Cryptic splice site mutations in the non-coding regions of the ABCA4 gene in Stargardt disease patients

Background. Mutations in the ABCA4 gene cause Stargardt disease and other blinding autosomal recessive retinal disorders. However, sequencing of the complete coding sequence in patients with clinical features of Stargardt disease sometimes fails to detect both mutations required to cause the disease. It has previously been hypothesized that mutations near rare alternate splice junctions in ABCA4 might cause disease by increasing the probability of mis-splicing at these sites. Deep next-generation sequencing of RNA extracted from human donor eyes revealed 15 alternate exons that are weakly expressed in normal human retina. Cryptic intronic splice sites, activation of which by mutations might lead to the formation of ABCA4 disease causing alleles, have been identified. Objective. To analyze the cryptic splice sites in the noncoding regions of the ABCA4 gene in Russian patients with Stargardt disease. Material and methods. High throughput parallel semiconductor sequencing of all previously described minor exons was performed on peripheral blood cells DNA from 29 patients with Stargardt disease. Results. In the group of patients under study we have identified only genetic variants in the cryptic splice sites that represent common polymorphisms and that cannot thus be interpreted as disease causing mutations. Our cohort of the patients lacked apparently disease causing intronic nucleotide substitutions that were previously identified in the North American and European populations. Conclusion. Failure to identify ABCA4 intronic mutations in this study may be explained by low representation of patients with one or no exonic mutations in our cohort and does not compromise the utility of intron cryptic splice sites sequencing as a part of Stargardt disease molecular genetic diagnostics.

болезнь Штаргардта, ген ABCA4, высокопроизводительное параллельное секвенирование ДНК, альтернативный сплайсинг, Stargardt disease, ABCA4 gene, next generation DNA sequencing, alternative splicing

1. Гудзенко С.В., Хлебникова О.В., Беклемищева Н.А., Поляков А.В. ДНК-диагностика наследственных абиотрофий сетчатки, обусловленных мутациями в гене ABCA4 // Медицинская генетика. - 2006. - (9). - С. 37-40.

2. Schindler E.I., Nylen E.L., Ko A.C. et al. Deducing the pathogenic contribution of recessive ABCA4 alleles in an outbred population // Hum. Mol. Genet. - 2010. - Vol. (19). - Р. 3693-3701.

3. Sheffield V.C., Stone E.M. Genomics and the eye // N. Engl. J. Med. - 2011. - Vol. (364). - Р. 1932-1942.

4. Braun T.A., Mullins R.F., Wagner A.H. et al. Non-exomic and synonymous variants in ABCA4 are an important cause of Stargardt disease // Hum. Mol. Genet. - 2013. - Vol. 22(25). - Р. 5136-5145.

5. Zernant J., Schubert C., Im K.M. et al. Analysis of the ABCA4 gene by next-generation sequencing // Invest. Ophthalmol. Vis. Sci. - 2011. - Vol. (52). - Р. 8479-8487.

6. Webster A.R., Heon E., Lotery A.J. et al. An analysis of allelic variation in the ABCA4 gene // Invest. Ophthalmol. Vis. Sci. - 2001. - Vol. (42). - Р. 1179-1189.

7. Bauwens M., De Zaeytijd J., Weisschuh N. et al. An augmented ABCA4 screen targeting noncoding regions reveals a deep intronic founder variant in Belgian Stargardt patients // Human mutation. - 2015. - Vol. 36(1). - Р. 39-42.

8. Bax N.M., Sangermano R., Roosing S. et al. Heterozygous Deep-Intronic Variants and Deletions in ABCA4 in Persons with Retinal Dystrophies and One Exonic ABCA4 Variant // Human mutation. - 2015. - Vol. 36(1). - Р. 43-47.

9. September A.V., Vorster A.A., Ramesar R.S., Greenberg L.J. Mutation spectrum and founder chromosomes for the ABCA4 gene in South African patients with Stargardt disease // Investigative ophthalmology & visual science. - 2004. - Vol. 45(6). - Р. 1705-1711.

10. Chacon-Camacho O.F., Granillo-Alvarez M., Ayala-Ramirez R., Zenteno J.C. ABCA4 mutational spectrum in Mexican patients with Stargardt disease: Identification of 12 novel mutations and evidence of a founder effect for the common p. A1773V mutation // Experimental eye research. - 2013. - Vol. (109). - Р. 77-82.

11. Maugeri A., Flothmann K., Hemmrich N. et al. The ABCA4 2588G> C Stargardt mutation: single origin and increasing frequency from South-West to North-East Europe // Eur. J. Hum. Genet. - 2002. - Vol. 10(3). - Р. 197-203.