Exon-by-exon screening for RB germline mutations using Heteroduplex-SSCP analysis

To disclose the nature of RB1 germline mutations in Chinese and to develop a practical and effective way for mutational screening.

Leukocyte DNA was prepared from 8 Chinese patients with hereditary retinoblastoma. PCR combined with nonisotopic heteroduplex-SSCP analysis was used to screen leukocyte DNA for RB1 germline mutations, exon-by-exon, without the use of restriction endonuclease digestion. The mutations were finally identified by sequencing. In order to testify the effectiveness of this method, the same method was used to detect other 17 samples which have been previously analyzed by other methods.

Heterozygous germline mutations were detected in the leukocyte DNA of 6 out of 8 Chinese patients: G del/codon 46, T del/codon 131, CAGAA del/codon 257-258, GCAgta-->GCAgca/donor of exon 16, C-->T/codon 661, and C-->T/codon 787. Heteroduplex-SSCP analysis may detect RB1 germline mutations in 68% (17/25) unselected patients, which is more effective than SSCP(56%) or heteroduplex analysis(64%) alone. The corrected RB1 mutation detection rate may be 80% if the samples were previously analyzed by Southern blotting, which is much higher than those reported on literature.

Mutations involving a few base pairs in RB1 gene are common in Chinese. Heteroduplex-SSCP analysis is more useful and effective than SSCP or heteroduplex analysis alone for the rapid screening for unknown mutations.