Multiplex ligation-dependent probe amplification (MLPA) screening in meningioma.
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ABSTRACT: Over the past 5 to 10 years, important advances were made in the understanding of meningioma biology. Progress in molecular genetics probably represents the most important accomplishment in the comprehensive knowledge of meningioma pathogenesis. Several genes could be identified as targets for mutation or inactivation. Additional chromosomal regions were found to be commonly deleted or amplified, suggesting the presence of further tumor suppressor genes or proto-oncogenes, respectively, in these regions. Histopathologically, the most important innovation is represented by the revised WHO classification in the year 2000. Meningioma grading criteria in the new classification scheme are more precise and objective, and should thus improve consistency in predicting tumor recurrence and aggressive behavior. This review focuses mainly on the advances in molecular biology that were achieved in recent years. It summarizes the most important aspects of meningioma classification as the basis to place biological observations into a correlative context, and, further, includes mechanisms of angiogenesis and edema formation as well as the role of hormone receptors in meningiomas.Journal of Neuropathology and Experimental Neurology 05/2004; 63(4):275-86. · 4.37 Impact Factor
Article: Meningioma: an update.[Show abstract] [Hide abstract]
ABSTRACT: Recent clinical and molecular research has shed new light on the biology of meningiomas--a common but understudied CNS neoplasm. This review will focus on recent advances and their significance for future research and treatment. Meningiomas represent the second most common brain tumor in adults, and while improved diagnostic modalities are available, these tumors remain underreported. Radiosurgery is an effective adjuvant therapy against meningioma; however, no effective chemotherapy exists. In addition to histologic grading and estimates of the extent of resection, biomarkers, such as progesterone receptor, cyclooxygenase 2, S100A5 and ornithine decarboxylase may be useful in predicting tumor recurrence and/or progression potential in patients with meningioma. On the genetic level, cytogenetic losses on chromosomes 1, 7, 10 and 14 and telomerase activation are observed in clinically aggressive meningioma, whereas monosomy 22 is a common early molecular event in tumor formation. Several candidate growth regulatory genes have been identified, including the Neurofibromatosis 2 (NF2), Tumor Suppressor in Lung Cancer-1 (TSLC1), Protein 4.1B, p53/MDM2 and S6-Kinase genes. The roles of these genes in meningioma formation and progression, as well as the clinical implications of these genetic changes, are discussed. The recent insights into the molecular biology and genetics of meningioma provide new avenues for basic science research aimed at understanding the mechanisms underlying meningioma formation and malignant progression. These advances may be useful in improving our ability to predict clinical outcome and developing targeted therapies to improve outcomes in patients with clinically aggressive meningiomas.Current Opinion in Neurology 01/2005; 17(6):687-92. · 5.73 Impact Factor
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ABSTRACT: The DAL-1/41B gene (differentially expressed in adenocarcinoma of the lung), located in the chromosome 18p11.3 region, belongs to the protein family 4.1 (membrane-associated proteins), which includes the product of the NF2 gene (merlin), and the proteins, ezrin, radixin, and moesin. DAL-1/4.1B is normally expressed at high levels in the brain, with lower levels in the kidney, intestine, and testis. DAL-1/4.1B is known to suppress growth in meningiomas and can be lost in about 60% of sporadic meningiomas as an early event in tumorigenesis; it is a critical growth regulator in the pathogenesis of neoplastic transformation. The similarity between the DAL-1/4.1B protein and merlin, with their high levels of expression in the brain and their recurrent loss in meningiomas, and the lack of previous DAL-1/4.1B mutational analysis reports initiated this mutational study of DAL-1/4.1B in a series of 83 meningiomas. We found the following sequence variations; Ala555Thr (G1663A in exon 13) and Thr950Lys (C2849A in exon 19) in two cases each, and one case with a 5pb deletion (del taaaa) in intron 18. A polymorphism in exon 14 (C2112T/Thr704Thr, also known as C2166T) was also identified; the tumoral allelic constitutions were heterozygous C/T in 15, homo- or hemizygous C in 67 and hemizygous T in one tumour. The low mutational frequency in our study discounts sequence variations in DAL-1/4.1B as the main mechanism underlying participation of this gene in the neoplastic transformation of meningiomas, and suggests that other inactivating mechanisms, such as epigenetic changes, may participate in DAL1/4.1B silencing.International Journal of Molecular Medicine 11/2005; 16(4):771-4. · 1.88 Impact Factor
Letter to the editor
Multiplex ligation-dependent probe amplification (MLPA)
screening in meningioma
Meningiomas, which develop from arachnoid meningeal
cells, represent up to 20e25% of all intracranial tumors,
with an incidence rate of approximately 6 per 100,000 in-
dividuals . Cytogenetically, the main alteration is partial
or total loss of a chromosome 22, found in 70% of the cases
. In addition, the loss of 1p and 14q are cytogenetic
changes associated with the progression of this type of
Molecular studies have shown that approximately half of
these tumors show allelic losses that affect the q12 band in
chromosome 22. The NF2 gene, located in 22q12.2 has
been implicated as a candidate gene in the genesis of me-
ningiomas, acting as a tumor suppressor gene. Alterations
in the NF2 gene appear in familial form, causing neurofi-
bromatosis type 2, which is characterized by schwannomas,
primarily of the eighth cranial nerve, and meningiomas.
Mutations of this gene (most of which are small insertions,
deletions, or missense mutations resulting in a truncated
and nonfunctional protein) have been detected in 60% of
The close association in meningiomas with NF2 gene
mutations and the allelic losses in chromosome 22 suggest
that NF2 is a tumor suppressor gene located in that chromo-
some and involved in this meningioma development .
This gene may also be inactivated by CpG island aberrant
promoter methylation . Loss of NF2 gene function oc-
curs in only one third of meningiomas with loss of hetero-
zygosity of chromosome 22, which suggests the existence
of a second tumor suppressor gene in this region. Some
of the potential candidates are SMARCB1 (alias INI1),
AP1B1 (alias BAM22), LARGE, and MN1 genes [5,6].
Among genes located outside chromosome 22 that may
be implicated in meningioma tumorigenesis are EPB41L3
(alias DAL-1/4.1B) on chromosome 18, IGSF4 (previously
TSLC1) on chromosome 11, TP53 on chromosome 17,
CDKN2A (alias p14ARF) on chromosome 9, TERT (alias
hTERT) on chromosome 5, TGFB1 (alias TGF-b) on chro-
mosome 19, and others [5e7].
Conventional methods based on exon scanning do not
detect large deletions or mutations in noncoding regions,
due to wild-type allele coamplification, as has been de-
scribed for the NF2 gene [8e10]. The multiplex ligation-
dependent probe amplification (MLPA) technique [11,12]
has proven to be a high-resolution gene-dosage assay for
the screening of large deletions and duplications. Previous
studies have confirmed the efficiency of MLPA as a rapid,
reliable, economical, and high-throughput method [13,14].
In this type of assay, the material that is amplified is not the
DNA of the sample, but the probes, after a hybridizatione
ligation step in a multiplex polymerase chain reaction
(PCR) reaction in which specific sequences are simulta-
neously quantified; in consequence, the amplification
depends on the presence of the target sequences in the
MLPA has been used to study abnormalities of 22q 
and NF2 patients in whom intragenic NF2 mutations had
not previously been found by exon scanning . We ap-
plied the MLPA technique to detect deletions and duplica-
tions in a large series of familial and sporadic newly
diagnosed meningiomas. DNA was isolated from the pe-
ripheral blood of five healthy donors as controls and from
54 frozen meningioma tumor samples, using the Wizard ge-
nomic DNA purification kit (Promega, Madison, WI). Nine
of the tumor samples were from patients with a known fam-
ily history of NF2.
Pathological diagnosis was established according to the
WHO classification . The study included 47 WHO grade
I and 7 WHO grade II meningiomas, corresponding to 39
transitional, 9 meningotheliomatous, and 6 other histologic
subtypes of meningioma.
For NF2 analysis, we used a commercial MLPA kit
(SALSA P044 NF2; MRC-Holland, Amsterdam, Nether-
lands). The kit includes single probes for the 17 coding
exons and two probes for the promoter region of the NF2
gene. As a control, it includes 12 probes for different chro-
mosomal locations. Information regarding the probe
sequences and ligation sites can be found at http://
www.mlpa.com. The MLPA protocol was performed as
previously described , using 50 ng of DNA from con-
trol and tumor samples. DNA denaturation and hybridiza-
tion of the SALSA probes was followed by a ligation
reaction and PCR. One microliter of the amplified sample
product was analyzed with the ABI 3100 Avant sequencer
(Applied Biosystems, Foster City, CA), using as an internal
size standard the ROX-500 GeneScan (ABI 401734). Suc-
cessful ligation reaction and identification of samples with
insufficient amounts of DNA were verified using MLPA
internal ligation-independent probes.
Data analysis was performed with MRC-Coffalyser ver-
sion 2 software (MRC-Holland, Amsterdam, Netherlands).
0165-4608/07/$ e see front matter ? 2007 Elsevier Inc. All rights reserved.
Cancer Genetics and Cytogenetics 173 (2007) 170e172
Intranormalization for sample data was first performed on
control probes, and then each tumor sample was normalized
on control probes using data from five control samples. Sin-
gle regression for control and tumor data slope correction
was performed. Normal ratio limits were set at 0.75 and
1.3. Statistical analysis was accomplished using the same
Deletions of the whole NF2 gene were detected in 26 out
of the 54 samples (48.1%) and partial losses in 14 of 54
(25.9%), for a combined 74% showing loss for this gene.
As expected, total or partial losses were higher in sporadic
meningiomas than in the NF2 cases. No specific association
between these results and WHO grading was found. The
partial deletions were diverse, with loss from the promoter
region to exon 7 being the most frequent, found in 5
(11.1%) of the 54 cases, followed by loss from the pro-
moter region to exon 1, in 3 cases (6.7%), one of which
had in addition a deletion in exon 17.
It is in partial losses that the MLPA shows its potential,
given that conventional methods of screening (e.g., single-
strand conformation polymorphism, automated sequencing,
or loss of heterozygosity) cannot detect the defects, because
they are masked by the wild-type allele. This is also appli-
cable to duplications. Of special interest is one sample that
showed deletion for all the probes on the NF2 gene and its
promoter region. The calculated ratio for each of the probes
was between 0.48 and 0.72, except for the probes of exons
9, 10, and 11, for which the calculated ratio was between
0.15 and 0.23. These findings demonstrate the capacity of
the MLPA technique to detect various deletions in each
one of the alleles of a sample; in this particular case, the to-
tal deletion of the NF2 gene in one of the alleles and the
loss of exons 9 to 11 in the other (Fig. 1).
We also found a case with probe ratios compatible with
NF2 gene duplication from the promoter region to exon 12.
A summary of the partial losses and gains for the NF2 gene
is given in Table 1. The combined P-value for all the probes
was 0.05, with 0.08 being the higher individual P-value for
the probes on exons 14 and 17, and also for the control
probe 15q24.3. The 14q13 probe showed deletion in 39
(52.7%) of the 55 tumor samples (P Z 0.07). These results,
together with the previously described frequent loss of this
region in meningioma , made us exclude the probe as
a control; we then analyzed it in the same way as the probes
corresponding to NF2 exons.
We also found 12 duplications for the control probes in
11 tumor samples: 6 samples with duplication of the 4q35
probe, 1 sample with duplication on 5q31.1, 3 samples
(6.7%) with duplication on 9q21.3, and 1 sample with
two duplications for the 11p12 probe. These data do not
seem to indicate a specific association between these dupli-
cations and tumorigenesis or tumor progression in meningi-
oma. Nevertheless, we cannot discard the possibility that
further studies may find genes with oncogenic capacity in
some of these regions.
Fig. 1. MLPA electrophoresis peak-area patterns. (A) Tumor sample showing deletion of the whole NF2 gene and the 14q13 probe. (B) The sizes of the
peaks in exons 9, 10, and 11 show a partial deletion in one allele of a tumor sample that also shows a total deletion of NF2 gene in the other allele. (C)
Normal peak-area pattern in a control sample. Arrows indicate probes with proportional loss. X axis: NF2 gene and control probes ranging from 139 to
400 base pairs with a progressive increase of 9 base pairs. Y axis: Automatic sequencer fluorescent intensity units.
NF2 alterations detected with MLPA in 54 familial and sporadic
del Pr/ex 11
del ex 5/ex 6
del Pr/ex 7
del Pr/ex 1
del Pr/ex 1 and del ex 17
dup ex 1/ex 12
del ex 9/ex 11
del Pr/ex 8
del Pr/ex 13
Abbreviations: del, deletion; dup, duplication; ex, exon; MLPA, multi-
plex ligation-dependent probe amplification; Pr, promoter region.
Letter to the editor / Cancer Genetics and Cytogenetics 173 (2007) 170e172
In summary, our study found a high frequency (74%) of
large alterations (deletions and duplications) in the NF2
gene in sporadic and familial meningiomas using MLPA,
a technique that has proven to be an accurate and simple
method for detecting alterations that escape other screening
methods. In the specific case of tumor samples, in which
the cytogenetic and molecular alterations are usually abun-
dant, we have confirmed that the MLPA technique can dis-
criminate between various possible losses that can be
present simultaneously in both alleles of a gene.
Acknowledgments and dedication
Bello, PhD (June 18, 1957eMarch 4, 2006). The study was
supported bythe followinggrant sponsors:Fondo deInvesti-
gaciones Sanitarias, Ministerio de Sanidad (03/0235 and 05/
0829), and Fundacio ´n MAPFRE Medicina.
Vı ´ctor Martı ´nez-Glez
Carmen Franco-Herna ´ndez
Jesu ´s Lomas
Carolina Pen ˜a-Granero
Jose ´ M. de Campos
Juan A. Rey
Laboratorio de Oncogene ´tica Molecular
Unidad de Investigacio ´n
Hospital Universitario La Paz
Paseo Castellana 261
28046 Madrid, Spain
E-mail address: firstname.lastname@example.org (J.A. Rey)
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