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

Riccardo Sangermano; Alejandro Garanto; Mubeen Khan; Esmee H Runhart; Miriam Bauwens; Nathalie M Bax; L Ingeborgh van den Born; Muhammad Imran Khan; Stéphanie S Cornelis; Joke B G M Verheij; Jan-Willem R Pott; Alberta A H J Thiadens; Caroline C W Klaver; Bernard Puech; Isabelle Meunier; Sarah Naessens; Gavin Arno; Ana Fakin; Keren J Carss; F Lucy Raymond; Andrew R Webster; Claire-Marie Dhaenens; Heidi Stöhr; Felix Grassmann; Bernhard H F Weber; Carel B Hoyng; Elfride De Baere; Silvia Albert; Rob W J Collin; Frans P M Cremers

doi:10.1038/s41436-018-0414-9

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

Riccardo Sangermano, Alejandro Garanto, Mubeen Khan, Esmee H Runhart, Miriam Bauwens, Nathalie M Bax, L Ingeborgh van den Born, Muhammad Imran Khan, Stéphanie S Cornelis, Joke B G M Verheij, Jan-Willem R Pott, Alberta A H J Thiadens, Caroline C W Klaver, Bernard Puech, Isabelle Meunier, Sarah Naessens, Gavin Arno, Ana Fakin, Keren J Carss, F Lucy RaymondAndrew R Webster, Claire-Marie Dhaenens, Heidi Stöhr, Felix Grassmann, Bernhard H F Weber, Carel B Hoyng, Elfride De Baere, Silvia Albert, Rob W J Collin, Frans P M Cremers

Medical retina and Uveitis

Research output: Contribution to journal › Article › Research › peer-review

Abstract

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.

Original language	English
Pages (from-to)	1751-1760
Number of pages	10
Journal	Genetics in medicine : official journal of the American College of Medical Genetics
Volume	21
Issue number	8
DOIs	https://doi.org/10.1038/s41436-018-0414-9
Publication status	Published - Aug 2019

Keywords

ATP-Binding Cassette Transporters/genetics
Adolescent
Adult
Aged
Child
Exons/genetics
HEK293 Cells
Humans
Introns/genetics
Middle Aged
Mutation/genetics
Oligonucleotides, Antisense/genetics
Pedigree
Polymorphism, Single Nucleotide/genetics
Protein Isoforms/genetics
RNA Splicing/genetics
Stargardt Disease/genetics
Young Adult

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10.1038/s41436-018-0414-9Licence: CC BY

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Sangermano, R., Garanto, A., Khan, M., Runhart, E. H., Bauwens, M., Bax, N. M., van den Born, L. I., Khan, M. I., Cornelis, S. S., Verheij, J. B. G. M., Pott, J-W. R., Thiadens, A. A. H. J., Klaver, C. C. W., Puech, B., Meunier, I., Naessens, S., Arno, G., Fakin, A., Carss, K. J., ... Cremers, F. P. M. (2019). Deep-intronic ABCA4 variants explain missing heritability in Stargardt disease and allow correction of splice defects by antisense oligonucleotides. Genetics in medicine : official journal of the American College of Medical Genetics, 21(8), 1751-1760. https://doi.org/10.1038/s41436-018-0414-9

@article{c5c5d59d903b42a5b47ec71f3a0cf6f3,

title = "Deep-intronic ABCA4 variants explain missing heritability in Stargardt disease and allow correction of splice defects by antisense oligonucleotides",

abstract = "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.",

keywords = "ATP-Binding Cassette Transporters/genetics, Adolescent, Adult, Aged, Child, Exons/genetics, HEK293 Cells, Humans, Introns/genetics, Middle Aged, Mutation/genetics, Oligonucleotides, Antisense/genetics, Pedigree, Polymorphism, Single Nucleotide/genetics, Protein Isoforms/genetics, RNA Splicing/genetics, Stargardt Disease/genetics, Young Adult",

author = "Riccardo Sangermano and Alejandro Garanto and Mubeen Khan and Runhart, {Esmee H} and Miriam Bauwens and Bax, {Nathalie M} and {van den Born}, {L Ingeborgh} and Khan, {Muhammad Imran} and Cornelis, {St{\'e}phanie S} and Verheij, {Joke B G M} and Pott, {Jan-Willem R} and Thiadens, {Alberta A H J} and Klaver, {Caroline C W} and Bernard Puech and Isabelle Meunier and Sarah Naessens and Gavin Arno and Ana Fakin and Carss, {Keren J} and Raymond, {F Lucy} and Webster, {Andrew R} and Claire-Marie Dhaenens and Heidi St{\"o}hr and Felix Grassmann and Weber, {Bernhard H F} and Hoyng, {Carel B} and {De Baere}, Elfride and Silvia Albert and Collin, {Rob W J} and Cremers, {Frans P M}",

year = "2019",

month = aug,

doi = "10.1038/s41436-018-0414-9",

language = "English",

volume = "21",

pages = "1751--1760",

number = "8",

}

Sangermano, R, Garanto, A, Khan, M, Runhart, EH, Bauwens, M, Bax, NM, van den Born, LI, Khan, MI, Cornelis, SS, Verheij, JBGM, Pott, J-WR, Thiadens, AAHJ, Klaver, CCW, Puech, B, Meunier, I, Naessens, S, Arno, G, Fakin, A, Carss, KJ, Raymond, FL, Webster, AR, Dhaenens, C-M, Stöhr, H, Grassmann, F, Weber, BHF, Hoyng, CB, De Baere, E, Albert, S, Collin, RWJ & Cremers, FPM 2019, 'Deep-intronic ABCA4 variants explain missing heritability in Stargardt disease and allow correction of splice defects by antisense oligonucleotides', Genetics in medicine : official journal of the American College of Medical Genetics, vol. 21, no. 8, pp. 1751-1760. https://doi.org/10.1038/s41436-018-0414-9

Deep-intronic ABCA4 variants explain missing heritability in Stargardt disease and allow correction of splice defects by antisense oligonucleotides. / Sangermano, Riccardo; Garanto, Alejandro; Khan, Mubeen et al.

In: Genetics in medicine : official journal of the American College of Medical Genetics, Vol. 21, No. 8, 08.2019, p. 1751-1760.

Research output: Contribution to journal › Article › Research › peer-review

TY - JOUR

T1 - Deep-intronic ABCA4 variants explain missing heritability in Stargardt disease and allow correction of splice defects by antisense oligonucleotides

AU - Sangermano, Riccardo

AU - Garanto, Alejandro

AU - Khan, Mubeen

AU - Runhart, Esmee H

AU - Bauwens, Miriam

AU - Bax, Nathalie M

AU - van den Born, L Ingeborgh

AU - Khan, Muhammad Imran

AU - Cornelis, Stéphanie S

AU - Verheij, Joke B G M

AU - Pott, Jan-Willem R

AU - Thiadens, Alberta A H J

AU - Klaver, Caroline C W

AU - Puech, Bernard

AU - Meunier, Isabelle

AU - Naessens, Sarah

AU - Arno, Gavin

AU - Fakin, Ana

AU - Carss, Keren J

AU - Raymond, F Lucy

AU - Webster, Andrew R

AU - Dhaenens, Claire-Marie

AU - Stöhr, Heidi

AU - Grassmann, Felix

AU - Weber, Bernhard H F

AU - Hoyng, Carel B

AU - De Baere, Elfride

AU - Albert, Silvia

AU - Collin, Rob W J

AU - Cremers, Frans P M

PY - 2019/8

Y1 - 2019/8

N2 - 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.

AB - 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.

KW - ATP-Binding Cassette Transporters/genetics

KW - Adolescent

KW - Adult

KW - Aged

KW - Child

KW - Exons/genetics

KW - HEK293 Cells

KW - Humans

KW - Introns/genetics

KW - Middle Aged

KW - Mutation/genetics

KW - Oligonucleotides, Antisense/genetics

KW - Pedigree

KW - Polymorphism, Single Nucleotide/genetics

KW - Protein Isoforms/genetics

KW - RNA Splicing/genetics

KW - Stargardt Disease/genetics

KW - Young Adult

U2 - 10.1038/s41436-018-0414-9

DO - 10.1038/s41436-018-0414-9

M3 - Article

C2 - 30643219

VL - 21

SP - 1751

EP - 1760

IS - 8

ER -

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

Abstract

Keywords

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