Novel Biomarkers for Limb Girdle Muscular Dystrophy (LGMD)

Abstract

Objective: To identify novel biomarkers as an alternative diagnostic tool for limb girdle muscular dystrophy (LGMD).

Background: LGMD encompasses a group of muscular dystrophies characterized by proximal muscles weakness, elevated CK levels and dystrophic findings on muscle biopsy. Heterozygous CAPN3 mutations are associated with autosomal dominant LGMD-4, while biallelic mutations can cause autosomal recessive LGMD-1. Diagnosis is currently often based on invasive methods requiring muscle biopsy or blood tests. In most cases Western blotting (WB) analysis from muscle biopsy is essential for a diagnosis, as muscle samples are currently the only known tissues to express the full-length CAPN3 isoform.

Methods: We analyzed CAPN3 in a cohort including 60 LGMD patients. Selected patients underwent a complete neurological examination, electromyography, muscle biopsy, and skin biopsies for primary fibroblasts isolation. The amount of CAPN3 was evaluated by WB analysis in muscle and skin tissues. The total RNA isolated from muscle, fibroblast and urine was processed, and cDNA was used for qualitative analysis. The expression of CAPN3 was investigated by qRT-PCR. The CAPN3 3D structure has been visualized and analyzed using PyMOL.

Results: Among our patients, seven different CAPN3 mutations were detected, of which two were novel. After sequencing CAPN3 transcripts from fibroblast and urine, we detected different CAPN3 isoforms surprisingly including the full-length transcript. We found comparable protein levels from fibroblasts and muscle tissue; in particular, patients harboring a novel CAPN3 mutation showed a 30% reduction in protein compared to controls from both tissues.

Conclusions: Our findings showed for the first time the presence of the CAPN3 full-length transcript in urine and skin samples. Moreover, we demonstrated surprisingly comparable CAPN3 protein levels between muscle and skin samples, thus allowing us to hypothesize the use of skin biopsy and probably of urine samples as an alternative less invasive method to assess the amount of CAPN3 when molecular diagnosis turns out to be inconclusive.

Keywords: CAPN3; RNA; limb girdle muscular dystrophy; western blotting analysis.

Figure 1

Met252Arg and c.1746-20C>G variants. (a) Electropherogram of intron 14 highlights the transition C > G in heterozygous state. (b) Electropherogram of exon 5 highlights the transition T > G in heterozygous state. (c) The alignment of orthologous proteins shows that the variation involves a highly conserved methionine (red arrow). In the 3D structure, the Met252 is shown in red (d), which is located at the level of an α-helix of domain II of calpain 3. Substitution of methionine with arginine at position 252 and measurement of the interaction between Arg252 and Asp402 are shown (e–h). (f) Met252, (g) Arg252, (h) the possible interaction with Arg252 and Asp402 (yellow arrow).

Figure 2

Western blot analysis in patient 1 and 3. (a) WB showing the expression levels of CAPN3 in muscle tissue of patients 1 (PT1) and 3 (PT3) and one normal control (NC). The bar graph (b) shows the expression fold change in CAPN3, calculated by setting the ratio of CAPN3 protein/GAPDH protein band intensities in theNC to 1. The bars show the mean ± SD (data are representative of n = 2 independent experiments).

Figure 3

Glu323*, Glu467del, and Asp753Asn variants. (a) The electropherogram of exon 11 highlights a deletion of three nucleotides in heterozygous state. (b) The electropherogram of exon 7 highlights the G > T transition (red arrow) in heterozygous state. (c) The electropherogram of exon 21 highlights the G > A transition (red arrow) in heterozygous state; the alignment of orthologous proteins shows that the variation involves a highly conserved aspartic acid (red arrow). (d–f) The Asp753Asn variant is shown in red, located at the level of an EF-hand of domain IV of calpain 3. Substitution of aspartic acid with asparagine at position 753 is shown.

Figure 4

Val176Met variant. (a) The electropherogram of exon 4 highlights the G > A transition (red arrow) in heterozygous state. (b) The alignment of orthologous proteins shows that the variation involves a highly conserved valine (red arrow). PyMOL 3D Structures of the calpain 3. In red, we highlighted the valine at position 176 (c,d).

Figure 5

Tyr820His variant. (a) The electropherogram of exon 24 highlights the T > C transition in heterozygous state. (b) Alignment of orthologous proteins shows that the variation involves a highly conserved tyrosine (red arrow). (c) Three-dimensional (3D) structure of CAPN3: domain I (green), domain II (white), domain III (blue) and domain IV (yellow). In red, the localization of p.Met819 within EF5-hand of CAPN3 domain IV is shown. Area (d), perimeter (e) and diameter size (f) in type I and type II muscle fibers in a normal control (NC) and PT6. Fibers sizes were measured from muscle sections from a total of 281 fibers in NC muscle and 258 fibers in PT6 muscle. Data represent mean ± SD. Asterisk (*) indicates p < 0.05; n.s.: not significant. (g) The percentage of type I and type II fibers in an NC and PT6. Muscle biopsy from a normal control (h) and PT6(i).

Figure 6

CAPN3 expression study in multiple tissues through long-range PCR, WB, and RT-qPCR. Long-range PCR (a) to amplify the CAPN3 cDNA region from exons 5 to 17 in white blood cells (WBC), fibroblast (F), muscle (M) and urine (U). PCR amplification of exons 13–17 region (b) in fibroblast (F), white blood cells (WBC), muscle (M), epithelial buccal swab (B) and urine (U). PCR amplification of exons 5–8 region (c) in fibroblast (F), white blood cells (WBC), muscle (M), epithelial buccal swab (B) and urine (U). Western blot analysis of CAPN3 on muscle and fibroblast samples (d) of normal control (NC) and patient 5 (PT5). Histogram bar showing similar protein reduction between muscle and skin (e). CAPN3 expression normalized respect to GAPDH expression (data are representative of n = 3 independent experiments). Full-length CAPN3 mRNA expression in muscle tissue and fibroblasts of normal control (NC) and PT5 (f). CAPN3 expression in muscle tissue of NC was set to 1. Relative expression levels were calculated relative to HPRT1 and ZNF80 mRNA levels (data are representative of n = 2 independent experiments).

Figure 7

Flowchart describing the performed analyses.