In vitro analysis of splice site mutations in the CLCN1 gene using the minigene assay.
Mutations in the chloride channel gene CLCN1 cause the allelic disorders Thomsen (dominant) and Becker (recessive) myotonia congenita (MC). The encoded protein, ClC-1, is the primary channel that mediates chloride (Cl-) conductance in skeletal muscle. Mutations in CLCN1 lower the channel's threshold voltage, leading to spontaneous action potentials that are not coupled to neuromuscular transmission and resulting in myotonia. Over 120 mutations in CLCN1 have been described, 10% of which are splicing defects. Biological specimens suitable for RNA extraction are not always available, but obtaining genomic DNA for analysis is easy and non-invasive. This is the first study to evaluate the pathogenic potential of novel splicing mutations using the minigene approach, which is based on genomic DNA analysis. Splicing mutations accounted for 23% of all pathogenic variants in our cohort of MC patients. Four were heterozygous mutations in four unrelated individuals, belonging to this cohort: c.563G>T in exon 5; c.1169-5T>G in intron 10; c.1251+1G>A in intron 11, and c.1931-2A>G in intron 16. These variants were expressed in HEK 293 cells, and aberrant splicing was verified by in vitro transcription and sequencing of the cDNA. Our findings confirm the need to further investigate the nature of rearrangements associated with this class of mutations and their effects on mature transcripts. In particular, splicing mutations predicted to generate in-frame transcripts may generate out-of-frame mRNA transcripts that do not produce functional ClC-1. Clinically, incomplete molecular evaluation could lead to delayed or faulty diagnosis.
Neurology Unit, Neuroscience Section, Department of Pathophysiology and Transplantation (DEPT), Dino Ferrari Centre, IRCCS Foundation Ca' Granda Ospedale Maggiore Policlinico, University of Milan, Via Sforza 35, 20122, Milan, Italy.
Gianna
G
Ulzi
Valeria A
VA
Sansone
Francesca
F
Magri
Stefania
S
Corti
Nereo
N
Bresolin
Giacomo P
GP
Comi
Sabrina
S
Lucchiari
D000293:Adolescent; D000328:Adult; D000483:Alleles; D002648:Child; D018118:Chloride Channels / Q000235:genetics*; D004252:DNA Mutational Analysis; D005260:Female; D023061:Gene Order; D006801:Humans; D066298:In Vitro Techniques; D008297:Male; D009154:Mutation; D009224:Myotonia Congenita / Q000235:genetics*; D022821:RNA Splice Sites; D012326:RNA Splicing; D055815:Young Adult
C086321:CLC-1 channel; D018118:Chloride Channels; D022821:RNA Splice Sites