FRAGILE X SYNDROME AND

POLYMERASE CHAIN REACTION

DETECTION

WHAT IS FRAGILE X SYNDROME?

Its a genetic condition that causes a range of developmental

problems including learning disabilities and cognitive
impairment.
Males are more severely affected by this disorder than females.
Affected individuals :
1. Delayed development of speech & language
2. Males have mild to moderate intellectual disability,
3. ~1/3rd of affected females are intellectually disabled.
4. Children may also have anxiety and hyperactive behavior
such as fidgeting or impulsive actions.
5. ~1/3rd of individuals have features of autism spectrum
disorders that affect communication and social interaction.

HOW COMMON IS FRAGILE X SYNDROME?

Fragile X syndrome occurs in approximately 1 in 4,000 males

And 1 in 8,000 females

CAUSES

Fragile X syndrome occurs as a result of amutationof thefragile

X mental retardation 1(FMR1)geneon theX chromosome, most
commonly an increase in the number of CGGtrinucleotide
repeatsin the5' untranslated regionofFMR1.
FXS can also occur as a result of point mutations affectingFMR1.
In unaffected individuals, theFMR1gene contains 544 repeats
of the CGGcodon, most commonly 29 or 30 repeats.Between 45
&
54
repeats
considered
a
"grey
zone",
with
a
premutationallelegenerally considered to be between 55 & 200
repeats in length.
Individuals with fragile X syndrome have a full mutation of
theFMR1allele, with over 200 repeats of the CGG codon.
In these individuals with a repeat expansion greater than 200,
there
ismethylationof
the
CGG
repeat
expansion
andFMR1promoter, leading to the silencing of theFMR1gene
and a lack of its product.

INHERITANCE
Fragile

syndrome
is
inherited
in
an
X-linked
dominant pattern.
In males, a mutation in the
only copy of a gene in each
cell causes the disorder.
In
women, theFMR1gene
premutation
on
the
X
chromosome can expand to
more than 200 CGG repeats in
cells that develop into eggs.
This means that women with
the premutation have an
increased risk of having a
child with fragile X syndrome,
premutation in men does not
expand to more than 200
repeats as it is passed to the
next generation.

SIGNS AND SYMPTOMS

Large, protruding ears.

Long face (vertical maxillary
excess)
High-arched palate.
Hyperextensible finger joints.
Double-jointed thumbs.
Flat feet.
Soft skin.
Post pubescentmacroorchidismLarge testis in men after puberty.
Low muscle tone.

DIAGNOSIS

Lab Tests for Fragile X, The FMR1 DNA Test can be

administered with two different lab procedures.
PCR analysis can determine the actual number of
CGG repeats (a pattern of DNA) that are present in
the Fragile X gene.
It is quite accurate in determining premutation and
normal gene repeat numbers.
However, PCR is less expensive and quicker than
Southern blot, and recent advances in technology
have increased its ability to identify Fragile X full
mutations.
PCR may thus be the only test used in the near
future.

ROLE OF POLYMERASE CHAIN REACTION

One of the frequently used tests is based on PCR amplification

of the FMR1 CGG-repeat region.
Moreover, because it is relatively easy to collect and transport
bloodspot samples on filter paper, they are often used for
molecular screening purposes.
Methylation-sensitive PCR is another commonly used method
for verifying the aforementioned methylation status.
PCR analysis can determine the actual number of CGG
repeats (a pattern of DNA) that are present in the Fragile X
gene.

METHYLATION SENSITIVE POLYMERASE

CHAIN REACTION.

It is a very specific method.

Although the sensitivity of this method has been reported to be
as high as 1 ng, many studies have reported the use of more
than 500 ng of DNA for a reliable assessment.
This is because the DNA sample is subject to significant
degradation during the bisulfite conversion process and
additional sample loss occurs during the subsequent removal
of the bisulfite and desulfonation steps.
Universally methylated or unmethylated genomic DNAs were
used as positive and negative methylation controls,
respectively, to determine the optimal conditions critical for the
success of MB-PCR.

STEPS INVOLVED

Universally methylated DNA was prepared by treating

lymphocyte DNA with M.SssI while universally unmethylated
DNA was prepared using nested whole genome amplification of
the same DNA sample with phi29 DNA polymerase.
Then, the optimized MB-PCR to examine the bloodspot
samples from 100 males.
Initialization step- This step consists of heating the reaction
to a temperature of 9496C (or 98C if extremely thermo
stable polymerases are used), which is held for 19 minutes.
Denaturation step: This step is the first regular cycling event
and consists of heating the reaction to 9498C for 2030
seconds. It causesDNA meltingof the DNA template by
disrupting the hydrogen bonds between complementary bases,
yielding single-stranded DNA molecules.

Annealing step: The reaction temperature is lowered to 50

65C for 2040 seconds allowing annealing of the primers to
the single-stranded DNA template.
If the temperature is too low, the primer could bind
imperfectly. If it is too high, the primer might not bind.
Typically the annealing temperature is about 35C below
the Tm of the primers used. The polymerase binds to the
primer-template hybrid and begins DNA formation.
Extension/elongation step: The temperature at this step
depends on the DNA polymerase used;Taq polymerasehas
its optimumactivitytemperature at 7580C, commonly a
temperature of 72C is used with this enzyme.
At this step the DNA polymerase synthesizes a new DNA
strand complimentary to the DNA template strand by adding
dNTPs that are complimentary to the template in 5' to 3.
The extension time depends both on the DNA polymerase
used and on the length of the DNA fragment to be amplified.
At its optimum temperature, the DNA polymerase will
polymerize a 1000 bases/minute.

Final elongation: This single step is occasionally

performed
at
a
temperature
of
7074C
(temperature needed for optimal activity for most
polymerases used in PCR) for 515 minutes after the
last PCR cycle to ensure that any remaining singlestranded DNA is fully extended.
Final hold: This step at 415C for an indefinite
time may be employed for short-term storage of the
reaction.

RESULTS
Molecular testing of 1399 males suspected of having fragile X
syndrome.
Methylation (+) Methylation ()
Screening PCR SBH MB-PCR
SBH MB-PCR
Venous blood Strong 219
0 0 21960
Weak 0
Failed 10* 10 10 0 0
Bloodspot Strong 1140
Weak # 16* 2 2 14 14
Failed #
14* 12 12 2 2
Total

1399

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