Rapid Analysis Of RB1 Gene Mutation By RT-PCR In A Southern Chinese Retinoblastoma Pedigree

BackgroundRetinoblastoma (RB) is the most common malignant intraocular tumor in children, comprising 4% of malignant tumors in children. Most cases are diagnosed before age 5 years with the presenting signs of leukocoria, strabismus, low-vision orbital cellulitis, unilateral mydriasis, heterochromia, and family history.Hereditary RB is an autosomal dominant trait with high penetrance (90%), caused by mutations in RBI gene which has been identified as the only pathological gene. RBI (OMIM 180200) is located at chromosome 13q14.1-q14.2, which is the first tumor suppressor gene to be cloned. Its ubiquitously normal expression of pRB is crucial for RB development. The RBI gene is relative longer gene organized into 27 exons spanning 198 kb of genomic DNA.There are two types of RB:bilateral RB, characterized with early onset and most of which are hereditary; unilateral RB, characterized with late onset and 15% of which are hereditary.92.7% of the bilateral and 14.6% of the unilateral RB patients are carriers of a predisposing germline mutation with high penetrance. Therefore, variomics study on RBI in either familial or nonfamilial patients is critical for accurate risk prediction, early diagnosis, early management, prognosis, genetic counseling, prenatal diagnosis, preimplantation genetic diagnosis, and gene therapy.ObjectivesTo investigate the genetic factors related to RB through rapid RT-PCR/sequencing and PCR/analysis of RBI gene in a Chinese RB pedigree, and to explore the genotype-phenotype relationship.PatientsA southern Chinese RB family, including 2 affected, living in Hangzhou, Zhejiang province, China. Ten biologically unrelated healthy controls with a similar ethnic background were matched.MethodsTotal RNA was extracted from 0.5 ml peripheral blood with a RiboPure blood kit for reverse transcriptase-polymerase reaction (RT-PCR) to analyze RBI transcripts. Genomic DNA was extracted from 5 ml whole blood following a standard protocol for PCR and direct sequencing to test RBI exons. DNA sequence analysis and variation identification were done by DNAssist and Chromas 2 software. Then compare with the authority databases such as Ensembl, NCBI, etc. Allele-specific PCR was used to verify the mutation.Results(1) The bilaterally affected son and the unilaterally affected father were both heterozygous for the nonsense mutation c.1363C>T (p.R455X) in exon 14 of RBI by RT-PCR/DNA sequencing. (2) PCR sequencing of exon 14 showed the heterozygous mutation g.76460C>T (c.1363C>T, p.R455X) in two patients, but unaffected family members and controls had no mutant signal.(3) The results of AS-PCR confirmed this variation. Only the two patients' genomic DNA amplified the mutant amplicons.Conclusions(1) RBI c.1363C>T (p.R455X) mutation was the pathological cause to this RB family.(2) The proband inherited RBI mutation from his affected father.(3) RT-PCR/DNA sequencing is the most efficient method to detect RBI gene mutations to date.