Unraveling Novel Pathogenic CAPN3 Variants in Limb-Girdle Muscular Dystrophy R1

Sukanya Banerjee and Bishan Dass Radotra^*

Department of Histopathology, Post Graduate Institute of Medical Education and Research, Chandigarh-160012, India

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Abstract

Limb-Girdle Muscular Dystrophy R1 (LGMDR1) is an autosomal recessive neuromuscular disorder caused by mutations in the calpain-3 (CAPN3) gene, which encodes a calcium-dependent intracellular protease. Various pathogenic mutations have been reported to date, typically leading to weakness of the pelvic and shoulder girdle muscles. However, the clinical and pathological features of LGMDR1 are often heterogeneous and overlap with those of other LGMD subtypes, making a definitive molecular diagnosis essential for understanding the disease pathogenesis. Immunoblot analysis of calpain-3 protein revealed either complete or partial loss of expression in patients with LGMD. Additionally, multiple CAPN3 variants were identified and evaluated using in silico pathogenicity prediction tools. Alongside previously known mutations, Sanger sequencing also revealed novel pathogenic variants in the CAPN3 gene. Identification of genetic variants in LGMDR1 patients helps explore the mechanisms of disease pathogenesis and may support more accurate diagnosis and prognostication.

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Introduction

LGMD is the most common form of muscular dystrophy worldwide¹ and accounts for approximately 40% of all LGMD cases across different regions², resulting from several variants in the CAPN3 gene³. The onset of symptoms ranges from 6 months to 14 years⁴. The most common phenotype of LGMDR1 is progressive muscle weakness and atrophy, typically accompanied by bilateral scapular winging and Achilles tendon contracture⁵. The clinical phenotype and laboratory data are not specific to LGMDR1 and may overlap with those of other types of LGMD; therefore, the disease cannot be diagnosed with certainty clinically or investigated at the biochemical level. Slowly progressive LGMDR1 can cause loss of ambulation during childhood and near-normal life expectancy⁶.

More than 440 different pathogenic variants of the calpain-3 gene have been reported to date, distributed along the entire length of the gene product⁷. The CAPN3 gene comprises 24 exons and is located on chromosome 15q15.2⁸; it is expressed as a 3.5-kb transcript and encodes a 94 kDa calpain-3 protein⁹.

Many pathogenic variants in the CAPN3 gene have been identified, including missense mutations, frameshift mutations, nonsense mutations, deletions/insertions, splice site mutations, and single-nucleotide variants. Out of these variants, missense variants are the most frequently dispersed throughout the whole CAPN3 gene^1,10-11. These mutations decrease the proteolytic activity of CAPN3 by impairing protein inter-domain interactions, leading to a complete or partial loss of CAPN3 protein^10,12-14. For example, a recent study by Massucco et al. (2024) mentioned heterozygous CAPN3 variant c.1478G>A (p.Arg493Gln) as a mild form of LGMDR1¹⁵. Another study by Feng et al. (2024) reported that the CAPN3 variant c.1783-72C>G, a novel pathogenic variant based on the American College of Medical Genetics (ACMG) guidelines, also highlights the importance of genetic counseling for these patients to understand disease progression¹⁶. Additionally, a study by Macias et al. (2021) emphasized the importance of targeted sequencing of the entire CAPN3 gene, which revealed that intronic mutations might play a role in understanding the etiology of disease in Polish LGMDR patients¹⁷.

Given the wide-ranging complexity and variability of CAPN3 mutations, this mini review aims to provide an up-to-date overview of the reported genetic variants and their clinical significance. This mini review takes a narrative approach, bringing together recent findings on CAPN3 variants and their clinical impact in LGMDR1. Studies were selected based on how they contribute to understanding the relationship between genetic changes, protein expression, and diagnostic insights.

Immunoblot-Based Assessment of Calpain-3 in LGMDR1

Calpain-3 protein expression in LGMDR1 patients is typically explored using immunoblotting techniques, which target the characteristic 94 kDa protein band. In most reported cases, this analysis reveals either a partial or complete loss of calpain-3 expression, supporting its role in disease pathogenesis¹⁸. This observation is consistent with previous studies, which also described reduced or undetectable calpain-3 protein levels at the 94 kDa position in affected individuals¹⁹. These findings underscore the importance of immunoblot analysis as a supportive diagnostic tool, particularly when combined with molecular testing for CAPN3 variants.

Spectrum of CAPN3 Variants in LGMDR1

Sanger sequencing remains a key method for identifying CAPN3 variants in patients with suspected LGMDR1. Recent studies have expanded the known mutation spectrum by reporting both novel and previously described pathogenic variants. Figure 3, modified from Fanin et al. (2007), provides an overview of the calpain-3 protein structure, highlighting its functional domains and mutation sites identified in earlier research.

Among the novel findings, a missense variant in exon 9 (c.1189T>C; p.Phe397Leu) has been reported and classified as likely pathogenic based on ACMG guidelines¹⁸. In-silico analysis suggested that this substitution, from phenylalanine to leucine, may affect the catalytic site of the protein and contribute to functional loss. Another novel variant, an insertion-deletion (c.1688delinsTC; p.Arg490Leufs*87) in exon 11, results in a frameshift leading to premature protein termination¹⁸. This change is predicted to disrupt domain III of the calpain-3 protein, which is involved in binding calcium and phospholipids. It has been classified as pathogenic according to ACMG criteria.

In addition to these novel variants, several known CAPN3 mutations have been detected. These include a missense variant (c.2338G>C; p.Asp780His), a nonsense mutation (c.1939G>T; p.Glu647Ter), and a splice-site variant (c.2051-1G>T), all of which are associated with LGMDR1 (Table 1)¹⁸. The c.2338G>C variant, found in three patients, has been described as a founder mutation in the Agarwal community in India²¹. This mutation may affect the calmodulin-like calcium-binding domain (domain IV) of the protein, as suggested by in silico predictions.

The nonsense variant c.1939G>T leads to early termination of the protein and is predicted to affect the linker region^22–23. Interestingly, these variants were also present in asymptomatic carrier parents, highlighting the importance of genetic counseling¹⁸.

Table 1: Variants in CAPN3 gene found in LGMDR1 patients, in-silico analysis and genotype-phenotype correlation

Genotype-phenotype correlation	Waddling gait; wasting of biceps, triceps and supraspinatus muscle; Calf with EDB (extensor digitorum brevis) muscle and Beevor sign were observed.	Waddling gait and scapular winging	Proximal weakness since 2 years	Problems in running since the last 6 years, difficulty in getting up	Difficulty in climbing stairs, getting up from squatting position. Waddling gait, scapular winging and adductor splay sign were observed	Patient needed support during walking, with presence of lordotic posture and was wheelchair bound
ACMG criteria and classification	PM1, PM2, PP2, PP3 (Likely pathogenic)	PM1, PM2, PP2, PP3, PP5 (Pathogenic)	PVS1, PM2, PM3, PP3, PP5 & PM1, PM2, PM3, PP2, PP3, PP5 (Pathogenic)	PVS1, PM2, PP3 (Pathogenic)	PM1, PM2, PP2, PP3, PP5 (Pathogenic)	PVS1, PM2, PP3, PP5 (Pathogenic)
Human Splice Site Finder	_	_	Acceptor splice site affected	_	_	_
Mutation Taster	Disease causing	Disease causing	Disease causing	Disease causing	Disease causing	Disease causing
Provean	del (-5.318)	del (-5.973)	del (-5.973)		del (-5.973)
PolyPhen	PD (0.929)	PD (1.000)	PD (1.000)		PD (1.000)
Mutation type	Missense	Missense	Splice site variant & missense	Insertion-deletion	Missense	Nonsense
HGVSp	p.Phe397Leu	p.Asp780His	p.Asp780His	p.Arg490Leufs*87	p.Asp780His	p.Glu647Ter
HGVSc	c.1189T>C	c.2338G>C	c.2338G>C & c.2051-1G>T	c.1688delinsTC	c.2338G>C	c.1939G>T
Genotype	Homo	Homo	Hetero	Homo	Homo	Homo
Exon	9	22	22 & splice site variant	11	22	17
Gene	CAPN3	CAPN3	CAPN3	CAPN3	CAPN3	CAPN3
Patient (Age/Sex)	1 (19/M)	2 (45/M)	3 (25/F)	4 (26/M)	5 (26/F)	6 (22/M)

Other studies have similarly contributed to the understanding of CAPN3 variant diversity. For example, Massucco et al. (2024) identified the c.1478G>A (p.Arg493Gln) variant, which may affect protein folding and is associated with a milder disease phenotype¹⁵. Feng et al. (2024) described the novel intronic variant c.1783-72C>G and a previously reported pathogenic mutation c.2120A>G, both of which support the relevance of intronic and regulatory sequences in LGMDR1¹⁶. Similarly, Macias et al. (2021) emphasized the significance of non-coding CAPN3 variants in modulating disease severity¹⁷.

Table 2 provides an overview of CAPN3 variants and their associated protein expression from previous literatures, highlighting how different mutation types relate to disease severity.

Table 2: Comparison of CAPN3 Variants and Protein Expression Across Selected Studies

Study	CAPN3   Variant(s)	Study Type	Protein   Expression	Phenotype   Severity
Banerjee et al., 2024 [18]	Novel: c.1189T>C, c.1688delinsTC Known: c.2338G>C, c.1939G>T, c.2051-1G>T	Original cohort, India	Partial or complete loss (by immunoblot)	Moderate to severe (early wheelchair use in indel/nonsense cases)
Fanin et al.,   2007 [13]	Multiple   known variants	Functional   study	Total loss in   null variants	Severe
Feng et al.,   2024 [16]	c.1783-72C>G   (novel intronic)	Case report	Not reported	Predicted exon   skipping

Genotype-Phenotype Correlation

Genotype-phenotype correlations in LGMDR1 are crucial for understanding the diverse clinical presentations observed in patients. Bevilacqua et al. (2020) identified nine genes—CAPN3, DYSF, SGCG, SGCA, SGCB, SGCD, FKRP, ANO5, TCAP, and GAA—as the most frequent contributors to recessive limb-girdle muscular weakness²⁵. In line with this, Banerjee et al. (2024) highlighted the impact of different CAPN3 pathogenic variants on clinical features in LGMDR1. Their findings suggested that patients harboring nonsense or insertion-deletion (indel) mutations had more severe disease progression, often becoming wheelchair users in early adulthood. Notably, one patient with a homozygous indel variant had complete absence of calpain-3 protein expression, underscoring the functional impact of such mutations¹⁸. This observation aligns with earlier work by Fanin et al. (2009), which reported that null variants typically result in total loss of calpain-3²³. In contrast, patients with missense variants generally exhibited milder symptoms, likely due to the variable effects these mutations have on protein structure and function^5,7. While these observations are informative, further studies on genotype-phenotype correlation are required in a large cohort of LGMDR1 patients¹⁸.

Future Directions

Identifying novel pathogenic variants in the CAPN3 gene may help determine the genetic cause of LGMD in patients who experience the disease from childhood to adulthood. Confirmation of diagnosis may prevent unnecessary treatments for such patients and aid in genetic counseling and the formulation of therapeutic interventions. Genetic factors may regulate the diseased phenotype of LGMDR1; therefore, genetic testing can help elucidate the molecular basis of the pathogenesis in these heterogeneous patients. Thus, genotype-phenotype correlations are required to explain the characteristics of a mutation and its effect on a patient’s phenotype with a genetic etiology. Expanding the CAPN3 variant database could aid in the development of targeted therapies for LGMDR1 patients. Broader genetic testing may also help detect the disease early and inform more effective therapeutic strategies. However, such studies involving mutation analysis of the CAPN3 gene should be carried out only after immunoblot analysis of the protein, which helps diagnose LGMDR1 in conjunction with clinical, pathological, and immunohistochemical findings. However, in most cases, genetic studies are sufficient to diagnose LGMDR1, which may avoid the need for a muscle biopsy.

Abbreviations

CAPN3 – Calpain-3

GAPDH – Glyceraldehyde 3-phosphate dehydrogenase

DYSF – Dysferlin

SGCG – Sarcoglycan gamma

SGCA – Sarcoglycan alpha

SGCB – Sarcoglycan beta

SGCD – Sarcoglycan delta

FKRP – Fukutin-related protein

ANO5 – Anoctamin 5

TCAP – Titin-cap

GAA – Alpha-glucosidase

Acknowledgements

All authors of the study by Banerjee et al., 2024 would like to acknowledge Post Graduate Institute of Medical Education and Research (PGIMER), Chandigarh and Indian Council of Medical Research (ICMR), New Delhi. The study was supported by Intramural Research Grant, PGIMER, Chandigarh (Endst No. 71/2-Edu-16/1265–1266).

Conflict of Interest

The authors have no relevant financial or non-financial interests to disclose.

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