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Stacey Sandell

Mutation detection – 9 th November 2009. Why is it important to measure gene dosage. A review of the methodologies available for detecting copy number changes and the advantages and disadvantages of each. Stacey Sandell. Why is gene dosage important?.

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Stacey Sandell

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  1. Mutation detection – 9th November 2009 Why is it important to measure gene dosage. A review of the methodologies available for detecting copy number changes and the advantages and disadvantages of each. Stacey Sandell

  2. Why is gene dosage important? • Detection of gene dosage may be required for therapeutic, prognostic or diagnostic purposes. • Primary defect in many disorders. • DMD, SMA, CMT/HNPP, a-thalassemia and growth hormone deficiency. • Significant defect in many other diseases. • BRCA1/2, HNPCC, PTEN, NF1 and many more. • Discrimination between deletions/duplications, UPD, and non-paternity. • Need to provide accurate recurrence risks • May give rise to different phenotypes. • Whole gene deletions can lead to a more severe phenotype. • Whole gene deletions may lead to a milder phenotype.

  3. Diseases which result from copy number changes • CMT/HNPP • The PMP22 gene is very sensitive to copy number. • 3 copies = AD CMT type 1 (severe cases 4 copies). • 1 copy = AD HNPP. • Caused by a reciprocal unequal crossing over event. • We currently test for this using MLPA.

  4. Diseases which result from copy number changes • SMA • 95-98% are homozygously deleted for exons 7 and 8 of SMN1. • 2-5% are compound heterozygous for the deletion and a point mutation. • Testing is technically difficult due to repeat elements and the pseudogene (SMN2 copy number fluctuates). • Carrier testing is complicated further by some carriers demonstrating a normal copy number. • 4% of the general population have 2 copies of SMN1 on one allele. • In 2% of SMA affected patients the point mutation has arisen de novo. • Testing is carried out using MLPA and a PCR restriction digest.

  5. Diseases which result from acquired copy number changes • Her-2 • ERBB2 (Her-2) is amplified in a substantial number of breast and ovarian cancers. • The gain in copy number is associated with an unfavourable prognosis. • However these patients may benefit from treatment with the anti-Her-2 antibody Herceptin. • FLT3 • Is over expressed in most B lineage and acute myeloid leukaemias (AML). • 2 types of mutation: • Internal tandem duplication (ITD) of varying size. • Point mutations. • Many studies have shown that the ITD is a poor prognostic indicator for both paediatric and adult patients.

  6. Detection Methods - 1 • There are well established detection methods for the detection of very small and very large deletions/duplications. • It is the medium sized deletions which are difficult to detect (single gene or partial gene) as they are missed by the techniques used to detect the very large and small. • Old techniques • Southern Blotting: • Low throughput and Laborious. • Large amount of DNA required. • Hard to detect 2/3 copy differences. • FISH: • Good at detecting known single gene deletions and duplications. • Able to detect low level mosaicism. • Low throughput. • Unlikely to detect partial gene deletions.

  7. Detection Methods - 2 • Long-range PCR: • Easy to design primers. • Ideal when same deletion/duplication is frequently seen (FLT3). • False negative result if the del/dup is not within the fragment. • Further analysis is required to characterise the del/dup. • Breakpoint PCR: • Usually used for identifying translocated regions of DNA particularly in oncology. • Can be used when the same breakpoints in a del/dup is observed e.g. BRCA1 exon 13 duplication confirmatory test.

  8. Detection Methods – Current techniques - 1 • MAPH (multiplex amplifiable probe hybridisation)

  9. Detection Methods – Current techniques - 2 • MLPA (multiplex ligation-dependant probe amplification)

  10. Advantages and disadvantages of MAPH and MLPA • MAPH • Probes are easier to design in-house. • Unlikely to be affected by SNPs • Higher contamination risk. • Slightly more DNA required. • More labour intensive • MLPA • Entirely liquid phase so amenable to automation and higher throughput. • No wash steps required. • Can be very sensitive to DNA quality, concentration and extraction method. • Affected by SNPs and it is best practice to confirm single exon deletions (either alternative method or sequence the probe binding site) • Lots of kits commercially available but expensive

  11. Detection Methods – Current techniques - 3 • QF-PCR • PCR products during the exponential phase, double with each cycle. • The amount of product should be proportional to the amount of starting template. • Fluorescent labelling of the primers and subsequent separation by electrophoresis is the most common method of detection.

  12. Detection Methods – Current techniques - 4 • Real-time PCR • Is a laboratory technique based on the polymerase chain reaction, which is used to amplify and simultaneously quantify a targeted DNA sequence. • The two most common methods of quantification are: • The use of fluorescent dyes that intercalate with double-stranded DNA. • Modified DNA oligonucleotide probes that fluoresce when either hybridised or released from the target DNA sequence. • SYBR green • SYBR green is an intercalating dye, the fluorescence of which is higher in the bound state than in the free state. • Cheaper than alternative methods. • Disadvantage – SYBR green binds to all dsDNA in the reaction including primer dimers and template DNA which can lead to slight inaccuracy.

  13. Detection Methods – Current techniques - 4 • Sequence specific probes example 1 - TaqMan probes. • Provide the most accurate and reliable probes • Expensive. • Potential for multiplexing. RNA based probes with fluorescent reporter at one end and a quencher of fluorescence at the other. In close proximity the quencher prevents the fluorescence of the reporter. The probe anneals to it’s complimentary sequence. During PCR the Taq polymerase displaces the TaqMan probe from the template. The fluorescence is measured.

  14. Detection Methods – Current techniques - 4 • Sequence specific probes example 2 – molecular beacons • There are 4 parts to a molecular beacon. • Loop – 18-30bp complimentary to target sequence. • Stem – 5-7bp both ends of the loop. • 5’ fluorophore • 3’ quencher When the molecular beacon is in it’s hairpin structure the quencher prevents the fluorophore from emitting light. When it unfolds in the presence of the Complimentary sequence it is able to fluoresce

  15. Detection Methods – Current techniques - 5 • Other methods • LOH studies using linked SNPs/microsatellites. • MLGA (multiplex ligation-dependent genome amplification). • Next generation sequencing. • Probably many more!

  16. References MLPA and MAPH: New techniques for detection of gene deletions. Loryn N. Sellner and Graham R. Taylor Human Mutation (2004) 23:413-419 Detection of large deletions in the LDL receptor gene with quantitative PCR methods. Dorte Damgaard et al BMC Med Genet (2005);6:15

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