Advantages of a next generation sequencing targeted approach for the molecular diagnosis of retinoblastoma

The molecular diagnosis of RB is a complex and articulate process that still represents an exciting challenge. Many resources and skills need to be involved to obtain satisfactory results. High-throughput technologies can actually offer new opportunities in relation to the amount of genes potentially analyzed, the number of samples examined and the quality of results. NGS is an innovative technology that is able to massive-parallel sequence millions of DNA segments with high definition capability. It has a wide diffusion in many fields of biomedical research, but diagnostic applications for genetic diseases are still in progress. We report our experience on a cohort of RB patients using a NGS approach on the Illumina MiSeq platform. The experiments required different timelines. The design of the target regions of RB1, carried out using DS, was performed in few hours. The preparation of the genomic library using the TSCA Illumina kit, was completed in two working days. One or two days were spent to run the samples on the MiSeq (48 samples were run all together in a first sequencing run using Panel A and the remaining 5 were run on a second experiment using Panel B). Few more days were required for results interpretation of the 53 RB patients using MiSeqReporter, AV and IGV2.3 software. Furthermore, all mutations identified by Miseq Reporter, were validated by Sanger sequencing using standard protocols. Of the two panels designed, Panel B has allowed to reach the full coverage of the target region, making the standard Sanger sequencing only a tool for confirming all detected variants. It was also calculated that the cost of NGS analysis for the entire RB1 gene, considering comparable devices cost, reagents expenses, operator’s worktime, would be 7 times less than the cost of a protocol entirely based on Sanger sequencing, allowing a strong decrease in costs and a large increase in the number of samples processed for each experiment [9, 17, 18].

NGS has allowed identifying all variants found in patients with BRB except one sample in which the variant was identified neither by Sanger nor by NGS sequencing. In this case we can speculate that the variant may be located outside the region under investigation. In fact, literature data show that 5 % of cases with bilateral involvement may have translocations, deep intronic splice site mutations, or low-level mosaic mutations, which may or may not be germline [8]. Twenty-four mutations were identified in the patients with RB: twelve nonsense, five frameshift and seven splice site mutations. As expected, eleven out of the twenty-four mutations found were newly discovered mutations, never reported before. Among these a rare case of trilateral RB with a new frameshift mutation in exon 2 was identified, differently from the current data reporting macroscopic deletions as the most frequent defects in this unusual disease [19‐22]. The nonsense mutation p.Arg787X was a known sequence variation found in the group of URB. The carrier was a female presenting, at the age of 17 months, with a left eye RB with loco-regional metastasis also involving lymphnodes and bone marrow. She was eye enucleated and treated with conventional and high-dose chemotherapy, followed by autologous bone marrow transplantation and radiotherapy. To date, she is alive and in good clinical conditions. p.Arg787X is a recurrent mutation commonly found in BRB as germline sequence variation, while in URB is more frequent as somatic mutation. Only four cases of URB carrying this germline mutation have been reported [23] including a patient with metastatic presentation [24]. These findings suggest that the phenotypic expression of p.Arg787X may reflect the variable penetrance of this defect, leading to the different pictures of the disease. Among the genomic abnormalities identified with RB1-custom aCGH method, four intragenic rearrangements and six large deletions involving genes adjacent to RB1 were revealed. Interestingly, the patients belonging to the first group had BRB, while the patients of the second group had mainly URB. These data fortify the hypothesis that deletion of genes essential for cell survival, adjacent to RB1, may cause less invasive tumors and, therefore, result in a higher frequency of unilateral disease [25, 26]. Patients with deletions greater than 5.7 Mb showed syndromic features with variable degree of intellectual disability ranging from moderate to severe. Patients with deletions smaller than 1.74 Mb had only RB. An unexpected exception was the case of the proband with BRB carrying an intragenic duplication from exon 18 to 23, lasting about 66 Kb. This patient presented with a clinical syndromic picture, characterized by macrosomia, nystagmus of the eye, macrocephaly and macroglossia evocative of the Beckwith-Wiedeman Syndrome. Molecular investigations revealed a normal methylation status and absence of microdeletions at the locus 11p15.5. Array-CGH did not show any genomic imbalances. In this cohort, 8 out 36 URB patients resulted positive either to NGS or aCGH, corresponding to a 22 % frequency. URB mutations are in fact infrequently found (15 %) in blood circulating cells in relationship to the known prevalence of somatic mutation in the target tissue. The 22 % frequency here reported is slightly different from what is reported in literature, however, the small number of patients in this cohort is not enough to establish a significant frequency reference.

Mutational mosaicism is an exciting challenge regarding molecular diagnostics as well as it is important in the genetic counseling setting. Low levels of mutational mosaicism have been identified in probands with bilateral disease and in individuals with unilateral disease who have affected children inheriting the mutation [8, 27]. Conventional investigations are unable to routinely detect low-rate mutated cells: currently, Sanger sequencing is able to disclose mosaicism only for rates above 20 %. Targeted mutation analysis is useful to study mosaic recurrent mutations in blood and can detect DNA variations below the limit of standard Sanger sequence analysis. This type of analysis, based on Allele Specific PCR (AS-PCR), however, investigates, only a limited number of recurrent point mutations [26]. A more recent study demonstrated that, using a deep semiconductor sequencing approach (Ion Torrent, Life Technology), the detection rate of targeted mutational mosaics can be revealed at a frequency down to 5 % [28]. In our study the capability of NGS in detecting low mosaicism frequency has been tested. Due to the absence of patients with RB1 mosaicism, three previously recognized samples, carriers of single-base substitution, single-base insertion and a complex rearrangement involving five-base and one-base double deletion respectively, were diluted with normal DNA at different concentration (10, 5, 1 %) and tested by NGS with MiSeq platform. As reported, all three mutations have been correctly detected at each different frequency for both coverage levels, independently of the variant type. When leading studies aimed at identifying low mosaicism frequencies, the major difficulty lies in accurately discriminating between a somatic variant and a false positive episode. Based on this, for all three studied mutations, it has been evaluated the frequency of false positive calls measured as the percentage of erroneously called bases at the target site. As shown, for all three types of variants studied, the frequency of false positive events has always been between 0 and 0.02 % for both sequencing runs. In particular, for the high coverage sequencing run, the false positive events never exceeded 0.02 %, far below the 1 % mosaicism variant frequency detected. This achievement, accompanied by a good coverage of the region of interest can accurately detect low mosaicism frequencies in biological samples, providing a reliable and sensitive method of screening. Validation experiments on mosaic biological samples are currently in progress.