Development and validation of a new screening strategy for SCA testing in Birmingham Chris Campbell
Spinocerebellar Ataxia • Patients with Autosomal Dominant Cerebellar Ataxia show poor coordination of movement and a wide-based, uncoordinated, unsteady gait. • SCAs 1,2,3,6 & 7 account for ~62% all ADCAs and are caused by a CAG expansion into an affected range. • SCAs caused by CAG expansion show anticipation – ‘earlier onset and increasing severity of disease in subsequent generations of a family’ • General correlations exist between earlier age of onset and more severe disease with increasing number of CAG repeats.
PCR for SCA 1, 2, 3, 6 & 7 alongside known +ve controls Products run on a denaturing polyacrylamide gel and silver stained Patients with 2 alleles in normal range Patients with 1 band Patients with borderline alleles ? Patients with 1 allele in affected range Repeat PCR Patients with 1 band - Homozygous Repeat PCR Report as affected Report as normal Previous testing strategy
Problems with PAGE The resolution means that it can be difficult to resolve 2 closely sized alleles Presence of heteroduplexes Even strong positives appear faint
Aims of the new strategy • Transfer SCA testing to capillary electrophoresis (Beckman CEQ8000) using by using fluorescent primers. • Increase the speed at which patients are tested and eliminate the current backlog. • Run pooled products on the Beckman reduce reaction volumes in order to reduce testing cost.
Sizing Alleles AIM To sequence 10 different sized alleles for each of the SCA genes. These can then be PCRed using fluorescently labelled primers and run through the Beckman CEQ8000 in order to generate a conversion table by which patient alleles can be sized. The alleles were derived from previously tested patients. RESULTS A total of 98 alleles (49 patients) were sequenced in order to generate the required validation data. - 8 or 9 sequenced alleles for each SCA - Difficulty in generating different sized alleles due to homogeneity e.g SCA2
Techniques used for isolating DNA from gels prior to sequencing • Isolation of DNA from an agarose gel Run PCR Products on 3% agarose gel containing Ethidium Bromide Excision of bands on a UV box using a scalpel and scoop QIAQuick gel extraction kit 1. Gel slice solubilised 2. DNA bound to spin column 3. Wash step using buffer 4. DNA eluted in 30µl elution buffer Extraction of DNA from gel slice using the QIAquick Gel Extraction Kit Check gel in order to assess DNA concentration Sequencing ABI 3730
6% or 8% dependent on the SCA gene being tested as repeat ranges differ significantly PCR products run on non-denaturing PAG 2. Isolation of DNA from a polyacrylamide gel PAG stained post electrophoresis in TE containing 0.0001% ethidium bromide for 20 mins Gel slices excised on UV light box DNA eluted in 10µl water overnight Re-PCR using 1µl eluted solution Check gel to assess concentration of products Sequencing ABI 3730
Fluorescent PCR • Labelled primers used to amplify sized alleles (PCR protocols received from Sheffield molecular genetics lab) for SCA 1, 2, 3, 6 and 7 • Products run through Beckman CEQ8000 SCA1 +ve patient
Fluorescent PCR validation • Each of the sized patient alleles run through the Beckman 5X so that an average Beckman product size is generated. • This size is used to generate a calibration curve against the actual CAG repeat number in each allele. For example in SCA 6: Number of sequenced alleles for SCA 6 = 8
10 patients Robot transfer SCA 1, 2 & 3 PCR mixes Pooling of Products Post PCR Using BIOMEK NX PCR A Load pooled PCR A products on Beckman SCA 6 & 7 PCR mixes PCR B Load pooled PCR B products on Beckman Automation • SCA 1, 2, 3 PCRs are labelled with different coloured labels and use the same PCR cycling conditions as do SCA 6 & 7 • Programme created on the Beckman Biomek NX so that 10 patients can be batched and screened for all 5 SCA genes.
Use of CE and robots significantly reduces testing time (well within 10 week reporting deadlines). • All 5 genes can be screened at once. • Reduced reaction volumes = reduced cost of testing. • Capillary eletrophoresis results were easier to interpret than PAGE with fewer technical artefacts.
TP-PCR If a patient has only one allele how can we guarantee that we are not missing a second allele because: • The second allele is very large and out of the range of the Beckman (certainly possible with SCA 7) or, • The second allele has significantly reduced amplification? TP–PCR is a technique that allows large alleles to be detected but not necessarily sized.
P3 P1 P2 FL Triplet repeat • Developed by Jon Warner, Edinburgh in 1996 (A general method for the detection of large CAG repeat expansions by fluorescent PCR. J Med Genet 1996, 33:1022-1026 ) • A general technique to detect large triplet expansions • P2 binds at multiple sites along the triplet repeat • A 10:1 molar ration of P3 to P2 ensures that P2 is exhausted in the early PCR cycles • A long extension time is used to allow complete extension of the larger sized products can conserve their representation.
TP-PCR Traces (FRDA) Normal patient (2 normal alleles) Large GAA expansion Large GAA expansion TP-PCR is currently being validated for use in the SCA testing strategy in Birmingham
SAMPLE RECIEPT SCA 1,2 & 3 @ 57°C – 96 WELL PLATE 1 SCA 6 & 7 @ 63 °C – 96 WELL PLATE 2 ON BECKMAN ROBOT 13µl REACTION 2 ALLELES, 1 ALLELE IN AFFECTED RANGE CAPIILIARY ELECTROPHORESIS 2 ALLELES IN NORMAL RANGE REPEAT IN INTERMEDIATE RANGE I ALLELE TP PCR EXPANSION SEEN NO EXPANSION SEEN SCA 1 – SEQUENCING OR SfaN1 RESTRICTION ANALYSIS REPEAT TO CONFIRM REPORT AS AFFECTED REPORT AS NORMAL Testing Strategy
Thanks • Sheffield molecular genetics lab for supplying the fluorescent PCR protocols. • Marcus Allen, Birmingham.