PMC6777637_01
Location of a base insertion (T) in the GLA genomic DNA sequence, corresponding to intron 5 of the GLA gene (c.801+2_801+3insT). The figure highlights the insertion of a thymine base at the splicing donor site of intron 5.
fgene-10-00783-g001.jpg
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f1
As seen in  Figure 1 , a base thymine was inserted at the position c.801+3, three nucleotides into the intron 5 after the coding position, located close to the 5'ss.
Aberrant mRNA transcripts formation caused by the insertion in the GLA gene. (A) Amplification of a cDNA fragment from patient and healthy volunteer by PCR, with primers cDNA 2F and cDNA 2R, showing formation of two fragments in the patient. (B) The diagram described the wild-type allele from healthy volunteer and the two aberrant alleles formed in the patient.
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f2
The electrophoresis showed two transcript fragments produced from the mutated GLA, presenting approximately 630 and 800 bp, instead of the 594 bp fragment corresponding to the normal allele, which was not observed in the patient ( Figure 2 ).
cDNA sequencing from two aberrant splicing events found in the patient, which occurs by the inclusion of intron fragments, forming cryptic exon into the mature transcript. (A) In the transcript 1, 37 bp were inserted corresponding to the first bases of the intron 5, plus the thymine insertion at position +3. The figure shows alteration of the open reading frame, promoting formation of a premature stop codon in the exon 6 (p.Leu268Valfs*43). (B) The 5' splice site was not recognized in the transcript 2, and the excision of intron 5 was not observed. There was an insertion of 217 bp corresponding to intron 5 plus a thymine, corresponding to the base insertion at the genomic DNA, promoting a frame-shift and a premature stop codon in the intron 5.
fgene-10-00783-g003.jpg
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f3
These results are shown in  Figure 3 .