Clinical, immunohistochemical, and genetic characterization of splice-altering biallelic DES variants: Therapeutic implications

Figures

• 

  Figure 1. Clinical presentation of the affected individual (P1) (A) Neuromuscular examination at 10 and 11 years of age confirmed scapular winging, decreased muscle mass throughout bilateral upper and lower extremities as well as axial, proximal, and distal weakness. (B) T1-weighted axial images from muscle MRI performed at age 10 years revealed slightly increased signaling in the semitendinosus muscle bilaterally (arrows) in the upper leg. In the lower leg, there was evidence of atrophy and increased signaling in the bilateral tibialis anterior muscle (asterisks), with a milder degree of increased signaling of the extensor digitorum longus and peroneus longus muscles (arrow heads). (C) Muscle ultrasound at 10 and 11 years of age showed mild-to-moderately increased echogenicity with granular appearance. BF, biceps femoris; F, femur; HS, hamstring; RF, rectus femoris; SM, semimembranosus; ST, semitendinosus. (D) Left ventricular outflow tract view demonstrating hyper-enhancement of the left ventricular epicardium extending from the epicardium to near transmural scar inferiorly. Yellow arrows indicate new large transmural scarring.

• 

  Figure 2. Histopathology of P1’s left rectus femoris muscle biopsy at 2 years of age and cardiac biopsy and explant at 13 years of age (A) Wide fiber size variation is noted including atrophic and hypertrophic fibers as well as increased internal nuclei in hematoxylin and eosin (H&E). However, in all other stains, including (B) nicotinamide adenine dinucleotide (NADH), (C) succinate dehydrogenase (SDH), (D and D′) Gomori trichrome, and (E) cytochrome c oxidase (COX), no evidence of myofibrillar disarray, inflammation, necrosis, regeneration, or endomysial fibrosis is observed. Electron microscopy (F) of rectus femoris at 2,950× demonstrates normal myofibrillar pattern without sarcolemmal folding, redundant or thickened basement membrane, or loss of plasma membrane. Cardiac biopsy at explant (G; scale bar, 100 μm) demonstrates considerable fiber size variation, increased vacuolation, endocardial fibrosis, and significant multifocal interstitial fibrosis in H&E staining.

• 

  Figure 3. Immunofluorescent staining and immunoblotting Left rectus femoris muscle biopsies from P1 at 2 years of age were stained for desmin, FHL1, and α-β-crystallin and compared with control tissue. While desmin protein staining is significantly decreased, FHL1 (A) and α-β-crystallin (B) staining appear relatively normal compared with control tissue. (C) Immunoblotting confirms decreased desmin staining in P1 skeletal and cardiac muscle compared with control tissue when normalized to tubulin as a loading control. CM, cardiac muscle; SM, skeletal muscle.

• 

  Figure 4. Segregation and characterization of DES variants (A) Pedigree showing segregation of the c.640-13T>A and c.1288-1 G>A variants. Filled symbol indicates individual affected with desminopathy. −, WT desmin allele. (B) Normal, WT splicing is depicted, as well as the inherited paternal and maternal alleles with altered splicing. The consequences of splicing on desmin protein are noted. Intron 2, 11-bp insertion, inherited from biological father results in new reading frame and premature stop codon. Intron 7 inclusion, p.Glu430GlyfsTer35, inherited from biological mother also results in new reading frame. Gray amino acids indicate proband desmin sequence while purple amino acids depict WT desmin. Black boxes highlight intronic insertions and red boxes highlight premature stop codons. Variants are present in trans (on separate chromosomes) supporting a recessive mode of inheritance. (C) Sashimi plot showing desmin splicing of exons 2–4 in control and P1 tissue showing the inclusion of the 11-bp exon extension.

• 

  Figure 5. Transdifferentiation protocol of control and P1 cells with RNA-seq analysis (A) Protocol outline for transdifferentiation of fibroblasts to myoblasts. Cells were collected for RNA extraction at various timepoints indicated. Green arrows indicate change of media and doxycycline (dox) respike. DFM, differentiation media; MPM, myoblast proliferation media. (B) PCR for MyoD and desmin expression in FC2 controls. Dox induction was initiated at 2.5 or 5 μg/mL in controls to determine optimal expression. (C) PCR for MyoD and desmin expression in P1 cells. P1 cells were induced at 3 μg/mL dox based on optimal expression in control cells. Cycloheximide (C) was used in P1 cells for 8 h at 50 μg/mL on the day of collection to account for potential nonsense-mediated decay with variants. MyoD-positive control, lentiviral plasmid with myoD gene sequence; desmin-positive control, human skeletal muscle myoblast (HSMM) cells. Graph shows qPCR expression of the intron 2 desmin variant normalized to beta-2-microglobin (B2M) expression over the first 10 days after doxycycline induction, indicating the highest variant expression at 7 days. (D) Sashmi plots of desmin RNA splicing of exons 2–4 in…

• 

  Figure 6. Differential expression analysis of FC2 control and P1 transdifferentiated cells (A) Lolliplots show the top five significantly different gene ontology processes in transdifferentiated FC2 control cells, highlighting genes in extracellular matrix organization and muscle system process over the course of 0–14 days of differentiation. Violin plots for FC2 control cells show extracellular matrix organization genes primarily decrease by 3 days after induction, while muscle system processes are primarily upregulated during the entirety of the experiment. (B) Lolliplots show the top five gene significantly different gene ontology processes in P1 transdifferentiated cells highlighting genes in extracellular matrix organization and muscle system process over the course of 0–14 days of differentiation. Violin plots for P1 cells show a similar trend to FC2 control, although extracellular matrix organization genes are downregulated more by 14 days.

• 

  Figure 7. PMO testing with minigene in HEK293T cells (A) Minigene design in the Exontrap pET01 vector. Vertical red line indicates location of variant. 1% agarose gel vector cDNA PCR reaction region showing expected sizing for WT and variant desmin expression depending on the transfected plasmid. Sanger sequencing of bands confirms 11 bp intron 2 inclusion but also shows leaky WT expression. (B) Complete skipping of exon 3 was noted with all 5 PMO options when HEK293T cells were transfected with WT or variant desmin plasmid DNA and treated with various PMOs.