PMC7522968_01
Genetic and phenotypic characterization of a family with a deep-intronic mutation in CAPN3. (A) Pedigree of a 37-year -old man (P1) from a consanguineous family. (B) Globally reduced muscle bulk with significand biceps atrophy. (C) Muscle MRI of lower extremities. (D) Sanger sequencing of CAPN3 in patient (P1) and unaffected mother of P1 (M1).
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F1
Muscle MRI of the lower extremities performed at age 37 years revealed diffuse muscle atrophy and fatty replacement of all upper and lower leg muscles except for isolated sparing of the tibialis posterior muscles (Fig. 1C). Targeted familial segregation studies confirmed that all three affected siblings (P1 and two affected brothers) were homozygous for the CAPN3 mutation, and unaffected relatives were found to be heterozygous for this mutation (Fig. 1D).
PMC7522968_02
Genetic and phenotypic characterization of a family with a deep-intronic mutation in CAPN3. (A) Pedigree of a 37-year -old man (P1) from a consanguineous family. (B) Globally reduced muscle bulk with significand biceps atrophy. (C) Muscle MRI of lower extremities. (D) Sanger sequencing of CAPN3 in patient (P1) and unaffected mother of P1 (M1).
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PMC7522968_02_nihms-1599862-f0001.jpg
F1
Muscle MRI of the lower extremities performed at age 37 years revealed diffuse muscle atrophy and fatty replacement of all upper and lower leg muscles except for isolated sparing of the tibialis posterior muscles (Fig. 1C). Targeted familial segregation studies confirmed that all three affected siblings (P1 and two affected brothers) were homozygous for the CAPN3 mutation, and unaffected relatives were found to be heterozygous for this mutation (Fig. 1D).
Confirmation of aberrant splicing of CAPN3 in muscle tissue from P1. (A) Model of the disrupted branch point preventing normal splicing resulting in an inclusion of a 389-bp exon extension in intron 6. (B) Representative gel electrophoresis of endpoint PCR using cDNA from muscle tissue. (C) Western blot of control and patient (P1) using a calpain 3 antibody on muscle tissue.
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F2
Studies performed on cDNA from RNA extracted from P1's muscle tissue confirmed that the mutation disrupts normal splicing and redirects splicing to an alternative upstream intronic splice acceptor site, resulting in an inclusion of a 389-bp exon extension in intron 6 (Fig. 2A). Gel electrophoresis of the PCR amplification of cDNA from P1's muscle tissue confirmed aberrant splicing of CAPN3 in P1 with an inclusion of the 389-bp exon extension, which is absent in control tissue (Fig. 2B), and results in an out-of-frame mRNA product with a premature truncation. Western blot of muscle tissue from P1 and control using an antibody against Exon 8 of calpain 3 (NCL-CALP-12A2 predictive of bands 94 and ~60kDa) showed complete absence of calpain 3 protein in P1 (Fig. 2C).
PMO treatment corrects the aberrant splicing in CAPN3. (A) PMO1 target sequence at the abrrant 3' splice acceptor. (B) Patient-derived fibroblasts (P1) and control fibroblasts (Control) were treated for 48 hours with PMO1. Representative gel electrophoresis of endpoint PCR after PMO1 treatment using primers located in exons 5 and 7, demonstrating suppression of the aberrant splice products and also a remaning 51-bp exon extension.
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F3
We designed phosphorodiamidate morpholino oligomers (PMOs) as splice modulators to block the new splice acceptor site (Fig. 3A). Representative gel electrophoresis of the PCR from fibroblasts after 48 hours of PMO1 treatment showed that the aberrant splicing was suppressed with high efficiency and splicing directed back to the normal splice site, despite of the still present mutant branch point (Fig. 3B, Lane 5).
Dosage effect of PMO1 on abrrant and native CAPN3 splicing. (A) Location of primers (arrows) and probe (line with no arrowheads) in CAPN3-E6E7 and CAPN3 E616. (B) Representative gel electrophoresis of endpoint RT-PCR following PMO treatments ranging from 0-2.50 muM. (C) Expression of the aberrant splice product (E616) relative to mock-treatede patient cells. (D) Expression of the native splice product (E6E7) relative to normal control cells.
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F4
To analyze the degree of suppression on the expression of mutation at the transcriptional level, we generated two sets of primers specific to the correct and aberrant splice sites (Fig. 4A). Representative gel electrophoresis of endpoint qPCR showed splice products (Fig. 4B) while clearly demonstrating the presence of the aberrant splice product detected with primers E6I6 (Fig. 4A, below) and of the correct splice product with primers E6E7 (Fig. 4A, above). Dose response expression of aberrant splice product (Primers E6I6) showed that more than 50% of aberrant splicing expression was suppressed using the PMO1 treatment at a concentration of 1.25 mum relative to the mock-treated patient cells (Fig. 4C). Additionally, over 90% of the normal splicing product (Primers E6E7) was recovered after treatment with PMO1 at the same concentration of 1.25 mum (Fig. 4D).