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Rapid Identification of Germline Mutations in Retinoblastoma by Protein Truncation Testing
Tony Tsai, MD;
Lilia Fulton, BA;
Barbara J. Smith, PhD;
Rachel L. Mueller, BA;
Gustavo A. Gonzalez, MD, PhD;
Marita S. Uusitalo, MD, PhD;
Joan M. O'Brien, MD
Arch Ophthalmol. 2004;122:239-248.
ABSTRACT
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Objective To demonstrate the utility of protein truncation testing (PTT) for rapid detection and sequencing of germline mutations in the retinoblastoma tumor suppressor gene (RB1).
Methods We performed PTT, a technique based on the in vitro synthesis of protein from amplified RNA, on 27 probands from 27 kindreds with hereditary retinoblastoma. In 4 kindreds, PTT was also performed on 1 additional affected relative. Ten unrelated patients without retinoblastoma were included as negative control subjects. All PTT-detected mutations were further analyzed by focused sequencing of genomic DNA. When no mutation was detected by PTT, we performed exon-by-exon sequencing, as well as cytogenetic analysis by Giemsa-trypsin-Giemsa banding and by fluorescent in situ hybridization for RB1. The results of proband testing were used for direct genetic testing by polymerase chain reaction and sequencing in 11 relatives from 7 of the 27 kindreds.
Results Of the probands tested, 19 (70%) of 27 tested positive for germline mutations by PTT. In 1 kindred, the proband had negative PTT results but an additional affected relative had positive PTT results. Focused DNA sequencing of 1 patient with positive PTT results from each of the 20 kindreds with positive PTT results revealed truncating mutations in 19 kindreds. Four demonstrated frameshift deletions, 6 had splice site mutations, and 9 showed nonsense mutations. Further analysis by genomic exon-by-exon sequencing and karyotype analysis of the 8 probands who tested negative for germline mutations by PTT revealed 1 splice site mutation, 2 missense mutations, and 1 chromosomal deletion. Focused sequencing based on positive PTT results was successfully used to confirm shared truncating mutations in additional affected family members in 2 kindreds. Using a multitiered approach to genetic testing, 23 (85%) of 27 kindreds had mutations identified and those detected by PTT received a positive result in as few as 7 days. In control subjects, PTT produced no false-positive results.
Conclusions Protein truncation testing is an effective, rapid single-modality screen for germline mutations in patients with retinoblastoma. When used as an initial screen, PTT can increase the yield of additional testing modalities, such as sequencing and chromosomal analysis, providing a timely and cost-effective approach for the diagnosis of heritable germline mutations in patients with retinoblastoma.
Clinical Relevance The clinical application of PTT in retinoblastoma will improve detection of germline retinoblastoma mutations, which will supply critical information for prognosis, treatment planning, follow-up care, and genetic counseling.
INTRODUCTION
Retinoblastoma (RB) affects approximately 1 in 20 000 children1-2 and accounts for 12% of infant cancers in the United States.3 The disease arises from loss or mutation of both alleles of the RB tumor suppressor gene (RB1). Retinoblastoma is divided into 2 types, heritable and nonheritable, according to the mechanism by which the 2 RB1 alleles are inactivated. In nonheritable RB, both alleles are inactivated somatically within a single retinal cell, resulting in a unilateral, unifocal tumor. In heritable RB, 1 RB1 allele is inactivated in the germline and loss or mutation of the second allele occurs somatically. Children with heritable RB typically develop bilateral, multifocal retinal tumors, although 15% of children with unilateral disease also harbor an underlying germline mutation.4-5
The distinction between heritable and nonheritable RB is critical for treatment planning and disease management. Children with heritable RB, in addition to experiencing bilateral vision impairment in early childhood, are at increased risk for developing primitive neuroectodermal tumors and second tumors later in life. While rare, primitive neuroectodermal tumors represent a significant threat because they are uniformly and rapidly fatal. Other tumors also represent a significant problem, with 50% of patients with heritable RB who survive childhood succumbing to second tumors within 50 years after diagnosis.6 Additionally, patients with heritable RB have a nearly 50% chance of passing this genetic defect on to their children.7-8 Heritable and nonheritable RB cases are not always clinically distinguishable, especially in patients with unilateral disease. In many cases, genetic testing for the presence of a germline RB1 mutation is the only means by which the diagnosis may be established with certainty.
Direct genetic testing for germline mutations that underlie heritable RB first became possible when RB1 was identified in 1986.9-11 Despite knowledge of the complete genomic sequence, 3 obstacles have made efficient RB1 genetic testing difficult: (1) RB1 is large, containing 27 exons that span approximately 180 000 bases.9-11 (2) No known mutation hot spots have emerged, despite the description of more than 230 different germline RB1 mutations.12-13 (3) The majority of mutations arise from small (< 10 base pair [bp]) sequence changes. These obstacles have traditionally necessitated detailed screening of all 27 RB1 exons and adjacent introns to locate a tumorigenic alteration. This kind of testing is costly, and the time required for obtaining a positive result often reduces clinical utility.
Analysis of published RB1 mutations, however, reveals that despite broad variation in mutation location and type, up to 90% of all RB1 mutations result in a premature stop codon.12 This commonality is well suited to detection by protein truncation testing (PTT) using in vitro transcription and translation (IVTT).14 In PTT, the in vitro synthesis of protein from amplified RNA screens the coding region of a gene for mutations that result in protein truncation.
Because PTT can rapidly detect truncating mutations from a peripheral blood sample, it has been applied with success in detecting mutations in genes responsible for several heritable forms of cancer, including colon cancer (FAP, HNPCC), breast cancer (BRCA1, BRCA2), tuberous sclerosis (TSC1, TSC2), and neurofibromatosis (NF1, NF2).15 As in RB1, the vast majority of the reported mutations in these genes result in a premature translation termination. One research group has applied PTT to detect RB1 mutations in tumor specimens. In that study, positive results were obtained in 5 of 35 malignant fibrous histiocytoma tumor samples (14%).16 However, PTT has yet to be applied to detect germline RB1 mutations in peripheral blood samples. Peripheral blood analysis has particular clinical utility in RB because these patients are often treated with ocular conservation and their tumor specimens are not readily available for genetic analysis.
Once a truncating mutation has been detected by PTT, the relative degree of protein truncation present pinpoints the site of the underlying mutation. This localizing information has the benefit of expediting mutation identification by obviating the need to screen the entire RB1 gene. Only a small fraction of the gene, determined by the results of PTT, requires sequencing. This benefit of PTT produces savings in time and effort required for mutation detection and provides genetic information in a time frame that is useful for patient counseling and for clinical decision making. With these advantages in mind, an RB1 genetic testing protocol using PTT was developed for clinical testing in the Ocular Oncology Unit at the University of California, San Francisco.
METHODS
PATIENT SELECTION
Twenty-seven patients with the clinical diagnosis of hereditary RB from 27 separate kindreds were enrolled with written informed consent as probands for RB1 genetic testing by PTT. All probands were established patients in the clinical practice of one ocular oncologist (J. O. B.) and were enrolled consecutively at the time of their regularly scheduled visits. All procedures, protocols, and consent forms were approved by the University of California, San Francisco, Committee on Human Research and followed the tenets of the Declaration of Helsinki. The clinical diagnosis of hereditary RB was determined by the presence of bilateral tumors, multifocal unilateral tumors, or a unilateral tumor concurrent with a positive family history of RB. Additionally, 13 relatives from 7 of the 27 kindreds were enrolled for familial testing. Seven of these family members were unaffected by RB; the remaining 6 had either unilateral or bilateral RB tumors. Ten unrelated patients with neither a personal nor a family history of RB were enrolled as negative control subjects.
GENERAL TESTING PROTOCOL
To maximize the advantages of PTT and to minimize the number of patients requiring full RB1 screening, 1 affected member from each kindred was selected as the proband for PTT. If PTT detected a germline mutation in this proband, targeted complementary DNA (cDNA) and genomic sequencing were undertaken to identify the precise DNA alteration. When a mutation was identified, direct testing of available relatives by targeted genomic sequencing was undertaken to determine if the proband's mutation was shared. If PTT did not detect a mutation in the proband, the test was repeated on a new blood sample from the same patient. Additionally, when available, another affected member of the kindred was selected as a second subject for PTT. If no second affected family member was available or if both affected family members had negative PTT results, then exons 2 to 27 and the promoter region were systematically sequenced from the proband's polymerase chain reaction (PCR)–amplified genomic DNA. When possible, a new blood sample was collected from patients displaying negative results using PTT. Cytogenetic analysis by Giemsa-trypsin-Giemsa (GTG) banding and by fluorescent in situ hybridization (FISH) for RB1 was performed.
PTT ANALYSIS
The general scheme for PTT is illustrated in Figure 1.
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Figure 1. General scheme for protein truncation testing. mRNA indicates messenger RNA; cDNA, complementary DNA.
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Isolation of RNA and Reverse Transcription
Within 2 hours after collection in heparin sodium, blood samples were transported to the laboratory at room temperature and incubated with gentle inversion for 4 hours at 37°C in 200 µg/mL of puromycin. Total RNA was then isolated from leukocytes using QIAamp RNA Blood Mini Kits (QIAGEN, Valencia, Calif) according to the manufacturer's protocol. Synthesis of first-strand cDNA was performed on 0.5 to 1.0 µg of total RNA with the Superscript First-Strand Synthesis System for RT-PCR (Gibco-BRL, Rockville, Md) using a primer mixture containing oligo dT and 2 RB1-specific antisense primers, RbRT (5'-GACTAACATTTCAAGTGGC-3'), which primes 128 bp downstream of the endogenous stop codon, and RbRT2 (5'-GAGGTAGATTTCAATGGCT-3'), which primes at amino acid 646 within the wild-type open reading frame. Reactions were performed at 42°C for 90 minutes.
PCR Amplification of RB1 Coding Region
The PCR amplification of the RB1 coding region was performed in 3 overlapping fragments. Each 50 µL reaction contained 3 to 5 µL of first-strand cDNA; 10 pmol each of the sense and antisense primers (Table 1); 10 µL of 5x Q-Solution (QIAGEN); 5 µL of 10x PCR buffer (QIAGEN); 4.0mM of magnesium chloride; 0.2mM each of dATP, dCTP, dGTP, and dTTP; and 2 units of Taq polymerase (QIAGEN). Sense primers contained the T7 promoter sequence, a eukaryotic translation initiation sequence (consensus Kozak sequence), and an N-terminal myc-tag, MEQKLISEEDLN (Figure 2). 17 Reactions were performed with an initial denaturation at 94°C for 3 minutes with 35 cycles of 94°C for 1 minute, 55°C for 1 minute, and 72°C for 3 minutes, followed by a final polymerization at 72°C for 7 minutes. We confirmed the reverse transcription PCR (RT-PCR) success by visualizing 5 µL of the PCR reaction with ethidium bromide staining after electrophoresis through a 0.8% agarose gel. Large DNA sequence deletions or insertions could be detected at this point by comparison with wild-type RT-PCR products.
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Figure 2. Polymerase chain reaction (PCR) modifications incorporated into reverse transcription PCR sense primers.17
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IVTT and Immunoprecipitation Purification
We performed IVTT with the TNT T7 Quick Coupled Transcription/Translation System (Promega, Madison, Wis) at 30°C for 90 minutes. Each 25 µL reaction contained 4 µL of RT-PCR product and 10 µCi (370 000 Bq) of 35S-methionine (Amersham, Piscataway, NJ). The IVTT products were immunoprecipitated by adding 10 µg of agarose-conjugated anti-c-myc (9E10) mouse Mab (Santa Cruz Biotechnology, Santa Cruz, Calif) and 1 mL of phosphate-buffered saline. After incubation with gentle inversion at 4°C for more than 2 hours, samples were pelleted and washed 4 times with phosphate-buffered saline.
Electrophoeretic Analysis and Autoradiography
Electrophoresis was performed through 12% sodium dodecyl sulfate–polyacrylamide gels after resuspension of the immunoprecipitated pellet in Laemmli loading buffer (Bio-Rad, Hercules, Calif) and denaturation at 95°C for 5 minutes. Autoradiographic visualization was performed after gels were fixed for 15 minutes in acetic acid–methonol-water (10:50:40), soaked for 15 minutes in Amplify fluorographic reagent (Amersham), vacuum dried at 65°C for 90 minutes, and exposed to film at -80°C for 1 to 4 days. A mutation was detected when visual comparison revealed a patient protein size smaller than that of wild-type proteins.
FOCUSED DNA SEQUENCING
Sequencing of cDNA
Templates for cDNA sequencing were generated by RT-PCR, using the same primers and conditions used in PTT (Table 1). These sequencing templates were purified from free nucleotides and primers using the QIAquick PCR Purification Kit (QIAGEN). Then, 2 to 4 overlapping primers from a set of 18 spanning the entire RB1 coding region (Table 2) were selected to target direct sequencing to the region most likely to harbor the truncating mutation, based on the size of the mutant protein relative to the size of the wild-type protein. Sequencing was performed on an ABI Prism 377 DNA sequencer (PE Biosystems, Foster City, Calif). Electropherograms of sequenced regions were compared visually with wild-type electropherograms for the presence of sequence anomalies resulting in premature stop codons. All cDNA mutations were confirmed by genomic DNA sequencing.
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Table 2. cDNA Sequencing Primers*
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Sequencing of Genomic DNA
Concurrent with the blood draw for PTT, whole blood was collected from patients for genomic DNA preparation and stored at -20°C. For genomic DNA isolation, 400 µL of this blood was thawed and processed using the QIAamp Blood Kit (QIAGEN) according to the manufacturer's protocol. Sequencing templates were generated from genomic DNA by PCR amplification of RB1 exons and their adjacent intronic sequences (Table 3). Specific exons were selected to direct sequencing to the region most likely to contain the premature stop codon based on either the results of cDNA sequencing or the size of truncated PTT products. For patients with hereditary RB in whom PTT results were negative, exons 2 to 27 and the promoter region (including -327 to -89)18 were amplified for sequencing. Exon 1 was not included because of difficulties with amplification in this guanine- and cytosine-rich region of RB1. Amplification products were purified from free nucleotides and primers using the QIAquick PCR Purification Kit (QIAGEN) according to the manufacturer's protocol. For most exons and adjacent intronic sequences, the same primers used for PCR amplification were used to sequence both strands of the purified templates. Exceptions are noted in Table 3.
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Table 3. Exon Amplification and Sequencing Primers
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Sequencing was performed on an ABI Prism 377 DNA sequencer (PE Biosystems). Electropherograms of sequenced exons and adjacent introns were compared visually with wild-type electropherograms for the presence of sequence anomalies. All mutations were confirmed by sequencing both strands of genomic DNA, by correlation with PTT truncated fragment size, and by repeat sequencing from a second genomic DNA preparation from the same patient. The RB1 reference sequence corresponded to GenBank accession number L11910.
HIGH-RESOLUTION CHROMOSOMAL ANALYSIS AND FISH FOR RB1
When an additional blood sample was available for patients in whom PTT results were negative, cytogenetic analysis by GTG banding and by FISH for RB1 were performed according to protocols of the Cytogenetics Laboratory at the Children's Hospital of Oakland, Oakland, Calif.
RESULTS
Of the 27 probands tested, 19 (70%) tested positive for germline mutations by PTT (Figure 3). Genomic DNA sequence confirmation on probands with positive PTT results revealed 3 patients with small frameshifting deletions, 6 with splice site mutations, and 9 with single base pair substitutions resulting in nonsense mutations (Figure 3 and Figure 4) (Table 4). The proband from the 1 remaining kindred (Rb231) demonstrated an approximately 20 kDa truncated protein product with PTT of the most 3' of the overlapping RT-PCR templates. This product would suggest an early termination codon near exon 18, but focused sequencing of both cDNA and genomic DNA that included splice sites and coding regions for exons 17 to 20 failed to reveal the truncating mutation.
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Figure 3. Protein truncation test gel demonstrating RB1 truncating mutations. The gel shows radioactively labeled proteins produced by in vitro transcription and translation from 6 different patients with retinoblastoma who were heterozygous for germline mutations in RB1. In each lane, the upper band represents the wild-type protein product and the lower band represents the truncated product (arrows). The homozygous wild-type control (WT) contains only 1 band.
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Figure 4. Electropherograms of RB1 mutations detected by protein truncation testing. Examples of sequence data confirming nonsense (A), splice site (B), and frameshift (C) mutations detected by protein truncation testing.
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Table 4. RB1 Mutations Identified by Protein Truncation Testing and Focused Sequencing*
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In 8 probands, the initial PTT results were negative. When repeated on a second blood sample, all of these probands had negative results a second time. For 3 of these probands (Rb204, Rb209, and Rb222), a second family member affected by RB was available for PTT (Rb205, Rb210, and Rb232, respectively). Protein truncation testing results were also negative for 2 of these other affected family members (Rb205 and Rb210). One second family member (Rb232) demonstrated positive PTT results that were subsequently identified as resulting from a frameshifting mutation (Table 4).
In all, 10 patients with hereditary RB had negative PTT results. Eight were probands and 2 were affected second family members in 2 of the 8 kindreds. All 10 of these were further analyzed by systematic sequencing of exons 2 to 27 and the promoter region. Systematic sequencing identified 2 probands with missense mutations (Rb177 and Rb222) and 1 proband (Rb219) with a splice site mutation that would result in an inframe deletion. Three of the 10 patients with negative PTT results, representing 3 different kindreds, had additional blood drawn for cytogenetic analysis. Cytogenetic analysis by GTG banding revealed 1 proband (Rb207) with an interstitial deletion of 1 chromosome 13 spanning RB1 (break points at 13q12 and 13q14) that was confirmed by FISH (Table 5).
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Table 5. RB1 Mutations Identified In Probands With Negative Protein Truncation Testing Results*
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The results of PTT were used for focused screening of 11 relatives in 7 kindreds. Three kindreds had multiple family members with RB, and in all 3, the affected family members shared a mutation with the affected proband. In 1 kindred with 2 affected siblings (Rb222 and Rb232), 2 germline mutations were identified. One sibling (Rb232) had positive PTT results as a second affected family member, and a truncating mutation was revealed by direct sequencing, a 4 base pair deletion in exon 18 (g. 150029-150032del). This is a frameshift mutation that produces a new stop codon in exon 19. Familial testing of the proband sibling found no mutation at this location. Systematic exon-by-exon sequencing revealed that both siblings shared a missense mutation (g.70243A>T), a single base change that causes the threonine at amino acid position 377 to be substituted with serine.
None of the 7 unaffected family members tested shared the causative mutation identified in an affected relative. Of the 10 control patients, 9 tested negative for germline mutations by PTT and 1 had an insufficient amount of total RNA isolated for analysis.
COMMENT
Despite the availability of many molecular diagnostic techniques for the detection of germline mutations in RB1, clinical application of this genetic testing has not been widespread. Available molecular techniques such as Southern blotting, single-strand conformation polymorphism analysis, heteroduplex analysis, restriction fragment length polymorphism analysis, and direct sequencing can be prohibitively time-consuming or expensive. Modalities that are performed efficiently or are commercially available, such as cytogenetic analysis, are able to detect only a small fraction of the total germline mutations that may be present in RB1. A single procedure is needed that can function as a rapid screen for a majority of RB1 germline mutations.
An ideal test for germline mutations in RB1 would incorporate the following features: (1) a noninvasive method of sample collection, (2) the capacity to detect all mutation types that may exist in RB1, (3) specificity for RB1 sequence alterations that are pathogenic (neutral variations and polymorphisms would not be detected), (4) rapid and cost-effective performance, and (5) sensitivity sufficient to assure that all negative results are true negative results. While it is unlikely that any single procedure can meet all of these criteria, to our knowledge, PTT fulfills more of these than any other genetic testing approach to date.
Samples for PTT are collected relatively noninvasively by phlebotomy. Because of its high sensitivity for truncating mutations, PTT requires only a small fraction of leukocytes to carry the mutation in order to detect alterations in patients with somatic mosaic RB. Since the sensitivity of PTT was 70% in probands with hereditary RB, one could not safely exclude the presence of a germline mutation in a patient with unilateral RB using PTT alone. In cases where no mutation is detected by PTT in the blood, primary analysis of tumor tissue followed by a secondary search for tumor-detected mutations in blood samples would allow for more confident diagnoses of nonhereditary RB.
Although PTT is not sensitive to all mutation types, it detects a broad range of the most common RB1 mutation types: small frameshift mutations, splice site alterations, and nonsense mutations. Protein truncation testing can also readily detect some rare mutation types that may be difficult to detect with genomic screening alone, such as an intronic mutation affecting a splice site adjacent to an exon. Genomic sequencing of exon and flanking intron DNA will miss deletions or insertions with break points deeper into the intronic sequences that result in messenger RNA truncated by the absence of 1 or more exons. While focused sequencing of cDNA and genomic DNA led to identification of truncating mutations in 19 (95%) of 20 kindreds, the presence of an internal intron mutation affecting splicing may explain why focused sequencing of genomic DNA did not identify a truncating mutation in the remaining kindred (Rb231).
Mutation types exist that PTT cannot detect, 2 of which, missense mutations and large chromosomal deletions, were found in this study on further analysis of probands with negative PTT results. Other known RB1 mutation types not detectable by PTT and not discovered among the negative samples in this study include promoter mutations, small in-frame deletions, and chromosomal translocations. Fortunately, the mutation types that evade detection by PTT represent a minority of RB1 germline mutations. Missense, promoter, and in-frame deletions have been estimated to represent 11% of RB1 germline mutations.12 With the prevalence of chromosomal alterations in patients with bilateral RB estimated at 7% to 8%,19 the remaining 80% of patients with nonsense, frameshift, and splice site mutations represent a rich opportunity for detecting a majority of RB1 germline mutations using PTT as a single modality.
In this study, 19 (70%) of 27 probands had a germline mutation detectable by PTT. By using PTT as an initial screen, one can be more selective and efficient in the use of labor-intensive tests for mutations undetectable by PTT. Even on the small scale of this study, limiting additional testing to those who had negative PTT results increased the yield of subsequent testing. Detailed molecular analysis results for missense mutations by genomic PCR and systematic sequencing of exons 2 to 27 and the promoter region were positive for 3 (30%) of 10 probands. Additional blood samples for cytogenetic analysis by GTG banding and FISH at the RB1 locus were positive for a large deletion in 1 of 3 probands whose PTT results were negative. With the prevalence of missense mutations and of cytogenetically detectable alterations estimated at 6% and 7% to 8%, respectively,12, 19 the detection rates in this study are boosted by first eliminating those patients who have PTT detectable mutations.
Many mutation detection schemes capable of detecting single base pair changes will also detect silent nonpathogenic alterations, false-positive results that must be distinguished from more functionally relevant alterations. Protein truncation testing has the advantage of detecting only mutation types that result in protein truncation, and these mutations are generally pathogenic. The truncating mutations reported in this study varied between kindreds; however, 16 (84%) of 19 mutations were predicted to disrupt the small pocket domain (Figure 5), and all were predicted to affect the nuclear localization signal contained in exon 25. These are regions of the RB protein that have demonstrated functional significance.20-22 No truncated RB1 protein products were detected in healthy control subjects. The absence of known truncating polymorphisms in RB1 supports the pathogenic likelihood of a positive PTT result.
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Figure 5. Locations of all mutations detected by protein truncation testing and confirmed by focused DNA sequencing. Exons are indicated by number, and the pocket region is shaded. Positions of nonsense mutations have solid black arrowheads, splice site mutations have gray arrowheads, and frameshift mutations have outlined arrowheads.
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The presence of missense and promoter mutations should always raise the possibility that they may be nontumorigenic polymorphisms. In this study, 2 missense mutations went undetected by PTT, as expected, but were discovered on systematic DNA sequencing. One occurred at the site of a previously defined polymorphism in exon 20.23 Whereas the described polymorphism at position g.156713 (C, 95%; A, 5%) results in no amino acid change (a silent polymorphism), the mutation identified in Rb177 (g.156713 C>T) results in alteration of an arginine codon (CGG) into a tryptophan codon (TGG). In the other missense mutation (Rb 222; g.70234A>T), an A-to-T substitution results in the alteration of the codon for amino acid 377 in exon 12 from threonine to serine. Both missense mutations are presumed to be tumorigenic, but functional analysis has not yet been performed for confirmation.
Perhaps the greatest advantage of PTT in RB1 genetic testing is its rapidity in detecting a positive result. Using the protocol applied in this study, a truncated protein product can be detected in as few as 7 days. The detection of a truncated protein by PTT signals the presence of a germline truncating mutation. This confirms the diagnosis of hereditary RB, but sequencing must be performed to define the exact mutation. The scope of the search is narrowed, however, because the size of the truncated protein relative to the wild-type product provides an estimate of the location of the premature stop codon. Therefore, sequencing either of cDNA or genomic DNA may be focused on a small segment of the RB1 gene. Positive PTT results in this study required 2 to 4 weeks for sequence confirmation with the aid of automated DNA sequencing.
Sequence confirmation provides the power to rapidly test both affected and unaffected relatives for a familial mutation. Testing of relatives is expedited because this testing can be focused on the presence or absence of the specific mutation detected in the proband. The PCR amplification from genomic DNA followed by sequencing of a single exon is all that is required. In this study, the results of all familial testing were consistent with the subject's clinical phenotype.
An unexpected result of familial testing is the discovery of a kindred in which both affected siblings share a missense mutation and 1 of these also carries a second truncating germline mutation (Rb222 and Rb232). Rarely, families affected by RB have been known to have individuals with different germline mutations.24 The possibility exists that the missense mutation shared is a nonpathogenic polymorphism, that the diagnosis of hereditary RB is incorrect (although the histopathologic features of the enucleated specimen are consistent with this diagnosis), or that a different pathogenic mutation shared by both affected siblings has gone undetected. Further genetic analysis of this kindred is in progress.
The cost savings of PTT lie in the reduced time for analysis, saving both laboratory time and technician labor. Material and reagent costs have been estimated at $200 to $250 per proband and $30 to $40 for each family member subsequently tested. This suggests that a 4-member family could be tested for fewer than $400 and that the results could be available in 2 to 4 weeks. This compares favorably with existing methods of genetic analysis commercially available, where testing a 4-member family could cost $3000 to $7500 and take 1 to 6 months.25 Published analysis of the relative costs of molecular and conventional RB screening shows that, while health care savings are present initially, savings increase geometrically as kindreds with known RB1 mutations grow.26
In summary, the advantage of PTT lies not in its ability to detect all RB1 germline mutations but rather in its capacity to detect the majority of mutations noninvasively, rapidly, and at low cost. No single genetic testing method currently available can detect all RB1 mutation types. The most effective and efficient strategies to date have used a series of nested tests. These protocols, some of which have used as many as 5 different testing modalities, have final sensitivities ranging up to 83%.27 Protein truncation testing is well suited as the first step in a more comprehensive testing protocol, because it delivers a rapid, single test sensitivity (70%) in the range previously provided only by multiple-modality testing, thereby allowing reservation of more intensive techniques for difficult cases. With a scheme of nested testing using PTT as the first line and limited exon-by-exon sequencing and karyotyping applied to individuals with negative PTT results to increase the sensitivity, the overall sensitivity is raised to 85% (23 of 27 kindreds with germline mutations identified).
Future implications of a more efficient method of genetic testing are broad. The clinical application of PTT in RB will improve detection of germline RB mutations, which will supply critical information for prognosis, treatment planning, follow-up care, and genetic counseling. In addition to providing an adjunct to the clinical diagnosis of germline RB, PTT may permit presymptomatic diagnosis and enable in utero screening. Identifying infants who inherit this disease for early medical intervention could maximize their survival and visual potential. Protein truncation testing will allow rapid expansion of the published RB mutation database, with potential benefits for understanding the molecular and genetic bases for variations in the phenotype of this disease. If specific genetic features predispose patients to better or worse clinical outcomes, patients could be stratified early in their disease course to identify those who require more aggressive clinical intervention.
The RB1 gene was the first tumor suppressor gene to be identified almost 2 decades ago. Despite this breakthrough, many families afflicted with this disease have never benefited from genetic testing. Protein truncation testing analysis is a tool that could make genetic testing more available, affordable, and practical. Undoubtedly, this screening technique will be replaced by more powerful approaches in future. Effective, efficient genetic screening will eventually be applied to broad populations at risk for cancer and for many other diseases with a genetic basis. Until that time, PTT will allow us to accumulate RB1 mutations and to correlate these with clinical information in an effort to better diagnose and treat this pediatric cancer syndrome.
AUTHOR INFORMATION
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Corresponding author and reprints: Joan M. O'Brien, MD, Director, Ocular Oncology Unit, Department of Ophthalmology, University of California San Francisco, 10 Koret Way, Box 0730, San Francisco, CA 94143.
Submitted for publication January 7, 2003; final revision received July 15, 2003; accepted August 21, 2003.
This study was supported by the Knights Templar Eye Foundation Inc, Chicago, Ill; the Giannini Foundation, San Francisco, Calif; That Man May See Inc, San Francisco; the Sand Hill Foundation, Menlo Park, Calif; a Physician-Scientist Award from Research to Prevent Blindness, New York, NY (Dr O'Brien); and grant EY13812 (Dr O'Brien) and core grant EY02162 from the National Eye Institute, Bethesda, Md.
We would like to thank Anny Shai, BSc, for expert technical assistance in automated DNA sequencing.
From the Ocular Oncology Unit, Department of Ophthalmology, University of California, San Francisco (Drs Tsai, Smith, Gonzalez, Uusitalo, and O'Brien and Mss Fulton and Mueller), and the Department of Ophthalmology, Helsinki University Central Hospital, Helsinki, Finland (Dr Uusitalo).
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