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Fragile X syndrome is a common form of mental retardation with an estimated
incidence of 1 per 2,000 to 4,000 in the general population. The physical
manifestations associated with the syndrome include macroorchidism, large
ears, a prominent jaw, moderate to severe mental retardation, and autistic-like
behavior. However, physical features are not always reliable indicators
of the presence of the condition, particularly in prepubertal children
and females. For years, investigators had noted that the phenotype of
fragile X syndrome cosegregated with an unusual disruption of the X chromosome.
Karyotyping of cells grown in folate-depleted cell culture media revealed
that many patients had a ³fragile² site on one of their X chromosomes
that appeared as a constriction on the distal long arm.
This genetic syndrome is of particular interest to clinicians and scientists
who want to understand brain development and function in children. Individuals
with fragile X syndrome manifest neurodevelopmental abnormalities that
include varying levels of cognitive dysfunction, particularly in the domains
of executive, visual-spatial, and visual motor abilities, and frequently
display behavioral symptoms of autism, attention-deficit/hyperactivity
disorder, and social anxiety. Morphological variations of brain structure
have been observed in this population and include abnormalities in the
cerebellar vermis, caudate, hippocampus, and lateral ventricles.
In 1991, Verkerk and colleagues identified a single gene that was associated
with symptoms of the disorder. The gene, known as fragile X mental retardation
gene 1 (FMR-1), exhibited a novel form of mutation that had not
been previously described. It was determined that a sequence of 3 nucleotides
(CGG) was repeated many times in patients. This region of the FMR-1
gene is highly variable in the general population, meaning that its length
is found to vary considerably from one person to the next. In normal individuals,
the repeated sequence ranges in size from 5 to approximately 50 repeats.
In premutated but unaffected patients, however, this sequence was found
to enlarge to up to 200 repeats, and in affected patients repetitions
of more than a thousand nucleotides can be seen. The stability of the
region depends directly on the length of the CGG region and probably also
on the presence of single interspersed AGG islets anchoring the CGG region.
The enlargement of this triplet repeat across generations is responsible
for the increasing severity of the disorder that is often seen over several
generations.
Although the CGG region is transcribed into RNA, it precedes the nucleotide
sequence that will be translated into protein (Fig.
1). Thus, the expansion is not exerting its effects by introducing
novel and destabilizing amino acids into the protein structure. This is
in contrast to several disorders caused by triplet repeat expansions such
as Huntington disease in which the expansion occurs within the coding
sequence and leads to the abnormal inclusion of a large series of glutamine
residues within the protein itself (see last monthıs column). This large
tract of glutamines is thought to disrupt the normal functioning of the
huntingtin protein.
How, then, does the expanded triplet repeat produce its effects within
the FMR-1 gene? When more than 200 CGG repeats are present, there
is a high likelihood that the promoter region of the FMR-1 gene
will be hypermethylated. Methylation is a chemical modification that occurs
in certain DNA regions and, in particular, to DNA enriched in CGG triplets.
When hypermethylation occurs, the enzyme necessary for transcription is
unable to bind to the promoter region and initiate transcription. The
end result is that no messenger RNA is produced, a condition referred
to as ³transcriptional silencing² of a gene.
What is the consequence of large stretches of CGG repeats? In vitro structural
studies have shown that nucleotide regions enriched in C and G nucleotides
tend to bend into a hairpin shape. This abnormal conformation becomes
increasingly stable as the length of the uninterrupted CGG region grows.
The increase in stability with length is also thought to explain why an
expansion of the triplet repeat is favored. In addition, this explains
why AGG islands could have a stabilizing effect on the CGG region by interrupting
the CGG sequence and shortening uninterrupted sequences and preventing
them from forming the hairpins. The hairpin structure is also known to
be a signal that leads to an increase in methylation that eventually blocks
FMR-1 transcription.
Exactly how and when the premutated allele expands into its full mutational
form is still a matter of debate. Interestingly, it occurs only when passed
through the female germ line. There is now evidence suggesting that the
expansion event occurs in a postzygotic stage early in embryogenesis.
This would explain the common mosaic status of many individuals with fragile
X syndrome. Mosaic status refers to the presence of different repeat lengths
in cells originating from various parts of the body. This makes it possible
for a male with the fragile X full mutation to have only premutated alleles
in his sperm or for monozygotic twins to have CGG expansions of different
sizes.
The FMR-1 gene is expressed at its highest levels in the brain
and testes, but it has been found in many other adult and fetal tissues.
Its protein product (FMRP) is mainly localized in the cytoplasm of cells,
particularly in Purkinje cells of the cerebellum and neurons of the hippocampus
and basal forebrain.
Current research efforts are devoted to understanding the normal function
of the protein. For example, the amino acid sequence that is found in
FMRP was compared with the amino acid sequence of other known proteins.
Several domains are present that are highly homologous to domains on other
proteins of known function (Fig. ). The similarities between various domains
of proteins suggested a similarity in their function as well. Three amino
acid domains (2 KH domains and an RGG box) have been located on FMRP.
Both the KH domains and RGG box are known to be important functional domains
in proteins that bind to RNA. FMRP was then shown to be able to bind to
RNA transcripts, as well as with the ribosomal subunit involved in the
translation of messages into proteins. Another domain, termed the nuclear
export signal, is found on FMRP and is a signal that leads to the export
from the nucleus into the cytoplasm of proteins that contain it. Thus,
FMRP may play a role in the transport of messages between the nucleus
and cytoplasm. In this manner, FMRP would participate in the translational
machinery that converts messenger RNA into protein.
Recently, 2 additional genes have been identified that are very similar
to FMR-1. The protein products of these genes, named FXR1P and
FXR2P, are highly homologous with FMRP at the amino acid level. Both of
these proteins have the KH domains and RGG box present in FMRP. FMRP has
the ability to interact with FXR1P and FXR2P, and these protein-protein
interactions may be important for the function of this new family of RNA-binding
proteins. However, different distributions of these proteins have been
found in various tissues, and they could signify different functions for
each of these proteins. Furthermore, the fact that the absence of FMRP
in fragile X males leads to mental retardation even in the presence of
normal FXR1P and FXR2P suggests at least partial independence of function.
It is clear that the absence of FMRP is responsible for abnormal brain
development within the cerebellar vermis, caudate, and hippocampus. What
remains to be determined is exactly how the absence of this gene leads
to the observed variations in brain morphology. The recent creation of
an experimental knockout mouse for FMR-1 gene may shed some light
on the underlying mechanisms and help explain how mutations of these proteins
result in abnormal brain development.
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