© 2001 Oxford University Press Human Molecular Genetics, 2001, Vol. 10, No.

20 2187–2194

Dynamic mutations: a decade of unstable expanded

repeats in human genetic disease
Robert I. Richards*

Department of Molecular Biosciences, The University of Adelaide, Adelaide, SA 5000, Australia and Centre for
Medical Genetics, Women’s and Children’s Hospital, North Adelaide, SA 5006, Australia

Received June 10, 2001; Accepted July 6, 2001

The term ‘dynamic mutation’ was introduced to distinguish the unique properties of expanding, unstable DNA
repeat sequences from other forms of mutation. The past decade has seen dynamic mutations uncovered as
the molecular basis for a growing number of human genetic diseases and for all of the characterized ‘rare’
chromosomal fragile sites. The common properties of the repeats in different diseases and fragile sites have
given insight into this unique form of DNA instability. While the dynamic mutation mechanism explains some
unusual genetic characteristics, unexpected findings have raised new questions and challenged some
assumptions about the pathways that lead from mutation to disease. This review will address the current
understanding of the molecular mechanisms involved in the dynamic mutation process and elaborate on the
pathogenic pathways that lead from expanded repeats to the diseases with which they are associated.

INTRODUCTION molecular mechanisms for both the genesis of expanded

Ten years ago the first reports appeared (1,2) of expanded
DNA repeat sequences being the molecular basis of human
genetic diseases—fragile X syndrome and spino-bulbar
muscular atrophy (Kennedy disease). In both cases a simple
DNA repeat sequence (CCG or CAG), which varied in copy
number in normal individuals, was found to be increased in
copy number beyond a certain threshold in affected individuals.
Given the idiosyncratic nature of these diseases, particularly
fragile X syndrome (3), it could have been that these expanded
repeats were manifestations of an uncommon mechanism with
characteristics peculiar and unique to their associated diseases.
Instead, however, an ever-growing list of human diseases and
chromosomal fragile sites has identified dynamic mutation as
an important mechanism in human genetics. Consequently,
both the molecular processes underlying this form of mutation
and the pathogenic pathways leading from the mutation to its
phenotype have been the subject of intense investigation. Over
the past decade numerous reviews have updated the progress
of these investigations (4–10). Rather than reiterate this
progress, the purpose of this review is to identify the generally
accepted principles underlying dynamic mutation and to
discuss those areas where the current understanding is either
lacking or clouded by apparently conflicting observations.
Comparative anatomy has long been a potent tool to develop an
understanding of the functional or mechanistic basis responsible
for a particular physical phenomenon. The application of
comparative molecular anatomy to the study of different
classes of chromosomal fragile sites and to the diseases caused
by DNA repeat expansion has led to the identification of a
number of common properties that most likely reflect common
repeats and the molecular pathways from repeat expansion to
disease phenotype. These general properties include: (i) mutation
manifests as a change (usually increase) in repeat copy number
with mutation rate related to the initial copy number of the
repeat; (ii) rare ‘founder’ events (such as loss of repeat inter-
ruption) lead to alleles with increased likelihood of undergoing
changes in repeat copy number; and (iii) the diseases caused by
repeat expansion exhibit a relationship between copy number
of the repeat and the severity and/or age-at-onset of symptoms.
These properties together account for anticipation, the
increasing severity/incidence and/or decreasing age-at-onset in
successive generations within an affected family. However, an
increasing number of exceptions and unexpected findings
suggest that early conclusions, particularly in regard to
possible common pathogenic mechanisms, may have been
premature.
MECHANISM OF DYNAMIC MUTATION
The process of dynamic mutation is affected by a variety of
elements and factors that have been divided into those directly
associated with the expanding repeat (cis-acting elements) and
those (trans-acting factors) whose interaction with the repeat
contributes to its instability. Examples of cis-acting factors
include the copy number and the composition of the repeat
(whether it is perfect or interrupted) (11). In general, higher copy
number alleles that are free of interruptions (perfect repeats) are
more unstable than those of lower copy number and/or contain
interruptions (imperfect repeats). While there is indirect
evidence for the existence of trans-acting factors (such as
parental gender bias in repeat instability), the identity of these

*To whom correspondence should be addressed at: Department of Molecular Biosciences, The University of Adelaide, Adelaide, SA 5000, Australia.
Tel: +618 8303 7541; Fax: +618 8303 4362; Email: robert.richards@adelaide.edu.au
2188 Human Molecular Genetics, 2001, Vol. 10, No. 20
Table 1. Founder effects in dynamic mutation diseases/fragile sites
Repeat Disease/fragile site
CUG Myotonic dystrophy
CCG Fragile X syndrome
CAG Huntington disease
CAG Spinocerebellar ataxia type 1 (SCA1)
CAG SCA3/Machado–Joseph disease
CAG Spino-bulbar muscular atrophy
CAG Dentatorubral pallidoluysian atrophy
AAG Friedreich ataxia
AT-rich 42 bp FRA10B
CAG SCA2
CAG SCA7
???? SCA4
CAG SCA6
aThese reports appeared before the expanded repeat mutation was identified.
factors remains elusive. Candidates include the proteins
involved in DNA replication such as FEN1 (Rad27), which is
known to have a role in Okazaki fragment metabolism (12).
What kinds of repeat sequences undergo dynamic
mutation?—Cis-acting factors
The first expanded repeats to be identified were the trinucleotides
CCG/CGG and CAG/CTG. Initially this was taken as evidence
that only trinucleotide repeats could undergo this form of
mutation (which was sometimes referred to as trinucleotide
repeat expansion). Furthermore, since these two repeats could
form secondary structures, it was assumed that this was also a
necessary condition for repeat instability (13). Subsequently,
six of the ten possible trinucleotide repeats have been found to
exhibit repeat expansion in humans (14), and, more recently, 5
and 6 bp microsatellite repeats (15,16) and 12, 33 and 42 bp
minisatellite repeats (17–19) have also been observed to
undergo repeat copy number expansion.
Founder effects are observed in association with dynamic
mutation as a higher frequency of particular alleles in the
affected population, compared with the unaffected population,
suggesting that these alleles are ‘at-risk’ for mutation. The
likelihood is that the initial mutation on these alleles is a rare
event that increases the instability of the repeat, setting in
process the subsequent expansion of these few founder
mutations. Founder effects (Table 1) are a common feature of
dynamic mutation and in many cases common flanking marker
haplotypes provide evidence of a relationship between the
expanded alleles and the longest normal (usually uninterrupted)
alleles in the population. Therefore a common molecular
mechanism appears to be responsible (Fig. 1).
At the FRAXA locus, detailed haplotyping studies, together
with sequence analysis of different repeat alleles, have provided
evidence in support of the view that certain alleles are predisposed
to instability (11,20). Similar analyses in other populations
suggest that such differences are likely to be population-specific
rather than indicative of a molecular pathway predisposed by
Reported founder effect (reference)
(71a,72)
(73)
(74)a
(75)
(76)
(77)
(78)
(79)
(19)
(80)
(81)
(82)a
(83)
cis-acting elements in and around the FRAXA repeat (21,22).
One possible explanation for the inconsistency here is that
chance has a role to play and that the differences between
populations merely reflect the different rare initial events that
have precipitated the dynamic mutation process. Given that
there are multiple possibilities for generating an unstable
repeat then different populations are likely to have different
combinations and/or frequencies of these unstable alleles.
Thus, in terms of understanding the mutation process, caution
is needed when extrapolating the results observed in one
population to the human species as a whole.
What factors contribute to repeat DNA expansion?—
Trans-acting factors
Gender bias in the instability of repeats during their transmission
from parent to offspring is a common property of dynamic
mutations. Most cases of the congenital form of myotonic
dystrophy are maternally inherited, while the juvenile onset
Huntington disease is generally from paternal inheritance. The
gender of the germ line clearly contributes to these biases;
however, the identity of the factors involved remains obscure.
Other factors that interact in some way with the repeat are
also thought to contribute to repeat instability. A variety of
circumstantial evidence (particularly from studies in model
systems) suggests that Okazaki fragments may have a role to
play in the expansion of DNA repeats. Several of the repeats
(e.g. CCG in FRAXA and CAG in DM) exhibit a bimodal
distribution of instability with the boundary between small
changes and large changes in copy number approximating
Okazaki fragment length. In Escherichia coli (which has a
substantially longer Okazaki fragment length), this boundary is
increased in copy number again to that approximating Okazaki
fragment length (23). The secondary structure of CAG/CTG
and CGG repeat sequences has been found to inhibit Okazaki
flap endonuclease (FEN1) binding and cleavage (24). In
addition, the FEN1 homologue, Rad27, has a role to play in
DNA repeat instability in yeast. Yeast Rad27 mutants are

Figure 1. Model for common mechanism of repeat expansion. A model for the relationship between expanded alleles and the longest normal (usually perfect)
repeat alleles at a dynamic mutation locus. Interrupted alleles are blue bars, uninterrupted (perfect repeat) alleles are red bars.
prone to both DNA breakage and copy number instability of
repeat sequences (25 and personal communication).
Contradicting the proposed role of DNA replication,
Kennedy and Shelbourne (26) found dramatic expansion of the
CAG repeat in the striatum of a transgenic mouse HD model.
These neurons are presumably no longer undergoing cell
division, indicating that expansion is occurring independently
of DNA replication. Similarly, Kotvun and McMurray (27)
have found that germ-line trinucleotide repeat sequence
expansion in transgenic mice also proceeded in the absence of
DNA replication via gap repair. Perhaps, given that DNA
instability is occurring at the repeat sequence in the absence of
replication, some form of transcriptional healing may be
contributing to the repeat expansion process (28). Transcrip-
tional healing has been invoked in the fragile site expression of
tandemly repeated small RNA transcripts (29). In further
support of a role for breakage and repair Manley et al. (30)
have found that Msh2 is required for CAG repeat somatic cell
instability in an HD transgenic mouse.
Age-dependent somatic cell instability of the CTG repeat
sequence has been found in myotonic dystrophy (31), although
here it is not confined to those cells that are affected in the
disease and in fact gives a growth advantage to lymphoblastoid
cell lines that have undergone expansion (32).
PATHOGENIC PATHWAYS
Loss of function/haploinsufficiency
Extinction of, or interference with transcription is one means
by which expanded repeats cause loss of gene function.
Examples are: fragile X syndrome, in which repeat expansion
brings about methylation of the FMR1 promoter region (33);
myoclonus epilepsy (EPM1) (34), in which repeat expansion
physically separates transcription factors in the cystatin B
Human Molecular Genetics, 2001, Vol. 10, No. 20 2189
promoter; and Friedreich ataxia, in which the intronic
expanded repeat sequence adopts a ‘sticky’ DNA structure that
interferes with transcription of the frataxin gene (35). While
these resultant diseases exhibit recessive (or X-linked) trans-
mission, the potential exists for dominant transmission where
reduction in the level or activity of a rate limiting gene product
can appear as haploinsufficiency. This appears to be the case
for the SIX5 gene in myotonic dystrophy (36,37), as the
expanded CTG repeat is located in the SIX5 promoter. The
reduction in effective ‘dosage’ of the SIX5 gene product
brought about by extinction of one allele is thought to
contribute to some aspects of pathology (see below).
Gain of function: the toxic polyglutamine hypothesis—old
concerns about a new dogma
Numerous expanded repeat sequences are located within
coding regions where they translate into expanded poly-
glutamine tracts in disease alleles (Table 2). This, together
with a substantial body of data demonstrating the toxicity of
polyglutamine in cells, has led to the view that the expanded
polyglutamine is a common, crucial component in the patho-
genesis of these diseases. In support of this view, an antibody,
1C2, raised against the normal length polyglutamine tract of
the TATA-box binding protein (TBP), was found to specifically
recognize the expanded polyglutamine in several of the
expanded repeat diseases, e.g. huntingtin, ataxin 1 and others
(38). This was taken as evidence that the expanded poly-
glutamine adopted a different conformation that in some way
contributed to its toxicity. The context in which the repeat
sequence was located was proposed to account for why such an
otherwise toxic conformation did not cause problems when
located within TBP. This theory is now somewhat flawed, as
the expanded polyglutamine tract in some alleles (n >50) of
TBP has recently been associated with neurodegenerative
disease (39,40). It is possible that yet another conformation of
2190 Human Molecular Genetics, 2001, Vol. 10, No. 20
Table 2. Polyglutamine (or not) in neurodegenerative disorders
Polyglutamine diseases Non-polyglutamine/expanded repeat, dominant spino-cerebellar ataxias
SBMA a SCA8 3′-UTR expanded CUG
HD a SCA10 intronic expanded 5 bp repeat
DRPLA a SCA12 5′-UTR expanded CAG
SCA1 a
SCA2 a
SCA3 (MJD) a
SCA6 Normal polyQ 4–16
Affected polyQ 21–27
SCA7 a
TBP Normal polyQ 27–44
Affected polyQ >50
*Typical normal polyQ, n < 40; typical affected polyQ, n > 36 (some overlap because of variable intermediate alleles).
the expanded polyglutamine tract is adopted once the repeat
sequence gets beyond an even greater threshold.
Nuclear inclusions were found in the affected neurons of HD
and SCA1 transgenic mouse models and HD patients (41),
prompting the assertion that these expanded polyglutamine-
containing nuclear inclusions cause neuronal dysfunction.
Klemment et al. (42) deleted the SCA1 self-association region
in transgenic mice and found that these mice still exhibited
ataxia and Purkinje cell pathology; however, no evidence of
nuclear aggregates was found. Other studies on the brains of
individuals affected with Huntington’s disease (43,44)
revealed that nuclear aggregates were more commonly found
in unaffected neurons than in the vulnerable striatal spiny
neurons. Consequently, it has now been proposed that rather
than being pathogenic, nuclear inclusions may even be protective
against the toxic effects of the expanded polyglutamine (45).
Further conflicting evidence to a common role for poly-
glutamine in pathogenesis is that disease alleles of the CAG
repeat in spinocerebellar ataxia 6 are well within the normal
range for other ‘polyglutamine diseases’. Functional assays on
expanded CAG alleles of the SCA6 associated gene, CACNA1A,
demonstrate increased Ca2+ transport (46) and thus make it
likely that this gain of function is a specific and unique cause of
pathogenesis for this disorder. Taken together, these exceptions
suggest that the notion of a common single pathogenic
pathway involving polyglutamine is unlikely.
As if contradictions with polyglutamine encoding genes are
not enough, a growing list of repeat-associated ataxias do not
even encode polyglutamine (Table 2). In SCA12, the expanded
CAG repeat is located within the 5′-untranslated region (5′-UTR)
of the PPP2R2B gene (47,48), while in SCA8 an expanded
CUG is located within the 3′-UTR of a transcript (although
there is controversy over whether this expansion is causative of
neurodegenerative disease) (49–51). In addition, an intronic
expanded 5 bp repeat was recently found to be associated with
SCA10 (15).
So the question must be asked whether expanded poly-
glutamine is a necessary and sufficient condition for those
diseases in which it has been identified as the disease-causing
agent. If it is, then how is the cellular specificity of this toxicity
accounted for when at least some of these proteins are widely
expressed (see 52)? One possible explanation for this conundrum
comes from the findings of Kennedy and Shelbourne (26),
which reveal affected cell-specific amplification of the
unstable CAG repeat in a mouse model of Huntington’s
disease. Individuals affected with Huntington’s disease have
also been found to exhibit somatic instability, which was most
pronounced in the affected areas of the brain (53,54). This
suggests that ongoing somatic mutation may contribute to
diseases caused by expanded repeats. This expansion may also
account for the relationship between germ-line repeat copy
number and age-at-onset for these diseases (7). The time taken
for the disease to manifest could represent how long it takes for
the disease-associated allele to reach a critical higher copy
number threshold in the affected cell population (Fig. 2).
What then is the evidence that polyglutamine has a role? The
principal data are from transgenic animal model studies,
particularly those from SCA1 transgenic mice. Mutation of the
ataxin-1 nuclear localization signal severely diminishes toxicity,
whereas abolition of sequences necessary for aggregation does
not (42). Crossing of SCA1 mice with a transgenic mouse
deficient in ubiquitin ligase results in more severe pathology
(55). For SCA2 and SCA6, nuclear localization does not appear
to be necessary, suggesting different pathways than that for
SCA1 (and HD) (56,57).
In an HD transgenic mouse model where transcription of the
transgene could be artificially turned on or off, expression of
the expanded CAG-containing gene was elegantly demon-
strated to be necessary for HD-like pathology (58). This animal
model provides the first indication that HD (and hopefully
other expanded CAG repeat diseases) is potentially treatable—
given that, in those patients at risk, the disease-causing gene
can either be switched off or the toxic gene product can be
cleared from the sensitive neurons.
Presumably other factors, such as modification of the poly-
glutamine tract and/or the protein context in which the
 Human Molecular Genetics, 2001, Vol. 10, No. 20 2191

Figure 2. Somatic mutation model for relationship between repeat copy number and age-at-onset. Somatic cell changes in copy number could contribute to the
relationship seen in certain neurodegenerative diseases between the copy number of the repeat locus and the age-at-onset of disease symptoms. The time taken (t)
for the repeat to reach a critical copy number threshold by means of incremental increases in repeat copy number would be determined by the inherited (germ-line)
copy number (green arrows) at birth (n). The greater is n the shorter is t. Time taken (t) to reach a disease-causing copy number is inversely proportional to ‘starting’
copy number (n), t = k/n (where k is determined by the repeat context).

polyglutamine is located, contribute to the cellular specificity
of toxicity. Examples of where this apparently is (59) or is not
(60) the case have been reported. The relative contribution of
various intrinsic and extrinsic factors is therefore likely to
differ between the different disease genes (and experimental
model systems). Genetic screens for factors that modify poly-
glutamine-generated pathology in Drosophila have produced a
large number and variety of candidates (61,62), and it will be
of great interest to see which (if any) of these has a role in
human polyglutamine disease.
Bias of ascertainment was invoked by Penrose to account for
(and discredit) the observation of anticipation seen by clinical
geneticists in myotonic dystrophy families (63). While
expanded DNA repeats now give a molecular basis (and
validation) for the phenomenon of anticipation, the relative
ease with which expanded CAG repeats can be screened for as
a cause of neurodegenerative disease may well have led to an
overemphasis of the relative importance of this form of
mutation and perhaps contributed to the view that expanded
CAG repeats necessarily have a common pathogenic pathway
(via their encoded expanded polyglutamines). While there is a
substantial body of evidence in support of a role for poly-
glutamine in some repeat expansion diseases there is a growing
list of exceptions and inconsistencies which suggest (at the
least) that this is not necessarily a common pathogenic
pathway exhibited by all neurodegenerative diseases in which
an expanded repeat (let alone an expanded CAG) is the
disease-causing mutation.
Further evidence for alternative pathways—multiple gene
effects
In myotonic dystrophy there is good evidence to support not
only a role for multiple genes, but also multiple pathways in
mediating the pathogenic effects of repeat expansion (for
review see 10). Three genes (DMPK, SIX5 and DMWD) are all
located in the immediate vicinity of the DM expanded CTG
repeat. At least three distinct pathogenic pathways are thought
to contribute to DM. (i) The expanded repeat affects the
splicing of the DMPK gene in which it is located (64). (ii) The
RNA transcript containing the expanded repeat is both
inappropriately compartmentalized and titrates a crucial
protein (muscleblind) (65). Expression of the CTG repeat in
another muscle-specific transcript is able to cause at least some
of the DM phenotype (myotonia and myopathy) (66). (iii) The
DM CTG repeat is also located in the promoter region of the
SIX5 gene and its expansion also interferes with the expression
of this gene. The SIX5 gene encodes a transcription factor
whose concentration is critical for eye, muscle and testicular
development (67). Heterozygous deletion of Six5 is sufficient
to cause ocular cataracts in transgenic mice (36,37), and
therefore the SIX5 haploinsufficiency brought about by the
DM CTG repeat expansion is likely to contribute to the DM
phenotype.
The FRAXE chromosomal fragile site is associated with a
mild form of mental retardation (68). The expanded CCG
repeat was first initially located in the 5′-UTR of a gene,
referred to as FMR2. The expression of FMR2 is extinguished
by repeat expansion and subsequent methylation of the CpG
island promoter region (69), in a manner analogous to the
silencing of the FMR1 gene at the FRAXA locus in fragile X
syndrome (33). It was therefore thought that the loss of FMR2
was responsible for the phenotype associated with FRAXE.
However, an additional gene, FMR3, has now been identified
that shares the same methylated CpG island (70). Expression
of FMR3 is also silenced by FRAXE full mutation and there-
fore this gene may also contribute to the phenotype.
2192 Human Molecular Genetics, 2001, Vol. 10, No. 20
Figure 3. Multiple pathogenic pathways for dynamic mutation diseases. Alternative and sometimes multiple pathways contribute to the complex pathology brought
about by expansion of the repeat sequences within or near genes.
CONCLUSIONS
Given that increasing the copy number of existing DNA repeat
sequence would appear to be a relatively simple process, in
reality the biological causes and consequences of such
phenomena can be remarkably complex. In fact, a major issue
in understanding both the process of dynamic mutation and the
pathway from genotype to phenotype is being able to distinguish
between cause and consequence. It certainly appears as though
there are multiple components to the mutation process and it is
clear that there can be multiple pathways to the resultant
disease (Fig. 3). So far dynamic mutations have been detected
in diseases (and at fragile site loci) with very high penetrance.
It will be intriguing to see whether DNA repeats also
contribute to multifactorial diseases and disease susceptibility.
ACKNOWLEDGEMENTS
I thank Keith Johnson, Catherine Freudenreich and Alec
Jeffreys for communicating data prior to publication and
Kathryn Friend, Sonia Dayan, Merran Finnis and Lynne
Hobson for constructive comments on drafts of this review. I
am grateful to Shelley Richards for her support and encourage-
ment. The preparation of this review was supported by grants
from the Anti-Cancer Foundation of South Australia and the
National Health and Medical Research Council of Australia.
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