PMC6434044_01
PMC6434044_02
Clinical findings and genetic tests of the patient.. a EEG performed at 4 years showed focal epileptic discharge with generalization in multiple foci. b Brain MRI (T1 weighted sagittal) performed at 4 years did not show any abnormal findings. c Brain MRS in the basal ganglia performed at 4 years did not show any abnormal glutamate/glutamine peaks (white arrow). d The patient carried a de novo hemizygous SLC9A6 mutation (NM_006359.2:c.1141-8C>A) that was confirmed by Sanger sequencing. e RT-PCR analysis identified multiple aberrant transcripts but no canonical transcripts in the patient, while it identified only canonical transcripts in control DNA (wild type). f: Transcript variants in the patient. Twenty-one percent of transcripts included intronic 6-bp nucleotides (a), 21% excluded exonic 28-bp nucleotides (b), 4% excluded exonic 30-bp nucleotides (c), and 54% skipped exon 10 (d)
41439_2019_46_Fig1_HTML.jpg
PMC6434044_02_41439_2019_46_Fig1_HTML.jpg
Fig1
An electroencephalography (EEG) performed at four years showed focal epileptic discharges with generalization in multiple foci (Fig. 1a). Brain magnetic resonance imaging (MRI) (performed at 1, 2, and 4 years of age) and magnetic resonance spectroscopy (MRS) (performed at 4 years of age) showed no abnormal findings (Fig. 1b, c). We identified a de novo hemizygous splicing mutation (c.1141-8C>A) in SLC9A6 (NM_006359.2), which was confirmed by Sanger sequencing using the SLC9A6-intron 9-Fwd (5'-TCCACATTTGCTCCCTTCT-3') and SLC9A6-exon 10-Rev (5'-ACCACATACTCAAAACCCAC-3') primer pair (Fig. 1d). We predicted that the mutation affected RNA splicing because it resulted in a new AG acceptor site six nucleotides upstream of the canonical acceptor site of exon 10. SLC9A6-exon 8-Fwd1 (5'-ACCAAATTACGGGAGTTCCA-3') and SLC9A6-exon 12-Rev2 (5'-CACCACCAAATACCCACAC-3') primer pair revealed the presence of multiple transcripts (Fig. 1e). SLC9A6 cDNA was then ligated into a TOPO cloning vector (Life Technologies) and transformed into TOP10 Competent Cells (Life Technologies), and 24 colonies were screened by extracting plasmid DNA using a QIAprep Spin Miniprep Kit (Qiagen, Hilden, Germany). Sanger sequencing of the plasmid clones identified four unique, aberrant transcripts but no canonical transcripts. Of the 24 cDNA transcripts screened, five (21%) had the six-nucleotide addition of the intronic sequence to the 5' end of exon 10 that was predicted by our in silico analysis (Fig. 1f, transcript (a)). We also identified five (21%) transcripts and one (4%) transcript with 5' exon 10 deletions of 28 and 30 nucleotides, respectively, as well as 13 transcripts (54%) with complete skipping of exon 10 (Fig. 1f, transcripts (b), (c), and (d)).