Circulation. 1995;91:513-520
(Circulation. 1995;91:513-520.)
© 1995 American Heart Association, Inc.
Unstable Triplet Repeat Diseases
Darren G. Monckton, PhD;
C. Thomas Caskey, MD
From the Department of Molecular and Human Genetics, Howard Hughes
Medical Institute and Human Genome Center, Baylor College of Medicine,
Houston, Tex.
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Abstract
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Abstract Seven inherited human disorders are now
associated
with the intragenic expansion of triplet repeat DNA
sequences.
These repeats demonstrate extreme instability in both
germline
and somatic tissue, accounting for the unusual genetic
inheritance
patterns and symptom variability associated with these
diseases.
Key Words: diagnosis genes molecular biology nucleotides hereditary diseases polymerase chain reaction
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Introduction
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In recent years, a new mode of molecular
mutation responsible
for inherited human disease has come to light. The
expansion
of trinucleotide repeat DNA sequences within the transcribed
regions
of genes has been demonstrated to be the underlying genetic
defect
in seven inherited human disorders: fragile X
syndrome,
1 2 3 spinal and bulbar muscular
atrophy (SBMA,
also known as Kennedy's
disease),
4 myotonic dystrophy
(DM),
5 6 7 Huntington's disease
(HD),
8
spinocerebellar ataxia type 1 (SCA1),
9 fragile X E mental
retardation
(FRAXE),
10 and dentatorubral pallidoluysian
atrophy (DRPLA).
11 12 In all cases, a simple
trinucleotide, tandemly repeated
DNA sequence exists in the transcribed
region of the gene (see
Table

). The triplet repeat shows
moderate levels of length variation
within the normal population.
However, in patient populations,
the repeat has expanded well beyond
the normal range, altering
the biological functioning of the gene. In
SBMA, HD, SCA1, and
DRPLA, the amplifications are quite small, whereas
for fragile
X syndrome, DM, and FRAXE, the amplifications may be very
large,
generating alleles containing many thousands of repeats. The
unusual
properties of triplet repeat DNA sequences account for the
wide-ranging
disease severity and nonmendelian inheritance patterns
associated
with these diseases.
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Triplet Repeats in Genes
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Fragile X Syndrome
Fragile X syndrome is the most common form
of mental retardation
in
males, with an incidence of

1 in 1500 males (see Reference 13).
It
also affects, although generally less severely, about 30% of
obligate
carrier females. The disease derives its name from the
observation
of a fragile site near the tip of the long arm of the X
chromosome,
which can be induced in patient's cells when grown under
specific
in vitro tissue culture conditions (see Reference 14). The
vast
majority of fragile X patients have been shown to carry an
expanded
CGG repeat array at the fragile X A site
(FRAXA).
1 2 3 The
FRAXA repeat lies
within the 5'
untranslated region (UTR) of
a gene termed fragile X mental retardation
1 (FMR1).
2 Normal
individuals have alleles with between 6
and 52 CGG repeats,
whereas fully affected fragile X patients have an
allele with
greater than 200 repeats.
3 Nonpenetrant
fragile X family members
carry so-called premutation alleles in the
range of 50 to 200
repeats
3 (for a detailed review of the
molecular genetics of
fragile X syndrome, see Warren and
Nelson).
13
Spinal and Bulbar Muscular Atrophy
SBMA is a sex-limited
dominant disorder associated with
progressive muscle weakness and atrophy in males (see Reference 4). The
underlying molecular defect has been found to be the expansion of a
coding CAG repeat within the first exon of the androgen receptor (AR)
gene.4 The repeat is polymorphic in the normal population,
with 12 to 33 CAG repeats and is approximately doubled in size in
patients with 40 to 62
repeats.4 15 16 17
Myotonic Dystrophy
DM is the most common form of inherited
muscular dystrophy in
adults, with an incidence of
1 in 8000 (see Reference 18). DM
patients carry a CTG expansion in the 3' UTR of a chromosome 19 gene
showing strong homology to serine-threonine protein kinases and termed
myotonin protein kinase (Mt-PK).5 6 7
Normal alleles fall
into the range of 5 to
40 repeats, whereas the vast majority of
individuals carrying a DM chromosome have from 50 to greater than 3000
repeats.5 6 7 Individuals with 50 to
200 repeats are
generally asymptomatic or have relatively mild symptoms, although they
are at increased risk of having affected
children5 6 7 (for
a detailed review of the molecular genetics of DM, see
Wieringa).18
Huntington's Disease
HD was the first disorder to be
genetically linked to a defined
chromosomal region near the telomere of the long arm of chromosome 4,
using DNA markers.19 After an intensive 10-year search,
the HD gene was finally isolated and found to be a novel gene of
unknown function, termed huntingtin.8 The vast majority of
HD patients were found to carry an expansion of a CAG repeat located
within the huntingtin open reading frame, which codes for a
polyglutamine tract.8 Normal alleles have in the range of
9 to 30 repeats and affected HD patients greater than 40 and up to 121
repeats.8 20 21 22
Spinocerebellar Ataxia Type 1
SCA1 is the first of the
inherited ataxias to be defined at the
molecular level. SCA1 has been found to be the result of an expansion
of a coding CAG, also polyglutamine, in a novel gene located on
chromosome 6.9 The repeat is polymorphic in the normal
population, with alleles in the range of 9 to 39 repeats, and expanded
in SCA1 patients, who have from 41 to 81
repeats.9 23 24
Fragile X E Mild Mental Retardation
A small number of
patients with mental retardation and a fragile X
chromosome do not carry the FRAXA expansion. A subset of these patients
has been shown instead to have an expansion at a second fragile site,
FRAXE,
600 kb distal to FRAXA.10 The FRAXE site is also
due to the expansion of a CGG repeat,10 although the
gene(s) affected by this expansion is currently not known. Once again,
this repeat is polymorphic in the normal population (6 to 25 repeats)
and expanded on FRAXE chromosomes (>100
repeats).10 25
FRAXE patients have a similar although generally milder phenotype than
FRAXA patients.10 25
Dentatorubral Pallidoluysian Atrophy
DRPLA is a rare
neurodegenerative autosomal dominant disorder
primarily affecting Japanese individuals. A number of human brain cDNAs
have been shown to contain triplet repeat sequences that are
polymorphic in length in the normal population.26 Using
these genes as candidates for the DRPLA gene, two Japanese groups
simultaneously identified DRPLA patient-specific expansions (49 to 75
repeats)11 12 in the brain cDNA B37, which had
previously
been shown to contain a coding CAG repeat polymorphic in the normal
population (9 to 23 repeats).26
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Genotype-Phenotype Correlations
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In all of the disorders described above, there is a positive
relation
between the severity of the symptoms and the size of the
expanded
array.
3 5 6 7 8 9 10 11 12 15 20 21 27 28 29 30 31 32
In fragile X
syndrome
and FRAXE, once the repeats reach a threshold size
(>220 repeats), all
males are fully affected and are said
to carry a "full mutation"
allele. In the other disorders, a
more progressive relation is seen.
Moreover, the age of onset
of symptoms is inversely correlated with the
size of the repeat.
Thus, patients with larger expansions tend to have
an earlier
age of onset and increasingly more severe symptoms. This is
particularly
striking for DM and HD, in which the very largest alleles
can
result in congenital DM (CDM, see Reference 30) and juvenile-onset
HD.
8 20 21 22 31
However, these correlations are by no means
absolute,
and large variations in phenotype still exist between
individuals
carrying similar-sized expansions. Thus, although the size
of
the expansion is a major influence on disease severity, the
precise
prognosis for any one patient is also related to other
as yet unknown
genetic and/or environmental factors.
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Molecular Diagnosis
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The cloning of the genes for these diseases has allowed the
development
of specific DNA-based diagnostic tests to determine the
allelic
status of individuals in affected pedigrees. For those diseases
associated
with relatively small expansions, for example, less than 150
repeats,
SBMA, HD, SCA1, and DRPLA, allelic status in all individuals
may
be determined rapidly by polymerase chain reaction (PCR)
amplification
of the region containing the repeats and sizing by
polyacrylamide
gel
electrophoresis.
4 8 9 11 12
For fragile
X syndrome, DM,
and FRAXE, a similar approach can also be used to
identify normal
alleles and small
expansions.
3 5 6 7 10
However, large, expanded
alleles are, due to technical limitations of
the technique,
not amplified using the PCR under standard conditions,
producing
an apparent null allele in patients. Large expansions in
patients
may be confirmed using traditional Southern blot length
analysis
of restriction endonucleasedigested genomic
DNA.
3 5 6 7 10
Accurate presymptomatic diagnosis is
especially important
for late onset neurodegenerative disorders in
which symptoms
may often not develop until after childbearing age.
The availability of highly specific DNA-based tests has allowed the
molecular evaluation of prior clinical diagnoses. In the majority of
cases, the molecular findings have supported the clinical evaluation.
However, a minority of patients do not show the expected expansions in
the relevant genes. It is probable that many of these cases
represent clinical misdiagnosis of these complex disorders (see
Reference 33). Retrospective molecular analysis has already
revealed that one family clinically identified as segregating for HD
does not carry an expansion in the huntingtin gene but instead has an
amplification in the DRPLA gene.34 Furthermore, it is
probable that DRPLA is the same disorder as that termed Haw River
syndrome in the United States; a simple DNA-based assay will allow this
assumption to be tested directly.35 Nevertheless, a
proportion of nonexpansion patients probably represents mutations
in other genes producing a very similar phenotype (so-called
phenocopies, see Reference 33), while others may represent as yet
unidentified mutations in the disease gene. Both of these possibilities
have now been observed for fragile X syndrome. Two families previously
identified as segregating fragile X syndrome actually have expansions
not at the FRAXA locus but at FRAXE.25 In addition, one
fragile X syndrome patient containing a point mutation in the FMR1 gene
and at least three patients with deletions encompassing the FMR1 gene
have been
identified.36 37 38 39
Although apparent phenocopies
have been seen for some of the other disorders, other nonexpansion
mutations in the disease gene or mutations in further genes have not
yet been reported.
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Hypermutable DNA and Unusual Genetics
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All of the triplet repeat diseases display the unusual genetic
feature
of anticipation, that is, the increasing severity and earlier
age
of onset of the disease in successive generations (see Fig
1
for an example of anticipation in a DM family). Before
the demonstration
of the molecular basis of these diseases, the
phenomenon of
anticipation was highly debated (see Reference 40).
However,
once the molecular basis of the diseases became known, a
potential
resolution of this phenomenon became apparent.

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Figure 1. Anticipation and repeat expansion in myotonic
dystrophy (DM). Shown are all affected individuals from a large,
five-generation DM family (for clarity, normal individuals in this
pedigree are not shown). Beside each individual are the allele sizes
for the CTG repeat in the DM gene. Each individual has one normal
allele, less than 30 repeats, and one disease-associated expanded
allele greater than 50 repeats. Sizes for the expanded alleles are
approximate since, due to somatic instability, a heterogeneous smear
was observed. Note how the repeat size and associated symptoms increase
in succeeding generations with one exception; individual IV-5 has
inherited an apparent reduction from his mother, III-4, although he
still has severe DM symptoms. Note also that the largest expansions
causing congenital DM are transmitted by females. Part of this pedigree
was presented previously in Reference 44.
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This was first demonstrated for fragile X syndrome, where the
particular form of anticipation observed is an increasing penetrance in
succeeding generations, known as the Sherman
paradox.41 42
One of the most interesting features of fragile X pedigrees is the
normal transmitting male (NTM), a normal obligate male carrier of the
fragile X chromosome whose daughters are also normal. However, the
daughters of NTMs are at high risk of transmitting the disease to their
own children.41 The Sherman paradox was resolved when it
was shown that the CGG repeat in the FMR1 gene was extremely unstable
and frequently enlarged on transmission from mother to
child.3 The increasing penetrance of the disease in
successive generations correlates with increasing size of the CGG
repeat. NTMs were shown to have inherited relatively small alleles in
the so-called premutation range.3 These alleles are
relatively stable in the male germline with no apparent expansion bias,
with the result that the offspring of NTMs are all normal. The
premutation alleles, though, do apparently demonstrate significant
instability in the female germline such that the grandchildren of NTMs
are at high risk of having an expanded disease causing full mutation
allele.3 It has since been shown that, despite large
expansions in other tissues, sperm from affected fragile X males only
contains alleles in the premutation range, consistent with a bias
against large expansions in the male germline.43
Similar expansion biases exist in the germline transmission of the
other triplet repeat disease genes to account for the phenomenon of
anticipation. DM displays very strong anticipation as well as parental
sex differences in
transmission5 7 28 40 44 45 46 47 48 49 50
(see Fig 1
). Smaller expanded alleles are unstable in the
germline of both
sexes, and classic adult-onset DM may be inherited from parents of
either sex. CDM, however, occurs in children inheriting very large
alleles and is usually transmitted only by affected mothers. Larger
expansions appear to be relatively stable in the male germline, and
alleles beyond 1000 repeats have not been observed in the sperm of
affected males51 (Monckton et al, unpublished
observations). Allele size, though, does not appear to be the only
determinant in the development of CDM. It has been noted that children
who inherit the DM chromosome from their mother are at greater risk
than if they had inherited a similar-sized allele from their
father.52 This suggests that the affected status of DM
mothers has an adverse in utero effect on their children, or
alternatively, that children inheriting larger alleles from their
father are protected in utero by a normal mother.
The intergenerational instabilities of SBMA, HD, SCA1, and DRPLA show
remarkable similarities to each other, although in contrast to fragile
X, anticipation is most strongly associated with paternal transmission.
The molecular data have indicated that the observed anticipation is
associated with an expansion bias on paternal
transmission.8 9 11 12 15 16 23 32 53
Relatively few
transmissions of the FRAXE expansion have been observed, but it appears
that the repeats are unstable in the germlines of both sexes and may
apparently expand and contract.10 25
Although germline mutation of expanded alleles generally leads to an
increase in allele size, reductions in allele size are occasionally
observed. For DM, at least, reductions can occasionally generate
alleles that fall into the normal allele size range. However, such DM
reversions are rare. Only four events have been observed during
pedigree analysis,28 54 55 while direct
sperm
analysis indicates that considerably less than 1% of gametes from
affected males carry revertant alleles (Monckton et al, unpublished
observation).
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Somatic Instability
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The high mutability of some of these expanded alleles is not
confined
to the germline. FRAXA, FRAXE, and DM display high levels of
somatic
instability, especially for larger
alleles.
3 5 6 7 10
In fact,
somatic instability may be so
high that gel electrophoresis
basedsize analysis of expanded
alleles in somatic tissue
usually results not in a discrete band but in
a heterogeneous
smear of multiple unresolved
fragments.
3 5 6 7 10
Intergenerational differences have been generally determined by
comparison of blood DNA between parent and child, and the differences
detected have often been assumed to reflect germline instability.
However, the demonstration of considerable somatic instability raises
doubts as to the actual timing of observed intergenerational
differences. In fact, for fragile X, there is now mounting evidence
that a significant proportion of the observed intergenerational
differences may arise during early
embryogenesis.56 57
Consistent with this theory is the observation that the smear seen in
different tissues is usually of a similar size and may even be shared
between monozygotic twins.56 Furthermore, FRAXA cell lines
established from single lymphoblasts with large expansions are stable
in vitro.57 Such limited early embryogenic instability has
been observed for two murine minisatellite sequences that are
frequently triallelic in adult
mice.58 59 60 Interestingly,
both of these minisatellites have short repeats, one GGCA and the other
GGGCA, which incorporate both the FRAXA and DM repeat sequences.
Detailed analysis of one of these minisatellites has shown that
instability is almost exclusively limited to the first two cell
divisions after fertilization.60 The factors that result
in the early embryonic instability of repeated DNA sequences are not
known.
DM also displays high levels of somatic instability, although in
contrast to FRAXA, there are often considerable intertissue differences
as well. In particular, it has been shown that the expansion size
observed in muscle is consistently larger than that observed in
circulating leukocytes.61 62 Whether this is relevant
to
the mainly muscle-related phenotype associated with DM is currently
unknown. Despite the observation of only minimal levels of variation in
fetal and neonatal tissue, it also has been speculated that DM also may
be unstable during early embryogenesis.18 51
Nonetheless,
recent results have demonstrated that the DM expansion continues to
enlarge throughout adult life (Wong et al, unpublished observations).
Moreover, there appears to be a significant bias toward increasing
allele size related directly to patient age. This phenomenon is likely
to have confounded attempts to reconcile the observed clinical
anticipation with increasing intergenerational allele length.
Approximately 7% of DM transmissions apparently involve a contraction
of the DM repeat.48 Paradoxically, however, the vast
majority of cases do not result in a later age of onset.48
Recent results have indicated that the observed "contractions"
may actually be artifacts of age-limited expansion in the children and
that the DM allele is even more biased toward germline expansion than
previously thought (Monckton et al, unpublished observations).
Of the other diseases, only HD has been demonstrated to show
significant levels of somatic instability, with the greatest levels of
variation seen between regions of HD brains.63 The major
differences are observed between the cerebellum, which has the smallest
alleles, and other regions of the brain.63 Whether the
observed somatic differences are relevant to HD pathogenesis is yet to
be determined.
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Pathogenesis
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In FRAXA, expansion of the CGG repeat into the full mutation
range
beyond 220 repeats leads to methylation of the repeat
and the nearby
CpG island.
64 65 66 Methylation in this
region
dramatically
reduces transcription of the FMR1 gene, and no
FMR1 protein product is
produced in affected males.
67 68 The
level of disease
expression in females is presumably determined
by X-inactivation
patterns; those cells in which the normal
X chromosome is inactivated
presumably do not produce any FMR1
protein. The effect of methylation
is clearly seen in a case
of monozygotic twin sisters who both carry a
fully methylated
expanded allele of the same size.
69 The
normal sister has a
normal active X chromosome in most of her cells. In
contrast,
her mentally retarded twin sister has cells mostly containing
inactivated
normal X chromosomes, and she consequently displays the
fragile
X phenotype. Thus, it is evident that the major fragile X
phenotype
results from an expansion-induced methylation event that
prevents
expression of the FMR1 gene product. The direct relation
between
loss of FMR1 function and fragile X has been further confirmed
in
four other patients, three of whom have deletions encompassing
the
FMR1 gene and one of whom contains a point mutation in the
FMR1
gene.
36 37 38 39 The FMR
protein is highly expressed in
brain
and gonadal tissues, consistent with the observed
phenotype.
70 71 72 73
Although the FMR1 protein has been shown
recently
to contain a conserved RNA binding
domain,
74 75 76
the specific
function of this domain, the normal function of FMR1, and
how
precisely a loss of FMR1 leads to mental retardation is still
unknown.
FRAXE mental retardation also appears to be the result of an
expansion-induced, methylation-mediated loss of
expression.10 However, the actual gene(s) affected is not
known, and it has not been completely ruled out that the 600 kb
proximal FMR1 gene is also affected in FRAXE patients.25
It has been suggested that DM pathogenesis also may be a result of
expansion-induced loss of function, and, consistent with this, the
level of message RNA derived from the affected chromosome appears to be
reduced.77 78 79 80 However,
one group has detected an apparent
increase in message in a CDM fetus.81 To date, no
mutations in the DM gene other than the repeat expansion have been
reported, and it remains to be clarified whether DM pathogenesis is
really a result of loss of function of one allele of the Mt-PK gene,
effects on other nearby genes, or a more complex, as yet undetermined
disease pathway. Recent in vitro analyses have confirmed that the Mt-PK
protein does indeed have kinase activity and can phosphorylate the
ß-subunit of the dihydropyridine receptor, although the in vivo
relevance of these observations has yet to be determined (Timchenko et
al, unpublished observations).
The AR functions normally as a hormone-activated, DNA-binding
transcriptional regulator and is highly expressed in the neurons
affected in SBMA (see Reference 82). However, although some SBMA males
do show some slight androgen insensitivity, SBMA does not result from
an expansion-associated loss of function of the AR. Deletions and other
clear loss of function mutations of the AR gene are already known to
cause testicular feminization in males and are not associated with
specific neuronal losses (see Reference 4). Consistent with this, AR
molecules carrying expanded arrays are able to bind hormone, although
they have slightly lower transregulatory capabilities.82
The CAG expansion in the AR gene must therefore result in a gain of
function, potentially mediated by abnormal
transregulation.82 Whatever the precise gain of function
is, it must be sex limited, since females carrying expanded arrays are
unaffected.
The remaining disorders are all autosomal dominants affecting both
sexes, with no mutations reported other than the triplet repeat
expansions. Remarkably, of the 10 possible trinucleotide DNA sequences,
only 2 have so far been identified as producing pathogenic expansions
in human genes. Note that of the 64 possible trinucleotide sequences,
most are degenerate at the double-stranded DNA sequence level; for
instance, a CGG repeat is equivalent to GCG and GGC repeats on the same
strand and CCG, CGC, and GCC repeats on the opposite strand. SBMA, DM,
HD, SCA1, and DRPLA are all associated with expansion of a CAG repeat
(the "CTG" expansion in DM is a "CAG" expansion on the
reverse strand). Moreover, in SBMA, SCA1, HD, and DRPLA, the CAG repeat
lies within the coding region and gives rise to a polyglutamine tract
in the resultant protein. It seems likely that SBMA, HD, SCA1, and
DRPLA all represent gains of function of the protein product
related to the expanded polyglutamine tract. All of the disorders
result in the death of specific populations of neurons, different for
each disease. How expansion of a polyglutamine tract may lead to
specific cell death is not known. Speculation includes abnormal
transglutamination-mediated83 and "glutamine
zipper"mediated84 formation of intramolecular
complexes. However, cellular aggregates are not a recognized feature of
these disorders, and the exact pathogenic action of expanded
polyglutamine tracts remains elusive.
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Molecular Mechanisms of Expansion
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There has been considerable speculation as to the molecular
mechanisms
responsible for the dramatic DNA triplet repeat expansions
observed
at these loci. Hypotheses have largely centered on replication
errors,
including replication slippage (see Fig 2

)
induced by the adoption
of novel secondary structures by the expanded
repeats or the
binding of repeat specific proteins (for example, see
Reference
85). More recently, it has been speculated that expansion may
be
a length-dependent product of lagging strand
synthesis.
86 It
has been suggested that once the array
reaches

80 repeats in
length, then one Okazaki fragment could be
entirely contained
within the array. Such a fragment composed entirely
of repeats,
not anchored by unique sequence DNA, could be free to slip
within
the array and produce large loopouts that, dependent on the
repair
process, could result in expansions.
86 Many have
ruled out
recombination-based mechanisms, since exchange of flanking
markers
has not been observed to accompany de novo length change
mutations;
in fact, FRAXA, DM, and HD all show high levels of linkage
disequilibrium
with nearby flanking markers, suggesting that full-scale
chromosomal
recombination rarely accompanies
mutation.
8 87 88 89 90
Nonetheless,
recent analyses at tandemly
repeated human minisatellite loci
have demonstrated that germline
length change mutation events
are biased toward repeat expansion and
frequently involve interallelic
exchange yet rarely involve exchange of
flanking markers in
a complex gene conversion mutation
process.
91 Thus, recombination-based
mechanisms, including
gene conversion and sister chromatid exchange,
51 cannot be
ruled out as possible mechanisms for triplet repeat
instability.
Indeed, one large germline reversion at the DM
locus has been
associated with a complex gene conversion event.
54
Analyses of occasional variant AGG repeats within the CGG FRAXA
array
recently have revealed another tantalizing correlation
with human
minisatellite variability.
92 Allelic variation at
both
FRAXA and at least three human minisatellites are highly
polarized
toward one end of the array, strongly suggesting the
involvement of
cis-acting sequence elements in the mutation
process.
91 92 The high levels of linkage
disequilibrium
observed for FRAXA,
DM, and
HD
8 87 88 89 90
have generally been
interpreted as
a result of the existence of a pool of larger normal
alleles
present only on specific "founder" haplotypes.
Alternatively,
the linkage disequilibrium may also reflect the
existence of
specific predisposing flanking
elements.
91 93
The biological
nature of any
cis-acting elements remains
speculative, but replication
origins would seem to be good potential
candidates.

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Figure 2. Replication slippage as a possible mechanism for
repeat expansion. During the replication process, the partially
synthesized DNA strand (a) may disassociate from the template strand.
If this happens as the 3' end is passing through the repeats (stripes),
it may reanneal out of register within the repeat template, producing a
single-stranded loopout (b). Further extension of the newly synthesized
strand will produce a double-stranded product containing a heteroduplex
within the repeats (c). Repair of the heteroduplex can result in
incorporation of the "slipped" region and repeat expansion (d).
Adoption of novel secondary structures or binding of repeat specific
proteins possibly may increase rates of replication slippage.
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 |
Other Fragile Sites
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In addition to the fragile sites associated with mental
retardation
on the X chromosome, the human genome contains many other
fragile
sites that may be induced under a variety of tissue culture
conditions
(see Reference 14). Recently, the molecular basis for a
chromosome
16 folate-sensitive fragile site has been identified as
expansion
of a CGG triplet, which, like fragile X A and E, results in
hypermethylation
around expanded alleles.
94 It is not
known whether nearby genes
are affected by this hypermethylation, and
no disorder has yet
been associated with this expansion. However, the
fragile site
is very rare, no individuals homozygous for the expansion
have
been identified, and loss of function of one autosomal allele
may
not be sufficient to express a phenotype. Thus, of the three
fragile
sites defined at the molecular level, all are associated
with expansion
of a CGG repeat. It will be of considerable interest
to discover the
basis of the other fragile sites and the mechanistic
basis of
chromosomal fragility on expanded alleles.
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Unstable DNA and Colon Cancer
|
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Recently, it has been discovered that hereditary nonpolyposis
colon
cancer (HNPCC) is associated with tumor-specific unstable DNA
(see
Reference 95). It has been observed that the simple tandem repeat
loci
commonly used in molecular linkage analyses are frequently mutated
in
tumor DNA. Increased mutation rates in such tumors have been
associated
with mutations in at least two genes involved in the DNA
mismatch
repair system. However, although some of the triplet repeat
loci
also show increased instability in HNPCC tumor tissue, the changes
observed
are relatively small additions and deletions, with no reports
of
dramatic expansions. There is no reason to suppose, and no evidence
to
support, the direct involvement of mutant alleles of these repair
loci
in the dramatic expansion of triplet repeats associated with
human
inherited disorders.
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Conclusions
|
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Until recently, DNA was thought to be a relatively stable molecule
with
new mutations arising relatively infrequently and being stably
inherited
upon further transmission. However, the last few years have
forced
us to reevaluate our ideas on the stability of DNA and the
association
of de novo mutation and human disease. The discovery of
hypermutable
triplet repeat loci that show unprecedented levels of
instability
in both the germline and somatic tissue has revolutionized
our
understanding of the disorders to which they give rise. In
particular,
unstable DNA provides a molecular explanation for the high
degree
of symptom variability and the unusual genetic features of the
disorders
with which it is associated. The cloning of the disease gene
and
the identification of expanding repeats, despite a critical
beginning,
nevertheless represent preliminary steps in a pathway to
the
understanding of the full disease process. Molecular analysis
provides
at least an immediate diagnostic tool for today's parents,
but
much still remains to be determined for effective therapies
to be
developed for tomorrow's patients.
 |
Acknowledgments
|
|---|
We would like to thank Jim Wyngaarden, Belinda Rossiter, Paul
Schofield,
Nissim Ben-Arie, and Tetsuo Ashizawa for helpful discussions
of
the manuscript. Dr Monckton is the Muscular Dystrophy Association
Sammy
Davis Jr Research Fellow, and Dr Caskey is a Howard Hughes
Medical
Institute Investigator.
 |
Footnotes
|
|---|
Reprint requests to Dr Monckton, Department of Molecular and
Human
Genetics, Baylor College of Medicine, One Baylor Plaza,
Houston, TX 77030.
Received August 15, 1994;
accepted October 18, 1994.
 |
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