Science in medicine
4314 The Journal of Clinical Investigation http://www.jci.org Volume 122 Number 12 December 2012
Genetics of fragile X
Fragile X syndrome (FXS), an X-linked condition first described
by Martin and Bell (1), is the leading cause of inherited intellec-
tual disability (ID). Estimates report that FXS affects approxi-
mately 1 in 2,500 to 5,000 men and 1 in 4,000 to 6,000 women
(2, 3). FXS is caused by mutations in the FMR1 gene, which is
located on the X chromosome and whose locus at Xq27.3 coin-
cides with the folate-sensitive fragile site (4, 5). Cytogenetic
methods (6) used in the past to diagnose FXS have been replaced
by molecular diagnostic of FMR1 DNA using Southern blot anal-
ysis and, more recently, PCR.
Affected men display varying degrees of symptoms ranging from
mild to severe. Due to compensation by the unaffected X chromo-
some, only one-third of female carriers with a full mutation (FM)
have ID; the majority have normal IQ, although learning difficul-
ties and emotional problems are common (7).
Identified in 1991 by positional cloning (8), the FMR1 gene
is characterized by the presence of a polymorphic CGG triplet
sequence in the 5′ UTR (8, 9). Expansion in this triplet sequence
gives rise to FXS, which is the prototype of unstable triplet
expansion disorders. The triplet variability defines four types of
alleles (Figure 1). Normal alleles have a number of CGG repeats,
ranging from 5 to 54, with a mode of 30. Premutation (PM) alle-
les have a number of CGG repeats, ranging from 55 to 200. PM
alleles are unstable and have a strong tendency to expand to FM
alleles upon maternal transmission. Expansion from a PM to
FM can occur with alleles as small as 56 CGGs (10). Alleles pos-
sessing between 45 and 54 CGG repeats, referred to as gray-zone
or intermediate alleles, are proposed to be precursors of PM alle-
les, potentially due to paternal and maternal meiotic instability
(11). The risk of a PM to FM transition depends on the CGG
repeat size, such that the expansion risk is nearly 100% for alleles
of >99 CGG repeats (11). A recent study (12) showed that the
number of AGG interruptions present in the CGG repeats cor-
relates inversely with the risk of expansion to a FM in the next
generation. The presence of AGG interruptions, in addition to
the CGG length, may thus better define the risk for transmis-
sion from a maternal PM to FM in the offspring.
FMR1 silencing is the consequence of rather complex epigenetic
modifications (13). In FXS, cytosines located approximately up to
1-kb upstream of the CGG repeat sequences, including the FMR1
promoter, are methylated (14, 15). Normal alleles are also methy-
lated in the FMR1 promoter region but not in close proximity to
the CGG repeat, which seems to be a “boundary” in the normal
allele that prevents methylation from spreading. This boundary
is missing in FM alleles, and the cytosines upstream of the CGG
repeat become methylated around the thirteenth week of embry-
onic development (16). As a consequence, gene transcription is
inhibited, leading to the absence of its protein product FMRP (17).
Of note, some alleles remain partially or even fully unmethylated
(UFM), despite containing >200 CGG repeats, but the differences
in methylation status are poorly understood.
In addition to altered methylation status, FXS alleles show
deacetylation of histones H3 and H4, reduced methylation
of lysine 4 (K4), and increased methylation of lysine 9 (K9) on
histone H3 (18). These epigenetic changes promote a heterochro-
matic configuration that excludes the binding of specific tran-
scription factors (19), thus turning gene expression off (20). The
rare UFM alleles notably maintain a normal or higher FMR1 tran-
scriptional activity, with reduced FMRP levels (21); acetylation of
histones H3 and H4 and methylation of lysine 9 on H3 of UFM
alleles are more similar to those of FM alleles, while the level of
methylation of lysines 4 and 27 on H3 are more similar to that of
normal alleles (18, 22).
Clinical diagnosis of fragile X in young and adult patients
Early diagnosis. A diagnosis of FXS is often made in young children of
approximately 3 years of age (23), who show delayed or absent speech.
Among the recurrent problems that may occur in children before 2
years of age are hypotonia, delayed motor milestones, hand flapping,
Fragile X syndrome: causes, diagnosis,
mechanisms, and therapeutics
Claudia Bagni,1,2,3 Flora Tassone,4,5 Giovanni Neri,6 and Randi Hagerman5,7
1Katholieke Universiteit Leuven, Center for Human Genetics, Leuven, Belgium. 2VIB Center for the Biology of Disease, Leuven, Belgium.
3Department of Biomedicine and Prevention, University of Rome ‘‘Tor Vergata,’’ Rome, Italy.
4Department of Biochemistry and Molecular Medicine, UCD, School of Medicine, Davis, California, USA. 5UC Davis MIND Institute,
UCD, Medical Center, Sacramento, California, USA. 6Institute of Medical Genetics, Catholic University, Rome, Italy.
7Department of Pediatrics, UCD, Davis Medical Center, Sacramento, California, USA.
Fragile X syndrome (FXS) is the most frequent form of inherited intellectual disability and is also
linked to other neurologic and psychiatric disorders. FXS is caused by a triplet expansion that
inhibits expression of the FMR1 gene; the gene product, FMRP, regulates mRNA metabolism in
the brain and thus controls the expression of key molecules involved in receptor signaling and
spine morphology. While there is no definitive cure for FXS, the understanding of FMRP function
has paved the way for rational treatment designs that could potentially reverse many of the neu-
robiological changes observed in FXS. Additionally, behavioral, pharmacological, and cognitive
interventions can raise the quality of life for both patients and their families.
Conflict of interest: Randi Hagerman has received grant support from Roche,
Novartis, Seaside Therapeutics, Forest, and Curemark to carry out clinical trials for
fragile X and/or ASD. She has also consulted with Novartis regarding clinical trials
in fragile X syndrome. Giovanni Neri received grant support from Novartis for an in
vitro study of AFQ056.
Citation for this article: J Clin Invest. 2012;122(12):4314–4322. doi:10.1172/JCI63141.
science in medicine
The Journal of Clinical Investigation http://www.jci.org Volume 122 Number 12 December 2012
poor eye contact, frequent emesis and/or otitis media, and irritabil-
ity. The behavior of boys with FXS typically includes attention deficit
hyperactivity disorder (ADHD), with significant impulsivity and anx-
iety, as well as behaviors that include repetitive language, hand biting,
hand stereotypies, rocking, and sometimes head banging (24, 25).
These behaviors, combined with social and language deficits, often
lead to a diagnosis of autism spectrum disorder (ASD) before the
diagnosis of FXS is made. Approximately 30% of boys with FXS meet
the diagnostic criteria for autism, and these children have the low-
est developmental and adaptive behavior scores of those with FXS.
A child with FXS is often described as hyperaroused; an imbalance
of the excitatory and inhibitory synaptic pathways described in the
“From RNA metabolism to synaptic receptor dysfunction” section may con-
tribute to such a clinical phenotype. Studies on postmortem brains
from patients with FM and lacking FMRP revealed a neuronal spine
After the proband is diagnosed with FXS, a subsequent cascade
testing of family members will identify relatives with either the PM or
FM. Since FXS is complex, consulting a genetic counselor to ensure
correct interpretation of all positive fragile X test results is advisable,
allowing the provision of appropriate clinical advice for those with
PM or FM (27). Relatives may also be at risk of having children with
ID and therefore should also be offered genetic counseling.
Other clinical observations in the family may reveal the FXS phenotype.
Sometimes a family history of ID, ASD, neurological problems (such
as tremor, ataxia, or dementia in one of the grandparents), or early
menopause (before 40 years of age) will lead the clinician to diag-
nose FXS in the family. The neurological problems described above
are associated with a recently identified neurodegenerative disorder
seen in PM carriers, the fragile X–associated tremor ataxia syndrome
(FXTAS) (28). FXTAS (Figure 1) has been reported to affect approxi-
mately 46% of male and 17% of female adult carriers (28).
Over the last few years, insight into the spectrum of phenotypes
in both the PM and the FM alleles has substantially increased.
In PM carriers, shyness, anxiety, social deficits, and ADHD are
some of the most common features observed, particularly in boys
(29–31). In addition, fragile X–associated primary ovarian insuf-
ficiency (FXPOI), defined by the cessation of menses prior to the
age of 40, occurs in approximately 20% of women with PM (32).
Late diagnosis. A late diagnosis may occur for older patients who
may have undergone genetic testing prior to the 1991 discovery
of the FMR1 gene (8) or for patients that carry a mild form of the
disease, showing atypical symptoms as often occurs for the UFM
alleles. Occasionally, individuals with FXS were institutionalized in
adolescence or adulthood, without a subsequent diagnostic study
to find the cause of their ID. In addition, the majority of women
with FM often do not have ID, and they are frequently diagnosed
with FM following the diagnosis of FXS in their children.
Screening for FXS alleles: early diagnosis
The results of a pilot newborn screening (NBS) study for FXS in
the United States, based on the screening of 11,217 newborns,
indicated that the observed prevalence of a PM allele is 1:188 in
females and 1:480 in males, while the prevalence of gray-zone
alleles (45–54 CGG repeats) is 1:70 in females and 1:107 in males
(33). PM prevalence was found to be different in various ethnic
groups; it was higher in people of mixed European descent com-
pared with that in African American and Hispanic people (for both
males and females) and shows a higher incidence for PM compared
with that in previous studies (34, 35). NBS for FMR1 mutations
is not currently included in the NBS program, mainly because it
may also identify FMR1 mutations that may not develop a severe
FXS due to partial inactivation (UFM) as well as carriers that may
develop FXTAS later in life. NBS has recently captured attention
with the introduction of targeted treatments with encouraging
results (36, 37) and the use of new PCR-based population screen-
ing approaches (34, 38–43). Of note, in a recent European study of
213 FXS prenatal diagnoses, 17.6% of those with a family history
of unknown ID were found to be FXS carriers (44).
Clinical crosstalk between fragile X and autism
The fragile X mutation is the most common single genetic cause
of autism, occurring in 1% to 6% of boys with ASD (7). Individ-
uals with ASD and FXS have a lower IQ and lower receptive and
The four alleles of the human FMR1 gene. According to the degree of CGG triplet expansion and the level of FMR1 mRNA transcription and
translation changes, four alleles are generated: normal, PM, UFM, and FM.
science in medicine
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