Cancer Genetics and Cytogenetics 129 (2001) 88–91

0165-4608/01/$ – see front matter © 2001 Elsevier Science Inc. All rights reserved.PII: S0165-4608(01)00430-7

Short communication

Analysis of p73 gene in meningiomas with deletion at 1p

Jesus Lomas

a

, M. Josefa Bello

a

, Dolores Arjona

a

, Pilar Gonzalez-Gomez

a

, M. Eva Alonso

a

,Jose M. de Campos

b

, Jesus Vaquero

c

, Pedro Ruiz-Barnes

d

, Jose L. Sarasa

e

,Cacilda Casartelli

f

, Juan A. Rey

a,*

a

Laboratorio de Oncogenética Molecular, Unidad de Investigación, Departamento de Cirugía Experimental, Hospital Universitario La Paz,Paseo Castellana 261, 28046 Madrid, Spain

b

Departamento de Neurocirugía, Hospital del Rio Hortega, Valladolid, Spain

c

Departmento de Neurocirugía, Clinica Puerta de Hierra, Madrid, Spain

d

Departamento de Neurocirugía, Fundaciòn Jimènez Diaz, Madrid, Spain

e

Departamento de Anatomía Patológica, Fundaciòn Jimènez Diaz, Madrid, Spain

f

Departmento de Genetica, Facultade de Medicina de Ribeirao Preto, USP, Ribeirao Preto, SP, Brazil

Received 11 January 2001; accepted 25 January 2001

Abstract

The p73 gene has been mapped to 1p36.33, a chromosome region that is frequently deleted in awide variety of neoplasms including meningiomas. The protein encoded by p73 shows structuraland functional similarities to p53 and may thus represent a candidate tumor suppressor gene. Todetermine whether p73 is involved in the development of meningiomas, we examined 30 meningi-oma samples with proven 1p deletion for mutations of p73. Sequence analysis of the entire codingregion of the p73 gene revealed previously reported polymorphisms in eight cases. A tumor-spe-cific missense mutation as a result of an A-to-G transition with an Asn204Ser change was found inone meningioma that nevertheless retained the normal allele. These results suggest that if p73plays a role in meningioma carcinogenesis, it must be in a manner different from the Knudson

two-hit model. © 2001 Elsevier Science Inc. All rights reserved.

1. Introduction

Meningiomas are tumors arising from cells of themeningeal coverings of the brain and spinal cord; they ac-count for 13–19% of all intracranial tumors and 25% of allintraspinal neoplasms [1]. Although the majority of menin-giomas are considered benign tumors of WHO grade I,about 10% of cases are classified as atypical (WHO gradeII) or anaplastic (WHO grade III), and are associated withhigh risk for local recurrence [1].

The cytogenetic and molecular findings in meningiomahave shown that a genetic model of malignant progressionmay account for the formation of some atypical and ana-plastic forms, as a result of a mutation accumulation process[2–9]. An early step in this model would be the inactivationof a chromosome 22-located gene, such as NF2 or other tu-mor suppressor genes [10]. Secondary abnormalities in-clude the alteration of as yet unidentified tumor suppressor

genes located primarily at 1p [11–15], 14q [9,16,17] andchromosome 10 [18], parallel to the genesis of the more ag-gressive meningioma forms. Initial loss of heterozygosity(LOH) studies suggested that 1p32 and 1p36 might representcritical domains [12,19,20], and recently high-resolutionLOH studies have demonstrated that up to four 1p genomicregions may carry tumor suppressor genes, non-randomlyinvolved in meningioma tumor progression [21]. There isthus a consensus that 1p36 may represent a crucial region incarcinogenesis, and that up to three tumor suppressor genesmay be located here [22].

A novel gene termed p73 was recently identified at thischromosomal region; p73 encodes a protein with significanthomology to the p53 tumor suppressor gene throughout itsDNA-binding, transactivation, and oligomerization domains[23]. Mutational analyses of p73 have been performed in awide variety of tumor types but, to our knowledge, only fivemutations have been identified [24–27]. As no data areavailable in meningioma, we performed mutation analysisof the entire coding region and intronic splice donor and ac-ceptor regions of the p73 gene. We used PCR-SSCP and di-

* Corresponding author. Fax.: +34-91-727-7050.

E-mail address:

jarey@hulp.insalud.es (J.A. Rey).

J. Lomas et al. / Cancer Genetics and Cytogenetics 129 (2001) 88–91

89

rect sequencing on meningiomas with previously proven 1pdeletions, including samples with and without 1p36 losses.We found one missense mutation, various silent mutationsand one intronic nucleotide change, implying that p73 is arare target for genetic alterations in meningiomas.

2. Materials and methods

2.1. DNA samples

Normal and tumor DNA from 30 patients with meningi-oma were selected on the basis of previously detected dele-tions at 1p in the tumors, as reported [21]. High molecularweight DNA had been extracted from normal and tumor tis-sues by standard methods [28], and pathological tissueswere classified by histologic examination and graded ac-cording to the WHO guidelines [1] as follows: 23 grade IIor grade III tumors and seven grade I samples.

2.2. PCR-SSCP analysis and direct sequencing of thep73 gene

Genomic PCR amplification was performed of all codingexons of the p73 gene and their splice site junction se-quences using the primers described by Yoshikawa et al.[29] (purchased from GENSET, SA, France). PCR condi-tions were 35 cycles of 94

�

C for 30 s, 55–68

�

C for 30 s, and72

�

C for 90 s, with a final extension of 7 min at 72

�

C. ThePCR products were loaded onto 6–12% nondenaturing poly-acrylamide gels (with or without 10% glycerol), electrophore-sed and silver stained as previously described [30]. Samplesthat displayed an altered PCR-SSCP pattern were reamplifiedby PCR with the same set of primers, and the PCR productswere sequenced using the ABI PRISM Big Dye TerminatorCycle Sequencing Kit (Perkin Elmer, Alameda, CA, USA).Each amplicon was sequenced bidirectionally.

3. Results and discussion

To determine whether p73 is involved in meningiomaprogression, we analyzed 30 samples with a previouslyproven deletion at 1p [21] for the presence of gene alter-ations at the p73 locus. Using PCR-SSCP methods, we de-tected aberrantly migrated bands in eight specimens (Fig. 1)and nucleotide sequence analysis revealed seven types ofchanges, summarized in Table 1. Consistent with previousreports dealing with mutation analysis of p73 in differentmalignancies [31–36], we detected several genomic poly-morphisms at intron 3 and exons 5 and 9, including a delGat E3/

�

12 position of intron 3 in one atypical meningioma.In five samples, a double nucleotide change was detected atexon 5, consisting of a G-to-A transition at nucleotide 438and C-to-T change at nucleotide 612, with no change in Pro146 and Asn 204, respectively. Two other meningiomasalso displayed a double nucleotide change affecting exon 9.They were defined as a C-to-T transition at 1008 positionand T-to-C variation at 1047, with no changes in Ala 336

and His 349. The most interesting finding was detected in acase (T-54) that presented a polymorphic change and a tu-mor-specific mutation, both involving exon 5. The poly-morphic variation was a T-to-C transition at nucleotide 519,with no change in Thr 173. The missense mutation wasidentified as a heterozygous A-to-G transition at nucleotide611, resulting in an Asn-to-Ser substitution at codon 204.This meningioma thus retained the wild-type allele, and themolecular implications of this amino acid change remainunknown. The change Asn204Ser we have identified is lo-cated in the DNA-binding domain of p73 and may representa crucial point of p73/DNA contacts, or have an importantrole in stabilizing the p73/DNA complex. Similar missensemutations of p53 have been found to exert a dominant nega-tive effect due to increased stability of the mutant proteincompared to that of the wild-type protein [37], but addi-tional studies should be carried out to verify this hypothesisin respect to p73.

As shown in previous reports on mutation detection ofp73, a low frequency of tumor specific changes has been de-scribed. In a series of 140 neuroblastomas, Ichimiya et al.[25] found one somatic and one germ-line mutation whichconsisted of Pro405Arg and Pro425Leu changes. Van Gele

Fig. 1. Exon 5 missense at position 611 of p73 gene (A-to-G: Asn204Serchange). SSCP analysis corresponding to normal (N) and tumoral (T)DNAs of case T-54 show the aberrantly shifted band. Direct sequence anal-ysis of the corresponding DNAs confirmed the nucleotide change in thetumor.

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et al. [24] identified one Merkel cell carcinoma displaying aheterozygous C-to-T transition at nucleotide 439, resultingin Ser110Leu substitution, whereas Peng et al. [26] de-scribed a five-nucleotide deletion in the DNA-binding do-main of the gene in one hepatocellular carcinoma. Finally,we previously described a primary brain lymphoma charac-terized by a Glu291Lys variation as a result of G-to-A tran-sition at nucleotide 871 [27]. In agreement with this limitedp73 mutation level in human cancer, we detected no muta-tions in 1p36 LOH positive meningiomas, as the sole casewith an amino acid change retained the normal p73 allele.These findings and the fact that no homozygous deletion ofthis gene has been found in any cancers suggest that p73seems not to follow the classical “two-hit” model for tumorsuppressor genes. Mechanisms other than mutation in p73may lead to tissue-specific up- or down-regulation of thegene, but the data available on p73 gene expression show abiallelic pattern in lung tumors [38], Merkel cell carcinoma[24], and breast tumors [36], while it was originally reportedto be expressed monoallelically in neuroblastoma [23].These controversial findings do not fit with the mechanismof silencing of one allele accompanied by loss of another asa general explanation for the complete inactivation of p73.

As an alternative, the alkaline phosphatase gene has beenrecently proposed as a candidate 1p36 located tumor sup-pressor gene in meningiomas, on the basis of an apparentlyhomozygous loss of expression in some WHO grade II andgrade III tumors [39]. The gene(s) at the various 1p criticalregions thus remain to be identified, as other candidategenes at this location have shown non preferential involve-ment in meningiomas development [40].

Acknowledgments

This work was supported by grant 00/0331 from Fondode Investigacion Sanitaria, Ministerio de Sanidad. Partialsupport for this research was provided by a grant from Pro-grama de Cooperación Científica con Iberoamérica, Minis-terio de Educación, Cultura y Deporte. M.E.A. is supportedby fellowship from Comunidad de Madrid.

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Table 1Mutation and polymorphisms identified in the p73 gene in meningiomas

Exon/intron Nucleotide change AA change Samples

3/

�

12 delG — T-325 438G

�

A/612C

�

T Pro146Pro/Asn204Asn T-6, T-14, T-16 T-79, T-83

5 519T

�

C/611A

�

G Thr173Thr/Asn204Ser T-549 1008C

�

T/1047T

�

C Ala336Ala/His349His T-11, T-30

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