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. 1996 May;58(5):940–949.

The spectrum of RB1 germ-line mutations in hereditary retinoblastoma.

D R Lohmann 1, B Brandt 1, W Höpping 1, E Passarge 1, B Horsthemke 1
PMCID: PMC1914612  PMID: 8651278

Abstract

We have searched for germ-line RB1 mutations in 119 patients with hereditary retinoblastoma. Previous investigations by Southern blot hybridization and PCR fragment-length analysis had revealed mutations in 48 patients. Here we report on the analysis of the remaining 71 patients. By applying heteroduplex analysis, nonisotopic SSCP, and direct sequencing, we detected germ-line mutations resulting in premature termination codons or disruption of splice signals in 51 (72%) of the 71 patients. Four patients also showed rare sequence variants. No region of the RB1 gene was preferentially involved in single base substitutions. Recurrent transitions were observed at most of the 14 codons within the RB1. No mutation was observed in exons 25-27, although this region contains two CGA codons. This suggests that mutations within the 3'-terminal region of the RB1 gene may not be oncogenic. When these data were combined with the results of our previous investigations, mutations were identified in a total of 99 (83%) of 119 patients. The spectrum comprises 15% large deletions, 26% small length alterations, and 42 % base substitutions. No correlation between the location of frameshift or nonsense mutations and phenotypic features, including age at diagnosis, the number of tumor foci, and manifestation of nonocular tumors was observed.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Blanquet V., Créau-Goldberg N., de Grouchy J., Turleau C. Molecular detection of constitutional deletions in patients with retinoblastoma. Am J Med Genet. 1991 Jun 1;39(3):355–361. doi: 10.1002/ajmg.1320390321. [DOI] [PubMed] [Google Scholar]
  2. Blanquet V., Gross M. S., Turleau C., Sénamaud-Beaufort C., Doz F., Besmond C. Three novel germline mutations in exons 8 and 18 of the retinoblastoma gene. Hum Mol Genet. 1994 Jul;3(7):1185–1186. doi: 10.1093/hmg/3.7.1185. [DOI] [PubMed] [Google Scholar]
  3. Blanquet V., Turleau C., Gross-Morand M. S., Sénamaud-Beaufort C., Doz F., Besmond C. Spectrum of germline mutations in the RB1 gene: a study of 232 patients with hereditary and non hereditary retinoblastoma. Hum Mol Genet. 1995 Mar;4(3):383–388. doi: 10.1093/hmg/4.3.383. [DOI] [PubMed] [Google Scholar]
  4. Blanquet V., Turleau C., Gross M. S., Goossens M., Besmond C. Identification of germline mutations in the RB1 gene by denaturant gradient gel electrophoresis and polymerase chain reaction direct sequencing. Hum Mol Genet. 1993 Jul;2(7):975–979. doi: 10.1093/hmg/2.7.975. [DOI] [PubMed] [Google Scholar]
  5. Cavenee W. K., Dryja T. P., Phillips R. A., Benedict W. F., Godbout R., Gallie B. L., Murphree A. L., Strong L. C., White R. L. Expression of recessive alleles by chromosomal mechanisms in retinoblastoma. 1983 Oct 27-Nov 2Nature. 305(5937):779–784. doi: 10.1038/305779a0. [DOI] [PubMed] [Google Scholar]
  6. Chillón M., Dörk T., Casals T., Giménez J., Fonknechten N., Will K., Ramos D., Nunes V., Estivill X. A novel donor splice site in intron 11 of the CFTR gene, created by mutation 1811+1.6kbA-->G, produces a new exon: high frequency in Spanish cystic fibrosis chromosomes and association with severe phenotype. Am J Hum Genet. 1995 Mar;56(3):623–629. [PMC free article] [PubMed] [Google Scholar]
  7. Chu C. S., Trapnell B. C., Curristin S., Cutting G. R., Crystal R. G. Genetic basis of variable exon 9 skipping in cystic fibrosis transmembrane conductance regulator mRNA. Nat Genet. 1993 Feb;3(2):151–156. doi: 10.1038/ng0293-151. [DOI] [PubMed] [Google Scholar]
  8. Cooper D. N., Krawczak M. The mutational spectrum of single base-pair substitutions causing human genetic disease: patterns and predictions. Hum Genet. 1990 Jun;85(1):55–74. doi: 10.1007/BF00276326. [DOI] [PubMed] [Google Scholar]
  9. Cowell J. K., Smith T., Bia B. Frequent constitutional C to T mutations in CGA-arginine codons in the RB1 gene produce premature stop codons in patients with bilateral (hereditary) retinoblastoma. Eur J Hum Genet. 1994;2(4):281–290. doi: 10.1159/000472372. [DOI] [PubMed] [Google Scholar]
  10. Dunn J. M., Phillips R. A., Zhu X., Becker A., Gallie B. L. Mutations in the RB1 gene and their effects on transcription. Mol Cell Biol. 1989 Nov;9(11):4596–4604. doi: 10.1128/mcb.9.11.4596. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Friend S. H., Bernards R., Rogelj S., Weinberg R. A., Rapaport J. M., Albert D. M., Dryja T. P. A human DNA segment with properties of the gene that predisposes to retinoblastoma and osteosarcoma. Nature. 1986 Oct 16;323(6089):643–646. doi: 10.1038/323643a0. [DOI] [PubMed] [Google Scholar]
  12. Ganguly A., Rock M. J., Prockop D. J. Conformation-sensitive gel electrophoresis for rapid detection of single-base differences in double-stranded PCR products and DNA fragments: evidence for solvent-induced bends in DNA heteroduplexes. Proc Natl Acad Sci U S A. 1993 Nov 1;90(21):10325–10329. doi: 10.1073/pnas.90.21.10325. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Greger V., Debus N., Lohmann D., Höpping W., Passarge E., Horsthemke B. Frequency and parental origin of hypermethylated RB1 alleles in retinoblastoma. Hum Genet. 1994 Nov;94(5):491–496. doi: 10.1007/BF00211013. [DOI] [PubMed] [Google Scholar]
  14. Greger V., Passarge E., Höpping W., Messmer E., Horsthemke B. Epigenetic changes may contribute to the formation and spontaneous regression of retinoblastoma. Hum Genet. 1989 Sep;83(2):155–158. doi: 10.1007/BF00286709. [DOI] [PubMed] [Google Scholar]
  15. Hamel P. A., Gallie B. L., Phillips R. A. The retinoblastoma protein and cell cycle regulation. Trends Genet. 1992 May;8(5):180–185. doi: 10.1016/0168-9525(92)90221-o. [DOI] [PubMed] [Google Scholar]
  16. Hashimoto T., Takahashi R., Yandell D. W., Xu H. J., Hu S. X., Gunnell S., Benedict W. F. Characterization of intragenic deletions in two sporadic germinal mutation cases of retinoblastoma resulting in abnormal gene expression. Oncogene. 1991 Mar;6(3):463–469. [PubMed] [Google Scholar]
  17. Hogg A., Bia B., Onadim Z., Cowell J. K. Molecular mechanisms of oncogenic mutations in tumors from patients with bilateral and unilateral retinoblastoma. Proc Natl Acad Sci U S A. 1993 Aug 1;90(15):7351–7355. doi: 10.1073/pnas.90.15.7351. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Hogg A., Onadim Z., Baird P. N., Cowell J. K. Detection of heterozygous mutations in the RB1 gene in retinoblastoma patients using single-strand conformation polymorphism analysis and polymerase chain reaction sequencing. Oncogene. 1992 Jul;7(7):1445–1451. [PubMed] [Google Scholar]
  19. Hu Q. J., Dyson N., Harlow E. The regions of the retinoblastoma protein needed for binding to adenovirus E1A or SV40 large T antigen are common sites for mutations. EMBO J. 1990 Apr;9(4):1147–1155. doi: 10.1002/j.1460-2075.1990.tb08221.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Huang S., Wang N. P., Tseng B. Y., Lee W. H., Lee E. H. Two distinct and frequently mutated regions of retinoblastoma protein are required for binding to SV40 T antigen. EMBO J. 1990 Jun;9(6):1815–1822. doi: 10.1002/j.1460-2075.1990.tb08306.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Huie M. L., Chen A. S., Tsujino S., Shanske S., DiMauro S., Engel A. G., Hirschhorn R. Aberrant splicing in adult onset glycogen storage disease type II (GSDII): molecular identification of an IVS1 (-13T-->G) mutation in a majority of patients and a novel IVS10 (+1GT-->CT) mutation. Hum Mol Genet. 1994 Dec;3(12):2231–2236. doi: 10.1093/hmg/3.12.2231. [DOI] [PubMed] [Google Scholar]
  22. Kloss K., Währisch P., Greger V., Messmer E., Fritze H., Höpping W., Passarge E., Horsthemke B. Characterization of deletions at the retinoblastoma locus in patients with bilateral retinoblastoma. Am J Med Genet. 1991 May 1;39(2):196–200. doi: 10.1002/ajmg.1320390215. [DOI] [PubMed] [Google Scholar]
  23. Knudson A. G., Jr Mutation and cancer: statistical study of retinoblastoma. Proc Natl Acad Sci U S A. 1971 Apr;68(4):820–823. doi: 10.1073/pnas.68.4.820. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Krawczak M., Reiss J., Cooper D. N. The mutational spectrum of single base-pair substitutions in mRNA splice junctions of human genes: causes and consequences. Hum Genet. 1992 Sep-Oct;90(1-2):41–54. doi: 10.1007/BF00210743. [DOI] [PubMed] [Google Scholar]
  25. Lohmann D. R., Brandt B., Höpping W., Passarge E., Horsthemke B. Spectrum of small length germline mutations in the RB1 gene. Hum Mol Genet. 1994 Dec;3(12):2187–2193. doi: 10.1093/hmg/3.12.2187. [DOI] [PubMed] [Google Scholar]
  26. Lohmann D., Horsthemke B., Gillessen-Kaesbach G., Stefani F. H., Höfler H. Detection of small RB1 gene deletions in retinoblastoma by multiplex PCR and high-resolution gel electrophoresis. Hum Genet. 1992 Apr;89(1):49–53. doi: 10.1007/BF00207041. [DOI] [PubMed] [Google Scholar]
  27. Nakai K., Sakamoto H. Construction of a novel database containing aberrant splicing mutations of mammalian genes. Gene. 1994 Apr 20;141(2):171–177. doi: 10.1016/0378-1119(94)90567-3. [DOI] [PubMed] [Google Scholar]
  28. Onadim Z., Hogg A., Baird P. N., Cowell J. K. Oncogenic point mutations in exon 20 of the RB1 gene in families showing incomplete penetrance and mild expression of the retinoblastoma phenotype. Proc Natl Acad Sci U S A. 1992 Jul 1;89(13):6177–6181. doi: 10.1073/pnas.89.13.6177. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Sakai T., Ohtani N., McGee T. L., Robbins P. D., Dryja T. P. Oncogenic germ-line mutations in Sp1 and ATF sites in the human retinoblastoma gene. Nature. 1991 Sep 5;353(6339):83–86. doi: 10.1038/353083a0. [DOI] [PubMed] [Google Scholar]
  30. Sakai T., Toguchida J., Ohtani N., Yandell D. W., Rapaport J. M., Dryja T. P. Allele-specific hypermethylation of the retinoblastoma tumor-suppressor gene. Am J Hum Genet. 1991 May;48(5):880–888. [PMC free article] [PubMed] [Google Scholar]
  31. Shapiro M. B., Senapathy P. RNA splice junctions of different classes of eukaryotes: sequence statistics and functional implications in gene expression. Nucleic Acids Res. 1987 Sep 11;15(17):7155–7174. doi: 10.1093/nar/15.17.7155. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Shimizu T., Toguchida J., Kato M. V., Kaneko A., Ishizaki K., Sasaki M. S. Detection of mutations of the RB1 gene in retinoblastoma patients by using exon-by-exon PCR-SSCP analysis. Am J Hum Genet. 1994 May;54(5):793–800. [PMC free article] [PubMed] [Google Scholar]
  33. Stirdivant S. M., Ahern J. D., Oliff A., Heimbrook D. C. Retinoblastoma protein binding properties are dependent on 4 cysteine residues in the protein binding pocket. J Biol Chem. 1992 Jul 25;267(21):14846–14851. [PubMed] [Google Scholar]
  34. Toguchida J., McGee T. L., Paterson J. C., Eagle J. R., Tucker S., Yandell D. W., Dryja T. P. Complete genomic sequence of the human retinoblastoma susceptibility gene. Genomics. 1993 Sep;17(3):535–543. doi: 10.1006/geno.1993.1368. [DOI] [PubMed] [Google Scholar]
  35. Weir-Thompson E., Condie A., Leonard R. C., Prosser J. A familial RB1 mutation detected by the HOT technique is homozygous in a second primary neoplasm. Oncogene. 1991 Dec;6(12):2353–2356. [PubMed] [Google Scholar]
  36. Yandell D. W., Campbell T. A., Dayton S. H., Petersen R., Walton D., Little J. B., McConkie-Rosell A., Buckley E. G., Dryja T. P. Oncogenic point mutations in the human retinoblastoma gene: their application to genetic counseling. N Engl J Med. 1989 Dec 21;321(25):1689–1695. doi: 10.1056/NEJM198912213212501. [DOI] [PubMed] [Google Scholar]
  37. Youssoufian H., Antonarakis S. E., Bell W., Griffin A. M., Kazazian H. H., Jr Nonsense and missense mutations in hemophilia A: estimate of the relative mutation rate at CG dinucleotides. Am J Hum Genet. 1988 May;42(5):718–725. [PMC free article] [PubMed] [Google Scholar]
  38. Youssoufian H., Kazazian H. H., Jr, Phillips D. G., Aronis S., Tsiftis G., Brown V. A., Antonarakis S. E. Recurrent mutations in haemophilia A give evidence for CpG mutation hotspots. 1986 Nov 27-Dec 3Nature. 324(6095):380–382. doi: 10.1038/324380a0. [DOI] [PubMed] [Google Scholar]

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