Progressive GAA.TTC repeat expansion in human cell lines.

Abstract

Trinucleotide repeat expansion is the genetic basis for a sizeable group of inherited neurological and neuromuscular disorders. Friedreich ataxia (FRDA) is a relentlessly progressive neurodegenerative disorder caused by GAA.TTC repeat expansion in the first intron of the FXN gene. The expanded repeat reduces FXN mRNA expression and the length of the repeat tract is proportional to disease severity. Somatic expansion of the GAA.TTC repeat sequence in disease-relevant tissues is thought to contribute to the progression of disease severity during patient aging. Previous models of GAA.TTC instability have not been able to produce substantial levels of expansion within an experimentally useful time frame, which has limited our understanding of the molecular basis for this expansion. Here, we present a novel model for studying GAA.TTC expansion in human cells. In our model system, uninterrupted GAA.TTC repeat sequences display high levels of genomic instability, with an overall tendency towards progressive expansion. Using this model, we characterize the relationship between repeat length and expansion. We identify the interval between 88 and 176 repeats as being an important length threshold where expansion rates dramatically increase. We show that expansion levels are affected by both the purity and orientation of the repeat tract within the genomic context. We further demonstrate that GAA.TTC expansion in our model is independent of cell division. Using unique reporter constructs, we identify transcription through the repeat tract as a major contributor to GAA.TTC expansion. Our findings provide novel insight into the mechanisms responsible for GAA.TTC expansion in human cells.

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Progressive GAA?TTC Repeat Expansion in Human Cell
Lines
Scott Ditch, Mimi C. Sammarco, Ayan Banerjee, Ed Grabczyk*
Department of Genetics, Louisiana State University Health Sciences Center, New Orleans, Louisiana, United States of America
Abstract
Trinucleotide repeat expansion is the genetic basis for a sizeable group of inherited neurological and neuromuscular
disorders. Friedreich ataxia (FRDA) is a relentlessly progressive neurodegenerative disorder caused by GAA?TTC repeat
expansion in the first intron of the FXN gene. The expanded repeat reduces FXN mRNA expression and the length of the
repeat tract is proportional to disease severity. Somatic expansion of the GAA?TTC repeat sequence in disease-relevant
tissues is thought to contribute to the progression of disease severity during patient aging. Previous models of GAA?TTC
instability have not been able to produce substantial levels of expansion within an experimentally useful time frame, which
has limited our understanding of the molecular basis for this expansion. Here, we present a novel model for studying
GAA?TTC expansion in human cells. In our model system, uninterrupted GAA?TTC repeat sequences display high levels of
genomic instability, with an overall tendency towards progressive expansion. Using this model, we characterize the
relationship between repeat length and expansion. We identify the interval between 88 and 176 repeats as being an
important length threshold where expansion rates dramatically increase. We show that expansion levels are affected by
both the purity and orientation of the repeat tract within the genomic context. We further demonstrate that GAA?TTC
expansion in our model is independent of cell division. Using unique reporter constructs, we identify transcription through
the repeat tract as a major contributor to GAA?TTC expansion. Our findings provide novel insight into the mechanisms
responsible for GAA?TTC expansion in human cells.
Citation: Ditch S, Sammarco MC, Banerjee A, Grabczyk E (2009) Progressive GAA?TTC Repeat Expansion in Human Cell Lines. PLoS Genet 5(10): e1000704.
doi:10.1371/journal.pgen.1000704
Editor: Harry Orr, University of Minnesota, United States of America
Received March 2, 2009; Accepted September 28, 2009; Published October 30, 2009
Copyright: ? 2009 Ditch et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported in part by a grant from the NIH (R01NS046567) and by a grant from the Friedreich’s Ataxia Research Alliance (FARA www.
curefa.org) to EG. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: egrabc@lsuhsc.edu
Introduction
Trinucleotide repeat disorders are caused by the expansion of
unstable tandem repeats to a pathogenic size above disease-specific
length thresholds [1–5]. Disease-associated trinucleotide repeat
arrays include CAG?CTG, CGG?CCG, and GAA?TTC sequenc-
es. Disease pathology in these disorders is often progressive and
usually involves a neurodegenerative phenotype. Friedreich ataxia
(FRDA) is a relentlessly progressive neurodegenerative disorder
caused by GAA?TTC repeat expansion within the first intron of
the frataxin (FXN) gene [6]. FRDA is autosomal recessive and is
the only currently known human disorder associated with
GAA?TTC repeat expansion. The normal range of GAA?TTC
sequences within this intronic region is between 6 and 36 repeats,
while affected individuals have expansions ranging from 120 to
1700 uninterrupted repeats, most commonly 600 to 900 triplets
[6–9]. Transcription-dependent structure formation by expanded
GAA?TTC repeats and/or heterochromatin-mediated gene si-
lencing have been proposed as likely causes of reduced FXN
expression in FRDA [10–16]. The length of the repeat tract
directly correlates with disease severity [17,18], but our current
understanding of the mechanisms governing GAA?TTC repeat
expansion in FRDA is incomplete and is the focus of this study.
While intronic GAA?TTC sequences within the normal size
range (,36 triplets) are stably maintained, uninterrupted pre-
mutation (36–120 triplets) and expanded (.120 triplets) alleles
display intergenerational and somatic instability, consisting of both
contraction and expansion [8,9,19–27]. Interruptions within the
repeat tract stabilize premutation alleles during germline trans-
mission [9] and in peripheral leukocytes from GAA?TTC carriers
[24], but the effects of interruptions on the stability of expanded
alleles have not been reported. The intergenerational dynamics of
GAA?TTC instability are dependent on the mode of inheritance;
paternal transmission results in a bias towards repeat contraction,
while maternal transmission leads to both repeat expansion and
contraction [19,20,22]. Somatic instability in FRDA appears to be
tissue-specific as to whether repeat contraction or expansion
predominates. Analysis of GAA?TTC allele size in multiple tissues
from FRDA patients found a general contraction bias during aging
in most tissues examined [26]. However, a bias towards age-
dependent expansion is seen in disease relevant tissues of FRDA
patients, notably the dorsal root ganglia (DRG) of the central
nervous system, which suggests that the somatic expansion bias in
these tissues directly contributes to disease progression [27].
The unstable nature of the disease-associated repeat sequences
is generally attributed to the ability of these sequences to adopt
non-B DNA structures [3,4], but the cellular processes potentiating
instability have yet to be fully elucidated. Studies using bacteria,
yeast, and patient cell lines demonstrated a strong association
between replication and GAA?TTC instability, consisting mostly
of contractions [24,28–30]. The somatic expansion bias within
post-mitotic neurons of the spinal cord suggests that mechanisms
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other than replication, such as transcription and/or post-
replicative DNA repair, could be the primary forces driving
GAA?TTC repeat expansion in FRDA patients. GAA?TTC
repeat sequences have been shown to adopt DNA triplex and
triplex-associated structures in vitro and in bacteria [11–14,31,32],
leading to stalled transcription within the promoter distal half of
the GAA?TTC repeat region [13,14]. The association between
transcription and structure formation by GAA?TTC repeat
sequences, coupled with the high levels of FXN expression in the
spinal cord [6], suggests that there may be a relationship between
transcription and GAA?TTC expansion. However, the contribu-
tion of transcription to GAA?TTC expansion within the genomic
context has not been thoroughly examined.
The current lack of information regarding the mechanisms
responsible for GAA?TTC expansion in FRDA is partly due to the
absence of a good experimental model for GAA?TTC repeat
expansion. Bacterial and yeast models of GAA?TTC instability
display a pronounced contraction bias [24,29,30]. While a recently
developed system was able to capture the rare GAA?TTC
expansion events in yeast by a selection scheme, this limited
analysis to single-event expansions [33]. Lymphoblastoid cell lines
derived from FRDA patients have proven to be inconsistent
models for the analysis of GAA?TTC instability and require
meticulous small-pool PCR techniques to analyze the rare
expansion events [23,34]. While mouse models have proven
valuable in reproducing the tissue-specific expansion seen in
FRDA patients [35,36], a more homogeneous and rapid system
readily capable of experimental manipulation would provide a
valuable tool for mechanistic studies of GAA?TTC repeat
expansion.
Here we present a novel model for studying trinucleotide repeat
expansion in human cells. In our model system, uninterrupted
GAA?TTC repeat sequences display high levels of genomic
instability, with an overall tendency towards progressive expan-
sion. Using this system, we characterize the relationship between
repeat length and expansion. We further differentiate key
mechanistic processes regarding GAA?TTC expansion in human
cells. We demonstrate that GAA?TTC repeat expansion in our
model is independent of cell division rates. Using unique reporter
constructs, we identify transcription through the repeat tract as a
major contributor to GAA?TTC repeat expansion.
Results
GAA?TTC Repeat Sequences Undergo Progressive
Expansion in Human Cell Lines
In order to analyze the dynamics of GAA?TTC repeat stability
within the context of the human genome, we utilized tandem
reporter constructs containing uninterrupted (GAA?TTC)nrepeat
arrays integrated into the genome of an HEK 293 host cell line
(Figure 1A). The tandem reporter constructs utilize two self-
cleaving ribozymes in order to isolate transcription elongation
through the insert region of the construct. These constructs have
been previously tested and characterized [37]. Stable cell lines
were established using Flp-recombinase mediated recombination,
which allowed for single-copy integration at a consistent
chromosomal location and orientation in all cell lines used in this
study. Antibiotic selection following construct transfection pro-
duced colonies derived from individual cells with an integrated
reporter construct (see Materials and Methods). Therefore, all cell
lines used in this study are single-cell clonal lineages. We
confirmed single-copy integration by Southern blot analysis (as
shown in Figure 1B). We constructed the (GAA?TTC)nrepeat
arrays using an in vitro ligation strategy [38] in order to circumvent
bacterial propagation, which often leads to the contraction of large
GAA?TTC sequences and could result in the preferential selection
of repeats stabilized by interruptions. The repeat inserts were
sequenced prior to transfection to ensure that the repeat tracts in
our cell lines were uninterrupted.
We analyzed the stability of GAA?TTC repeat sequences within
our cell lines (Figure 1B and 1C). A (GAA?TTC)352insert was
initially chosen in order to analyze the stability of a repeat allele
within the size range expected to be unstable in FRDA. A cell line
made with a (GAA?TTC)352insert was serially passaged over a 10
week period. Genomic DNA samples were isolated at Day 0 (W0)
and after 1, 2, 4, and 10 weeks in culture (W1–W10). Subsequent
sizing of the GAA?TTC insert by Southern blot (Figure 1B) and
PCR (Figure 1C) analyses showed the progressive expansion of the
GAA?TTC repeat insert as a function of time in culture.
Expansion was detected as early as W1 (Figure 1B and 1C).
Southern blot analysis confirmed that the observed instability is
restricted to the (GAA?TTC)nregion of the integrated construct
(Figure 1B). EcoRV digestion of the genomic DNA cuts the
integrated tandem construct upstream of the GAA?TTC insert
region and between the 59 hRLUC and 39 hRLUC region of the
construct (Figure 1A). Our results show that the expansion is
localized to the 59 hRLUC probe region containing the
GAA?TTC insert, while the 39 hRLUC probe region remains
stable (Figure 1B). PCR analysis of the GAA?TTC insert
reproduced the results obtained by the Southern blot analysis
and showed a gain of roughly 119 triplets at W4 (Figure 1C) and a
gain of 284 triplets at W10 (Figure 1C). PCR and sequencing
analysis of the DNA sequence flanking the GAA?TTC repeat
region showed that instability is restricted to the repeat tract, while
the flanking sequence remains unaffected (Figure S1 and Figure
S2). PCR analysis allows for a more efficient and accurate analysis
for GAA?TTC insert sizing and will be used for sizing analysis
throughout this study.
We wanted to further characterize the dynamics of GAA?TTC
instability within the cell population during culturing by analyzing
the size distribution of individual GAA?TTC repeat alleles in that
population. End-point dilution of the parental (GAA?TTC)352cell
Author Summary
The human genome is comprised of the DNA base
sequences used by the cell as a blueprint to direct proper
cellular function. Changes in this sequence, known as
genomic instability, often interfere with vital cellular
functions, resulting in genetic disorders. Repetitive DNA
sequences are particularly susceptible to genomic insta-
bility. Trinucleotide repeat disorders are caused by three
base repeat sequences that increase in size when passed
from parent to child and during aging. Trinucleotide
repeat expansion results in disease when the size of the
repeat sequence increases into the pathogenic size range.
Our understanding of the mechanisms responsible for
these repeat length changes is incomplete and modeling
repeat expansion in human cells has proven difficult. Here,
we have developed a unique human cellular model of
GAA?TTC trinucleotide repeat expansion, the causative
mutation in Friedreich ataxia. Using this model, we
characterize GAA?TTC expansion in human cells and
identify gene transcription as a key regulator of GAA?TTC
repeat expansion. The findings of this study provide novel
insight into the mechanisms contributing to trinucleotide
repeat expansion in human cells and present new
implications for certain therapeutic approaches in Frie-
dreich ataxia.
GAA?TTC Expansion in Human Cells
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line at W4 (Figure 1B and 1C) produced colonies derived from
individual cells within the population. Size analysis of GAA?TTC
sequences from these clonal cell isolates revealed a mixed pool of
GAA?TTC repeat alleles, ranging in size from 264 to 1000 repeat
units (Figure 1D). Of 9 clones, 8 represented expansion events
relative to the transfected (GAA?TTC)352insert and one deletion
product was detected (lane 1 in Figure 1D). In several of these
colonies, multiple amplification products were detected. The
detection of different sized alleles in the individual colonies is likely
due to continued instability during the growth of the colony. The
mixed distribution of repeat alleles in Figure 1D illustrates the
mosaicism of individual GAA?TTC repeat alleles within the cell
population, which is a characteristic commonly seen in the somatic
tissues of FRDA patients [26,27]. The wide-ranging instability of
individual repeat alleles illustrated in Figure 1D suggests that the
progressive expansion displayed in Figure 1B and 1C does not
represent the uniform expansion of every allele in the cell
population. Figure 1B and 1C likely represent an expansion bias
among the majority of repeat alleles, with larger and smaller
outlier alleles within the population. PCR and Southern blot
analysis of a large pool of mixed sized repeat alleles is prone to
detect the most common alleles in that population, therefore the
products shown in Figure 1B and 1C are likely a reflection of this
tendency. It is important to note that there is no selective pressure
acting on the GAA?TTC repeat inserts within the integrated
reporter construct during culturing. Therefore, there is unlikely to
be any sampling bias favoring repeat expansion over deletion.
GAA?TTC Expansion Is Repeat Length–Dependent
We next analyzed the relationship between repeat length and
stability within our cell lines (Figure 2). The duration between
construct transfection and initial insert sizing varies among the
different clones (see Materials and Methods). The repeat lengths
used throughout this report are in reference to the transfected
insert sequence and do not account for any gains in repeat size
between transfection and the beginning of the time-course
experiments. Time-course analysis of repeat stability was per-
formed using cell lines harboring 11, 44, 88, 176, and 1000
GAA?TTC repeats (Figure 2A). (GAA?TTC)11inserts are within
the normal size range of GAA?TTC repeats found within the first
Figure 1. Model of GAA?TTC repeat expansion in human cell lines. (A) Tandem reporter construct designed to isolate transcription
elongation through GAA?TTC repeat insert sequences. Single copy integration of the construct into the genome of the host cell line is facilitated via
conservative, site-specific recombination using Flp-recombinase. (B) Southern blot analysis of GAA?TTC repeat expansion using the template DNA
isolated from a human cell line with a (GAA?TTC)352insert after 0, 1, 2, 4, and 10 weeks (W0–W10) in culture. M: 1 Kb plus size standard. EcoRV
digestion of the genomic DNA cuts the tandem construct upstream of the GAA?TTC repeat region and between the 59 hRLUC and 39 hRLUC regions.
59 hRLUC probe is specific to the 59 side of the hRLUC reporter in the tandem construct containing the GAA?TTC insert region. 39 hRLUC probe is
specific to the 39 region of the hRLUC expression cassette in the tandem construct and is not associated with the GAA?TTC repeat region. (C) PCR
analysis of a (GAA?TTC)352repeat insert isolated at W0, W1, W2, W4, and W10 from Figure 1B. PCR amplification adds 438 bp to the GAA?TTC insert
(59: 338 bp+(GAA)n+100 bp: 39). M: 1 Kb plus size standard (D) PCR analysis of GAA?TTC repeat inserts from clonal cell isolates derived from an end-
point dilution of the (GAA?TTC)352parental cell line at W4 in Figure 1B and 1C. PCR amplification adds 438 bp to the GAA?TTC insert. M: 1 Kb plus size
standard.
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intron of the FXN allele and repeats within this range have been
shown to be below the reported initiation threshold for instability
in simple replication models and human cells [23,24,38]. As
expected, the (GAA?TTC)11insert sequence remained stable over
the 4 week time-course as indicated by the tight banding pattern of
the PCR amplification product at each time-point (Figure 2A).
The (GAA?TTC)44underwent a modest level of expansion, which
is in agreement with the initiation threshold for instability reported
by others [23,24] (Figure 2A). PCR mobility profile analysis
(Figure 2B) further illustrates the stability of the (GAA?TTC)11
insert and the modest expansion of the (GAA?TTC)44repeat insert
during the time-course experiments. Substantial expansion of the
(GAA?TTC)88and (GAA?TTC)176inserts was observed during the
4 week time-course with the larger (GAA?TTC)176 sequence
demonstrating a more rapid expansion when compared to the
(GAA?TTC)88sequence (Figure 2A). PCR mobility profile analysis
of the (GAA?TTC)88sequence showed a gain of 9 triplets when
comparing the peak intensities of the amplified products at W0
and W4, while the (GAA?TTC)176sequence showed a gain of 140
triplets over the same time period (Figure 2B). These results
demonstrate that GAA?TTC repeat sequences undergo a length-
dependent increase in expansion rate in our cellular model.
Previous analysis using peripheral blood samples of FRDA
patients revealed a contraction bias in GAA?TTC sequences
greater than 500 repeats in length [23], while post-mortem
analysis of GAA?TTC sequences ranging from 350–1030 repeats
in the dorsal root ganglia of FRDA patients demonstrated an age-
dependent expansion bias [27]. To analyze the stability dynamics
of larger GAA?TTC sequences within our cell lines, we performed
time-course experiments using a cell line isolated from the end-
Figure 2. Expansion is specific to GAA?TTC repeats and is repeat length–dependent. (A) PCR analysis of (GAA?TTC)11, (GAA?TTC)44,
(GAA?TTC)88, (GAA?TTC)176, and (GAA?TTC)1000inserts isolated at W0, W2, W3, and W4. The tetramer insert sequence is a 2.1 Kb semi-repetitive
sequence controlling for the effects insert size on stability. PCR amplification adds 438 bp to the GAA?TTC and tetramer inserts. M: 1 Kb plus size
standard. (B) Profile analysis of the insert PCR product mobility distribution. Profile analysis of inserts isolated at W0 and W4 are shown. The x-axis
represents product signal intensity. The y-axis represents product mobility during electrophoresis. Dashed lines mark the mobility of the peak
amplification signals at W0 and W4. Profile analysis was performed using Kodak Molecular Imaging software.
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point dilution of the (GAA?TTC)352cell line (lane 9 in Figure 1D).
This cell line contained a (GAA?TTC)1000 insert sequence
(Figure 2A). Sizing of the insert sequence showed the continued
expansion of the (GAA?TTC)1000insert from W0 to W4 (Figure 2A
and 2B), confirming that larger GAA?TTC inserts continue to
expand in our system.
To determine if the observed expansion is due to the length of
the DNA sequence inserted into poly-linker region of our
construct, rather than sequence composition, we analyzed the
stability of a semi-repetitive 2.1 kilobase (kb) tetramer insert
sequence (Figure 2A). The tetramer sequence is composed of four
identical non-repetitive 529 bp fragments and is equivalent in size
to a (GAA?TTC)700 insert sequence [37]. We have previously
shown that this tetramer insert has a neutral affect on transcription
through the insert region of the reporter constructs [37]. The
tetramer sequence remained stable over the duration of the time-
course (Figure 2A and 2B), indicating that the observed expansion
of GAA?TTC repeat sequences in these cell lines is not solely a
function of insert length. This suggests that GAA?TTC expansion
in our cell lines must be due to certain properties intrinsic to these
triplet repeats.
Repeat Interruptions Reduce GAA?TTC Expansion Levels
Previous studies have shown that interruptions in the purity of
the GAA?TTC repeat tract have a stabilizing effect on these
sequences [9,24], possibly by interfering with the formation of
secondary structures by the repeat. Time-course analysis of repeat
stability was performed on four individual cell lines with
(GAA?TTC)176 insert sequences. The mean increase in repeat
size among three of these cell lines was 61.1611.6 triplets, while
the fourth cell line gained only 12 triplets after 3 weeks in culture
(Figure 3A). Sequencing of the first three inserts did not detect any
interruptions. Sequencing of the GAA?TTC insert region within
the fourth cell line identified two separate interrupting point
mutations (Figure S3). An ART mutation was detected approx-
imately 118 triplets into the repeat region from the 59 end and a
TRG mutation was detected 31 triplets into the repeat region
from the 39 end (Figure S3). Expansion of the repeat sequence
either upstream or downstream of the interruption distributes the
base signal when sequencing from a polymorphic population, as
represented in Figure S3. These shifts can mask the detection of
potential interruptions during automated sequencing, with muta-
tions towards the edges of a repetitive run being more readily
detected. Careful visual inspection of the primary sequencing data
is needed when seeking to identify potential interruptions within
unstable tandem repeat arrays. The identification of multiple
interruptions within the GAA?TTC sequence in this cell line
suggests that even a couple of point mutations that interrupt the
purity of the repeat sequence can greatly reduce the rate of repeat
expansion.
To further analyze the effects of interruptions on GAA?TTC
repeat expansion, we created a cell line containing a repeat insert
with an interrupting hexamer (TCAATT) that creates an Mfe I
restriction endonuclease recognition site situated between two
repetitive tracts, (GAA)88MfeI(GAA)90. Sequencing did not detect
any interruptions in the either of the two repeat tracts flanking the
interruption within this construct, but visual inspection of the
sequencing chromatogram confirmed the presence of the inter-
rupting hexamer (Figure S3). Stability analysis revealed that this
interrupting hexamer sequence reduces the rate of expansion
when compared to uninterrupted (GAA?TTC)176insert sequences
(Figure 3B). The (GAA)88MfeI(GAA)90 insert gained only 22
triplets after 3 weeks in culture, compared to the 61.1611.6 mean
triplet gain by the uninterrupted (GAA?TTC)176insert sequences.
Figure 3. Expansion levels are affected by the purity of the
repeat sequence and the repeat orientation. (A) PCR analysis of
GAA?TTC expansion within an uninterrupted (GAA?TTC)176insert at W0
and W3 compared to a (GAA?TTC)176 insert with two interrupting
mutations identified by sequencing analysis. An ART mutation was
detected approximately 118 triplets into the repeat region from the 59
end and a TRG mutation was detected 31 triplets into the repeat
region from the 39 end. PCR amplification adds 438 bp to the GAA?TTC
insert. M: 1 Kb plus size standard. (B) PCR analysis of GAA?TTC
expansion within an uninterrupted (GAA?TTC)176 insert at W0, W2,
and W3 compared to a (GAA?TTC)88MfeI(GAA?TTC)90insert sequence
containing an interrupting hexamer sequence located between two
repeat tracts. The CAATTG hexamer is the recognition sequence for the
Mfe I restriction endonuclease. PCR amplification adds 438 bp to the
GAA?TTC insert. M: 1 Kb plus size standard. (C) Mfe I digestion of the
PCR amplification products from the (GAA?TTC)88MfeI(GAA?TTC)90time-
course at W0, W2, and W3. PCR amplification adds 338 bp to the 59 end
of the GAA?TTC insert and 100 bp to the 39 end. Mfe I digestion yields
two distinct fragments representing the promoter proximal
(GAA?TTC)88repeat tract (59 GAA) and the distal (GAA?TTC)90repeat
tract (39 GAA). The residual full-length product (Uncut) is due to
incomplete digestion. M: 1 Kb plus size standard. (D) PCR sizing analysis
of a (GAA?TTC)176insert at W0 and W3 compared to a reverse oriented
(CTT?AAG)176insert sequence at the same time-points. PCR amplifica-
tion adds 438 bp to the repeat insert. M: 1 Kb plus size standard. A
representative gel from an n=2 is shown.
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