Cancer Genetics and Cytogenetics 138 (2002) 27–31

0165-4608/02/$ – see front matter © 2002 Elsevier Science Inc. All rights reserved.

PII: S0165-4608(02)00577-0

Identification of novel mutations in the

RB1

gene inMexican patients with retinoblastoma

Maricela Rodríguez

a,b,

*, Mauricio Salcedo

c

, Marina González

b

, Ramón Coral-Vazquez

b

,Fabio Salamanca

b

, Diego Arenas

b

a

Laboratorio de Metabolismo de Lípidos, Unidad de Investigación en Nutrición, Hospital de Pediatría,

b

Laboratorio de Genética Molecular, Unidad de Investigación Médica en Genética Humana, Hospital de Pediatría, Centro Médico Nacional Siglo XXI,

México D.F., Mexico

c

Laboratorio de Oncología Genómica, Unidad de Investigación en Oncología, Hospital de Oncología,

Received 24 July 2001; received in revised form 20 March 2002; accepted 20 March 2002

Abstract

Retinoblastoma (RB) is a childhood tumor of the eye with an average incidence of one case in every15,000–20,000 live births and occurs in sporadic or hereditary form. This cancer results from loss or inac-

tivation of the

RB1

gene located at 13q14.1. This gene encodes for a 110 Kd nuclear phosphoprotein(

pRB

) that plays a major role in cell proliferation control. Different types of mutations in the

RB1

genehave been reported, but point mutations are the most common. There are no molecular studies on

RB1

gene mutation in Mexican patients. In this study, 19 patients with bilateral or unilateral RB were analyzed.Genetic and cytogenetic studies were carried out. Detection of

RB1

gene mutations was done using single-strand conformational polymorphism (SSCP). Five conformational polymorphisms were identified in dif-ferent exons. In all cases, SSCP sequence showed new non-described mutations that produced a frame-shift on the open reading frame. The identification of mutations in the

RB1

gene contributes to basicknowledge of this neoplasia and permits the possibility to offer adequate genetic counseling to relatives at

risk. © 2002 Elsevier Science Inc. All rights reserved.

1. Introduction

Retinoblastoma (RB) is a childhood tumor of the eye withan average incidence of one case in every 15,000–20,000live births and occurs in sporadic or hereditary form. Mostclinical retinoblastoma phenotypes can be explained by the

mutational inactivation of two

RB1

alleles [1]. Among RBtumors, 70% are of the nonhereditary type and the disease isunilateral. Approximately 30% of patients appear to havehereditary RB that may be manifested by unilateral or bilat-eral disease. Patients with hereditary RB who inherit a mu-tant allele of the

RB1

gene are usually affected by bilateralmultifocal tumors; the onset of such tumors is earlier thanthat of nonhereditary tumors [2]. This cancer results fromsuccessive loss or inactivation of the

RB1

gene located in13q14.1 [3,4]. This gene encodes a nuclear phosphoprotein(

pRB

) of 110 Kd, which plays a major role in cell prolifera-

tion control through cell cycle-regulated phosphorylation/dephosphorylation of the protein [5].

Different types of mutations in the

RB1

gene have beenreported; these mutations constitute nearly the entire spec-trum of genetic alterations and range from large deletions tosingle-base changes; point mutations are the most common[6–9]. Even though there are many cases of RB in Mexico

(the highest incidence was reported in 1991, 6.23

�

10

6

, ap-proximately one case/month) [10], there are no molecularstudies on

RB1

gene mutations. Although the diagnosis ofmost hereditary RB tumors can be made on clinicalgrounds, the development of sensitive and reliable genetictests for germline

RB1

gene mutation detection could helpin the identification of carriers, obviate the need for manyexaminations, and potentially decrease cost of treatment [11].

In the present study, 19 patients with bilateral or unilat-eral RB were analyzed. Genetic, cytogenetic, and molecularstudies were carried out. Detection of

RB1

gene mutationswas done in each patient, using single-strand conforma-tional polymorphism (SSCP) and sequence. Five new previ-ously undescribed mutations that produced a frameshift onthe open reading frame were observed. This work contrib-

* Corresponding author. Laboratorio de Genética Molecular, Unidad deInvestigación Médica en Genética Humana, Hospital de Pediatría, CentroMédico Nacional Siglo XXI, Av., Cuauhtémoc 330, Col. Doctores, 06725México D.F., Mexico. Tel.:

�

52-55-56276941; fax:

�

52-55-57610952.

E-mail address

: giraviol@yahoo.es (M. Rodríguez).

28

M. Rodríguez et al. / Cancer Genetics and Cytogenetics 138 (2002) 27–31

utes to the analysis of

RB1

gene mutations in Mexican pa-tients with RB.

2. Material and methods

2.1. Patients

Samples of RB tissue and peripheral blood cells were ob-tained from 19 Mexican patients with unilateral or bilateraltumors; the patients’ parents and grandparents were Mexi-can (mestizos). Diagnosis of RB was established by histo-logic criteria and standard ophthalmologic procedures. Thepatients were chosen from the Pediatric Hospital’s Ophthal-mology and Pathology Services at the XXI Century Na-tional Medical Center of the Mexican Institute of Social Se-curity (IMSS) in Mexico City.

All patients’ parents gave informed consent. The Ethicaland Academic Committees of the Pediatric Hospital ap-proved the studies. The genetic and molecular studies werecarried out at the Unidad de Investigacion Médica enGenética Humana.

2.2. Cytogenetic analysis

Chromosomes from leukocyte cultures using standardtechniques [12] and high-resolution chromosomes with pro-cedures described by Viegas and Dutrillaux [13] were ob-tained. G banding of chromosomes was obtained accordingby M. Seabright [14]. One hundred metaphases from eachpatient were analyzed.

2.3. Isolation of genomic of leukocyte and tumor DNA

Genomic DNA from leukocytes of peripheral blood andtumors was isolated. Leukocyte DNA purification was car-ried out according to the method described by Kempter[15], except that DNA was additionally purified by phenolextractions. Genomic DNA of the tumors was obtained fromformalin-fixed, paraffin-embedded tumors as described byWright and Manos [16]. The quality and quantity of theDNA preparation was ascertained by spectrophotometryand agarose gel electrophoresis criteria.

2.4. Detection of mutations in

RB1

Leukocyte and tumor DNA from each patient was ampli-fied by polymerase chain reaction (PCR) using the followingthree sets of duplex reactions for exons: 3/20, 8/18, and 19/23.The sequences of the primers were described by Lohmannet al. [6]. PCR reactions were performed in 20

�

L of total vol-ume covered with 40

�

L mineral oil. The reaction mixturecontained 50–100 ng DNA, 2.5 units

Taq

polymerase, 0.2mM of each dNTP, and 20 pmol of each primer.

Initial denaturation was performed at 94

�

C for 5 minutes,followed by 35 cycles of denaturation at 94

�

C for 1 minute,annealing at 55

�

C for 30 seconds, and extension at 72

�

C for30 seconds, with final extension for 5 minutes at 72

�

C. PCRproducts were analyzed by electrophoresis on 2% agarosegel stained with ethidium bromide.

2.5. SSCP analysis

PCR products of the six exons of the

RB1

gene were ana-lyzed by SSCP following the PCR conditions previously de-scribed. For these amplifications, 2

�

Ci of [

�

32

P] dCTP and0.02 mM of dCTP were used. Aliquots of PCR reactionwere denaturalized and electrophorosed on a 6% or 8% de-naturing polyacrylamide gel with or without 10% glycerol.The gel was run at 20–30 W constant power in a 4

�

C coldroom for 5–7 hours. Autoradiography of dried gel was per-formed for 2–12 h.

2.6. DNA sequencing

Exons that showed mobility shifts were amplified as pre-viously described. PCR products were purified by agarosegel electrophoresis and recovered as described by Koenen[17]. The purified products were sequenced with asymmet-ric PCR using BigDye kit and analyzed in an automated se-quencer ABI PRISM 377 (Applied Biosystems) (InstrumentCore Facility, Medical Research Coordination, IMSS).

3. Results

In this work, genetic, molecular, and cytogenetic analysis of19 Mexican patients with unilateral or bilateral RB was per-formed. Eight patients were females and 11 were males. Twelvepatients had unilateral RB and 7 had bilateral RB; only onepatient (RB-5) had late RB. Three patients had a familial his-tory, of which two had bilateral RB and one had unilateral RB.

Cytogenetic studies were carried out in 10 patients withRB and in six first-grade relatives, analyzing 100 metaphasesfor each case. Only one patient (RB-3) had an abnormalchromosome number and a polymorphism in chromosome 1,which was not associated with this neoplasia. The patient’schromosomal complement was 47,XXY.

SSCP analysis showed conformational polymorphismsin distinct exons of three patients. The RB-2 patient hadconformational polymorphisms in exons 8 and 20 of his tu-mor DNA. Fig. 1A only shows the polymorphism in exon20. The RB-8 patient presented a polymorphism in exon 20of his leukocyte DNA. The analysis of tumor DNA of thispatient was not carried out because her eye was not enucle-ated. The RB-12 patient showed the polymorphic band inexon 18 of his tumor DNA. These polymorphisms were se-quenced and all presented different point mutations (Table1). RB-2 patient had an insertion of an adenine in the AACtriplet replacing Asn 304Lys in exon 8; this mutation causeda stop codon. Interesting, the same patient had another mu-tation G

→

T in the triplet GCC replacing Ser 704Ala inexon 20 (Fig. 1C). The RB-8 patient had a 156697delT inthe TAT triplet in exon 20; this mutation also caused a stopcodon (Table 1). The RB-12 patient had two different muta-tions in exon 18, T

→

G in the CTT codon, the mutation a si-lent polymorphism, whereas the other mutation was a dele-tion of a T in the TCA triplet replacing Ser 613His (Figs. 1Band 1D).

M. Rodríguez et al. / Cancer Genetics and Cytogenetics 138 (2002) 27–31

29

4. Discussion

Although RB is a frequent intraocular neoplasia in Mexico,there are few studies on Mexican patients; one was a geneticand cytogenetic study [18], and the remaining were epidemio-logic studies [10,19]. Thus, molecular studies in RB had notbeen performed in Mexico. Mutation analysis of RB is con-sidered important for genetic counseling purposes as well as

for understanding the molecular mechanisms leading to tu-mors with different degrees of penetrance or expressivity. Forthis reason, it is important to define with precision the dif-ferent mutations in the

RB1

gene of Mexican patients. The per-centages of Mexican patients with unilateral (63%) and bi-lateral (34%) RB tumors were similar to worldwide studies[2,18,20]. By cytogenetic analysis, it was not possible to iden-tify any deletions in the 13q14.1 region of these patients,

Fig. 1. Molecular analysis of two patients with RB. (A) Conformational polymorphism by SSCP of exon 20 of tumor DNA; (B) conformational polymorphism bySSCP of exon 18 of tumor DNA; (C) partial sequence of exon 20 indicating 156704G→T; and (D) partial sequence of exon 18 indicating 150001delT.

Table 1Novel mutations found in

RB1

in Mexican patients with retinoblastoma

Patient DNA source Exon Description Putative consequence Phenotype

RB-2 Tumor 8 59704insA Asn304Lys Bilat RBRB-2 Tumor 20 156704G

→

T Ser704Ala Bilat RBRB-8 PBC 20 156697delT Tyr702Tyr Unilat RBRB-12 Tumor 18 150001delT Ser613His Unilat RBRB-12 Tumor 18 150084G

→

T Leu640Leu Unilat RB

Abbreviations

: Bilat RB, bilaterol retinoblastoma; del, deletion; ins, insertion; PBC, peripheral blood cells; unilat RB, unilateral retinoblastoma.

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M. Rodríguez et al. / Cancer Genetics and Cytogenetics 138 (2002) 27–31

probably because sample size was small. Furthermore, theseresults support that the

RB1

gene is mainly affected bysmall mutations. Only one patient (RB-3) had an abnormalkaryotype associated with Klinefelter syndrome.

One patient (RB-16) with unilateral RB had a familialhistory, probably a case of irregular dominance [21,22], inwhich the mutation was not fully expressed but segregatedfrom germ-line in the family. Seven patients had bilateralRB, and five had no familial history. In these cases, proba-bly de-novo mutations appeared in germinal cells of one ofthe parents, more probably in the paternal allele or duringthe early development of the patient [23].

A wide spectrum of mutations ranging from point muta-tions to large deletions has been described in the

RB1

gene[6,7,9,24–26]. In the present study, six exons (3, 8, 18, 19,20, and 23) were chosen because these regions concentratethe majority of point mutations and the codifying region ofthe pocket domain of

pRB

protein [7,25], the main func-tional domain.

Point mutations in 3 of 19 (15.7%) patients were foundin exons 8, 18, and 20. The RB-2 patient had two differentpoint mutations, bilateral RB, and did not have familial his-tory for the disease. These two mutations were identifiedonly in the tumor DNA (somatic mutation). One possibilityis that this patient possessed a mosaicism for any of thesemutations. Mosaicism is not rare in familial RB: Sippel etal. [27] estimated that approximately 10% of familial RBcases possess mosaicism in at least one member of eachfamily. Another possibility is that this patient could have adouble heterozygous mutation.

The RB-12 patient with unilateral RB and without famil-iar history for this disease had a nonsense mutation in exon18; additionally, the mutation was identified only in the tu-mor DNA. The second mutation is probably located in otherexons or in the promoter region of the

RB1

gene, whichwere not analyzed. The last mutation identified was in theexon 20 of leukocyte DNA of patient RB-8B and generateda stop codon; this patient had unilateral RB lacking familialhistory; we did not study the tumor because the patient re-sponded favorably to chemotherapy and her eye was notenucleated. Because this patient had a constitutional muta-tion, her progeny may have a probability of 50% to inheritthis mutation.

In this work, we determined the type of mutation in someregions of the

RB1

gene and cytogenetic analysis. Point mu-tations were recognized in three of 19 patients. This is thefirst report in Mexico that characterizes mutations in the

RB1

gene in RB of Mexican patients.Additionally, this type of work is important because

identification of mutations contributes to basic knowledgeof this neoplasia and allows for the possibility of improvinggenetic counseling for relatives at risk. Furthermore, thisstudy contributes to understand the molecular mechanismsleading to tumors with different degrees of penetrance orexpressivity. Finally, the mutations herein described havenot been previously reported [28].

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