Manchester - National Genetics Reference Laboratories

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Figure 9 illustrates some typical results. Most of the odds ratios for the shown ligation products strongly favour the normal hypothesis and are consequently shaded in with a green backround. However the ligation product for BRCA1 Ex 13 is showing high DQs, consequently the fit is better to the duplicated hypothesis than either the normal or deleted hypotheses. Comparing the relative probabilities of fit to the duplicated and normal hypotheses the relative likelihood is calculated as 146:1 in favour of the sample being duplicated than the result being a normal outlier experienced by chance. Fig 9: The spreadsheets also incorporate a graphical representation of the results in the form of a histogram. This is located to the right of each test sample on the RESULTS sheet. Fig 10 illustrates a histogram from the HNPCC MLPA spreadsheet summarising the mean DQ data. This particular sample gave normal odds ratios for all ligation products except that for hMLH1 exon 2 which appeared to be deleted. This was subsequently confirmed by further analysis. FIG 10: INTERNAL QUALITY CONTROL Internal quality control is an important consideration for all diagnostic laboratories. Although the extra tiers of analysis given in the spreadsheets assist in the analysis of MLPA dosage data, the meaning and significance of some results will still remain a matter of professional judgement. The precise limits of what is an acceptable result must remain the responsibility of each laboratory to determine, however what follows are guidelines that have been found to be useful locally. 1) In order to be an acceptable result the “Quality” value (standard deviation of the control ligation products) should have a value of = 20:1 odds then the sample can be judged to be normal. 3) If two or more contiguous ligation products favour a deleted or duplicated hypothesis with >= 1:20 odds then the sample can be judged to be abnormal provided the remaining ligation products give odds ratios for the normal hypothesis of >= 20:1 odds. It still remains good NGRL(Manchester) MLPA analysis spreadsheet instructions v10-06
practice to confirm any mutations by repeating the analysis on a replicate MLPA analysis or on an affected first degree relative 4) If a single ligation product gives a >= 1:20 odds ratio in favour of deletion/duplication then further evidence must be obtained to confirm this result. Firstly the sample should be sequenced to establish that there are no mutations present beneath the ligation product hybridisation sites (this has been a frequently cause of a ligation product which appears deleted). Secondly the deletion/duplication should either (i) be confirmed using a separate assay e.g. long PCR, dosage PCR (ii) be confirmed on a separate DNA sample (not just a new replicate MLPA assay) or (iii) be confirmed on an affected first degree relative. 5) Where a sample otherwise appears normal yet the odds ratios for normality drop below >= 20:1 for some test ligation products this can be accepted to be a normal result provided (i) no one normal odds ratio drops below 5:1 in favour of normality (ii) that contiguous exons are not involved (iii) that no more than 10% of test ligation products are involved (iv) that none of the mean DQs falls into the transitional range (0.7-0.8 or 1.2-1.3). Please note that in the presence of a deletion the normal:duplication comparison is meaningless and will yield equivocal odds the same applies for the normal:deletion comparison for a duplicated sample. SPREADSHEETS FOR NEW MLPA ASSAYS If there is an MLPA assay in use in your laboratory for which you would like us to design a spreadsheet please contact me by email at andrew.wallace@cmmc.nhs.uk CONFIGURATION OF EXISTING SPREADSHEETS If there is an existing spreadsheet for which the data entry page is not compatible with your fragment analysis output, I can usually quite simply alter these to suit your requirements. In order to do this please email to me at andrew.wallace@cmmc.nhs.uk a sample of your data which you would like to import directly into the spreadsheets MODIFICATIONS TO EXISTING PROBE SETS MRC-Holland review their kits and often change the probe combinations in response to their customer demand. If you notice that this has occurred and the currently available spreadsheet will no longer analyse the data please let me know by email at andrew.wallace@cmmc.nhs.uk as I can usually make minor modifications quite easily. ACKNOWLEDGEMENTS The quality control measure using the standard deviation of the control ligation products was developed by Dr Ruth Charlton, Regional Genetics Service, Leeds NGRL(Manchester) MLPA analysis spreadsheet instructions v10-06
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Manchester - National Genetics Reference Laboratories

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