A Novel Missense Variant Associated with A Splicing Defect in A Myopathic Form of PGK1 Deficiency in The Spanish Population

Phosphoglycerate kinase (PGK)1 deficiency is an X-linked inherited disease associated with different clinical presentations, sometimes as myopathic affectation without hemolytic anemia. We present a 40-year-old male with a mild psychomotor delay and mild mental retardation, who developed progressive exercise intolerance, cramps and sporadic episodes of rhabdomyolysis but no hematological features. A genetic study was carried out by a next-generation sequencing (NGS) panel of 32 genes associated with inherited metabolic myopathies. We identified a missense variant in the PGK1 gene c.1114G > A (p.Gly372Ser) located in the last nucleotide of exon 9. cDNA studies demonstrated abnormalities in mRNA splicing because this change abolishes the exon 9 donor site. This novel variant is the first variant associated with a myopathic form of PGK1 deficiency in the Spanish population.


PGK Enzyme
The phosphoglycerate kinase (PGK) is a key enzyme for adenosine triphosphate (ATP) generation in the glycolysis. It catalyzes the reversible conversion of 1,3-bisphosphoglycerate (1,3-BPG) to 3-phosphoglycerate (3-PG), by the transfer of the high-energy phosphate from position 1 of 1,3-BPG to adenosine diphosphate (ADP) [1]. The 1,3-BPG may be metabolized to 2,3-bisphosphoglycerate (2,3-BPG) by bisphosphoglycerate mutase (BPGM), an enzyme only present in erythrocytes, and then the phosphate at position 2 is removed by bisphosphoglycerate phosphatase (BPGP) to 3-PG. The end product of both reactions is the same, i.e., 3-PG, but PGK also generates a molecule of ATP, while the Neonatal anemia (Hb: 7.3g/dL) and progressive neurological impairment leading to mental retardation (7 years). The decrease in PGK activity is more closely related to a loss of enzyme stability than to a decrease in catalytic function.

Case Report
A 40-year-old male was referred to the Department of Neurology of Navarra's Hospital after suffering two episodes of rhabdomyolysis in the last 2 years without an apparent trigger, complicated with acute renal failure, requiring hemodialysis. He was the eldest of two siblings born to non-consanguineous parents of Spanish origin and was studied in childhood for mild psychomotor delay with frequent falls and a first episode of rhabdomyolysis triggered by exercise (learning to swim) at the age of seven without reaching a specific diagnosis (we have not been able to access previous studies that included a muscle biopsy). From adolescence, he developed a progressive clinical picture of fatigue, exercise intolerance, cramps and recurrent episodes of myoglobinuria. No "second wind" phenomenon was reported and there was no previous history of seizures or other central nervous system (CNS) dysfunctions. Laboratory studies demonstrated increased resting serum creatine kinase levels (x7-10) and normal red cell count in consecutive analyses.
At the time of our neurological evaluation, the physical examination revealed mild cognitive disability, global muscle amyotrophy, facial weakness with bilateral ptosis, proximal and symmetrical weakness in the limbs and neck flexors, without scapular winging or contractures. No evidence of Parkinsonism or tremor. Cerebral and muscle magnetic resonance imaging, abdominal ultrasound and echocardiogram were normal. The analysis showed a normal acylcarnitine profile and reduced lactate production in exercise test. On suspicion of metabolic myopathy, in particular a muscle glycogenoses, a biceps brachialis muscle biopsy was performed without showing significant findings. No vacuoles or glycogen accumulation were observed, and phosphorylase activity was normal. A metabolic myopathies panel was performed.

Metabolic Myopathies Panel
This study was performed in accordance with the declaration of Helsinki and was approved by the Ethics Committee of Complejo Hospitalario of Navarra. Patient inclusion and case publication were approved by the Ethics Committee of Complejo Hospitalario of Navarra. Proyect 2017/68. Blood was collected from the patient and relatives after obtaining informed consent. DNA was extracted using a MagNa Pure Compact Nucleic Acid Isolation Kit I and MagNA Pure Compact Instrument (Roche Molecular Diagnostics, Pleasanton, CA, USA).
A customized next-generation sequencing (NGS) panel of 32 genes associated with inherited metabolic myopathies was designed using AmpliseqTM and sequencing with the PGM-Ion Torrent platform (Life Techonologies) ( Table S1). The alignment of the sequences (ref. CRCh37/hg19) and detection of variants was performed in Torrent Suite (TMAP-variantCaller plugin). The annotation and prioritization of variants has been carried out through the integration of own scripts with Annovar [47]. The theoretical coverage of this panel reaches 99.21%. This analysis did not cover all of target regions due to characteristics inherent to this methodology.

Clinical and Methodological Validation
Variant prioritization was performed assuming an autosomal recessive or recessive X-linked inheritance following the next steps: Available databases were used to determine the complete sequence of the gene and design the most appropriate primers for the PCR: University of California Santa Cruz USCS Genome Browser (https://genome.ucsc.edu/) and Primer3plus (http://www.bioinformatics.nl/cgi-bin/ primer3plus/primer3plus.cgi/). The sequences of the upstream and downstream primers used to amplify the genomic region surrounding the variant of interest located in exon 9 of the PGK1 gene were 5 -GGTCCTGAAAGCAGCAAGAA-3 and 5 -CTCCCCAACCCAAAAGGTAG-3 , respectively.

cDNA Analysis of PGK1
Peripheral blood mononuclear cells (PBMCs) were isolated from whole blood of the index case and a healthy control by Ficoll gradient centrifugation according to the manufacturer's instructions (Ficoll-Paque PLUS, GE Healthcare).
Total RNA was isolated from PBMCs using TRIzol Reagent (Invitrogen, Thermo Fisher Scientific). The cDNAs were synthesized from 1 µg of total RNA using random hexamers with the SuperScript IV-First Strand Synthesis System kit (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA) in a total volume of 20 µL.
Subsequently, cDNA of PGK1 was amplified and Sanger sequenced using specific primers surrounding the exon 9 of PGK1. The sequence of the upstream primer, located in the exon 7-8 junction of PGK1, was 5 -GTGCTCAACAACATGGAGAT-3 , and the sequence of the downstream primer located inside the exon 11 of PGK1 was 5 -TAAATATTGCTGAGAGCATCCA-3 . Amplicons were visualized on a 1% agarose gel using gel red and were purified from the gel with IllustraTM GFXTM PCR DNA and Gel Band Purification Kit (GE Healthcare) to be analyzed separately by direct sequencing (ABI 3500 Genetic Analyzer, Applied Biosystems, Warrington, UK) using a Big Dye Terminator Cycle Sequencing Kit (Applied Biosystems, Warrington, UK). The chromatograms were analyzed with Chromas 2.3 (Technelysium Pty Ltd.).

Enzyme Activity of PGK1
The blood enzymatic activity of PGK was analyzed by molecular absorption spectrometry. The enzymatic analysis of RBCs was performed as described previously by the method of the International Committee for Standardization in Hematology (ICSH) [48].

Results
To identify the genetic cause of the patient's myopathy, we performed a customized NGS panel including 32 genes associated with metabolic myopathies. We found 174 variants-Of which, only one variant was prioritized after applying the established criteria, a hemizygous c.1114G > A substitution that predicts a (p.Gly372Ser) missense mutation in the PGK1 gene (NM_000291). This variant was absent in the gnomAD and the 1000 genomes databases and was categorized as deleterious by in silico predictors such as SIFT, PolyPhen2, LRT, PROVEAN, MutationTaster, CADDPhred, GERP and PhyloP. The variant was registered in the ClinVar database as an uncertain significance (VUS) [49]. Following the American College of Medical Genetics and Genomics (ACMG) guidelines for the interpretation of sequence variants [50], the variant was classified as likely pathogenic.
The Gly-372 residue is located adjacent to the catalytic site of the enzyme [19] and is highly conserved in this protein family among different species ( Figure 1A). Moreover, the variant falls in the last nucleotide of 3 at the end of exon 9, so that in silico analysis using Human Splice Finder and Mutation Taster also suggested that the variant would alter the splicing process, since this change abolishes the exon 9 donor site.
3 variant was absent in the gnomAD and the 1000 genomes databases and was categorized as deleterious by in silico predictors such as SIFT, PolyPhen2, LRT, PROVEAN, MutationTaster, CADDPhred, GERP and PhyloP. The variant was registered in the ClinVar database as an uncertain significance (VUS) [49]. Following the American College of Medical Genetics and Genomics (ACMG) guidelines for the interpretation of sequence variants [50], the variant was classified as likely pathogenic.
The Gly-372 residue is located adjacent to the catalytic site of the enzyme [19] and is highly conserved in this protein family among different species ( Figure 1A). Moreover, the variant falls in the last nucleotide of 3' at the end of exon 9, so that in silico analysis using Human Splice Finder and Mutation Taster also suggested that the variant would alter the splicing process, since this change abolishes the exon 9 donor site.
The presence of the hemizygous variant c.1114G > A (p.G372S) in the PGK1 gene in the patient was confirmed by Sanger sequencing. The asymptomatic parents were analyzed for the variant, which was found to be heterozygous in the patient's mother and absent in the father ( Figure 1B    The presence of the hemizygous variant c.1114G > A (p.G372S) in the PGK1 gene in the patient was confirmed by Sanger sequencing. The asymptomatic parents were analyzed for the variant, which was found to be heterozygous in the patient's mother and absent in the father (Figures 1B and 2).
The analysis of the patient's blood cDNA revealed the presence of two abnormal transcripts: (i) One apparently more abundant species showing a skipping of the exon 9, and (ii) a barely detectable band corresponding to the transcript with a retention of intron 9 (Figure 3). cDNA analysis also reveals that normal splicing is present in the patient, although the missense variant is observed in the sequence of this transcript.
The enzyme activity of PGK was decreased in the patient's blood, 39 UI/g Hb (normal range 197-343).

Figure 2.
Family pedigree illustrating the patient´s family, showing that the patient mother is a carrier of the variant. The four sisters of the patient's mother and the patient´s sister have not been analyzed for their carrier status.
The analysis of the patient's blood cDNA revealed the presence of two abnormal transcripts: i) One apparently more abundant species showing a skipping of the exon 9, and ii) a barely detectable band corresponding to the transcript with a retention of intron 9 ( Figure 3). cDNA analysis also reveals that normal splicing is present in the patient, although the missense variant is observed in the sequence of this transcript.
The enzyme activity of PGK was decreased in the patient's blood, 39 UI/g Hb (normal range 197-343).   Family pedigree illustrating the patient´s family, showing that the patient mother is a carrier of the variant. The four sisters of the patient's mother and the patient´s sister have not been analyzed for their carrier status.
The analysis of the patient's blood cDNA revealed the presence of two abnormal transcripts: i) One apparently more abundant species showing a skipping of the exon 9, and ii) a barely detectable band corresponding to the transcript with a retention of intron 9 (Figure 3). cDNA analysis also reveals that normal splicing is present in the patient, although the missense variant is observed in the sequence of this transcript.
The enzyme activity of PGK was decreased in the patient's blood, 39 UI/g Hb (normal range 197-343).

Discussion
We identified a novel hemizygous missense variant associated with PGK1 deficiency, c.1114G > A (p.G372S) in a male adult patient presenting with exercise intolerance and several episodes of rhabdomyolysis, ptosis, muscle weakness and mild cognitive disability.
The variant is probably the cause of the clinical phenotype, since (i) it was the only prioritized variant after analyzing a customized NGS panel including 32 genes associated with metabolic myopathies; (ii) it was absent in several population databases; (iii) and although it was found as a VUS in the ClinVar database, the ACMG guidelines classified the variant as 'likely pathogenic'; (iv) the amino acid residue is located adjacent to the catalytic site of the enzyme and is highly evolutionary conserved; (v) the 11/11 predictors of pathogenicity indicated the variant is deleterious; (vi) the variant was located at the last nucleotide of the 3 end of exon 9, and predictors of aberrant splicing suggested that the variant affects mRNA maturation, and this fact was demonstrated by the patient's cDNA studies ( Figure 3); (vii) the PGK enzyme activity in the patient's blood cells was strongly decreased.
The variant c.1114G > A (p.G372S) is the first mutation associated with a myopathic form of PGK1 deficiency in the Spanish population; two other Spanish patients who were previously reported with PGK1 deficiency with different mutations in the PGK1 gene presented hemolytic anemia and CNS involvement, with no signs of myopathy ( Table 1).
The c.1114G > A variant in our patient, despite being located in the last nucleotide of the 3 end exonic region of exon 9 of the PGK1 gene, was demonstrated to affect mRNA maturation by cDNA studies on the patient's blood cells. We showed that the mutation c.1114A > G leads to the expression of an apparent main aberrant cDNA-PGK1 species in which exon 9 skipping occurred. In addition, besides the canonical transcript carrying the predicted missense mutation, another longer abnormal transcript was detected which includes a retention of the entire intron 9 of the gene (Figure 3). In summary, c.1114G > A that was initially predicted to lead to a missense change, actually, also gave rise to a splicing defect, resulting in two structural aberrant transcripts of the PGK1 gene that generate a premature stop codon (PTC) (p.G313Vfs * 20 due to exon 9 skipping and p.G372Gfs * 11 due to intron 9 retention).
There are five other demonstrated splicing variants of PGK1 that have been reported so far, namely, IVS4 + 1G > T (North Carolina), p.Gly213=, p.Glu252Ala (Antwerp), IVS7 + 3A > G and IVS7 + 5G > A (Fukuroi) [25,36,37,[39][40][41]. In addition, two splicing defects have been described lacking the identification of the genetic mutation [13,14]. All of these cases were reported in patients with the myopathic form of PGK1 deficiency and with no hemolytic anemia, in a similar manner to our patient. However, there are five other patients with the myopathic phenotype-Four of them were carriers of missense mutations, namely, p.Cys108Tyr, p.Ile253Thr (Hamamatsu), p.Asp315Asn (Creteil) and p.Thr378Pro (Afula) [19,24,28,42]. The fifth was a male hemizygous for a frameshift variant with a four base pair deletion in exon 6 that predicts the formation of a truncated protein (p.Gly213Glufs * 21, Fukui) [35]. Interestingly, three of these missense mutations (Hamamatsu, Creteil and Afula) are located at the beginning or at the end of the corresponding exons similarly to the mutation described here (Figure 4) but a splicing defect was not experimentally discarded, as it was ruled out for other previously reported missense variants localized in these critical regions for mRNA splicing [11].
Regarding genotype-phenotype correlation in PGK1 deficiency, all the mutations that affect splicing have been associated with myopathic forms and absence of hemolytic anemia. Although we cannot state that all myopathic forms without anemia are associated with splicing variants, it seems conceivable to consider this hypothesis, since some reported missense mutations located in critical exonic regions for splicing were not discarded as being involved in the abnormal splicing of PGK1. This is no new concept, because a large and growing number of variants located in protein coding exons have primary disease-causing effects by disrupting splicing-For example, Spinal Muscular Atrophy (SMA) is caused by the loss of SMN1 genes and the C to T substitution in exon 7 of the SMN2 gene that promotes exon skipping in SMN2 and could not compensate the loss of SMN1 [51].

6
A patient recently reported to harbor a missense variant of PGK1 (c.649G > A, p.Val217Ile) and intellectual disability, mild cerebral and cerebellar atrophy and peculiar episodes of muscle weakness of unknown etiology, but without hemolytic anemia, had a residual enzymatic activity of 78-91% in RBCs [38]. This finding would suggest this missense variant, apparently not involved in splicing, is a neutral polymorphism. The correlation between variants that apparently are the most damaging at the protein level such as splicing or frameshift and milder forms of PGK1 deficiency are more complicated to explain; in fact, the levels of residual enzyme activity in these patients are very low (Table 1), and it was shown that these individuals have a much lower amount of enzyme. Recent studies of RNA massive sequencing indicate that the number of events of alternative splicing and produced isoforms are much more abundant than had been estimated previously [52], and that alternative transcripts of PGK1 could even exist that have not been detected to date. This mechanism could explain the phenotype of the frameshift deletion (Fukui variant), where a third part of the protein is missing but surprisingly the mutation is compatible with life and enzymatic activity is detected in erythrocytes and muscle.
In addition, two cases, i.e., PGK Antwerp and our patient, both harboring a predicted missense variant that leads to a combined alteration of the splicing process and the expression of a mutated canonical transcript, showed only less severe myopathic forms. Moreover, in our patient, we showed that the canonical transcript harbors the change c.1114G > A, which is predicted to substitute a Gly residue by Ser in position 372 of the enzyme that is localized adjacent to the catalytic site. Therefore, it would be expected to express a more severe phenotype. Although, it has still to be characterized at the enzymatic level, it is known that G372, G373 and G374 are important residues in the enzyme, coordinating the nucleotide substrate and stabilizing contacts with G395, thus maintaining the closed catalytically active conformation [11].
Previously, it has been attempted to associate intermediate metabolites accumulated in PGK-deficient red cells with hemolysis. These intermediaries, such as 2,3-BPG, inhibit different A patient recently reported to harbor a missense variant of PGK1 (c.649G > A, p.Val217Ile) and intellectual disability, mild cerebral and cerebellar atrophy and peculiar episodes of muscle weakness of unknown etiology, but without hemolytic anemia, had a residual enzymatic activity of 78-91% in RBCs [38]. This finding would suggest this missense variant, apparently not involved in splicing, is a neutral polymorphism.
The correlation between variants that apparently are the most damaging at the protein level such as splicing or frameshift and milder forms of PGK1 deficiency are more complicated to explain; in fact, the levels of residual enzyme activity in these patients are very low (Table 1), and it was shown that these individuals have a much lower amount of enzyme. Recent studies of RNA massive sequencing indicate that the number of events of alternative splicing and produced isoforms are much more abundant than had been estimated previously [52], and that alternative transcripts of PGK1 could even exist that have not been detected to date. This mechanism could explain the phenotype of the frameshift deletion (Fukui variant), where a third part of the protein is missing but surprisingly the mutation is compatible with life and enzymatic activity is detected in erythrocytes and muscle.
In addition, two cases, i.e., PGK Antwerp and our patient, both harboring a predicted missense variant that leads to a combined alteration of the splicing process and the expression of a mutated canonical transcript, showed only less severe myopathic forms. Moreover, in our patient, we showed that the canonical transcript harbors the change c.1114G > A, which is predicted to substitute a Gly residue by Ser in position 372 of the enzyme that is localized adjacent to the catalytic site. Therefore, it would be expected to express a more severe phenotype. Although, it has still to be characterized at the enzymatic level, it is known that G372, G373 and G374 are important residues in the enzyme, coordinating the nucleotide substrate and stabilizing contacts with G395, thus maintaining the closed catalytically active conformation [11].
Previously, it has been attempted to associate intermediate metabolites accumulated in PGK-deficient red cells with hemolysis. These intermediaries, such as 2,3-BPG, inhibit different enzymes of glycolysis and others pathways, such as 6-phosphogluconate dehydrogenase [37], promoting hemolysis. A significant increase in 2,3-BPG in red cells was described in a few patients with hemolytic anemia related to PGK deficiency (PGK Barcelona, PGK Matsue, PGK Amiens/New York and PGK Uppsala), because it has not been quantified in most of the described cases. However, in PGK Creteil, which is not associated with hemolytic anemia, a moderate increase in 2,3-BPG levels in RBCs was described [9]. Perhaps the correlation of the phenotype should not be established with the enzyme activity itself, but with the stability of the protein and its tendency to form aggregates as already raised in 2014 by Pey et al. [53]. From the variants studied by this group, those causing severe affectation to protein stability and mild catalytic efficiency (Barcelona, Matsue, Michigan, Murcia and Kyoto) were associated with hemolytic anemia and neurological disorders but not myopathy. We propose that the aggregates themselves could accumulate and cause damage in different tissues, the in case of RBCs in the form of hemolytic anemia. The splicing or deletion variants would protect from this toxic deposit, which would explain why none of the described splicing variants are associated with hemolytic anemia. In these patients, the clinical picture resembles that of muscular glycogenosis-in which, the symptoms are exacerbated with exercise due to the inability of the mutant enzyme to cope with energy demand. The limitation in establishing this new idea about the phenotype-genotype association may be due to the fact that we could not check how the p.Gly372Ser missense variant affects stability and/or enzymatic activity in vitro and also the variability of the results described so far in terms of the methods used for the identification of variants and functional studies of enzymatic activities. Some of these studies are from 50 years ago; the first case of PGK deficiency was described in 1968 [12].
From the point of view of CNS involvement, the phenotype is heterogeneous. Most of the myopathic forms without hemolytic anemia are associated with mild mental retardation but not with other neurological manifestations. However, the forms characterized by hemolytic anemia are described in patients with a wide range of neurological disturbances ranging from no neurological problem to mental retardation, Parkinson's [1] or retinal dystrophy [31]. Considering the muscular symptoms, phenotypic heterogeneity is also found. Myopathy may be underestimated in the cases described at an earlier age due to its later onset.
However, although the mechanism underlying this clinical heterogeneity of PGK1 deficiency remains unknown, we can conclude with dichotomization in myopathic forms without anemia and hemolytic forms. Finally, we highlight that patients with myopathic forms of PGK1 deficiency might be underdiagnosed due to the absence of hemolytic anemia.