case_images: PMC11319185_01
case_images: PMC11319185_02
case_images: SMA Molecular Diagnostic Testing. SMA deletion/duplication analysis via ddPCR from Patient 1 (A), Patient 2 (B), and Patient 3 (C). Droplet clusters correspond to populations of nucleotide targets; SMN1, SMN2, or RPP30 (control). Resulting CNV of SMN1 and SMN2 for each sample (D). Sanger sequencing electropherogram of SMN1 from Patient 1 (E) and Patient 2 (F). Arrow notes c.5C>G missense variant.
case_images: fgene-15-1406819-g001.jpg
case_images: PMC11319185_02_fgene-15-1406819-g001.jpg
case_images: F1
case_images: Patient 3 was tested with Patient 1 and 2 as a control. ddPCR deletion/duplication analysis of Patients 1 and 2 revealed SMN1 positive clusters, as compared to Patient 3 which did not have an SMN1 positive cluster (Figures 1A-C). Absolute copy number analysis revealed that Patients 1 and 2 had one copy of SMN1 and two copies of SMN2 (Figure 1D) as compared to Patient 3 which had zero copies of SMN1 and two copies of SMN2 (Figure 1D). Sanger sequencing detected the c.5C>G variant in Patients 1 and 2 (Figures 1E, F). Using this additional information, a family pedigree was constructed (Supplemental Figure S1). The affected patients inherited the allele with the deletion from their father and the allele with the missense variant from their mother (Supplemental Figure S1).
case_images: Immunofluorescence Staining of SMN in Fibroblasts and Quantification of Nuclear Gems. Immunofluorescence staining for SMN (red) and DAPI (blue) in patient-specific fibroblasts; (A) SMA Carrier, (B) Patient 3, (C) Patient 1, and (D) Patient 2. The arrow denotes gems. x20 magnification. Bar = 100 uM. Inset with representative SMN staining (red) of single nuclei. Identified gems were quantified and normalized to total nuclei (E); SMA Carrier: mean = 5.17, n = 35, standard error from the mean (SEM) = +- 0.56, Patient 1: n = 52, mean = 3.11, SEM = +- 0.30, Patient 2: mean = 3.00, n = 50, SEM = +- 0.21, Patient 3: mean = 1.41, n = 58, SEM = +- 0.31. A one-way ANOVA was performed to reveal there was a statistically significant difference in mean gems per nuclei between the fibroblast cultures [F(3, 191) = (19.84), p-value < 0.00001].
case_images: fgene-15-1406819-g002.jpg
case_images: PMC11319185_02_fgene-15-1406819-g002.jpg
case_images: F2
case_images: SMN levels in fibroblasts from Patient 1 and Patient 2 are higher than SMN levels in fibroblasts from Patient 3 (Figures 2A-D). Furthermore, like the fibroblasts from the SMA carrier control, the fibroblasts from Patient 1 and Patient 2 are positive for multiple nuclear gems per nuclei (Figures 2A-D). This is compared to a mean of 5.2 gems per nuclei in the SMA carrier control fibroblasts and 1.4 gems per nuclei in Patient 3 fibroblasts (Figure 2E).