Identification of three novel RB1 mutations in Brazilian patients with retinoblastoma by "exon by exon" PCR mediated SSCP analysis

Abstract

Aims: To carry out a retrospective study, screening for mutations of the entire coding region of RB1 and adjacent intronic regions in patients with retinoblastoma.

Methods: Mutation screening in DNA extracts of formalin fixed, paraffin wax embedded tissues of 28 patients using combined "exon by exon" polymerase chain reaction mediated single strand conformational polymorphism analysis, followed by DNA sequencing.

Results: Eleven mutations were found in 10 patients. Ten mutations consisted of single base substitutions; 10 were localised in exonic regions (eight nonsense, one missense, and one frameshift) and another one in the intron-exon splicing region. Three novel mutations were identified: a 2 bp insertion in exon 2 (g.5506-5507insAG, R73fsX77), a G to A transition affecting the last invariant nucleotide of intron 13 (g.76429G>A), and a T to C transition in exon 20 (g.156795T>C, L688P). In addition, eight C to T transitions, resulting in stop codons, were found in five different CGA codons (g.64348C>T, g.76430C>T, g.78238C>T, g.78250C>T, and g.150037C>T). Although specific mutation hotspots have not been identified in the literature, eight of the 11 mutations occurred in CGA codons and seven fell within the E1A binding domains (codons 393-572 and 646-772), whereas five were of both types-in CGA codons within E1A binding domains.

Conclusions: CGA codons and E1A binding domains are apparently more frequent mutational targets and should be initially screened in patients with retinoblastoma. Paraffin wax embedded samples proved to be valuable sources of DNA for retrospective studies, providing useful information for genetic counselling.

Figure 1

(A) Single strand conformational polymorphism (SSCP) analyses of exon 14 and adjacent intronic regions. Amplified products of tumour DNA of patient HC1 show a different migration pattern when compared with the normal control (C) and other patients. (B) Comparative DNA sequencing between a control and patient HC1. C/F, normal control with the forward primer; C/R, normal control with the reverse primer; HC1/F, DNA of patient HC1 with the forward primer; HC1/R, DNA of patient HC1 with the reverse primer. In the box, the letter “N” indicates simultaneous presence of the normal base (G and C in forward and reverse strands respectively) and A and T, respectively, in patient HC1. This mutation was seen both in tumour and blood DNA samples, confirming its constitutional origin. Note that patient HC27 shows a normal SSCP pattern, although DNA sequencing identified a transition one base apart (table 2).

Figure 2

(A) Single strand conformational polymorphism (SSCP) analyses of exon 17 and adjacent introns. Tumour DNA was obtained from both eyes of patient HC9 and analysed separately (HC9R, DNA from right eye; HC9L, DNA from left eye). HC9R shows a different migration pattern when compared with the normal control (C) and other patients, whereas HC9L shows a mixed pattern between normal and HC9R. (B) Comparative DNA sequencing between control, HC9R, and HC9L. C/F, normal control with the forward primer; HC9R/F, tumour DNA from the right eye with the forward primer; HC9L/F, tumour DNA from the left eye with the forward primer. Sequencing confirmed the SSCP patterns, showing only T in HC9R and the presence of C (normal base) and T (mutated base) in HC9L (indicated by “N”). These data were confirmed using reverse sequencing (not shown).