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

}Premutation range alleles}}

}Normal range alleles}

}

}

}

}

}

- 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