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
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
Keywords
MLPA • EXT1 • EXT2 • hereditary multiple exostoses • HME • mutation detection
Abstract
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.
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
Keywords
MLPA • EXT1 • EXT2 • hereditary multiple exostoses • HME • mutation detection
Abstract
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
of Medical Genetics, University of Antwerp (Belgium) & The Genetic Unit ,The Rizzoli Orthopaedic
Institute, Bologna, Italy
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| 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]
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]
http://www.mlpa.com/pages/indexpag.html
If you are laboratory please contact P.C.W.Hogendoorn, M.D. Ph.D. / Wim Wuyts Ph.D. directly
concerning setting up this testing.
If you are laboratory please contact P.C.W.Hogendoorn, M.D. Ph.D. / Wim Wuyts Ph.D. directly
concerning setting up this testing.
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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
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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By the Health On the Net Foundation. Click here to verify.# HON Conduct 282463 and is linked on the NIH
National Library of Medicine, Directory of Health Organizations (SIS) website, as well as the link for Patient
Information on The Diseases Database a cross-referenced index of human disease, and the Intute: health & life
sciences a free online service providing access to the very best Web resources for education and research
located in the UK
The MHE Research Foundation is proud to be working with the EuroBoNeT consortium, a European Commission
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