In this study, the clinical and genetic data of a Chinese family were collected. The case study was approved by the Ethics Committee of the First Affiliated Hospital and the College of Clinical Medicine of Henan University of Science and Technology, Luoyang, China (ethical approval number: 2022-03-B127). Informed consent was acquired from the patient (or relative/guardian) AND that patient (or relative/guardian) consented to the publishing of all images, clinical data, and other data included in the manuscript.

The proband (a 33-year-old male) was born to non-consanguineous Chinese parents. He presented with progressive weakness, decreased stride frequencies, increased unsteadiness in both lower limbs, and, at the age of 15, needed knee support while standing. Over time, he began to fall to the ground frequently and required help to rise. Three years ago, he could not walk, comb his hair, or get dressed unaided. The weakness and atrophy of the proximal limbs, a wide-based gait, hyperlordosis, scapular winging, and anterior pelvic tilt were found through physical examination. The strength was measured with the Medical Research Council muscle testing/MRC scale (MacAvoy and Green, 2007): deltoid 2/5, biceps/triceps 3/5, quadriceps and biceps femoris 3/5, and gluteus maximus 2/5. The facial and bulbar muscles were not involved. The cardiovascular and respiratory systems were normal. At 16 years of age, his blood analysis results showed elevated creatine kinase levels of 3,000 U/L (normal value < 150 U/L), which decreased to 1,700 U/L at 21. Electromyography (EMG) showed severe generalized myopathy with low-amplitude short-duration motor units. Nerve conduction was spared. The electrocardiogram (ECG) and ultrasound cardiogram (UCG) results were normal. He is now wheelchair-dependent and unable to take care of himself. Due to regular follow-up, the proband has a stable mood and sleeps well. The patient was treated with oral inosine and functional exercise. He consequently developed concomitant hyperthyroidism, and he was put on oral propylthiouracil tablets.

The sister of the proband was a 36-year-old woman. She complained of paroxysmal myalgia after exercise and hyperCKemia for 7 years. Progressively, she presented with proximal lower limb weakness. Her physical examination showed bilateral atrophy of the proximal lower limbs, scapular winging, bilateral calf hypertrophy, and slight weakness (the MRC scale: deltoid 4+/5, musculus biceps brachii 5−/5, forearm flexors 5/5, forearm extensor 4/5, iliopsoas and gluteus maximus 5−/5, pronator and supinator 5/5) and a positive Gower’s maneuver result. Her creatine kinase level was 1,800 U/L at the first evaluation and 3,000 U/L at the 1-month follow-up. EMG results showed features of myopathy. Within 3 years of follow-up, she had difficulty climbing the stairs and needed to hang on to the railing with both hands. On physical examination, the patient’s deltoid muscle, proximal thigh, and right calf were atrophic (Figure 1A). The proximal limb weakness progressively aggravated (the MRC scale: deltoid 4/5, musculus biceps brachii 4/5, forearm flexors 4/5, forearm extensor 3/5, iliopsoas and gluteus maximus 4-/5, pronator and supinator 5/5). Muscular MRI of the lower limb showed that the glutei/iliopsoas muscles, vastus lateralis/gracilis, adductors/biceps, femoris/semitendinosus and semimembranosus muscles, and gastrocnemius/soleus were affected (Figure 1B). Muscle fibers became thinner, accompanied by fat infiltration and edema. She had no sleeping or emotional disorder. She took vitamin E tablets and trimetazidine hydrochloride tablets orally. Meanwhile, she was prescribed functional training.

Samples of the quadriceps femoris muscle of patient Ⅱ-3 were collected for muscle biopsies according to the conventional methods.

Peripheral blood samples were collected from all the members of the family, and genomic DNA was extracted. Agilent SureSelect Human All Exon V6 reagent was used for capture and enrichment, and Illumina NovaSeq 6000 was applied for whole-exon gene sequencing in the proband. The results were aligned to the human reference genome hg19. Variations were annotated according to the American College of Medical Genetics and Genomics (ACMG) guidelines (Richards et al., 2015). Some databases, including the Human Gene Mutation Database (HGMD), ClinVar database, 1,000 Genomes, Exome Aggregation Consortium (ExAC), and genome Aggregation Database (gnomAD), were applied to determine the known or potential novel mutations. Predictions for the pathogenicity of mutations were performed with SIFT, PolyPhen-2, and MutationTaster.

Sanger sequencing to predict and analyze the mutation splicing sites. The primers were designed to target CAPN3 (Forward: 5′-CTC​CAA​GTG​CCT​TCT​GAA​TGA​CCA​CAG​GCG-3′; Reverse: 5′-CAT​CTC​GTA​GCT​GTT​GAT​GGT​GCC​GGA​CTG-3′). Genetic validation was performed at the Institute of Neurology, the First Affiliated Hospital of Fujian Medical University.

Muscle biopsy histology was performed in patient Ⅱ-3 (Figure 2). Hematoxylin and eosin (H&E) show dystrophic features with fiber size variation, scattered small circular atrophic muscle fibers, and partial compensatory hypertrophic muscle fibers. Nuclear translocation was observed in some muscle fibers. The denatured necrotic muscle fibers were observed with a small amount of mononuclear phagocyte infiltration. The inflammatory cell infiltration was not observed in the connective tissue and adipose tissue. Adenosine triphosphatase (ATPase) staining indicated that the fibers of type Ⅰ and type Ⅱ showed mosaic distribution, and no grouping phenomenon was observed. Gomori’s trichrome (GT) staining showed no typical or atypical ragged-red fibers (RRF) and rimmed vacuole (RV) in muscle fibers, and periodic acid-Schiff (PAS) staining showed no obvious glycogen increase in muscle fibers. No special changes were observed in succinic dehydrogenase (SDH) staining and cytochrome c oxidase (COX) staining. We concluded that pathological changes were consistent with muscular dystrophy. Limb-girdle muscular dystrophy was suggested, and further gene sequencing was recommended.

The family pedigree was obtained (Figure 3A). The phenotypes of the proband and his sister were different (Table 1). However, the gene analysis results showed that two siblings possessed two identical mutations in the CAPN3 gene that were derived from their parents (Table 2). The mutation c.1783-72 C>G from the father (Figure 3B) was not previously reported. According to the ACMG guidelines (Richards et al., 2015), its pathogenicity grade is PM3+PP1+PP3+PP4, which may be a “likely pathogenic” mutation. Another mutation, c.2120 A>G, from the mother, leading to amino acid changes (p.D707G) (Figure 3C), is the LGMD2A/R1 pathogenic mutation reported in the HGMD Professional and ClinVar databases.

The cDNA of the proband’s sister was amplified based on the result of the WES. Sanger sequencing showed that exon 2 had a skip (16 bp total) near the variant site chr15-42698052-C-G that validated the mutation c.1783-72 C >G detected by WES (Figure 3D).