The advantages of AmplideX® Technologies

JSeptember 1st 2013 Jane Papadaki Markley Global Director, Sales & Marketing

Overview

• Fragile X and Molecular Diagnosis

• AmplideX® - breakthrough technology

- Amplification of FMs and mosaics

- Exquisitive sensitivity

- Resolves female zygosity

- Reveals AGG interuptions

- Accuracy in sizing

• Correlation with Southern Blot

• Publications

• Educational Programme – No More Southern Blots

• Setting up the assay in your lab

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Fragile X Syndrome

• Fragile X Syndrome (FXS) is caused by expansion of CGG repeats in the FMR1 gene. It is the leading cause of hereditary mental retardation

• Fragile X carriers may be at risk for fragile X-associated tremor/ataxia syndrome (FXTAS), a form of age-related cognitive and motor dysfunction, or primary ovarian insufficiency (FXPOI), a cause of premature menopause.

• FXS and other FMR1 disorders impact an estimated 1.5 million individuals in the US, and affect a broad range of populations and ages.

• New therapies and targeted drugs for treating FXS are in development.

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Fragile X Molecular Analyses

to study FXS and related disorders.

Methylation

Transcription

None

Reduced / no RNA Reduced/ no Protein FXS

Elevated RNA Slightly Reduced Protein

FXTAS FXPOI

Defect Consequence Outcomes

Normal RNA Normal Protein Normal

Expansion of CGG repeats and methylation status of the FMR1 gene are used to study FXS and related disorders. PCR and Southern blot are the techniques applied 4

Limitations of current approaches

• In house PCRs

- rarely able to amplify above 100-150 repeats

- Frequently miss low abundance mosaics

- Unable to confirm heterozygous females

- Unable to reveal AGG interruptions

Because laboratory PCR methods have been inadequate FMR1 molecular analyses relies heavily on Southern blot, but this also has shortcomings.

• Southern blotting

- Is costly, laborious, time –consuming (~1 week )

- Requires large amounts of input g DNA (5 -10ug)

- May provide inaccurate CGG repeat quantification

- May have poor sensitivity to detect mosaic alleles

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Why AmplideX® technology is a breakthrough

- Amplification of full mutations and mosaics

- Sensitivity higher than Southern blots

- Resolving Zygosity

- Revealing AGG Interruptions

- Sizing accuracy

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Amplification of full mutations and mosaics

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A B

AmplideX® FMR1 PCR Kit – Routinely Amplify Greater than 1000 CGG Repeats

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Sensitivity higher than Southern blots

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Detection Sensitivity Compared to Southern Blot

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Southern Blot: LOD ~5% full mutation FMR1 PCR: LOD ~1% full mutation (400 pg 940 CGG gDNA, ~120 copies)

By input mass, this PCR is ~875-fold more sensitive than Southern blot

Asuragen’s FMR1 PCR is ~5-fold More Sensitive than Southern Blotting for Detection of Full Mutation Alleles

Enables Detection to as Low as 1% Full Mutation Allele

% FM Allele

0%

1% 400 pg FM + 39.6 ng NOR

3%

1.2 ng FM + 38.8 ng NOR

5% 2 ng FM +38 ng

NOR

Analytical titration of two clinical samples: A male full mutation (FM) allele in a background of a 31 CGG male normal (NOR) allele High sensitivity detection may enable molecular analysis of unconventional samples, archived materials, or simply those with limited DNA available.

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Resolving Zygosity

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Zygosity Resolution

♂ ♀ ♀ ♀

(CGG)30 (CGG)30

Homozygote?

OR

(CGG)5000 (CGG)30

Heterozygote with one unamplifiable FMR1 allele?

Resolving female homozygosity is the key to reducing the incidence of Southern blot reflex testing

20% of female samples are biologically homozygous

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Repeat Primed PCR Amplicons

Zygosity Resolution

FMR1 Forward Primer

FMR1 Reverse Primer

CGGCGGCGGCGGCGGCGGCGGCGGCGGCGGCGGCGGCGGCGGCGGCGGCGGCGGCGGCGG

Gene-specific primer full length

product

50 CGG

100 CGG 150 CGG

200 CGG

CGG primers

CGG Repeat Primed (RP) PCR queries the triplet repeat structure of the FMR1 gene

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CGG Repeat Primed PCR Produces an Unmistakable Signature for Heterozygous Alleles

Repeat primed peak profile Full length 336 CGG product

Heterozygous

Homozygous

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Revealing AGG Interruptions

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30/30 CGG AGG AGG

30/54 CGG

Is 54 CGG without AGGs at a higher risk for expansion than 54 CGG with AGGs? (e.g. Fernandez-Carvajal, et al., Expansion of an FMR1 grey-zone allele to a full mutation in two generations. J Mol Diagn, 2009. 11(4): p. 306-10.

30/53 CGG

Note that the number of CGG repeat peaks provides an unbiased,

extremely accurate determination of the number

of allele triplet repeats

AGG Mapping Yields More Comprehensive Genotypic Information

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AGG interruptions refine the risk of expansion to a full mutation from moderately sized PM alleles

• 267 mothers (55-175 CGG) • 373 transmissions • 296 expansions to full mutations • AGG mapping using AmplideX technology (enabled by Chen et al., 2010)

• Reclassifies risk of expansion to full mutation

o 75 CGG, 0 AGG=77% o 75 CGG, 2 AGG=12%

AmplideX PCR profile

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AGG interruptions refine the risk of expansion to a full mutation from moderately sized PM alleles

• 267 mothers (55-175 CGG) • 373 transmissions • 296 expansions to full mutations • AGG mapping using AmplideX technology (enabled by Chen et al., 2010)

• Reclassifies risk of expansion to full mutation

o 75 CGG, 0 AGG=77% o 75 CGG, 2 AGG=12%

AmplideX PCR profile

“We conclude that failure to account for AGG interruptions can result in profound errors in predicted risk for fragile X syndrome.”

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Sizing Accuracy

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Sizing Accuracy enabled with Process Control

199 119 54 29/31 20

• Peak selection is based on the highest or center peak within each allele • Peaks up to 119 CGG have been sequenced verified • Matching peak selection to DNA sequencing results enables more

accurate algorithms for CGG repeat quantification

CGG Repeat Length is calibrated using a pooled cell line control matched to DNA sequencing

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Correlation with Southern Blot

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Summary of independent publications validating AmplideX PCR concordance with fragile X reference methods

2012 2012

GTMB 2012

Publications

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AmplideX® PCR reagent validation in peer-reviewed publications

Genet Med. 2011 Jun;13(6):528-38.

J Mol Diagn. 2010 Sep;12(5):589-600.

Clin Chem. 2010 Mar;56(3):399-408

AmplideX PCR products have been validated and incorporated in clinical research applications in 10 peer-reviewed publications since 2010, and more pending.

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AmplideX® PCR reagent validation and applications described in peer-reviewed publications

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No More Southern Blots Educational Campaign

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Educational Campaign for FMR1 testing

Series of 4 educational webinars with KOL speakers Co-sponsored with AMP 1,531 registrants 808 attendees 761 new leads >4,000 website hits

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No More Southern Blots educationa website

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Setting up the assay in you lab

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A Comprehensive Product Solution for FMR1 CGG Sizing*

Component Volume Description

Human FMR1 F,R FAM-Primers 50 µL A proprietary FAM-labeled primer pair for human FMR1 gene amplification by PCR

FMR1 CGG Primer 50 µL An unlabeled CGG primer for probing the number of CGG repeats by PCR amplification

GC-Rich AMP Buffer 1.2 mL A buffered solution containing PCR amplification components optimized for GC-rich DNA

GC-Rich Polymerase Mix 5 µL An optimized mixture of polymerases for the amplification of GC-rich DNA

Diluent 1 mL A proprietary solution used in PCR mastermix preparation

ROX 1000 Size Ladder 200 µl ROX labeled DNA fragments for sizing from 79 to 1007 bp

AmplideX® protocols, CE instrument modules and GeneMapper configuration files are not part of the research assay. *For Research Use Only. Not For Use in Diagnostic Procedures.

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AmplideX® FMR1 reagents support a simple PCR mastermix setup and analysis workflow

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Required Equipment and Materials

• Thermal Cycler

- ABI9700 , ABI Veriti, PTC-200 Eppendorf Mastercycler, BR C1000 /Biometra

• Capillary electrophoresis

- ABI3130 (xL) / ABI3730 (xL) / ABI3500 (xL)

- POP 7

- 36cm or 50c

- Calibrated with DS-30 or DS-31 dye set

• Pipettes

- standard, multichannel, and repeat, need 2 mL pipettor

• Analysis Software

- GeneMapper v4.0/4.1 from Life Technologies

- Genemarker v 1.95 or higher from Softgenetics

• Reagents and Supplies

- Hi-Di Formamide, standard PCR plastics, CE plates

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Training Sample P3 example (80% 23ccg, 20% 940cgg)

23 cgg, gene-specific peak

> 200 cgg, gene-specific peak

cgg stutter peaks

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Training Sample P4 example (process control)

Clearly identified peaks for 20, 29, 31, 54, 119, ~200 cgg

20

119

54 29, 31

~200

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Next steps, recommended

First PCR / CE: - amplify archived samples

- inject amplified PCR and training material PCR with default conditions

- send .fsa files to Asuragen for review

- Asuragen will recommend adjustments in injection conditions, if necessary

Analysis Webex: - data analysis training will be set once the first data are received and reviewed by Asuragen

Second PCR/CE: - continued evaluation per lab requirements

- contact Asuragen for support, data, review, etc

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Training

• Training Webex

• PCR and CE verification

- Reagent handling tips

- Confirmation of sample preparation

- Injection setting confirmation

- Training material samples available (P3 and P4)

• Data analysis support (Webex #2 and ongoing support)

- Step-by-step gene-mapper analysis with prepared modules

- Review of sample files

- Confirmation of mobility and correction factors

- Use of macro/processor for training and validation support

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