Cost-effective molecular inversion probe-based ABCA4 sequencing reveals deep-intronic variants in Stargardt disease

Mubeen Khan; Stéphanie S. Cornelis; Muhammad Imran Khan; Duaa Elmelik; Eline Manders; Sem Bakker; Ronny Derks; Kornelia Neveling; Maartje van de Vorst; Christian Gilissen; Isabelle Meunier; Sabine Defoort; Bernard Puech; Aurore Devos; Heidi L. Schulz; Heidi Stöhr; Felix Grassmann; Bernhard H.F. Weber; Claire Marie Dhaenens; Frans P.M. Cremers

doi:10.1002/humu.23787

Cost-effective molecular inversion probe-based ABCA4 sequencing reveals deep-intronic variants in Stargardt disease

Mubeen Khan, Stéphanie S. Cornelis, Muhammad Imran Khan, Duaa Elmelik, Eline Manders, Sem Bakker, Ronny Derks, Kornelia Neveling, Maartje van de Vorst, Christian Gilissen, Isabelle Meunier, Sabine Defoort, Bernard Puech, Aurore Devos, Heidi L. Schulz, Heidi Stöhr, Felix Grassmann, Bernhard H.F. Weber, Claire Marie Dhaenens, Frans P.M. Cremers^*

^*Corresponding author for this work

Medical Sciences

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

35 Citations (Scopus)

Abstract

Purpose: Stargardt disease (STGD1) is caused by biallelic mutations in ABCA4, but many patients are genetically unsolved due to insensitive mutation-scanning methods. We aimed to develop a cost-effective sequencing method for ABCA4 exons and regions carrying known causal deep-intronic variants. Methods: Fifty exons and 12 regions containing 14 deep-intronic variants of ABCA4 were sequenced using double-tiled single molecule Molecular Inversion Probe (smMIP)-based next-generation sequencing. DNAs of 16 STGD1 cases carrying 29 ABCA4 alleles and of four healthy persons were sequenced using 483 smMIPs. Thereafter, DNAs of 411 STGD1 cases with one or no ABCA4 variant were sequenced. The effect of novel noncoding variants on splicing was analyzed using in vitro splice assays. Results: Thirty-four ABCA4 variants previously identified in 16 STGD1 cases were reliably identified. In 155/411 probands (38%), two causal variants were identified. We identified 11 deep-intronic variants present in 62 alleles. Two known and two new noncanonical splice site variants showed splice defects, and one novel deep-intronic variant (c.4539+2065C>G) resulted in a 170-nt mRNA pseudoexon insertion (p.[Arg1514Lysfs*35,=]). Conclusions: smMIPs-based sequence analysis of coding and selected noncoding regions of ABCA4 enabled cost-effective mutation detection in STGD1 cases in previously unsolved cases.

Original language	English
Pages (from-to)	1749-1759
Number of pages	11
Journal	Human Mutation
Volume	40
Issue number	10
Early online date	18 Jun 2019
DOIs	https://doi.org/10.1002/humu.23787
Publication status	Published - Oct 2019

Bibliographical note

ACKNOWLEDGEMENTS
We thank Béatrice Bocquet, Hélène Dollfus, Isabelle Drumare, Emeline Gorecki, Christian P. Hamel, Karsten Hufendiek, Cord Huchzermeyer, Herbert Jägle, Ulrich Kellner, Valérie Pelletier, Yaumara Perdromo, Charlene Piriou, Philipp Rating, Klaus Rüther, Eric Souied, Georg Spital, and Xavier Zanlonghi for their cooperation and ascertaining STGD1 cases. SmMIP hybridization, library preparation and NextSeq 500 sequencing was performed at the Genome Technology Center, Department of Human Genetics, Radboud University Medical Center, Nijmegen. This work was supported by the Retina UK grant GR591 (to F. P. M. C. and S. Albert), a Fighting Blindness Ireland grant (to F. P. M. C., J. Farrar, and S. Roosing), the FP7‐PEOPLE‐2012‐ITN programme EyeTN, agreement 317472 (to F. P. M. C.), the Foundation Fighting Blindness USA, grant no. PPA‐0517‐0717‐RAD (to A. Garanto, F. P. M. C., R.W.J. Collin), the Rotterdamse Stichting Blindenbelangen, the Stichting Blindenhulp, and the Stichting tot Verbetering van het Lot der Blinden (to F. P. M. C.), and by the Landelijke Stichting voor Blinden en Slechtzienden, Macula Degeneratie fonds and the Stichting Blinden‐Penning that contributed through Uitzicht 2016‐12 (to F. P. M. C.). This work was also supported by the Algemene Nederlandse Vereniging ter Voorkoming van Blindheid and Landelijke Stichting voor Blinden en Slechtzienden that contributed through UitZicht 2014‐13, together with the Rotterdamse Stichting Blindenbelangen, Stichting Blindenhulp and the Stichting tot Verbetering van het Lot der Blinden (to F. P. M. C.). The funding organizations had no role in the design or conduct of this research, and provided unrestricted grants.

Keywords

ABCA4
deep-intronic variants
next generation sequencing
smMIPs
Stargardt disease

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Khan, M., Cornelis, S. S., Khan, M. I., Elmelik, D., Manders, E., Bakker, S., Derks, R., Neveling, K., van de Vorst, M., Gilissen, C., Meunier, I., Defoort, S., Puech, B., Devos, A., Schulz, H. L., Stöhr, H., Grassmann, F., Weber, B. H. F., Dhaenens, C. M., & Cremers, F. P. M. (2019). Cost-effective molecular inversion probe-based ABCA4 sequencing reveals deep-intronic variants in Stargardt disease. Human Mutation, 40(10), 1749-1759. https://doi.org/10.1002/humu.23787

Khan, M, Cornelis, SS, Khan, MI, Elmelik, D, Manders, E, Bakker, S, Derks, R, Neveling, K, van de Vorst, M, Gilissen, C, Meunier, I, Defoort, S, Puech, B, Devos, A, Schulz, HL, Stöhr, H, Grassmann, F, Weber, BHF, Dhaenens, CM & Cremers, FPM 2019, 'Cost-effective molecular inversion probe-based ABCA4 sequencing reveals deep-intronic variants in Stargardt disease', Human Mutation, vol. 40, no. 10, pp. 1749-1759. https://doi.org/10.1002/humu.23787

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title = "Cost-effective molecular inversion probe-based ABCA4 sequencing reveals deep-intronic variants in Stargardt disease",

abstract = "Purpose: Stargardt disease (STGD1) is caused by biallelic mutations in ABCA4, but many patients are genetically unsolved due to insensitive mutation-scanning methods. We aimed to develop a cost-effective sequencing method for ABCA4 exons and regions carrying known causal deep-intronic variants. Methods: Fifty exons and 12 regions containing 14 deep-intronic variants of ABCA4 were sequenced using double-tiled single molecule Molecular Inversion Probe (smMIP)-based next-generation sequencing. DNAs of 16 STGD1 cases carrying 29 ABCA4 alleles and of four healthy persons were sequenced using 483 smMIPs. Thereafter, DNAs of 411 STGD1 cases with one or no ABCA4 variant were sequenced. The effect of novel noncoding variants on splicing was analyzed using in vitro splice assays. Results: Thirty-four ABCA4 variants previously identified in 16 STGD1 cases were reliably identified. In 155/411 probands (38%), two causal variants were identified. We identified 11 deep-intronic variants present in 62 alleles. Two known and two new noncanonical splice site variants showed splice defects, and one novel deep-intronic variant (c.4539+2065C>G) resulted in a 170-nt mRNA pseudoexon insertion (p.[Arg1514Lysfs*35,=]). Conclusions: smMIPs-based sequence analysis of coding and selected noncoding regions of ABCA4 enabled cost-effective mutation detection in STGD1 cases in previously unsolved cases.",

keywords = "ABCA4, deep-intronic variants, next generation sequencing, smMIPs, Stargardt disease",

author = "Mubeen Khan and Cornelis, {St{\'e}phanie S.} and Khan, {Muhammad Imran} and Duaa Elmelik and Eline Manders and Sem Bakker and Ronny Derks and Kornelia Neveling and {van de Vorst}, Maartje and Christian Gilissen and Isabelle Meunier and Sabine Defoort and Bernard Puech and Aurore Devos and Schulz, {Heidi L.} and Heidi St{\"o}hr and Felix Grassmann and Weber, {Bernhard H.F.} and Dhaenens, {Claire Marie} and Cremers, {Frans P.M.}",

note = "ACKNOWLEDGEMENTS We thank B{\'e}atrice Bocquet, H{\'e}l{\`e}ne Dollfus, Isabelle Drumare, Emeline Gorecki, Christian P. Hamel, Karsten Hufendiek, Cord Huchzermeyer, Herbert J{\"a}gle, Ulrich Kellner, Val{\'e}rie Pelletier, Yaumara Perdromo, Charlene Piriou, Philipp Rating, Klaus R{\"u}ther, Eric Souied, Georg Spital, and Xavier Zanlonghi for their cooperation and ascertaining STGD1 cases. SmMIP hybridization, library preparation and NextSeq 500 sequencing was performed at the Genome Technology Center, Department of Human Genetics, Radboud University Medical Center, Nijmegen. This work was supported by the Retina UK grant GR591 (to F. P. M. C. and S. Albert), a Fighting Blindness Ireland grant (to F. P. M. C., J. Farrar, and S. Roosing), the FP7‐PEOPLE‐2012‐ITN programme EyeTN, agreement 317472 (to F. P. M. C.), the Foundation Fighting Blindness USA, grant no. PPA‐0517‐0717‐RAD (to A. Garanto, F. P. M. C., R.W.J. Collin), the Rotterdamse Stichting Blindenbelangen, the Stichting Blindenhulp, and the Stichting tot Verbetering van het Lot der Blinden (to F. P. M. C.), and by the Landelijke Stichting voor Blinden en Slechtzienden, Macula Degeneratie fonds and the Stichting Blinden‐Penning that contributed through Uitzicht 2016‐12 (to F. P. M. C.). This work was also supported by the Algemene Nederlandse Vereniging ter Voorkoming van Blindheid and Landelijke Stichting voor Blinden en Slechtzienden that contributed through UitZicht 2014‐13, together with the Rotterdamse Stichting Blindenbelangen, Stichting Blindenhulp and the Stichting tot Verbetering van het Lot der Blinden (to F. P. M. C.). The funding organizations had no role in the design or conduct of this research, and provided unrestricted grants.",

year = "2019",

month = oct,

doi = "10.1002/humu.23787",

language = "English",

volume = "40",

pages = "1749--1759",

journal = "Human Mutation",

issn = "1059-7794",

publisher = "Wiley-Liss Inc.",

number = "10",

}

TY - JOUR

T1 - Cost-effective molecular inversion probe-based ABCA4 sequencing reveals deep-intronic variants in Stargardt disease

AU - Khan, Mubeen

AU - Cornelis, Stéphanie S.

AU - Khan, Muhammad Imran

AU - Elmelik, Duaa

AU - Manders, Eline

AU - Bakker, Sem

AU - Derks, Ronny

AU - Neveling, Kornelia

AU - van de Vorst, Maartje

AU - Gilissen, Christian

AU - Meunier, Isabelle

AU - Defoort, Sabine

AU - Puech, Bernard

AU - Devos, Aurore

AU - Schulz, Heidi L.

AU - Stöhr, Heidi

AU - Grassmann, Felix

AU - Weber, Bernhard H.F.

AU - Dhaenens, Claire Marie

AU - Cremers, Frans P.M.

N1 - ACKNOWLEDGEMENTS We thank Béatrice Bocquet, Hélène Dollfus, Isabelle Drumare, Emeline Gorecki, Christian P. Hamel, Karsten Hufendiek, Cord Huchzermeyer, Herbert Jägle, Ulrich Kellner, Valérie Pelletier, Yaumara Perdromo, Charlene Piriou, Philipp Rating, Klaus Rüther, Eric Souied, Georg Spital, and Xavier Zanlonghi for their cooperation and ascertaining STGD1 cases. SmMIP hybridization, library preparation and NextSeq 500 sequencing was performed at the Genome Technology Center, Department of Human Genetics, Radboud University Medical Center, Nijmegen. This work was supported by the Retina UK grant GR591 (to F. P. M. C. and S. Albert), a Fighting Blindness Ireland grant (to F. P. M. C., J. Farrar, and S. Roosing), the FP7‐PEOPLE‐2012‐ITN programme EyeTN, agreement 317472 (to F. P. M. C.), the Foundation Fighting Blindness USA, grant no. PPA‐0517‐0717‐RAD (to A. Garanto, F. P. M. C., R.W.J. Collin), the Rotterdamse Stichting Blindenbelangen, the Stichting Blindenhulp, and the Stichting tot Verbetering van het Lot der Blinden (to F. P. M. C.), and by the Landelijke Stichting voor Blinden en Slechtzienden, Macula Degeneratie fonds and the Stichting Blinden‐Penning that contributed through Uitzicht 2016‐12 (to F. P. M. C.). This work was also supported by the Algemene Nederlandse Vereniging ter Voorkoming van Blindheid and Landelijke Stichting voor Blinden en Slechtzienden that contributed through UitZicht 2014‐13, together with the Rotterdamse Stichting Blindenbelangen, Stichting Blindenhulp and the Stichting tot Verbetering van het Lot der Blinden (to F. P. M. C.). The funding organizations had no role in the design or conduct of this research, and provided unrestricted grants.

PY - 2019/10

Y1 - 2019/10

N2 - Purpose: Stargardt disease (STGD1) is caused by biallelic mutations in ABCA4, but many patients are genetically unsolved due to insensitive mutation-scanning methods. We aimed to develop a cost-effective sequencing method for ABCA4 exons and regions carrying known causal deep-intronic variants. Methods: Fifty exons and 12 regions containing 14 deep-intronic variants of ABCA4 were sequenced using double-tiled single molecule Molecular Inversion Probe (smMIP)-based next-generation sequencing. DNAs of 16 STGD1 cases carrying 29 ABCA4 alleles and of four healthy persons were sequenced using 483 smMIPs. Thereafter, DNAs of 411 STGD1 cases with one or no ABCA4 variant were sequenced. The effect of novel noncoding variants on splicing was analyzed using in vitro splice assays. Results: Thirty-four ABCA4 variants previously identified in 16 STGD1 cases were reliably identified. In 155/411 probands (38%), two causal variants were identified. We identified 11 deep-intronic variants present in 62 alleles. Two known and two new noncanonical splice site variants showed splice defects, and one novel deep-intronic variant (c.4539+2065C>G) resulted in a 170-nt mRNA pseudoexon insertion (p.[Arg1514Lysfs*35,=]). Conclusions: smMIPs-based sequence analysis of coding and selected noncoding regions of ABCA4 enabled cost-effective mutation detection in STGD1 cases in previously unsolved cases.

AB - Purpose: Stargardt disease (STGD1) is caused by biallelic mutations in ABCA4, but many patients are genetically unsolved due to insensitive mutation-scanning methods. We aimed to develop a cost-effective sequencing method for ABCA4 exons and regions carrying known causal deep-intronic variants. Methods: Fifty exons and 12 regions containing 14 deep-intronic variants of ABCA4 were sequenced using double-tiled single molecule Molecular Inversion Probe (smMIP)-based next-generation sequencing. DNAs of 16 STGD1 cases carrying 29 ABCA4 alleles and of four healthy persons were sequenced using 483 smMIPs. Thereafter, DNAs of 411 STGD1 cases with one or no ABCA4 variant were sequenced. The effect of novel noncoding variants on splicing was analyzed using in vitro splice assays. Results: Thirty-four ABCA4 variants previously identified in 16 STGD1 cases were reliably identified. In 155/411 probands (38%), two causal variants were identified. We identified 11 deep-intronic variants present in 62 alleles. Two known and two new noncanonical splice site variants showed splice defects, and one novel deep-intronic variant (c.4539+2065C>G) resulted in a 170-nt mRNA pseudoexon insertion (p.[Arg1514Lysfs*35,=]). Conclusions: smMIPs-based sequence analysis of coding and selected noncoding regions of ABCA4 enabled cost-effective mutation detection in STGD1 cases in previously unsolved cases.

KW - ABCA4

KW - deep-intronic variants

KW - next generation sequencing

KW - smMIPs

KW - Stargardt disease

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U2 - 10.1002/humu.23787

DO - 10.1002/humu.23787

M3 - Article

C2 - 31212395

AN - SCOPUS:85071606043

SN - 1059-7794

VL - 40

SP - 1749

EP - 1759

JO - Human Mutation

JF - Human Mutation

IS - 10

ER -

Cost-effective molecular inversion probe-based ABCA4 sequencing reveals deep-intronic variants in Stargardt disease

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Bibliographical note

Keywords

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