LGMDR14, a rare autosomal recessive subtype of LGMD, is attributed to genetic mutations in the POMT2 gene [4]. Here, we presented a summary of the clinical and genetic characteristics of three Chinese patients with adult-onset LGMDR14. The onset of muscle symptoms occurred in late adulthood in our three patients. According to Panicucci et al., the median age of onset for LGMDR14 was 4 years [10]. However, the onset age in our patients was all after the age of 20, which suggested that adult onset LGMD type also deserved attention. All of these patients had common clinical features, including slowly progressive proximal muscle weakness, elevated CK levels and myogenic impairment on EMG. However, patient 2 had severe cardiac involvement as well as mild cognitive impairment. Cardiac muscle and brain cells also underwent glycosylation of α-DG, which could explain the common incidence of cardiac and cognitive involvement in LGMDR14 due to α-DG glycosylation abnormalities [7]. These findings underscored the importance of regular cardiac and cognitive assessments in LGMDR14 management. In terms of muscle pathology, all three patients showed significant myogenic damage and a reduction in α-DG, which was consistent with the pathological characteristics of reported LGMDR14 patients. In addition, patient 1 exhibited moth-eaten muscle fibers and patient 3 showed a preponderance of type I fibers and atrophy in size of type II fibers (Fig. 1C, G, H), which have never been described in previous reports. These suggested that there could be myofibril network disorder and confusion in the distribution of muscle fiber types. Moreover, we identified three new unreported mutations and studied the splicing and pathogenicity mechanisms of the mutations in detail.
For patient 1, we firstly identified the splicing mutation c.1006 + 1G > A in LGMDR14 and conducted a detailed analysis about the effect of splicing mechanism. According to our results, the splicing mutation c.1006 + 1G > A induced the retention of the first 26 bp of intron 8 by inducing recognition of new donor splice sites in POMT2 pre-mRNA (Fig. 3B-D). Aside from shortened transcripts, transcripts that contain exon skipping and intron retention may also be subject to degradation by NMD [20]. By utilizing pyrosequencing, we also discovered that the splicing mutation c.1006 + 1G > A induced the effects of NMD (Fig. 3E). The HGMD database reported a total of 17 intron mutations in the POMT2 gene, but few studies have elucidated their specific splicing mechanism. Yanagisawa et al. identified two c.248 + 5G > C and c.1333-14G > A intronic mutations in the two CMD patients [21]. They studied that c.248 + 5G > C led to a 72-bp insertion between exons 1 and 2 and c.1333-14G > A resulted in a 12-bp retention between exons 12 and 13 [21]. In previous researches, the c.1006 + 1G > A mutation was confirmed to be associated with WWS and cobblestone lissencephaly in two patients [15, 16]. Reeuwijk et al.firstly identified the c.1006 + 1G > A homozygous mutation in a male child who manifested with severe WWS [15]. And Devisme et al.reported the other patient with cobblestone lissencephaly who carried c.1006 + 1G > A and c.1168_1172 delCATA complex heterozygous mutations [16]. Compared with patient 1, both of previous two patients had more severe clinical manifestations. These results showed that the same mutation site could appear in different phenotypes of POMT2-associated diseases, which reinforced the complexity between genotype and phenotype.
Three novel variants, c.700_701insCT(p.V234Afs*8), c.812C > T(p.S271L) and c.170G > A(p.W57*) have not been reported in POMT2-related disorders previously, which further expanded the mutation spectrum of POMT2 gene. The c.700_701insCT was a frameshift mutation. At the protein level, the mutation c.700_701insCT resulted in p.V234Afs*8. Additionally, c.700_701insCT led to the production of truncated protein which could cause NMD effects according to our pyrosequencing analysis (Fig. 3F). Patient 3 was found to harbor compound heterozygous mutations, including c.812C > T( p.S271L) and c.170G > A(p.W57*). The amino acids at positions p.S271 and p.W57 displayed marked conservation and were anticipated to induce significant damage based on computational predictions (Fig. 3A). In 3D model, the c.812C > T(p.S271L) caused the 271th amino acid and its surrounding side chain to shift, resulting in the loss of two hydrogen bonds between SER271 and THR274 (Fig. 4B). The nonsense mutation c.170G > A(p.W57*) caused the premature appearance of stop codons, leading to the formation of truncated protein, which could generate severe consequences for protein function. Therefore, we concluded that the c.700_701insCT, c.812C > T and c.170G > A mutations were deleterious.
POMT2, the enzyme catalyzing the initial step of α-DG O-mannosylation, was implicated in severe phenotypic manifestations [16]. The earliest documented case of POMT2 mutation was associated with WWS, which exhibited the most severe phenotype [15]. Recent advancements in genetic testing have revealed a spectrum of milder POMT2-related phenotypes, such as CMD and LGMDR14. However, due to the pleiotropic effects of genes, a definitive connection between genotype and phenotype was not evident, and a range of phenotypes could be induced by the identical mutation site in different individuals. Our results validated the hypothesis that the clinical phenotype in POMT2-related disorders was influenced by the type and location of mutations [18, 22]. In LGMDR14 patients, the presence of at least one missense mutation was common. This was likely due to the fact that missense mutations often resulted in a residual level of POMT2 enzyme activity, which could help improve the clinical phenotype [21, 22]. Conversely, individuals with WWS typically exhibited two "null" mutations, leading to severe protein functional defects such as truncated proteins and the involvement of crucial residues for enzymatic activity. These factors contributed to the exacerbation of the clinical phenotype in patients [15]. Considering that patient 1 had a point mutation c.295C > T which correlated with less impact on protein function and an intron mutation c.1006 + 1G > A which caused truncated protein, we hypothesized that patient 1 may exhibit a relatively mild phenotype. Similarly, according to this hypothesis, patients 2 and 3 each carried a mild mutation, c.1261C > T (p.R421W)and c.812C > T (p.S271L), respectively, and a severe mutation, c.700_701insCT (p.V234Afs*8) and c.170G > A (p.W57*), respectively. Therefore, they both exhibited a relatively mild phenotype compared with WWS. However, Panicucci et al. demonstrated that 18% of the POMT2 variants linked to LGMDR14 were also found in more severe disorders, such as CMD and WWS [10]. This suggested that predicting the phenotype based solely on the genotype could be challenging at times.
Our ability to correlate genotype with phenotype was constrained by the abundance of compound heterozygous mutations that vary in type and location [23]. After mutations occur in the POMT2 gene, two possible outcomes could be observed: (1) a total lack or partial deletion of the POMT2 protein and (2) the synthesis of a complete protein with either a substitution or insertion of amino acids[21]. Alterations in protein structure could have diverse effects on functionality, potentially leading to the development of various diseases. Therefore, investigating the impact of POMT2 mutations on the 3D protein structure was imperative for comprehending the clinical manifestations of diverse mutations. In our analysis, the mutations c.1006 + 1G > A, c.700_701insCT and c.170G > A all produced truncated proteins that may impair the normal structure and function of POMT2. To better elucidate the genotype-phenotypic relationship among the three patients, we used molecular dynamics simulations to analyze the effects of truncated proteins on function and stability. We conducted a 100 ns simulation process to simulate the complete atomic molecular dynamics of three mutant proteins and the wild-type protein. The RMSD values of MT1 and MT3 were larger than WT, indicating the reduction of protein stability. RMSF showed that the MT1 and MT2 had different degrees of influence on the N-terminal stability of POMT2 protein. Moreover, the three variants also affected the number of hydrogen bonds and their major secondary structures. These changes may affect the binding ability and catalytic function of the protein with ligands and other molecules, thus affecting the O-mannoylation of α-DG catalyzed by POMT2. This method simulated the 3D helical structure of the three mutants and wild type protein well, and used the variation information to monitor the changes of the trimer structures. Molecular dynamics simulations, to a certain extent, could help establish a link between genotype and phenotype, which was a good tool to evaluate their complexity and has not been applied in previous POMT2-related studies.