title: Screening for RB1 mutations in tumor tissue using denaturing high performance liquid chromatography, multiplex ligation-dependent probe amplification, and loss of heterozygosity analysis.
abstract: Retinoblastoma is a malignant retinal neoplasm arising in infancy as a result of inactivating mutations in both alleles of the retinoblastoma susceptibility gene, RB1. Identification of the causative RB1 mutations in a patient assists in the clinical management of the affected patient and risk assessment of family members, principally on the basis of whether there is a germline mutation. In this paper, we describe our experience with molecular analysis of RB1 mutations in tumor and nontumor samples from 18 retinoblastoma patients, using multiplex ligation dependent probe amplification (MLPA) to detect large deletions or duplications, microsatellite analysis to detect loss of heterozygosity (LOH), and denaturing high performance liquid chromatography (D-HPLC) analysis to detect point mutations and small insertions or deletions. We found LOH in 71% of all cases, and 83% of these were due to acquired isodisomy rather than chromosomal deletions. Small mutations identified by D-HPLC accounted for 78% of the non-LOH mutations, and large deletions/duplications detected by MLPA accounted for the remaining 22%. We give the first report of a large, multiexon duplication in RB1 of exons 8 to 18.
authors.affiliation: Molecular Gentics, Princess Margaret Hospital for Children, Roberts Road, Subiaco 6009, Perth, Western Australia. geyer101@tartarus.uwa.edu.au
authors.forename: Loryn N
authors.initials: LN
authors.lastname: Sellner
authors.forename: Edward
authors.initials: E
authors.lastname: Edkins
authors.forename: Nicholas
authors.initials: N
authors.lastname: Smith
mesh_terms: D002648:Child; D002675:Child, Preschool; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D004252:DNA Mutational Analysis / Q000379:methods; D017931:DNA Primers / Q000737:chemistry; D004273:DNA, Neoplasm / Q000737:chemistry / Q000235:genetics; D005820:Genetic Testing / Q000379:methods*; D006801:Humans; D007223:Infant; D019656:Loss of Heterozygosity; D018895:Microsatellite Repeats; D021141:Nucleic Acid Amplification Techniques; D009691:Nucleic Acid Denaturation; D019572:Retinal Neoplasms / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D012175:Retinoblastoma / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D016160:Retinoblastoma Protein / Q000235:genetics* / Q000378:metabolism
chemical_list: D017931:DNA Primers; D004273:DNA, Neoplasm; D016160:Retinoblastoma Protein