identifying and phenotyping an enu derived mouse model for … · 2013-11-07 · master’s of...
TRANSCRIPT
Identifying and Phenotyping an ENU Derived Mouse Model for MYH9-Related Disease
by
Elizabeth Sara Lefebvre Berndl
A thesis submitted in conformity with the requirements for the degree of Master’s of Applied Science
Graduate Department of the Institute of Biomaterials and Biomedical Engineering
University of Toronto
© Elizabeth Sara Lefebvre Berndl (2012)
ii
Identifying and Phenotyping an ENU Derived Mouse Model of MYH9-Related Disease
Master’s of Applied Science, 2012
Elizabeth Sara Lefebvre Berndl
Institute for Biomaterials and Biomedical Engineering
University of Toronto
Abstract
A dominant ENU screen produced mouse line 7238 with large platelets. Sequence capture and
Next Generation sequencing identified a mutation in Myh9 at Q1443L [1]. Mice were tested for
aspects of MYH9-Related Disease (MYH9RD), a rare human condition caused by mutations
within MYH9; macrothrombocytopenia and neutrophil inclusions are found in almost all cases,
while deafness, cataracts and renal disease have variable penetrance and severity.
Myh9Q1443L/+
and Myh9Q1443L/Q1443L
animals have neutrophil inclusions [1] and increased cataracts
at 2, 6 and 12 months; Myh9Q1443L/Q1443L
animals at 12 months have changes in kidney output [2].
Immunofluoresence showed changes in protein expression in glomeruli at two months.
This is the first ENU mouse model identified by a sequence capture mechanism, and the first
mouse line to produce a point mutation within the Myh9 gene [1,2]. This mouse models
MYH9RD, and is an invaluable tool to understand the role of this protein, and to determine
mechanisms underlying this disease.
iii
Acknowledgements
To my PI, Dr. Bill Stanford who provided support, suggestions, and corrections throughout my
research and writing,
To Dr. Dwayne Barber, who was available to give advice and direction on this project while Bill
was on sabbatical,
To Dr. Robert Harrison, and Dr. Trecia Brown, who did the initial ABR analysis on so many
mice,
And do my husband, David Calabrese, who has always supported me, loved me and kept me
going when I wanted to give up,
Thank you all so very much. Without you, I wouldn’t have made it through.
Lizz.
iv
Table of Contents
Abstract ........................................................................................................................................... ii Acknowledgements ........................................................................................................................ iii Table of Contents ........................................................................................................................... iv List of Tables ................................................................................................................................. vi
Abbreviations ................................................................................................................................. ix 1 Overview .....................................................................................................................................1 2 Background .................................................................................................................................3
2.1 N-Ethylnitrosourea (ENU) Mutagenesis..............................................................................3
2.2 Haematopoiesis and thrombopoiesis....................................................................................5 2.3 Thrombocytopenia ...............................................................................................................8
2.4 Giant Platelet Disorders .......................................................................................................9 2.5 MYH9 .................................................................................................................................10
2.5.1 MYH9 Structure and Function ...............................................................................10 2.5.2 MYH9 Related Disease (MYH9RD) .....................................................................12 2.5.3 Platelets and MYH9 ................................................................................................15
2.5.4 Neutrophils and MYH9 ..........................................................................................15 2.5.5 Cochleae and MYH9 ..............................................................................................16
2.5.6 Kidneys and MYH9 ................................................................................................18 2.5.7 Lenses and MYH9 ..................................................................................................22 2.5.8 Cellular and Mouse Models of Myh9.....................................................................24
2.6 Genome Sequencing ..........................................................................................................26
2.6.1 Sanger Sequencing .................................................................................................26
2.6.2 Next Generation Sequencing .................................................................................26 2.6.3 Sequence Capture Techniques ...............................................................................28
3 Objectives and Hypotheses .......................................................................................................29 4 Materials and Methods ..............................................................................................................30
4.1 ENU mutagenesis and line creation ...................................................................................30
4.2 Haematological testing of mice .........................................................................................30 4.3 Next Generation Sequencing .............................................................................................30
4.4 Gene Expression Analysis .................................................................................................31 4.5 Tail Bleed assay .................................................................................................................31
4.6 Neutrophil Isolation and Staining ......................................................................................31 4.7 Auditory Brainstem Response ...........................................................................................32 4.8 Tissue Sectioning ...............................................................................................................33 4.9 Immunofluorescent staining of tissues...............................................................................33 4.10 Statistics .............................................................................................................................33
v
5 Results .......................................................................................................................................34
5.1 Establishment and Phenotyping of the 7238 Mouse line ...................................................34 5.2 Next Generation Sequencing Identified Two Novel ENU Mutations in the 7238
Mouse Line ........................................................................................................................35
5.3 The T77599238A Mutation in the Myh9 Gene Causes a Q1443L Mutation in the
Associated NMHCIIA Protein within a Highly Conserved Region and Causes a
Change in Amino Acid Class .............................................................................................40 5.4 The C77710527A Mutation between the Myh9 and Txn2 Genes does not Effect
Expression Levels of Either Gene......................................................................................42
5.5 Myh9Q1443L
Mice have Neutrophil Inclusions and Increased Bleeding Tendancy .............43 5.6 Myh9
Q1443L Mice do not Exhibit Changes in Hearing ........................................................45
5.7 Myh9Q1443L
Mice have Increased Serum Urea and Creatine Levels, Highly Increased
Rate of Cataract Formation, and Decreased Body Fat Percentage ....................................47 5.8 Optimization of Staining Protocol for α-NMHCIIA ..........................................................49 5.9 Myh9
Q1443L Mice have Altered Kidney Morphology and Expression Patterns in
NMHCIIB ..........................................................................................................................57
6 Discussion .................................................................................................................................59
7 Conclusions and Future Directions ...........................................................................................66 8 References .................................................................................................................................68
vi
List of Tables
Table 2-1: Known Myh9 Mutations in Humans ........................................................................... 14
Table 5-1: Loci of interest found via sequence selection and next generation sequencing of four
7238 animals. ................................................................................................................................ 39
Table 5-2: Primer sets for Sanger Sequencing .............................................................................. 40
vii
List of Figures
Figure 2-1: N-ethyl-N-nitrosourea (ENU) and ENU screening. ..................................................... 4
Figure 2-2: Simplified differentiation path for the haematological lineage. .................................. 7
Figure 2-3: Basic structure of Class II myosins. ........................................................................... 11
Figure 2-4: A family with an undisclosed Myh9 mutation. .......................................................... 13
Figure 2-5: Hearing and the cochlea ............................................................................................. 17
Figure 2-6: Excretion and the Kidney ........................................................................................... 20
Figure 2-7: Structure of the Human Eye and Lens ....................................................................... 23
Figure 2-8: Solexa sequencing. ..................................................................................................... 27
Figure 2-9: NimbleGen’s Sequence capture technology ............................................................. 28
Figure 5-1: Phenotyping of the 7238 line. .................................................................................... 35
Figure 5-2: The region of interest for the 7238 line. ..................................................................... 37
Figure 5-3: Identifying sites for further study on the 7328 line .................................................... 38
Figure 5-4: The Myh9 mutation found in the 7238 line is novel, and produces an amino acid
change in a highly conserved region. ............................................................................................ 41
Figure 5-5: The mutation in the Txn2-Myh9 intergenic region is unlikely to cause the 7238
phenotype. ..................................................................................................................................... 43
Figure 5-6: Additional Hematopoietic phenotypes in Myh9Q1443L
mice ....................................... 44
Figure 5-7: Minimum hearing thresholds of female mice. ........................................................... 46
Figure 5-8: Renal, Optical and Morphological phenotypes. ......................................................... 48
Figure 5-9: α-NMHCIIA antibody optimization .......................................................................... 50
viii
Figure 5-10: Blocking optimization .............................................................................................. 51
Figure 5-11: Antigen retrieval optimization ................................................................................. 52
Figure 5-12: Tissue preparation optimization. .............................................................................. 53
Figure 5-13: Tissue preparation optimization (cont) .................................................................... 55
Figure 5-14: α-NMHCIIB optimization ........................................................................................ 56
Figure 5-15: Kidney morphology and protein expression in Myh9Q1443L
mice. ........................... 58
ix
Abbreviations
129 – 129S1/SvImJ ..................................... 8
ABR – Auditory Brainstem Response ...... 25
B6 – C57BL/6J ........................................... 8
BAC – Bacterial Artificial Chromosome .. 34
CLP – Common Lymphoid Progenitors ... 11
CMP – Common Myeloid Progenitors ..... 11
DMS – Demarcation Membrane System .. 12
ENU – N-ethylnitrosourea .......................... 7
EPO – Erythropoietin ................................ 12
FSGS – Focal Segmental
Glomerulosclerosis ................................ 28
GSA - Goat Serum Albumin ..................... 55
HSC – Haematopoietic Stem Cell............. 11
MEP – Megakaryocyte-Erythroid Progenitor
............................................................... 12
MK – Megakaryocyte ............................... 11
MPV - Mean Platelet Volume................... 14
MYH9RD – MYH9-Related Disease ......... 7
NBF - Neutral Buffered Formalin............. 55
NMHCIIA – Non-Muscle Myosin Heavy
Chain IIA ............................................... 15
SNP – Single Nucleotide Polymorphism .... 8
TPO – Thromopoietin ............................... 12
TTFD – Temporarily Terminated,
Fluorescently Labelled dNTP ................ 34
WT – Wild Type ....................................... 32
1
1 Overview
The original goal of this project was to use N-ethylnitrosourea (ENU) mutagenesis to discover
novel proteins or genes involved in the maintenance and control of multi-lineage hematopoietic
cells. These proteins would then, in turn, be used to aid in the discovery and development of
novel drugs and treatments that could be used in a variety of clinical settings, from heart attack
and stroke prevention, to reducing the immunological effects of chemotherapy.
The Stanford lab has successfully produced four mouse lines with highly penetrant and heritable
phenotypes: AnkE924X
which produces a truncated protein, leading to increased red blood cells in
the heterozygous mice, and severely decreased RBCs in homozygous mice [3]; Jak2K915X
also
produces a truncated mutation, leading to increased platelet levels in heterozygous mice, and is
homozygous lethal at approximately E14.5, a full day later than null Jak2 mice [4]; 7238, the
main topic of this report, was identified as having thrombocytopenia in the heterozygous mice,
and severe thrombocytopenia in the homozygous mice; and 7325 was found to have enhanced
platelet recovery following chemotherapy.
The minimum region of interest for 7238 was a 1.7Mb region of chromosome 15, which
included the first exon and the regulatory region of Myh9, a type II non-muscle myosin involved
in proplatelet extension. This exon had previously been sequenced and found to have no
mutations, therefore it was posited that the mutation may be present in the regulatory region of
Myh9.
The 7238 mouse line was later identified to have macrothrombocytopenia, with normal numbers
of platelets in the heterozygous mice, and a decrease in quantity in the homozygotes. In humans,
mutations in the MYH9 gene are known to cause macrothrombocytopenia [5-10], and a group of
symptoms which are together referred to a MYH9-Related Diseases (MYH9RD) [11].
Next generation sequencing was used to sequence the minimum region of interest as well as the
entire Myh9 gene, a total of 4.25Mb. From this, 275 novel variants were found, 19 of which
were located in the exons or the exon borders. Only one of these sites caused an amino acid
change – Q1443L in Myh9. This site was the only one of the 19 found to be a true ENU
mutation; all the others were novel single nucleotide polymorphisms (SNPs) between the
2
C57BL/6J (B6) and 129S1/SvImJ (129) strains [1]. Further crossovers indicated that only one of
the 256 intronic or intergenic variants was still within the region of interest, found in the region
between Txn2 and Myh9. QPCR analysis indicates that this mutation does not affect the
regulation of Txn2.
The 7238 mouse line was found to be caused by a Myh9Q1443L
mutation, and is a potential model
for MYH9RD. There are more than 40 known human mutations in MYH9, all but one of which
cause macrothrombocytopenia and neutrophil inclusions visible by immunofluorescence. Also
present at varying penetrances are cataracts, sensorineural deafness, and nephritis.
Therefore, this mouse line is potentially capable of modelling all aspects of MYH9RD, providing
a novel tool to determine how this protein causes the observed phenotypes.
3
2 Background
2.1 N-Ethylnitrosourea (ENU) Mutagenesis
ENU (Figure 2-1A) has been used as a mutagen in both plants [12] and animals [13] since at
least 1964, causing sterility in Arabidopsis thalana (mustard) seeds [12], and mutagenic and
teratogenic effects in rats exposed to ENU in utero [13]. In 1979, ENU was first used to induce
mutations in the sperm of mice, and was found to be approximately 87 times the spontaneous
mutation rate [14]. This mutagen has since been used extensively in the Drosophila, C. elgans,
and Danio rerio (zebra fish) [15-20], as well as in the mouse [21-25].
ENU is an alkylating agent, capable of transferring its active ethyl group to a reactive nitrogen or
oxygen on the base of a nucleotide. Because they each have three reactive groups, adenines and
thymines are more likely to be affected than cytosine and guanine. An analysis of 62 known
mutations in 1999 found that 87% of known ENU mutations occurred at an A/T pairing, most
commonly being transversions (to T/A, 44%), and transitions (to G/C, 38%) [22].
The mutation rate of ENU is dependent upon the dose injected into the male: linear up to a single
dose of 100mg/kg, and then plateauing at 200mg/kg [26]. However, multiple doses at lower
concentrations have been found to both reduce the fatality rate of the males, and increase the
mutation load to up to 2.2 times that seen at a single 250mg/kg dose [25]. At a common ENU
dose of 2x100mg/kg [28], the mutation rate has been reported to be 1 mutation in 1.01 x106 base
pairs, and the functional mutation rate at 1 in 1.82 x 106 base pairs [27]. Silent mutations,
conservative and non-conservative missense mutations, truncation mutations, loss of stop
mutations, and splicing mutations have all been observed due to ENU mutagenesis because it
produces point mutations, which can cause all of the above effects [22,27].
4
Figure 2-1: N-ethyl-N-nitrosourea (ENU) and ENU screening.
A) Chemical structure of ENU. The reactive ethyl group at the bottom of the structure
binds to a reactive nitrogen or oxygen in DNA, causing a point mutation. B) A dominant
screen was used, as shown. Male F0 mice are injected with ENU, and bred to females of a
different strain. G1 mice of interest, shown with red stars, are bred to WT mice of the F0
female strain. Mice of interest in later generations are bred to mice of the F0 female
strain, and their DNA is sent for genetic mapping. C) Genetic mapping uses known
polymorphisms between two mouse strains. In the example shown here, the mutation
must lie between SNP 4 and SNP 6, where all five mice could be heterozygous.
A typical dominant ENU screen involves mutagenizing the sperm of male mice of one strain, and
breeding to females of another strain in the F0 generation (Figure 2-1B). G1 offspring are
screened for phenotypes of interest, and carriers are backcrossed to the F0 female strain to
determine heritability and penetrance. For generations G2 and beyond, DNA of phenodeviants
undergoes a genomic panel of SNPs and microsatellites to determine the region of interest in
5
which the mutation causing the phenotype is located (Figure 2-1C). The F0 male is both the
source of the mutation and the only source of non-F0 female DNA. Because of this,
phenodeviants will have overlapping areas which are heterozygous for the two parental strains
indicating the region of interest. As the region of interest is specified, additional variations,
usually SNPs, are inserted between the microsatellites, as they are generally more frequent. In
Figure 2-1C, the region of interest is between 4 and 6, as it is possible that the mutation is
located several base pairs after the polymorphism at 4, or several before the one at 6.
Although ENU mutagenesis is random, it is also possible to screen for a series of mice with
mutations within the same gene. This technique sequences the gene of interest at the G1 stage
rather than completing a physiological screen [22,29]. By producing an allelic series of
mutations within a particular gene, it is possible to determine the relationship between mutation,
structure, and dysfunction. This is a particularly useful system when a protein is present in
multiple tissues or organ systems, allowing the net effect of a mutation to be observed at the
organism level.
2.2 Haematopoiesis and thrombopoiesis
Haematopoiesis is the process of producing the cellular component of blood. This process takes
place primarily in the medulla (bone marrow), but can also occur in the spleen and the fetal liver,
where the haematopoietic stem cells (HSC) are located, and involves the production of common
lymphoid progenitors (CLP) and common myeloid progenitors (CMP). The CLP produces B-
cells, T-cells and Natural Killer cells – the primary components of the adaptive immune system.
The CMP differentiate into erythrocytes, granulocytes, and megakaryocytes, which are
responsible for oxygen transport, innate immunity, and blood clotting, respectively.
Thrombopoiesis is the process of shedding platelets, or anuclear thrombocytes, from
megakaryocytes (MKs), and is specific to mammals. This process, like all haematopoiesis, takes
place primarily in the bone marrow, but MKs are also found in the lungs, liver, kidney, and heart
[29]. During differentiation from HSC, these cells pass through the CMP stage, and then, due to
up-regulation of the transcription factor GATA-1 and down-regulation of PU.1, differentiate into
6
MK-erythroid progenitors (MEPs) [30]. The ultimate fate of the MEP is determined by the
presence of one of two cytokines: erythropoietin (EPO) drives red blood cell production, while
thromopoietin (TPO) induces the cells to differentiate into MKs [29-31].
Exposure to TPO drives MEPs to differentiate into megakaryoblasts, which can be classified as
either burst-forming units (BFU-MK), or the more mature colony forming units (CFU-MK) [32].
At this stage megakaryoblasts lose the ability to divide, while retaining the ability for DNA
replication, a process found exclusively in MKs called endomitosis. During each round of
endomitosis, the quantity of DNA in the maturing MK doubles – with 2N to 4N in immature
MKs, and a mode of 16N in mature MKs, although some MKs have been recorded to have as
much as 128N [30,33]. As the MK undergoes endomitosis, the cystolic volume increases rapidly
and it produces the proteins required for platelet function – including platelet gylcoproteins and
clotting factors [30] – as platelets lack significant protein synthesis [29].
The shedding of platelets produces 2000 to 5000 platelets from each MK and involves the
formation of the demarcation membrane system (DMS) [34], followed by budding of the
proplatelets [35]. The DMS is formed by the invagination of the plasma membrane, forming
“pre-packages” of platelets, containing all the required proteins, organelles, clotting factors, and
other components required for proper function [34]. Once assembled, these packages are
transferred to the outer membrane of the MK by microtubules, and the DMS is evaginated,
forming pseudopods [34]. These pseudopods grow into branched structures, called proplatelets,
providing an increased number of sites where platelets can shed from the structure. As the
proplatelets grow, additional packages of platelet components are shifted out from the main
volume of the MK by the microtubules [34]. To release the proplatelets into the blood stream, the
microtubules twist back on themselves, and pinch off the platelets [35].
7
Figure 2-2: Simplified differentiation path for the haematological lineage.
CLP differentiates into the components of the adaptive immune system. Myeloblasts
differentiate into the components of the innate immune system. MEPs differentiate into
thrombocytes and erythrocytes, the anuclear haematological components.
Platelet differentiation takes about four to five days, from HSC to mature platelet [33]; in times
of extreme stress or after large quantities of blood loss, differentiation can occur within 24 to 48
hours [30]. Differentiated MKs exist in the bone marrow, releasing platelets into the blood at the
sinusoidal lumen. There is also some evidence that proplatelet separation can occur in the
bloodstream, and that some MKs migrate to the lung, become lodged in the alveolar capillary
beds, and complete shedding there [32]. Although some platelets are consumed in hemostatic
maintenance, most platelets survive for seven to 10 days in the blood. Those that remain are
identified via an unknown mechanism for destruction by the macrophages in the spleen.
Severely damaged MKs can also be removed by hepatic macrophages [36].
8
2.3 Thrombocytopenia
Thrombocytopenia occurs when there is a below normal concentration of platelets in the blood.
For humans, normal ranges are 150 000 to 450 000/µL, and with a mean platelet volume (MPV)
of 7 to 10.5fL [37]. Observable effects of thrombocytopenia include increased bleeding and
bruising, such as nose bleeds, bleeding gums or menorrhagia (long or heavy menstrual bleeding)
when the count decreases to the 80 000 to 100 000/µL range, and below 10 000/µL spontaneous
bleeding, often in the form of purpura (pin-point haemorrhages), can be observed in the skin [37-
39].
Thrombocytopenia is due to either decreased platelet production, or increased platelet removal,
and can be either acquired or inherited. Acquired cases of thrombocytopenia can be due to
antibodies that develop in the blood, diseases such as HIV/AIDS or von Willebrand Disease, or
various drugs [39]. Antibodies against platelets have been known to develop shortly after a viral
infection in children; the theorized mechanism is that viral proteins cross-react with a platelet
surface antigen. AIDS patients also develop autoimmune antibodies that can target platelets, as
can individuals with Hepatitis C [40]. Drugs such as heparin and chemotherapeutics can cause
transient thrombocytopenia to develop. Chemotherapeutics target rapidly dividing cells, such as
HSCs and platelet levels will return to normal only after the drug is stopped [41,42]. Both type I
and type II Heparin induced thrombocytopenia causes an increased rate of platelet destruction;
type I patients have their platelet levels return to normal while still on the drug, while type II
individuals present with thrombocytopenia until the drug stimulus is removed [43].
Thrombocytopenia is also common in the third trimester of pregnancy, with an average 10%
decrease in platelet density [44], however it is possible that this is a physiological change related
to pregnancy, rather than disease. Pregnant women who have thrombocytopenia and increased
platelet volume during pregnancy are at an increased risk of pre-eclampsia and hypertension
[37].
Inherited thrombocytopenia is caused by genetic defects at various stages of platelet
differentiation, function, or destruction. These mutations hinder the correct production of
platelets, cause them to aggregate and remove them from the functional pool, or cause the early
destruction of the platelets [39]. Among the genes involved in platelet formation is MYH9,
9
mutations in which cause both thrombocytopenia and giant platelets, along with several other
haematological and non-haematological phenotypes.
2.4 Giant Platelet Disorders
Giant platelet disorders are characterized by abnormally large platelets, and are often associated
with increased bleeding and bruising tendencies; females are often diagnosed at a higher rate
than males due to menorrhagia. There are several disorders associated with giant platelets
including Bernard-Soulier Syndrome, Gray Platelet Syndrome, Von Willebrand Disease type 2B,
and MYH9-Related Disease. The actual incidence of many of these syndromes is unknown due
to misdiagnosis, under reporting and the unavailability of genetic tests [45,46].
Bernard-Soulier Syndrome is a recessive bleeding disorder with a known incidence of at least 1
in a million [45]. There are currently eight known mutations associated with this disease, causing
misfolding in GPIb, GPIX or GPV. All affected individuals were found to be homozygous, and
obligate carriers (parents and/or offspring) were found to be heterozygous. In addition to giant
platelets, many affected individuals exhibit thrombocytopenia, and decreased expression of
GPIbα, GPIX and GPV [47].
Gray Platelet Syndrome is characterized by large agranular platelets and mild thrombocytopenia.
It is generally an autosomal recessive disorder, caused by three known mutations in the NBEAL2
protein [48,49], however a similar phenotype has been associated with X-linked inheritance of
mutations within GATA1 [50]. NBEAL2, a protein with no previously known function, was
found to be obligate for plateltes α-granule biogenesis, and GATA1 is known to be required for
thrombopoiesis, and likely is an upstream regulator for NBEAL1.
Von Willebrand disease type 2B (VWD2B) is associated with a gain of function mutation in the
GPIbα-binding domain of Von Willebrand Factor (VWF), preventing platelet aggregation [51].
However, VWF has also been found to be involved in megakaryopoiesis, and individuals with
VWD2B also can present with thrombocytopenia, giant platelets, and spontaneous platelet
aggregates. There are at least eight known sites of mutation within VWF, with a variety of
10
observed phenotypes [52]. Montreal Platelet Syndrome, characterized by giant platelets, has
recently been found to be caused by a V1316M mutation in VWF [53].
2.5 MYH9
2.5.1 MYH9 Structure and Function
MYH9 is located on human chromosome 22 between 35.01 and 35.11Mb and on mouse
chromosome 15 between 77.59 and 77.67Mb, and encodes for a 216kDa protein, Non-Muscle
Myosin Heavy Chain II A (NMHCIIA) which is exclusive to mammals. Of the just over 40
known human myosins, Class II myosins are the largest class, with 15 known members [54].
This class of myosin is a “conventional” myosin which works with actin to provide contractile
forces both in the “muscle myosins”, found in skeletal, smooth and cardiac muscles, and within
cells in the three “non-muscle myosins” [55].
Structurally, Class II myosins are hexameric, with two identical heavy chains, two 20kDa
regulatory light chains and two 17kDa essential light chains [5,54-56] (Figure 2-3A). The heavy
chain is composed of three main regions – the head, the neck, and the tail. The head is the active
region, and contains the ATP hydrolysis domain, the motor domain, and is the region that binds
to actin; type II myosins always move the head toward the (+) end of actin [57]. The neck region
is where both the regulatory and essential light chains bind to the heavy chain. Regulatory light
chains regulate Ca2+
binding in the head, and increased phosphorylation of this chain enhances
motor domain activity, while essential light chains stabilize the heavy chain [58,59]. The tail
domain dimerizes with the tail domain of an identical heavy chain tail, and also binds to
molecules to be transferred within the cell as “cargo” [60,61] (Figure 2-3B).
There are currently three known forms of NMHCII, producing isoforms A (MYH9), B (MYH10)
and C (MYH14) [62,63]. These isoforms are variably expressed both temporally and by tissue,
with IIA found primarily in embryonic liver, kidney and eye, and in the adult lung, cochlea,
blood and placenta. IIB is found primarily in the embryonic central nervous system, and in adult
lung, brain, and smooth and cardiac muscle. IIC is highly expressed in the cochlea, and
11
mutations in this gene are associated with hereditary deafness in humans [64,65] but is otherwise
expressed constitutively [56,66].
Figure 2-3: Basic structure of Class II myosins.
A) Hexameric structure with two sets of light chains (in yellow and orange), and one set
of heavy chains (in green, red, and blue). B) The heavy chain has three components: the
head (green), the neck (red) and the tail (blue).
The function of NMHCIIA is often studied in tandem with that of IIB and IIC, as they often co-
localize. IIA has the highest level of ATPase activity of the three, indicating that it moves actin
filaments (or along actin filaments) most quickly, and is involved in rapid changes within cells
[55]. All three non-muscle myosins have been found to be involved in cell adhesion, motility,
and polarity [55,59,67,68]. More specifically, IIA is often found near the leading edge of
moving cells or at the extending edges of spreading cells, while IIB is more often found at the
trailing edges of cells [55,68]. IIA is uniquely involved in cellular response to mechanical stress,
and in cellular durotaxis (the movement toward stiffer substances) [55].
On the IIA protein there is only one known phosphorylation site, a serine near the end of the α-
helical tail. In T lymphocytes at rest, this site is known to be phosphorylated, however the cause
and effect between de-phosphorylation and T-lymphocyte activation is uncertain [68]. In IIB
12
proteins, phosphorylation aids in the filamentation of the proteins, a situation not observed in
IIA.
Phosphorylation of the light regulatory chains of IIA increases the motor domain activity of IIA
when it is associated with actin, and is completed by three known kinases, including Rho-
activated kinase (ROCK) [59]. In MKs, this increased IIA activity is known to decrease the rate
of proplatelet formation.
2.5.2 MYH9 Related Disease (MYH9RD)
In humans, mutations in the MYH9 gene cause a range of phenotypes that have historically been
referred to as May-Hegglin anomaly, and Epstein, Sebastien, Fechtner, and Alport-like
syndromes [11,69,70], but are now collectively referred to as MYH9-Related Disease [11].
MYH9RD presents with macrothrombocytopenia, and neutrophil inclusions in all cases, and
variable presence of increased bleeding and bruising, bilateral sensorineural deafness, nephritis,
and cataracts [5-10]. There is a general correlation between location of mutation and disease
severity. Mutations within the ATPase binding region, particularly mutations at R702, are
generally more severe, and with those in the tail domain are more often restricted to
haematopoietic phenotypes [8,71,72].
The presence or absence of aural, renal and ocular phenotypes is variable not only between
mutation locations, but also within families with the same mutation, as seen in Figure 2-4. In this
family, both II-II and III-III were identified as having menorrhagia and genetic screening
identified an unspecified mutation in MYH9 in five of six genetically related individuals tested,
with three (I-I, II-III and III-I) being previously undiagnosed. Of these five individuals, all have
macrothrombocytopenia, but there is a variable penetrance of both bleeding tendency and
deafness. This is typical of families with MYH9 mutations [73-77].
13
Figure 2-4: A family with an undisclosed Myh9 mutation.
Both II-II and III-III presented with menorrhagia. Note the inconsistent phenotypes,
particularly between II-III and III-III. Modified from Althaus and Greinacher, 2009.
There are 44 known mutations in MHY9, with a variety of reported phenotypes (Table 2-1)
[7,8,11,69-75,77,79-88]. These disorders are generally caused by a single point mutation, with
75% of known families having a mutation at one of four mutational hotspots – specifically
R702C/H, D1424H/N/Y, E1841K and R1993X [7].
14
Table 2-1: Known Myh9 Mutations in Humans
Known mutations in MYH9 with the earliest citation, exon location and phenotypic
components reported in patients for each. (+): the phenotype has been found in at least
one individual with the mutation; (-): the phenotype has never been found in individuals
with the mutation; (?): no information given. (†): Found only as a double hetrozygote. Exon Mutation Hematological Aural Renal Optical
1 W33C † [87] + ? ? ?
1 P35A † [87] + ? ? ?
1 N76-S81del [89] ? ? ? ?
1 N93K [69][82] + - - -
1 A95T [82] + - - -
1 A95N [75] + + + -
1 S96L [74] + + + -
10 K373N [7,55] + - - -
16 R702C [69] + + + +
16 R702H [7] + + + +
16 R705H [80] - + ? ?
16 Q706E [84] + ? ? ?
16 R718W [8] + + + +
21 K910Q †[11] + + + -
24 G1055-Q1068del [89] + + + -
24 E1066-A1072del [11,89] + + + +
24 E1066-A1072dup [71] + - - +
24 E1084del [89] + - - -
25 V1092-R1162del [85] + - + -
25 D1114P [7] + - + -
26 T1155A [8] + + + +
26 T1155I [79] + - - -
26 R1162T [88] + ? ? ?
26 R1165C [69] + + + +
26 R1165L [82] + + + -
26 L1205-Q1207del [82] + - - -
30 R1400W† [74] + - + -
30 D1424Y [82] + + + +
30 D1424N [81] + + + +
30 D1424H [69] + + + +
30 D1447V [8] + ? ? ?
30 D1447H [86] + ? ? ?
31 V1516L [90] + ? + +
31 V1516M [72] + - ? ?
32 R1557L [72] + - ? ?
37 I1816V [83] + + + -
38 E1841K [79] + - + +
40 G1924RfsX21 [72,82] + - - -
40 D1925TfsX23 [8] + - - -
40 P1927fs [81] + - - -
40 V1930CfsX18 [72] + + ? ?
40 R1933EfsX15 [8,72,79] + + + -
40 D1941MfsX7[8,82,83] + ? ? ?
40 E1945X [11] + - + -
15
2.5.3 Platelets and MYH9
The basic structure and function of platelets has already been discussed. MYH9 is a negative
regulator of platelet production, ensuring that platelets remain attached to the MK until they are
fully matured [5,59,73]. As the only non-muscle myosin present in MKs [56], they are implicitly
involved in the DMS invagination, PPF, and the final release of platelets into the blood stream. A
MK with deficient or mutated MYH9 is incapable of properly restricting the budding of platelets,
causing premature release from the MKs [59]. Similarly, a MK with over expressed MYH9 will
have platelet release delayed by one to two days [91,92].
Deficient or mutated MYH9 is associated with giant platelets, as observed in both mice [93] and
humans [5-10]. It is likely that the giant platelets occur due to the insufficient restriction by
NMHCIIA, allowing the platelets separate from the MK before they are reduced to their optimal
size. In normal populations, the MPV is in the range of 7.0 to 12.3fL [11,37]. In various studies
of patients with MYH9RD, observed MPVs are recorded as either beyond the maximal
measurement of 13fL, or other values beyond the normal range, with one patient reported to have
an MPV of 18.6fL [11].
2.5.4 Neutrophils and MYH9
Neutrophils are the primary immune cell that detects and destroys microorganisms [94]. These
hematopoietic cells are leukocytes, more specifically granulocytes, derived from the common
myeloid progenitor [95]. Granulocytes, consisting of neutrophils, eosinophils and basophils, can
be visually identified by their multi-lobed nuclei and the presence of small “granules” within the
cytoplasm. These granules are actually secretory vesicles required for the granulocytes’
functions [94].
Neutrophils are the most common type of leukocyte, making up almost 50% of the total 3.5x109
to 11.7x109 leukocytes/L in human blood [96]. These normal levels are dependent on the
gender, time the blood was taken, and the genetic background of the patient [96,97]. Neutrophils
have extremely short life spans – 8 to 12 hours – after which they undergo apoptosis [98]. When
neutrophils are recruited to sites of injury or infection, they transmigrate through the blood vessel
to the interstitial tissue, where they migrate toward the damaged area, directed by
chemoattractants released by bacteria, dying cells, or inflamed tissue [99]. At the site of damage,
16
neutrophils identify their target, and proceed to ingest microorganisms with non-self IgG factors,
and release microbicidal molecules stored in their granules [94,99].
Patients with MYH9RD have been known to possess neutrophil inclusions since the disease was
first observed by May [100] and Hegglin [101]. These inclusions are visible by May-Grünwald-
Geimsa staining under light microscopy as light blue ovoids. It was later discovered that
neutrophils of MYH9RD patients contain similar ovoids when stained with fluorescently labelled
α-NMHCIIA [81,83] compared to the staining in normal neutrophils, which is uniform
throughout the cytoplasm [83,102]. The exact role of NMHCIIA in neutrophils is uncertain, but
is known to include the normal functions of NMHCIIA– providing contractile forces to the cells
during motion and cellular replication [55,102].
2.5.5 Cochleae and MYH9
Hearing is a complicated sense that requires sound waves to be translated into electrical signals
which can be processed by the brain. This is done by the ear (Figure 2-5A) which is made of
three components: the outer, middle, and inner ear. The outer ear is made up of the pinna (the
visible component of the ear), and the auditory canal. The pinna reflects sound waves into the
auditory canal, causing the tympanic membrane to vibrate. Beyond the tympanic membrane is
the middle ear, which also contains the ossicles: the mallus, incus, and stapes (commonly called
the hammer, anvil and stirrup). These bones amplify the vibrations of the tympanic membrane,
increasing the pressure caused by the sound waves, and transmitting them through the oval
window into the fluid filled cochlea. The cochlea and the vestibular system, which is involved in
balance, make up the inner ear [103].
17
Figure 2-5: Hearing and the cochlea
A) The anatomy of the human ear, showing the outer (orange), middle (pink), and inner
ear (blue). B) A cross section of a cochlear turn, showing the three compartments and the
location of the organ of Corti and the cochlear nerve. C) A diagram showing the
relationship between distance from the base of the cochlea and the tone frequency (in Hz)
the region of the cochlea is sensitive to – the closer to the apex, the lower the tone.
The cochlea is a spiral shaped bony structure that resembles a sea shell with three and a half
rotations in humans. The cross-section of a cochlear turn (Figure 2-5B) shows that there are
three cavities within the space: the scala vestibuli and scala tympani are filled with perilymph,
and are connected at the apex of the cochlea; and the scala media, or cochlear duct, which is
filled with endolymph and contains the organ of Corti. The scalae media and vestibuli are
separated by Reissner’s membrane, which is two cells thick, approximately 12µm. The
membrane between the scalae media and tympani is called the basilar membrane, and the organ
18
of Corti rests against this structure [103]. As the basilar membrane extends from the oval
window to the apex of the cochlea, the membrane reduces in stiffness – this change is what
allows the different tonal frequencies to be perceived (Figure 2-5C), with higher frequencies in
the stiff zone near the oval window, and lower frequencies in the flexible areas near the apex of
the cochlea [104].
The stapes rests against the oval window and induces waves in the lymph inside the scala
vestibuli. These waves travel through the lymph, toward the apex of the cochlea, and are easily
transmitted through Reissner’s membrane to the scala media. The wave travels along the scala
media to the point where the change in pressure due to the wave is sufficient to move the basilar
membrane, and therefore move the hair cells located within the organ of Corti [104]. This
movement activates the cochlear nerve, sending an electrical signal to the brain. The wave in the
scala vestibuli continues to the apex, and, if it has sufficient force, down the scala tympani,
where the pressure is absorbed by the round window [103].
MYH9 is known to be expressed in the organ of Corti, spiral membrane, and in Reissner’s
membrane [80], however its role has not as yet been elucidated. It is possible that MYH9
expression in the organ of Corti is related to its role in durotaxis, the preferential motion of cells
toward stiffer materials [55].
Bilateral sensorineural hearing loss or deafness, which is observed in patients with MYH9RD,
occurs when the cochlea, the auditory nerve, or the brain are unable to properly create, transmit,
or interpret electrical signals [105]. Although it is possible to measure the electric potential
directly at the cochlear nerve, it is somewhat invasive. Because it is less invasive, Auditory
Brainstem Response (ABR), which uses electrodes placed under the skin of the scalp, is the
preferred method of observing electrical signals from the cochlea to the brain [106].
2.5.6 Kidneys and MYH9
The primary role of the kidney is to remove water soluble waste molecules from the blood for
excretion in urine as part of the excretory system. To do this effectively, the kidneys together
receive approximately 25% of the cardiac output, filtering the blood at a rate of 1L/min [107].
The functional components of the kidneys are nephrons, with more than one million in each
19
kidney. A nephron consists of a glomerulus, proximal convoluted and straight tubules, the loop
of Henle, the distal tubule, and initial collecting tubule (Figure 2-6A). The glomerulus is
surrounded by Bowman’s capsule, with and a capillary tuft and its surrounding cells inside [108].
Unfiltered blood enters the glomerulus (Figure 2-6B) through the afferent arteriole, and splits
into eight lobules to form a capillary tuft. Before exiting the glomerulus, the lobules merge into
the efferent arteriole, through which filtered blood exits. Filtration occurs through the basement
membrane of the capillaries into the space between Bowman’s capsule and the tuft – called
Bowman’s Space; small particles, such as water, salts, urea, glucose, and amino acids pass freely
through the membrane, as do small proteins, under 30kDa [107]. Filtration is also aided by the
podocytes surrounding the capillary basement membrane (Figure 2-6B). Podocytes form
extensions, called foot processes, that interdigitate, leaving narrow slits, called the slit
diaphragm, through which the filtrate must pass [108] (Figure 2-6C). The foot processes are
negatively charged, and prevent other negative molecules, particularly proteins such as serum
albumin, from leaving the blood stream [109].
20
Figure 2-6: Excretion and the Kidney
A) The components of the nephron. B) Detailed view of the glomerulus. C) SEM of a
glomerular capillary enclosed by a podocyte. Note the interdigited foot processes
(Pavenstädt, 2003).
The liquid contained in Bowman’s capsule is the precursor for urine, and leaves the glomerulus
through the proximal tubule. The proximal and distal tubules, and the loop of Henle are
surrounded by a capillary bed, with arterial capillaries surrounding the proximal tubule and the
descending arm of the loop of Henle, and venous capillaries surrounding the distal tubule and the
ascending arm. As the glomerular filtrate travels through the proximal tubule, water, glucose,
21
and amino acids return to the blood through the intertubular capillaries and the arterial vasa recta
(Figure 2-6A) [107]. The descending arm of the loop of Henle is permeable to water, but not
salts. Osmotic pressure due to proteins in the blood forces the water into the blood, increasing
the salt concentration in the urine. The ascending arm (surrounded by the venous vasa recta) is
permeable to salt, but not water, allowing the body to passively recover it. The distal tubule is
capable of active transport of salt against the gradient for times when salt conservation is
required. At the end of the distal tubule is the collecting tubule which leads to the bladder for
storage.
MYH9 expression is predominant in the glomeruli, more specifically within the podocytes [110],
and patients with MYH9RD, particularly those with mutations at R702, will often develop
proteinuria at a young age, and go into renal failure before they reach their mid-twenties [8,110].
Because these patients often also have increased bleeding, very few biopsies are done, however,
a patient with proteinuria with biopsies taken at 9 and 11 years old showed a progression of focal
segmental glomerulosclerosis (FSGS), or scarring of the glomeruli, with a decrease in NMHCIIA
expression levels within her podocytes [110].
Both proteinuria and FSGS are consistent with damage to the podocyte. Loss of cytoskeletal
function prevents proper podocyte filtration, allowing albumin to pass into Bowman’s capsule –
the prime cause of proteinuria. FSGS is associated with regions of the glomerular tuft being
exposed directly to Bowman’s space; although neighbouring podocytes attempt to maintain
hemostasis by increasing their filtration, this creates a feedback loop leading to the entire
nephron attempting to filter through the parietal space, and inflammation and scarring to render
the nephron irreparably damaged [109].
In 2008, several groups identified MYH9 as a common locus for chronic kidney disease among
the African American population [111-113]. However further studies indicated that a SNP
located within APOL1, located approximately 40kb upstream of MYH9 has a much higher
association with renal disease in this population than the ones discussed in the earlier articles
[114].
22
2.5.7 Lenses and MYH9
The function of the eye is to transform light into usable data in the brain; focussing the light, and
then determining light intensity and colour. The structure of the eye evolved to optimize this
function, using multiple transparent structures of differing density to control how and where light
is detected (Figure 2-7A). Light enters the eye through the cornea, which causes the light to
refract, and then through the pupil. The pupil’s size is controlled by the iris, which dilates to
reduce the amount of light entering the eye, and contracts to maximize the light entering the eye.
The light then passes through the lens, a biconvex structure, which focuses the light so that it hits
the retina on the interior of the eye. The eye can focus on either near or far objects by changing
the curvature of the lens [115]. Within the eye, rods and cones are responsible for light/dark
perception and colour vision, respectively. Rods are more common at the outer edges of the
retina, and absent in the fovea, an area located just below the optical nerve, where precision
seeing takes place; while cones are present throughout the retina, being least common at the
outer edge; neither rods nor cones are located at the egress of the optical nerve –the blind spot
[116].
The development of the eye begins with the formation of the lens pit, into which the lens
develops. These early lens cells close over the pit to form the cornea on the anterior side, at the
posterior side – at the back of the pit – the cells lose their nuclei and elongate, and the cells
closing the pit become the epithelium of the lens . The elongated cells and the epithilium make
the primary lens [115,117]. The secondary lens is constantly developing throughout life, formed
from the divisions of the epithelial cells on the anterior side of the primary lens. Secondary lens
cells are metabolically inactive, anucleic, have minimal numbers of organelles, and are
compacted against the existing primary and secondary cells (Figure 2-7B). The cells of the
secondary lens are approximately one third protein by wet weight. Crystallins are highly stable,
water soluble proteins which act to aid in cellular transparency, but also have known roles as
stress proteins and in metabolism. Oxidative stress is minimized by inhibiting the glutathione
redox cycle, catalases, and the mercaptopuric pathways. As individuals age, the lenses develop
from a nearly spherical structure with a soft, plastic-like texture at birth to an oblong structure
with a glass-like consistancy [115,117]. The cytoskeletal system within the lens is responsible
23
for co-ordinating the structural and biochemical changes that occur as the secondary lens
develops, and is highly involved in lens fibre differentiation [118].
Figure 2-7: Structure of the Human Eye and Lens
A) Light passes through the cornea, into the eye. The lens contracts to focus light through
the vitreous gel onto the retina. The retina contains rods and cones which convert light
into signals passed through the optical nerve to the brain. B) A diagram of the lens
showing the primary lens, made up of the nucleus and the cortex, and the secondary lens,
made of the anterior capsule, the maturing cells at the equator, and the compacting lens
fibers
24
Cataracts, which are a component of MYH9RD, are a loss of transparency in the lens and can be
caused by trauma, toxic effects, metabolic disorders, or genetics [119]. The best understood type
of genetic cataract in humans is congenital cataracts – those that occur in patients less than one
year old [120,121]. These patients often have mutations in the crystallin or connexin gene
families. Crystallins have light scattering properties required for the lens to correctly focus light,
and connexins allow for communication between lens fibres; both of these are integral for correct
function of the lens [117]. Patients with MYH9RD generally develop cataracts in their mid
twenties [8], after years of lens fibre differentiation. The only current treatment for cataracts is
surgical removal of the affected lens(es), followed by implantation of polymer lenses which have
similar structure and functionality [120,122].
2.5.8 Cellular and Mouse Models of Myh9
The role of NMHCIIA in the adult eye, kidney and cochlea are not well understood, however
much research has gone into the role of IIA in platelet production. When driven to
thrombopoiesis, ES cells null for Myh9 produce more proplatelets, and produce them earlier,
than their wild type counterparts in vitro [8]. Conversely, ES cells overexpressing Myh9 reduces
proplatelet production [5]. Mice with conditionally knocked-down Myh9 in MKs have larger
proplatelet extensions than MKs from their wild type litter mates. Furthermore, the MKs of the
knock down mice have three to four times more proplatelet extensions than the MKs of their
wild type litter mates [91]. These indicate that Myh9 is a negative regulator of platelet
formation, and is responsible for ensuring that platelets and proplatelets are not released from the
MKs prematurely.
Myh9-/-
mice have been found to be embryonic lethal by E7.5, due to defects in cell adhesion,
indicating that this gene is highly involved in differentiation and/or embryogenesis. Of the live
births from heterozygous mating, 53% were heterozygous, however with a lethal homozygous
condition, 66% of the offspring are expected to be heterozygous, suggesting that there is some
embryonic lethality in the Myh9+/-
mice as well. When tested for the classic indications of
MYH9RD, the Myh9+/-
mice showed normal platelet size, no visible neutrophil inclusions by
immunofluorescence, and no abnormal kidney damage compared to the wild type controls. Two
25
of the six Myh9+/-
mice tested showed hearing loss compared to the wild type mice, however,
overall this is statistically insignificant [67,93].
To determine the role of Myh9 in MKs and thrombocytes, an MK-specific knock down mouse
line was developed. This mouse line expressed normal quantities of Myh9 in all tissues except in
MKs and platelets; in platelets the protein level was reduced to about 3% of that in the control
animals. The platelet count in the Myh9 deficient animals was significantly lower than the wild
type (WT) and heterozygous mice, and the platelet area was doubled, indicating
macrothrombocytopenia. In tail bleed tests, the wild type and heterozygous mice were not
statistically different with 78 and 135s clot times, while all of the Myh9 KO mice bled for the
maximally allowed 600s. This was confirmed by clot retraction tests, which showed that
platelets lacking Myh9 were incapable of forming a clot within 5 hours [123].
A conditional knockout of Myh9 in the podocytes was also developed, and has also provided
interesting information about its role in this structure [124]. This knockout line was developed
on the B6 background, and knockouts were found to have normal blood pressure, albumin and
creatine levels. This can be explained in part by the protective kidney phenotype associated with
the B6 background [90]. Electron microscopy of the kidney showed that although there were no
structural differences in the podocyte feet between WT and untreated KOs, when treated with
doxorubicin hydrochloride (a podocyte stressor to which B6 mice are immune) the foot
processes of the KO mice widened and/or fused. The treated KO mice also developed
glomerulosclerosis.
Although all of these models are useful to study the general function of Myh9, none is capable of
displaying the phenotypes caused by MYH9RD, because the currently known mutations causing
MYH9RD cause a change in the structure of NMHCIIA, while retaining the majority of the
protein. This cannot be mimicked by haploinsufficiency, as seen in the absence of
haematological or renal phenotype in the Myh9-/-
mice; or by targeted deletion, which only
effects one of the multiple systems affected by MYH9RD.
26
2.6 Genome Sequencing
2.6.1 Sanger Sequencing
DNA sequencing by its most common method, the Sanger method, was first completed in 1977
[125]. In this process, ddNTPs are used to terminate DNA extension, and samples are then
separated by electrophoresis to determine the sequence. Originally, four reactions were
completed – each with the same DNA, polymerase, and a full complement of dNTPs, along with
one of ddATP, ddTTP, ddGTP or ddCTP. After the polymerase reaction was completed, the
reactions were run out in parallel on a gel, and the sequence could be read from the bottom of the
gel to the top, knowing that the presence of a band indicated that amplification was terminated
by the presence of that particular nucleotide [125]. This method was improved upon first by the
discovery of polymerase chain reaction [126], and then by the conjugation of fluorescently
labelled dyes to the ddNTPs [127]. These two advances allowed a single reaction to sequence a
much longer region, and for sequencing to become more automated. Typical Sanger sequencing
is now completed in 96- or 384-well plates, with samples separated by capillary electrophoresis.
2.6.2 Next Generation Sequencing
The next challenge for sequencing was to improve throughput by parallelizing the process.
There are currently at least six processes for Next Generation sequencing [128], all of which are
considered to be “non-Sanger” sequencing methods, as they do not use ddNTP termination and
electrophoresis to determine the sequence. Solexa sequencing – now called TruSeq technology
by Illumina [129] – is one of these methods (Figure 2-8).
27
Figure 2-8: Solexa sequencing.
DNA (purple) in ~200bp sections have adapters (aqua and orange) added to them and are
then amplified on a glass plate. Multiple rounds of TTDFs are added, allowing hundreds
of clusters to be sequenced concurrently.
This type of sequencing requires the addition of adapters to the DNA in order to attach it to the
solid surface on which the sequencing occurs. Several rounds of DNA amplification occurs on
the glass plate to produce identical clusters. Sequencing then begins with the addition of
temporarily terminated, fluorescently labelled dNTPs (TTFDs). The first nucleotide of each
cluster will bind, and the unbound TTFDs are removed. A laser excites the TTFDs, and the first
nucleotide of each cluster is sequenced. The fluorescent label and the termination are both
removed from the first nucleotide, and a new batch of TTFDs is exposed to the clusters, allowing
the next nucleotide to be sequenced [130]. This process produces thousands of sequences
between 25 and 400 nucleotides long, which must then be aligned by computer using a reference
sequence to determine the complete sequence.
Solexa sequencing has previously been used in tandem with bacterial artificial chromosomes
(BACs) to identify an ENU mutation [23]. In this process, the 2.2Mb region of interest was
separated into a series of 15 overlapping BACs, each of which was sequenced by Solexa. Two
28
mutations were found within the region, one of which was the causative mutation for the ENU
phenotype.
2.6.3 Sequence Capture Techniques
Using BACs to sequence large area requires the time consuming process of obtaining minimally
overlapping BACs, as BACs can hold a maximum of 300kb of DNA [131]. NimbleGen chips,
developed by Roche in 2007, were originally capable of isolating up to 5Mb of specific DNA
[132-134], but now can capture up to 50Mb [135] (Figure 2-9). In this procedure, genomic DNA
is sheared to produce fragments of approximately 200bp, and then incubated with the chip,
which is covered with 25-mers which are unique to the region of interest. All DNA that does not
bind to the chip is discarded, and the bound DNA is eluted, ready for sequencing. The
NimbleGen system is therefore a significant improvement over BACs, reducing the preparation
time and streamlining the process for Next Generation Sequencing.
Figure 2-9: NimbleGen’s Sequence capture technology
DNA (pink and purple) is sheared and exposed to a chip with 25-mers specific to the
region of interest (purple). Non specific DNA is washed away and specific DNA is
eluted for further processing.
29
3 Objectives and Hypotheses
The purpose of this ENU screen, organized by the Centre for Modelling Human Diseases, was to
find novel genes involved in haematopoiesis in order to better elucidate the mechanism of
differentiation, and to provide therapeutic targets for heart disease, and post-cancer treatment.
This is not where this project went. It did, however, live up to the centre’s name.
The attained objective of this project was to produce a mouse line that is capable of truly
modelling a human disease for which there had previously been no accurate model. The
overarching goal was to show that ENU mutation can be used to produce novel and useful
mouse models that mimic human diseases.
In order to show that in the 7238 strain, the genetic cause of the macrothrombocytopenia
phenotype had to be determined. The majority of human genetic disorders are caused by a single
error in a single gene. However, ENU mutation causes a mutational rate of approximately 1 in
106 base pairs. In order for this mouse strain to be useful, the phenotype must be caused by a
single ENU induced point mutation.
Once it was found that the 7238 phenotype was caused by a Myh9Q1443L
point mutation, the other
effects of this mutation became of interest. MYH9RD causes not only the relatively benign
condition of macrothrombocytopenia and neutrophil inclusions, but can also cause premature
bilateral sensorineural deafness, cataracts and renal disease. These additional aspects of
MYH9RD were studied to determine if the Myh9Q1443L
mouse line displays multiple
phenotypes associated with human MYH9RD.
The function of MYH9, and its dysfunction in MYH9RD, is nominally understood in platelets,
but poorly understood in the other organs affected by MYH9RD. Equally, the roles of other
Class II Non-Muscle Myosins (MYH10 and MYH14) are currently unknown in these tissues.
Immunofluorescence of these three proteins was therefore completed to observe potential
changes in the expression patterns of NMMII caused by Myh9 mutation.
30
4 Materials and Methods
4.1 ENU mutagenesis and line creation
Healthy, male 129S1/SVImJ (129) mice were injected with 100mg/kg of N-ethyl-nitrosourea, and
harem bred with two healthy C57BL/6J (B6) females once sterility had passed (~60 days).
Offspring were screened, as described below, to determine if there was a hematological
phenotype of interest. Mice with phenotypes were mated to B6 mice, and offspring were
screened to determine heritability. Back crosses were completed to determine the chromosomal
region of interest, and intercrosses were completed to determine the homozygous phenotype. All
mice were maintained at Mt. Sinai or Toronto Centre for Phenogenomics, and handled according
to the ethical guidelines established by the Canadian Council on Animal Care, with protocols
approved by the University of Toronto, and the site at which the tests were completed.
4.2 Haematological testing of mice
Peripheral blood (20-30µL) from the saphenous vein was collected in EDTA coated capillary
tubes and transferred to (Eppendorf) tubes. Complete blood counts were performed by either the
Ac-T Differential Haematology Analyser with veterinary software (Beckman-Coulter) or the
Hemavet Haematological Analyser (Drew Scientific). Mice were bled at 6 and 8 weeks.
4.3 Next Generation Sequencing
Genomic DNA was isolated from tail clips, and then purified using Qiagen DNA Clean-up Kit,
and sent to the Ontario Institute for Cancer Research. The DNA was sheared to ~500bp and
isolated using a custom NimbleGen chip specific to interval from 77.5 to 81.2Mb on mouse
chromosome 15. Following a 70h incubation and washes to remove the un-bound DNA, the
DNA of interest was eluted by a wash in 95°C water. The eluted sample was then sequenced
using the Illumina GAII platform. The resulting sequence was aligned to the NCBI37 mouse
reference, providing 7144 variations.
31
4.4 Gene Expression Analysis
Kidneys were removed from mice, placed in (Eppendorf) tubes, flash-frozen on dry ice with
isopropanol, and transferred to -80°C for storage. Once all samples were collected, ~30µg of
tissue was placed in polypropylene microvial (Biospec) tube washed with DEPC-water, filled
with 250µL of autoclaved 1mm glass beads (Cole Parmer), 350µL of RA1 from the NucleoSpin
RNA II Kit (Macherey-Nagel), and 3.5µL of β-mercaptoethanol. Tissue was beat 3x30s at
maximum frequency, and put on ice between sessions. Samples and beads were transferred to
the NucleoSpin filter, and the manufacturer’s protocol was followed from step 3.
RNA was quantified by Nanodrop (ThermoFisher), and converted to cDNA using 200µg of
DNA, SuperScriptII (Invitrogen) and random hexamers, and then purified by ethanol
precipitation. Quantitative PCR (QPCR) was completed on the Lightcycler 480 (Roche), using a
10µL reaction volume and 1µM primers. Standard curves were generated using 10-fold dilutions
of liver cDNA, prepared as above, to determine the efficiency of each primer pair.
4.5 Tail Bleed assay
Unclipped male mice aged 15 weeks were anaesthetized with 1-2% isoflurane in oxygen. A
0.5cm length of tail was removed with a scalpel, and the tail was placed in 0.9% saline warmed
to 37°C. Time to clotting was measured; if bleeding had not arrested after 600s, the experiment
was stopped to prevent animal injury. All tails were cauterized before the animals were returned
to their cages.
4.6 Neutrophil Isolation and Staining
Negative immunogenic isolation was used to purify neutrophils from whole blood [136]. In
brief, whole blood is collected from anaesthetized mice by cardiac puncture and transferred to
10mL dextran (1.25% w/v) in saline and inverted. After resting for 30min, the leukocyte-rich
supernatant is collected, washed with PBS (-/-) with 0.5% BSA, centrifuged (6 min, 300xg, 4°C),
and resuspended in 1mL of PBS/BSA. An aliquot is used to complete a leukocyte count.
32
Leukocytes are incubated with α-CD2 (1.5 µg/106 lymphocytes), α-CD5 (2 µg/10
6 lymphocytes),
α-CD45R (10 µg/106 lymphocytes), α-F4/80 antigen (2 µg/10
6 monocytes), and α-ICAM-1 (0.6
µg/106 leukocytes) for 30min at 4°C. Excess antibody is removed by addition of 8mL of
PBS/0.5%BSA and centrifugation. Cells are then resuspended to 4mL and washed Dynabeads in
a 1:1 ratio with the leukocytes are added. Cells are incubated, rotating, for 30min at 4°C, then
separated at room temperature using a magnetic apparatus. Labelled cells will remain in the tube,
while the desired neutrophils are eluted. Erythrocytes are removed by lysis in a hypotonic
solution (7mL of 0.2% NaCl solution inverted 10X), followed by neutrophil rescue by
hypertonic solution (7mL 1.6% NaCl and 0.1% glucose, inverted once). Cells are centrifuged,
and then resuspended in 1mL of PBS/BSA. Using a Cytospin and MegaFunnels
(ThermoFisher), neutrophils are mounted on slides, and fixed using Cytospray (ThermoFisher).
Cells are fixed at room temperature with acetone cooled to -20°C for 3 min, then blocked for 1h
at RT with 3% w/v skim milk in TBS. Slides are probed with 1:300 dilution of Rabbit α-
NMHCIIA (BioTechnologies, Inc, BT-567) in a humidified chamber for 1h, washed, and stained
for 1h with 1:200 α-Rabbit 488 Alexafluor, and 1:5000 Dapi at RT in the dark. Slides are
washed, and then sealed with Mowiol.
4.7 Auditory Brainstem Response
Mice were anaesthetized with 1-2% isoflurane in oxygen (induced in a chamber and maintained
using a facemask) and placed in a sound isolated box. Skin needle electrodes were placed under
the skin at the top of the head (vertex) and behind the ear area (mastoid) for ABR measurement,
and a soft tipped ear tube which presents sound stimuli was inserted into the ear. Mice were
exposed to pure tones at frequencies between 4kHz and 24kHz for 50ms, at volumes of 0 - 80dB
in 10dB increments. Frequency-response curves were generated for each tone and volume
combination using the average of 512 measured outputs of tone stimuli and a compensatory filter
was created. Tone stimuli were calibrated by adjusting the gain at each particular stimulus
frequency. Frequency modulated stimuli were generated with MATLAB then calibrated by
applying the compensatory filter to each stimulus.
33
4.8 Tissue Sectioning
Organs were preserved by several different methods. Whole animals were perfused with 4%
PFA, and organs dissected out for storage. From sacrificed animals, organs were drop-fixed in
NBF (24h) or in Bouin’s Solution (6h). Once fixed, organs can be immediately dehydrated and
embedded in paraffin blocks. Alternately, the organs can go through a sucrose gradient,
followed by embedding into OTC for cryosectioning. Flash frozen organs were embedded in
OTC, then flash frozen in liquid nitrogen. Paraffin-embedded sections sections are cut to 6µm
thickness; while cryosections are cut to 10µm.
4.9 Immunofluorescent staining of tissues
Paraffin embedded tissues were deparaffinised through a xylene/ethanol gradient, to water.
Some slides underwent antigen retrieval. Cryosectioned tissues were thawed at 37°C. Flash
frozen tissues were fixed for five minutes in acetone. From this point, all slides were treated
identically. Slides were blocked for 1 hour at room temperature, then exposed to primary
antibodies overnight at 4°C. Slides were then washed in TBS, and exposed to secondary
antibodies for 1 hour, in the dark, at room temperature. Slides were washed in TBS, then water,
and cover-slipped with Mowiol. Images were taken using a Leica DMIRE2 microscope, using
OpenLab software. Colour corrections and merging was completed with ImageJ software.
4.10 Statistics
In general, ANOVAs were completed using GraphPad Prism, with Bonferroni’s post-test.
Certain data sets were analysed as non-parametric data sets; these were analysed using a
Kruskal-Wallis test, the non-parametric equivalent of an ANOVA, with Dunn’s post-test.
Significance was set at p<0.05 for all analyses.
34
5 Results
5.1 Establishment and Phenotyping of the 7238 Mouse line
Mice from the G1 generation underwent a panel of tests, including hematological testing, to
identify phenotypic variants. The founder of the 7238 line was identified as having platelet
counts of 601x103 platelets/µL at 9 weeks, and 580x10
3/µL at 12 weeks on the AcTDiff
Hematology machine, both more than 2 standard deviations below the normal WT levels.
Subsequent breeding found that both 7238/+ and 7238/7238 mice were viable, and these mice
were screened at 6 and 8 weeks (Figure 5-1A). Platelet counts were 1031±169x103/µL,
599±126x103/µL, and 245±109x10
3/µL (p<0.0001) for WT, 7238/+ and 7238/7238 respectively,
indicating a gene-dosage dependent thrombocytopenia. Therefore, platelet count was used as the
primary phenotyping tool for counts taken with the AcTDiff machine. When tested with the
Hemavet machine, platelet counts of 788±171x103/µL, 904±171x10
3/µL, and 506±127x10
3/µL
(p<0.0001) for WT, 7238/+ and 7238/7238, indicating that only the 7238/7238 mice had
thrombocytopenia, and that a different phenotyping mechanism would be required when using
this machine. The mean platelet volumes (MPV) using the Hemovet were 4.7±0.2fL, 6.3±.03fL,
and 7.3±0.2fL (p<0.0001) for WT, 7238/+ and 7238/7238 (Figure 5-1B); these values have very
little overlap between phenotyping, and both provided an excellent phenotyping tool, and an
indication of the causative gene.
To determine the viability of the phenotypes, 20 heterozygous breedings were analysed to
determine the ratio of offspring that survived to the 6-week blood test, comprising 486 animals.
Of these, 27.6% were WT, 53.5% were 7238/+ and 18.9% were 7238/7238 (p<0.01) (Figure 5-
1C). The expected phenotype ratio of heterozygous breedings is 1:2:1; assuming that the WT
mice survived at a normal rate, the observed number of 7238/+ mice was appropriate. However,
there were approximately 40 fewer 7238/7238 animals present than the number of WT animals
suggested should be present, indicating that approximately 30% of the 7238/7238 animals did
not survive to 6 weeks of age. Because timed matings were not performed for this line, it is
undetermined whether the 7238/7238 mice were lost during gestation, pre-weaning, or post-
weaning.
35
Figure 5-1: Phenotyping of the 7238 line.
A) Platelet count phenotype as measured on the AcTdiff Hemocytometer. B) Platelet
count and platelet volume phenotypes, as measured with the Hemavet hemocytometer.
A,B) n=100, p>0.0001 for all comparisons. C) Genotypes of pups surviving to
hematological testing (6 weeks) from 20 heterozygous breedings. p>0.001.
5.2 Next Generation Sequencing Identified Two Novel ENU Mutations in the 7238 Mouse Line
Gross mapping of the 7238 line was completed using microsatellites, and indicated an initial
32Mbp region between 61.8 and 94Mb of chromosome 15 (Figure 5-2A). Further crossovers
36
determined a region of interest between 77.655Mb and 79.08Mb, which includes part of Myh9.
Sequence capture arrays were designed for a 4.25Mb region, between 77.2 and 81.6Mb on
chromosome 15, which included the entire Myh9 gene. Genomic DNA from four mice was used
for sequence capture, followed by next generation sequencing; two of the mice were confirmed
7238/+ between 77.655 and 79.08Mb, and two were confirmed 7238/7238 within the same
region (Figure 5-2B).
A total of 7 144 sites were flagged with at least one mouse having differences from the B6
consensus sequence (Figure 5-3). Of the initial sites flagged, 4 433 were present in all of the
animals tested. All known SNPs between mouse strains were removed, leaving 274 novel sites of
interest. These sites were screened based on their location, leaving 18 sites which were either
exonic, or within 60bp of an exon.
Analysis of the coding region of Myh9 provided a potential exonic mutation, T77599238A, that
was found in both the 7238/7238 samples and one of the 7238/+ samples. This mutation would
cause a Q1443L mutation within the Myh9 coding region. Because this site was beyond the
region for which the samples were tested prior to next generation sequencing, all four were tested
at 77.468Mb (Figure 5-2B). The remaining “7238/+” mouse was in fact WT for the region
surrounding the Myh9 exonic mutation, indicating that this site required further investigation.
The 256 intronic and intergenic sites present in all four mice were not screened directly;
however, a crossover between Myh9Q1443L and the SNP at 77.782Mb (Figure 5-2C) removed
all but one intergenic site, at 77.711Mb, from contention.
This now accounts for the 20 sites of interest, which can be broken down into one intergenic site,
8 sites in the exon border region, four in the untranslated regions of the first or last exons, six
synonymous mutations, and one non-synonymous mutation – Myh9Q1443L (Table 5-1).
37
Figure 5-2: The region of interest for the 7238 line.
A) Gross mapping was determined using microsatellites. B) Fine detail mapping of mice
sent for Solexa sequencing. Note that the genotype at rs31919996 (at 77.468Mb) was
determined after the samples were sent for Solexa sequencing. C) A cross-over
discovered during mapping indicating a cross-over between Myh9Q1443L and 77.78Mb,
and confirmed by breeding.
.
38
Figure 5-3: Identifying sites for further study on the 7328 line
Sites of interest from the initial list of sites flagged by Next Generation Sequencing were
identified using the flow chart above. Boxes in red indicate sites that underwent further
sequencing.
Sequencing primers were designed around each mutation (Table 5-2), and both B6 and 129 DNA
were sent for Sanger sequencing to determine if the locus was a novel SNP, or an ENU mutation.
Novel SNPs would have the variation present in one of the two parental lines, while ENU
mutations would not have the variation in either parental line. Of the 20, only Myh9Q1443L and
the intergenic site between Myh9 and Txn2 were found to be novel. Because the Myh9Q1443L
causes a mutation in amino acid expression, it is the primary site of interest.
7 144 flagged sites from Next Generation
Sequencing
4 433 sites present in all animals tested
274 sites not associated with
previously known SNPs
18 sites exonic or near intron-exon
boundary
256 intronic and intragenic sites
1 site of interest after additional crossover
1 site present in 3 of 4 animals tested -
matches genotyping
39
Table 5-1: Loci of interest found via sequence selection and next generation sequencing
of four 7238 animals.
Primers were designed for each locus, and the parental mouse lines were sequenced. The
Myh9Q1443L mutation and the intergenic Txn2/Myh9 loci were found to be novel, the
remaining 18 were novel B6/129 SNPs.
40
Table 5-2: Primer sets for Sanger Sequencing
These primer sets were designed to sequence the regions surrounding the 20 loci of
interest indicated in Table 5-1. (†) - Both of the Slc16a8 sites were located within a 400bp
region and were therefore sequenced using the same primer set.
5.3 The T77599238A Mutation in the Myh9 Gene Causes a Q1443L Mutation in the Associated NMHCIIA Protein within a Highly Conserved Region and Causes a Change in Amino Acid Class
Sanger sequencing of the Myh9Q1443L site in both B6 and 129 lines indicated that this mutation
is novel to this strain, and not a previously unknown SNP. The 7238/+ and 7238/7238 mice
possess one and two copies of the T mutation, respectively (Figure 5-4A). This mutation was
Sanger sequenced in more than 120 mice with 99% accuracy between MPV phenotype and Myh9
genotype. More than 500 mice have since been genotyped via TaqMan with 100% accuracy.
41
Figure 5-4: The Myh9 mutation found in the 7238 line is novel, and produces an amino
acid change in a highly conserved region.
A) Neither the C57Bl/6J nor the 129S1/SvImJ parental strains of 7238 posses the
variation, but all phenotypically 7238/+ and 7238/7238 mice do. B) The amino acid
coding region for the 7238/7238 mouse strain, the wild type mouse strain, a panel of 10
other mammalian species, and human variants D1447V/H. Amino acids are colour coded
by class; green – polar; red – non-polar; yellow – basic; blue – acidic.
The amino acid structure of NMHCIIA in the region of this mutation is very highly conserved
through a panel of 11 species (Figure 5-4B). These 11 species were aligned using ClustalW2
[137], and have homologies ranging between 84 and 99 percent. The cat comparisons all have
84-85% homology; however, this protein is not fully sequenced, with 229 amino acids
undetermined. Excluding the cat, the weakest homology is between horse and gorilla NMHCIIA,
at 92%; mouse and humans have a 97% homology. Although there is slight variation in the
exact amino acids used, all species have identical patterns of amino acid class (polar, non-polar,
acidic and basic) for the sequence between mouse amino acid 1401 and 1490. In the 7238/7238
mutant a highly conserved glutamine (polar) is changed to a non-polar leucine. Similarly, there
are known human mutations at 1447 where the highly conserved aspartic acid is mutated to
42
either a non-polar valine, or a polar histidine [8,55]. Also located nearby is a very common
D1424Y/N/H mutation group [69,81,82] which also has the same pattern of changing classes
from acidic to non-polar, polar, and basic, respectively. Because the most likely cause of the
7238 phenotype is the Q1443L mutation, the line is referred to as Myh9Q1443L
for the remainder
of this thesis.
5.4 The C77710527A Mutation between the Myh9 and Txn2 Genes does not Effect Expression Levels of Either Gene
The ENU mutation, C77710527A is located in the intergenic region between Txn2 and Myh9,
both of which code on the reverse strand of chromosome 15 in the mouse. This site is
approximately 350kb upstream of the Myh9 start site, and 350kb downstream of the end of the
Txn2 coding region (Figure 5-5A). Txn2 is a member of the thioredoxin family, which is
responsible for reducing disulphide bonds, act as electron donors for ribonucleotide reductase,
are involved in regulating apoptosis. TXN2 is found primarily in the mitochondria and is highly
involved in both mitochondrial apoptosis and null mice have been used as models for spina
bifida [78,138].
To determine whether this mutation has caused any change in the expression level of Txn2 or
Myh9, QPCR was completed (Figure 5-5B). In addition, the expression levels of Myh10 and
Myh14, which code for NMHCIIB and IIC, the related non-muscle myosins, were also
determined. For both Myh9 and Txn2, there were slight, non-statistically significant changes in
expression level – Myh9 levels increased by about 10-15%, while Txn2 levels decreased by less
than 5% between the WT and either the Myh9Q1443L/+
or Myh9Q1443L/Q1443L
mice. The levels of
Myh10 and Myh14 both increased at statistically significant levels between those same groups,
with 35 to 50% increase in RNA expression.
43
Figure 5-5: The mutation in the Txn2-Myh9 intergenic region is unlikely to cause the
7238 phenotype.
A) Location of the C77710527A mutation relative to the Txn2 and Myh9 genes on the
mouse genome. B) Relative expression of genes from kidney tissue. N= 6-7, bars
indicate p<0.001. The 35-50% increase observed in Myh10 and Myh14 expression is
statistically significant, but may not be biologically significant.
5.5 Myh9Q1443L
Mice have Neutrophil Inclusions and Increased Bleeding Tendancy
Beyond the macrothrombocytopenia, the other hematological phenotypes associated with
MYH9RD are neutrophil inclusions and bleeding or bruising tendencies. To determine whether
the Myh9Q1443L
line has inclusions, neutrophils were isolated from the blood of mice, fixed to
slides and then stained with α-NMHCIIA. Normal neutrophils have uniform staining for
NMHCIIA throughout the cytoplasm, with no staining visible within the nuclear region, wheras
44
inclusions appear as regions of intense staining (Figure 5-6A). It was found that approximately
15% of all WT samples had some form of inclusion present, while they were found in 40% of the
Myh9Q1443L/+
neutrophils, and 45% of the Myh9Q1443L/Q1443L
samples (p<0.05 and p<0.01,
respectively).
Figure 5-6: Additional Hematopoietic phenotypes in Myh9Q1443L
mice
A) Neutrophil inclusions occur in both the Myh9Q1443/+
and Myh9Q1443L/Q1443L
animals. Myh9Q1443/+
inclusions are larger, while Myh9Q1443L/Q1443L inclusions are much smaller. For both groups,
inclusions are located primarily at the cell membrane. B) Tailbleed assays indicate a partially penetrant
bleeding phenotype is present in Myh9Q1443L/Q1443L
animals. * p<0.05; **p<0.01
45
To determine whether the Myh9Q1443L
strain showed altered bleeding times, mice were also tested
for the amount of time it took for a mouse to produce a clot after 0.5cm of its tail was clipped
(Figure 5-6B). WT mice took an average of 90 ± 15s to stop bleeding, the Myh9Q1443L/+
mice
took 152 ± 37s, and the Myh9Q1443L/Q1443L
mice took 308 ± 64s, giving statistically significant
differences between the Myh9Q1443L/Q1443L
mice and both the WT (p<0.01) and Myh9Q1443L/+
(p<0.05) groups. However, it is also important to determine how the samples are grouped. The
WT mice were in a single cluster between approximately 15 and 150s, with an outlier at about
350s. The Myh9Q1443L/+
mice were primarily in two clusters: one between 20 and 140s, the
second at 580 to 600s. Finally, the Myh9Q1443L/Q1443L
mice were in two distinct clusters, with the
first group having ten mice with bleeding times less than 200s, and the second group of eight
having the maximum tail bleed time allowed by the experiment, 600s. The presence of this
cluster of animals with long bleeding times indicates that this phenotype is robust, but only
partially penetrant.
5.6 Myh9Q1443L
Mice do not Exhibit Changes in Hearing
To determine whether the mouse line exhibited signs of hearing loss, auditory brainstem
responses (ABRs) were measured. We used pure tone stimuli at 4, 8, 16 and 24kHz. Each
mouse was tested with either three or four of the tones, however not all tests produced results due
to signal interference. Mice of each genotype at ages 10- (n=8,6,7; total data points =26,21,23),
27- (n=11,7,9; total =32,20,25), and 54-weeks (n=16,8,13; total=38,26,33) were tested and ABR
thresholds, the minimum volume detected by the mice, were determined. Useable data from
each tone were plotted (Figure 5-7A). Data was unusable if the electrical response from the
mouse was conflated with muscular response; this occurred when the mouse had a gasping
response under anesthetic.
With age, the ABR thresholds for all mouse genotypes increased, which is typical for the mouse.
Comparing WT, Myh9Q1443/+
, and Myh9Q1443L/Q1443L
mice, there appears to be little difference
between ABR thresholds, with statistical significance (p<0.05) only between the 10 week-old
WT and Myh9Q1443/+
mice at 4kHz. The older Myh9Q1443/+
and Myh9Q1443L/Q1443L
animals had
46
more variable responses, both by tone, as well as between animals, as seen by an increase in
error bar size.
Figure 5-7: Minimum hearing thresholds of female mice.
Mice were tested with tones of 4, 8, 16 and 24kHz at 10 weeks (n=8,6,7; total data points
=26, 21, 23), 27 weeks (n=11, 7, 9; total =32, 20, 24) and 54 weeks (n=16, 8, 13;
total=38,26,33). Hearing was tested at volumes between 10 and 80dB; mice that were
non-responsive at 80dB were recorded as having MHRs of 90dB. A) Minimum
thresholds by tone. At 10 weeks, 4kHz, there is a statistically significant difference
between the thresholds of WT and Myh9Q1443L/+ animals. B) Survival style curve
showing the fraction of mice able to hear at or above a specific volume. There are no
statistical differences between the genotypes.
47
For further analysis, threshold data for all tones were grouped together, and survival-style curves
were produced, plotting fraction of animals able to hear at or above specific volumes (Figure 5-
7B). At all time points, there was no statistical change in trend. A Wilcoxon test showed that no
groups were statistically different, however the differences between WT and Myh9Q1443/+
mice at
10-weeks, and between WT and Myh9Q1443L/Q1443L
mice at 54-weeks may have been trending
towards significance.
5.7 Myh9Q1443L
Mice have Increased Serum Urea and Creatine Levels, Highly Increased Rate of Cataract Formation, and Decreased Body Fat Percentage
Studies were done to determine whether the Myh9Q1443L
strain was susceptible to renal or optical
phenotypes associated with MYH9RD. Serum urea and serum creatinine were determined for
mice at 2 and 12 months. At 2 months, there were no differences between the genotypes (data
not shown), but at 12 months, both urea and creatinine were increased (Figure 5-8A, B). The
presence of at least one cataract was determined at 2, 6 and 12 month time points (Figure 5-8C).
Presence of cataracts increased with age for all genotypes, with a dose-response increase
between genotypes at all time points. No WT mice were found to have cataracts at 2 or 6 months
of age, but nearly 20% had developed at least one by 12 months; Myh9Q1443L/+
mice with
cataracts increased from 10% at 2 months to more than 60% at 12 months; 20% of
Myh9Q1443L/Q1443L
mice had cataracts at the 2 month time point, increasing to nearly 80% by 12
months.
At 2, 6 and 12 month time points total body mass, body fat percentages, and the masses of
kidneys and livers were determined (Figure 5-8D-G). There were no differences found at 2 or 6
months for either males or females (data not shown). At 12 months, female Myh9Q1443L/Q1443L
mice had significantly lower total body mass (p<0.001 vs WT, p<0.05 vs Myh9Q1443L/+
), body fat
percentage (p<0.001 vs WT and Myh9Q1443L/+
), kidney mass (p<0.001 vs WT and Myh9Q1443L/+
)
and liver mass (p<0.05 vs WT). Similar results were found for 12 month males (data not
shown), with the exception of body fat percentage, which could not be completed due to the
48
large size of the WT mice. Visual inspection of sacrificed 12 month males indicates a similar
pattern of body fat percentage as in the females.
Figure 5-8: Renal, Optical and Morphological phenotypes.
A,B) Serum urea and serum creatine of 12 month old males show statistically significant increases
between the WT and homozygous animals, indicating damage to the kidney. C) Cataract presence was
determined for 2, 6, and 12 month old animals, with Myh9Q1443L
animals having a much greater rate of
cataracts than their WT counterparts. D-G) Total body mass, liver mass, kidney mass, and body fat
percentage for 12 month old females. (*p<0.05, **p<0.001)
49
5.8 Optimization of Staining Protocol for α-NMHCIIA
To ensure that all immunofluorescent images on tissue sections were optimal for both structure
and staining, each step was completed under various conditions. The first step completed was to
determine optimal staining of the α-NMHCIIA antibody. Kidneys and livers were drop-fixed in
Neutral Buffered Formalin (NBF) for 24 hours, then transferred to 70% ethanol prior to paraffin
embedding and sectioning. Slides were deparaffinized with a xylene/ethanol gradient (Robertson,
2008), and blocked for 1h at room temperature in 10% goat serum albumin (GSA) in TBS. Slides
were stained with Rabbit α-NMHCIIA antibody at dilutions of 1:100, 1:200, and 1:500 (Figure
5-9). The kidney tissue with 1:100 dilution (Figure 5-9A) appeared to have clumping of
antibody, while at 1:200 dilution (Figure 5-9C), the intensity of staining in the glomerulus and
Bowman’s capsule was slightly increased. At 1:500 dilution, the overall intensity of staining
was decreased (Figure 5-9E). In the liver, the antibody stains primarily on the interior side of the
cells, with an increased intensity at 1:200 (Figure 5-9D), and no staining observed at 1:500
(Figure 5-9F). Because of the increased staining of the glomerular region in the kidney, and
more intense staining in the liver, a 1:200 dilution of α-NMHCIIA was determined to be optimal.
To determine correct blocking of the samples, slides from the set described above were
deparaffinized, and blocked with 5% or 10% GSA in TBS, or in 10% GSA in TBST for 1h at
room temperature, then stained with 1:200 Rabbit α-NMHCIIA, and 1:200 Goat α-Rabbit 488
Alexafluor. The slides blocked with 5% GSA in TBS showed poor glomerular staining (Figure
5-10A), and was lacking specific staining in the liver (Figure 5-10B). With a 10% GSA in TBS
block there was specific staining within the glomerular region (Figure 5-10C), and within the
liver (Figure 5-10D). When the slides were blocked with 10% GSA in TBST, the kidney showed
increased non-specific staining in the tubules (Figure 5-10E), and the liver was unstained (Figure
5-10F). With specific staining in both the glomerulus and in the liver, the 10% GSA in TBS was
chosen as the optimal blocking protocol for immuofluorescence.
50
Figure 5-9: α-NMHCIIA antibody optimization
WT Kidneys (A, C, E) and livers (B, D, F) were drop fixed in NBF, and paraffin sections
were prepared. Slides were then stained with α-NMHCIIA at dilutions of 1:100 (A, B),
1:200 (C, D) or 1:500(E, F).
While preparing paraffin coated slides, many proteins are modified by the NBF fixative, the
paraffin, or the xylene used in the deparaffinization steps [139]. To improve the staining quality,
the antigens of interest may be returned to their native states using either chemical or enzymatic
methods after deparaffinization, and before blocking. For chemical retrieval, slides were placed
in solutions of sodium citrate (10mM, pH6.0) (Figure 5-11A,B), Tris-EDTA (10mM, pH 9.0)
(Figure 5-11C,D) and EDTA (1mM, pH 8.0) (Figure 5-11E) were either heated in a microwave
for 10 minutes, (Figure 5-11A,C,E) or heated in 50mL tubes in a beaker of water heated to 95°C
on a hot plate for 20 min (Figure 5-11B,D). Enzymatic retrieval (Figure 5-11F) used a 0.05%
trypsin solution incubated at 37°C in a humidified chamber for 15 minutes. Slides were co-
51
stained with Rabbit α-NMHCIIA and α-Rabbit 488 Alexafluor. Although several of the methods
had non-specific staining, staining was best for the Tris-EDTA solution on the hotplate (Figure
5-10D), and the microwave-heated EDTA solution (Figure 5-11E). The boiled Tris-EDTA
solution had specific glomerular expression of NMHCIIA, with very little tubular staining, while
the staining for the microwaved EDTA solution was not specific for the glomerulus, therefore
Tris-EDTA was selected as the best antigen retrieval method. Another effect of the chemical
antigen retrieval methods was that during the heating, some of the samples separated from the
slide, particularly in the testis and the eye (data not shown).
Figure 5-10: Blocking optimization
Kidney (A, C, E) and liver (B, D, F) sections were prepared identically and blocked for
1h at room temperature in 5% GSA in TBS (A,B), 10% GSA in TBS (C, D) or 10% GSA
in TBST (E, F), then probed with 1:200 Rabbit α-NMHCIIA and 1:200 Goat α-Rabbit
488 Alexafluor.
52
Figure 5-11: Antigen retrieval optimization
Antigen retrieval was attempted by microwaving (A, C, E) and heating on a hot plate (B,
D) sodium citrate (A, B), Tris-EDTA (C, D), and EDTA (E), and by enzymatic (F)
methods. Optimal staining was observed for the Tris-EDTA solution on the hot plate (D).
Because antigen retrieval was not entirely successful, tissue samples were prepared using four
different methods: drop fixing organs in NBF as described above (treatment i); flash freezing
organs in liquid nitrogen, and then cryosectioning (treatment ii); paraformaldehyde perfusion,
then paraffin sectioning (treatment iii); or paraformaldehyde perfusion, then applying a sucrose
gradient (15% for 24h then 30% for 24h) to the tissues, followed by cryosectioning (treatment
53
iv). Slides were either deparaffinized (i and iii) or brought to room temperature (ii and vi), then
blocked, and stained with α-NMHCIIA (Figure 5-12A,C,E) or 1:500 α-NMHCIIB (Figure 5-
12B,D,E) overnight, and then probed with secondary antibodies (1:200 488 α-Rabbit; 1:500 555
α-Mouse) and DAPI. Kidney (Figure 5-12A,B), liver (Figure 5-12C,D) and eye (Figure 5-12E,F)
tissues were prepared.
Figure 5-12: Tissue preparation optimization.
Kidney (A, B), liver (C,D) and eye (E,F) tissues were sectioned by four different
techniques: drop fixing and paraffin sections (i); flashfreezing and cryosections (ii);
paraformaldehyde perfusion and paraffin sections (iii); and paraformaldehyde perfusion
and cryosections (iv). Tissue sections were stained with DAPI and either α-NMHCIIA
(A,C,E) or α-NMHCIIB (B,D,F).
54
Staining in the drop-fixed slides (Figure 5-12, treatment i) was comparable with those previously
observed. Flash frozen slides (Figure 5-12, treatment ii) had much more intense staining, with
particularly strong staining in the kidney and eye (Figure 5-12Bii, Eii, Fii), and a different
NMHCIIA pattern than in treatment i (Figure 5-12Cii). Upon close inspection the nuclei of all
tissues from treatment ii appear smeared, and non-cohesive. Treatment iii has reduced staining
in all samples compared to treatment i. Treatment vi has staining intensity between the levels
observed in treatments i and ii, but also had the benefit of having normal appearing nuclei. No
usable slides were produced from the eye in treatment vi. Treatments i and vi were selected as
preferred. Because perfusion (treatment iii) was found to stain more poorly than drop fixing
(treatment i), the drop-fixation technique was selected.
To determine the optimal solution for drop fixing tissue samples, NBF was compared to Bouin’s
Solution for both paraffin sections and cryosections. NBF drop fixation was completed as
described above; for Bouin’s solution, organs were drop fixed for 6 hours, and rinsed in PSB for
12h twice. Each set of organs was then divided, with half being placed in 70% ethanol for
paraffin embedding, and the other half going through a sucrose gradient for cryosectioning.
Deparaffinization, and blocking are as described above; slides were co-stained for α-NMHCIIA
and α-NMHCIIB using previously described concentrations.
Slides prepared in this manner included kidneys (Figure 5-13A-D) and eyes (Figure 5-13E-H)
fixed with either NBF (Figure 5-13A,B,E,F) or Bouin’s solution (Figure 5-13C,D,G,H). Slides
were then prepared using either paraffin embedding (Figure 5-13A,C,E,G) or cryosectioning
(Figure 5-13B,D,F,H). Paraffin sections for both fixes and both tissues showed little to no
specific staining, while the cryosections showed clear NMHCIIA staining (green), and some
NMHCIIB staining (red) in the kidneys. The frozen NBF sample (Figure 5-13B) showed much
more change in morphology than the Bouin fixed sample (Figure 5-13D), however in the eye,
there was more ripping and tearing in the Bouin’s sample than in the NBF sample (Figure 5-
13H,F). As there was no strong morphological difference to the glomerulus, and due to the
difficulty associated with preparing eye slides, NBF was selected as fixative over Bouin’s
solution.
55
Figure 5-13: Tissue preparation optimization (cont)
Kidneys – A to D – and eyes – E to G – were fixed with either Neutral Buffered
Formamide (NBF) – A, B, E, F – or Bouin’s Fixative – C, D, G, H. Sections were then
prepared after embedding in either paraffin – A, C, E, G – or OTC (cryosections) – B, D,
F, H. Slides were stained for α-NMHCIIA (green), α-NMHCIIB (red) and with DAPI.
NBF fixed cryosections were determined to give the optimal staining.
With the slide preparation confirmed, the optimal co-staining levels for Mouse α-NMHCIIB and
Goat α-Mouse 594 Alexafluor were determined. Using NBF fixed cryosections, kidney slides
were prepared with primary antibodies at 1:200 (Figure 5-14B,D) or 1:500 (Figure 5-14C,E), and
56
secondary antibodies at 1:200 (Figure 5-14B,C) or 1:500 (5-14D,E). A control slide, with 1:200
Goat α-Mouse 594 Alexafluor only was used to calibrate and adjust the images (Figure 5-14A).
Both slides stained using a 1:200 dilution of secondary antibody (Figure 5-14B,C) produced
similar looking images, with a broad range of intensities observed. For the sample stained with
1:200 primary and 1:500 secondary (Figure 5-14D), there appears to be a high level of
background, or non-specific staining, and very few regions of high intensity. The slide stained at
1:500 for both primary and secondary antibodies (Figure 5-14E) had low levels of staining,
though it did appear to be specific.
Figure 5-14: α-NMHCIIB optimization
NBF-fixed Kidney cryosections sections were either unstained – A – or stained overnight
with Mouse α-NMHCIIB at dilutions of either 1/200 – B, D – or 1/500 – C, E. A
secondary stain, Goat α-Mouse 594 Alexafluor was added for 1h at dilutions of either
1/200 – B, C – or 1/500 – D, E. C chosen as optimal.
57
5.9 Myh9Q1443L
Mice have Altered Kidney Morphology and Expression Patterns in NMHCIIB
Kidneys of 14 week old male mice of each genotype were co-stained for NMHCIIA and IIB.
WT mice (Figure 5-15A) had highly cellular glomeruli, and generally possessed a thin
Bowman’s capsule and a small, well defined Bowman’s space. For WT samples, expression
within the glomeruli was primarily for NMHCIIA, with intense areas in the podocytes, which
surrounded the capillaries in the glomeruli. NMHCIIB was also present in the capillary area for
most glomeruli, however, the expression was less consistent than the IIA, and fewer regions of
IIB expression were observed. Tubules in the WT mice contained both NMHCIIA and IIB,
however there was generally more IIA than IIB. Kidney of Myh9Q1443L/+
mice (Figure 5-15B)
showed some indications of damage, with increased Bowman’s space (top), or thickening of the
Bowman’s capsule (bottom) present in some of the glomeruli observed. Expression for
NMHCIIA and IIB was similar to in the WT, with NMHCIIB expression in the tubules slightly
increased (Figure 5-15B, middle) compared to WT samples. In Myh9Q1443L/Q1443L
mice,
significant changes in glomerular morphology were observed (Figure 5-15C), with many
glomeruli having increased Bowman’s space (top, middle) and thickened Bowman’s capsules
(top, bottom). The size of the glomeruli was also increased compared to WT and Myh9Q1443L/+
mice. Expression in Myh9Q1443L/Q1443L
glomeruli showed increased NMHCIIB, however, the
location of this increased expression did not appear to be consistent, with some glomeruli having
increased expression in the membrane surrounding the Bowman’s capsule (top, bottom), and
others having increased staining around the capillaries (middle, bottom). There also appeared to
be increased NMHCIIB expression in some tubules (bottom).
58
Figure 5-15: Kidney morphology and protein expression in Myh9Q1443L
mice.
Glomeruli of WT (A), Myh9Q1443L/+
(B) and Myh9Q1443L/Q1443L
(C) animals stained with α-
NMHCIIA(green), α-NMHCIIB (red), and Dapi (blue). Changes in morphology,
particularly increased Bowman’s space are present Myh9Q1443L/Q1443L
glomeruli, as is an
increase in expression of NMHCIIB.
59
6 Discussion
The 7238 mouse line was initially indentified with thrombocytopenia with a gene-dose response
using the AcTDiff machine, while the Hemovet machine indicated giant platelets, also with a
gene-dose response. The likely reason for this difference in values is the mechanism by which
each machine identifies different blood components. The AcTDiff machine differentiates
between red blood cells and platelets solely based on volume [140], therefore, any component
larger than a specific size is classified as a red blood cell or a reticulocyte. Since platelets are in
the 103/µL range, and red blood cells are in the 10
6/µL range, no significant change in RBC
counts would be observed, even with significant mis-identification of platelets as RBCs. The
Hemavet machine uses “Focused Flow” technology which more accurately distinguishes
between cell types [141], and therefore is less likely to mis-classify giant platelets as red blood
cells or reticulocytes. Determining that the phenotype was giant platelets, not a reduced number
of platelets, also helped indentify Myh9 as a candidate gene.
Next generation sequencing was used to identify 275 sites of interest in a 4.25Mb region of
chromosome 15. Of the 20 sites selected for sequencing due to their location within the gene, 18
were found to be previously unknown SNPs between the B6 and 129 strains. Two sites were
found to be ENU mutations. C77710527A is located between the Txn2 and Myh9 genes, and
T77599238A, which causes a Q1443L mutation within NMHCIIA. The remaining 255 sites have
not been sequenced, but have a high probability of being novel B6/129 SNPs. This data
indicates the power of next generation sequencing. A more complete experimental design,
which would include DNA samples from both parental strains, would enable each SNP between
the strains to be identified for the region, and would reduce the requirement of Sanger
sequencing to determine whether a site of interest was due to a novel mutation or a previously
unknown SNP. This sequencing also confirms the mutation rate of ENU, with two mutations in
a 4.25Mb region, giving a rate of at least one mutation in about 2.12Mb. This is quite
comparable to the one mutation in 1.83Mb that has been previously reported [27]. However,
since only 20 of the 275 sites of interest were confirmed, the remaining 255 sites may contain
more ENU sites, further increasing the rate of ENU mutations present in this experiment.
60
The Myh9Q1443L mutation and the 7238 phenotype have a nearly perfect correlation. But
correlation does not ensure causation, particularly since this mutation has not previously been
associated with MYH9RD. However, since MYH9 was determined to be the causative gene for
MYH9RD in 2000 (referred to as May-Hegglin, Sebastien, Epstein and Fetchner’s Syndromes at
the time), 43 different causative mutations have been found (Table 2-1). The rate of discovery
appears to have slowed only slightly in the 11 years since the link between the disease and this
gene was discovered, with 10 published since 2009 [71,72,75,87,89]. It is therefore possible that
although not a currently known MYH9RD mutation, it may at some point be observed in a
human patient.
Despite not being a described MYH9RD mutation, Myh9Q1443L is similar to known MYH9RD
mutations located nearby, in that it is associated with a change in amino acid type. Individuals
with the D1447V/H mutation are not well described for the presence or absence of aural, renal
and optical phenotypes, however phenotypes in all three of those areas have been found in
individuals with D1424Y/N/H mutations.
The intergenic location of the C77710527A mutation indicates that it is unlikely to cause the
phenotype observed in the Myh9Q1443L
mice. In the intergenic regions, mutations causing
phenotypes would have to interfere with a regulatory region, such as a promoter, repressor or
enhancer element. Promotor regions, such as TATA boxes, are usually found 50bp upstream of
the start site [142], while most enhancer regions are generally found within 100kb of a gene
[143]. Since this mutation is well beyond 100kb from the start site of either gene, it is highly
unlikely that this mutation could change the expression pattern of either gene.
Myh9Q1443L
mice were found to have most of the components associated with MYH9RD: giant
platelets, neutrophil inclusions, increased bleeding and bruising, increased serum and creatinine
levels, and increased rates of cataracts. They also have a phenotype not currently associated with
MYH9RD – a decrease in body fat accumulation in older individuals.
The rate of neutrophil inclusions in the WT mice tested was 15%, which is higher than would be
expected. Neutrophil inclusions are often observed under Giemsa staining when the organism is
fighting a disease [144-147], though it has not been reported whether this has also be observed
with α-NMHCIIA staining. It is therefore possible that the WT neutrophils with inclusions
61
indicate a disease or infection present in the mouse colony. Since the room in which the mice
are caged is known to have Mouse Hepatitis Virus, this underlying disease may play a role in the
increased neutrophil rate in the WT mice. Despite that, the rate of neutrophil inclusions in the
Myh9Q1443L/+
and Myh9Q1443L/Q1443L
mice is increased compared to the WT mice. In human cases,
neutrophil inclusions are almost always described as either “present” or “absent”, however in the
one study where the percentage of neutrophils with inclusions is indicated, two families have
inclusions at 10-25%, another is reported as “~100%” and the last had no observed inclusions
[72]. This indicates that within the human MYH9RD population, there is variability of
neutrophil inclusion rates, which may be determined by the location of the mutation.
Increased bleeding and bruising is often associated with MYH9RD. Figure 5-5B shows that there
is clearly a poor clotting phenotype associated with the Myh9Q1443L
allele that is partially
penetrant. It also shows that this phenotype is gene dose responsive, as 12.5% of the
Myh9Q1443L/+
mice were unable to clot, while 44% of the Myh9Q1443L/Q1443L
animals failed to clot
within the 600s allotted.
No significant hearing loss was observed in Myh9Q1443L
mice, however the location of the
mutation appears to be highly related to the presence of any hearing loss phenotype in humans.
In a comparison of hearing loss in 108 consecutive MYH9RD patients, 55% of individuals with
tail domain mutations (beyond aa 873) had hearing loss, compared to 81% with motor domain
mutations [8]. When further separated into individual mutations, of the 31 known tail domain
sites, patients encoding 26 mutations have been tested for hearing loss, and 14 of those sites have
never been associated with hearing loss in the literature (Table 2-1). It is therefore possible that
the Myh9Q1443L
mutation does not affect the protein function in the cochlea. Additionally, the B6
mouse strain is known to have progressive hearing loss starting at about 3 months [148-152], and
it is therefore possible that hearing defects due to the Myh9 mutation may be masked by hearing
loss due to strain effects.
Increased serum urea and creatinine levels are indicative of renal damage, specifically incorrect
filtration at the glomeruli [109,144,145,153,154]. These results, particularly at 2 and 6 months
are consistent with the podocyte-specific Myh9 knockout, which showed no differences between
the WT and KO at 9 months [124]. Further to this, mice with a B6 background are known to
62
have a protective kidney phenotype [90,155], where damage or removal of up to 7/8 of B6
kidney results in no changes in proteinuria or blood pressure. These, along with the serum
values done in our study, are all indicators of glomerulosclerosis.
The presence of presenile cataracts is a major phenotypic component of MYH9RD [8], with
approximately 16% of tested individuals developing cataracts, with an average onset at 24 years
of age. Myh9Q1443L
mice appear to have a higher rate of cataract development than discussed in
the above paper, however, it is difficult to do a comparison due to different aging patterns
between humans and mice. Additionally, B6 mice are known to develop cataracts [156], with 5-
15% showing some form of ocular abnormality by adulthood [157]. This known susceptibility to
cataract development may have caused a compounding effect with the Myh9 mutation, leading to
an unexpected increase in the rate of cataracts in this mouse line.
The B6 mouse line was selected for this ENU project because its genomic sequence has been
completed, it is one of the more commonly used lines, and because blood parameters for this line
are well documented. However, the B6 line is known to develop early hearing loss, have a
highly protective kidney phenotype, and develop cataracts, which is sub-optimal for determining
the presence or absence of the auditory, renal and optical phenotypes associated with mutations
in Myh9.
The observed decrease in body mass in Myh9Q1443L/Q1443L
mice at 12 months has no known
related human phenotype, as no humans have been found to be homozygous for any MYH9RD
allele. This decrease in mass and body fat percentage may indicate that this protein may play a
role in metabolism or fat storage, revealing additional functions of NMHCIIA.
Determining the best concentration of α-NMHCIIA antibody was chosen as the first step of
immunofluoresence optimization, as this protein is the focus of the research. Optimizing this
antibody ensures that all images taken afterward will have a minimum quality of NMHCIIA
staining. The increased intensity of the staining in the glomerular region was important due to
known NMHCIIA expression in the podocytes, and the preferential expression of NMHCIIA
observed in the liver mimics the preferential expression observed in motile cells [158].
63
Blocking with 10% GSA in TBS was determined to be the best treatment, as there was less non-
specific binding than observed in the blocking with TBST, and better binding than with 5%
GSA. Four different types of antigen retrieval were attempted, with some improvement
observed when boiling slides in a Tris-EDTA solution on a hotplate. An unfortunate side effect
of heating the slides in this manner was that in many cases, the tissue was unable to adhere to the
glass as well, and much of the tissue was lost either during antigen retrieval, or during staining.
To determine an alternative to antigen retrieval, several different methods of tissue preparation
were attempted, comparing pre-fixing to post-fixing and paraffin sections to cryosections.
Overall, the best staining was found using fresh-frozen sections, however these tissues also had
damaged nuclei, and abnormal staining patterns, particularly in the liver, compared to all other
fixing techniques. The two optimal methods appeared to be drop-fixing in paraffin (treatment i)
and perfusion followed by cryosectioning (treatment vi), as they had normal nuclear structure,
minimal background staining, and a range of expression levels within the tissues. Further,
because the staining from the drop fixed paraffin samples (treatment i) had better staining than
the perfused paraffin samples (treatment iii), it was decided that further testing would drop fix all
samples, and then make both paraffin and cryosections.
Poor staining of both NMHCIIA and IIB in all paraffin sections prevented this sample
preparation technique from being selected. There were some morphological changes observed in
the samples which were drop fixed, followed by cryosectioning. It may be that the change in
osmotic pressure causes the cells to swell, and then partially dissociate with each other.
Fortunately, most of the morphological changes were limited to the proximal and distal tubules –
areas with very little NMHCIIA or IIB activity, leaving the glomeruli in good form. In the eye,
there is some difficulty in cutting through the lens without causing it to shear off, fold over, rip,
or tear. In general, this type of damage can be alleviated by placing only one eye in each
cryosectioning mold, reducing the chance that the bladeskips, and damages the lens. Despite
this, there was still more damage observed in the eye fixed in Bouin’s solution than in NBF.
Because of this, and the preference that all organs be prepared in the same manner, NBF and
cryosectioning were chosen as the preferred method of sample preparation.
64
Optimizing the staining of the α-NMHCIIB antibody found that similar levels of intensity and
range could be observed for both slides using a 1:200 dilution of the secondary antibody. Since
there was no improvement in using more α-NMHCIIB antibody, a 1:500 dilution was selected as
the more economical method.
Changes in both morphology and nonmuscle myosin expression in the kidney shows that defects
in NMHCIIA are associated with damage to the kidney, and that there is a compensation
mechanism between members of this family within this organ. In cannot be determined if the
mutated NMHCIIA causes the damage, or if mutated proteins are less capable of repairing
damage that occurs during the normal course of kidney function. The increase in NMHCIIB
expression appears to be in response to the kidney damage, rather than a decrease in NMHCIIA
expression, as IIA expression appears very consistent between genotypes. Despite the increased
glomerular damage, there is no change in kidney function for these mice at the 2 month time
point.
During the writing of this thesis, a paper presenting MYH9 mouse models for the three most
commonly observed mutations was published [159]. The models for D1424N and E1841K
produced viable heterozygotes and homozygotes, while the R702C mouse model was viable only
as a heterozygote. These mice also show the variability of the MYH9RD phenotype; cataracts
were observed in both heterozygous and homozygous D1424N animals, and in heterozygous
E1841K; hearing loss was only reported in the E1841K heterozygotes; and all five groups had
indications of kidney damage.
The lack of cataract and hearing phenotype in the R702C mice is particularly interesting given
that in humans, the R702C mutations are generally indicative that the affected individual will
have all three non-hematalogical phenotypes [8,110]. Further, although all five mouse genotypes
display renal damage at the cellular level, it is different from the type of damage observed in the
Myh9Q1443L
mice, in that the mice in Zhang’s paper are described as having focal
glomerulosclerosis, while the Myh9Q1443L
mice exhibit various structural changes, focused
around Bowman’s Capsule.
Finally, since we have been studying the Myh9Q1443L
mouse line since 2005, we have had ample
opportunity to observe phenotypes in aged mice, as well as those not previously observed in
65
humans. These mouse lines will be of great use to better understand the mechanism behind
MYH9RD, but do not eclipse the work done on the Myh9Q1443L
mouse line, as there are truly
observable differences between the phenotypes of their lines, and that presented in this thesis.
66
7 Conclusions and Future Directions
The Myh9Q1443L
mouse line is the first ENU mouse line to be identified via sequence capture
technology. Sequence capture is a still improving technology, having had a maximum capture
region of 5Mb in 2008, and 50Mb currently. This, combined with the decreasing cost of next
generation sequencing, has made random mutagenesis a more efficient and effective research
tool. A larger capture region means fewer crossovers are required, greatly reducing the number
of generations needed to accurately identify a causative mutation, and increasing the throughput
of random mutagenesis screens.
The Myh9Q1443L
mouse line was the first mouse model of MYH9RD to be presented. Identifying
mouse models for MYH9RD will allow for a better understanding of how MYH9 mutations
cause the phenotypes affecting individuals, as well as providing a starting point to develop
treatments that may be able to prevent the worst aspects of the disease. This is particularly
important due to the complications of incomplete penetrance of the aural, renal and optical
phenotypes observed in patients with tail domain mutations.
The Myh9Q1443L
mouse line presents with abnormally large platelets, increased bleeding,
neutrophil inclusions, a high incidence of cataract formation, and damage to the kidneys, with no
change in renal output. There are currently no indications of a hearing defect. However,
because of the known characteristics of the B6 line – early hearing loss, highly protective kidney
phenotype, and increased rate of cataract formation – the phenotypes may be better observed on
a different background, such as 129 or DBA/2J.
A complete study how genotype effects the expression of Myh9, 10 and 14 RNA and NMHCIIA,
–IIB and –IIC in various tissues would be extremely valuable, particularly to determine if there
are any additional systems that undergo changes, but have not produced an obvious phenotype in
humans. One such system may be the metabolic system, due to the extreme changes in body fat
composition in homozygous Myh9Q1443L
mice. This study would also help to determine if either
NMHCIIB or –IIC is capable of a complete or partial rescue of damaged NMHCIIA.
67
A better understanding of the mechanistic effects of the mutation could be obtained by modelling
the protein structure, and determining how this tail domain mutation changes the shape and
binding characteristics of the protein.
Interactions between NMHCIIA, IIB, and IIC could also be studied by intercrossing the knock
out and knock down lines of NMHCIIB and IIC with the Myh9Q1443L
line. This would provide a
better understanding of which, if any, of these myosins are capable of rescuing the system when
another myosin is broken or missing. It would also be useful to determine if the addition of
normal NMHCIIA could stop, or even reverse, the damage caused by Myh9Q1443L
protein. This
would be a more complicated series of experiments, as a conditional knock-in mouse line for
normal Myh9 would first have to be produced.
Although this mouse line shows that mouse models of MYH9RD can fully recapitulate the
human MYH9RD phenotype, and that there are strong changes in both RNA and protein
expression, it is a tool that has just been discovered. Because this line exists, it is possible to ask
more specific questions about the role of MYH9, the molecular causes of MYH9RD, and what, if
anything, can be done to reduce the severity of this condition for afflicted individuals.
68
8 References
(1) An ENU-Derived Mouse Model of MYH9-Related Disease Identified by Massively Multiple
Parallel Sequencing. ; 06/12; Barcelona, Spain; 2010.
(2) Anderson N,M., Berndl E, Berberovic Z, Reheman A, Brown T,A., Bailey M,L., et al.
Myh9Q1443L Is a Novel Mouse Model of MYH9-Related Disorders. ASH Annual Meeting
Abstracts 2010 11/19;116(21):2527-2527.
(3) Hughes MR, Anderson N, Maltby S, Wong J, Berberovic Z, Birkenmeier CS, et al. A novel
ENU-generated truncation mutation lacking the spectrin-binding and C-terminal regulatory
domains of Ank1 models severe hemolytic hereditary spherocytosis. Experimental Hematology
2011 03;39(3):305-320.e2; 305-320.e2.
(4) Neubauer H, Cumano A, Müller M, Wu H, Huffstadt U, Pfeffer K. Jak2 deficiency defines an
essential developmental checkpoint in definitive hematopoiesis. Cell 1998 05/01;93(3):397-409.
(5) Chen Z, Shivdasani R,A. Regulation of platelet biogenesis: insights from the May-Hegglin
anomaly and other MYH9-related disorders. J Thromb Haemost 2009 07;7 Suppl 1:272-276.
(6) Dong F, Li S, Pujol-Moix N, Luban NL, Shin SW, Seo JH, et al. Genotype-phenotype
correlation in MYH9-related thrombocytopenia. Br J Haematol 2005;130(4):620-627.
(7) Heath KE, Campos-Barros A, Toren A, Rozenfeld-Granot G, Carlsson LE, Savige J, et al.
Nonmuscle Myosin Heavy Chain IIA Mutations Define a Spectrum of Autosomal Dominant
Macrothrombocytopenias: May-Hegglin Anomaly and Fechtner, Sebastian, Epstein, and Alport-
Like Syndromes. The American Journal of Human Genetics 2001 11;69(5):1033-1045.
(8) Pecci A, Panza E, Pujol-Moix N, Klersy C, Bari FD, Bozzi V, et al. Position of nonmuscle
myosin heavy chain IIA (NMMHC-IIA) mutations predicts the natural history of MYH9-related
disease. Hum Mutat 2008;29(3):409-417.
69
(9) Peterson L, Rao K, Crosson J, White J. Fechtner syndrome--a variant of Alport's syndrome
with leukocyte inclusions and macrothrombocytopenia. Blood 1985 02/01;65(2):397-406.
(10) Kashtan C,E., Michael A,F. Alport syndrome. Kidney Int 1996 11;50(5):1445-1463.
(11) Seri M, Pecci A, Di Bari F, Cusano R, Savino M, Panza E, et al. MYH9-related disease:
May-Hegglin anomaly, Sebastian syndrome, Fechtner syndrome, and Epstein syndrome are not
distinct entities but represent a variable expression of a single illness. Medicine (Baltimore) 2003
05;82(3):203-215.
(12) Velemínsky J, Gichner T, Pokorny V. The effect of inhibitors on the mutagenic activity of
N-methyl-N-nitrosourea and N-ethyl-N-Nitrosourea in Arabidopsis thaliana. Biol Plant 1965
07/01;7(4):325-329.
(13) Druckrey H, Ivankavic S, Preussmann R. Teratogenic and Carcinogenic Effects in the
Offspring after Single Injection of Ethylnitrosourea to Pregnant Rats. Nature 1966
06/25;210(5043):1378-1379.
(14) Russell W,L., Kelly E,M., Hunsicker P,R., Bangham J,W., Maddux S,C., Phipps E,L.
Specific-locus test shows ethylnitrosourea to be the most potent mutagen in the mouse. Proc Natl
Acad Sci U S A 1979 11;76(11):5818-5819.
(15) Fossett NG, Arbour-Reily P, Kilroy G, McDaniel M, Mahmoud J, Tucker AB, et al.
Analysis of ENU-induced mutations at the Adh locus in Drosophila melanogaster. Mutation
Research/Fundamental and Molecular Mechanisms of Mutagenesis 1990 07;231(1):73-85.
(16) Pastink A, Vreeken C, Nivard M, Searles LL, Vogel EW. Sequence Analysis of N-Ethyl-N-
Nitrosourea-Induced vermilion Mutations in Drosophila melanogaster. Genetics
1989;123(1):123-129.
(17) De Stasio E, Lephoto C, Azuma L, Holst C, Stanislaus D, Uttam J. Characterization of
Revertants of unc-93(e1500) in Caenorhabditis elegans Induced by N-ethyl-N-nitrosourea.
Genetics 1997 10/01;147(2):597-608.
70
(18) Huang CG, Agre P, Strange K, Lamitina T. Isolation of C. elegans Deletion Mutants
Following ENU Mutagenesis and Thermostable Restriction Enzyme PCR Screening. MB
2006;32(1):083-086.
(19) Haffter P, Granato M, Brand M, Mullins MC, Hammerschmidt M, Kane DA, et al. The
identification of genes with unique and essential functions in the development of the zebrafish,
Danio rerio. Development 1996 12/01;123(1):1-36.
(20) Mullins MC, Hammerschmidt M, Haffter P, Nüsslein-Volhard C. Large-scale mutagenesis
in the zebrafish: in search of genes controlling development in a vertebrate. Current Biology
1994 03/01;4(3):189-202.
(21) Nolan PM, Hugill A, Cox RD. ENU mutagenesis in the mouse: Application to human
genetic disease. Brief Funct Genomic Proteomic 2002 01/01;1(3):278-289.
(22) Justice MJ, Noveroske JK, Weber JS, Zheng B, Bradley A. Mouse ENU Mutagenesis. Hum
Mol Genet 1999 09/01;8(10):1955-1963.
(23) Zhang Z, Alpert D, Francis R, Chatterjee B, Yu Q, Tansey T, et al. Massively parallel
sequencing identifies the gene Megf8 with ENU-induced mutation causing heterotaxy. Proc Natl
Acad Sci U S A 2009 03/03;106(9):3219-3224.
(24) Graw J, Jung M, Löster J, Klopp N, Soewarto D, Fella C, et al. Mutation in the
[beta]A3/A1-Crystallin Encoding Gene Cryba1 Causes a Dominant Cataract in the Mouse.
Genomics 1999 11/15;62(1):67-73.
(25) Hitotsumachi S, Carpenter D,A., Russell W,L. Dose-repetition increases the mutagenic
effectiveness of N-ethyl-N-nitrosourea in mouse spermatogonia. Proc Natl Acad Sci U S A 1985
10;82(19):6619-6621.
(26) Russell W,L., Hunsicker P,R., Raymer G,D., Steele M,H., Stelzner K,F., Thompson H,M.
Dose--response curve for ethylnitrosourea-induced specific-locus mutations in mouse
spermatogonia. Proc Natl Acad Sci U S A 1982 06;79(11):3589-3591.
71
(27) Quwailid MM, Hugill A, Dear N, Vizor L, Wells S, Horner E, et al. A gene-driven ENU-
based approach to generating an allelic series in any gene. Mamm Genome 2004 08;15(8):585-
591.
(28) Nolan PM, Peters J, Strivens M, Rogers D, Hagan J, Spurr N, et al. A systematic, genome-
wide, phenotype-driven mutagenesis programme for gene function studies in the mouse. Nat
Genet 2000;25(4):440-443.
(29) Eldor A, Vlodavsky I, Deutsch V, Levine RF. Megakaryocyte function and dysfunction.
Baillière's Clinical Haematology 1989 07;2(3):543-568.
(30) Deutsch VR, Tomer A. Megakaryocyte development and platelet production. Br J Haematol
2006;134(5):453-466.
(31) Lok S, Kaushansky K, Holly RD, Kuijper JL, Lofton-Day CE, Oort PJ, et al. Cloning and
expression of murine thrombopoietin cDNA and stimulation of platelet production in vivo.
Nature 1994 06/16;369(6481):565-568.
(32) Italiano J,E., Hartwig J,H. Megakaryocite Development and Platelet Formation. In:
Michelson AD, editor. Platelets Boston: Academic Press; 2002. p. 21-35.
(33) Tomer A, Harker L, Burstein S. Purification of human megakaryocytes by fluorescence-
activated cell sorting. Blood 1987 12/01;70(6):1735-1742.
(34) Chang Y, Bluteau D, Debili N, Vainchenker W. From hematopoietic stem cells to platelets.
J Thromb Haemost 2007 07;5 Suppl 1:318-327.
(35) Italiano J,E., Patel-Hett S, Hartwig J,H. Mechanics of proplatelet elaboration. J Thromb
Haemost 2007 07;5 Suppl 1:18-23.
(36) Batar P, Dale G,L. Platelet Turnover and Aging. In: Michelson AD, editor. Platelets Boston:
Academic Press; 2002. p. 53-64.
(37) Giles C. The Platelet Count and Mean Platelet Volume. Br J Haematol 1981 05/01;48(1):31-
37.
72
(38) George J. Overview of Platelet Disorders: Thrombocytopenia and Platelet Dysfunction.
2009; .
(39) Michelson A,D. The Clinical Appreoach to Disorders of Platelet Number and Function. In:
Michelson AD, editor. Platelets Boston: Academic Press; 2002. p. 541-545.
(40) Bussel J,B. Immune Thrombocytopenic Purpura. In: Michelson AD, editor. Platelets
Boston: Academic Press; 2002. p. 547-557.
(41) Chenaille P, Steward S, Ashmun R, Jackson C. Prolonged thrombocytosis in mice after 5-
fluorouracil results from failure to down-regulate megakaryocyte concentration. An experimental
model that dissociates regulation of megakaryocyte size and DNA content from megakaryocyte
concentration. Blood 1990 08/01;76(3):508-515.
(42) Radley JM, Scurfield G. Effects of 5-Fluorouracil on Mouse Bone Marrow. Br J Haematol
1979;43(3):341-351.
(43) Chong B,H. Heparin-Induced Thrombocytopenia. In: Michelson AD, editor. Platelets
Boston: Academic Press; 2002. p. 571-591.
(44) Chan H,H.W., Gill K,K., Kelton J,G. Thrombocytopenia in Pregnancy. In: Michelson AD,
editor. Platelets Boston: Academic Press; 2002. p. 621-633.
(45) Mhawech P, Saleem A. Inherited Giant Platelet Disorders. Am J Clin Pathol 2000
02/01;113(2):176-190.
(46) Nurden P. Congenital disorders associated with platelet dysfunctions. Thrombosis &
Haemostasis 2008;99(2):253-253.
(47) Savoia A, Pastore A, De Rocco D, Civaschi E, Di Stazio M, Bottega R, et al. Clinical and
genetic aspects of Bernard-Soulier syndrome: searching for genotype/phenotype correlations.
Haematologica 2011 03/01;96(3):417-423.
73
(48) Albers C,A., Cvejic A, Favier R, Bouwmans E,E., Alessi M, Bertone P, et al. Exome
sequencing identifies NBEAL2 as the causative gene for gray platelet syndrome. Nat Genet
2011;43(8):735-737.
(49) Kahr W,H.A., Hinckley J, Li L, Schwertz H, Christensen H, Rowley J,W., et al. Mutations
in NBEAL2, encoding a BEACH protein, cause gray platelet syndrome. Nat Genet
2011;43(8):738-740.
(50) Tubman VN, Levine JE, Campagna DR, Monahan-Earley R, Dvorak AM, Neufeld EJ, et al.
X-linked gray platelet syndrome due to a GATA1 Arg216Gln mutation. Blood 2007
04/15;109(8):3297-3299.
(51) Nurden P, Gobbi G, Nurden A, Enouf J, Youlyouz-Marfak I, Carubbi C, et al. Abnormal
VWF modifies megakaryocytopoiesis: studies of platelets and megakaryocyte cultures from
patients with von Willebrand disease type 2B. Blood 2010 04/01;115(13):2649-2656.
(52) Federici AB, Mannucci PM, Castaman G, Baronciani L, Bucciarelli P, Canciani MT, et al.
Clinical and molecular predictors of thrombocytopenia and risk of bleeding in patients with von
Willebrand disease type 2B: a cohort study of 67 patients. Blood 2009 01/15;113(3):526-534.
(53) Jackson SC, Sinclair GD, Cloutier S, Duan Z, Rand ML, Poon M. The Montreal platelet
syndrome kindred has type 2B von Willebrand disease with the VWF V1316M mutation. Blood
2009 04/02;113(14):3348-3351.
(54) Berg JS, Powell BC, Cheney RE. A Millennial Myosin Census. Mol Biol Cell 2001
04/01;12(4):780-794.
(55) Vicente-Manzanares M, Ma X, Adelstein RS, Horwitz AR. Non-muscle myosin II takes
centre stage in cell adhesion and migration. Nat Rev Mol Cell Biol 2009 11;10(11):778-790.
(56) Marigo V, Nigro A, Pecci A, Montanaro D, Di Stazio M, Balduini CL, et al. Correlation
between the clinical phenotype of MYH9-related disease and tissue distribution of class II
nonmuscle myosin heavy chains. Genomics 2004 06;83(6):1125-1133.
74
(57) Homma K, Yoshimura M, Saito J, Ikebe R, Ikebe M. The core of the motor domain
determines the direction of myosin movement. Nature 2001;412(6849):831-834.
(58) Sweeney HL, Bowman BF, Stull JT. Myosin light chain phosphorylation in vertebrate
striated muscle: regulation and function. American Journal of Physiology - Cell Physiology 1993
05/01;264(5):C1085-C1085 -C1095.
(59) Chen Z, Naveiras O, Balduini A, Mammoto A, Conti MA, Adelstein RS, et al. The May-
Hegglin anomaly gene MYH9 is a negative regulator of platelet biogenesis modulated by the
Rho-ROCK pathway. Blood 2007 07/01;110(1):171-179.
(60) Hirano Y, Hatano T, Takahashi A, Toriyama M, Inagaki N, Hakoshima T. Structural basis
of cargo recognition by the myosin-X MyTH4-FERM domain. EMBO J 2011
07/06;30(13):2734-2747.
(61) Buss F, Kendrick-Jones J. Multifunctional myosin VI has a multitude of cargoes.
Proceedings of the National Academy of Sciences 2011 04/12;108(15):5927-5928.
(62) Katsuragawa Y, Yanagisawa M, Inoue A, Masaki T. Two distinct nonmuscle myosin-
heavy-chain mRNAs are differentially expressed in various chicken tissues. European Journal of
Biochemistry 1989 10/01;184(3):611-616.
(63) Golomb E, Ma X, Jana SS, Preston YA, Kawamoto S, Shoham NG, et al. Identification and
Characterization of Nonmuscle Myosin II-C, a New Member of the Myosin II Family. J Biol
Chem 2004 01/23;279(4):2800-2808.
(64) Donaudy F, Snoeckx R, Pfister M, Zenner H, Blin N, Di Stazio M, et al. Nonmuscle Myosin
Heavy-Chain Gene MYH14 Is Expressed in Cochlea and Mutated in Patients Affected by
Autosomal Dominant Hearing Impairment (DFNA4). The American Journal of Human Genetics
2004 04;74(4):770-776.
(65) Yang T, Pfister M, Blin N, Zenner H,P., Pusch C,M., Smith RJ,H. Genetic heterogeneity of
deafness phenotypes linked to DFNA4. American Journal of Medical Genetics Part A 2005
11/15;139A(1):9-12.
75
(66) Su AI, Wiltshire T, Batalov S, Lapp H, Ching KA, Block D, et al. A gene atlas of the mouse
and human protein-encoding transcriptomes. Proc Natl Acad Sci U S A 2004
04/20;101(16):6062-6067.
(67) Conti MA, Even-Ram S, Liu C, Yamada KM, Adelstein RS. Defects in Cell Adhesion and
the Visceral Endoderm following Ablation of Nonmuscle Myosin Heavy Chain II-A in Mice. J
Biol Chem 2004 10/01;279(40):41263-41266.
(68) Bresnick A,R. Molecular mechanisms of nonmuscle myosin-II regulation. Curr Opin Cell
Biol 1999 02/01;11(1):26-33.
(69) The May-Hegglin/Fechtner SC. Mutations in MYH9 result in the May-Hegglin anomaly,
and Fechtner and Sebastian syndromes. Nat Genet 2000;26(1):103-105.
(70) Seri M, Savino M, Bordo D, Cusano R, Rocca B, Meloni I, et al. Epstein syndrome: another
renal disorder with mutations in the nonmuscle myosin heavy chain 9 gene. Hum Genet 2002
02/01;110(2):182-186.
(71) De Rocco D, Pujol-Moix N, Pecci A, Faletra F, Bozzi V, Balduini CL, et al. Identification
of the first duplication in MYH9-related disease: A hot spot for unequal crossing-over within
exon 24 of the MYH9 gene. European Journal of Medical Genetics 2009 07/;52(4):191-194.
(72) Pecci A, Panza E, Rocco DD, Pujol-Moix N, Girotto G, Podda L, et al. MYH9 related
disease: four novel mutations of the tail domain of myosin-9 correlating with a mild clinical
phenotype. Eur J Haematol 2010;9999(9999).
(73) Althaus K, Greinacher A. MYH9-Related Platelet Disorders. Semin Thromb Hemost
2009;35(2):189-203.
(74) Arrondel C, Vodovar N, Knebelmann B, Grünfeld J, Gubler M, Antignac C, et al.
Expression of the Nonmuscle Myosin Heavy Chain IIA in the Human Kidney and Screening for
MYH9 Mutations in Epstein and Fechtner Syndromes. Journal of the American Society of
Nephrology 2002 01/01;13(1):65-74.
76
(75) de Rocco D, Heller P,G., Girotto G, Pastore A, Glembotsky A,C., Marta R,F., et al. MYH9
related disease: a novel missense Ala95Asp mutation of the MYH9 gene. Platelets 2009
12;20(8):598-602.
(76) Epstein CJ, Sahud MA, Piel CF, Goodman JR, Bernfield MR, Kushner JH, et al. Hereditary
macrothrombocytopathia, nephritis and deafness. Am J Med 1972 03;52(3):299-310.
(77) Ma ES, Wong CL, Shek TW, Hui S. Hematologic and genetic characterization of an
MYH9-related disorder in a Chinese family. Haematologica 2006 07;91(7):1002-1003.
(78) Arnér ES,J., Holmgren A. Physiological functions of thioredoxin and thioredoxin reductase.
European Journal of Biochemistry 2000 10/01;267(20):6102-6109.
(79) Kelley MJ, Jawien W, Ortel TL, Korczak JF. Mutation of MYH9, encoding non-muscle
myosin heavy chain A, in May-Hegglin anomaly. Nat Genet 2000;26(1):106-108.
(80) Lalwani AK, Goldstein JA, Kelley MJ, Luxford W, Castelein CM, Mhatre AN. Human
Nonsyndromic Hereditary Deafness DFNA17 Is Due to a Mutation in Nonmuscle Myosin
MYH9. The American Journal of Human Genetics 2000 11;67(5):1121-1128.
(81) Kunishima S, Kojima T, Matsushita T, Tanaka T, Tsurusawa M, Furukawa Y, et al.
Mutations in the NMMHC-A gene cause autosomal dominant macrothrombocytopenia with
leukocyte inclusions (May-Hegglin anomaly/Sebastian syndrome). Blood 2001
02/15;97(4):1147-1149.
(82) Kunishima S, Matsushita T, Kojima T, Amemiya N, Choi YM, Hosaka N, et al.
Identification of six novel MYH9 mutations and genotype-phenotype relationships in autosomal
dominant macrothrombocytopenia with leukocyte inclusions. J Hum Genet 2001
12/20;46(12):722-729.
(83) Kunishima S, Matsushita T, Kojima T, Sako M, Kimura F, Jo E, et al. Immunofluorescence
Analysis of Neutrophil Nonmuscle Myosin Heavy Chain-A in MYH9 Disorders: Association of
Subcellular Localization with MYH9 Mutations. Lab Invest 2004;83(1):115-122.
77
(84) Otsubo K, Kanegane H, Nomura K, Ogawa J, Miyawaki T, Kunishima S. Identification of a
novel MYH9 mutation in a patient with May-Hegglin anomaly. Pediatr Blood Cancer 2006
12;47(7):968-969.
(85) Kunishima S, Matsushita T, Hamaguchi M, Saito H. Identification and characterization of
the first large deletion of the MYH9 gene associated with MYH9 disorders. Eur J Haematol 2008
06/01;80(6):540-544.
(86) Burt R,A., Joseph J,E., Milliken S, Collinge J,E., Kile B,T. Description of a novel mutation
leading to MYH9-related disease. Throm Res 2008;122(6):861-863.
(87) Miyajima Y, Kunishima S. Identification of the first in cis mutations in MYH9 disorder. Eur
J Haematol 2009 04/01;82(4):288-291.
(88) Savoia A, De Rocco D, Panza E, Bozzi V, Scandellari R, Loffredo G, et al. Heavy chain
myosin 9-related disease (MYH9 -RD): Neutrophil inclusions of myosin-9 as a pathognomonic
sign of the disorder. Thromb Haemost 2010.
(89) Miyazaki K, Kunishima S, Fujii W, Higashihara M. Identification of three in-frame deletion
mutations in MYH9 disorders suggesting an important hot spot for small rearrangements in
MYH9 exon 24. Eur J Haematol 2009 09;83(3):230-234.
(90) Ma L, Fogo A,B. Model of robust induction of glomerulosclerosis in mice: Importance of
genetic background. Kidney Int 2003 07;64(1):350-355.
(91) Eckly A, Strassel C, Freund M, Cazenave J, Lanza F, Gachet C, et al. Abnormal
megakaryocyte morphology and proplatelet formation in mice with megakaryocyte-restricted
MYH9 inactivation. Blood 2009 04/02;113(14):3182-3189.
(92) Eckly A, Rinckel J-, Laeuffer P, Cazenave J-, Lanza F, Gachet C, et al. Proplatelet
formation deficit and megakaryocyte death contribute to thrombocytopenia in Myh9 knockout
mice. Journal of Thrombosis and Haemostasis 2010 10/01;8(10):2243-2251.
(93) Matsushita T, Hayashi H, Kunishima S, Hayashi M, Ikejiri M, Takeshita K, et al. Targeted
disruption of mouse ortholog of the human MYH9 responsible for macrothrombocytopenia with
78
different organ involvement: hematological, nephrological, and otological studies of
heterozygous KO mice. Biochem Biophys Res Commun 2004 12/24;325(4):1163-1171.
(94) Nathan C. Neutrophils and immunity: challenges and opportunities. Nat Rev Immunol 2006
03;6(3):173-182.
(95) Orkin SH, Zon LI. Hematopoiesis: An Evolving Paradigm for Stem Cell Biology. Cell 2008
02;132(4):631-644.
(96) Bain B,J., England J,M. Normal haematological values: sex difference in neutrophil count.
Br Med J 1975 02/08;1(5953):306-309.
(97) Haddy T,B. Benign ethnic neutropenia: What is a normal absolute neutrophil count? J Lab
Clin Med 1999;133(1):15-22.
(98) Hänsch G,M. Polymorphonuclear neutrophils: basic facts and new insights. 2008; .
(99) Witko-Sarsat V, Rieu P, Descamps-Latscha B, Lesavre P, Halbwachs-Mecarelli L.
Neutrophils: Molecules, Functions and Pathophysiological Aspects. Lab Invest 2000;80(5):617-
653.
(100) May R. Leukozyteneinschlüsse. Dtch Arc Klin Med. 1909;96:1-6.
(101) Hegglin R. Gleichzeitige konstitutionelle Veränderungen an Neutrophilen und
Thrombozyten. Helv Med Acta 1945;12:439-440.
(102) Maupin P, Phillips CL, Adelstein RS, Pollard TD. Differential localization of myosin-II
isozymes in human cultured cells and blood cells. J Cell Sci 1994 11/01;107(11):3077-3090.
(103) Harrison R,V. Anatomy and Physiology of the Auditory Periphery. In: Burkard R, Don M,
Eggermont J, editors. Auditory Evoked Potentials: Basic principles and clinical applications
Philadelphia: Lippincott, Williams & Wilkins; 2007. p. 140-158.
(104) Ehret G. Stiffness gradient along the basilar membrane as a basis for spatial frequency
analysis within the cochlea. J Acoust Soc Am 1978 12;64(6):1723-1726.
79
(105) Jasmin L. Sensorineural deafness - Overview. 2009; .
(106) Eggermont J,J. Electric and Magnetic Field of Synchormous Neural Activity. In: Burkard
R, Don M, Eggermont JJ, editors. Auditory Evoked Potentials: Basic principles and clinical
applications Philadelphia: Lippincott, Williams & Wilkins; 2007. p. 2-21.
(107) Lifschitz M,D. Renal Physiology. In: Forland M, editor. Nephrology New York: Medical
Examination Publishing; 1983. p. 21-32.
(108) Bannayan G. The Structure of the Kidney. In: Forland M, editor. Nephrology New York:
Medical Examination Publishing; 1983. p. 1-20.
(109) Pavenstädt H, Kriz W, Kretzler M. Cell Biology of the Glomerular Podocyte. Physiol Rev
2003 01/01;83(1):253-307.
(110) Sekine T, Konno M, Sasaki S, Moritani S, Miura T, Wong W, et al. Patients with Epstein-
Fechtner syndromes owing to MYH9 R702 mutations develop progressive proteinuric renal
disease. Kidney Int 2010 03/03.
(111) Kao WL, Klag MJ, Meoni LA, Reich D, Berthier-Schaad Y, Li M, et al. MYH9 is
associated with nondiabetic end-stage renal disease in African Americans. Nat Genet 2008
10;40(10):1185-1192.
(112) Kopp JB, Smith MW, Nelson GW, Johnson RC, Freedman BI, Bowden DW, et al. MYH9
is a major-effect risk gene for focal segmental glomerulosclerosis. Nat Genet 2008
10;40(10):1175-1184.
(113) Freedman B,I., Hicks P,J., Bostrom M,A., Cunningham M,E., Liu Y, Divers J, et al.
Polymorphisms in the non-muscle myosin heavy chain 9 gene (MYH9) are strongly associated
with end-stage renal disease historically attributed to hypertension in African Americans. Kidney
Int 2009 01/28;75(7):736-745.
(114) Genovese G, Friedman DJ, Ross MD, Lecordier L, Uzureau P, Freedman BI, et al.
Association of Trypanolytic ApoL1 Variants with Kidney Disease in African Americans.
Science 2010 07;329(5993):841-845.
80
(115) Tabbara K. Anatomy & Embryology of the Eye. In: Vaughan D, Asbury T, Tabbara K,
editors. General Ophthalmology Norwalk: Appleton & Lange; 1989. p. 1-18.
(116) Wassle H, Boycott BB. Functional architecture of the mammalian retina. Physiol Rev 1991
04/01;71(2):447-480.
(117) Francis PJ, Berry V, Moore AT, Bhattacharya S. Lens biology: development and human
cataractogenesis. Trends in Genetics 1999 05/01;15(5):191-196.
(118) Wride MA. Cellular and molecular features of lens differentiation: a review of recent
advances. Differentiation 1996 12;61(2):77-93.
(119) Shock J. Lens. In: Vaughan D, Asbury T, Tabbara K, editors. General Ophthalmology
Norwalk: Appleton & Lange; 1989. p. 144-152.
(120) Leske M, Sperduto R,D. The epidemiology of senile cataracts: a review. Am J Epidemiol
1983;118(2):152-165.
(121) Hejtmancik JF. Congenital cataracts and their molecular genetics. Semin Cell Dev Biol
2008 04;19(2):134-149.
(122) Denoyer A, Le Lez M, Majzoub S, Pisella P. Quality of vision after cataract surgery after
Tecnis Z9000 intraocular lens implantation: Effect of contrast sensitivity and wavefront
aberration improvements on the quality of daily vision. Journal of Cataract & Refractive Surgery
2007 02;33(2):210-216.
(123) Léon C, Eckly A, Hechler B, Aleil B, Freund M, Ravanat C, et al. Megakaryocyte-
restricted MYH9 inactivation dramatically affects hemostasis while preserving platelet
aggregation and secretion. Blood 2007 11/01;110(9):3183-3191.
(124) Johnstone DB, Zhang J, George B, Léon C, Gachet C, Wong H, et al. Podocyte-specific
deletion of Myh9 encoding non-muscle myosin heavy chain 2A predisposes mice to
glomerulopathy. Mol Cell Biol 2011 03/14:MCB.05234-11; MCB.05234-11.
81
(125) Sanger F, Nicklen S, Coulson AR. DNA sequencing with chain-terminating inhibitors.
Proc Natl Acad Sci U S A 1977 12;74(12):5463-5467.
(126) Saiki R,K., Scharf S, Faloona F, Mullis K,B., Horn G,T., Erlich H,A., et al. Enzymatic
amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of
sickle cell anemia. Science 1985 12/20;230(4732):1350-1354.
(127) Smith LM, Sanders JZ, Kaiser RJ, Hughes P, Dodd C, Connell CR, et al. Fluorescence
detection in automated DNA sequence analysis. Nature 1986 06/12;321(6071):674-679.
(128) Schuster S,C. Next-generation sequencing transforms today's biology. Nat Meth 2008
01;5(1):16-18.
(129) Illumina. Illumina - Solexa Technology. 2011; .
(130) Illumina. SS_DNAsequencing.pdf (application/pdf Object). 2007; .
(131) Chalhoub B, Belcram H, Caboche M. Efficient cloning of plant genomes into bacterial
artificial chromosome (BAC) libraries with larger and more uniform insert size. Plant
Biotechnology Journal 2004 05/01;2(3):181-188.
(132) Okou D,T., Steinberg KM, Middle C, Cutler D,J., Albert T,J., Zwick M,E. Microarray-
based genomic selection for high-throughput resequencing. Nat Meth 2007 11;4(11):907-909.
(133) Albert T,J., Molla M,N., Muzny D,M., Nazareth L, Wheeler D, Song X, et al. Direct
selection of human genomic loci by microarray hybridization. Nat Methods 2007 11;4(11):903-
905.
(134) Hodges E, Xuan Z, Balija V, Kramer M, Molla M,N., Smith S,W., et al. Genome-wide in
situ exon capture for selective resequencing. Nat Genet 2007 12;39(12):1522-1527.
(135) Roche. NimbleGen Sequence Capture: Genetic Discovery Made Easy. 2010.
(136) Cotter MJ, Norman KE, Hellewell PG, Ridger VC. A Novel Method for Isolation of
Neutrophils from Murine Blood Using Negative Immunomagnetic Separation. Am J Pathol 2001
08/01;159(2):473-481.
82
(137) Goujon M, McWilliam H, Li W, Valentin F, Squizzato S, Paern J, et al. A new
bioinformatics analysis tools framework at EMBL-EBI. Nucleic Acids Res 2010 05/03.
(138) Wen S, Lu W, Zhu H, Yang W, Shaw G,M., Lammer E,J., et al. Genetic polymorphisms in
the thioredoxin 2 (TXN2) gene and risk for spina bifida. American Journal of Medical Genetics
Part A 2009 02/01;149A(2):155-160.
(139) Abcam. IHC-Paraffin Protocol. 2011.
(140) Beckman Coulter I. AcTDiff Analyser - Reference. 2010 08.
(141) Drew Scientific. HemaVet 950. 2011.
(142) Smale ST, Kadonaga JT. The RNA Polymerase II Core Promoter. Annu Rev Biochem
2003 06;72(1):449-479.
(143) Arnosti D, Kulkarni M. Transcriptional enhancers: Intelligent enhanceosomes or flexible
billboards? J Cell Biochem 2005;94(5):890-898.
(144) Weiner W, Topley E. Döhle Bodies in the Leucocytes of Patients with Burns. J Clin Pathol
1955 11/01;8(4):324-328.
(145) Itoga T, Laszlo J. Döhle Bodies and Other Granulocytic Alterations during Chemotherapy
with Cyclophosphamide. Blood 1962 12/01;20(6):668-674.
(146) Cawley J, Hayhoe F. The Inclusions of the May-Hegglin Anomaly and Döhle Bodies of
Infection: an Ultrastructural Comparison. Br J Haematol 1972 04/01;22(4):491-496.
(147) Hurd ER, Jasin HE, Gilliam JN. Correlation of disease activity and Clq-binding immune
complexes with the neutrophil inclusions which form in the presence of SLE sera. Clin Exp
Immunol 1980 05;40(2):283-291.
(148) Willott J,F. Effects of aging, hearing loss, and anatomical location on thresholds of inferior
colliculus neurons in C57BL/6 and CBA mice. J Neurophysiol 1986;56(2):391-391.
83
(149) Willott J,F., Aitkin L,M., McFadden S,L. Plasticity of auditory cortex associated with
sensorineural hearing loss in adult C57BL/6J mice. J Comp Neurol 1993;329(3):402-411.
(150) Mikaelian DO, Warfield D, Norris O. Genetic Progressive Hearing Loss in the C57/M6
Mouse: Relation of Behaviorial Responses to Cochlear Anatomy. Acta Otolaryngol
1974;77(1):327-334.
(151) Mikaelian D,O. Development and degeneration of hearing in the C57/b16 mouse: relation
of electrophysiologic responses from the round window and cochlear nucleus to cochlear
anatomy and behavioral responses. Laryngoscope 1979;89(1):1-15.
(152) Spongr V,P., Flood D,G., Frisina R,D., Salvi R,J. Quantitative measures of hair cell loss in
CBA and C57BL/6 mice throughout their life spans. J Acoust Soc Am 1997;101:3546-3546.
(153) Wharram BL, Goyal M, Wiggins JE, Sanden SK, Hussain S, Filipiak WE, et al. Podocyte
Depletion Causes Glomerulosclerosis: Diphtheria Toxin-Induced Podocyte Depletion in Rats
Expressing Human Diphtheria Toxin Receptor Transgene. Journal of the American Society of
Nephrology 2005 10/01;16(10):2941-2952.
(154) Chen H, Liu Z, Zeng C, Li S, Wang Q, Li L. Podocyte Lesions in Patients With Obesity-
Related Glomerulopathy. American Journal of Kidney Diseases 2006 11;48(5):772-779.
(155) Kren S, Hostetter TH. The course of the remnant kidney model in mice. Kidney Int 1999
07;56(1):333-337.
(156) Smith R, Sundberg J. Inbred C57 Black Mice : Microphthalmia and Ocular Infections.
JAX Notes 1995 Fall(463).
(157) Robinson M,J., Cobb M,H. Mitogen-activated protein kinase pathways. Curr Opin Cell
Biol 1997 04;9(2):180-186.
(158) Kolega J. Cytoplasmic dynamics of myosin IIA and IIB: spatial 'sorting' of isoforms in
locomoting cells. J Cell Sci 1998 08/01;111(15):2085-2095.
84
(159) Zhang Y, Conti MA, Malide D, Dong F, Wang A, Shmist YA, et al. Mouse models of
MYH9-related disease: mutations in nonmuscle myosin II-A. Blood 2011.