ClinVar Genomic variation as it relates to human health

Published functional studies indicate this variant leads to aberrant splicing, demonstrating a damaging effect (PMID: 34906502); In silico analysis supports a deleterious effect on splicing; This variant is associated with the following publications: (PMID: 25914343, 28967462, 34426522, 25987006, 34906502, 35628876)

This variant was observed in the ICSL laboratory as part of a predisposition screen in an ostensibly healthy population. It had not been previously curated by ICSL or reported in the Human Gene Mutation Database (HGMD: prior to June 1st, 2018), and was therefore a candidate for classification through an automated scoring system. Utilizing variant allele frequency, disease prevalence and penetrance estimates, and inheritance mode, an automated score was calculated to assess if this variant is too frequent to cause the disease. Based on the score and internal cut-off values, a variant classified as likely benign is not then subjected to further curation. The score for this variant resulted in a classification of likely benign for this disease.

PYGM: PS3:Moderate, PP3

Variant summary: PYGM c.660G>A (p.Gln220Gln) alters a conserved nucleotide located close to a canonical splice site and therefore could affect mRNA splicing, leading to a significantly altered protein sequence. A recent publication reported experimental evidence that this variant affects mRNA splicing, demonstrating in-frame exon 5 skipping and out-of-frame exon 4-5 skipping, from whole blood derived RNA samples that were isolated from heterozygous carriers, however, the degree of mis-splicing (i.e. whether complete or partial) was not specified (Bournazos_2022). The variant allele was found at a frequency of 0.0025 in 1614170 control chromosomes, predominantly at a frequency of 0.0067 within the South Asian subpopulation in the gnomAD database, including 2 homozygotes. The observed variant frequency within South Asian control individuals in the gnomAD database is approximately 1.9 fold of the estimated maximal expected allele frequency for a pathogenic variant in PYGM causing Glycogen Storage Disease, Type V phenotype (0.0035). c.660G>A has been reported in the literature in individuals affected with Glycogen Storage Disease, Type V (McArdle disease) (Zoccolella_2015, Inal-Gultekin_2017); however, in two of these reported patients who were from the same family, two co-occurring pathogenic variants were also present (although the phase was not specified), thus potentially explaining the phenotype (Inal-Gultekin_2017). Additionally, the variant was reported in a case of Limb-girdle muscular dystrophy in the homozygous state (Barbosa-Gouveia_2022) and a case of metabolic disease in the heterozgous state (Abolhassani_2024). These data do not allow any conclusion about variant significance. The following publications have been ascertained in the context of this evaluation (PMID: 38374194, 35628876, 34906502, 28967462, 25987006). ClinVar contains an entry for this variant (Variation ID: 197777). Based on the evidence outlined above, the variant was classified as uncertain significance.

Detected two abnormal splicing events: (1) In-frame exon 5 skipping (r.529_660del). This event removes 44 amino acids from the glycogen phosphorylase domain of PYGM (p.(Met177_Gln220del)), of which 26 residues are conserved to Caenorhabditis elegans, (2) Exon 4 and exon 5 skipping (r.425_660del). This event induces a frameshift and encodes a premature termination codon (p.(Ala142Glyfs*32)). These transcripts are predicted to be targeted by nonsense-mediated decay (NMD). Any mis-spliced PYGM transcripts that escape NMD encode PYGM protein lacking 701 amino acids from the C-terminus, including 697 residues from the glycogen phosphorylase domain.

The Gln220= variant in PYGM does not lead to an amino acid exchange in the protein, but according to calculations by the prediction program varSEAK it could very likely lead to the generation of a new cryptic 5' donor splice site that would change the splice mechanism in intron 5. The variant was described in combination with another PYGM variant in a case report in a patient with symptoms of McArdle disease and multiple sclerosis [Zoccolella(2015) Neurol Sci 36(9):1721-3]. Toe Walking has various causes, ranging from idiopathic or habitual reasons to an underlying neuromuscular disease. The most observed form of toe walking is idiopathic toe walking (ITW) - a diagnosis of exclusion. ITW occurs in about 5% of children after their second birthday and is a common problem in pediatric orthopedics. In about 70% of these cases, there is spontaneous remission within six months of the onset of ITW. If the toe walk persists, one can assume the presence of a non-idiopathic form of toe walk (n-ITW). In n-ITW, the causes of the abnormal gait are neurological or myogenic. Differential diagnoses such as infantile cerebral palsy, muscular dystrophy, spinal amyotrophy and hereditary motor-sensory neuropathy as well as rare metabolic disorders of the musculature must be considered (Pomarino et al., 2018). In our clinical ITW consultation, we screen children with n-ITW for a genetic form of tiptoe gait using next generation sequencing for gene variants in 49 genes. These are genes in which gene variants can lead to neuromuscular diseases in which an association with toe-tapping gait has been reported or can be suspected due to patients’ clinical symptoms. To the best of our knowledge, this is the first study in which several patients with toe walking displayed heterozygosity for pathogenic or likely pathogenic PYGM mutations and mild symptoms of the metabolic muscle disease McArdle. The findings of our research are in line with recently published observations in heterozygous family members patients with McArdle disease. We should mention that some of the patients in our cohort harbored heterozygous variants in other genes of our gene panel. However, the numbers in this study were too small to workout any resulting combined genetic effects. It is concluded that genetic conditions can contribute to the development of toe walking. Apparently, even a slight genetic weakening of the muscles can lead to changes to the gait pattern. Future studies must show how the pathomechanism can be explained for the PYGM variants and whether there are new therapeutic approaches to be developed based on this research.