GYG1 gene mutations in a family with polyglucosan body myopathy

Abstract

Defects in enzymes involved in glycogen metabolism result in glycogen storage diseases (GSDs), which may affect the skeletal and sometimes also the cardiac muscle. The most frequent abnormality causing GSDs is glycogen storage, whereas other and uncommon forms of GSD are due to a perturbation of the branching structure of glycogen. These latter GSDs are characterized by an accumulation of polyglucosan (PG),(1) an abnormal polysaccharide with few branched points and excessively long peripheral chains. PG is accumulated in PG bodies that can be easily identified in muscle by their typical features using histopathologic (strong periodic acid-Schiff [PAS] reaction, resistance to diastase digestion) and ultrastructural analyses.

Figure. 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.