The Evolution of Skeletal Muscle Performance Through Gene Duplication

DOI 10.1002/bies.200900110 Review article

The evolution of skeletal muscle performance:
gene duplication and divergence of human
sarcomeric a-actinins
Monkol Lek,1,2 Kate G. R. Quinlan,1,2 and Kathryn N. North1,2*
1
Institute for Neuroscience and Muscle Research, The Children’s Hospital at Westmead, Sydney, 2145 NSW, Australia
2
Discipline of Paediatrics and Child Health, Faculty of Medicine, University of Sydney, Sydney, 2006 NSW, Australia

In humans, there are two skeletal muscle a-actinins, expressed and function as cytoskeletal proteins. a-Actinin-4
encoded by ACTN2 and ACTN3, and the ACTN3 genotype has unique functions in kidney tissue(7) and cancer inva-
is associated with human athletic performance. Remark- sion,(8–10) while a-actinin-1 is highly expressed at focal
ably, approximately 1 billion people worldwide are defi-
cient in a-actinin-3 due to the common ACTN3 R577X adhesions and adherens junctions.(8) The skeletal muscle or
polymorphism. The a-actinins are an ancient family of sarcomeric a-actinins, a-actinin-2 and a-actinin-3, are highly
actin-binding proteins with structural, signalling and expressed in muscle where they act as major structural
metabolic functions. The skeletal muscle a-actinins components of the contractile apparatus at the Z-line.(11)
diverged 250–300 million years ago, and ACTN3 has a-Actinin-2 is more widely expressed, and is found in all
since developed restricted expression in fast muscle
fibres. Despite ACTN2 and ACTN3 retaining considerable human skeletal muscle fibres and cardiac muscle fibres, as
sequence similarity, it is likely that following duplication well as the brain. The closely related isoform, a-actinin-3 has
there was a divergence in function explaining why a more specialised expression pattern and is expressed only
a-actinin-2 cannot completely compensate for the in fast glycolytic skeletal muscle fibres, with low levels of
absence of a-actinin-3. This paper focuses on the role expression in the brain.(12) Although these two sarcomeric a-
of skeletal muscle a-actinins, and how possible changes
in functions between these duplicates fit in the context of actinins diverged 250–300 million years ago following a
gene duplication paradigms. duplication event, they have retained considerable sequence
similarity. At the amino acid level, human a-actinin-2 and a-
Keywords: gene duplication; muscle performance; skeletal actinin-3 are 80% identical and 90% similar.(12)
muscle actinin Remarkably, an estimated one billion humans worldwide
are completely deficient in a-actinin-3, due to homozygosity
for a common nonsense polymorphism (R577X) in the
Introduction ACTN3 gene.(13,14) The absence of a-actinin-3 is not
associated with an obvious disease phenotype, suggesting
The a-actinins are an ancient family of actin-binding proteins that other proteins are able to largely compensate for the
that play a key role in the maintenance and regulation of the absence of a-actinin-3 in the fast fibres of humans. The most
cytoskeleton.(1) The a-actinins have homologues in slime likely candidate as a compensatory protein is a-actinin-2.
mould,(2) fungi(3) and metazoans, but, surprisingly, are not However, given that a-actinin-3 has been highly conserved
present in plants.(4) Amongst the metazoans, vertebrates during vertebrate evolution, it is unlikely to be completely
possess four a-actinin genes (ACTN1-4) postulated to arise functionally redundant.(4,5) This has been confirmed by
from a single invertebrate ancestral gene.(4,5) All members of demonstrating that the loss of a-actinin-3 influences the
the a-actinin family share a distinct domain topology function of human skeletal muscle. ACTN3 genotype is
consisting of an actin-binding domain, a central rod domain strongly associated with elite athletic performance; the
and a C-terminal EF hand domain(1) (Fig. 1A). The frequency of the ACTN3 577XX null genotype associated
distinguishing feature that separates vertebrate from inverte- with a-actinin-3 deficiency is significantly lower in Caucasian
brate a-actinins is splicing of the EF-hand domain to create sprint/power athletes compared to controls.(15) This associa-
distinct functional forms.(6) tion has been independently replicated in studies of Greek,(16)
In mammals there are four a-actinins.(4,5) Both of the non- Russian,(17) Spanish,(18) American(19) and Israeli(20) athletes.
muscle a-actinins, a-actinin-1 and -4, are ubiquitously In non-athlete human populations, there have been several
studies demonstrating differences in muscle strength and
power between ACTN3 genotypes. The ACTN3 577XX
*Correspondence to: K. North, Institute for Neuroscience and Muscle
Research, The Children’s Hospital at Westmead, Sydney, 2145 NSW, Australia. genotype is associated with lower strength(21,22) and reduced
E-mail: kathryn@chw.edu.au speed; in a study of Greek adolescent boys (n ¼ 525),

BioEssays 32:17–25, ß 2009 Wiley Periodicals, Inc. 17

Review article M. Lek, K. G. R.Quinlan and K. N. North

absence of a-actinin-3. This paper focuses on the role of
the sarcomeric a-actinins in skeletal muscle and how possible
changes in functions between these duplicates fit in the
context of gene duplication paradigms.

a-Actinin gene family – created by two
rounds of duplication
The evolution from invertebrate to more complex vertebrate
Figure 1. Molecular models of a-actinin visualised using UCSF
Chimera. A: Ribbon representation of a-actinin dimer (1SJJ). The
species is thought to have occurred due to whole genome
actin binding, rod and EF-hand domains are coloured red, orange and duplication.(27) A study of developmental gene families such as
blue, respectively. B: Surface representation of a-actinin rod domain the Hox genes,(28) which occur as single genes in invertebrates
(1HCI), aligned with spectrin repeat 1 on the left and spectrin repeat 4 and as tetra-paralogues in vertebrates,(29) supports this
on the right. The colours grey, green and purple represent identical,
hypothesis; the two rounds of whole genome duplication are
conservative and non-conservative residue substitutions when a-
actinin-2 and -3 vertebrate sequences are aligned. thought to have occurred in the basal vertebrates before bony
fishes diverged. The first round occurred near the divergence of
XX individuals took significantly longer to complete a 40-m jawless fish (e.g. lamprey and hagfish), while the second
sprint.(23) occurred near the divergence of cartilaginous fish (e.g. sharks
Although the loss of a-actinin-3 has detrimental effects on and rays).(30) Not all gene families follow the 1:4 rule, and this
sprint performance, a-actinin-3 deficiency appears to be can be explained by chromosome segment loss and duplication
beneficial in certain circumstances. There has been strong or gene loss after each round of duplication.(31) The a-actinin
positive selection for the X allele during recent human family follows the 1:4 rule with only one gene in invertebrates
evolution in Europe and Asia.(13) ACTN3 is one of only two and four genes in vertebrates.(4) This suggests that during
genes (the other is CASP12) for which strong evidence exists evolution there may have been intermediate species with only
for recent positive selection of a null allele in human one sarcomeric a-actinin and one non-muscle a-actinin. In fact,
populations.(24) The CASP12 null allele, which has been lamprey has two a-actinin genes and the elephant shark has at
positively selected for in non-African populations, results in least three a-actinin genes (Lek, unpublished).
the expression of a truncated protein that decreases the risk The developmental genes that follow the 1:4 rule are
of developing sepsis.(25) Recent studies in an Actn3 knockout forced, shortly after duplication, to diverge in function to avoid
(KO) mouse model demonstrate that a-actinin-3 deficiency the fate of most gene duplicates, i.e. gene loss. We propose
results in upregulation of a-actinin-2, suggesting that genetic that similar forces have acted on the a-actinins. This paradigm
feedback regulates sarcomeric a-actinin expression.(26) In of gene duplication and divergence resulting in alterations in
fast, glycolytic 2B muscle fibres expressing a-actinin-2 in the protein function and expression patterns has emerged as an
absence of a-actinin-3, there is a shift towards a slow fibre important concept in gene evolution.(32) The paradigm allows
phenotype with increased fatigue resistance, and an increase for two general scenarios, neo-functionalisation and sub-
in oxidative enzyme activity. The shift towards more efficient functionalisation. There are two main characteristics that
oxidative metabolism may underlie the selective advantage of distinguish between these two scenarios(33) (Fig. 2). First, in
the ACTN3 R577X allele during evolution.(13) Together, these sub-functionalisation both duplicates are subjected to adap-
results suggest that a-actinin-2 and a-actinin-3 perform tive changes, while in neo-functionalisation only one duplicate
overlapping but distinct functional roles and that the is subjected to adaptive changes with the other maintaining
phenotypic effects of a-actinin-3 deficiency are due primarily ancestral function through purifying selection. Hence, in the
to functional differences between a-actinin-2 and -3. sub-functionalisation model, both duplicates develop an
The sarcomeric a-actinins are well characterised in terms optimised ancestral function.(34) A corollary to the paradigm
of their structure, function and interactions and thus provide is that the pre-duplicate gene in the sub-functionalisation
an opportunity to apply the concepts and paradigms of gene model can perform the role of both duplicate genes, while the
duplication to structural proteins belonging to a more complex pre-duplicate gene in the neo-functionalisation model can
system. On this basis, we have reviewed what is known about only perform the role of one of the duplicate genes. The
sarcomeric a-actinin structure, mechanical properties, func- enzymes GAL1/3,(35) HST1/SIR2,(36) dihydroflavonol-4-
tional isoforms, tissue expression patterns and important a- reductase (DFR)(34) and RNASE1/1B(37) provide good
actinin interaction partners expressed in muscle, since examples of how this corollary can be tested.
differences in these functions may help to explain the Functional divergence after duplication comes in two
phenotypic differences observed in the presence and forms: (i) divergence of the protein coding sequence, resulting

18 BioEssays 32:17–25, ß 2009 Wiley Periodicals, Inc.

M. Lek, K. G. R.Quinlan and K. N. North Review article

a-actinins, which allow them to function as a major component
of the contractile apparatus. This adaptation allows the
sarcomeric a-actinins to remain rigid when extension and
torsion forces are applied, while still maintaining flexibility
along the length of the rod domain. Molecular dynamics
simulation involving the a-actinin-2 rod domain in monomer
and dimer forms has identified charged residues as important
for extension rigidity and flexibility, while aromatic
residue stacking is important for torsional rigidity.(46) The
sequence divergence of the rod domain (Fig. 1B) contributes
to the majority of the sequence changes as a whole between
the sarcomeric a-actinins.(47) Therefore, any substitutions
that involve charged or aromatic residues may slightly alter
the mechanical properties of a-actinin-3 relative to a-actinin-
2. a-Actinin-3 has developed restricted expression in fast
glycolytic muscle fibres(12) that are responsible for the
generation of force at high velocity; we postulate that changes
in the residues within the rod domain have allowed a-actinin-3
to confer to the fast muscle fibres the extra strength and
flexibility required to generate additional power.

Figure 2. Using sarcomeric a-actinin as an example, after duplica-
tion of a gene capable of multiple interactions/functions, there are two
possible distinct scenarios besides gene loss. A: Sub-functionalisa-
Functional divergence in isoforms
tion, where one interaction site is optimised in each of the copies.
B: Neo-functionalisation, where one copy retains the ancestral inter- The sarcomeric a-actinins have maintained a difference in
action sites while the other is free to evolve new interaction sites. alternative splice isoforms. a-Actinin-2 has two known
isoforms, which arise through mutual exclusive splicing of
in changes in protein structure, function and interactions; and exon 8 or exon 19 in the actin binding or EF hand domains,
(ii) divergence in regulatory sequence resulting in altered respectively. In contrast, a-actinin-3 has no known alternate
expression patterns.(32) The following sections review the splice isoforms. The ACTN2 alternative splicing in exon 8,
functional divergence between the sarcomeric a-actinins in which is also conserved in Drosophila melanogaster and
the context of gene duplication paradigms. Caenorhabditis elegans,(48) encodes a brain-specific
isoform in humans(49) and a non-muscle isoform in
D. melanogaster.(50) Despite the conservation of this isoform
Conservation of structure in both vertebrates and non-vertebrates, little is known about
its function. However, studies of disease mutations in ACTN4
The numerous structural studies on the sarcomeric a-
exon 8, which cause focal and segmental glomerulosclerosis
actinins(38–41) and the non-muscle a-actinins(42–44) suggest
(FSGS),(51) provide potential insight into the function of the
that the high similarity between the a-actinins at the protein
brain-specific isoform of ACTN2. The ACTN4 K255E mutation
sequence level has allowed them to maintain an almost
resulted in a loss of calcium-mediated actin binding along with
identical tertiary and quaternary protein structure. This is not
an increased affinity for actin.(7) Since a-actinin-2 plays a role
unexpected since proteins with sequence identity as low as
in regulating dendritic spine morphology and density, the brain
30% retain relatively similar structures.(45) This conservation
isoform may contribute to this function through the regulation
of structure suggests that any functional divergence between
of actin binding.(52)
the sarcomeric a-actinins is likely to be due to changes in
The alternative splicing of exon 19 that allows a-actinins to
surface or mechanical properties introduced through slightly
switch between calcium-sensitive and -insensitive isoforms is
altered physiochemical properties of the substituting residues.
a known functional difference between the sarcomeric and
non-muscle a-actinins.(6) The zebra fish and chicken ACTN2
Possible divergence in mechanical have retained alternative splicing at exon 19 and remain an
properties exception to this functional difference.(53) There is no
evidence to date to suggest that these isoforms of
The sarcomeric a-actinins have adapted specialised a-actinin-2 are expressed in skeletal muscle; however, the
mechanical properties since diverging from the cytoskeletal existence of these isoforms suggests that the pre-duplicated



sarcomeric a-actinin was a multi-functional gene and, with channels and receptors and some studies have shown
following duplication, the functions mediated by alternative that a-actinins are able to modulate their function,(86–89)
splicing were lost in a-actinin-3 but were retained in a-actinin- suggesting the sarcomeric a-actinins play more than just a
2 in a species-specific manner. The loss of function in structural role at the membrane. Finally, interactions
a-actinin-3 could then allow adaptive changes in a-actinin-3, with metabolic proteins, fructose 1,6-bisphosphatase
such as optimisation of interaction sites, which previously (FBPase),(90) glycogen phosphorylase(91) and glycogen
were in conflict with another function in the pre-duplicated synthase(92) may help to explain the shift to a more oxidative
gene. phenotype observed in the Actn3 KO mouse.(13) Interestingly,
the vast majority of these interactions are mediated by the four
spectrin repeats that make up the a-actinin rod domain; this is
Divergence in protein interaction and the region where the majority of the sequence differences
interaction networks occur between the sarcomeric a-actinins.(47) Taken together,
these data suggest that there are likely to be interaction
The sarcomeric a-actinins interact with a diverse range of partners that have higher binding affinity for either a-actinin-2
proteins in skeletal muscle with the spectrin repeats mediating or -3. In addition, some of these preferential interactions may
the majority of these interactions. Any minor changes to these be fibre-type specific which may help to explain why a-actinin-
protein interactions, due to sequence divergence or changes 2 cannot completely compensate for a-actinin-3. Many of the
in overall a-actinin levels, may affect the overall properties a- studies that identified these a-actinin binding partners have
actinin-containing complexes, both at the Z-line and within the not distinguished between a-actinin-2 and -3 or even between
sarcomere. First, interactions with structural proteins actin,(54) sarcomeric and non-muscle a-actinins; the few that have were
titin(55,56) and myotilin(57,58) may contribute to the overall unable to show differences in binding affinity.(70–72,93–95)
contractile properties at the Z line. Second, interactions with However, this may be expected given the high similarity
proteins with protein interaction domains (i.e. PDZ and LIM between the sarcomeric a-actinins; future studies will need to
domains) such as ZASP (59,60) ALP (61) hCLIM,(62) MLP(63) and
, , use assays designed for the specific purpose of detecting
myopodin(64) play a role in maintaining structural integrity and small differences in binding affinity.
responding to physiological stimuli.(65–68) Third, interactions Sarcomeric a-actinins interact with a diverse range of
with calsarcins(69–72) may influence the calcineurin pathway, proteins,(96–98) forming a highly connected network of protein
which is responsible for muscle hypertrophy(73,74) and fibre interactions most likely derived from the pre-duplicated gene.
type switching.(75) Interactions with calcineurin via calsarcins The ability of sarcomeric a-actinins to homodimerise and
may explain the significant decrease in type 2A (fast oxidative) heterodimerise(99) adds a degree of evolutionary freedom for
muscle fibre size observed in the Actn3 KO mouse and the asymmetric divergence of each protein interaction
enhanced endurance capacity common to both Actn3(13) and network, which may result in new or specialised function of
calsarcin-2 KO mice.(76) In addition, interaction with the the complexes formed (Fig. 3).(100) In the Actn3 KO mouse,
calcineurin pathway may explain why ACTN3 577RR humans absence of a-actinin-3 results in accumulations of desmin,
have a greater proportion of 2X (fast glycolytic) muscle fibres myotilin and glycogen phosphorylase in fast glycolytic type 2B
compared to those with a-actinin-3 deficiency (577XX).(21) fibres (North, unpublished). This suggests that the loss of a-
Fourth, interactions with signalling proteins, G protein- actinin-3 results in destabilisation of complexes that require
coupled receptor kinases (GRKs),(77) phospholipase D2 either the a-actinin-3 homodimers or heterodimers for
(PLD2),(78) protein kinase N (PKN),(79) rabphilin-3A(80) and stability.
PI 3-kinase,(81) may allow structures scaffolded by the
sarcomeric a-actinins to respond and amplify physiological
stimuli. However, the exact role of interactions with signalling Divergence in tissue expression patterns
proteins may be hard to elucidate due to their generalised
functions. Fifth, interactions with membrane proteins, dystro- In addition to divergence in protein sequence, the altered
phin(82) and integrin,(83,84) which provide the structural link tissue-specific expression patterns observed in the sarco-
between the sarcomere and the basal lamina, contribute to meric a-actinins suggests a divergence in regulatory regions.
maintaining membrane integrity during muscle contractions. In humans, a-actinin-2 is expressed in both skeletal and
There is one study in humans that demonstrated that ACTN3 cardiac muscle along with both the grey and white matter in
577XX individuals had lower creatine kinase (CK) levels at the brain; while a-actinin-3 expression is restricted to skeletal
baseline, suggesting that they were less prone to muscle muscle and low levels in grey matter in brain.(12) Within human
damage.(85) However, this study did not normalise for the skeletal muscle, a-actinin-2 is expressed in all fibres, while
lower muscle mass typically seen in XX individuals, which a-actinin-3 is restricted to fast, glycolytic type 2 fibres.(101)
could also explain the lower CK levels. a-Actinins also interact A detailed analysis of mouse skeletal muscle revealed



Figure 3. The asymmetric divergence of protein interaction networks after duplication allows for an increase in complexity and possible novel
functions. Before duplication, dimerisation allowed for larger complexes. After duplication and divergence of protein interaction networks,
homodimiseration allows for larger complexes with slightly altered functions, while heterodimersation increases complexity and addition of
possible novel functions.

a-actinin-2 expression in type 1, 2A, 2X and 60% of 2B provides evidence against the functional redundancy of
fibres, while a-actinin-3 was exclusively expressed in type 2B a-actinin-3.
(fast glycolytic) fibres. However, it is important to note that Besides humans with the ACTN3 577XX genotype (who
mouse muscles are composed of predominately fast fibres have a-actinin-2 as their only sarcomeric a-actinin), chickens
(80%), while human muscle has a roughly equal mixture of are the only other species identified with only one sarcomeric
fast and slow fibres,(12) which suggests the loss of a-actinin-3 a-actinin(98); however, the mechanism of loss appears to be
in mice may have a greater impact on muscle performance. different. In humans the loss is due to a null mutation within
The variation in tissue expression patterns suggests that, the coding region of the pre-existing ACTN3 gene.(14) In
after gene duplication, degenerative changes in ACTN3 contrast, an analysis of chicken chromosome synteny against
regulatory regions have restricted its expression compared to mammalian chromosomes revealed loss of whole chromo-
ACTN2, rather than novel changes in ACTN2 regulatory some segments(102) as part of the genome streamlining
regions allowing for a greater range of tissue expression. The process known to have occurred in the dinosaur and bird
restricted expression profile of a-actinin-3 has simultaneously lineage.(103) It is possible that the chicken ACTN3 gene was
relieved evolutionary constraints placed on functions that are loss during this streamlining process. Furthermore, the draft
not required in fast muscle fibres and has allowed specialised genome of zebra finch also contains three a-actinin genes,
functions in fast fibres that may be disadvantageous in other while in contrast the lizard genome contains four a-actinin
tissues. genes, which adds further support to this mechanism (Lek,
unpublished).

The evolutionary need for ACTN3
The possible contribution of gene dosage
There is no overt disease phenotype in humans who are a-
actinin-3 deficient, which is likely largely due to compensation Genetic backup or robustness is an alternative evolutionary
by a-actinin-2. Conversely, the only known ACTN2 disease- pressure that can also maintain genes after duplication. Both
causing mutation, Q7R, results in a dilated cardiomyopa- duplicate genes may be required to act as a genetic backup in
thy(63) with no obvious skeletal muscle or brain phenotype. case one gene acquires a deleterious mutation or alterna-
This suggests that the ACTN2 Q7R mutation only affects tively to produce higher levels of protein.(104,105) Highly
function in the heart where a-actinin-3 is not expressed. An connected nodes in protein interaction networks such as the
analysis of haplotypes containing the R577X polymorphism sarcomeric a-actinins require a backup, as deletion of this hub
showed a recent and rapid expansion amongst the European node causes greater disruption to networks compared to
and Asian populations, suggesting that the loss of a-actinin-3 deletions of lowly connected nodes.(106) In addition, in the
results in a more efficient muscle metabolism may be Actn3 KO mouse, there is an upregulation of a-actinin-2 to
advantageous in the Eurasian environment.(13) In combina- compensate for the loss of a-actinin-3, so that a-actinin-2 is
tion these data raise the question as to whether two expressed in all muscle fibres in the mouse, similar to that
sarcomeric a-actinins are necessary or, more specifically, seen in ACTN3 577XX humans.(13) It is still not known
whether a-actinin-3 is functionally redundant. However, the whether the absolute amount of sarcomeric a-actinins is
increasing number of studies showing an association maintained as a consequence of this upregulation. In addition,
between ACTN3 genotype and human skeletal muscle it is possible (but not yet known) that a-actinin-1(107)
performance and conservation amongst vertebrates(4) and -4(108) are also upregulated to maintain the total amount



of a-actinin. We cannot exclude ‘dosage effect’ as a possibility
to explain the observed phenotypic differences as there are
various diseases caused by altered protein levels.(104) Studies
that involve quantitative measurements of muscle function
reveal a dosage-effect pattern in which the RX genotype lies
between RR and XX genotypes.(21–23,109) However, other
studies have not shown any pattern to suggest a dosage
effect amongst the ACTN3 genotypes.(110–112) The discre-
pancy in these studies can be explained by confounding
factors such as age, lifestyle and genetic background. Further
studies involving the heterozygous Actn3 KO mouse will
remove these confounding factors and thus provide further
insight into whether a-actinin levels contribute to overall
muscle function.

Conclusions

The gene duplication that gave rise to ACTN2 and ACTN3 is
Figure 4. Duplication and divergence model proposed by this
thought to have occurred during the second round of whole
paper. Before duplication the ancestral sarcomeric a-actinin had
genome duplication within vertebrate evolution.(30) Shortly the functions of both ACTN2 and ACTN3 in terms of tissue expression
after duplication, a-actinin-2 and a-actinin-3 most likely had a and functional isoforms. After duplication, ACTN2 has conserved
similar protein interaction network. However, we know from most of the functions of the preduplicated gene, while ACTN3 has
other protein families that have arisen through gene lost many of these functions, which may have allowed it to optimise
function in fast fibres.
duplication events that, over time, interaction sites may be
lost or optimised (sub-functionalisation) or new interaction
sites may evolve (neo-functionalisation).(33) We can differ- (another member of the spectrin superfamily) has also
entiate between these two possibilities by studying the evolved a new function since duplication and divergence,
reconstructed pre-duplicated sarcomeric a-actinins. The allowing for labile spectrin tetramers thought to be advanta-
sub-functionalisation model is more likely if the pre-duplicated geous in human red blood cells.(113) Further studies are
gene can perform the functions of both ACTN2 and ACTN3. required to define the specialised function(s) of ACTN3 and to
This may have been necessary to allow a-actinin-2 and -3 to determine total a-actinin levels in muscle fibres of individuals
specialise in muscle fibres or tissues with contrasting with different ACTN3 genotypes. This will determine whether
characteristics, also known as escape from adaptive conflict. loss of specialised a-actinin-3 function or merely gene dosage
Further, specialisation is not limited to optimisation of a protein can explain the variations in skeletal muscle performance
interaction, it can also be associated with weakening or loss of associated with a-actinin-3 deficiency.
an interaction. Conversely, the neo-functionalisation model is
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