QPCR (a), microarray (b) and small RNA sequencing results (c) for <i>MIR204</i> cells.

<p>(a) Average normalized relative quantity (NRQ) for miR-204-5p and miR-204-3p in MIR204WT (CC genotype, black bars, clones 1–3) and MIR204SNP cells (TT genotype, white bars, clones 1–3) normalized for transduction efficiency. (b) Average normalized expression values (NEV) for significantly differently expressed genes between MIR204WT (CC genotype, black bars, clones 4–5) and MIR204SNP (TT genotype, white bars, clones 4–6) cells. (c) Log2 fold changes of miRNAs significantly differently expressed in MIR204SNP cells (clones 4–6) compared to MIR204WT cells (clones 4–5). Error bars in panels (a-b) represent standard deviation of biological replicates, error bars in (c) represent standard error as calculated by DESeq2.</p

MFE secondary structure predictions of hsa-mir-204 and hsa-mir-618 as generated by miRVaS.

<p>(a-b) MFE structure for hsa-mir-204 with variant rs7861254, (a) wild type miRNA, (b) variant miRNA. As the variant is located 107 nt outside the hairpin, flanking regions of 150 nt were included for the predictions. Centroid and MEA predictions with this flank size also showed large structural changes (but different changes), while predictions with flanks of 200 nt resulted in minor changes far away from the hairpin. (c-d) MFE structure of hsa-mir-618 for variant rs2682818, (c) wild type miRNA, (d) variant miRNA. The variant is predicted to induce a shift of the first base of the miR-618 sequence into an internal loop. Flanks of 100 nt were used for this prediction. Predictions were also run for the hairpin with flank sizes of 50 nt, 150 nt and 200 nt and centroid and MEA structures: all predicted the same change within the hairpin. Color scheme: magenta: mature miRNA, orange: seed region, dark blue: terminal loop, cyan: hairpin. The variant is colored in red and indicated by an arrow. Structural changes induced by the variant are colored in black.</p

Additional file 2: of STUB1/CHIP mutations cause Gordon Holmes syndrome as part of a widespread multisystemic neurodegeneration: evidence from four novel mutations

Western blots of CHIP in mutation carriers. (DOCX 241 kb

Overview of patients with <i>SCN9A</i> variants and their corresponding <i>SCN1A</i> mutation.

*<p>In <0.3% controls. FS, febrile seizures; AFS, afebrile seizures; TLE, temporal lobe epilepsy; GSW, generalized spike wave; IGE, idiopathic generalized epilepsy; SMEB-MA, Dravet syndrome without myoclonic seizures and ataxia.</p><p>Reference sequences used are: SCN9A (NP 002968) and SCN1A (Swiss-Prot P35498).</p><p>AD, autosomal dominant; P, paternal; M, maternal; n.a., parents not available.</p

<i>SCN9A</i> is mutated in multiple patients with febrile seizures (FS) and Dravet syndrome.

<p>(A) Phenotypic profile and secondary structure locations of all variants found in <i>SCN9A</i>. Red text, variants in FS patients; blue text, variants in Dravet syndrome patients; black text, variants in both phenotypes; *variants also found in controls. (B) Amino acids from the UCSC genome browser (<a href="http://genome.ucsc.edu/" target="_blank">http://genome.ucsc.edu/</a>) showing conservation across 8 species for FS and Dravet syndrome variants (red) found in <i>SCN9A</i>. The human Na_v1.7 protein shares identity of 97% to rhesus, 92% to rat, 92% to mouse, 94% to cow, 94% to dog, 93% to rabbit, and 75% to chicken.</p

Reduced electroconvulsive seizure thresholds of <i>Scn9a</i> knockin mice compared to wild-type littermate controls.

<p>Convulsive current curves generated by testing (A) male B6;129-<i>Scn9a</i>^N641Y/N641Y, B6;129-<i>Scn9a</i>^N641Y/+, and B6;129-<i>Scn9a</i>^+/+ mice to minimal clonus electroconvulsive seizures (B6;129-<i>Scn9a</i>^N641Y/N641Y vs B6;129-<i>Scn9a</i>^N641Y/+<i>p</i> = 0.008; B6;129-<i>Scn9a</i>^N641Y/N641Y vs B6;129-<i>Scn9a</i>^+/+<i>p</i> = 0.001; B6;129-<i>Scn9a</i>^N641Y/+ vs B6;129-<i>Scn9a</i>^+/+<i>p</i> = 0.093) and (B) female B6;129-<i>Scn9a</i>^N641Y/N641Y, B6;129-<i>Scn9a</i>^N641Y/+, and B6;129-<i>Scn9a</i>^+/+ mice to minimal tonic hindlimb extension electroconvulsive seizures (B6;129-<i>Scn9a</i>^N641Y/N641Y vs B6;129-<i>Scn9a</i>^N641Y/+<i>p</i><0.001; B6;129-<i>Scn9a</i>^N641Y/N641Y vs B6;129-<i>Scn9a</i>^+/+<i>p</i><0.001; B6;129-<i>Scn9a</i>^N641Y/+ vs B6;129-<i>Scn9a</i>^+/+<i>p</i> = 0.227). Convulsive current data are expressed in terms of 1-seizure probability (1-P_seizure) for a given stimulus (mA). Individual data points shown for homozygote (closed square), heterozygote (x), and wild-type (closed circle) mice are used to construct curves indicated by black solid, red dashed, and blue dotted lines, respectively.</p

Increased corneal kindling acquisition rates of <i>Scn9a</i> knockin mice compared to wild-type littermate controls.

<p>Male N5F2 mice separated by genotype (n = 8–15) were stimulated with corneal electrodes twice daily until four consecutive Racine Stage 4 or 5 secondarily generalized seizures were elicited. The effect of <i>Scn9a</i>-N641Y on kindling acquisition is shown in (A) for B6.129-Scn9a^+/+, B6.129-Scn9a^N641Y/+, and B6.129-Scn9a^N641Y/N641Y mice; results are expressed as the average seizure score per genotype observed after each stimulation. (B) The number of stimulations required to reach the first fully generalized Racine Stage 4–5 seizure, regraphed with p-values from the data in (A), is 9.89±0.93 (B6.129-Scn9a^+/+, clear bar), 5.63±0.92 (B6.129-Scn9a^N641Y/N641Y, black bar), and 6.93±0.89 (B6.129-Scn9a^N641Y/+, pink bar), left panel; the number of stimulations required to reach a fully kindled mouse defined as four consecutive Racine Stage 4–5 seizures, regraphed with p-values from the data in (A), is 14.56±0.88 (B6.129-Scn9a^+/+, clear bar), 11.13±1.2 (B6.129-Scn9a^N641Y/N641Y, black bar), 12.64±0.86 (B6.129-Scn9a^N641Y/+, pink bar), right panel.</p

Utah Dravet syndrome patient #34302 harbors mutations in both <i>SCN9A</i> and <i>SCN1A</i>.

<p>Sequence chromatograms of wild-type (top panel) and mutant (middle panel) clones of <i>SCN1A</i> exon 15 reveals a frameshift mutation p.N892fsX2 (c.2675delA); sequence chromatogram of genomic DNA shows a heterozygous p.L1123F (c.3369G>T) in exon 17 of <i>SCN9A</i> exon (bottom panel).</p

Generation of <i>Scn9a</i>-N641Y knockin mice.

<p>Schematic representation of the (A) wild-type allele, (B) targeting construct introduced into embryonic stem (ES) cells. Numbered boxes denote exons; *, p.N641Y missense change introduced into exon 11; PCRa and PCRb, primers used to screen ES cell DNA for homologous recombination; S and probe, denotes SspI sites and probe used in genomic Southern blot of ES cells; ACN cassette, Cre-recombinase gene (<i>Cre</i>) driven by the testes-specific promoter from the angiotensin-converting enzyme gene (<i>tACE</i>); <i>Cre</i> is linked to the <i>Neo</i>^r selectable marker driven by the mouse RNA polymerase II large subunit gene (<i>polII</i>); the entire cassette is flanked by 34 bp loxP sites oriented in parallel. TK, HSV-TK gene for negative selection of ES cells. (C) following <i>Cre</i>-mediated self-excision in the chimeric mouse germline, a single loxP site and the point mutation remain. (D) Southern blot of three SspI cut ES cell clones followed by hybridization of probe yields an 8.4 kb endogenous band and a 7.2 kb targeted band (horizontal arrows); vertical arrow denotes clone used to make mouse. (E) LightScanner normalized melting peaks used to genotype <i>SCN9A</i>^+/+, <i>SCN9A</i>^N641Y/+, and <i>SCN9A</i>^N641Y/N641Y mice. (F) PCR used to verify self-excision of the ACN cassette. Amplicons generated by primers flanking remaining 34 bp loxP site in intron 10 yield distinct <i>SCN9A</i>^+/+ (left), <i>SCN9A</i>^N641Y/+ (center), and <i>SCN9A</i>^N641Y/N641Y (right, denoted by arrow) bands on 2% agarose.</p

Additional file 1: Table S1. of Sporadic late-onset nemaline myopathy: clinico-pathological characteristics and review of 76 cases

Genetic variants identified in patients with SLONM using NGS and a panel of 283 genes. (DOC 72 kb