MLPA: 10 questions and answers

Last update: December 2, 2015

MLPA, Multiplex Ligation Probe Amplification

MLPA in GeneMarker (courtesy Wikimedia commons)

MLPA: Q&A

MLPA is acronym for Multiplex Ligation Probe Amplification, which is a special molecular technique based on PCR. It is used to scan large deletions/duplications/multiplications which are not detectable by standard sequencing (either NGS or Sanger). The alternative to this technique is the qPCR (quantitative PCR).

  1. Is it necessary to always perform MLPA?

No, it isn’t always indicated. Multiplex Ligation Probe Amplification is usually indicated whenever large deletions/duplications have been already described in the literature. Indeed there are genes in which such kind of mutations have never been reported and are very unlikely to occur (like, for instance, in several genes associated to autosomal dominantly inherited disorders caused by gain-of-function mutations).

  1. At which stage it is recommended to do MLPA?

Frequently, and intelligently, MLPA is done only as second step, i.e. in case sequencing is negative. Monogenic disorders are mostly caused by point mutations or small deletions/insertions of few nucleotides which can be easily detected by sequencing, whereas large deletions/duplications are rarer. Anyway, there are some exceptions: in a few genes most mutations are consistent with large deletions or duplications (see for instance the SMN1 gene). In such cases Multiplex Ligation Probe Amplification is indicated as the primary (or sole) diagnostic step.

  1. For which genes MLPA must be done?

There are no general rules to select genes for MLPA testing. In principle it is of help to check the mutational spectrum of each gene in major databases like Genereview, HGMD, ClinVar, dbVar, DECIPHER or Ensembl. If a certain gene-disease association has been described just recently, any kind of mutation might be expected. By contrast, whenever the mutation spectrum is well known and large deletions/duplications have not been reported, MLPA may be considered of secondary importance.

  1. When is MLPA indicated and when qPCR?

The answer is very easy: qPCR can be done whenever the MLPA commercial kit isn’t available. It is important to underline that most MLPA analyses worldwide are done utilizing the commercial kits from MRC-Holland: to see if the kit for a certain gene is available, it is enough to do a fast search on the MRC-Holland website.

  1. What’s best: MLPA or qPCR?

As said above, qPCR is usually the only possible choice whenever the MLPA commercial kit is not available. Both methodologies, if well done, may give excellent results. As a less attractive side, qPCR must be specifically designed by the lab and its sensitivity/specificity and coverage are therefore strictly operator- and budget*-dependent. By contrast, MLPA is provided as a tried and tested commercial kit and as such may be easier and faster (although a minimum of practice, especially in the pre-amplification phase and in the software calibration, remains necessary).

*qPCR, if done exclusively for one single sample, is much more expensive than MLPA, especially for big genes, for which a high number of amplicons must be designed.

  1. Does MLPA cover the entire gene?

It depends. Once again it is necessary to refer to the manufacturer to verify how many and which exons are covered by the kit. In most cases there’s full coverage, but for some genes it is possible to test just selected exons. This information can be easily found in the manufacturer’s fact sheets.

  1. Does MLPA detect only large deletions and duplications?

No. There is another useful application of MLPA, which allows to detect also the methylation status in certain genes. Through the use of specifically modified kits (MS-MLPA: Mehtylation-Specific MLPA) it is possible to do the testing for several imprinting disorders, from Beckwith-Wiedemann syndrome to Angelman and Prader-Willi syndromes. It is just necessary to remember that the mutational spectrum of imprinting disorders is very complex and that a negative MS-MLPA result alone does not allow to exclude the diagnosis.

  1. What’s the level of sensitivity and specificity of MLPA analyses?

False positives and false negatives are possible with any diagnostic technique, although MLPA analysis can be considered safe enough. The manufacturer MRC-Holland recommends to always perform the confirmation of positive results through an alternative method such as qPCR. Such recommendation, however, remains neglected by most laboratories, mainly because of the basic reliability of MLPA and for obvious financial reasons. However, the confirmation by qPCR may be necessary whenever the MLPA points to a results of a single exon deletion.

  1. Why is it necessary to confirm the deletion of a single exon with an alternative method?

Being based on PCR, MLPA is subject to some typical failures of this technique, like, for instance, the non-pairing between a primer and the patient’s DNA with a consequent allele drop-out. If the patient is carrier of a polymorphism right within the binding sequence of a MLPA primer, it is possible that the amplification of that particular DNA trait won’t take place, giving so a results of deletion, which will actually be a false positive. Thus, whenever MLPA points to a single exon deletion, it is good practice to consider the confirmation testing by qPCR.

  1. MLPA or FISH?

Some mutations which are detectable by FISH (Fluorescence In Situ Hybridization) may be detectable also by MLPA and vice versa. It is basically a questions of size. For very large deletions or duplications MLPA may simply detect the presence of the mutation, whereas the FISH may also be able to define the entire extension of the mutation.

Posted in Academia, Technohub and tagged , , , , .

Leave a Reply

Your email address will not be published. Required fields are marked *