Identification of a pathogenic mutation in ATP2A1 via in silico analysis of exome data for cryptic aberrant splice sites

Background Pathogenic mutations causing aberrant splicing are often difficult to detect. Standard variant analysis of next‐generation sequence (NGS) data focuses on canonical splice sites. Noncanonical splice sites are more difficult to ascertain. Methods We developed a bioinformatics pipeline that...

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Bibliographic Details
Published in:Molecular genetics & genomic medicine 2019-03, Vol.7 (3), p.e552-n/a
Main Authors: Bruels, Christine C., Li, Chengcheng, Mendoza, Tonatiuh, Khan, Jamillah, Reddy, Hemakumar M., Estrella, Elicia A., Ghosh, Partha S., Darras, Basil T., Lidov, Hart G. W., Pacak, Christina A., Kunkel, Louis M., Modave, François, Draper, Isabelle, Kang, Peter B.
Format: Article
Language:English
Subjects:
Aberrant RNA splicing
Aberration
Algorithms
Animals
ATP2A1
Bioinformatics
Brody myopathy
Ca2-transporting ATPase
Calcium (reticular)
Cells, Cultured
Computational Biology - methods
cryptic variants
Data processing
Drosophila
Drosophila melanogaster
Endoplasmic reticulum
Exome Sequencing - methods
Genetic screening
Genetic Testing - methods
Humans
Immunoblotting
Immunohistochemistry
Insects
Lethality
Male
Muscle, Skeletal - metabolism
Mutation
Myoblasts
Myopathy
Myotonia
Myotonia Congenita - genetics
Myotonia Congenita - pathology
Original
Phenotype
Proteins
Quadriceps muscle
RNA Splice Sites - genetics
RNA-mediated interference
Sarcoplasmic Reticulum Calcium-Transporting ATPases - genetics
Sarcoplasmic Reticulum Calcium-Transporting ATPases - metabolism
Splicing
Young Adult
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Summary:Background Pathogenic mutations causing aberrant splicing are often difficult to detect. Standard variant analysis of next‐generation sequence (NGS) data focuses on canonical splice sites. Noncanonical splice sites are more difficult to ascertain. Methods We developed a bioinformatics pipeline that screens existing NGS data for potentially aberrant novel essential splice sites (PANESS) and performed a pilot study on a family with a myotonic disorder. Further analyses were performed via qRT‐PCR, immunoblotting, and immunohistochemistry. RNAi knockdown studies were performed in Drosophila to model the gene deficiency. Results The PANESS pipeline identified a homozygous ATP2A1 variant (NC_000016.9:g.28905928G>A; NM_004320.4:c.1287G>A:p.(Glu429=)) that was predicted to cause the omission of exon 11. Aberrant splicing of ATP2A1 was confirmed via qRT‐PCR, and abnormal expression of the protein product sarcoplasmic/endoplasmic reticulum Ca++ ATPase 1 (SERCA1) was demonstrated in quadriceps femoris tissue from the proband. Ubiquitous knockdown of SERCA led to lethality in Drosophila, as did knockdown targeting differentiating or fusing myoblasts. Conclusions This study confirms the potential of novel in silico algorithms to detect cryptic mutations in existing NGS data; expands the phenotypic spectrum of ATP2A1 mutations beyond classic Brody myopathy; and suggests that genetic testing of ATP2A1 should be considered in patients with clinical myotonia. We developed a novel bioinformatics pipeline to identify potentially aberrant novel essential splice site (PANESS) mutations in next‐generation sequencing data, and used it successfully to identify a pathogenic splice site mutation in ATP2A1 for a family with a myotonic phenotype. The pathogenic nature of the mutation was confirmed via RT‐PCR, western blot, and immunohistochemistry, along with functional studies in Drosophila.
ISSN:2324-9269
2324-9269
DOI:10.1002/mgg3.552