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
SN - 1098-3600
VL - 21
SP - 1751
EP - 1760
JO - Genetics in medicine : official journal of the American College of Medical Genetics
JF - Genetics in medicine : official journal of the American College of Medical Genetics
IS - 8
ER -