Multiplex ligation-dependent probe amplification (MLPA)

Overview

Multiplex ligation-dependent probe amplification (MLPA) is commonly used to diagnose conditions that are predominantly caused by exon-level copy number variation (deletions and duplications), for example spinal muscular atrophy.

Alternatively, it can be used in combination with massively parallel sequencing (sometimes referred to as next-generation sequencing) to examine genes that may contain single nucleotide variants or copy number variants.

MLPA may also be used:

to test family members for known familial deletions or duplications;
to refine the breakpoints of intragenic copy number variants detected by microarrays;
occasionally, to test for specific single nucleotide variants (for which probes are included in the kit); and
in methylation-specific MLPA (MS-MLPA), to determine the methylation status of imprinted or promoter regions at which aberrant methylation is associated with disease. For clinical examples, see Genomic imprinting.

MLPA uses pairs of oligonucleotide probes – small molecules of DNA that are designed to hybridise with a specific genomic sequence. Multiple pairs of probes can be multiplexed in the same reaction, for example to screen for all exons of a gene.
The probes bind only where their exact target sequence is present. If a pair of probes bind immediately next to each other, their ends ligate (join together).
There is one primer binding site on the end of each of the probes in the pair. Polymerase chain reaction (PCR) amplification therefore only occurs if the pair of probes have ligated.
Amplification products are separated by length using capillary electrophoresis.
The amount of amplification product is proportional to the amount of target sequence present in the sample. The pattern of amplification products from a patient sample can be compared with a reference sample of a known copy number to detect dosage abnormalities in the sample (for example, exon deletions or duplications).

MS-MLPA can be used to determine the methylation status of an imprinted or promoter region.
Patient DNA is used in two separate reactions: one reaction is a standard MLPA reaction (as above); for the second reaction, prior to the MLPA, a methylation-sensitive endonuclease (restriction digest enzyme) is applied.
Methylated regions of DNA are protected from digestion by the enzyme, and therefore amplification products result. Unmethylated regions are digested (broken down) and therefore no amplification products are formed.
MLPA peak heights are compared between the reaction that has been exposed to the methylation-sensitive endonuclease, and the reaction that has not. Peaks that occur in both show where methylation occurs. Those that occur in only the undigested reaction are not methylated.
Both CNVs and methylation status can be determined.

For a more detailed look at how MLPA works, see the resources section below.

MLPA:

is the gold standard for exon-level CNV detection (alongside some microarrays);
is robust, fast and cost-effective;
provides a high resolution and accurate detection of single exon deletions and duplications;
can be designed to include specific single nucleotide variants;
can be modified to detect methylation (through MS-MLPA); and
has a higher throughput and is less laborious than fluorescent in situ hybridisation (FISH).

Commercial MLPA kits are not available for all genes.
There are a limited number of target regions per kit (though digital MLPA may be able to overcome this).
Where a duplication is detected, the location or orientation of the duplicated material is not known.
MLPA will not detect balanced rearrangements.
MLPA may not detect mosaicism.
MLPA may miss small or intronic deletions that are located outside of the regions targeted by the probes in the kit.
If the deletion or duplication extends beyond the region targeted by the probes in the kit, a secondary method may be required to investigate this (for example, microarray).
MLPA does not detect single nucleotide variants (unless specifically targeted by MLPA probes).

MLPA is used to detect exon-level CNVs (deletions and duplications).
MLPA uses DNA probes that only bind when the exact target sequence is present. The amount of PCR application that occurs is directly proportional to the amount of target sequence present in the sample.
MS-MLPA can be used to determine the methylation status of an imprinted or promoter region.

References:

Stuppia L, Antonucci I, Palka G and others. ‘Use of the MLPA assay in the Molecular Diagnosis of Gene Copy Number Alterations in Human Genetic Diseases‘. International Journal of Molecular Sciences 2012: volume 13, issue 3, pages 3,245–3,276. DOI: 10.3390/ijms13033245