Home Molecular Testing Methods Multiplex Ligation-Dependent Probe Amplification (MLPA) Test: Gene Deletions, Duplications, and Results

Multiplex Ligation-Dependent Probe Amplification (MLPA) Test: Gene Deletions, Duplications, and Results

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Vita Library
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MLPA testing detects targeted gene deletions and duplications; learn how the assay works, how results are interpreted, why findings need confirmation, and what a negative result means.

Multiplex ligation-dependent probe amplification, usually called MLPA, is a targeted molecular test used to measure the number of copies of selected DNA regions. It is especially useful for finding exon-level deletions and duplications that may be too small for a conventional chromosome study and may not be detected reliably by routine sequence analysis. An MLPA test may examine every exon of one gene, several clinically related genes, or a defined chromosome region, depending on the probe set. The result is interpreted by comparing the patient’s signal pattern with reference samples that have the expected copy number. A reduced signal can indicate a deletion, while an increased signal can indicate a duplication. However, MLPA does not read the complete DNA sequence, survey the whole genome, or show exactly where duplicated material is located. Understanding the assay’s target list, quality controls, and confirmation strategy is therefore essential before using a result for diagnosis, treatment, reproductive planning, or family testing.

  • MLPA is primarily a targeted copy-number test for deletions and duplications in selected genes or chromosome regions.
  • It can detect changes involving a single exon when a suitable probe is present.
  • A sequence variant beneath a probe may imitate a deletion, especially when only one probe is abnormal.
  • MLPA usually cannot define deletion breakpoints, duplication orientation, or balanced rearrangements.
  • Positive and borderline findings often require confirmation with an independent method or a second probe set.

Table of Contents

What an MLPA Test Measures

MLPA measures relative DNA dosage at locations represented by probes in a particular assay. In most inherited-disease applications, a person is expected to have two copies of an autosomal target: one inherited from each biological parent. A heterozygous deletion may reduce the relative signal to approximately half of the expected level, while a duplication may raise it above the normal two-copy level. For X-linked genes, expected dosage depends on chromosomal sex and the laboratory’s analytical model.

The test is often described as a deletion/duplication assay, but its scope is more precise than that phrase suggests. MLPA does not continuously scan every base across a gene. Instead, it samples specific points. A common gene-focused probe mix includes one probe for each coding exon and additional reference probes elsewhere in the genome. If the probe for exon 12 shows a reproducibly reduced dosage while neighboring probes are normal, the assay may suggest a deletion involving exon 12. If several adjacent exon probes are reduced, the pattern may indicate a multi-exon deletion.

This targeted design gives MLPA high practical resolution where probes have been placed. It may reveal a single-exon copy-number change that is difficult to identify by Sanger sequencing and that may be below the resolution of some chromosome-level methods. It is therefore an important form of deletion and duplication testing. However, the test cannot establish what happens between probes. A deletion can extend beyond the last abnormal probe, and a small deletion located entirely between probes may be missed.

MLPA is not the same as DNA sequencing. Standard sequencing is designed primarily to identify changes in the order of DNA bases, such as substitutions and small insertions or deletions. MLPA is designed primarily to identify relative gains and losses of targeted DNA segments. Many comprehensive hereditary tests combine sequencing with dosage analysis because either method alone may leave clinically important variant classes unexamined.

MLPA is also not a genome-wide chromosomal microarray. A microarray evaluates copy-number patterns across large portions of the genome and may discover an unexpected deletion or duplication outside the gene initially suspected. MLPA answers a narrower question with dense coverage of selected targets. This distinction matters when a result is negative: a normal MLPA result means that no reportable dosage change was found at the tested probes, not that the entire genome or even every base of the gene is normal.

When MLPA Is Used

A clinician may order MLPA when the suspected disorder is commonly caused by exon-level or whole-gene deletions and duplications. It may be used as a first-line assay, as a companion to sequencing, or as a focused follow-up to another test. The best position in the testing pathway depends on the disease, gene architecture, available assay, family history, and laboratory protocol.

One well-known use is testing the DMD gene in Duchenne and Becker muscular dystrophy. Large intragenic deletions and duplications account for a substantial proportion of disease-causing variants in this gene. Probe sets covering the coding exons can identify the affected region and help determine whether the change disrupts the reading frame. That information may support diagnosis and, in some situations, help assess eligibility for variant-specific therapies. MLPA cannot by itself characterize every complex rearrangement, so unusual patterns may need further study.

MLPA is also used in SMN1 and SMN2 analysis for spinal muscular atrophy. Here, the assay must distinguish highly similar genes and estimate clinically important copy numbers. A finding of absent functional SMN1 copies can support a diagnosis, while carrier testing may involve more complex interpretation because some people have two SMN1 copies on one chromosome and none on the other. Copy number alone does not always resolve that configuration, and residual carrier risk can remain.

Hereditary cancer testing may use MLPA to examine genes such as BRCA1, BRCA2, MLH1, MSH2, MSH6, PMS2, or EPCAM for exon-level and whole-gene rearrangements. Sequence analysis may find a pathogenic single-nucleotide variant, but it can miss a deletion of one or more exons unless the test includes a validated copy-number component. Conversely, a normal MLPA result does not exclude pathogenic sequence variants. Other applications include dosage analysis in genes associated with neuromuscular, metabolic, renal, cardiac, hearing, connective-tissue, neurodevelopmental, and ophthalmic disorders. Some probe mixes assess recurrent microdeletion or subtelomeric regions. Others help refine a known copy-number change detected by another method. For example, a genome-wide test may identify a larger deletion, and MLPA may clarify whether particular exons are involved in relatives or additional samples.

MLPA may be especially useful for familial variant testing when a deletion or duplication has already been characterized in an affected relative and the selected probe mix reliably covers the altered region. Targeted testing can then determine whether another relative carries the same dosage change. The laboratory must still confirm that the familial change is detectable with the chosen probes; knowing only the gene name may not be enough.

How MLPA Works in the Laboratory

MLPA derives its specificity from paired probes that bind immediately next to each other on the patient’s DNA. Although the full workflow includes several technical controls and normalization steps, the central process can be understood in five stages.

First, genomic DNA is denatured so that its two strands separate. A mixture containing many probe pairs is then added. Each target has a left probe oligonucleotide and a right probe oligonucleotide designed to hybridize to adjacent sequences. Different probe pairs contain unique length-adjusting sequences, allowing their amplified products to be distinguished later.

Second, correctly hybridized probe halves are joined by a ligase. Ligation occurs efficiently only when the two probes are positioned properly at their target. The ligated molecule represents the presence of that targeted sequence. A mismatch near the ligation site can interfere with this step, which contributes to specificity but can also create an important interpretive pitfall.

Third, all ligated probes are amplified together using a common pair of PCR primers. Because each probe contains the same universal primer-binding sequences, dozens of targets can be amplified in one tube. The probes, rather than the patient’s genomic regions themselves, are the principal templates for amplification. This design reduces the need to optimize a separate primer pair for every exon.

Fourth, the fluorescently labeled amplification products are separated by length using capillary electrophoresis. The instrument produces a series of peaks. Each peak corresponds to a particular probe, and its height or area reflects the amount of amplification product generated for that target.

Fifth, software compares the patient’s peak pattern with reference probes within the sample and with multiple reference samples that have expected copy numbers. Normalization is essential because raw peak height depends on many factors beyond genomic dosage. The analysis produces relative values, often called dosage quotients or dosage ratios, rather than an absolute count obtained by simply reading one peak.

The exact normal and abnormal ranges are assay- and laboratory-specific. A simplified teaching model might place a heterozygous deletion near 0.5, a normal diploid target near 1.0, and a single-copy duplication near 1.5 after normalization. Actual reporting thresholds are established through validation and may differ among probe mixes, targets, specimens, and software versions. Borderline values require review of the complete pattern rather than mechanical classification by one universal cutoff.

How Deletion, Duplication, and Borderline Results Are Read

A positive deletion result means that one or more targeted regions produced a lower relative signal consistent with reduced copy number and that the laboratory considered the finding reportable. The report should identify the gene and affected probes or exons, describe whether the change is heterozygous, hemizygous, or another state, and explain its clinical classification when possible.

A deletion involving several consecutive probes is generally more persuasive than an isolated low signal because adjacent probes provide internal support for the same event. Even so, the assay usually does not reveal the exact breakpoints. A report such as “deletion of exons 3–7” ordinarily means that probes for exons 3 through 7 showed loss while flanking tested probes did not. The physical deletion could begin within an intron before exon 3 and end within an intron after exon 7, or it could have a more complex structure.

A positive duplication result means that one or more targets have increased dosage relative to reference samples. MLPA can identify which probes show gain but generally cannot determine where the extra copy is located, whether it is in tandem, what its orientation is, or whether additional rearrangement has occurred. These details can change functional interpretation. A duplication of exons may disrupt a transcript, preserve a reading frame, or represent part of a complex event. RNA studies, breakpoint analysis, long-read sequencing, genomic sequencing, or another structural method may sometimes be needed.

An MLPA result may be described as borderline, equivocal, suggestive, or indeterminate when the dosage ratio is near a threshold, technical variation is present, or the signal pattern does not fit a straightforward copy-number model. Borderline results are not automatically weak positives. They may reflect low-level mosaicism, poor sample quality, a sequence change beneath a probe, contamination, a complex rearrangement, or ordinary analytical variation. Repeating the assay from the same DNA can test reproducibility, but confirmation from a new extraction or independent method provides stronger evidence.

The number of abnormal probes matters. A consistent multi-probe pattern across neighboring exons often supports a genuine deletion or duplication. A single-probe abnormality deserves particular caution. It may represent a true single-exon copy-number change, but it can also occur because a small sequence variant interferes with probe binding or ligation. The laboratory may sequence the probe-binding region or use a second probe targeting a different sequence within the same exon.

Clinical classification is separate from analytical detection. Once a dosage change is confirmed, the laboratory considers gene-disease validity, inheritance, predicted effect, population frequency, published cases, database evidence, and the patient’s phenotype. A copy-number change can be pathogenic, likely pathogenic, uncertain, likely benign, or benign. “Deletion” does not automatically mean disease-causing, and “duplication” does not automatically mean harmless.

In autosomal dominant disease, a de novo pathogenic deletion may strongly support diagnosis, but parental testing is required to establish whether it is truly new. In recessive disease, a single heterozygous pathogenic event may prompt a search for another variant on the opposite chromosome. In X-linked disease, the same dosage change can have different implications depending on sex chromosomes, X-inactivation, and family relationships.

Limitations, False Signals, and Quality Concerns

The most important limitation is target dependence. MLPA detects only regions represented by probes. A normal signal at exon 5 does not prove that every nucleotide in exon 5 and its surrounding introns is normal. Small sequence variants, deep intronic variants, regulatory changes, and copy-number changes outside probe targets may be missed.

Most MLPA assays do not detect balanced translocations or inversions because these rearrangements can preserve copy number. They also do not usually reveal the chromosomal position or orientation of duplicated material. A balanced rearrangement that disrupts a gene may therefore require karyotyping, FISH, genome sequencing, or another structural assay.

Sequence variation at a probe-binding or ligation site can reduce probe hybridization or ligation. The resulting low peak may resemble a heterozygous deletion even though the DNA segment is physically present. This risk is particularly important for a one-probe finding. Conversely, a probe may not detect a deletion whose breakpoint leaves its binding sequence intact.

DNA quality affects performance. Degraded DNA, contaminants, insufficient or excessive input, incomplete denaturation, and extraction-related differences can distort peak patterns. Formalin-fixed paraffin-embedded tissue is often fragmented and chemically modified, so assays using tumor tissue require method-specific validation and cautious review. Blood, saliva, buccal material, prenatal samples, cultured cells, and tissue are not automatically interchangeable.

Mosaic copy-number changes can be difficult to detect because the abnormal signal is diluted by cells with the usual copy number. The lower the abnormal cell fraction, the closer the ratio moves toward the normal range. Detectability depends on assay precision, probe behavior, specimen type, and laboratory threshold. A normal blood result may not exclude tissue-limited mosaicism.

A test’s stated scope should be read carefully. Commercial probe mixes are versioned, and target content can change. Laboratories may add custom probes, omit poorly performing probes, or report only a subset of included targets. The report, assay description, or test directory should identify clinically relevant coverage and major limitations.

Confirming an MLPA Finding

Confirmation helps distinguish a genuine genomic copy-number change from probe-specific or technical effects. The optimal method depends on the number of abnormal probes, suspected event size, genomic region, disease mechanism, specimen, and clinical consequence.

A second MLPA probe mix can provide confirmation when it targets different sequences near the same region. It is not truly independent if both assays use a probe with the same binding or ligation site. For a single-exon deletion, a confirmation assay should ideally use another probe within that exon or a different technology.

Quantitative PCR, long-range PCR, droplet or digital PCR, targeted next-generation sequencing, array comparative genomic hybridization, SNP microarray, or genome sequencing may be appropriate. Sanger sequencing can determine whether a nucleotide variant under the MLPA probe caused signal dropout. Long-range PCR and breakpoint sequencing can sometimes define the structure of a deletion or duplication.

The confirmation question should be explicit. There are at least three different goals:

  1. Confirm that copy number is abnormal. An orthogonal dosage method addresses whether the deletion or duplication is real.
  2. Define the event’s boundaries and structure. Breakpoint analysis, long-read sequencing, or genomic methods may show where the event begins and ends and how duplicated material is arranged.
  3. Determine clinical significance. Family testing, transcript studies, phenotype correlation, and variant classification address whether the confirmed event explains disease.

Testing a second specimen can be valuable when sample identity, contamination, culture artifact, or mosaicism is a concern. For prenatal or tumor testing, the confirmation plan should account for specimen-specific issues, including maternal cell contamination, tumor purity, and normal-cell admixture.

A confirmed pathogenic finding should be documented at a level that permits reliable testing of relatives. Exon labels alone may be insufficient if transcript numbering differs among laboratories. Reports should state the reference transcript, affected region, method, and any available breakpoint description. Families should retain the original report because future testing may depend on those details.

Not every laboratory automatically confirms every multi-probe finding with another platform. Some validated workflows consider a coherent multi-probe pattern sufficient analytically, while emphasizing orthogonal confirmation for single-probe or unusual results. The report should state whether confirmation was performed and what evidence supports the call.

Negative Results, Family Testing, and Next Steps

A negative MLPA result means that the laboratory did not detect a reportable copy-number abnormality among the probes analyzed under the assay’s validated conditions. It does not exclude all genetic disease, all variants in the tested gene, or all copy-number variants.

The next step after a negative result depends on what was already tested. If MLPA was performed without sequencing, sequence analysis may still be needed for substitutions and small insertions or deletions. If both sequencing and MLPA were negative, a broader multigene panel, exome sequencing, genome sequencing, RNA analysis, repeat-expansion testing, methylation testing, or a genome-wide copy-number assay may be considered according to the phenotype.

A negative result is most informative when the test was designed to detect a known familial deletion or duplication and included probes within that exact region. In that setting, a relative who tests negative usually has not inherited the targeted familial event, although the report’s technical limitations still apply. When no familial variant is known, a negative result leaves a wider range of untested possibilities.

For a positive inherited-disease result, genetic counseling can help address inheritance, medical management, reproductive options, and testing of relatives. Testing biological parents may show whether the change was inherited or de novo. In recessive disease, phasing may be needed to determine whether two variants are on opposite chromosome copies. For X-linked findings, counseling should account for sex chromosomes, carrier implications, and variable expression.

A result of uncertain significance should not generally be used as the sole basis for irreversible medical decisions. Segregation testing may provide evidence, but testing unaffected relatives without a clear analytical plan can produce ambiguous information. The laboratory or genetics team can advise which family members would be most informative.

When clinical suspicion remains high, re-evaluation is reasonable. Gene-disease knowledge, variant classification, probe design, and testing technology evolve. A previous “negative deletion/duplication test” may have used a method with incomplete exon coverage or limited mosaic sensitivity. Obtaining the original laboratory report is more useful than relying on a summary in the medical record.

Methylation-Specific MLPA

Methylation-specific MLPA, abbreviated MS-MLPA, extends the method so that it can assess both copy number and methylation at selected sites. It is not simply a more sensitive form of standard MLPA. It answers an additional epigenetic question by comparing an undigested reaction with a reaction exposed to a methylation-sensitive restriction enzyme.

In the digested reaction, unmethylated target sites are cut, preventing the corresponding ligated probes from producing the usual signal. Methylated sites are protected from digestion and retain signal. Comparing digested and undigested reactions allows the laboratory to estimate methylation at the targeted restriction sites. The undigested portion is used for copy-number analysis.

MS-MLPA is used in selected imprinting disorders and tumor applications. It may help evaluate regions associated with disorders such as Prader-Willi, Angelman, Beckwith-Wiedemann, or Silver-Russell syndromes, depending on the assay. It can also assess promoter methylation in certain cancer-related genes. The clinical meaning depends on the locus, parent-of-origin pattern, tissue, and mechanism.

A methylation abnormality does not always identify its underlying cause. The same abnormal profile may result from a deletion, uniparental disomy, an imprinting-center defect, a pathogenic sequence variant affecting imprint establishment, or a broader multilocus disturbance. Additional testing may be required to separate these mechanisms. The general principles of a methylation genetic test therefore extend beyond the MLPA trace itself.

MS-MLPA has its own technical limitations. It evaluates only targeted restriction sites, may be affected by incomplete digestion or DNA quality, and may have limited sensitivity for low-level mosaicism. Copy-number and methylation findings should be interpreted together because a deletion can alter the apparent dosage and methylation pattern. Maternal cell contamination and tissue-specific methylation are important considerations in prenatal or mosaic disorders.

Whether standard MLPA or MS-MLPA is ordered, the most useful result is one interpreted in the context of the patient’s phenotype, family history, specimen, and complementary testing. The technique is powerful because it answers a focused dosage question efficiently—not because it replaces sequencing, genome-wide analysis, or clinical judgment.

References

Disclaimer

This article provides general education about MLPA and is not a substitute for interpretation by the testing laboratory, a genetics professional, or the clinician responsible for care. Assay coverage, thresholds, confirmation practices, and report language vary among laboratories and probe mixes. Medical or reproductive decisions should be based on the complete clinical evaluation and a confirmed, appropriately classified result.

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