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Vol.9 No.11 November 2003
Slipping while sleeping? Trinucleotide
repeat expansions in germ cells
Christopher E. Pearson
Program of Genetics and Genomic Biology, The Hospital for Sick Children, 555 University Avenue, Elm Wing 11-135, Toronto,
Ontario, Canada M5G 1X8
Trinucleotide expansions cause at least 30 diseases
including Huntington’s disease (HD). Many are inherited
predominantly through paternal transmissions, which
are probably the result of germ-cell-specific mutations.
A recent study of testicular germ cells in HD patients
revealed that expansions occur in diploid cells before
the completion of meiosis. Therefore, expansions are
not limited to the late-haploid spermatids, in which the
genome is ‘sleeping’. These results have implications
both for research aimed at understanding the transmission of this serious mutation and for developing
new therapies for the disease.
Expansion
of
gene-specific
CAG-,
CTGand
CGG-trinucleotide repeats cause multiple diseases,
including Huntington’s disease (HD), myotonic dystrophy
type 1 (DM1) and fragile X mental retardation (FRAXA).
Many are inherited in a non-mendelian manner known
as genetic anticipation, in which disease symptoms are
more severe and/or are evident at an earlier age with
successive generations. Genetic anticipation is thought
to arise through dynamic mutations displayed by the
repeats, in which larger repeat tracts are more susceptible
to subsequent mutations than progenitor alleles. Each
disease displays different levels of repeat instability in
somatic tissues and during parent– offspring transmissions (instability in patients is reviewed in Ref. [1]).
Whereas expansions and contractions both during postfertilization cell divisions and throughout life can contribute to somatic instability (Figure 1a), transmitted
instability must involve germline mutations (Figure 1b).
Many of these diseases are inherited predominantly
through either paternal or maternal transmissions;
however, the mechanism of this parent-of-origin effect
for any one of the diseases has, until recently, remained
elusive [2]. Huntington’s disease shows a paternal bias for
transmission of repeat expansions. By contrast, in FRAXA,
disease-associated expansions occur only through maternal
transmissions. Myotonic dystrophy – like HD – shows a
paternal bias for expansions of progenitor CTG/CAG
lengths of 100 or less (in the range of HD alleles). For
longer alleles of 200– 600 repeats (which do not occur in
HD), DM1 shows both a paternal and maternal bias for
expansions; tract lengths of . 600 are maternally transmitted to exceedingly large lengths of 1000– 4000 repeats
Corresponding author: Christopher E. Pearson (cepearson@genet.sickkids.on.ca).
(reviewed in Ref. [1]). Each parent-of-origin mutation bias
is driven by complex processes specific to sperm or oocyte
development. Expansion mediated by DNA slippage
between repeat strands [3] could occur in proliferating,
arrested meiotic cells or in the relatively dormant haploid
germ cells (Figure 2). Knowing when expansion events
occur during spermatogenesis in HD patients is crucial to
understanding this important mutation and its transmission in affected families. A recent study reported that
expansion events occurred in pre-meiotic germ cells of
male HD patients [2].
Cellular and DNA metabolism through human
gametogenesis
Knowing when expansions occur during germ-cell development will suggest which DNA metabolic process mediates instability. Mitotic and DNA metabolic activities
change throughout the stages of gametogenesis (Figure 1b).
Many replication, repair or recombination genes show
altered or specialized expression during gametogenesis
[4– 9]. If expansions occur during replication, the larger
number of mitotic divisions in male gametogenesis could
explain the paternal bias for HD mutation. Following
spermatogonial proliferation and meiosis, the genome of
the haploid spermatids is re-packaged with protamine,
thereby entering a relatively inactive or ‘sleeping’ state,
and eventually becoming spermatozoa. If the mismatch
repair proteins MSH2 and MSH3 are required for
CAGCTG expansions in humans (and possibly protection
from deletions), as has been reported in transgenic mice
[10 –13], then expansion events must occur in the germcell stages that contain MSH2 and MSH3. In humans
and mice, MSH2 levels are high in mitotically active
spermatogonia and decrease from meiosis onwards to
immunohistochemically undetectable levels in round and
elongating spermatids [5,7]. Interestingly, the level of
MSH3 expression was low in spermatogonia but increased
during meiosis – peaking in pachytene spermatocytes –
then decreased in later stages [7]. Together, these results
suggest that expansion mutations occur in replicating
spermatogonia.
CAG expansions in germ cells of HD patients
Analysis of testicular cells of human HD patients with
(CAG)50 (presumed the progenitor allele) in the blood
revealed that high frequencies of repeat expansions of
considerable magnitude were present in mitotic diploid
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Review
TRENDS in Molecular Medicine
Brain
Heart
(a)
Somatic instability
(mitotic and non-mitotic tissues)
via replication, genome
maintenance and repair
Kidney
491
Vol.9 No.11 November 2003
Blood
Brain
HD, DM1
DRPLA, SCA1,
SCA2, SCA3,
SCA8 and FRAXA
(CAG)n-N
Heart
Blood
Testis
Skeletal muscle
Skeletal muscle
Kidney
Skin
(b)
Skin
Female embryo
Male embryo
16 divisions
16 divisions
~ Day 5 - 12,
germ cell segregation
primordial germ cells
Replication errors,
repair associated
with DNA replication
and genome
maintenance
~ Day 5 - 12,
germ cell segregation
primordial germ cells
13 divisions
17 divisions
Max. # of germ cells
(~ 6.8 million)
In
utero
Oogonium
Beginning of
meiosis I
Primary oocytes (2 x 2C)
arrested at dictyotene
stage of prophase
# of germ cells
(~ 99 million)
Birth
Repair associated
with genome maintenance
Puberty
Release from
dictyotene arrest
1 division
Replication errors,
repair associated
with DNA replication
and genome maintenance
Ovulation
Spermatogonia (2C)
1 division
per
16 days
2 divisions
Primary spermatocyte (2 x 2C)
Repair associated
Meiosis I
with recombination, ~74 days
Secondary spermatocyte (2 x 1C)
meiosis and genome
maintenance
Meiosis II
Completion of
meiosis I
Secondary oocytes (2C + pb)
with polar body
Beginning
of meiosis II
Round spermatid (1C)
Postmeiotic
repair
Elongating spermatid
(1C)
Histone
Ovum (2 x 1C + pb)
blocked at metaphase II
Protamine
Spermatozoa (1C)
(c)
Spermatozoa
Ovum
blocked at
metaphase II
gm-repair
Protamine
to histone
2 pronuclei
(2 x 1C)
Completion
of maternal
meiosis II
Gonomeric
replication
2 pronuclei
(2 x 1C)
gm-repair
rec-repair meiosis & gm-repair
Envelope
breakdown
2 pronuclei
(2 x 2C)
Division
Embryo
(4C)
repl. & gm-repair rec-repair & gm-repair
2 cell embryo
(2C)
repl. & gm-repair
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germ cells before meiosis [2]. Cells at defined stages of
spermatogenesis were isolated by laser capture microdissection, which circumvents many of the difficulties
of other isolation procedures [14]. Repeat lengths were
assessed by the highly sensitive single-molecule PCR.
This instability could have occurred in at least two
phases; expansion products were evident at similar frequencies in both pre-meiotic and post-meiotic cells.
However, the proportion of large expansions in postmeiotic spermatids or spermatozoa was greater, indicating
that expansion events occurred before and possibly after
(or during) meiosis. The frequency of expanded alleles in
either pre-meiotic cells or in a mixture of pre-meiotic and
meiotic cells was 87% and 88%, respectively. Post-meiotic
spermatids and spermatozoa showed a similar expansion
frequency of 75%. (The slight reduction might indicate
a failure of some pre-meiotic or meiotic expansions to
progress.) The progenitor (CAG)50 showed a range of
mutant length changes: pre-meiotic cells showed expansion products of up to 125 repeats and tracts as short as
43 repeats, with a median of 77 repeats. Post-meiotic cells
showed a relatively continuous range of expansions products of up to 127 repeats and tracts as short as 43 repeats,
with a median of 87 repeats. Thus, in HD male germ cells,
expansions occur before and possibly after meiosis.
Data from HD patients revealed that extensive expansions of a (CAG)50 occurred in diploid germ cells before
meiosis. Similar to the results from HD patients, a DM1
transgenic mouse showed expansions arising in diploid
spermatogonia [11]; however, these did not occur in postmeiotic spermatids or spermatozoa (G. Gourdon, pers.
commun.). Results from another DM1 mouse also showed
that expansions occur in pre-meiotic proliferating spermatogonia [15]. Together with data from humans, these
results contrast with conclusions reported for a transgenic
HD mouse, which suggest that expansions occur only
in late-haploid stages of spermatogenesis, specifically in
elongating spermatids when the genome is becoming
dormant [10,16]. In this case, a mouse with a (CAG)117
tract showed modest increases of 1 –8 repeats. The discrepancy with results from the HD mouse can be explained
in several ways [2,11,15]. In addition to DNA-sequence
context differences between HD patients and mice, cellular
and DNA metabolism during spermatogenesis differ
between the two species [4,6]. For example, in humans,
Vol.9 No.11 November 2003
the greater number of mitotic divisions and the extended
time before and during spermatogenesis increases the
potential for mutations compared with mice. Inter-species
differences in repair gene expression in germ cells
might also contribute [4,5,7]. Moreover, humans could be
exposed to very different mutagens than mice, many of
which might affect either DNA or repair systems
in gametes [17]. These inter-species differences might
also explain the inability of any mouse model to recapitulate the large expansion increments observed in some
human transmissions.
There are clear differences in transmitted repeat
instability between the HD mice [10,16] and HD patients
[2]; similar differences exist between the HD mice
and other transgenic mice [11,15]. The differences
between transgenic mice could reveal important cis- and
trans-acting factors. Both within and among the various
HD, DM1 and other transgenic mice, there are differences
in transmitted parental mutation biases (if at all evident)
[10 –13,15]. Such differences might reflect context-specific
(chromosomal integration site) differences [15], similar to
the different human parental mutation biases between
diseases such as HD and spinocerebellar ataxia (SCA8;
both are associated with CAG repeats [1]). In addition,
the length of the integrated repeat might determine the
parent mutation bias pattern in transgenic mice, similar to
the variable parent-of-origin effects displayed by different
CTG lengths in human DM1 families (see earlier) [1].
Mouse-strain-specific differences might also account for
variable murine mutation patterns: unsuspected mutations in repair genes within the supposed wild-type
embryonic stem cells, such as those occurring in the
polymerase iota (pol i) gene in the 129-derived strains
[18], might unknowingly contribute to repeat instability.
Importantly, high levels of the error-prone DNA polymerases i, k, l, h and b, as well as pol e, are present in
mouse testes at various stages of spermatogenesis, before,
during and after meiosis, and, in some cases, in round
spermatids ([8,9] and Refs therein). Outside of mitotic
spermatogonia, the replicative polymerases a and d are
present at higher levels coincident with meiotic DNA
synthesis ([8] and Refs therein). Strain-specific defects in
polymerases or other repair genes might influence the
interpretation of transmitted or somatic instabilities
observed in transgenic mice.
Figure 1. Somatic and germline DNA metabolic processes in humans. (a) Repeat instability in patients suffering from a disease associated with a trinucleotide repeat can
display both pre-natal and post-natal tissue-specific repeat instabilities. Tract-length heterogeneity is evident in both proliferating and terminally differentiated patient
tissues (reviewed in Ref. [1]). Huntington’s disease patients show CAG-length mosaicism in sperm and brain tissues [25]. (b) Both male and female germ-cell segregation
is thought to occur between day 5– 12 post-fertilization, although the timing has not been rigorously established. Male germ-stem-cell production involves , 30 in utero
mitotic cell divisions as well as post-pubertal spermatogonial cell divisions throughout life (1 every 16 days over a spermatogenic cycle of 74 days). Assuming puberty
begins at 14 years of age, the number of accumulated divisions (mitotic only) through spermatogenesis for a 30-year-old male is , 400 divisions ½¼ 30 þ ðð30 2 14 2 ð74 4
365ÞÞ £ ð365 4 16 divisions per year))]. There are various possible sources of error in the estimates of the number of replicative cell divisions: estimations of the number of
stem cells and the number of divisions to produce them made no allowance for the extensive cell degeneration (apoptosis) known to occur. Therefore, the true number of
replicative divisions might be higher. Female germ-cell production, which is complete before birth (5 months gestation), involves only 24–33 mitotic cell divisions. Oogenic
meiosis begins in utero and is arrested in dictyotene for years, resuming only minutes before ovulation, after which meiosis I is complete. Meiosis II then begins and is not
completed until fertilization. In contrast to post-meiotic male germ cells, arrested and/or resting oocytes are primed with DNA repair and recombination activities [4,26],
which can lead to age-related CAG instability in SCA1 transgenic mice [33]. (c) Fertilization and the steps leading to the two-cell embryo, which includes the formation of
two pronuclei, the completion of maternal meiosis II, decondensation of the paternal genome, DNA repair, gonomeric DNA replication within two haploid pronuclei, the
breakdown of pronuclear envelopes, syngamy and cleavage [34]. Gonomeric duplication is the only haploid DNA replication in the diploid metazoan life cycle. DNA metabolic processes occurring during each developmental stage are indicated by graded shading. Abbreviations: HD, Huntington’s disease; DM1, myotonic dystrophy type 1;
DRPLA, dentaturbral-pallidoluysian atrophy; SCA, spinocerebellar ataxia; FRAXA, fragile X mental retardation; C, ploidy; pb, polar body; gm-repair, genome maintenance
repair; rec-repair, recombination-associated repair; repl., replication errors and replication-associated repair. Information compiled from Refs [4,6,17 and 34] and references
therein. These processes can vary greatly between different species.
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Vol.9 No.11 November 2003
(a)
Expansion of Huntington CAG repeat during spermatogenesis
Meiosis I
Embryo
PGC
Birth
Replicating
Puberty
Primary Secondary
RST
SC
SC
SG
Arrested Replicating/pre-meiotic
Progenitor or expanded?
Meiosis II
SZ
Post-meiotic
Meiotic
Expanded
EST
Expanded
Expansions must have occurred at any time during
this period, prior to meiosis
Continued expansions might occur
(b)
n=n
n<N
DNA structure
(CTG)n
(CTG)n
(CTG)N
(CTG)n
(CAG)N
Source
Aberrant strand annealing
in non-replicating DNA or
between sister chromatids
Intermediates of replication errors
or
of nick/gap-repair
Cell stage
(CAG)n
Proliferating
(non-replicating DNA)
and
non-proliferating cells
Proliferating
(replicating and non-replicating DNA)
and
non-proliferating cells
Processes
(CAG)n
(CAG)n
Genome-maintenance
repair and/or
recombination
Replication errors
Replication-associated repair
Genome-maintenance repair
and/or recombination
Double strand break
Blocked replication forks
Damage induced
Protein induced
Proliferating
(replicating and
non-replicating DNA)
and
non-proliferating cells at nicks
Replication errors
Replication-associated repair
Genome-maintenance repair
and/or recombination
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Figure 2. Expansion in male HD germ cells, and candidate processes for instability. (a) Instability can occur in primordial germ cells through to fertilization and post-natal
growth, encompassing processes associated with proliferating, arrested, meiotic and dormant haploid states. As expansion products are evident in human pre-meiotic
diploid cells, the initial mutation event must involve DNA replication-fork errors, replication-associated repair or genome-maintenance repair [2]. Further expansions can
involve meiotic recombination or post-meiotic genome maintenance. The high number of life-long spermatogonial cell divisions might drive the paternal mutation bias of
HD. (b) All models of expansion involve slipped-strand DNA structures formed by the repeat tracts as the putative mutagenic intermediates of repeat instability (reviewed
in Ref. [3]). Slippage in non-replicating DNA, or improper annealing between sister chromatids can result in structures that have the same number of repeats in the two
strands (n ¼ n). Slippage during replication or during nick- and/or gap-repair can contain either an excess of CAG repeats or an excess of CTG repeats (n , N). Slip-outs of
CAG and CTG are biophysically distinct, forming single-stranded loops and intrastrand hairpins, respectively [3]. Double-strand breaks within the repeats can arise during
the resolution of blocked replication forks, or passively (damage), or can be induced by specific proteins. Slipped DNAs can also form at double-strand breaks. Each of
these DNA structures might be recognized and processed differentially. DNA repair can contribute to instability by differentially processing or neglecting to process slipped
DNAs. Slipped-structures might escape processing, resulting in the maintenance of both strand lengths. Alternatively, they might be processed correctly to either one
strand, or undergo error-prone processing, resulting in novel tract lengths. Exactly how repair might contribute to instability is an area of intense research. Abbreviations:
PGC, primordial germ cell; SG, spermatogonia; SC, spermatocyte; RST, round spermatid; EST, elongating spermatid; SZ, spermatozoa; n and N, repeat numbers in which
n , N; HD, Huntington’s disease.
Mechanistic implications
Results from HD patients suggest various DNA metabolic
processes that could mediate instability. Huntington CAG
expansion events must occur at some point between
segregation of the primordial germ cells, development to
puberty, or during the life-long post-pubertal spermatogonial stem-cell divisions (Figure 2a). A second phase of
expansion might occur after meiosis and possibly through
to elongating spermatids. Primordial germ cells and
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spermatogonia undergo genome replication and are subject to DNA replication errors, repair associated with
replication, and genome maintenance repair (Figures 1b
and 2a). The mitotically inactive post-natal pre-pubertal
stages are subject to genome maintenance repair. Later
stages undergo meiotic recombination, genome maintenance repair and, ultimately, chromatin decondensation
(coincident with dormancy) (Figure 1b). The absence of
considerable instability in non-germ somatic tissues of
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HD patients argues against the occurrence of expansion
events in early embryos and favors their occurrence during
late-fetal, post-natal or post-pubertal stages.
If HD CAG expansion events occur only during the
post-pubertal cycling of spermatogonia, sperm produced at
advanced ages would have larger expansions than sperm
produced at the onset of puberty. A limitation of CAG
expansions to cycling spermatogonia would be expected to
show a paternal age effect on the progress of CAG expansions in HD families, as has been observed in several
transgenic mice [11,15,19]. Analysis of pre-meiotic
male germ cells in a DM1 mouse, over the course of
seven weeks to 11 months, revealed continuous expansions, suggesting that expansions occur in cycling spermatogonia (G. Gourdon, pers. commun.). An effect of paternal
age on transmitted CAG instability has only been reported
for dentaturbral-pallidoluysian (DRPLA) families [19]. In
HD, such an association has only been suspected based
upon the inverse relationship between age of disease onset
and paternal age (this study was performed before the
discovery that HD was caused by an unstable CAG tract
[20]). However, subsequent analyses failed to reveal such
an association at the molecular level [21,22]. The inability
to detect a paternal age effect in HD-affected fathers with
CAG instability (through analysis of sperm CAG instability or parent-to-child mutations) is likely to be the result of:
(i) a paucity of transmissions by older HD parents, (ii) the
paucity of sperm samples from the same individual taken
over long enough periods of time, or (iii) other complex
variables. Thus, although clinical data [20– 22] indicate a
probable paternal age effect on HD CAG instability, it is
difficult to prove its association with mutations occurring
during spermatogonial cycling.
Rather than narrowing the potential processes that
might mediate CAG expansions, the results from HD
patients [2] have broadened the spectrum relative to
some previous murine studies [10,16]. As expansion
products are evident in human pre-meiotic diploid cells,
the initial mutation event must involve DNA replicationfork errors, replication-associated repair or genomemaintenance repair. Further expansions might involve
recombination or post-meiotic genome maintenance. As
shown in Figure 2b, CAG expansions can arise through
various processes. Instability via replication can be complex and could vary between different developmental
stages and/or tissues [1,23]. There is evidence to suggest
that instability involves DNA repair proteins, which might
be associated with replication fork errors, genome maintenance, or recombination [24]. Many aspects of genome
maintenance repair are active in mitotic, inter-mitotic and
non-proliferating cells. All models of expansion involve
slipped-strand DNA structures that are formed by repeat
tracts as the putative mutagenic intermediates of instability (reviewed in Ref. [3]). Deciphering the formation and
processing of these mutagenic intermediates will be
crucial to understanding instability (Figure 2b).
Caution should be taken when extrapolating or generalizing the results from HD patient to other HD patient
tissues (such as the brain [25]) or to other trinucleotideassociated diseases. Notably, tissue-specific somatic instability in HD and other diseases (Figure 1a) might occur by
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Vol.9 No.11 November 2003
processes that are unrelated to germ cells [23,26,27]. In
DM1 and SCA8, which, like HD, are caused by an unstable
CTG or CAG repeat, families show a maternal bias rather
than a paternal bias for large expansions. Furthermore,
in FRAXA, maternal oocyte-specific CGG expansions
probably occur soon after germ-cell segregation, whereas,
in males, the inherited expansion undergoes germ-cellspecific CGG contractions and selective growth, resulting
in spermatozoa with only pre-mutation tract lengths [28].
A similar process leading to deletions in sperm might occur
in SCA8 patients [29]. Thus, it is probable that HD
instability mechanisms are similar in some respects to
certain disease loci, but vastly different to others.
Future studies
Although we are far from curing diseases caused by trinucleotide expansions, understanding the mechanism(s),
tissue specificity, and developmental timing of repeat
expansions and contractions is crucial to achieving such
goals. The following sections cover only those avenues
directed towards DNA.
Transmission
If HD CAG expansion events occur only during the postpubertal cycling of spermatogonia, sperm samples isolated
at the onset of puberty would have smaller expansions
than samples isolated later in life. If this is true, early
pubertal semen could be stored for the eventual purposes
of in vitro fertilization, which would lead to transmissions
with minimal expansions or no expansions for those
fertilizations derived from sperm harboring the normal
HD allele. Such measures could limit the contribution of
germline CAG instability to genetic anticipation.
Somatic
In HD, like most trinucleotide repeat diseases, the mutant
gene product (protein and/or RNA) exerts a clinically
dominant effect mediated by a gain-of-function. Thus, at
the somatic level, a cure or treatment for the individual
might require replacing the mutant gene with a wild-type
gene. One way of ‘replacing’ the mutant gene with a
normal gene would be to modulate specifically the instability of the expanded allele in cells to harbor only contractions to shorter non-diseased repeat lengths. Treatment
of cells either in vitro or within the patient with a compound that might modulate instability towards contractions could effectively transmute a diseased allele to become
a normal allele. An important criterion of such a compound
is that it should affect instability only at the expanded
diseased locus and not have deleterious effects upon the
rest of the genome. Ablating a repair gene or system could
have dire consequences, including genome-wide instability and induction of tumorigenesis [13,30]. Targeting the
activity of a particular gene product involved in DNA
metabolism to both a specific tissue and genic locus (the
expanded disease locus) should be a goal of future studies
[31]. Another approach would be to isolate and/or selectively grow somatic (stem) cells that have repeat contractions, and these could be transplanted into patients.
Incidentally, the process of repeat contraction and selective growth advantage appears to occur naturally in the
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TRENDS in Molecular Medicine
germ cells of FRAXA [28] and SCA8 males [29], and in the
cerebellar cortex of HD, DM1, SCA1, SCA2, SCA3 and
DRPLA patients (for review, see Ref. [1]).
Concluding remarks
In the case of Huntington CAG expansion mutation,
Arnheim and colleagues have revealed both the window of
human sperm development and the breadth of the DNA
metabolic processes, which contribute to the CAG dynamic
mutations that drive genetic anticipation. Huntington
CAG expansion events in male germ cells occur before
meiosis at a point following segregation of the primordial
germ cells, through to the life-long post-pubertal spermatogonial stem-cell divisions. Future research should focus
on delimiting both the specific timing of instability and
the DNA metabolic process(es), as well as identifying the
development- and tissue-specific factors and mechanisms
that are involved in disease-associated instability. Hopefully, such studies will keep pace with the advances of
modulating repeat instability and stem-cell transplantation.
Note added in proof
Incidentally, somatic CAG instability in the brains of HD
patients might not occur by the assumed process of genome
maintenance repair [27], as recent evidence has revealed
increased cell proliferation and neurogenesis in HD brains
[32]. Also, spontaneous locus-0specific CTG expansions in
cultured DM1 patient fibroblasts occurred only during
proliferation, and these were specifically enhanced by
chemicals that perturb replication-fork progression [31].
Acknowledgements
I thank John D. Cleary and many of my colleagues for preparation of
Figures 1 and 2. Research in the Pearson laboratory is supported by grants
from the Canadian Institutes of Health Research (CIHR), the Muscular
Dystrophy Association USA, and the Fragile X Research Foundation
Canada. C.E.P. is a CIHR Scholar and a Canadian Genetic Disease
Network Scholar.
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