MLPA

Multiplex ligation dependent probe amplification (MLPA) is a method that measures the copy number of many DNA targets in a single reaction. It finds deletions and duplications of whole exons and genes, which sequencing and some other tests can miss, and it can also measure methylation (methylation specific MLPA).

For each target, two short probes bind side by side to the DNA and are joined by a ligase enzyme only when they have attached to the correct sequence. The joined probes are amplified with one pair of universal primers, so all targets are amplified with equal efficiency. The products are separated by capillary electrophoresis, and the height or area of each peak is compared with control samples.

A peak ratio of about 1.0 means two copies, 0.5 means one copy (a heterozygous deletion), and 1.5 means three copies (a duplication).

A kit contains probes for one gene or region, such as BRCA1, MLH1, DMD or the region on chromosome 22q11.2. Each reaction measures up to about 50 targets. The test needs 50 to 100 ng of DNA, takes 1 to 2 days to run, and a result is reported in 1 to 3 weeks.

Large deletions and duplications cause a notable share of genetic disease: about 60 to 65% of Duchenne and Becker muscular dystrophy variants are deletions and 5 to 10% are duplications. About 5 to 10% of pathogenic variants in BRCA1 and 1 to 2% in BRCA2 are exon level rearrangements, and more in some other genes.

Indications.

• Detection of exon deletions and duplications in a known gene when sequencing is negative or incomplete: BRCA1, BRCA2, mismatch repair genes (MLH1, MSH2, MSH6, PMS2), APC, LDLR, NF1, DMD, and many others.
• Duchenne and Becker muscular dystrophy: first line test in male patients, and carrier testing in females.
• Spinal muscular atrophy: SMN1 deletion and SMN2 copy number, and carrier testing.
• Microdeletion and microduplication syndromes: 22q11.2 deletion, Williams syndrome, Smith-Magenis, and subtelomeric rearrangements.
• Imprinting disorders: methylation specific MLPA for Prader-Willi, Angelman, Beckwith-Wiedemann and Silver-Russell syndromes.
• Charcot-Marie-Tooth disease type 1A (PMP22 duplication) and hereditary neuropathy with pressure palsies (PMP22 deletion).
• Cascade testing of relatives with a known deletion or duplication, and prenatal diagnosis.

Not indicated for balanced rearrangements, small point variants, or genome wide screening, which need other tests.

Specimen requirements.

• Genomic DNA from EDTA blood, with good purity, at a defined concentration, and free of contaminants such as phenol, heparin and ethanol, which interfere with ligation.
• Prenatal samples (chorionic villi, amniotic cells), with a maternal blood sample to exclude maternal contamination.
• At least 3 to 5 healthy reference samples in each run, extracted in the same way as the test samples.
• Clinical details, the gene or syndrome in question, and the family variant where known.

Normal appearances.

All probe ratios fall between 0.7 and 1.3 (some kits use 0.8 to 1.2), so there is no loss or gain of copy number in the targets. The pattern is normalised to reference probes and to control samples in the run.

Interpretation.

• Heterozygous deletion: ratios of about 0.5 (under 0.7) for one or more adjacent probes.
• Homozygous deletion: ratios near zero, with no peak.
• Duplication: ratios of about 1.5 (over 1.3). Triplication: about 2.0.
• A deletion or duplication of several adjacent exons is more reliable than a single exon change, and a single probe change is checked with an independent method.
• Inheritance: the result is compared with that of the parents, and a de novo change is common in microdeletion syndromes.
• Methylation specific MLPA: loss of the unmethylated maternal or paternal allele points to the type of defect (deletion, uniparental disomy, or imprinting defect).
• Gene dosage: SMN1 copy number of 0 means spinal muscular atrophy, and a copy number of 1 means a carrier. SMN2 copy number predicts severity.
• Effect on the reading frame in DMD (in frame or out-of-frame) predicts Becker or Duchenne phenotype and eligibility for exon skipping treatments.
• Results are reported using internationally agreed variant nomenclature, with the exons affected, and are interpreted in the clinical setting.

Critical values and findings needing immediate action.

• A deletion or duplication in a cancer gene (BRCA1, BRCA2, mismatch repair genes): referral for surveillance and risk reducing options, and cascade testing.
• An SMN1 homozygous deletion in an infant with weakness: urgent referral for treatment, since early treatment gives much better outcomes.
• A deletion in DMD in a boy with weakness: referral to the neuromuscular team, with a plan for steroid treatment and cardiac and respiratory monitoring.
• A prenatal finding of a deletion: urgent genetic counselling.

Limitations and artefacts.

• A single nucleotide variant or small change in the probe binding site stops probe binding and gives a false deletion signal, so single probe results are confirmed by another method (sequencing, long range PCR or a different MLPA probe).
• Poor quality DNA, differences in DNA extraction between samples and controls, and contaminants cause wide variation in the ratios and false positives.
• Results from samples with mosaicism or low level changes can fall between ratios and are hard to call.
• MLPA detects only the targets covered by the probes. It does not show where the breakpoints lie, nor whether a duplication is in tandem, and it does not find balanced translocations or inversions.
• The result needs interpretation for the effect on the reading frame and gene function.
• Maternal cell contamination in prenatal samples can mask a deletion.
• Probe sets and thresholds differ between kits, so each laboratory validates its own limits.
• Cost is low compared with other tests, but a separate kit is needed for each gene or syndrome.

Special groups.

• Boys with muscle weakness and high creatine kinase: MLPA of DMD is the first genetic test.
• Female relatives of boys with DMD or families with exon deletions: carrier testing.
• Couples at risk of spinal muscular atrophy: carrier screening for SMN1 copy number, noting that two copies on one chromosome can hide a carrier.
• Families with hereditary cancer: testing of relatives for the known deletion.
• Pregnancies at risk: prenatal diagnosis with an accompanying maternal sample.
• Regions: MLPA is a relatively low cost, robust method that can be used in regional laboratories. It is useful for the common exon deletions of DMD, SMN1 and cancer genes where sequencing is not available. Quality of DNA and of controls is the main concern.

Follow up tests.

• Sequencing of the gene when MLPA is normal and suspicion is high.
• Confirmation of single exon changes by long range PCR, a second MLPA kit or microarray.
• Chromosomal microarray for larger deletions and duplications.
• Parental testing to find inheritance and de novo origin.
• Methylation tests, microsatellite analysis and uniparental disomy testing in imprinting disorders.
• Genetic counselling, cascade testing and prenatal diagnosis.
• Specialist referral and surveillance.