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. 2019 Sep 24;10:896. doi: 10.3389/fgene.2019.00896

Figure 1.

Figure 1

Clinical and molecular features of the patients with homozygous NFASC mutations at the splice acceptor of intron 25. The mutation leads to skipping of exon 26 in transcript NM_001005388.2 which encodes for NF186. (A) Pedigree of the family. The two affected individuals with a homozygous c.3020-1G > A mutation in NFASC are marked with filled symbols. The two unaffected siblings, of whom one has passed away from a severe infection, as well as the healthy parents are marked with dots indicating heterozygous carrier status. Carrier status of family members was identified using both ES and Sanger sequencing. (B) Facial photographs of the two affected individuals showing no dysmorphology. (C) Magnetic resonance imaging (MRI) of the brain in both patients. T2-weighted scans show in the sister (III:1) mild atrophy of the cerebellar vermis as indicated by arrows on axonal as well as sagittal images. Normal findings were seen in the brother (III:4). (D) Schematic representation of NFASC exons and RefGene transcript isoforms are shown. Transcript structures are adapted from ProteinPainter application of St. Jude PeCan Data Portal (https://pecan.stjude.cloud/proteinpaint). Black arrow shows the location of the mutation at the splice acceptor site of intron 25 according to transcript NM_001005388.2. Green and red arrows represent forward and reverse primers for reverse transcription PCR and quantitative PCR, respectively. (E) Reverse transcription followed by PCR amplification of region between exons 25 and 27 shows a single short fragment (241 bp) in the affected individual compared to the fragment (358 bp) obtained from RNA sample extracted from control fibroblasts C2. The carrier parents have both short and long fragments. RNA extracted from EBV transformed blood cells C1 does not show any NFASC expression. NTC, no template control (F) Sanger sequencing of short fragments confirms skipping of exon 26. (G) mRNA isolated from fibroblasts that are obtained from skin biopsies of the patient III:4, parents and two controls were used for RT-qPCR. Expression of exon 26 containing transcript isoform NF186 is not detected in patient III:4. The expression level of this isoform in the parents is half compared to the mean expression level of controls when the expression levels normalized to against total NFASC isoforms. Significance is indicated as *p < 0.05 and **p < 0.005.