July 2019
Volume 60, Issue 9
Free
ARVO Annual Meeting Abstract  |   July 2019
Targeted identification of reported deep-intronic variants in ABCA4 in 224 French Stargardt disease cases
Author Affiliations & Notes
  • Claire-Marie DHAENENS
    University Lille, Inserm UMR-S 1172, CHU Lille, Biochemistry and Molecular Biology Department - UF Génopathies,, Lille, France
  • Mubeen Khan
    Department of Human Genetics, Radboud University Medical Center, Nijmegen, Netherlands
  • Aurore Devos
    University Lille, Inserm UMR-S 1172, CHU Lille, Biochemistry and Molecular Biology Department - UF Génopathies,, Lille, France
  • Charlene Piriou
    University Lille, Inserm UMR-S 1172, CHU Lille, Biochemistry and Molecular Biology Department - UF Génopathies,, Lille, France
  • Duaa ELMELIK
    Department of Human Genetics, Radboud University Medical Center, Nijmegen, Netherlands
  • Eline Manders
    Department of Human Genetics, Radboud University Medical Center, Nijmegen, Netherlands
  • Emeline Goreki
    University Lille, Inserm UMR-S 1172, CHU Lille, Biochemistry and Molecular Biology Department - UF Génopathies,, Lille, France
  • Yaumara Perdomo
    Centre de Référence pour les affections rares en génétique ophtalmologique, CARGO, Filière Sensgene Hôpitaux Universitaires de Strasbourg, Strasbourg, France
  • Helene Dollfus
    Centre de Référence pour les affections rares en génétique ophtalmologique, CARGO, Filière Sensgene Hôpitaux Universitaires de Strasbourg, Strasbourg, France
  • Xavier Zanlonghi
    Clinique Jules Verne, Nantes, France
  • Béatrice Bocquet
    Institut des Neurosciences de Montpellier, INSERM, Université de Montpellier, Montpellier, France
  • Isabelle Anne Meunier
    Institut des Neurosciences de Montpellier, INSERM, Université de Montpellier, Montpellier, France
  • Bernard Puech
    Service d’exploration de la vision et neuro-ophtalmologie, CHRU de Lille, Lille, France
  • Sabine defoort
    Service d’exploration de la vision et neuro-ophtalmologie, CHRU de Lille, Lille, France
  • Frans P Cremers
    Department of Human Genetics, Radboud University Medical Center, Nijmegen, Netherlands
  • Footnotes
    Commercial Relationships   Claire-Marie DHAENENS, None; Mubeen Khan, None; Aurore Devos, None; Charlene Piriou, None; Duaa ELMELIK, None; Eline Manders, None; Emeline Goreki, None; Yaumara Perdomo, None; Helene Dollfus, None; Xavier Zanlonghi, None; Béatrice Bocquet, None; Isabelle Meunier, None; Bernard Puech, None; Sabine defoort, None; Frans Cremers, None
  • Footnotes
    Support  Retina UK grant GR59
Investigative Ophthalmology & Visual Science July 2019, Vol.60, 4930. doi:
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      Claire-Marie DHAENENS, Mubeen Khan, Aurore Devos, Charlene Piriou, Duaa ELMELIK, Eline Manders, Emeline Goreki, Yaumara Perdomo, Helene Dollfus, Xavier Zanlonghi, Béatrice Bocquet, Isabelle Anne Meunier, Bernard Puech, Sabine defoort, Frans P Cremers; Targeted identification of reported deep-intronic variants in ABCA4 in 224 French Stargardt disease cases. Invest. Ophthalmol. Vis. Sci. 2019;60(9):4930.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose : In the last two decades, ABCA4 has been tested in 1087 French Stargardt disease (STGD1) cases by employing high-throughput analyses. Many remained unsolved because the two causal mutations in ABCA4 gene were not found. Recently, a frequent coding (p.Asn1868Ile) and a non-canonical splice site variant (c.5461-10T>C), were deemed causative. In addition, several causal deep-intronic variants were recently identified. Therefore, we aimed to search for these variants in 224 French STGD1 cases to improve the genetic diagnosis for patients in Lille.

Methods : We ascertained 135 monoallelic STGD1 probands and 89 STGD1 cases with no ABCA4 variants. The 50 ABCA4 exons and 12 selected intronic regions were sequenced using 513 single molecule inversion probes (smMIPs). The smMIPs were pooled, phosphorylated and hybridized to 100 ng of genomic DNA of each patient. Sequencing analysis was performed using one NextSeq 500 high output module. Novel non-canonical splice site and deep-intronic variants were tested for splice defects using in vitrosplice assays in HEK393T cells employing ABCA4 midigenes.

Results : smMIPs-based sequencing solved 71 (31.7%) STGD1 cases and found one ABCA4 variant in 63 (28.1%) probands. 31 (13.8%) probands carried p.(Asn1868Ile) in trans with a severe mutation and are likely solved. The latter showed a late age at onset (average 47.8 yrs) and consisted of more females (22/9, 70.9%) than males. In addition, we found 42 deep intronic variants in 40 (17.8%) probands of which c.4253+43G>A (n=17) and c.5196+1137G>A (n=13) were the most frequent. A novel deep-intronic variant, c.4539+2065C>G, showed a 170-nt pseudoexon insertion in the mRNA when tested in ABCA4 midigenes. Similarly, three novel non-canonical splice-site variants, c.4128G>C, c.4539G>A and c.4849G>A led to splice defects, i.e. a 12-nt exon 27 elongation and skipping of exon 30 and 35, respectively.

Conclusions : By employing a smMIPs-based sequencing method, we could (likely) solve ~45.5% of previously genotyped STGD1 probands. The high proportion (17.8%) of STGD1 cases with deep intronic variants highlights the relevance to investigate ABCA4i ntronic regions in standard genotyping. The smMIPs platform is very cost-effective and proved its efficiency for the analysis of coding and intronic regions. In follow-up studies, the entire ABCA4 gene will be sequenced employing smMIPs.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.

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