SUPPLEMENTARY MATERIALS AND METHODS
Molecular Measures
Blood was collected in EDTA vacutainers, and genomic DNA (gDNA)
isolated from 5 ml of peripheral blood leukocytes using Gentra Puregene Blood
Kit (Qiagen, Germantown, MD, USA). The gDNA concentration was measured
using the Nanodrop Spectrophotometer (Thermo Scientific, Wilmington, DE,
USA). Southern blot and PCR analysis were conducted as previously
described.1,2 CGG size was calculated using FluorChem 8900 software (Alpha
Innotech, San Leandro, CA, USA).
To determine the presence and locations of the AGG interruptions within
each FMR1 allele, PCR was performed using 100 ng of gDNA, and a
combination of a two-tier PCR approach using a CGG linker primer as previously
described1 and the Human FMR1 PCR kit (Asuragen Inc., Austin, TX, USA),
based on manufacturer recommendations and as previously described. 3,4 The
latter was performed in 15 µl final reaction volume. DNA was amplified using the
ABI GeneAmp 9700 thermal cycler (Applied Biosystems, Carlsbad, CA, USA)
under the following conditions: 95°C for 5 minutes; followed by 10 cycles of 97°C
for 35 seconds, 62°C for 35 seconds 68°C for 4 minutes; 20 cycles of 97°C for 35
seconds, 62°C for 35 seconds 68°C for 4 minutes and an additional 20 seconds
per cycle; 72°C extension for 10 minutes and finally incubation at 4°C. Details
are as previously described.1,3
Electrophoresis
Two microliters of PCR product were denatured with 2 µl of ROX 1000
size marker (Asuragen Inc.) in Hi-Di Formamide (Applied Biosystems) to a total
volume of 15 µl for 2 minutes at 95°C, followed by 3 minutes at 4°C. The singlestranded FAM-6 labeled PCR product was detected by capillary electrophoresis
using the ABI 3100 Genetic Analyzer (Applied Biosystems). Fragments were
separated through a 36-cm capillary array using POP4 polymer (Applied
Biosystems) following manufacturer’s recommendations with the following
changes to default run parameters: injection for 20 seconds at 1,250 volts, and a
run time of 50 minutes.
Determination of AGG Interruptions
Genescan files were analyzed using Peakscanner Software (v1.0)
(Applied Biosystems). The full fragment band(s) amplified from the C and F
primers were first identified, the size of which was determined by comparing to
ROX1000 size standard (Asuragen Inc.) and using an in-house DNA ladder
comprising PCR alleles previously sequenced (20, 29, 30, 31, 55, 75, 88, and
108 CGG repeats in length). The remaining peaks (here referred to as CGG
peaks) mark the presence of CGG repeats in the amplicon, each being the result
of the CGG linker primer annealing to a CGG5 region and elongating towards the
F primer. In females, the CGG peak can occur from amplification of either of the
two X chromosomes. The first CGG peak detected by electropherogram
corresponds to the fifth CGG unit from the F primer, the second peak detected
corresponds to the sixth CGG unit, and so on. The presence of a CGG peak that
is twice the height (intensity) of a neighboring peak is the result of amplification
on the FMR1 allele on both chromosomes. The presence of one CGG peak and
one AGG interruption at the same location (female) will result in a peak with the
intensity expected for one allele (half the height of the previously described peak).
Likewise, the absence of a CGG repeat and the presence of either one (male) or
two (female) AGG interruptions results in a decrease in CGG peak intensity to
baseline levels. The presence of CGG peaks and their corresponding height thus
allows the entire locus to be characterized so that the location of CGG and AGG
segments are precisely determined. Using this assay, the initial scanned
fluorescence signal corresponds to the 5’ end of the amplicon (details are as
previously described).3,4
The total CGG length is characterized as the number of CGG repeats,
plus AGG interruptions if present; and corresponds to the CGG length reported
out by PCR analysis. The pure CGG stretch is characterized as the longest
segment of repeating CGG units within the total CGG length that does not
contain an interrupting AGG. In premutation alleles, the longest pure CGG
stretch is characteristically on the 3’ end of the microsatellite locus, with AGG
interruptions typically occurring within the first 9 to 32 trinucleotide units. 5
Eci I Digestion
The presence of AGG interruptions was confirmed in a subset of samples by
digestion with the EciI restriction enzyme (New England Biolabs Inc., Ipswich,
MA, USA). The recognition site for EciI cuts 11 nucleotides after the adenine of
the AGG interruption (5’-GGCGGA (N)11 -3’).6 For this assay, gene-specific PCR
was performed using the Human FMR1 PCR kit without the addition of the CGG
linker primer, and using the following PCR thermal cycler conditions (15 µl total
volume): 98°C for 5 minutes followed by 25 cycles of 97°C for 35 seconds, 62°C
for 35 seconds, 72° for 4 minutes; a final extension was performed at 72°C for 10
minutes, followed by short-term storage at 4°C. Following PCR amplification, 10
µl of PCR product was digested with 0.6 ul EciI (New England Biolabs) in 20 µl
total reaction volume for 4 hours at 37°C, followed by heat inactivation of the
enzyme for 20 minutes at 67°C. Both digested and undigested samples were run
on an agarose gel using 1X Bionic buffer (Sigma Aldrich, St. Louis, MO, USA) for
improved resolution.2 Fragments were separated for 15 minutes at 80 volts
followed by 70 minutes at 150 volts. The gel was stained using Sybr Gold
(Invitrogen, Carlsbad, CA, USA) and visualized with the FluorChem 8900 (Alpha
Innotech). Size ranges were determined and compared to the results from
capillary electrophoresis separation for data consistency (Supplementary
Figure 1).
Haplotype Markers
Four markers flanking the FMR1 microsatellite were analyzed in
premutation carrier mothers and offspring, as necessary and with sample
availability: DXS548, FRAXAC1, ATL1 (rs4949), and IVS10 (rs25714). The
DXS548 and FRAXAC1 loci were amplified as described7,8 with minor
modifications. Briefly, PCR was performed using 100 ng of gDNA, Amplitaq Gold
polymerase with Buffer II (Applied Biosystems), 2.5mM MgCl, 200 M of each
dNTP (Roche, Indianapolis, IN, USA), 0.2 M of each primer (DXS548: (forward)
5’ 6-FAM-GAA TAG TCT CTG GGG TGG ATC TC 3’; (reverse) 5’ AGA GCT
TCA CTA TGC AAT GGA ATC 3’) and (FRAXAC1: (forward) 5’ 6- FAM-GAT
CTA ATC AAC ATC TAT AGA CTT TAT T 3’; (reverse) 5’ GAT GAG AGT CAC
TTG AAG CTG G 3’). Thermal cycling conditions for DXS548 and FRAXAC1
were as follows: 95C for 7.5 minutes, 10 cycles of 98C for 30 seconds, 60C for
1.5 minutes, 72C for 1.5 minutes; 22 cycles of 98C for 30 seconds, 55C for 1.5
minutes, 72C for 1.5 minutes; and a final extension for 10 minutes at 72C.
SNPs ATL1 and IVS10 were amplified using Taqman Universal Master Mix
according to manufacturer’s recommendations. FRAXAC1 and DXS548 PCR
products were separated using capillary electrophoresis on an ABI 3100 Genetic
Analyzer (Applied Biosystems) using standard run parameters and POP7
polymerase with LIZ 500 size standard (Applied Biosystems). Raw data was
analyzed using Peakscanner (Applied Biosystems). ATL1 and IVS10+14 were
analyzed using the ABI 7900HT with SDS software (Applied Biosystems).
Statistical Methods
The association of SNPs rs4949 and rs25714 with maternal total CGG
repeat number or pure CGG stretch was analyzed using ANOVA models, using
one observation from each mother. The association of each SNP with number of
maternal AGG interruptions was analyzed using Fisher's exact test. All statistical
analyses were conducted using R, version 2.13.0.9
The association between haplotype and interspersion pattern was
analyzed using chi-square tests, with p-values calculated via Monte Carlo
simulation.
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chain reaction-based screening method for identification of all expanded
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Filipovic-Sadic S, Sah S, Chen L, et al. A novel FMR1 PCR method for the
routine detection of low abundance expanded alleles and full mutations in
fragile X syndrome. Clin Chem. Mar 2010;56(3):399-408.
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Chen L, Hadd A, Sah S, et al. An information-rich CGG repeat primed
PCR that detects the full range of fragile X expanded alleles and
minimizes the need for southern blot analysis. J Mol Diagn. Sep
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Yrigollen CM, Tassone F, Durbin-Johnson B, Tassone F. The role of AGG
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Eichler EE, Hammond HA, Macpherson JN, Ward PA, Nelson DL.
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biased polarity for the loss of AGG interruptions. Hum Mol Genet. Dec
1995;4(12):2199-2208.
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Tassone F, Beilina A, Carosi C, et al. Elevated FMR1 mRNA in
premutation carriers is due to increased transcription. RNA. Apr
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Riggins GJ, Sherman SL, Oostra BA, et al. Characterization of a highly
polymorphic dinucleotide repeat 150 KB proximal to the fragile X site. Am
J Med Genet. Apr 15-May 1 1992;43(1-2):237-243.
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Zhong N, Dobkin C, Brown WT. A complex mutable polymorphism located
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