Screening of RB1 gene mutations in Chinese patients with retinoblastoma and preliminary exploration of genotype–phenotype correlations

Patients

This study recruited a total of 85 unrelated RB probands, including 37 (43.5%) boys and 48 (56.5%) girls, who were diagnosed between January 2012 and October 2013 in a Chinese cooperative group consisting of the Children’s Hospital of Fudan University and the Eye & ENT Hospital of Fudan University. The genetic testing results, using Sanger sequencing, of 35 of these patients were reported in a previous paper published in Chinese [22], but new findings emerged using the complementary MLPA method. The combined results are discussed in the present paper. The patients included 39 cases of bilateral RB and 46 cases of unilateral RB. Clinical features, including laterality, age at diagnosis, and International Classification of Intraocular Retinoblastoma (ICRB) staging at diagnosis (Appendix 1), were recorded. After the study protocol was approved by the Institutional Review Board of Fudan University, informed consent for genetic testing was obtained from the guardians of children affected with RB. The study protocol was in accordance with the provisions of the Declaration of Helsinki and the ARVO (the Association for Research in Vision and Ophthalmology) statement on human subjects. Peripheral blood samples (3 ml) were collected from the probands in EDTA (EDTA) anticoagulant tubes. Blood samples were stored at −20 °C until DNA was extracted.

RB1 gene sequencing

Genomic DNA was isolated from peripheral blood leukocytes using the QIAamp DNA Blood Mini Kit (Qiagen, Düsseldorf, Germany), according to the manufacturer’s instructions. Extracted DNA was measured with spectrophotometry (NanoDrop 2000; Thermo Scientific, Wilmington, DE) to ensure a concentration of 40 ng/µl.

Primers were designed according to a previous study by Abouzeid et al., with some modifications, using the Oligo software (National Biosciences, Plymouth, MN) [23]. Twenty-four pairs of primers were generated, which covered all coding exons and at least 50 bp flanking intronic sequences of each exon. PCR reactions were performed in a thermal cycler (ABI 2700), in a total volume of 10 µl containing 40 ng of genomic DNA, 2 picomoles of each primer, 0.2 µl of Taq DNA polymerase, and 1 µl of 10× buffer. Dimethyl sulfoxide (DMSO, 10%) was added to the exon 1 amplification system. Reactions were performed for the first 14 cycles of denaturation at 95 °C for 30 s, annealing at a specific temperature gradient (65 °C, −0.5 °C per cycle) for 30 s, extension at 72 °C for 40s; the second 20 cycles of denaturation at 95 °C for 30 s, annealing at 58 °C for 30 s, extension at 72 °C for 40 s, and a final extension step at 72 °C for 5 min. The PCR products were qualified with agarose gel electrophoresis.

After the unincorporated deoxynucleoside triphosphates and primers were removed using Exonuclease I and shrimp alkaline phosphatase, the PCR products were directly sequenced using the ABI BigDye Terminator v 3.1 Sequencing Standard Kit (Carlsbad, CA) and run on an ABI 3500 Genetic Analyzer. The sequence data were analyzed by comparison to the consensus sequence of the RB1 gene (NM_000321), using Mutation Surveyor (V 4.0) software (SoftGenetics; State College, PA). Additional information was obtained from the literature, the RB1 Gene Database (LOVD, 2010), the Human Gene Mutation Database (HGMD, 2007), and the Thousand Genomes Project (Thousand Genome Consortium, 2010). The functional consequences of the mutations were predicted by bioinformatics tools, SIFT, and PolyPhen-2.

Multiplex ligation-dependent probe amplification analysis

MLPA analysis was applied in bilateral probands who did not show germ-line mutations in DNA sequencing and in unilateral probands whose guardians demanded additional genetic examinations, considering the earlier age at diagnosis, which was less than 1 year old, although the DNA sequencing results had been negative. Gross deletion and duplication of RB1 were examined using methylation-specific (MS)-MLPA analysis, the commercially available SALSA MLPA P047 RB1 probemix (MRC-Holland, Amsterdam, The Netherlands). This P047-C1 RB1 probemix contains probes for 26 of the 27 RB1 exons and 13 reference probes. This method was used to detect loss of heterozygosity (LOH) status of the RB1 gene located on 13q14. The procedure was based on the manufacturer's protocol. Each MS-MLPA reaction was subjected to further analysis on an ABI 3730 automated sequencer (Applied Biosystems). The data were analyzed with GeneMarker v1.91 (SoftGenetics) according to the manufacturer’s instructions. The relative value of probes compared to the reference control was calculated. Threshold ratios for deletion and duplication were set at 0.75 and 1.35, respectively. The peak ratios for all target loci in a 1:1 ratio indicated normal copy numbers. The reduced or amplified signal of probes with <0.75 or >1.35 indicated loss or gain, respectively, of the copy number in these probe loci.

Statistical analysis

Data were analyzed using SPSS software (version 16.0, Chicago, IL). Statistical analysis was performed with one-way ANOVA, the chi-square test, and Fisher’s exact test. p<0.05 was considered to indicate statistical significance.

Null mutation

A null mutation is a pathogenic mutational type, which refers to those with mRNA harboring a premature translational-termination codon, including whole allele deletions, deletions involving one or more exons, and splice mutations, small frame-shifting intraexonic insertions and deletions, and nonsense mutations [24,25]. Our group identified 25 distinct null mutations in 31 (36.5%) of the 85 patients (Table 1). Of these mutations, the c.1735C>T mutation was a recurrent nonsense mutation identified in three patients. Six null mutations had not been reported before. The 31 patients with RB with an average age of 10.7 months at diagnosis included one patient (3.2%) with unilateral RB and 30 patients (96.8%) with bilateral RB (Table 2). Of the 61 eyes studied in this group, 20 (32.8%) presented with group E ICRB followed by groups C, D, B, and A ICRB, and the other four (6.6%) presented with extraocular extension at diagnosis (Table 3).

In-frame mutations

In-frame mutations stand for those not truncating based on prediction, such as indels (small insertions and deletions) in frame, and missense mutations [24]. We identified nine missense mutations in nine probands, of which one was a new mutation. Predicting the effects of these non-synonymous variants on protein function indicated ‘damaging’ designations for six patients with bilateral RB and ‘tolerated’ for three patients with unilateral RB (Table 1). The nine probands with an average age of 13.5 months at diagnosis involved 66.7% (6/9) bilateral and 33.3% (3/9) unilateral patients (Table 2). Of the 15 eyes, eight (53.3%) presented with group E ICRB, followed by groups D, A/B, and C ICRB, and one (6.7%) presented with extraocular extension (Table 3).

Negative genetic testing results

This group represented patients with negative genetic testing results. There were 45 cases, including three (6.67%) bilateral and 42 (93.3%) unilateral. The mean age at diagnosis was 27.3 months. Of the 48 eyes, 23 (47.9%) presented with group E ICRB, followed by groups D/C, B, and A ICRB. Sixteen of the 48 (33.3%) presented with extraocular extension (Table 3).

Genotype–phenotype correlations and implications for genetic counseling

In our study, all mutations were classified based on the prediction whether the transcripts harbored a premature translational-termination codon. Transcripts from alleles of the RB1 gene with null mutations have been shown to be subject to degradation by nonsense-mediated decay (NMD) [25-27], resulting in no detectable protein. Therefore, a null mutation is a comparatively more severe mutation than an in-frame mutation. Of the 31 probands with null mutations, 96.8% had bilateral RB, while only 66.7% of the probands with in-frame mutations developed bilateral RB (Table 2). Early onset retinoblastoma was seen more with null mutations (average age at diagnosis was 10.7 months) than in-frame mutations (average age at diagnosis was 13.5 months). These results are consistent with reports from Canada and India [24,28], and confirm that null mutations tend to the severe phenotype (early onset and bilateral RB). However, ICRB staging, an additional clinical feature, did not seem to be correlated with genotype. Group E was the most frequent staging at diagnosis in our study, which had no remarkable differences between patients with null mutations and in-frame mutations. We believe that tumor staging might be more related to access to care rather than the type of mutation.

Interestingly, cases with negative genetic testing results, which were mostly non-hereditary (42 unilateral and 3 bilateral probands), seemed to present with more serious clinical staging at diagnosis, with 16 (33.3%) extraocular extension tumors. The finding that advanced tumors tend to occur in non-hereditary cases might be explained by two hypotheses. First, most of the hereditary cases were bilateral RB, and the growth of the retinoblastoma tumors in the two eyes was not completely synchronous. The first-discovered eye disease in bilateral probands likely contributed to early diagnosis of another eye disease. Second, the growth processes of retinoblastoma tumors might have certain differences between patients with and without germ-line RB1 mutation, resulting in faster tumor growth in probands with negative genetic testing. A recent study reported that a previously unrecognized subtype of retinoblastoma tumor with intact RB1 alleles had high-level v-myc avian myelocytomatosis viral oncogene neuroblastoma derived homolog (MYCN) oncogene amplification, which might initiate retinoblastoma with intact RB1 alleles [29]. This suggests that the underlying mechanism involved in the initiation or development of retinoblastoma tumor could be investigated in future studies.

Distribution of the RB1 gene mutation

Our RB1 mutational distribution results revealed that RB1 mutations were scattered along the genomic sequence (Figure 1), lacking hot spots clustered in the important functional domains—A/B pocket regions. Richter et al. reported that 13 of 15 missense mutations of the RB1 gene gathered in the A/B pocket and the intervening spacer region, confirming that in-frame mutations favored the critical regions [24]. However, only five of the nine missense mutations appeared in the A/B pocket domains in our study. All five missense mutations that appeared in the A/B pocket domains were identified in bilateral probands, while only one of the four missense mutations that appeared in other regions were identified in bilateral probands, suggesting that in-frame mutations in A/B pocket domains could be considered to have more complete penetrance. Mutations occurring in different regions along the RB1 gene still need to be investigated in a study with an expanded sample size.

Incomplete detection rate

We reported a detection rate of 78.6% in our previous paper by using single DNA sequencing. Here, a method combining DNA sequencing and MLPA increased the detection rate to 92.3% in bilateral RB, which is consistent with an earlier study that reported that the RB1 mutation detection rate was 92.6% via a combination of full sequencing and deletions/duplication analysis of RB1 [30]. The remarkable enhancing of the effectiveness of detecting gross deletions attributable to MLPA was 20% (8/40)), which is slightly higher than in previous studies [31]. However, RB1 defects can also result from hypermethylation of the promoter region and different levels of mosaic mutations, as evidenced by reports that highly sensitive allele-specific PCR increased the sensitivity from 92.6% to 94.8% for detecting mosaic RB1 mutations [30]. Detecting methylation and mosaic mutations of RB1 would be interesting in future studies.

In conclusion, we developed the molecular detection project to optimize the clinical management of all patients with retinoblastoma. This study reports our results involving 31 patients who had been identified with null mutations and nine patients identified with in-frame mutations. The different groups had different clinical outcomes, and the type of mutations was related to age of onset and laterality, but not related to staging of the tumor, which has implications for genetic counseling. In addition, 20% of the total mutations were detected with MLPA, which confirms that combining sequencing and MLPA is a convenient, reliable, and powerful method for evaluating RB1 gene mutations in patients with retinoblastoma.