Dataset Information

A deep intronic splice variant advises reexamination of presumably dominant SPG7 Cases.

ABSTRACT:

Objective

To identify causative mutations in a patient affected by ataxia and spastic paraplegia.

Methods

Whole-exome sequencing (WES) and whole-genome sequencing (WGS) were performed using patient's DNA sample. RT-PCR and cDNA Sanger sequencing were performed on RNA extracted from patient's fibroblasts, as well as western blot.

Results

A novel missense variant in SPG7 (c.2195T> C; p.Leu732Pro) was first found by whole-exome sequencing (WES), while the second, also unreported, deep intronic variant (c.286 + 853A>G) was identified by whole-genome sequencing (WGS). RT-PCR confirmed the in silico predictions showing that this variant activated a cryptic splice site, inducing the inclusion of a pseudoexon into the mRNA sequence, which encoded a premature stop codon. Western blot showed decreased SPG7 levels in patient's fibroblasts.

Interpretation

Identification of a deep intronic variant in SPG7, which could only have been detected by performing WGS, led to a diagnosis in this HSP patient. This case challenges the notion of an autosomal dominant inheritance for SPG7, and illustrates the importance of performing WGS subsequently or alternatively to WES to find additional mutations, especially in patients carrying one variant in a gene causing a predominantly autosomal recessive disease.

SUBMITTER: Verdura E

PROVIDER: S-EPMC6952318 | biostudies-literature | 2020 Jan

REPOSITORIES: biostudies-literature

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Publications

A deep intronic splice variant advises reexamination of presumably dominant SPG7 Cases.

Verdura Edgard E Schlüter Agatha A Fernández-Eulate Gorka G Ramos-Martín Raquel R Zulaica Miren M Planas-Serra Laura L Ruiz Montserrat M Fourcade Stéphane S Casasnovas Carlos C López de Munain Adolfo A Pujol Aurora A

Annals of clinical and translational neurology 20191218 1

<h4>Objective</h4>To identify causative mutations in a patient affected by ataxia and spastic paraplegia.<h4>Methods</h4>Whole-exome sequencing (WES) and whole-genome sequencing (WGS) were performed using patient's DNA sample. RT-PCR and cDNA Sanger sequencing were performed on RNA extracted from patient's fibroblasts, as well as western blot.<h4>Results</h4>A novel missense variant in SPG7 (c.2195T> C; p.Leu732Pro) was first found by whole-exome sequencing (WES), while the second, also unreport ...[more]

PMID: 31854126

Similar Datasets

Project description:Antisense therapeutics such as splice-modulating antisense oligonucleotides (ASOs) are promising tools to treat diseases caused by splice-altering intronic variants. However, their testing in animal models is hampered by the generally poor sequence conservation of the intervening sequences between human and other species. Here we aimed to model in the mouse a recurrent, deep-intronic, splice-activating, COL6A1 variant, associated with a severe form of Collagen VI-related muscular dystrophies (COL6-RDs), for the purpose of testing human-ready antisense therapeutics in vivo. The variant, c.930+189C>T, creates a donor splice site and inserts a 72-nt-long pseudoexon, which, when translated, acts in a dominant-negative manner, but which can be skipped with ASOs. We created a unique humanized mouse allele (designated as "h"), in which a 1.9 kb of the mouse genomic region encoding the amino-terminus (N-) of the triple helical (TH) domain of collagen a1(VI) was swapped for the human orthologous sequence. In addition, we also created an allele that carries the c.930+189C>T variant on the same humanized knock-in sequence (designated as "h+189T"). We show that in both models, the human exons are spliced seamlessly with the mouse exons to generate a chimeric mouse-human collagen a1(VI) protein. In homozygous Col6a1 h+189T/h+189T mice, the pseudoexon is expressed at levels comparable to those observed in heterozygous patients' muscle biopsies. While Col6a1h/h mice do not show any phenotype compared to wildtype animals, Col6a1 h/h+189T and Col6a1 h+189T/h+189T mice have smaller muscle masses and display grip strength deficits detectable as early as 4 weeks of age. The pathogenic h+189T humanized knock-in mouse allele thus recapitulates the pathogenic splicing defects seen in patients' biopsies and allows testing of human-ready precision antisense therapeutics aimed at skipping the pseudoexon. Given that the COL6A1 N-TH region is a hot-spot for COL6-RD variants, the humanized knock-in mouse model can be utilized as a template to introduce other COL6A1 pathogenic variants. This unique humanized mouse model thus represents a valuable tool for the development of antisense therapeutics for COL6-RDs.

Dataset Information

A deep intronic splice variant advises reexamination of presumably dominant SPG7 Cases.

Objective

Methods

Results

Interpretation

Publications

A deep intronic splice variant advises reexamination of presumably dominant SPG7 Cases.

Similar Datasets

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