TY - JOUR
T1 - Identification of three novel RB1 mutations in Brazilian patients with retinoblastoma by "exon by exon" PCR mediated SSCP analysis
AU - Braggio, E.
AU - Bonvicino, C. R.
AU - Vargas, F. R.
AU - Ferman, S.
AU - Eisenberg, A. L.A.
AU - Seuánez, H. N.
PY - 2004/6
Y1 - 2004/6
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=2642574983&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=2642574983&partnerID=8YFLogxK
U2 - 10.1136/jcp.2003.014423
DO - 10.1136/jcp.2003.014423
M3 - Article
C2 - 15166261
AN - SCOPUS:2642574983
SN - 0021-9746
VL - 57
SP - 585
EP - 590
JO - Journal of clinical pathology
JF - Journal of clinical pathology
IS - 6
ER -