This study [2015(916)] was approved by the Ethics Committee of the Peking University First Hospital. Written informed consent was obtained from the individuals and legal guardians for participation in the study as well as for the publication of any potentially identifiable images or data included in this article.

The inclusion criteria for α-DGP were described in our previous study (Song et al., 2020). In this study, patients with molecular genetic results indicating POMT2 variants were enrolled, and patients diagnosed with other types of CMD or with other gene variants were excluded.

The patients’ clinical symptoms, physical examination results, and laboratory and molecular genetic tests were analyzed retrospectively.

Genomic DNA was extracted from peripheral blood lymphocytes of the patients and their parents for whole exome sequencing (WES). Sequencing analysis was based on the reference sequence of POMT2 (NM_013382.5), and the candidate variant was confirmed by Sanger sequencing in family members. We used some disease association databases, including the Human Gene Mutation Database (HGMD¹), Leiden Open Variation Database (LOVD²), ClinVar database³, and 1,000 Genomes⁴ to identify pathogenic, clinically reported or novel mutations. If WES data revealed differences in the number of sequence reads between the patients and control samples, the change was considered to be a copy number variant (CNV), which was then confirmed by quantitative polymerase chain reaction (qPCR). The pathogenicity of novel or clinically unreported variants was predicted by using three kinds of bioinformatics software: MutationTaster⁵, PolyPhen2⁶, and Provean⁷.

Open biopsy of the quadriceps or biceps brachii was performed, and the samples were fixed in isopentane precooled in liquid nitrogen. Routine histochemical staining was performed. Frozen sections (6 μm) from the muscle biopsy specimens were fixed with 4% paraformaldehyde at room temperature for 10 min. Non-specific binding was reduced by a 1-h incubation with 10% goat serum (Solarbio, China) in phosphate-buffered saline (PBS). The sections were then incubated at 4°C overnight with α-DG glycosylation antibody, IIH6C4 (Millipore, United States, 1:200), and β-DG antibody (Abcam, United Kingdom, 1:200), washed three times with PBS and then incubated with a goat anti-mouse fluorescent antibody (Life Technologies Corporation, United States, 1:500) at room temperature for 1 h. Immunofluorescence staining was observed by using an Olympus Fluoview FV10i (Olympus, Japan).

A hybrid minigene splicing assay in vitro was employed to evaluate the transcriptional effect of the variant on splicing. Wild-type and mutant minigene constructs comprising a genomic fragment spanning exon 17 to the exon 20 of POMT2 were synthesized and cloned into the pcDNA 3.1 plasmid (Ge et al., 2019); the mutant minigene contains the variant c.1891G > C. Wild-type and mutant minigenes were transiently transfected into HEK-293 cells using Lipofectamine 3000 transfection reagent (Thermo Fisher Scientific, United States), and the cells were then incubated for 72 h before isolation of total RNA using TRIzol. cDNA was synthesized using 1 μg of RNA with a reverse transcription system kit (Promega, A3500, United States) and specific primers, including primers upstream in exon 17 (forward 5′-GGTCAATGACACAGATTTCCGA-3′) and downstream in exon 20 (reverse 5′-TAGGCAGTTCCCAGGAGCA-3′).

A total of 11 POMT2-related α-DGP patients diagnosed by WES were enrolled. They come from non-consanguineous families, including eight males and three females. Among them, patients 4a and 5a were siblings, as were patients 7b and 8b. The patients were divided into three groups based on their clinical manifestations: MEB-like, CMD-ID, and LGMD2N. The clinical characteristics of these 11 patients were summarized in Table 1.

The MEB-like group includes patients 4a, 5a, and 10. Their age of onset was between 3 and 8 months old, manifesting as psychomotor developmental delay, microcephaly, and joint contractures. Their maximum mobile ability was only supported or unsupported sitting. Patients 4a and 5a were only able to speak simple words; patient 10 could pronounce two syllables unconsciously. In addition, the patients were only able to understand simple instructions. All of them had no abnormal history of birth. Physical examination revealed obvious myopathy appearances, small head circumferences, and prominently decreased muscle strength in the proximal limbs. In patient 4a, multiple joint contractures including interphalangeal, knee, and ankle joints and severe scoliosis occurred from 4 years old. Patient 5a presented with pseudohypertrophy of the forearm and gastrocnemius and knee joint contracture at the age of 5 years. Patient 10 showed a high palate arch and knee joint contracture at the age of 2 years. However, eye involvement in three children was not obvious, with only patient 10 exhibiting esotropia. Their serum creatine kinase (CK) levels were moderately elevated and fluctuated between 3,609 and 5,200 IU/L. Brain MRI showed cerebellar and brainstem dysplasia in patients 4a and 10 and obvious polymicrogyria in patient 10 (Figure 1). As the clinical and MRI data of these three patients were similar to MEB, but milder than it, we considered their phenotype to be MEB-like.

The CMD-ID group includes patients 1, 2, 7b, 8b, and 11. Their age of onset was between shortly after birth and 5 months of age. The symptoms initially manifested as motor developmental delays with different degrees of severity. The maximum motor capacity of patient 1 was running and jumping but worse than that of normal children of the same age; patient 2 could only sit without support. Patients 7b, 8b, and 11 were able to walk alone, albeit unstably, and patient 7b had lost his ambulant capacity at the age of 10 years due to severe knee and ankle joint contractures. All five patients exhibited intellectual disability. Patients 1 and 7b had difficulty learning at school and poor reading ability. Patients 2, 8b, and 11 were only able to understand simple instructions. Although patient 1’s language development was generally normal, the remaining patients could only speak single words or short sentences. Physical examination revealed muscle weakness, which was dominant in the proximal lower limbs. Patient 7b exhibited pseudohypertrophy in the gastrocnemius muscle with a positive Gowers’ sign and obvious knee and ankle joint contractures at the age of 10 years. Patient 2 displayed slight esotropia; the remaining patients showed no ocular involvement. Their CK levels ranged from 2,000 to 9,080 IU/L. Brain MRI of these patients showed no structural abnormalities, though patients 1 and 2 revealed an enlarged subarachnoid space or ventricle, and patients 7b and 8b showed abnormal white matter signals.

The LGMD2N group includes patients 3, 6, and 9. Their onset age ranged from 1 to 4 years old, with initial manifestations of abnormal or unstable gait and limb muscle weakness that were relatively milder than those of the patients above. They were susceptible to fatigue, although their daily activities were not affected, and they had poorer running and jumping abilities than normal individuals of the same age. All of them had difficulties learning at school and poor reading skills. The language development of the three patients was generally normal, but patient 6 had mild dysarthria, and patient 9 had poor language expression. Physical examination at the age of last follow-up revealed small head circumferences and slightly decreased muscle strength, which was proximally dominant, with no joint contractures. The patellar tendon reflex of patient 6 was weakened and that of patients 3 and 9 was not drawn out. They had no ocular involvement currently. CK levels ranged from 1,362 to 8,000 IU/L in these patients. Brain MRI indicated normal cerebral structure, though the posterior horns of the lateral ventricle of patient 6 were enlarged slightly, and patient 9 had abnormal white matter signals. Considering the late onset age, mild clinical symptoms, unlimited daily activities and lack of brain structural abnormalities of the three patients, we categorized them into the LGMD group. Notably, we found that all patients had learning difficulties through follow-up, as it was difficult for them to understand the contents of courses, and they had poor mathematical calculation and memory skills.

A total of 15 different POMT2 variants, including 9 novel variants and 6 known variants were identified in these 11 patients. No pathogenic CNVs were found. The genetic data for the 11 patients were summarized in Table 1. We performed pathogenicity prediction for the 11 unreported variants using bioinformatics software. Most of the variants are missense changes in highly conservative regions. Nine of these 11 variants are predicted to be deleterious or likely pathogenic, but the pathogenicity of 2 missense variants, c.1891G > C and c.874G > C, are uncertain (Table 2). Base G at position c.1891 is the last base in exon 18 of the POMT2 gene, and a nucleotide change at this position would possibly affect the splicing of the transcript. The position c.874G is not particularly conservative, and this change may be benign based on the prediction of software. In order to confirm the pathogenicity of these two variants, we have conducted further research.

Hematoxylin–eosin (HE) staining of the left biceps brachii biopsy of patient 6 showed muscular dystrophic changes with slightly increased variation in muscle fiber diameter, necrosis, and regeneration of muscle fibers accompanied by inflammatory cell infiltration (Figure 2A). In addition, obviously proliferated lipids and connective tissue in the partial perimysium, increased variation in muscle fiber diameter, and some necrotic muscle fibers were detected in the muscle biopsy of the right quadriceps of patient 7b (Figure 2B). Based on immunofluorescence staining with an IIH6C4 antibody, the expression of glycosylated α-DG in the muscle cell membrane from patients was significantly defective compared to that in the control (Figures 2A1–C3). The staining of β-DG antibody was normal in both patients and the healthy control (Figures 2A4–C4).

A minigene of POMT2 carrying the c.1891G > C variant was constructed to verify the transcriptional consequences of the splice site change. An electropherogram of PCR products revealed the predicted size 402-bp band in the wild-type control, whereas a larger 1,500-bp band was observed in the mutant (Figure 3A). Sequencing of the PCR product showed that the intron between exons 18 and 19 was spliced retention, which suggested that the variant c.1891G > C affects splicing. Furthermore, we found a termination codon when analyzing the sequence of the retention intron 18, which probably resulted in the premature termination of the protein translation (Figure 3B).