MLPA multiplex ligation-dependent probe amplification was introduced by the Microbiology Research Center-Holland in January
2002, and has become a rapidly growing technique used in the detection of exon deletions
Research Article
Two-color multiplex ligation-dependent probe amplification: Detecting genomic rearrangements in hereditary
multiple exostoses  
Full Text PDF Link
Stefan J. White 1 *, Geraldine R. Vink 1, Marjolein Kriek 1, Wim Wuyts 2, Jan Schouten 3, Bert Bakker 1, Martijn H. Breuning 1,
Johan T. den Dunnen 1
1Center for Human and Clinical Genetics, Leiden University Medical Center, Leiden, The Netherlands
2Department of Medical Genetics, University of Antwerp, Antwerp, Belgium
3MRC-Holland, Amsterdam, The Netherlands

Communicated by Graham R. Taylor
Funded by:
Leiden University Medical Center
MLPA • EXT1 • EXT2 • hereditary multiple exostoses • HME • mutation detection
Genomic deletions and duplications play an important role in the etiology of human disease. Versatile tests are required to
detect these rearrangements, both in research and diagnostic settings. Multiplex ligation-dependent probe amplification (MLPA)
is such a technique, allowing the rapid and precise quantification of up to 40 sequences within a nucleic acid sample using a one-
tube assay. Current MLPA probe design, however, involves time-consuming and costly steps for probe generation. To bypass
these limitations we set out to use chemically synthesized oligonucleotide probes only. The inherent limitations of this approach
are related to oligonucleotide length, and thus the number of probes that can be combined in one assay is also limited. This
problem was tackled by designing a two-color assay, combining two sets of probes, each amplified by primers labeled with a
different fluorophore. In this way we successfully combined 28 probes in a single reaction. The assay designed was used to
screen for the presence of deletions and duplications in patients with hereditary multiple exostoses (HME). Screening 18
patients without detectable point mutations in the EXT1 and EXT2 genes revealed five cases with deletions of one or more
exons: four in EXT1 and one in EXT2. Our results show that a two-color MLPA assay using only synthetic oligonucleotides
provides an attractive alternative for probe design. The approach is especially suited for cases in which the number of patients
to be tested is limited, making it financially unattractive to invest in cloning. Hum Mutat 24:86-92, 2004. © 2004 Wiley-Liss, Inc.
Please note that MLPA for clinical genetic testing MHE / MO  / HME is offered at : Department of Medical Genetics,
University of Antwerp (Belgium)
& The Genetic Unit ,The Rizzoli Orthopaedic Institute, Bologna, Italy
MLPA multiplex ligation-dependent probe amplification
Mutation screening of EXT1 and EXT2 by direct sequence analysis and MLPA in patients with multiple
osteochondromas: splice site mutations and exonic deletions account for more than half of the mutations.
Full text
PDF Link

Vink GR, White SJ, Gabelic S, Hogendoorn PC, Breuning MH, Bakker E.
Center for Human and Clinical Genetics, Leiden University Medical Center, Leiden, The Netherlands.

Multiple osteochondromas (MO) is an autosomal dominant condition, caused by mutations in either the EXT1 or the EXT2 gene.
The DNA of a cohort of 35 patients, clinically suspected to be affected with MO, was screened for mutations by a combination of
direct sequence analysis and multiplex ligation-dependent probe amplification (MLPA). In this cohort, 26 pathogenic gene
alterations were found (74%). With sequence analysis mutations were detected in 22 patients (63%). In total, 10 mutations
were detected in the EXT1 and 12 in the EXT2 gene. The number of the splice site mutations detected was larger than expected
from the literature. In addition, with the MLPA four deletions of one or more exons were found in this cohort. Two patients, of
whom one had a negative family history, showed deletions of exon 1 of the EXT1 gene, which is possibly a deletion hot spot. In
patients suspected to be affected by MO, we recommend a quantitative analysis such as MLPA, followed by direct sequence
analysis for the screening of the EXT1 and EXT2 genes.

PMID: 15586175 [PubMed - indexed for MEDLINE]
If you are laboratory please contact P.C.W.Hogendoorn, M.D. Ph.D. / Wim Wuyts Ph.D. directly
concerning setting up this testing.
Press Release 1 / 19 / 08
Mutation Screening of EXT1 and EXT2 by Denaturing High-Performance Liquid Chromatography, Direct Sequencing
Analysis, Fluorescence in Situ Hybridization, and a New Multiplex Ligation-Dependent Probe Amplification Probe Set
in Patients with Multiple Osteochondromas
Ivy Jennes*, Mark M. Entius{dagger}, Els Van Hul*, Alessandro Parra{ddagger}, Luca Sangiorgi{ddagger} and Wim Wuyts*
To Read this publication Click Here
Both Luca Sangiorgi, M.D., Ph.D. and Wim Wuyts, Ph.D. are members of our Scientific & Medical Advisory Board  and our foundation helped
support this research
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