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www.nomoresouthernblots.comAn educational website on the topic of Fragile X Testing
FMR1 testing: Current technologies & newadvances in CGG repeat quantification & categorization
Sue Richards, PhD, FACMGOregon Health & Science UniversityKnight Diagnostic Laboratories
Portland, OR
Disclosures
� Honorarium from Asuragen for this webinar
� Previous consultant for Abbott on FX product
� Director of academic laboratory providing FX testing
� ACMG Lab QA member, develop FX guidelines
� CAP/ACMG committee member for FX PT
Today’s Discussion
� Introduction to Fragile X
� Fragile X testing methods
� Triplet Repeat Primed Testing Methods
� Applications and strategies� Screening
� Diagnosis
Fragile X Syndrome
� Most common inherited form of mental retardation.
� Incidence 1:4000 males and 1:8000 females.
� Carrier 1.3%
� Strom et al. Genet in Med 2007;9:46-51
� Affected males have mental retardation, characteristic physical features and behavior.
� Affected females exhibit a less severe phenotype.
� Found in all populations.
Diagnostic Yield
� Common diagnoses of developmental delay/mental retardation
� Down syndrome 9.2%
� Microdeletion 22q11.2 2.4%
� Williams-Beuren syndrome 1.3%
� Fragile X 1.2%
� Cohen syndrome 0.7%
� Monosomy 1p36.3 0.6%
Rauch et al. Am J Med Genet A. 2006 140:2063-74
Neuroanatomical Aspects
� Immature dendritic spines
� Higher dendritic spine density
Koukoui and Chaudhuri. Brain Res Brain Res Rev. 2007 53:27-38
a b
Figure: Golgi-impregnated dendrites from (a) an FXS
individual and (b) unaffected control. Staining reveals
significantly longer spines and fewer short dendrites
compared to control. Original source: Irwin et al., (2000).
Molecular Defect
� Trinucleotide repeat (CGG) at the 5' untranslated region (UTR) in FMR1
� A small expansion (premutation) associated with increased mRNA
� FXTAS, POI
� RNA Gain of function mechanism
� A large expansion associated with methylation, inactivating gene expression.
� Protein loss of function mechanismXq27.3
55
DNA Methylation Affects Expression
GENE RNA PROTEIN
GENE + METHYLATION
(CGG)n
(5-44 repeats)
55-200(55-200)
Repeat Number Classification
ACMG Standards and Guidelines
Classification Repeat Range Purpose
Normal 5-44 repeats Rules out diagnosis of Fragile X
syndrome/carrier status
Intermediate 45-54 repeats Not affected but unstable, could
eventually expand to a pre-mutation,
then full mutation
Premutation 55-200 repeats Carrier and at risk for expansion in next
generation (females). At risk for
primary ovarian insufficiency (POI) or
ataxia (FXTAS).
Full mutation >200-230 repeats Gene is methylated and inactive; confirms diagnosis of Fragile X syndrome
Mosaic Both pre-mutation (unmethylated) and
full mutation (methylated) present.
Severity of symptoms cannot be
predicted, but may be milder.
Jacquemont, JAMA 2004
Penetrance is age related
Fragile X-associated Tremor/Ataxia Syndrome (FXTAS)
� Symptoms
� Late-onset, progressive cerebellarataxia/intention tremor
� Short-term memory loss, executive function deficits, cognitive decline
� Lower-limb proximal muscle weakness, and autonomic dysfunction
� Affects 20-40% male PM carriers & 8% female PM carriers >50 y.o.
� Genetics
� FMR1 premutation
� mRNA accumulation; gain of function
Distribution of Subjects
0
20
40
60
80
100
120
140
160
180
18 - 29 30 - 39 40 - 49 50+
Age at Interview
Nu
mb
er
of
Su
bje
cts
Full Mutation
Premutation
Control
Proportion of Subjects Experiencing POF
0 0 0 0
0.05
0.11
0.22
0.26
0.020 0 0
0
0.05
0.1
0.15
0.2
0.25
0.3
18 - 29 30 - 39 40 - 49 50+
Age at Interview
Pro
po
rtio
n o
f S
ub
jects
Primary Ovarian Insufficiency (POI)
� Cessation of menses before age 40
� 21% of females with premutations
� Odds ratio for POI increases with increasing repeat size
� 59-79 6.9
� 80-99 25.1
� >100 16.4
Sherman, 2005
Risk of Expansion byPre-mutation Size
Number of Maternal Pre-
Mutation CGG Repeats
Approximate % Risk of
expansion
Approximate % Risk that
a Son Will be Affected
with Fragile X Syndrome
56-59 14% 7%
60-69 20% 10%
70-79 58% 29%
80-89 72% 36%
90-99 94% 47%
> 100 100% 50%
Adapted originally from Warren & Nelson 1994; modified according to Nolin et al. 1996.
GENEReviews at www.genetests.org, FMR1-Related Disorders.
Role for AGG repeats
Fragile X Testing Combines Southern blot and PCR Analysis
� Southern blot analysis
� 80-800+ repeats
� Full mutations
� Methylation
� Difficult to size accurately
� PCR Analysis
� Sizes normal/premutation allele
� Difficult to amplify through CGG repeat
Southern Blot
Restriction Digest
Electrophoresis
Transfer to membrane
Anneal Probe
Detect
Schematic and workflow for Southern blot analysis
Fragile X Methylation Assay by Southern Blot
Ps
t1
Ea
g1
Ps
t1
Ec
oR
1
Ec
oR
1
probe
5.2 kb
2.8 kb
pE5.1
(CG
G)
5.2 kb = methylated
2.8 kb = unmethylated
Methylation-
Sensitive RE
Fragile X Methylation Analysis
• Southern Analysis usingmethylation-sensitive and insensitive enzymes• Premutations are not methylated• Full mutations are methylated and inactivate the FMR1 gene•Detects ~80->1000 repeats•Difficult to accurately size•Expensive, laborious, takes time•Would be desirable to reduce number of southerns or eliminate
N N P E N E
Legend: N= Normal; P= Premutation; E= expansion
Southern blot image for different sample types
PCR Analysis of the FMR1 (CGG)n Repeat
FMR1 EXON 1
CGG CGG CGG CGG CGG CGG CGG
Translation
StartForward
Primer
Reverse
Primer
Note that the FMR1 region is very difficult to amplify using PCR.
A premutation allele of 80 CGG repeats is ~440 bp and ~95% GC.
Fragile X PCR History and Methods
� PCR and agarose gel with radiolabeledprobes
� PCR and capillary electrophoresis using dye labeled primers
� CGG repeat primed PCR methods for screening and testing
Fragile X PCR Analysis Using Gel Blotting
• PCR for detection of normal size alleles and premutation size alleles
• Will not detect full size
expansionsNot automated
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}Normal range alleles}
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- Grey Zone
OLD HISTORICAL METHOD
Fragile X PCR Analysis Using Labeled Primer Extension
3’ 5’
5’ 3’
5’Forward primer reverse primer FL
Start (ATG) Stop
5’ 3’
(CGG)n triplet repeat region
FMR1
(CGG)n 17 exons
Fragile X Testing PCR
• PCR plus capillary electrophoresis
• Commercially available reagents
• Two injection conditions per sample used
• Sizes normal/premutation alleles
• Amplification into full mutation range possible
Electropherogram showing fragment sizes, markers, and reference ladder.Used by Fragile Xperts, Amos et al 2008
Male – Normal 42 CGG Repeats
Sample Plot from GeneMapper Software Analysis of Large Repeat Run Data
Sample Plot from GeneMapper Software Analysis of Small Repeat Run Data
Chr. Y (170 bps) Chr. X (203 bps) TR Allele (319 bps) 42 repeatsY
X 42
Male – 118 CGG Repeats
Sample Plot from GeneMapper Software Analysis of Large Repeat Run Data
Chr. Y (170 bp)
Chr. X (203 bp)
TR Allele (547 bp)
118 Repeats
Sample Plot from GeneMapper Software Analysis of Small Repeat Run Data
See large run
Male-Full mutation
Two CE runs are use to determine the FM peak relative to the gender control. The control peaks were detected in the small repeat run and the FM allele was identified in the long repeat run.
Female-Full mutation
A similar detection capability was achieved for female FM mutations which have a shorter allele and a longer allele.
The ability to use PCR for identification of a range of allele types influenced the capabilities of broader screening
Diagnostic Testing Strategy
PCR analysis
Normal
Stop, report
Premutation,Full mutation,
homozygous female
Southern analysis
Criteria for Population Screen
� Common disorder
� Fragile X, 1:2000
� Practical test
� PCR, not Southern
� Intervention
� Family planning
� Early behavioral intervention
� Treatment?
Population ScreenPCR only
� Carrier Screening� Pre-mutation alleles
� Females planning a pregnancy, high-risk family members, POI, FX-ataxia
� PCR with current technology� High pre-mutation occasionally missed
� Apparent homozygous females
� Newborn Screening� Full mutation alleles
� Easier in males
� Apparent homozygous females
� Methylated alleles (males only)
� Complex testing strategies have been proposed
Desirable Test Performance
� High sensitivity and specificity
� Detection of normal, premutation and full mutation alleles
� Screening tool for expansions
� Detection of mosaics
� Detection of homozygous females
� Decreased use of southern
� Rapid, cost effective, and ease of workflow for lab, robust, reproducible, high throughput
Recent Publications Incorporate a CGG Primer for Key Improvements in Workflow and Analysis
2008 Tassone, F. et al, A rapid polymerase chain reaction-based screening method for identification of all expanded alleles of the fragile X (FMR1) gene in newborn and high-risk populations.J Mol Diagn, 2008. 10, p. 43-9.
2-primer PCR and agarosegel analysis
2010 Lyon, E., et al.,A Simple, High-Throughput Assay for Fragile X Expanded Alleles Using Triple Repeat Primed PCR and Capillary Electrophoresis.J Mol Diagn. 2010.12:505-11.
2-primer PCR and CE
2010 Chen, L., 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, 2010. 12(5): p. 589-600.
3-primer PCR and CE
Allelic Sizing as a Basis for Triplet Primed PCR
Yu P, Sinitsyna I, Lyon E. A precise sizing method for fragile X (CGG) in repeats
(November 2006). [Abstract]. Journal of Molecular Diagnostics, 8(5), 627.
� Allelic ladder (every 3bp)� Accurate sizing� ABI � Within CGG repeat rather than over the CGG repeat
� Full mutation female used to generate allelic ladder
� PCR amplification for expanded alleles
E. Lyon, ARUP
FX Screening Concept: FMR1 Primer 2 ASR—No Sizing, Results “Normal” or “Expanded”
Expanded allele (> 55 CGG repeats)
Normal allele
Normal allele
Normal allele
Expanded allele (> 55 CGG repeats)
Ladder motif
Ladder motif
E. Lyon, ARUP
Female, Normal
Female, expanded
Male, expanded
Expanded Allele Detection
� Single PCR � Capillary electrophoresis
� Characteristic “stutter” easy to read
� High throughput
� Non-expanded alleles� Stuttering only up to the largest allele size present in the sample
� Expanded alleles (detected if present)� Females (resolve “apparent homozygous”)
� Males
� Mosaics � Pre-mutation/full mutation
� Normal/full mutation
FMR1 Primer 2 ASRControl Samples
50 repeatsMale, pre-mutation,
118 repeats
Female, normal,
29/30 repeats
Female, normal,
30 repeats
E. LyonARUP
Expansion
No expansion
No expansion
Female, Full-mutations
30/~435
39/~335,
19/~600
LyonARUP
39/~335
30,~435
19,~60019,~600
FMR1 Primer 2 ASR
55 Repeats
Mosaic
Full Mutation
LyonARUP
Mosaic
Full mutation
A Second Repeat Primed Method
� Uses 3 primers, 2 flanking and 1 within the CGG repeat
� Detects full mutation expansions
� Sizes normal and premutation repeats
� Can be run with or without the CGG repeat primer
� Also available commercially as ASR
� No gender markers
Repeat Primed PCR
� The method detects both full length products and individual CGG repeats
CGG primed amplicons Full length product>200 CGG50 CGG
100 CGG150 CGG
200 CGG
FMR1Forward Primer
FMR1 Reverse Primer
CGGCGGCGGCGGCGGCGGCGGCGGCGGCGGCGGCGGCGGCGGCGGCGGCGGCGGCGGCGG
CGG primers
CGG repeat primed assay detects FM in male samples
Adapted from Chen et al, JMD, 2010
NA06852
NA07862
FM>200CGG
FM>200 CGG
30 CGG(full length)
The electropherogram yields information on the size from the full length amplicon and the presence of CGG amplicons for different alleles.
CGG repeat primed assay detects FM and resolves zygosity in female samples
Adapted from Chen et al, JMD, 2010
30 CGG
30 CGG
29 CGG
FM>200 CGG
FM >200 CGG
Expanded CGG repeats
No expanded CGG peaks
Expanded CGG repeats
The absence of CGG primed amplicons beyond the size of the detected allele indicates the lack of a longer, heterozygous allele
The presence of CGG primed amplicons beyond the 30 CGG allele indicates the detection of a longer, heterozygous allele
“DNA from a normal
male and a full mutation
male were mixed in
varying amounts to
simulate a mosaic
sample containing 1-50%
full mutation allele…The
full mutation allele was
detectable in samples
with the equivalent of
1% of the expanded
allele.”
“DNA from a normal
male and a full mutation
male were mixed in
varying amounts to
simulate a mosaic
sample containing 1-50%
full mutation allele…The
full mutation allele was
detectable in samples
with the equivalent of
1% of the expanded
allele.”
Data Courtesy Dr. Cathy Stolle
Interrupting AGG Sequences may Stabilize the CGG
Repeat Region
The spacing of AGG interruptions can be determined
AGG interruptions may be useful in predicting
premutation expansions
A pilot study will be useful in determining the clinical significance of the AGG interruption
Courtesy Asuragen
Expanded Allele Detection for Females
� Principle published in 2008
� Agarose gel
� Detect by smears
–Tassone et al. Journal of Molecular Diagnostics Jan 2008, 10:43-49
Sample (female )
(DNA or blood spot)
First round PCR screen
and band sizing
(c and f primers)
Single band?
Second PCR screen
(c and chimeric primers)
Smear?
Southern blot
analysis
No. samples
50,000
30,000
20,000
19,985
15
finished
no
yes
finished
no
yes
Proposed Screening Strategyfor Males and Females
Sample (female or male)
(DNA or blood spot)
Screen
(Fx-screening primers)
Expanded
-DNA-
Diagnostic test
50,000
660
650 carrier
~10 affected
Finishedno yes
Lyon et al., 2010
Summary
� Two repeat primed PCR methods presented
� PCR (within repeat) and CE has high sensitivity and specificity
� All expanded alleles detected can be reflexed to diagnostic test
� Suitable for carrier screening
� Suitable for newborn screening with blood spots
More FX webinars to come…
� The clinical use of AGG repeat interruption (Liz Berry Kravis)
� A PCR-based methylation analysis (Elaine Lyon)
� Clinical studies of NBS for FX (Brad Coffee)
Thank you!
Elaine Lyon, ARUPMonique Johnson, OHSUDean Westberry, OHSUAndrew Hadd, Asuragen
QUESTIONS?
www.nomoresouthernblots.comAn educational website on the topic of Fragile X Testing