Pyrosequencing: The Dundee Experience

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Pyrosequencing The Dundee Experience

• Arlene Stewart
• Jennifer Dannfald
• SGLC Meeting 2008

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Pyrosequencing

• The technique is built on real-time monitoring of
  DNA synthesis that upon nucleotide incorporation
  ends in a detectable light signal
  (bioluminescence)
• The signal can be quantitatively connected to
  the number of bases added

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Pyrosequencing assay
Sequencing primer
PCR primer
Region of interest
Biotinylated PCR primer
Can read a nucleotide sequence starting from the
first nucleotide next to a sequencing primer,
allowing the design of small PCR products -useful
for degraded DNA samples, particularly DNA from
paraffin-embedded tissue, in which DNA is
typically fragmented into short fragments
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Advantages

• The PyroMark Q24 also allows automatic analysis
  and easy overview of results
• Pyrogram traces easily saved or printed, one
  by-one or in batch mode
• The method is sensitive, with a detection limit
  of approximately 5 mutant alleles.
• It is more cost effective than BigDye Terminator
  sequencing in terms of both reagents and labour
  time

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BRAF

• MMR mutation analysis is presently costly and
  labour intensive. There is a need for more
  sensitive and specific criteria to select
  patients for mutation analysis
• BRAF p.V600E mutation may be used as a selection
  tool for mutation analysis. It has been shown
  that this mutation is characteristic for sporadic
  colon tumours with MSI. Not observed in HNPCC
  tumours

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BRAF

• Potential to use BRAF as a pre-screening tool as
  only BRAF negative cases need to be screened for
  MLH1/MSH2 mutations and have IHC done
• The DNA extracted from tumours represents a
  mixture of tumour cells and non-neoplastic cells.
  To detect a minority of mutant BRAF alleles among
  abundant wild-type alleles, we performed a DNA
  sequencing assay using Pyrosequencing

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BRAF p.V600E

• We designed our BRAF Pyrosequencing assay to give
  a product of only 78bp.
• With dideoxy sequencing 50 failed to amplify,
  whereas with Pyrosequencing all samples amplified
• We found that the quantification of mutant
  alleles by Pyrosequencing was precise and useful
  for assay validation, monitoring, and quality
  assurance

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PyroMark Q24
BRAF p.V600E (c.1799 TgtA) mutation detected
No mutation detected
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Results

• The results from pyrosequencing were 100
  concordant with those from dideoxy sequencing
• Pyrosequencing offers a specific, sensitive,
  rapid and cost-effective alternative to dideoxy
  sequencing for the detection of BRAF V600E
  mutation in clinical tumour specimens.

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Results- overall

• 24/79 samples with MSI detected had p.V600E
  detected 30
• For validation
• - 20 samples with MMR gene mutation None had
  p.V600E
• - 31 with no MSI detected tested
  One had p.V600E

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Over 55 year olds

• 22/50 with MSI detected had p.V600E
• 44
• Almost half MSI ve tumours screened for MMR gene
  mutations (in over 55s) are sporadic
• 3 of the 50 samples had MMR gene mutation (6). 1
  had endometrial cancer at 56, 2 had multiple
  primaries

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Under 55s

• 29 MSI ve screened, detected 2 with p.V600E,
  both had loss of PMS2 staining sent for
  sequencing, waiting on results
• 8/29 (28) had MMR gene mutations detected
• Highlighting the importance of selecting a
  younger family member for tumour studies

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Conclusion

• Over 30 of tumours with MSI would not have
  needed MMR gene sequencing or MLPA analysis
• MLH1 sequencing cost 800 (based on a band 6
  minimum salary). Equivalent to 2215 WelScot units
• Reduce IHC (Histopathologist) workload

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Future work

• For next 6 months add BRAF test to all tumours
  along with IHC/MSI
• - 150 patients
• If results are consistent with work carried out
  so far, from July 09 MSI ve samples that have
  BRAF p.V600E detected will not have IHC or MMR
  gene mutation analysis performed

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Haemochromatosis

• One of the most common autosomal recessive
  disorders.
• Affects 1/200 to 1/400 people in Caucasian
  populations
• Untreated can lead to iron overload
• Majority of cases a result of two mutations,
  p.C282Y and p.H63D, found in the HFE gene on
  chromosome 6p.

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Dundee ARMS PCR

• Tests for common HMCRS mutations p.C282Y and
  p.H63D
• Rare mutation p.S65C lies under reverse primer
  for amplifying codon 63.
• Potential false result if the presence of p.S65C
  affects the ARMS amplification

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HMCRS and Pyrosequencer

• Primers designed to avoid p.S65C mutation
• 48 EMQN/NEQAS samples validated for both p.C282Y
  and p.H63D
• Pyrosequencer results concordant with previous
  ARMS result for all samples
• Cost per sample is around 1.39

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Results - ARMS
Control 2
Control 1
Patient 1
Patient 2
Patient 3
1
2
3
1 HGH control bands 2 C282Y bands 3 H63D
bands
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Results - Pyrosequencer
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Results - Pyrosequencer
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Conclusion

• Pyrosequencing technology allows fast, cheap and
  non-laborious genotyping of common mutations for
  HMCRS
• Validation of methods proves accurate results