Figure Genetic characterization of the novel GYG1 gene mutation (A) GYG1_cDNA sequence and position of primers used. Genetic characterization of the novel.

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Figure Genetic characterization of the novel GYG1 gene mutation (A) GYG1_cDNA sequence and position of primers used. Genetic characterization of the novel GYG1 gene mutation (A) GYG1_cDNA sequence and position of primers used. (B) Product amplified by PCR on the GYG1 coding sequence using 3 different primers pairs (from left to right: Fr II [465F/1053+165R], Fr I [1-63F/538R], and Fr U [1-63F/1053+165R]) in the sample of a patient (the cousin) and control. FR II corresponds to the 3′ half of the gene (from exon 5 to exon 7), Fr I to the 5′ region of GYG1 cDNA (from exon 1 to exon 4), and Fr U to the whole cDNA (from exon 1 to exon 7). At 3 different annealing temperatures (60°C, 62°C, and 64°C), the patient's sample showed a number of bands of different sizes. These PCR products are all longer than the control band, suggesting the insertion of a cryptic exon. To verify this hypothesis, each fragment was isolated and cloned (TA cloning kit; Invitrogen Corporation, Carlsbad, CA). (C) Sequencing of each clone showed that there are 2 different cDNAs, encoded by either allele's cDNA: allele 1 shows the skipping of exon 2, while allele 2 shows the insertion of an intronic fragment. (D) Sequence analysis of the critical region corresponding to allele 1 shows that in the patient's sample, exon 2 is skipped. (E) Sequence analysis of the critical region corresponding to allele 2 shows that in the patient's sample, there is an insertion between exon 4 and exon 5. By BLAST search, we mapped this sequence in intron 4 (chr 3: 148.717.897–148.717.963). A set of primers was designed to amplify the intronic region on genomic DNA to characterize the sequence of this intron retention. The sequence analysis shows a single nucleotide variant (chr 3: g.148.717.967C>G) that enhances the 5′ splice site of this cryptic exon; the variant is novel and also absent in the EXAC database. To confirm that this cryptic exon is not a secondary extra product of splicing, we designed a PCR protocol to amplify allele 2 alone, with the forward primer mapped to the intronic sequence: no band was detected in the control sample, while a strong band was present in the patient's sample. Marina Fanin et al. Neurol Genet 2015;1:e21 © 2015 American Academy of Neurology