Sandeepa Chauhan, Madhumita Roy Chowdhury, Neerja Gupta, Sheffali Gulati*,
BK Thelma#, Anjali Dabral#, Madhulika Kabra
Genetic Unit, Department of Pediatrics, *Department of Pediatrics, AIIMS, New Delhi
#Department of Genetics, University of Delhi South Campus, New Delhi
Abstract
Results
Fragile X syndrome is the most common inherited mental
impairment, affecting roughly 1 in 4,000 males and 1 in
7,000 females, arising from expansion of CGG repeats in the
FMR1 gene.
Our laboratory strategy was to do initial screening by PCR
method and to test PCR positive cases and family members
including at-risk females by Southern blotting. Recently, we
have started using AmplideX FMR1 PCR kit to validate our
results.
Eight patients clinically diagnosed with Fragile X Syndrome
were screened by PCR. Southern blotting was done to
confirm the result and to detect the carrier status of 25
females in these eight families. AmplideX FMR1 PCR Kit was
used to detect the accurate size of alleles up to 200 CGG.
Out of eight males, all were found to be affected by all three
techniques used. Out of 25 females, full mutation (FM) was
found in 3, premutation (PM) in 13 and 9 females were
normal
Confirmed by southern blotting
PCR amplification
In PCR simultaneous amplification of
FRAXA and FRAXE triplet repeats is
carried out using 100 ng of DNA in 25 µL
reaction
Fragile X arises from expansion of CGG repeats in the FMR1
gene. Generally, in normal individuals there are 28 to 32
repeats. Expansions between 55 to 200 (PM) are associated
with autism spectrum disorders, reproductive disorders in
females (FXPOI), and cognitive/motor disorders in both older
males and females (FXTAS). An expansion of repeats to >200
(FM) results in inactivation of the gene through methylation
of CpG islands.
FMR1 Gene
Objective
Applying AmplideX FMR1 PCR Kit to detect the actual
number of CGG repeats in the FMR1 gene to identify the
carrier status of females and validating the results with
Southern blot.
1
PCR
Unable to detect
pre mutation
Unable to detect full
mutation in females
Southern blotting was done by using
PstI enzyme for Premutation and
EcoRI and EagI for Full mutation
The test protocol involves three key sets of procedures:
PCR master mix setup and thermal cycling
Capillary electrophoresis
Fragment sizing analysis
Data interpretation
Conversion peak size to CGG repeat length
After capillary electrophoresis, the size of the target
amplicon is derived from comparison to a co-injected size
standard, e.g. ROX 1000 Size Ladder The AmplideX FMR1
PCR Kit incorporates two correction factors for conversion
of size in base pairs to the number of CGG repeats for each
allele
The size of each peak may be converted to repeat length
by the equation
Peaki  c0
CGGi 
m0
Peaki - size in base pairs of a given product peak, c0 - size
correction factor, m0 - mobility correction factor for each
CGG repeat
Size and mobility correction factors for standard
instrument configurations
Configuration
3130, 36 cm
c0
229.4
m0
2.965
Female with two normal X
Normal male with
no AGG
interruption
AGG
interruption
in both X in
2 locations
29 CGG
Male FM mosaic
with all peak
populations >200
Female 29/29 CGG
homozygous
Premutation
Female 108-125 CGG
125 CGG
108 CGG
AGG
interruption
Female heterozygous
Normal/FM with 2 AGG interruption
FM >200
Premutation78 CGG
Two X, with one normal X
and one pre-mutation
mosaic X. 2 AGG
interruptions on 1 X
Full Mutation:3
Premutation: 13
Normal: 9
Comparison of PCR, Southern blotting
and AmplideX FMR1
Southern Blotting
Can not interpret
accurate CGG
repeats,
Can not detect
interrupting AGG
sequences
AmplideX™ FMR1 PCR Kit Protocol
Material and Methods
Eight patients clinically diagnosed with Fragile X Syndrome
were initially screened by PCR. Southern blotting was done
to confirm the result and to detect the carrier status of 25
females in these eight families. AmplideX FMR1 PCR Kit was
further used to detect the accurate size of alleles up to 200
CGG, identification of FM alleles >200 CGG and a
characteristic product peak profile that resolves zygosity in
females samples.
Confirmed by southern
blotting
Confirmed by AmplideX FMR1
PCR Kit
S no
N – Normal
FM – Full mutation
PM – Pre mutation
PM
Confirmed by AmplideX FMR1 PCR Kit
Total no. of
females: 25
Southern Blot
5.2Kb
PCR
Positive
Total
families
screened :8
2 pairs of primers, 20 pmole of FXD and FXE for amplification
of the FRAXA CGG repeat and 35 pmole of 598 and 603 for
amplification of FRAXE CCG repeat are used. In a normal
individual two distinct bands are be visible, the upper band
is of FRAXE and a lower band is of FRAXA. Individual
showing absence of any of these bands indicates fragile X
positive case. FRAXE positive case is extremely rare thus
this band acts as a control band.
N N FM N FM FM N N
Introduction
Total no.of
males:8
AmplideX FMR1
accurately detects
full mutations having
200 CGG repeats to
1300
Can detect
interrupting AGG
sequences
resolves female
zygosity
2
No radioisotopes
Usage of
radioisotopes,
which are
hazardous
No radioisotopes
3
Easy to perform
Labor intensive
Easy to perform
4
Rapid
Time consuming
Less time consuming
than Southern
blotting
Conclusion
The FMR1 CGG Primer is specific for CGG repeats and will
not hybridize to AGG sequences commonly found in FMR1
alleles.
Signal intensity dips in the CGG RP PCR profile correspond
to the presence of interspersed AGG. These AGG
“interruptions” are thought to confer DNA stability and to
reduce the risk of expansion in the next generation
intermediate and PM alleles because AGG “interruptions”
are thought to confer DNA stability and to reduce the risk
of expansion in the next generation
Therefore, the risk of CGG repeat expansion for mothers
with AGG “interruptions” may be lower than mothers with
the same number of repeats but without at least one
AGG.
Hence helpful in genetic counselling in the families with
Fragile X