Deep-intronic ABCA4 variants explain missing heritability in Stargardt disease and allow correction of splice defects by antisense oligonucleotides.,Genetics in Medicine

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Deep-intronic ABCA4 variants explain missing heritability in Stargardt disease and allow correction of splice defects by antisense oligonucleotides.
Genetics in Medicine ( IF 6.6 ) Pub Date : 2019-01-15 , DOI: 10.1038/s41436-018-0414-9
Riccardo Sangermano _{1,

2} , Alejandro Garanto _{1,

3} , Mubeen Khan _{1,

3} , Esmee H Runhart _{3,

4} , Miriam Bauwens ₅ , Nathalie M Bax _{3,

4} , L Ingeborgh van den Born ₆ , Muhammad Imran Khan _{1,

2} , Stéphanie S Cornelis _{1,

3} , Joke B G M Verheij ₇ , Jan-Willem R Pott ₈ , Alberta A H J Thiadens ₉ , Caroline C W Klaver _{4,

9} , Bernard Puech ₁₀ , Isabelle Meunier _{11,

12} , Sarah Naessens ₅ , Gavin Arno _{13,

14} , Ana Fakin _{13,

14} , Keren J Carss _{15,

16} , F Lucy Raymond _{16,

17} , Andrew R Webster _{13,

14} , Claire-Marie Dhaenens ₁₈ , Heidi Stöhr ₁₉ , Felix Grassmann _{19,

20} , Bernhard H F Weber ₁₉ , Carel B Hoyng _{3,

4} , Elfride De Baere ₅ , Silvia Albert _{1,

3} , Rob W J Collin _{1,

3} , Frans P M Cremers _{1,

3}

Affiliation

Department of Human Genetics, Radboud University Medical Center, Nijmegen, The Netherlands.
Radboud Institute for Molecular Life Sciences, Radboud University Medical Center, Nijmegen, The Netherlands.
Donders Institute for Brain, Cognition and Behaviour, Radboud University Medical Center, Nijmegen, The Netherlands.
Department of Ophthalmology, Radboud University Medical Center, Nijmegen, The Netherlands.
Center for Medical Genetics, Ghent University and Ghent University Hospital, Ghent, Belgium.
The Rotterdam Eye Hospital and the Rotterdam Ophthalmic Institute, Rotterdam, The Netherlands.
Department of Medical Genetics, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands.
Department of Ophthalmology, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands.
Department of Ophthalmology, Erasmus Medical Center, Rotterdam, The Netherlands.
Service d'Exploration de la Vision CHU, Lille, France.
Institute for Neurosciences of Montpellier INSERM U1051, University of Montpellier, Montpellier, France.
Centre d'Etude du Polymorphisme Humain, Fondation Jean Dausset, Paris, France.
UCL Institute of Ophthalmology, London, UK.
Moorfields Eye Hospital, London, UK.
Department of Haematology, University of Cambridge, Cambridge, UK.
NIHR BioResource, Cambridge University Hospitals NHS Foundation Trust, Cambridge Biomedical Campus, Cambridge, UK.
Department of Medical Genetics, Cambridge Institute for Medical Research, University of Cambridge, Cambridge, UK.
Univ. Lille, Inserm UMR-S 1172, CHU Lille, Biochemistry and Molecular Biology Department - UF Génopathies, Lille, France.
Institut für Humangenetik, Universität Regensburg, Regensburg, Germany.
Department of Medical Epidemiology and Biostatistics, Karolinska Institutet, Stockholm, Sweden.

PURPOSE Using exome sequencing, the underlying variants in many persons with autosomal recessive diseases remain undetected. We explored autosomal recessive Stargardt disease (STGD1) as a model to identify the missing heritability. METHODS Sequencing of ABCA4 was performed in 8 STGD1 cases with one variant and p.Asn1868Ile in trans, 25 cases with one variant, and 3 cases with no ABCA4 variant. The effect of intronic variants was analyzed using in vitro splice assays in HEK293T cells and patient-derived fibroblasts. Antisense oligonucleotides were used to correct splice defects. RESULTS In 24 of the probands (67%), one known and five novel deep-intronic variants were found. The five novel variants resulted in messenger RNA pseudoexon inclusions, due to strengthening of cryptic splice sites or by disrupting a splicing silencer motif. Variant c.769-784C>T showed partial insertion of a pseudoexon and was found in cis with c.5603A>T (p.Asn1868Ile), so its causal role could not be fully established. Variant c.4253+43G>A resulted in partial skipping of exon 28. Remarkably, antisense oligonucleotides targeting the aberrant splice processes resulted in (partial) correction of all splicing defects. CONCLUSION Our data demonstrate the importance of assessing noncoding variants in genetic diseases, and show the great potential of splice modulation therapy for deep-intronic variants.

中文翻译：

深内含子 ABCA4 变体解释了 Stargardt 病中缺失的遗传力，并允许通过反义寡核苷酸纠正剪接缺陷。

目的使用外显子组测序，许多常染色体隐性遗传病患者的潜在变异仍未被发现。我们探索了常染色体隐性遗传 Stargardt 病 (STGD1) 作为识别缺失遗传力的模型。方法对8例STGD1有1个变异和p.Asn1868Ile反式进行ABCA4测序，25例有1个变异，3例无ABCA4变异。在 HEK293T 细胞和患者来源的成纤维细胞中使用体外剪接试验分析了内含子变体的影响。反义寡核苷酸用于纠正剪接缺陷。结果在 24 名先证者 (67%) 中，发现了一种已知的和五种新的深内含子变异。由于隐蔽剪接位点的加强或通过破坏剪接沉默基序，这五种新变体导致信使 RNA 假外显子包涵体。变体 C. 769-784C>T 显示部分插入了假外显子，并且在顺式中发现了 c.5603A>T (p.Asn1868Ile)，因此无法完全确定其因果关系。变体 c.4253+43G>A 导致外显子 28 的部分跳跃。值得注意的是，针对异常剪接过程的反义寡核苷酸导致所有剪接缺陷的（部分）校正。结论我们的数据证明了评估非编码变异在遗传疾病中的重要性，并显示了剪接调制疗法对深内含子变异的巨大潜力。针对异常剪接过程的反义寡核苷酸导致所有剪接缺陷的（部分）校正。结论我们的数据证明了评估非编码变异在遗传疾病中的重要性，并显示了剪接调制疗法对深内含子变异的巨大潜力。针对异常剪接过程的反义寡核苷酸导致所有剪接缺陷的（部分）校正。结论我们的数据证明了评估遗传疾病中非编码变异的重要性，并显示了剪接调制疗法对深内含子变异的巨大潜力。

更新日期：2019-01-26

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