Novel heterozygous mutations in the PGAM2 gene with negative exercise testing.

Pathogenic variants in the PGAM2 gene are associated with glycogen storage disease type X (GSDX) and is characterized by exercise induced muscle cramping, weakness, myoglobinuria, and often tubular aggregates in skeletal muscle. We report here a patient diagnosed with GSDX at 52 years of age with a normal increase in post-exercise lactate with both anaerobic and aerobic exercise. Genetic testing found two novel PGAM2 variants (c.426C > A, p.Tyr142Ter and c.533delG, p.Gly178Alafs*31).

Six novel mutations in the myophosphorylase gene in patients with McArdle disease and a family with pseudo-dominant inheritance pattern.

McArdle disease is an autosomal recessive glycogenosis due to deficiency of the enzyme myophosphorylase. It results from homozygous or compound heterozygous mutations in the gene for this enzyme, PYGM. We report six novel mutations in the PYGM gene based upon sequencing data including three missense mutations (p.D51G, p.P398L, and p.N648Y), one nonsense mutation (p.Y75X), one frame-shift mutation (p.Y114SfsX181), and one amino acid deletion (p.Y53del) in six patients with McArdle disease. We also report on a Caucasian family that appeared to transmit McArdle disease in an autosomal dominant manner. In order to evaluate the potential pathogenicity of the sequence variants, we performed in silico analysis using PolyPhen-2 and SIFT BLink, along with species conservation analysis using UCSC Genome Browser. The above mutations were all predicted to be disease associated with high probability and with at least the same level of certainty as several confirmed mutations. The current data add to the list of pathogenic mutations in the PYGM gene associated with McArdle disease.

Statin therapy depresses total body fat oxidation in the absence of genetic limitations to fat oxidation.

Cholesterol lowering drugs are associated with myopathic side effects in 7% of those on therapy, which is reversible in most, but not all patients. This study tested the hypothesis that total body fat oxidation (TBFO) is reduced by statins in patients with genetic deficiencies in FO, determined by white blood cells (FOwbc) and by molecular analysis of common deficiencies, and would cause intolerance in some patients. Six patients on statin therapy without myopathic side effects (tolerant) and 7 patients who had previously developed statin-induced myopathic symptoms (intolerant) (age = 58 +/- 8.25 yrs, ht. = 169 +/- 11 cm, and wt. = 75.4 +/- 14.2 kg) were tested for TBFO (Respiratory Exchange Ratio, RER) pre- and during exercise. FOwbc was not significantly different between tolerant and intolerant (0.261 +/- 0.078 vs. 0.296 +/- 0.042 nmol/h per 10(9) wbc), or normals (0.27 +/- 0.09 nmol/h per 10(9) wbc) and no common molecular abnormalities were found. Pre-exercise RER (0.73 +/- 0.05 vs. 0.84 +/- 0.05) was significantly lower in the intolerant group and the VO2 at RER = 1.0 (1.27 +/- 0.32 vs. 1.87 +/- 0.60 L/min) greater than the tolerant. Post-exercise lactates were not different between groups. Although dietary fat intake was not different, blood lipoprotein levels, particularly triglycerides were 35% lower in tolerant than previously intolerant. TBFO and blood lipoproteins were reduced in tolerant patients in spite of the absence of genetic limitations, but not in the intolerant group as hypothesized. Although not conclusive, these data suggest the need for a prospective study of the effects of statins on fat oxidation.

The distribution of white blood cell fat oxidation in health and disease.

Fat oxidation is important for maintaining health and for supplying energy for exercise. We have proposed that the predisposition for individual rates of fat oxidation is determined genetically but may be modulated by acute exercise or exercise training. The purpose of this study was to examine cellular fat oxidation in white blood cells (WBC) using [9,10-3H]palmitic acid. Sedentary controls free of symptoms (SED-C, n=32), were compared with known carnitine palmitoyltransferase (CPT) II-deficient patients (n =2), patients with fatiguing diseases (chronic fatigue syndrome, CFS, n=6; multiple sclerosis, MS, n=31), obesity (OB, n=5), eating disorders (ED, n=16), sedentary individuals prior to and after exercise (SED-Ex, n=12), exercise-trained sedentary individuals (SED-Tr, n=12), and elite runners (ER, n=5). Fat oxidation in WBC for all subjects was normally distributed (mean=0.270 +/- 0.090 nmol/h per 10(9) WBC) and ranged from 0.09 nmol/h per 10(9) WBC in CPT II-deficient patients to 0.59 nmol/h per 10(9) WBC in ER. There were no significant sex or acute exercise effects on WBC fat oxidation. Patients with MS, OB or ED were not different from SED-C; however, in CPT II-deficient patients, fat oxidation was low, while that of CFS patients was high. Exercise training in SED-C resulted in a 16% increase in fat oxidation but in ER it was still 97% higher than in SED-C. We propose that while WBC fat oxidation is not significantly affected by sex or acute exercise, and only by 15-20% with training, genetic factors play a role in determining both high and low fat oxidation in certain groups of individuals. The genetic predisposition for individual rates of fat oxidation may be easily measured using WBC fat oxidation, as has been shown for CPT II-deficient patients and for elite runners. Ranges of WBC fat oxidation that are abnormally low (<20 nmol/h per 10(9) WBC, normal 20-35) or high (>35 nmol/h per 10(9) WBC) are proposed based on genetic factors evaluated in this study.

Heterozygosity: an expanding role in proteomics.

The human genome sequence provides the framework for understanding the biology of human cell function. The next step is to intensify the investigation of protein function in the context of complex biological systems. Cellular functions are carried out by molecular complexes acting in concert rather than by single molecules or single reactions. Parallels have been drawn between scale-free nonbiologic networks and functionally interconnected metabolic pathways in the cell. Modeling of metabolic networks, in which functional modules or subnetworks represent individual related pathways, will lead to the prediction of protein function in the larger context of a complex system. Depending on the robustness of these metabolic networks, single-gene defects alone or in combination with other gene defects and the environment have the potential for invoking a spectrum of alterations in the integrity of a given network. The overall purpose of this review is to highlight the importance of simple heterozygosity for one pathogenic mutation or combinatorial heterozygosity for two or more mutations within or between individual genes in altering the stability of metabolic networks. Several forms of heterozygosity are considered, e.g., intra- and interallelic heterozygosity and double heterozygosity. The concepts of synergistic heterozygosity, loss of heterozygosity, and mitochondrial DNA heteroplasmy also are discussed in relation to the quantitative effects of coexisting mutations on the phenotypic expression of disease.

The clinical laboratory evaluation of the patient with noninflammatory myopathy.

The investigations used to diagnose an inflammatory muscle disease include history and physical examination, evaluation of serum levels of enzymes derived from skeletal muscle, electromyography, magnetic resonance imaging, and muscle histology. The evaluation of patients who may have noninflammatory myopathy includes, but is not limited to, these methods. Additional tools that may be useful include measurements of additional biochemistries, the forearm ischemic exercise test, magnetic resonance spectroscopy, and special tests on muscle tissue. Reports published in the past year have improved and expanded our understanding of the numerous noninflammatory myopathies and how these tools can be used more effectively.

Mitochondrial DNA depletion associated with partial complex II and IV deficiencies and 3-methylglutaconic aciduria.

We report a patient with mitochondrial DNA depletion, partial complex II and IV deficiencies, and 3-methylglutaconic aciduria. Complex II deficiency has not been previously observed in mitochondrial DNA depletion syndromes. The observation of 3-methylglutaconic and 3-methylglutaric acidurias may be a useful indicator of a defect in respiratory chain function caused by mitochondrial DNA depletion.

46,XX gonadal dysgenesis, short stature, and recurrent metabolic acidosis in two sisters.

Gonadal (ovarian) dysgenesis in 46,XX individuals is genetically heterogeneous. We report on two sisters who, in addition to primary ovarian failure, have marked short stature and recurrent episodes of dehydration with metabolic acidosis. Studies performed during one of these episodes suggested mitochondrial dysfunction; however, results of biochemical analysis of electron transport chain activity in skeletal muscle and mitochondrial DNA studies were normal. We discuss the phenotype in relation to previously described conditions of 46,XX gonadal dysgenesis. We suggest this constellation of findings represents a new syndrome.

Succinate dehydrogenase deficiency.

BACKGROUND: Partial succinate dehydrogenase deficiency (15% to 50% of normal reference enzyme activity) in skeletal muscle causes mitochondrial myopathy with various symptoms, for example, brain involvement, cardiomyopathy, and/or exercise intolerance. The deficiency may be isolated or may coexist with other respiratory-chain enzyme defects. The histopathologic assessment of succinate dehydrogenase activity in muscle biopsies of patients with suspected mitochondrial myopathies has focused on the finding of increased staining, usually in ragged-red fibers, rather than on reduced staining. OBJECTIVES: To determine the prevalence of muscle succinate dehydrogenase deficiency among patients with respiratory-chain defects and to determine whether the reduced activity is present histochemically and is comparable to the quantitative reduction found in muscle homogenates. PATIENTS AND METHODS: One hundred eight muscle biopsies were evaluated from patients with suspected mitochondrial myopathies by qualitative histochemical analysis and quantitative biochemical analyses of respiratory-chain enzymes using standard methodologies. RESULTS: Fifty-two patients had defects in respiratory-chain complexes; of these patients, 12 (23%) had partial deficiencies in succinate dehydrogenase activity either alone or together with reductions in other enzymes. The reduced activity was detectable histochemically in muscle biopsies with residual enzyme activity of up to 34% of the normal reference activity, while 2 biopsies with higher residual activity (49% and 68% of normal) could not be distinguished from normal biopsies. CONCLUSIONS: Of the patients with respiratory-chain enzyme defects, 23% had partial deficiencies of succinate dehydrogenase activity in muscle biopsies. This reduction could be detected histochemically in biopsies in most cases. The marked prevalence of succinate dehydrogenase deficiency among patients with respiratory-chain defects and its detection initially by histochemical analysis are important findings.

Synergistic heterozygosity: disease resulting from multiple partial defects in one or more metabolic pathways.

Inborn errors of metabolism show considerable variation in the severity of symptoms. This is often ascribed to the differential effects of specific mutations on gene/enzyme function; however, such genotype/phenotype correlations are usually imprecise. In addition, in some patients with clinical and biochemical findings consistent with a defect in a particular metabolic pathway, it is ultimately impossible to arrive at a precise enzymatic diagnosis. In this situation, we have increasingly been identifying concurrent partial defects in more than one pathway, or at multiple steps in one pathway. In this study, we present the clinical, biochemical, and molecular findings from several patients showing multiple partial defects in energy metabolism. These patients show clinical symptoms consistent with a defect in the affected pathways even though they do not have a complete deficiency in any one enzyme. We hypothesize that such patients are exhibiting clinically significant reductions in energy metabolism related to the compound effects of these partial defects, a phenomenon we term "synergistic heterozygosity." Based on the frequencies of known disorders of energy metabolism, we propose that this may represent a previously unrecognized, relatively common mechanism of disease of potentially great clinical relevance.

A variable myopathy associated with heterozygosity for the R503C mutation in the carnitine palmitoyltransferase II gene.

Adult-onset carnitine palmitoyltransferase II (CPT II) deficiency is an autosomal recessive disease characterized by muscle pain and stiffness with rhabdomyolysis and myoglobinuria in severe cases. Exercise, fasting, viral infection, anesthesia, or extremes in temperature may trigger symptoms. A 54-year-old woman exhibited a 35-year history of progressive weakness and myopathic symptoms. CPT II activity in the patient's lymphoblasts, cultured skin fibroblasts, and skeletal muscle was reduced to 47, 43, and 13% of normal, respectively. Respiratory chain enzymes were also reduced in muscle ranging from 22 to 49% of their respective normal reference means. beta-oxidation enzymes in fibroblasts ranged from 29 to 63% of normal. The patient, her father, and her 26-year-old son were all heterozygous for the R503C mutation. The patient's son has a lifelong history of myopathic symptoms while his grandfather only had mild weakness during childhood. Analysis of the V368I and M647V polymorphisms in the CPT2 gene showed that the mutant allele is linked to 368I and 647M in this family and that the normal allele is linked to 647V in the affected patient and her son, and to 647M in the patient's father. While the variability in CPT2 gene haplotypes may contribute to the phenotypic complexities in this family, it is also possible that an additional gene defect in the transport of mitochondrial proteins contributes to the complex phenotype in the patient. We present biochemical and molecular evidence for vertical transmission of a variable myopathy caused by heterozygosity for a single mutation, R503C, in the CPT2 gene.

The molecular diagnosis of metabolic myopathies.

The metabolic myopathies are distinguished by extensive clinical and genetic heterogeneity within and between individual disorders. There are a number of explanations for the variability observed that go beyond single gene mutations or degrees of heteroplasmy in the case of mitochondrial DNA mutations. Some of the contributing factors include protein subunit interactions, tissue-specificity, modifying genetic factors, and environmental triggers. Advances in the molecular analysis of metabolic myopathies during the last decade have not only improved the diagnosis of individual disorders but also helped to characterize the contributing factors that make these disorders so complex.

Biochemical and molecular correlations in carnitine palmitoyltransferase II deficiency.

Carnitine palmitoyltransferase II (CPT II) deficiency is the most common lipid myopathy in adults and is characterized by exercise-induced pain, stiffness, and myoglobinuria. Retrospective analysis of patients with CPT II deficiency has made it possible to correlate the presence of disease-causing mutations in the CPT2 gene with residual CPT activity in muscle. We present evidence that the ratio of CPT II activity to citrate synthase activity in the skeletal muscle of patients presumed to have CPT II deficiency is important for predicting whether the patient has one, two, or no mutations in the CPT2 gene. This finding will assist in the future correlation of the phenotype with the genotype and in identifying manifesting heterozygotes.

Novel mutations associated with carnitine palmitoyltransferase II deficiency.

The most common form of carnitine palmitoyltransferase II (CPT II) deficiency occurs in adults and is characterized by muscle pain, stiffness, and myoglobinuria, triggered by exercise, fasting, or other metabolic stress. This study reports the molecular heterogeneity of CPT2 mutations and their biochemical consequences among a series of 59 individuals who were suspected of having CPT II deficiency based on the decreased CPT activity observed in muscle or leukocytes samples, clinical findings, or referral for mutation analysis from other laboratories. Only 19 subjects were considered to be at particularly high risk of CPT II deficiency based on review of their clinical symptoms and residual CPT activity. The samples were initially screened for 11 mutations with allele-specific oligonucleotides (ASO). Extensive sequence analysis was subsequently performed on 14 samples which either had a CPT2 mutation detected by ASO screening or the residual CPT activity was below that observed in ASO positive samples. Three known (P50H, S113L, and F448L) and three novel mutations were identified among 13 individuals in this study. A single nucleotide polymorphism was also identified 11 bp distal to the CPT2 polyadenylation site that will be useful for linkage analysis. Two of the new mutations were single nucleotide missense mutations, R503C and G549D, that occurred in highly conserved regions of the CPT isoforms, and the third was a frameshift mutation, 413 delAG, caused by a 2-bp deletion upstream of a previously identified missense mutation, F448L. The 413 delAG mutation was the second most common mutation identified in our study (20% of mutant alleles) and all individuals with the mutation were of Ashkenazi Jewish ancestry suggesting a defined ethnic origin for the mutation. Despite rigorous mutation analysis, six of 13 individuals identified with CPT2 mutations remained as heterozygotes. We propose that heterozygosity for certain CPT2 mutations, S113L and R503C, is sufficient to render individuals at risk of clinical symptoms.