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Comparative Study
. 2003 Feb;72(2):253-69.
doi: 10.1086/345651. Epub 2002 Dec 18.

Sensitive and efficient detection of RB1 gene mutations enhances care for families with retinoblastoma

Suzanne Richter  1 , Kirk Vandezande, Ning Chen, Katherine Zhang, Joanne Sutherland, Julie Anderson, Liping Han, Rachel Panton, Patricia Branco, Brenda Gallie
Affiliations
Comparative Study

Sensitive and efficient detection of RB1 gene mutations enhances care for families with retinoblastoma

Suzanne Richter et al. Am J Hum Genet. 2003 Feb.

Abstract

Timely molecular diagnosis of RB1 mutations enables earlier treatment, lower risk, and better health outcomes for patients with retinoblastoma; empowers families to make informed family-planning decisions; and costs less than conventional surveillance. However, complexity has hindered clinical implementation of molecular diagnosis. The majority of RB1 mutations are unique and distributed throughout the RB1 gene, with no real hot spots. We devised a sensitive and efficient strategy to identify RB1 mutations that combines quantitative multiplex polymerase chain reaction (QM-PCR), double-exon sequencing, and promoter-targeted methylation-sensitive PCR. Optimization of test order by stochastic dynamic programming and the development of allele-specific PCR for four recurrent point mutations decreased the estimated turnaround time to <3 wk and decreased direct costs by one-third. The multistep method reported here detected 89% (199/224) of mutations in bilaterally affected probands and both mutant alleles in 84% (112/134) of tumors from unilaterally affected probands. For 23 of 27 exons and the promoter region, QM-PCR was a highly accurate measure of deletions and insertions (accuracy 95%). By revealing those family members who did not carry the mutation found in the related proband, molecular analysis enabled 97 at-risk children from 20 representative families to avoid 313 surveillance examinations under anesthetic and 852 clinic visits. The average savings in direct costs from clinical examinations avoided by children in these families substantially exceeded the cost of molecular testing. Moreover, health care savings continue to accrue, as children in succeeding generations avoid unnecessary repeated anaesthetics and examinations.

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Figures

Figure 1
Figure 1
The distribution, within the RB1 cDNA and promoter, of 111 different missense mutations and small insertions and deletions in the germline of 165 persons. Ninety-five families had unique mutations. Fourteen recurrent mutations that affected 70 independent families are shown in black; 14 in-frame mutations are outlined in black; R661W is both recurrent (all seven occurrences in germline) and in-frame, indicated by a wide black border.
Figure 2
Figure 2
The distribution within the RB1 cDNA and promoter of 41 splicing mutations and large deletions identified in the germline of 65 probands with retinoblastoma. The mutations in black occurred in more than one independent family; the number of occurrences is in brackets.
Figure 3
Figure 3
Mutation detection using QM-PCR. A, Detection of a multiexonic deletion spanning at least exons 4–11, while exons 19 and 25 remain two-copy. B, Detection of a 2-bp deletion in exon 22 by QM-PCR.
Figure 4
Figure 4
Multiplex AS4-PCR for rapid detection of recurrent mutations in RB1. Four samples from patients with retinoblastoma (patients A–D) and two control normal samples were tested by AS4-PCR containing primers specific for the recurrent mutations R455X (730 bp), R579X (287 bp), R358X (251 bp), and R251X (202 bp). Strong amplification occurred when the mutation identified by the specific PCR primer was present. Unaffected individual 1 and patient D also show low levels of cross-hybridization to the wild-type alleles with the primers for R455X and R358X.
Figure 5
Figure 5
Impact of molecular RB1 testing. A, Histograms of the number of examinations avoided because of molecular results. Clinic examinations are shown in white bars, EUAs in black bars. B, Histogram of direct surveillance costs saved for 20 families because of molecular RB1 testing. Mean savings was Can$6,591 per family (horizontal line). Families with bilateral and familial retinoblastoma are shown in white bars, and families with unilateral nonfamilial disease are shown in bars with oblique hatches. The molecular strategy cost more than conventional surveillance for two families in which all assays failed to show the proband’s mutation (dotted bars) and for six families in which at-risk family members were few in number or significantly older than the proband.
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References

Electronic-Database Information

    1. GenBank, http://www.ncbi.nlm.nih.gov/Genbank/ (for RB1 [accession number L11910])
    1. Online Mendelian Inheritance in Man (OMIM), http://www.ncbi.nlm.nih.gov/Omim/ (for retinoblastoma [MIM 180200])
    1. RB1 Gene Mutation Database, http://www.d-lohmann.de/Rb/mutations.html

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