To document 2 apparently incongruous clinical disorders occurring in the same infant: congenital myopathy with myophosphorylase deficiency (McArdle disease) and mitochondrial hepatopathy with liver failure and mitochondrial DNA depletion.
An infant girl born to consanguineous Moroccan parents had severe congenital hypotonia and hepatomegaly, developed liver failure, and died at 5 months of age. We studied muscle and liver biopsy specimens histochemically and biochemically, and we sequenced the whole coding regions of the deoxyguanosine kinase (dGK) and myophosphorylase (PYGM) genes.
Muscle biopsy specimens showed subsarcolemmal glycogen accumulation and negative histochemical reaction for phosphorylase. Liver biopsy specimens showed micronodular cirrhosis and massive mitochondrial proliferation. Biochemical analysis showed phosphorylase deficiency in muscle and cytochrome c oxidase deficiency in liver. We identified a novel homozygous missense G-to-A mutation at codon 456 in exon 11 of PYGM, as well as a homozygous 4–base pair GATT duplication (nucleotides 763-766) in exon 6 of dGK, which produces a frame shift and a premature TGA stop codon at nucleotides 766 to 768, resulting in a truncated 255–amino acid protein. Both mutations were absent in 100 healthy individuals.
Our data further expand the genetic heterogeneity in patients with McArdle disease; confirm the strong relationship between mitochondrial DNA depletion syndrome, liver involvement, and dGK mutations; and suggest that genetic "double trouble" should be considered in patients with unusual severe phenotypes.
McARDLE DISEASE is an autosomal recessive metabolic myopathy caused by mutations in the gene (PYGM) encoding the muscle-specific isoenzyme of glycogen phosphorylase.1 The disease typically presents in adolescents or young adults with exercise intolerance, exercise-induced cramps, and recurrent myoglobinuria. Although this clinical picture is stereotypical, more than 30 mutations have been identified in the PYGM gene.1
Mitochondrial DNA (mtDNA) depletion syndrome (MDS) encompasses a clinically heterogeneous group of disorders characterized by severe reduction in mtDNA copy number.2 Primary mtDNA depletion is inherited as an autosomal recessive trait and may affect single organs, typically muscle or liver, or multiple tissues.3 Mutations in the deoxyguanosine kinase (dGK) gene predominate in patients with the hepatic form of MDS,4,5 whereas patients with myopathic MDS often have mutations in the thymidine kinase 2 (TK2) gene.6,7
Herein, we describe an infant who harbored 2 distinct homozygous mutations, one in PYGM and the other in dGK, an example of genetic "double trouble."
This patient, studied in Bordeaux, France, was described briefly in 1993,8 and we have been unable to obtain a more detailed clinical history. She was born at term of consanguineous (first cousins) Moroccan parents after an uneventful pregnancy. At birth, she was severely hypotonic, with thin muscles. She also had hepatomegaly and developed jaundice and ascites. Laboratory investigations showed metabolic acidosis, hypoglycemia, hyperlactacidemia (89 mg/dL [9.9 mmol/L]; normal, <20 mg/dL [<2.2 mmol/L]), and "increased creatine kinase levels." Electrocardiogram was normal. The child developed liver failure, became comatose, and died at 5 months of age. Two brothers and one sister had died at 5, 9, and 1 month of age after similar clinical courses and with similar findings on liver biopsy. Two brothers were in good health.
We obtained frozen biopsy specimens from muscle and liver and performed histochemical staining for phosphorylase in muscle and for succinate dehydrogenase and cytochrome c oxidase in liver.9 Biochemical studies included analyses of mitochondrial respiratory chain enzymes in muscle, liver, and cultured skin fibroblasts10 and of glycogenolytic and glycolytic enzymes in muscle.11
Southern blot analysis and quantification of mtDNA were performed as described.2 The entire coding regions of the PYGM and dGK genes were amplified and sequenced directly, as reported previously.4,12
The presence of the dGK mutation was confirmed by polymerase chain reaction–restriction fragment length polymorphism analysis, as described previously.5 For the PYGM mutation, DNA was amplified by means of the following primers: forward 5′-TTCCTGGGTCTGGTTCTAGC-3′, reverse mismatched 5′-CTCGGAGTGGATGCGCGGCA-3′. The polymerase chain reaction conditions were 94°C for 3 minutes, followed by 35 cycles at 94°C for 1 minute, 65°C for 1 minute, 72°C for 1 minute, and a final extension step at 72°C for 7 minutes. Aliquots of polymerase chain reaction products were digested with SpHI restriction endonuclease and electrophoresed in 2% agarose gel.
The muscle biopsy specimen showed increased variability in fiber size, subsarcolemmal glycogen accumulation, and negative histochemical reaction for phosphorylase. There were no ragged-red or cytochrome c oxidase–negative fibers. Phosphorylase activity in muscle extracts was undetectable, whereas mitochondrial respiratory chain enzyme activities were normal.
The liver biopsy specimens showed micronodular cirrhosis, microvesicular steatosis, cholestasis, and hepatocellular damage, with fatty degeneration, giant hepatocytes, and rosette formations. Glycogen was slightly decreased on periodic acid–Schiff staining. Electron microscopy demonstrated oncocytelike hepatocytes containing abnormal mitochondria with sparse cristae, granular matrix, and, in some cases, dense or vesicular inclusions. The activities of respiratory chain complexes containing mtDNA-encoded subunits (expecially complex IV) were decreased in liver.
Southern blot analysis showed severe reduction of the mtDNA–nuclear DNA ratio in the liver, indicating 90% depletion of mtDNA (Figure 1).
Autoradiograph of Southern blot of total muscle DNA from the patient (P) and age-matched control subjects (C). The DNA was hybridized simultaneously with 2 probes labeled with sodium phosphate 32, one for human mitochondrial DNA (mtDNA) and the other (a cloned fragment of the human 18S ribosomal RNA gene) for nuclear DNA (nDNA); kb indicates kilobase.
By sequencing the entire coding region of the PYGM gene, we identified a novel homozygous G-to-A transition in exon 11, changing a highly conserved valine to methionine at codon 456 (V456M) (Figure 2A). The presence of the mutation was confirmed by restriction fragment length polymorphism analysis (Figure 2B) and was not detectable in 100 controls, including 20 North African Arabic individuals.
A, Electropherogram of the region of the gene (PYGM) encoding the muscle-specific isoenzyme of glycogen phosphorylase, encompassing the A1366G mutation (underlined). Reverse complement sequence. B, Polymerase chain reaction–restriction fragment length polymorphism analysis of the V456M mutation. 1 Indicates control subject; 2, patient; 3, uncut (undigested); bp, base pairs. C, Electropherogram of the 763-to-766 GATT duplication (underlined).
We also detected a previously described homozygous 4–base pair GATT duplication (nucleotides 763-766) in exon 6 of the dGK gene5 (Figure 2C). The duplication produces a frame shift and a premature TGA stop codon at nucleotides 766 to 768, resulting in a truncated, 255–amino acid protein.
This infant had a most unusual clinical association: myopathy caused by myophosphorylase deficiency (McArdle disease) and hepatic failure with mtDNA depletion. Both conditions were confirmed at the molecular level: we found a novel A1366G change in the PYGM gene and a previously described GATT duplication in the dGK gene.
More than 30 different mutations in the PYGM gene have been reported in patients with McArdle disease.1 We consider the novel V456M change identified in our case to be pathogenic for several reasons. First, it was consistent with muscle biopsy specimen and biochemical data (glycogen storage and lack of phosphorylase activity). Second, it was the only nucleotide alteration in the coding region and adjacent exon-intron boundaries of the PYGM gene. Third, the valine at position 456 is highly conserved in different species, suggesting a critical role of this amino acid for the normal function of the enzyme. Fourth, the mutation was absent in a group of 100 controls, including 20 ethnically matched individuals. Assuming that the severe congenital hypotonia in this child was due entirely to her glycogenosis, this presentation would correspond to the rare infantile myopathic variant of McArdle disease.1,13
Mitochondrial DNA depletion syndrome differs from other mitochondrial disorders because it is a quantitative rather than a qualitative defect. The low amount of mtDNA in some tissues presumably impairs the synthesis of all respiratory chain components containing mtDNA-encoded subunits.2 Mitochondrial DNA depletion syndrome is not uncommon,14 onset is in infancy or early childhood, and clinical expression can be multisystemic or limited to individual tissues.
Primary mtDNA depletion is inherited as an autosomal recessive trait, and mutations in 2 genes have been identified in many—but not all—patients. Mutations in the TK2 gene have been associated with the myopathic form of MDS6,7 and mutations in the dGK gene with the hepatocerebral form,4,5 confirming the importance of the nucleotide pool homeostasis in mtDNA maintenance and stability. The dGK enzyme efficiently phosphorylates deoxyguanosine and deoxyadenosine to the corresponding deoxynucleotide monophosphates, whereas TK2 catalyzes the transfer of a phosphate group from adenosine triphosphate to thymidine or deoxycytidine.15 The theoretical net effect of mutations in these 2 genes is nucleotide pool imbalance, leading to inefficient mtDNA replication, hence depletion. The liver appears particularly vulnerable to dGK mutations, as all reported cases had severe hepatopathy as a common clinical feature.
Our data further expand the genetic heterogeneity of McArdle disease, even in its rare infantile variant, as 2 other infants with congenital myopathy were homozygous for the "common" R49X mutation found in white subjects.1
Furthermore, this is the second family described with severe infantile hepatopathy and the GATT duplication in the dGK gene, possibly a common molecular defect in hepatic MSD.
Although genetic double trouble is rare, it should be kept in mind when there is a complex and apparently incongruous clinical presentation (such as muscle glycogenosis and mitochondrial hepatopathy in this case), especially if there is consanguinity in the family and inbreeding in the population.
Corresponding author and reprints: Salvatore DiMauro, MD, 4-420 College of Physicians and Surgeons, 630 W 168th St, New York, NY 10032 (e-mail: firstname.lastname@example.org).
Accepted for publication February 11, 2003.
Author contributions: Study concept and design (Drs Mancuso, Filosto, and DiMauro); acquisition of data (Drs Mancuso, Filosto, Tsujino, Lamperti, Shanske, Coquet, and Desnuelle); analysis and interpretation of data (Dr Filosto); drafting of the manuscript (Drs Mancuso, Filosto, Tsujino, Lamperti, and Coquet); critical revision of the manuscript for important intellectual content (Drs Shanske, Desnuelle, and DiMauro); obtained funding (Dr DiMauro); administrative, technical, and material support (Dr Mancuso); study supervision (Drs Filosto, Tsujino, Shanske, and Desnuelle).
This study was supported by grants NS11766 and PO1HD 32062 from the National Institutes of Health, Bethesda, Md, and by a grant from the Muscular Dystrophy Association, Tucson, Ariz. Dr Mancuso was supported by the Department of Neuroscience, University of Pisa, Pisa, Italy, and Dr Filosto by the Department of Neurological Science and Vision, University of Verona, Verona, Italy.
Mancuso M, Filosto M, Tsujino S, Lamperti C, Shanske S, Coquet M, Desnuelle C, DiMauro S. Muscle Glycogenosis and Mitochondrial Hepatopathy in an Infant With Mutations in Both the Myophosphorylase and Deoxyguanosine Kinase Genes. Arch Neurol. 2003;60(10):1445-1447. doi:10.1001/archneur.60.10.1445