marker contains
11 fragments for size determination of fragments between 72 bp to 1353 bp. In the
following lanes, 8uL of the digested products from the samples were loaded. Controls for
no DNA, mutant, heterozygous and homozygous were loaded into separate lanes.
Electrophoresis was set at 90 V for 75 minutes (Power Pac 200-Biorad). Next, the gel
was stained in the absence of light in a 10 mg/ul concentration of ethidium bromide for
10 minutes and de-stained in water for 2 minutes. An image of the gel was captured
(Fluorchem HD2- Cell Biosciences).
28
Figure 10: Phi-X174/Hae III Marker and 1Kb Plus Gene Ruler. After polymerization, the
acrylamide gel was loaded with the Phi-X174/Hae III Marker or 1Kb Plus Gene Ruler
and the digested products. Observations for bands at location 265 and 354 (wildtype
allele) and bands at 354 only (mutated allele) were made on the gel.
MgCl2 Titration
In a 200 uL thin-walled PCR tube (25 uL/reaction) several reagents were
mixed: nanopure water at 5.5 uL, 5 uL, or 4.5 uL, 2X GoTaq Buffer (10uL), 25 mM
magnesium chloride at 3.5uL, 4.5uL, or 5uL, dNTP (1uL), 10 uM Primer Mix (2uL),
GoTaq (1uL), and 2.0 uL of genomic DNA at 5ng/uL or 10ng/uL obtained from the
specimen. The samples were then placed in the Thermocycler (Gene Amp PCR SystemPerkin Elmer) and proceeded using the following program: initial denaturation at 95 °C
for 60 seconds, 35 cycles of 95 °C for 10 seconds, 60 °C for 8 seconds, 72 °C for 60
seconds, final extension at 72 °C for 10 minutes. The restriction digestion was performed
29
on the amplicon of the PCR product and the samples were then pipetted into
polyacrylamide gels and electrophoresed.
Data Analysis
The data such as bar graphs and tables in this study were analyzed using
Microsoft Office Excel and are shown in Chapter 3.
30
Chapter 3: Results
Genotyping
Shown below in Figure 11, is a photograph of an ethidium bromide stained 7.5 %
polyacrylamide gel electrophoresis. A gel was created to determine the 9000-9004 and
9013 GNE gene genotype for each mouse. The restriction enzyme NlaIII, cut at 265 and
354 for the wild-type allele and only cut at 354 for the mutant allele.
Figure 11: 7.5 % polyacrylamide gel electrophoresis for the 9000-9004 and 9013 mice.
Lane one is empty, lane two is the 1kb marker, lane three is the positive heterozygous
control, lane four is the 9000 mouse identified as heterozygous, lane five is the 9001
mouse identified as mutant, lane six is the 9002 mouse identified as wild-type, lane six is
the 9003 mouse identified as heterozygous.
Figure 12 is a photograph of an ethidium bromide stained 7.5 % polyacrylamide
gel electrophoresis created to show which magnesium chloride (25 mM MgCl2)
concentration (3.5uL, 4.5uL, 5uL) worked best using Go Taq, 2X Go Taq Buffer, and
dNTPs in PCR to amplify the region of interest. This gel was created to ascertain which
31
DNA concentration (10ng/uL or 5ng/uL) worked best with each of the three different
magnesium concentrations in the magnesium titration. The bands all showed that the
mouse was homozygous for the M712T mutation.
Figure 12: 7.5 % polyacrylamide gel electrophoresis showing effects of using different
MgCl2 (25mM) concentration on PCR. Lane one is empty. Lane two had 4.5uL of
MgCl2 and 10ng/uL of template. Lane three had 4.0uL of MgCl2 and 10ng/uL of
template. Lane four had 3.5uL of MgCl2 and 10ng/uL of template. Lane five is the PhiX
174 Hae III marker. Lane six had 4.5uL of MgCl2 and 5ng/uL of template. Lane seven
had 4.0uL of MgCl2 and 5ng/uL of template. Lane eight had 3.5uL of MgCl2 and 5ng/uL
of template. Lane nine is the 1kb gene marker. The bands all showed that the mouse was
homozygous for the M712T mutation. The restriction enzyme NlaIII only cut at 354 for
the mutant allele.
Mouse Weights and Grip Strength Force
The mice in this study were examined twice per week over the course of three
consecutive weeks. In total, there were six trial periods. All trial periods were conducted
in the morning. The investigator was blinded to the genotypes of the test subjects, this
avoided human bias.
32
Table 1 shows the results of the heterozygous (HT) mice. Column One shows the
trial period. Column Two shows the genotype. Column Three shows the mouse
identification number. Column Four shows the ear cut of the mouse. Column Five shows
the starting weight of each mouse. Column Six shows the sex of the mouse. Column
Seven shows the results of the forelimb test number one. Column Eight shows the results
of the forelimb test number two after a 60 second resting period. In Column Nine, the
resting time is shown in seconds. Column 10 shows the average strength of the two
forelimb tests for each mouse. Column 11 shows the standard deviation between the two
forelimb tests for each mouse. Column 12 shows the combined forelimb and hindlimb
test. Column 13 shows the second test of the combined forelimb and hindlimb test. The
resting time for the combined forelimb and hindlimb are shown in column 14. Test
results for the combined forelimb and hindlimb are shown in column 15. The last column
shows the standard deviation of the two tests for the combined forelimb and hind limb.
The measurements for the wild-type (WT) mice are shown in Table 2 and in Table 3 one
can locate the measurements for the mutant (MT) mice.
The change across each of the three experimental groups can be seen in Table 4.
Column One shows the genotype of each group. In Column Two, the average of all the
forelimb grip strength measurements for one group was calculated using Microsoft Excel.
The average of the combined forelimb and hindlimb grip strength measurements were
also calculated for each group. The mean starting weight for each group is shown in
column four. The mean ending weight is shown in column five. The difference in the
total from the starting weight to the ending weight is shown in the last column. The
33
weight difference was calculated to further explain the effects of genotype on grip
strength.
Trials
Genotype
1 HT
1 HT
1 HT
1 HT
1 HT
1 HT
2 HT
2 HT
2 HT
2 HT
2 HT
2 HT
3 HT
3 HT
3 HT
3 HT
3 HT
3 HT
4 HT
4 HT
4 HT
4 HT
4 HT
4 HT
5 HT
5 HT
5 HT
5 HT
5 HT
5 HT
6 HT
6 HT
6 HT
6 HT
6 HT
6 HT
Mice
Mouse ID Weight (g)
7088 N
7074 L
6858 N
7079 N
7084 N
7062 L
7088 N
7074 L
6858 N
7079 N
7084 N
7062 L
7088 N
7074 L
6858 N
7079 N
7084 N
7062 L
7088 N
7074 L
6858 N
7079 N
7084 N
7062 L
7088 N
7074 L
6858 N
7079 N
7084 N
7062 L
7088 N
7074 L
6858 N
7079 N
7084 N
7062 L
35.2 M
32.76 M
51.24 M
28.18 F
29.75 F
38.89 F
35.2 M
32.76 M
51.24 M
28.18 F
29.75 F
38.89 F
35.2 M
32.76 M
51.24 M
28.18 F
29.75 F
38.89 F
35.2 M
32.76 M
51.24 M
28.18 F
29.75 F
38.89 F
34.64 M
32.89 M
48.27 M
28.24 F
26.38 F
38.89 F
34.64 M
32.89 M
48.27 M
28.24 F
26.38 F
38.89 F
Sex
Forelimb Forelimb
Strength
Forelimb Forelimb Resting Average
and
and
Resting Average Strength
Standard
Test #1 Test #2
Time Strength
Hindlimb Hindlimb Time Strength Standard
Deviatio
(kg)
(kg)
(sec)
(kg)
Test #1 Test #2
(sec)
(kg)
Deviation
n
(kg)
(kg)
0.29
0.25
60
0.27 0.028284
0.31
0.46
60
0.385 0.10606602
0.25
0.23
60
0.24 0.014142
0.35
0.9
60
0.625 0.38890873
0.22
0.43
60
0.325 0.148492
0.31
0.42
60
0.365 0.07778175
0.07
0.16
60
0.115 0.06364
0.35
0.24
60
0.295 0.07778175
0.15
0.13
60
0.14 0.014142
0.24
0.52
60
0.38 0.1979899
0.16
0.28
60
0.22 0.084853
0.56
0.26
60
0.41 0.21213203
0.08
0.075
60
0.0775 0.003536
0.16
0.105
60
0.1325 0.03889087
0.02
0.105
60
0.0625 0.060104
0.095
0.145
60
0.12 0.03535534
0.33
0.16
60
0.245 0.120208
0.135
0.12
60
0.1275 0.0106066
0.07
0.2
60
0.135 0.091924
0.095
0.165
60
0.13 0.04949747
0.12
0.035
60
0.0775 0.060104
0.15
0.145
60
0.1475 0.00353553
0.075
0.04
60
0.0575 0.024749
0.28
0.185
60
0.2325 0.06717514
0.02
0.025
60
0.0225 0.003536
0.13
0.16
60
0.145 0.0212132
0.045
0.045
60
0.045
0
0.12
0.15
60
0.135 0.0212132
0.055
0.045
60
0.05 0.007071
0.22
0.2
60
0.21 0.01414214
0.12
0.03
60
0.075 0.06364
0.17
0.33
60
0.25 0.11313708
0.05
0.015
60
0.0325 0.024749
0.145
0.18
60
0.1625 0.02474874
0.095
0.03
60
0.0625 0.045962
0.245
0.26
60
0.2525 0.0106066
0.075
0.045
60
0.06 0.021213
0.17
0.19
60
0.18 0.01414214
0.045
0.075
60
0.06 0.021213
0.18
0.155
60
0.1675 0.01767767
0.085
0.075
60
0.08 0.007071
0.14
0.215
60
0.1775 0.05303301
0.115
0.045
60
0.08 0.049497
0.255
0.255
60
0.255
0
0.085
0.07
60
0.0775 0.010607
0.33
0.22
60
0.275 0.07778175
0.05
0.15
60
0.1 0.070711
0.325
0.24
60
0.2825 0.06010408
0.02
0.01
60
0.015 0.007071
0.175
0.155
60
0.165 0.01414214
0.05
0.015
60
0.0325 0.024749
0.12
0.095
60
0.1075 0.01767767
0.115
0.145
60
0.13 0.021213
0.195
0.235
60
0.215 0.02828427
0.025
0.045
60
0.035 0.014142
0.175
0.17
60
0.1725 0.00353553
0.055
0.05
60
0.0525 0.003536
0.17
0.16
60
0.165 0.00707107
0.05
0.055
60
0.0525 0.003536
0.215
0.27
60
0.2425 0.03889087
0.05
0.035
60
0.0425 0.010607
0.135
0.125
60
0.13 0.00707107
0.025
0.06
60
0.0425 0.024749
0.12
0.135
60
0.1275 0.0106066
0.06
0.095
60
0.0775 0.024749
0.215
0.26
60
0.2375 0.03181981
0.075
0.06
60
0.0675 0.010607
0.16
0.11
60
0.135 0.03535534
0.04
0.03
60
0.035 0.007071
0.105
0.16
60
0.1325 0.03889087
0.11
0.12
60
0.115 0.007071
0.13
0.14
60
0.135 0.00707107
Table 1: Heterozygous forelimb and combined forelimb and hindlimb grip strength
measurements.
34
Trials
Genotype
1 WT
1 WT
1 WT
1 WT
1 WT
1 WT
2 WT
2 WT
2 WT
2 WT
2 WT
2 WT
3 WT
3 WT
3 WT
3 WT
3 WT
3 WT
4 WT
4 WT
4 WT
4 WT
4 WT
4 WT
5 WT
5 WT
5 WT
5 WT
5 WT
5 WT
6 WT
6 WT
6 WT
6 WT
6 WT
6 WT
Mice
Mouse ID Weight (g)
7064 N
7082 R
7083 L
7067 N
9006 N
9007 B
7064 N
7082 R
7083 L
7067 N
9006 N
9007 B
7064 N
7082 R
7083 L
7067 N
9006 N
9007 B
7064 N
7082 R
7083 L
7067 N
9006 N
9007 B
7064 N
7082 R
7083 L
7067 N
9006 N
9007 B
7064 N
7082 R
7083 L
7067 N
9006 N
9007 B
31.43 M
35.22 M
35.42 M
28.55 F
31.08 F
29.61 F
31.43 M
35.22 M
35.42 M
28.55 F
31.08 F
29.61 F
31.43 M
35.22 M
35.42 M
28.55 F
31.08 F
29.61 F
31.43 M
35.22 M
35.42 M
28.55 F
31.08 F
29.61 F
31.92 M
34.65 M
33.58 M
26.95 F
30.17 F
30.94 F
31.92 M
34.65 M
33.58 M
26.95 F
30.17 F
30.94 F
Sex
Forelimb Forelimb Resting
Test #1 Test #2
Time
(kg)
(kg)
(sec)
0.42
0.41
0.03
0.07
0.29
0.18
0.025
0.075
0.015
0.095
0.05
0.075
0.045
0.01
0.02
0.085
0.04
0.02
0.055
0.06
0.08
0.05
0.02
0.025
0.025
0.095
0.135
0.07
0.075
0.105
0.03
0.035
0.07
0.06
0.12
0.135
0.12
0.28
0.03
0.37
0.34
0.11
0.015
0.12
0.04
0.11
0.145
0.095
0.05
0.035
0.015
0.075
0.06
0.04
0.065
0.075
0.035
0.01
0.02
0.05
0.035
0.02
0.02
0.04
0.22
0.07
0.045
0.02
0.025
0.065
0.05
0.095
60
60
60
60
60
60
60
60
60
60
60
60
60
60
60
60
60
60
60
60
60
60
60
60
60
60
60
60
60
60
60
60
60
60
60
60
Forelimb Forelimb
Strength
Strength
Average
and
and
Resting Average
Standard
Standard
Strength
Hindlimb Hindlimb Time
Strength
Deviatio
Deviatio
(kg)
Test #1 Test #2
(sec)
(kg)
n
n
(kg)
(kg)
0.27 0.212132
0.98
0.65
60
0.815 0.233345
0.345 0.091924
0.39
0.4
60
0.395 0.007071
0.03
0
0.43
0.32
60
0.375 0.077782
0.22 0.212132
0.64
0.82
60
0.73 0.127279
0.315 0.035355
0.32
0.71
60
0.515 0.275772
0.145 0.049497
0.36
0.35
60
0.355 0.007071
0.02 0.007071
0.27
0.265
60
0.2675 0.003536
0.0975 0.03182
0.155
0.2
60
0.1775 0.03182
0.0275 0.017678
0.105
0.13
60
0.1175 0.017678
0.1025 0.010607
0.23
0.12
60
0.175 0.077782
0.0975 0.067175
0.105
0.14
60
0.1225 0.024749
0.085 0.014142
0.215
0.18
60
0.1975 0.024749
0.0475 0.003536
0.155
0.425
60
0.29 0.190919
0.0225 0.017678
0.125
0.08
60
0.1025 0.03182
0.0175 0.003536
0.15
0.155
60
0.1525 0.003536
0.08 0.007071
0.22
0.14
60
0.18 0.056569
0.05 0.014142
0.165
0.27
60
0.2175 0.074246
0.03 0.014142
0.145
0.2
60
0.1725 0.038891
0.06 0.007071
0.19
0.29
60
0.24 0.070711
0.0675 0.010607
0.25
0.185
60
0.2175 0.045962
0.0575 0.03182
0.205
0.018
60
0.1115 0.132229
0.03 0.028284
0.24
0.25
60
0.245 0.007071
0.02
0
0.085
0.09
60
0.0875 0.003536
0.0375 0.017678
0.165
0.125
60
0.145 0.028284
0.03 0.007071
0.19
0.185
60
0.1875 0.003536
0.0575 0.053033
0.19
0.185
60
0.1875 0.003536
0.0775 0.081317
0.22
0.145
60
0.1825 0.053033
0.055 0.021213
0.195
0.235
60
0.215 0.028284
0.1475 0.10253
0.18
0.145
60
0.1625 0.024749
0.0875 0.024749
0.17
0.175
60
0.1725 0.003536
0.0375 0.010607
0.105
0.11
60
0.1075 0.003536
0.0275 0.010607
0.16
0.2
60
0.18 0.028284
0.0475 0.03182
0.195
0.205
60
0.2 0.007071
0.0625 0.003536
0.135
0.19
60
0.1625 0.038891
0.085 0.049497
0.225
0.17
60
0.1975 0.038891
0.115 0.028284
0.27
0.195
60
0.2325 0.053033
Table 2: Wildtype forelimb and combined forelimb and hindlimb grip strength
measurements.
35
Trials
Genotype
1 MT
1 MT
1 MT
1 MT
1 MT
1 MT
2 MT
2 MT
2 MT
2 MT
2 MT
2 MT
3 MT
3 MT
3 MT
3 MT
3 MT
3 MT
4 MT
4 MT
4 MT
4 MT
4 MT
5 MT
5 MT
5 MT
5 MT
5 MT
5 MT
6 MT
6 MT
6 MT
6 MT
6 MT
6 MT
Mice
Mouse ID Weight (g)
7075 B
8006 N
8008 L
9005 L
7087 B
9012 N
7075 B
8006 N
8008 L
9005 L
7087 B
9012 N
7075 B
8006 N
8008 L
9005 L
7087 B
9012 N
7075 B
8006 N
9005 L
7087 B
9012 N
7075 B
8006 N
8008 L
9005 L
7087 B
9012 N
7075 B
8006 N
8008 L
9005 L
7087 B
9012 N
29.34 M
32.15 M
32.74 M
34.2 M
32.85 F
30.34 F
29.34 M
32.15 M
32.74 M
34.2 M
32.85 F
30.34 F
29.34 M
32.15 M
32.74 M
34.2 M
32.85 F
30.34 F
29.34 M
32.15 M
34.2 M
32.85 F
30.34 F
28.19 M
31.53 M
32.1 M
35.43 M
30.61 F
29.32 F
28.19 M
31.53 M
32.1 M
35.43 M
30.61 F
29.32 F
Sex
Forelimb Forelimb
Strength
Strength
Forelimb Forelimb Resting Average
and
and
Resting Average
Standard
Standard
Test #1 Test #2
Time Strength
Hindlimb Hindlimb Time
Strength
Deviatio
Deviatio
(kg)
(kg)
(sec)
(kg)
Test #1 Test #2
(sec)
(kg)
n
n
(kg)
(kg)
0.25
0.38
60
0.315 0.091924
0.38
0.23
60
0.305 0.106066
0.23
0.35
60
0.29 0.084853
0.42
0.37
60
0.395 0.035355
0.34
0.43
60
0.385 0.06364
0.37
0.35
60
0.36 0.014142
0.2
0.07
60
0.135 0.091924
0.35
0.48
60
0.415 0.091924
0.17
0.37
60
0.27 0.141421
0.28
0.2
60
0.24 0.056569
0.1
0.1
60
0.1
0
0.31
0.51
60
0.41 0.141421
0.005
0.02
60
0.0125 0.010607
0.15
0.155
60
0.1525 0.003536
0.115
0.235
60
0.175 0.084853
0.205
0.135
60
0.17 0.049497
0.11
0.305
60
0.2075 0.137886
0.18
0.225
60
0.2025 0.03182
0.09
0.04
60
0.065 0.035355
0.105
0.255
60
0.18 0.106066
0.05
0.025
60
0.0375 0.017678
0.34
0.17
60
0.255 0.120208
0.055
0.045
60
0.05 0.007071
0.295
0.145
60
0.22 0.106066
0.075
0.095
60
0.085 0.014142
0.11
0.31
60
0.21 0.141421
0.105
0.125
60
0.115 0.014142
0.25
0.245
60
0.2475 0.003536
0.115
0.04
60
0.0775 0.053033
0.165
0.28
60
0.2225 0.081317
0.055
0.08
60
0.0675 0.017678
0.325
0.27
60
0.2975 0.038891
0.05
0.035
60
0.0425 0.010607
0.205
0.245
60
0.225 0.028284
0.085
0.035
60
0.06 0.035355
0.14
0.295
60
0.2175 0.109602
0.27
0.135
60
0.2025 0.095459
0.32
0.19
60
0.255 0.091924
0.11
0.025
60
0.0675 0.060104
0.105
0.215
60
0.16 0.077782
0.025
0.025
60
0.025
0
0.13
0.075
60
0.1025 0.038891
0.08
0.135
60
0.1075 0.038891
0.22
0.25
60
0.235 0.021213
0.025
0.055
60
0.04 0.021213
0.15
0.12
60
0.135 0.021213
0.08
0.175
60
0.1275 0.067175
0.18
0.15
60
0.165 0.021213
0.135
0.005
60
0.07 0.091924
0.235
0.18
60
0.2075 0.038891
0.075
0.075
60
0.075
0
0.17
0.16
60
0.165 0.007071
0.12
0.09
60
0.105 0.021213
0.31
0.115
60
0.2125 0.137886
0.125
0.1
60
0.1125 0.017678
0.225
0.195
60
0.21 0.021213
0.14
0.09
60
0.115 0.035355
0.23
0.135
60
0.1825 0.067175
0.185
0.195
60
0.19 0.007071
0.125
0.175
60
0.15 0.035355
0.05
0.03
60
0.04 0.014142
0.12
0.18
60
0.15 0.042426
0.08
0.05
60
0.065 0.021213
0.255
0.135
60
0.195 0.084853
0.075
0.075
60
0.075
0
0.24
0.23
60
0.235 0.007071
0.085
0.09
60
0.0875 0.003536
0.27
0.29
60
0.28 0.014142
0.065
0.065
60
0.065
0
0.175
0.16
60
0.1675 0.010607
Table 3: Mutant forelimb and combined forelimb and hindlimb grip strength
measurements.
Genotype
WT
MT
HT
Avg Forelimb (kg)
Avg Forelimb+Hindlimb (kg)
Start Weight (g)
End Weight (g)
Difference (g)
0.09
0.24
31.89
31.37
0.52
0.12
0.23
31.94
31.2
0.74
0.09
0.22
36
34.88
1.12
Table 4: Correlation of genotype, grip strength measurements and weight in mice.
Forelimb and Combined Forelimb and Hindlimb Grip Strength Results
Figure 13 contains a bar graph that shows a comparison between the average
forelimb measurements of the heterozygous, homozygous mutant and wild-type mice.
The force is produced during the pull on the grid. The heterozygous mice averaged 0.09
kg, homozygous mutant averaged 0.11kg, and the wild-type averaged 0.09kg. The
average of the combined forelimb and hindlimb measurements can be seen in Figure 14.
36
In Figure 14, the heterozygous mice averaged 0.22kg, homozygous mutant averaged
0.23kg, and the wild-type averaged 0.24kg.
Figure 13: Average forelimb measurements of heterozygous, homozygous mutant and
wild-type mice.
Figure 14: Average forelimb and hind limb measurements of heterozygous, homozygous
mutant and wild-type mice.
Three separate bar graphs were created to compare the average forelimb grip
strength measurements of the three groups. In Figure 15, the forelimb measurements of
37
the heterozygous and mutant mice were compared. The p-value was 0.16, which means
that there is a trend towards correlation, but the correlation is not statistically significant
between the heterozygous and mutant group. In trial five, the mutant mice averaged 0.1
kg, whereas the heterozygous mice averaged approximately 0.06 kg.
Forelimb: Heterozygous Mice and
Mutant Mice
0.3
Kilograms (kg)
0.25
0.2
0.15
HT Forelimb
0.1
MT Forelimb
0.05
0
1
2
3
4
5
6
Trial
Figure 15: Average forelimb measurements of the heterozygous and mutant mice. The xaxis represents each of the six trial periods, and the y-axis represents the muscle force
(kg) of the mice in grasping the metal grid on the force transducer.
The average forelimb measurements of the heterozygous and wild-type mice were
compared against each other in Figure 16. The p-value was 0.56, which means that there
is no significant difference between the heterozygous and wild-type groups. The main
difference is seen in trial two (heterozygous 0.11kg, wild-type 0.07kg).
38
Forelimb: Heterozygous Mice and
Wildtype Mice
0.3
Kilograms (kg)
0.25
0.2
0.15
HT Forelimb
0.1
WT Forelimb
0.05
0
1
2
3
4
5
6
Trial
Figure 16: Average forelimb measurements of the heterozygous and wild-type mice. The
x-axis represents each of the six trial periods, and the y-axis represents the muscle force
(kg) of the mice in grasping the metal grid on the force transducer.
The average forelimb measurements of the mutant and wild-type mice were
compared in Figure 17. The p-value was 0.06, which means that there could be a
correlation between the mutant and wild-type mice but perhaps the correlation is not
significant. The main difference is seen in trial four (mutant 0.09kg, wild-type 0.05kg).
Forelimb: Mutant Mice and Wildtype
Mice
0.3
Kilograms (kg)
0.25
0.2
0.15
MT Forelimb
0.1
WT Forelimb
0.05
0
1
2
3
4
5
6
Trial
Figure 17: Average forelimb measurements of the mutant and wild-type mice. The xaxis represents each of the six trial periods, and the y-axis represents the muscle force
(kg) of the mice in grasping the metal grid on the force transducer.
39
Three other bar graphs were created to compare the average combined forelimb
and hindlimb grip strength measurements of the three groups. In Figure 18, the average
combined forelimb (F) and hindlimb (H) measurements of the heterozygous and mutant
mice were compared against each other. In this case, all-mouse strength were measured.
The p-value was 0.60, which means that there is no significant difference between the
two heterozygous and mutant groups. The main difference is seen in trial one, where the
heterozygous mice averaged approximately 0.41 kg, whereas the mutant mice averaged
approximately 0.35 kg.
Forelimb + Hindlimb: Heterozygous
Mice and Mutant Mice
Kilograms (kg)
0.5
0.4
0.3
HT F+H
0.2
MT F+H
0.1
0
1
2
3
4
5
6
Trial
Figure 18: Average combined forelimb and hind limb measurements of the heterozygous
and mutant mice. The x-axis represents each of the six trial periods, and the y-axis
represents the muscle force (kg) of the mice in grasping the metal grid with all four limbs
on the force transducer.
The average combined forelimb and hindlimb measurements of the heterozygous
and wild-type mice were compared against each other in Figure 19. The p-value was
0.56, which means that there is no significant difference between the heterozygous and
wild-type groups. The main difference is seen in trial one (heterozygous 0.41kg, wildtype 0.53kg). The average for trial one in the heterozygous group was higher (0.41kg)
40
and the averages for trial two, three, four and five were lower (0.15kg, 0.19kg, 0.22kg,
0.18kg, 0.15kg).
Forelimb + Hindlimb: Heterozygous
Mice and Wildtype Mice
0.7
Kilograms (kg)
0.6
0.5
0.4
0.3
HT F+H
0.2
WT F+H
0.1
0
1
2
3
4
5
6
Trial
Figure 19: Average combined forelimb and hindlimb measurements of the heterozygous
and wild-type mice. The x-axis represents each of the six trial periods. The y-axis
represents the muscle force (kg) of the mice in grasping the metal grid using all four
limbs.
The average combined forelimb and hindlimb measurements of the mutant and
wild-type mice were compared in Figure 20. The p-value was 0.60, which means that
there is no significant difference between the mutant and wild-type groups. The main
difference is seen in trial one (mutant 0.35kg, wild-type 0.53kg). The average for trial one
in the wild-type group was higher (0.53kg) and the averages for trial two, three, four and
five was lower (0.18kg, 0.19kg, 0.17kg, 0.18kg, 0.18kg).
41
Forelimb + Hindlimb: Mutant Mice
and Wildtype Mice
0.7
Kilograms (kg)
0.6
0.5
0.4
0.3
MT F+H
0.2
WT F+H
0.1
0
1
2
3
4
5
6
Trial
Figure 20: Average combined forelimb and hindlimb measurements of the mutant and
wild-type mice. The x-axis represents each of the six trial periods, and the y-axis
represents the muscle force (kg) of the mice in grasping the metal grid with all four limbs
at once.
42
Chapter 4: Discussion
Grip Strength Tests
The results presented in Chapter 3 did not reveal a statistically significant (p-value
0.05) difference between the groups. The bar graphs that represented the averages of the
grip-strength tests over the course of the six trial periods, did not disclose that the mutant
mice showed progressive muscle weakness compared to the wild-type and heterozygous
groups. I expected the mutant mice to show significantly reduced grip-strength, which is
why I hypothesized that the mutant group would perform significantly worse than both
the heterozygous and the wild-type groups. As shown in Figure 17, this was not the case.
In Figure 17, the mutant group in all six trials had slightly higher forelimb grip-strength
than the wild-type. The p-value in Figure 17 was 0.06, which means that there was a
trend towards correlation between the mutant and wild-type mice but perhaps the
correlation was not statistically significant. This was not expected as the mutant mice
were predicted to show evidence of muscle wasting and the results indicate otherwise.
However, the results may not be entirely conclusive, because some of the mice were
studied when they were too young for measurement. This is because two 26 week old
mice were included in the study as no more 42 plus week mutant mice were available to
have a mutant group of six.
In order to further evaluate muscle weakness, the three groups were weighed
before the start of trial one and at the end of trial six. When looking at the difference
between the starting weight and the ending weight (See Table 4) all three groups had
slightly lost weight but the results were not statistically significant. The similar values
between the mutant group and the wild-type group indicated that the homozygous mice
43
did not show any muscle loss as a result of the M712T mutation. The similar numbers
seen in all three groups also suggested that the small loss in weight was not affected by
the M712T mutation or the use of the grip strength meter itself.
Interestingly, all the bar graphs in this experiment showed higher, and in some
cases double, the grip strength measurements in the first trial compared to the other five
trials. The results are contrary to the idea that during the first trials, the mice sometimes
display an initial period of little participation (De Luca, 2009). To prevent this, the mice
in this experiment were exposed to the grip strength meter a week prior to the recorded
first trial. The tests thereafter were repeated no more than twice per week to avoid
habitual bias. Perhaps, the scores were higher in the first trial period because the mice
were then tired of practicing on the grip-strength meter, and made less of an effort in the
five subsequent trials. Environmental factors such as unusual noises and unexpected
movements are another explanation for higher measurements in the first trial for all
testing groups. For example, perhaps the other rodents in other modules were making
loud noises which could have made the mice anxious and therefore resulted in greater
grip-strength. Sudden movements from the other mice in the same module, who were not
being tested, also could have startled the mice being tested giving rise to greater gripstrength in the first trial. Given that there was a trend towards correlation between the
mutant group having higher limb measurements compared to the wild-type (control)
group, it can be inferred that the mice homozygous for the M712T mutation did not show
evidence of muscle wasting. However, based on the investigators subjective observations
of the mice, the homozygous population had significantly lethargic activity when
44
compared to the heterozygous and wild-type groups. Nonetheless, these differences were
not illustrated by the grip-strength measurements.
Some of the homozygous GneM712T/M712T mice in the study showed signs of
kidney abnormalities. One homozygous mouse in particular (7080R), who was not
included in the trials, was observed to be extremely lethargic and double the body size of
the wild-type mice (See Appendix I, II). Following sacrifice, upon dissection of the
mouse, the gross anatomy of the kidneys appeared swollen with small red dots (See
Appendix III, IV). Such small red dots have been identified as surface petechial
hemorrhages in other studies related to M712T mouse models (Kakani et al., 2012). The
mouse also had tumors and vast amounts of serum within the body cavities. In another
study by Huizing et al. (2013), the M712T mutation was created in exon 12, and a neo
cassette (under the PGK promoter) flanked by flippase recombinase target (FRT) sites
was inserted. LoxP sites were inserted before exon 12 and after the PGK-neo gene. When
nine pairs of the GNE heterozygous mice were mated, 101 offspring were obtained. Of
the 101 mice 26 homozygous mutated mice were produced. However, only one male with
the homozygous genotype survived and showed no muscle pathology. Instead of showing
muscle pathology, signs of severe glomerular hematuria and podocytopathy, including
effacement of the podocyte foot processes and segmental splitting of the glomerular
basement membrane (GBM) were evinced. Huizing and colleagues speculated that the
kidney disorder is due to hyposialylation of specific membrane glycoproteins. This would
demonstrate the significance of sialic acid synthesis in kidney development and function.
Future Directions
45
If this or similar mouse models of HIBM are to be tested in the future, several
experimental changes should be considered. Future studies should involve a different
grip-strength meter that has the ability to easily zero in quickly and reliably. Another idea
would be to use a pull bar instead of a grid. A very thin bar is not recommended because
the required grasp might be too tight. A thick bar might have resulted in weak gripstrength values. The most appropriate bar would be 1-2mm in diameter and composed of
non-flexible metal, allowing an efficient grasp that can be easily broken by the operator
at the end of each trial (De Luca et al., 2009). Also a different protocol with more than
six trial periods would be recommended. This would allow for more trends to appear on
bar graphs.
For more accurate evaluation at least six to eight mice per group are generally
needed if statistical significance is to be reached (De Luca et al., 2009). In this study six
mice per group were used because the smallest group, the homozygotes, had only six
individuals. Breeding the mice to generate homozygous mutants was one of the major
challenges I encountered when conducting this study. This was not surprising because
homozygous mice died earlier than other mice in previous studies. Sometimes the mice in
the present study did not mate with each other, or they seemed uninterested in doing so.
Sunflower seeds were sometimes placed in the breeding cages to encourage reproduction.
The small litter sizes were most likely due to deficiency in sialic acid as identified in
prior research by Galeano et al. (2007) and Valles-Ayoub et al. (2013). Mutant pups
were also observed to die sooner after birth than pups with other genotypes. As a result of
not having enough homozygous mutant mice, the mutant group was not age-matched.
Two homozygous mice were 26 weeks old and the other four were about 42 weeks old.
46
Having two younger mutant mice in the study might explain why the grip-strength values
were comparable and in some cases slightly higher than the wild-type values. Also, it is a
possibility that 42 weeks might be too late to accurately reflect the human disorder in the
M712T homozygous mice. Instead, the mice should be tested at 20 and 31 weeks which
would be more similar to the human version of the disorder when age of onset is
adolescence or early adulthood. In the future, to increase litter sizes, breeding cages
should contain one male heterozygote and two female heterozygotes. The females can
later be separated from males, in case the male mice are aggressive and destructive
towards the pups. It would be interesting to see whether increasing the group size of the
colony and having age matched groups would alter the experimental results.
Additional tests should be part of any prospective experiment regarding the mice
homozygous for the M712T mutation. Histological tests should be used to determine if
the mice suffer from the same muscle wasting pattern as humans. Mouse tissues should
be collected, stained with Hematoxylin and eosin (H&E) following standard procedures
(American Histolabs) and then viewed under a microscope. H&E-stained muscle tissues
of the GneM712T/M712T mice and the wild-type and homozygous littermates should be
compared for histological differences. Observations should also be confirmed at an
ultrastructural level to validate the presence of rimmed vacuoles in muscles similar to
those of individuals affected with HIBM. Finally, mice in poor health or those showing
signs of inadequate self-care could be studied by analysis of organ necropsies, focusing
on the gross anatomy of the kidney. The serum of these mice should also be tested for
levels of creatine and muscle enzymes (creatine kinase and lactate dehydrogenase). Also,
47
testing sera for immunoglobulin levels should rule out the involvement of inflammation
in the phenotype.
The genotyping method should be improved in order to generate more reliable
data in future experiments. During the genotyping procedure, the bands were not as clear
and bright as they could have been. For determining the cause of the unclear bands,
different conditions from my own should be conducted in the future. For example,
genotyping could be improved by changing PCR reaction conditions to improve PCR
efficiency, and by decreasing voltage during electrophoresis.
Conclusion
The aim of this project was to determine whether this particular M712T mouse
animal model, reflects the HIBM disorder well enough to allow testing of different
treatments. I conducted grip-strength tests to assess muscle force between wild-type,
heterozygous, and homozygous mice. Specifically, this research study was conducted in
order to measure progressive muscle weakness as a primary phenotype screen in mice
that are homozygous for the M712T allele. The results of the grip-strength meter did not
show that the homozygous mice had decrease grip strength compared to the heterozygous
and homozygous groups. Instead, in some graphs the opposite was shown where the
mutant mice had slightly higher grip-strength compared to the wild-type group.
Unexpectedly, some of the diseased/dissected homozygous mice also showed signs of
abnormal kidneys. The grip-strength protocol could be improved upon or there simply is
no progressive muscle weakness in the homozygous M712T mice when studied at those
ages. In conclusion, if an adequate mouse model for HIBM is discovered, it could be used
in future experiments to assess various treatment options for the disorder.
48
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Appendix I
Photograph of the ventral view of the homozygous mouse (7080R).
57
Appendix II
Photograph of the cranial view of the homozygous mouse (7080R).
58
Appendix III
Gross anatomy of the homozygous (7080R) mouse kidney still attached to body.
59
Appendix IV
Photograph of both 7080R mouse’s kidneys.
60