Massively parallel sequencing identifies the gene Megf8 with ENU-induced mutation causing heterotaxy

Zhen Zhang, Deanne Alpert, Richard Francis, Bishwanath Chatterjee, Qing Yu, Terry Tansey, Steven L. Sabol, Cheng Cui, Yongli Bai, Maxim Koriabine, Yuko Yoshinaga, Jan Fang Cheng, Feng Chen, Joel Martin, Wendy Schackwitz, Teresa M. Gunn, Ken Kramer, Pieter J. De Jong, Len A. Pennacchio, Cecilia W. Lo

Research output: Contribution to journalArticle

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Abstract

Forward genetic screens with ENU (N-ethyl-N-nitrosourea) mutagenesis can facilitate gene discovery, but mutation identification is often difficult. We present the first study in which an ENU-induced mutation was identified by massively parallel DNA sequencing. This mutation causes heterotaxy and complex congenital heart defects and was mapped to a 2.2-Mb interval on mouse chromosome 7. Massively parallel sequencing of the entire 2.2-Mb interval identified 2 single-base substitutions, one in an intergenic region and a second causing replacement of a highly conserved cysteine with arginine (C193R) in the gene Megf8. Megf8 is evolutionarily conserved from human to fruit fly, and is observed to be ubiquitously expressed. Morpholino knockdown of Megf8 in zebrafish embryos resulted in a high incidence of heterotaxy, indicating a conserved role in laterality specification. Megf8C193R mouse mutants show normal breaking of symmetry at the node, but Nodal signaling failed to be propagated to the left lateral plate mesoderm. Videomicroscopy showed nodal cilia motility, which is required for left-right patterning, is unaffected. Although this protein is predicted to have receptor function based on its amino acid sequence, surprisingly confocal imaging showed it is translocated into the nucleus, where it is colocalized with Gfi1b and Baf60C, two proteins involved in chromatin remodeling. Overall, through the recovery of an ENU-induced mutation, we uncovered Megf8 as an essential regulator of left-right patterning.

Original languageEnglish
Pages (from-to)3219-3224
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume106
Issue number9
DOIs
StatePublished - Mar 3 2009
Externally publishedYes

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Ethylnitrosourea
High-Throughput Nucleotide Sequencing
Mutation
Genes
Morpholinos
Video Microscopy
Intergenic DNA
Chromosomes, Human, Pair 7
Chromatin Assembly and Disassembly
Congenital Heart Defects
Cilia
Genetic Association Studies
Mesoderm
Zebrafish
DNA Sequence Analysis
Mutagenesis
Diptera
Cysteine
Arginine
Amino Acid Sequence

All Science Journal Classification (ASJC) codes

  • General

Cite this

Massively parallel sequencing identifies the gene Megf8 with ENU-induced mutation causing heterotaxy. / Zhang, Zhen; Alpert, Deanne; Francis, Richard; Chatterjee, Bishwanath; Yu, Qing; Tansey, Terry; Sabol, Steven L.; Cui, Cheng; Bai, Yongli; Koriabine, Maxim; Yoshinaga, Yuko; Cheng, Jan Fang; Chen, Feng; Martin, Joel; Schackwitz, Wendy; Gunn, Teresa M.; Kramer, Ken; De Jong, Pieter J.; Pennacchio, Len A.; Lo, Cecilia W.

In: Proceedings of the National Academy of Sciences of the United States of America, Vol. 106, No. 9, 03.03.2009, p. 3219-3224.

Research output: Contribution to journalArticle

Zhang, Z, Alpert, D, Francis, R, Chatterjee, B, Yu, Q, Tansey, T, Sabol, SL, Cui, C, Bai, Y, Koriabine, M, Yoshinaga, Y, Cheng, JF, Chen, F, Martin, J, Schackwitz, W, Gunn, TM, Kramer, K, De Jong, PJ, Pennacchio, LA & Lo, CW 2009, 'Massively parallel sequencing identifies the gene Megf8 with ENU-induced mutation causing heterotaxy', Proceedings of the National Academy of Sciences of the United States of America, vol. 106, no. 9, pp. 3219-3224. https://doi.org/10.1073/pnas.0813400106
Zhang, Zhen ; Alpert, Deanne ; Francis, Richard ; Chatterjee, Bishwanath ; Yu, Qing ; Tansey, Terry ; Sabol, Steven L. ; Cui, Cheng ; Bai, Yongli ; Koriabine, Maxim ; Yoshinaga, Yuko ; Cheng, Jan Fang ; Chen, Feng ; Martin, Joel ; Schackwitz, Wendy ; Gunn, Teresa M. ; Kramer, Ken ; De Jong, Pieter J. ; Pennacchio, Len A. ; Lo, Cecilia W. / Massively parallel sequencing identifies the gene Megf8 with ENU-induced mutation causing heterotaxy. In: Proceedings of the National Academy of Sciences of the United States of America. 2009 ; Vol. 106, No. 9. pp. 3219-3224.
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abstract = "Forward genetic screens with ENU (N-ethyl-N-nitrosourea) mutagenesis can facilitate gene discovery, but mutation identification is often difficult. We present the first study in which an ENU-induced mutation was identified by massively parallel DNA sequencing. This mutation causes heterotaxy and complex congenital heart defects and was mapped to a 2.2-Mb interval on mouse chromosome 7. Massively parallel sequencing of the entire 2.2-Mb interval identified 2 single-base substitutions, one in an intergenic region and a second causing replacement of a highly conserved cysteine with arginine (C193R) in the gene Megf8. Megf8 is evolutionarily conserved from human to fruit fly, and is observed to be ubiquitously expressed. Morpholino knockdown of Megf8 in zebrafish embryos resulted in a high incidence of heterotaxy, indicating a conserved role in laterality specification. Megf8C193R mouse mutants show normal breaking of symmetry at the node, but Nodal signaling failed to be propagated to the left lateral plate mesoderm. Videomicroscopy showed nodal cilia motility, which is required for left-right patterning, is unaffected. Although this protein is predicted to have receptor function based on its amino acid sequence, surprisingly confocal imaging showed it is translocated into the nucleus, where it is colocalized with Gfi1b and Baf60C, two proteins involved in chromatin remodeling. Overall, through the recovery of an ENU-induced mutation, we uncovered Megf8 as an essential regulator of left-right patterning.",
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AU - Chatterjee, Bishwanath

AU - Yu, Qing

AU - Tansey, Terry

AU - Sabol, Steven L.

AU - Cui, Cheng

AU - Bai, Yongli

AU - Koriabine, Maxim

AU - Yoshinaga, Yuko

AU - Cheng, Jan Fang

AU - Chen, Feng

AU - Martin, Joel

AU - Schackwitz, Wendy

AU - Gunn, Teresa M.

AU - Kramer, Ken

AU - De Jong, Pieter J.

AU - Pennacchio, Len A.

AU - Lo, Cecilia W.

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N2 - Forward genetic screens with ENU (N-ethyl-N-nitrosourea) mutagenesis can facilitate gene discovery, but mutation identification is often difficult. We present the first study in which an ENU-induced mutation was identified by massively parallel DNA sequencing. This mutation causes heterotaxy and complex congenital heart defects and was mapped to a 2.2-Mb interval on mouse chromosome 7. Massively parallel sequencing of the entire 2.2-Mb interval identified 2 single-base substitutions, one in an intergenic region and a second causing replacement of a highly conserved cysteine with arginine (C193R) in the gene Megf8. Megf8 is evolutionarily conserved from human to fruit fly, and is observed to be ubiquitously expressed. Morpholino knockdown of Megf8 in zebrafish embryos resulted in a high incidence of heterotaxy, indicating a conserved role in laterality specification. Megf8C193R mouse mutants show normal breaking of symmetry at the node, but Nodal signaling failed to be propagated to the left lateral plate mesoderm. Videomicroscopy showed nodal cilia motility, which is required for left-right patterning, is unaffected. Although this protein is predicted to have receptor function based on its amino acid sequence, surprisingly confocal imaging showed it is translocated into the nucleus, where it is colocalized with Gfi1b and Baf60C, two proteins involved in chromatin remodeling. Overall, through the recovery of an ENU-induced mutation, we uncovered Megf8 as an essential regulator of left-right patterning.

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