Screening for RB1 Gene Alleles in Saudi Retinoblastoma Patients

Abstract

Retinoblastoma (Rb) is the most common intraocular tumor which occurs in infantile retina and may result from germ-line or somatic mutation. In Saudi Arabia, Rb is diagnosed at an incidence of approximately 25 cases every year. However, there is no information on the nature of mutations in the RB1 gene. The major aim of this study was to identify germ line mutations in the RB1 gene in Saudi Rb patients by sequencing the entire RB1 gene. The study was conducted on 20 Rb patients and 20 normal healthy controls. In addition, 7 affected tissue and normal tissue samples were obtained from the same patients. The demographic data of each patient and control were recorded. Blood was used to extract DNA, and following polymerase chain reaction, the 27 exons of the RB1 gene were sequenced. Multiplex Ligation-dependent Probe Amplification (MLPA) studies were carried out to search for long deletions in the patients and control. Mutations were identified in 11/20 (55%) of the patients in the germ line and somatic cell DNA. In the germ line DNA the mutations identified were in exons 3, 4, 5, 18 and 19. These included small deletions resulting in frameshift (in exon 3: c.373delG; in exon 4: c.409delG; in exon 5: 504- 505delAT), base substitutions resulting in nonsense mutation and producing premature termination (in exon 18: c.1735C>T, p.R579X), and a point mutation resulting in substitution of an amino acid (c.1862G>A, p.R621H). A silent mutation was also identified (c.1917G>A). Five patients had large deletions. One of these has deletion of exons 6-9 and four patients had exon 24-26 deletions. The patients in whom RB1 mutation was not identified may be having a large deletion which could not be identified during sequencing. In the somatic cell DNA (from the tissue), the mutations identified were nonsense mutations resulting in premature termination (in exon 8: c.751C>T, p.R251X and in exon 14: c.1363C>T, p.R455X) small deletions (in exon 5: 504-505delAT); and a large deletion of exons 24-26. A silent mutation was also identified in the normal tissue. Of these mutations four were novel mutations and further studies are necessary to identify their effect on the functional aspects of the resultant Rb protein (pRb). In the intronic regions sequenced, ten single nucleotide polymorphisms (SNPs) were identified. These were in the introns 1, 2, 3, 4, 25 and 26. Two of the SNPs associated significantly with retinoblastoma. rs2252544 in intron 1, showed a statistically significant difference in the genotype frequency in the Rb patients and controls (p<0.05), while rs1981434 also in the intron 1 showed a highly significant (p<0.0001) difference in both the genotype and allele frequencies. In conclusion, sequencing was considered as an effective though expensive method for screening. It is suggested that there is a need to develop such screening methods that will identify the mutation without screening the entire gene in each Rb patient. Further studies are warranted to have a more clear view of RB1 mutations involved in Rb development in Saudis.