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Laboratory Sciences
February 2004

Rapid Identification of Germline Mutations in Retinoblastoma by ProteinTruncation Testing

Author Affiliations

From the Ocular Oncology Unit, Department of Ophthalmology, Universityof California, San Francisco (Drs Tsai, Smith, Gonzalez, Uusitalo, and O'Brienand Mss Fulton and Mueller), and the Department of Ophthalmology, HelsinkiUniversity Central Hospital, Helsinki, Finland (Dr Uusitalo).

Arch Ophthalmol. 2004;122(2):239-248. doi:10.1001/archopht.122.2.239

Objective  To demonstrate the utility of protein truncation testing (PTT) for rapiddetection and sequencing of germline mutations in the retinoblastoma tumorsuppressor gene (RB1).

Methods  We performed PTT, a technique based on the in vitro synthesis of proteinfrom amplified RNA, on 27 probands from 27 kindreds with hereditary retinoblastoma.In 4 kindreds, PTT was also performed on 1 additional affected relative. Tenunrelated patients without retinoblastoma were included as negative controlsubjects. All PTT-detected mutations were further analyzed by focused sequencingof genomic DNA. When no mutation was detected by PTT, we performed exon-by-exonsequencing, as well as cytogenetic analysis by Giemsa-trypsin-Giemsa bandingand by fluorescent in situ hybridization for RB1. Theresults of proband testing were used for direct genetic testing by polymerasechain reaction and sequencing in 11 relatives from 7 of the 27 kindreds.

Results  Of the probands tested, 19 (70%) of 27 tested positive for germlinemutations by PTT. In 1 kindred, the proband had negative PTT results but anadditional affected relative had positive PTT results. Focused DNA sequencingof 1 patient with positive PTT results from each of the 20 kindreds with positivePTT results revealed truncating mutations in 19 kindreds. Four demonstratedframeshift deletions, 6 had splice site mutations, and 9 showed nonsense mutations.Further analysis by genomic exon-by-exon sequencing and karyotype analysisof the 8 probands who tested negative for germline mutations by PTT revealed1 splice site mutation, 2 missense mutations, and 1 chromosomal deletion.Focused sequencing based on positive PTT results was successfully used toconfirm shared truncating mutations in additional affected family membersin 2 kindreds. Using a multitiered approach to genetic testing, 23 (85%) of27 kindreds had mutations identified and those detected by PTT received apositive result in as few as 7 days. In control subjects, PTT produced nofalse-positive results.

Conclusions  Protein truncation testing is an effective, rapid single-modality screenfor germline mutations in patients with retinoblastoma. When used as an initialscreen, PTT can increase the yield of additional testing modalities, suchas sequencing and chromosomal analysis, providing a timely and cost-effectiveapproach for the diagnosis of heritable germline mutations in patients withretinoblastoma.

Clinical Relevance  The clinical application of PTT in retinoblastoma will improve detectionof germline retinoblastoma mutations, which will supply critical informationfor prognosis, treatment planning, follow-up care, and genetic counseling.