Multiplex ligation-dependent probe amplification

Definition

Multiplex ligation-dependent probe amplification (MLPA) is a technique used by laboratories to detect an abnormal number of chromosomes, gene deletions, gene duplications, and gene expansions.

Description

Multiplex ligation-dependent probe amplification (MLPA) was first described by Schouten and his colleagues in 2002. The technique is designed to detect genetic issues ranging from an abnormal number of chromosomes to the location of changes in the "spelling" of genes. The technique is a multiplex assay in that it allows simultaneous testing of multiple genetic sequences.

Chromosomes are the gene-containing structures found in all of the body's cells. In each cell, there are 46 chromosomes that come in 23 pairs. Bodies are so specific that the exact amount of genetic information contained in the 46 chromosomes is required to grow and develop properly; having any more or any less genetic information causes abnormal physical and mental growth and development.

Genes are the blueprints that carry the instructions for making all of the proteins a cell needs and determine traits, such as hair and eye color. Genes contain the inherited information that is passed on from parents to children. Each gene has a specific spelling made of the base pairs, including C, G, A, and T. If the spelling of a gene is changed, or mutated, it can cause abnormalities in the way the gene functions. Missing genetic information from the loss of one normal base pair to the loss of most of a chromosome is referred to as a deletion. A copy of genetic information that results in a redundant piece of genetic information is known as a duplication. A gene expansion refers to a genetic abnormality caused by a sequence of base pairs that is repeated too many times in a section of a gene. Detection of chromosomal abnormalities , deletions, duplications, and expansions of genetic material is an important part of diagnosing genetic conditions and diseases.

The technique of MLPA uses the spelling of the base pairs to determine if portions of chromosomes or genes are changed or mutated. The technique begins with probes that attach to the base pairs of genes and chromosomes in a specific location. The probes are divided into halves that attach around a specific gene or chromosomal location and then attach to each other. This attachment to the area around a gene or chromosome is called hybridization, and the "glue" that attaches the two probe halves to each other is the enzyme ligase. In order to help count the number of probe halves present at the end of the procedure, the probes are made in a series of different lengths. If two probe halves have properly attached to their gene or chromosomal location and each other, they serve as a target for the next step. If the probes are attached to the wrong gene area or a mismatched probe half, they will not participate in the next part of the procedure.

In the next step, a technique called polymerase chain reaction (PCR) is used to make many copies of the correctly paired and attached probe halves. The PCR will not make copies of probes attached or paired incorrectly. Finally, the properly attached and matched probes of each size are separated by size and counted. The amount of each probe size provides the information whether a small area of the gene (called an exon) or the chromosome contains a deletion, duplication, or expansion. Since there are multiple probe sizes with different gene targets, 40 or more gene exons can be tested for abnormalities at the same time.

Indications

The technique of MPLA may be indicated when a genetic condition, disease, syndrome, or disorder involving a chromosome abnormality, gene deletion, gene duplication, or gene amplification is suspected. Possible testing options include chromosomal disorders, such as Down syndrome , conditions caused by rearrangements of a chromosome, genetic conditions caused by gene deletions and duplications, such as Duchenne's muscular dystrophy , certain forms of inherited cancer predisposition, and diseases caused by gene amplification, such as fragile X syndrome .

Samples

MLPA can be run on many bodily fluids and tissues, including blood, human embryonic stem cells, amniocytes, chorionic villi, and formalin-fixed paraffin-embedded tissue. The MLPA method requires that DNA samples obtained from the fluids and tissues be samples of uniform quality, which reduces false positives.

Results

The MLPA test can provide information about chromosomal abnormalities and gene duplications, deletions, or expansions. If a chromosome abnormality, gene duplication, deletion, or expansion is found, it may indicate predisposition for, diagnosis of, or carrier status for a specific genetic disorder or condition.

Benefits

MLPA is a quick technique that requires a smaller amount of DNA than other testing methods. The test exhibits good sensitivity and specificity when probes are made correctly and proper reaction conditions are used. Several genetic locations can be tested in the same procedure and, accordingly, large genes made of many exons can be tested in a quick and efficient manner. Data from the technique are reproducible. DNA degradation to small fragments does not influence results, and thus testing can be done on formalin-fixed paraffin-embedded tissue.

Limitations

Currently, MLPA requires the creation of labor-intensive probes for each new gene or chromosome to be examined before the test can be run. Kits of developed probes are sold, but are not certified by the U.S. Food and Drug Administration (FDA) for use in diagnostic procedures at this time. Currently, the probe kits are labeled for research purposes and to demonstrate the possibilities of the MLPA technique. Accordingly, results detected through MLPA should be verified with an independent method, such as G-banding chromosome analysis or Southern blots. Although MLPA can determine the approximate location of a genetic anomaly within an exon, unless special probes for specific mutations are designed, the test cannot determine the exact mutation of base pair or pairs.

Alternative tests

Currently, there are several testing methods that provide the same information as the MLPA technique. Standard chromosome analysis, comparative genomic hybridization (CGH), fluorescent in situ hybridization (FISH), BAC arrays, Southern blots, and loss of heterozygosity (LOH) assays are used to detect chromosomal abnormalities. Some genetic mutations and expansions can be detected with multiplex polymerase chain reaction (PCR) assays, fluorescent in situ hybridization (FISH), and/or Southern blotting.

Resources

PERIODICALS

Schouten, J. P., C. J. McElgunn, R. Waaijer, D. Zwijnenburg, F. Diepvens, and G. Pals. "Relative Quantification of 40 Nucleic Acid Sequences by Multiplex Ligation-dependent Probe Amplification." Nucleic Acids Res. 2002 June 15; 30(12): e57.

WEBSITES

"MLPA Info." MRC-Holland. (April 21, 2005.) <http://www.mrc-holland.com/mlpa_info.htm>.

National Genetics Reference Laboratories Information on Multiplex Ligation-dependent Probe Amplification. (April 21, 2005.) <http://www.ngrl.org.uk/Wessex/mlpa.htm>.

Dawn Jacob Laney, MS, CGC