Phosphoglucomutase 1 Deficiency Research Articles

In Vitro Skeletal Muscle Model of PGM1 Deficiency Reveals Altered Energy Homeostasis.

Phosphoglucomutase 1 (PGM1) is a key enzyme for the regulation of energy metabolism from glycogen and glycolysis, as it catalyzes the interconversion of glucose 1-phosphate and glucose 6-phosphate. PGM1 deficiency is an autosomal recessive disorder characterized by a highly heterogenous clinical spectrum, including hypoglycemia, cleft palate, liver dysfunction, growth delay, exercise intolerance, and dilated cardiomyopathy. Abnormal protein glycosylation has been observed in this disease. Oral supplementation with D-galactose efficiently restores protein glycosylation by replenishing the lacking pool of UDP-galactose, and rescues some symptoms, such as hypoglycemia, hepatopathy, and growth delay. However, D-galactose effects on skeletal muscle and heart symptoms remain unclear. In this study, we established an in vitro muscle model for PGM1 deficiency to investigate the role of PGM1 and the effect of D-galactose on nucleotide sugars and energy metabolism. Genome-editing of C2C12 myoblasts via CRISPR/Cas9 resulted in Pgm1 (mouse homologue of human PGM1, according to updated nomenclature) knockout clones, which showed impaired maturation to myotubes. No difference was found for steady-state levels of nucleotide sugars, while dynamic flux analysis based on 13C6-galactose suggested a block in the use of galactose for energy production in knockout myoblasts. Subsequent analyses revealed a lower basal respiration and mitochondrial ATP production capacity in the knockout myoblasts and myotubes, which were not restored by D-galactose. In conclusion, an in vitro mouse muscle cell model has been established to study the muscle-specific metabolic mechanisms in PGM1 deficiency, which suggested that galactose was unable to restore the reduced energy production capacity.

A new D-galactose treatment monitoring index for PGM1-CDG.

Phosphoglucomutase 1 (PGM1) catalyzes the interconversion of glucose-6-phosphate to glucose-1-phosphate and is a key enzyme of glycolysis, glycogenesis, and glycogenolysis. PGM1 deficiency (OMIM: 614921) was initially defined as a glycogen storage disorder (type XIV), and later re-classified as a PGM1-congenital disorder of glycosylation (PGM1-CDG). Serum transferrin (Tf) glycan isoform analysis by liquid chromatography-mass spectrometry (LC-MS) is used as a primary diagnostic screen tool, and reveals a very unique CDG profile described as a mixture of CDG-type I and CDG-type II patterns. Oral d-galactose supplementation shows significant clinical and metabolic improvements, which are indicated by the Tf glycan isoform normalization over time in patients with PGM1-CDG. Thus, there is a need for biomarkers to guide d-galactose dosage in patients in order to maintain effective and safe drug levels. Here, we present a simplified algorithm called PGM1-CDG Treatment Monitoring Index (PGM1-TMI) for assessing the response of PGM1-CDG patients to d-galactose supplementation. For our single-center cohort of 16 PGM1-CDG patients, the Tf glycan profile analysis provided the biochemical diagnosis in all of them. In addition, the PGM1-TMI was reduced in PGM1-CDG patients under d-galactose supplementation as compared with their corresponding values before treatment, indicating that glycosylation proceeds towards normalization. PGM1-TMI allows tracking Tf glycan isoform normalization over time when the patients are on d-galactose supplementation.

Phosphoglucomutase-1 deficiency: Early presentation, metabolic management and detection in neonatal blood spots

Phosphoglucomutase 1 deficiency is a congenital disorder of glycosylation (CDG) with multiorgan involvement affecting carbohydrate metabolism, N-glycosylation and energy production. The metabolic management consists of dietary D-galactose supplementation that ameliorates hypoglycemia, hepatic dysfunction, endocrine anomalies and growth delay. Previous studies suggest that D-galactose administration in juvenile patients leads to more significant and long-lasting effects, stressing the urge of neonatal diagnosis (0–6 months of age).Here, we detail the early clinical presentation of PGM1-CDG in eleven infantile patients, and applied the modified Beutler test for screening of PGM1-CDG in neonatal dried blood spots (DBSs).All eleven infants presented episodic hypoglycemia and elevated transaminases, along with cleft palate and growth delay (10/11), muscle involvement (8/11), neurologic involvement (5/11), cardiac defects (2/11). Standard dietary measures for suspected lactose intolerance in four patients prior to diagnosis led to worsening of hypoglycemia, hepatic failure and recurrent diarrhea, which resolved upon D-galactose supplementation. To investigate possible differences in early vs. late clinical presentation, we performed the first systematic literature review for PGM1-CDG, which highlighted respiratory and gastrointestinal symptoms as significantly more diagnosed in neonatal age.The modified Butler-test successfully identified PGM1-CDG in DBSs from seven patients, including for the first time Guthrie cards from newborn screening, confirming the possibility of future inclusion of PGM1-CDG in neonatal screening programs.In conclusion, severe infantile morbidity of PGM1-CDG due to delayed diagnosis could be prevented by raising awareness on its early presentation and by inclusion in newborn screening programs, enabling early treatments and galactose-based metabolic management.

Structural basis for substrate and product recognition in human phosphoglucomutase-1 (PGM1) isoform 2, a member of the \u03b1-d-phosphohexomutase superfamily

Human phosphoglucomutase 1 (PGM1) is an evolutionary conserved enzyme that belongs to the ubiquitous and ancient α-d-phosphohexomutases, a large enzyme superfamily with members in all three domains of life. PGM1 catalyzes the bi-directional interconversion between α-d-glucose 1-phosphate (G1P) and α-d-glucose 6-phosphate (G6P), a reaction that is essential for normal carbohydrate metabolism and also important in the cytoplasmic biosynthesis of nucleotide sugars needed for glycan biosynthesis. Clinical studies have shown that mutations in the PGM1 gene may cause PGM1 deficiency, an inborn error of metabolism previously classified as a glycogen storage disease, and PGM1 deficiency was recently also shown to be a congenital disorder of glycosylation. Here we present three crystal structures of the isoform 2 variant of PGM1, both as a free enzyme and in complex with its substrate and product. The structures show the longer N-terminal of this PGM1 variant, and the ligand complex structures reveal for the first time the detailed structural basis for both G1P substrate and G6P product recognition by human PGM1. We also show that PGM1 and the paralogous gene PGM5 are the results of a gene duplication event in a common ancestor of jawed vertebrates, and, importantly, that both PGM1 isoforms are conserved and of functional significance in all vertebrates. Our finding that PGM1 encodes two equally conserved and functionally important isoforms in the human organism should be taken into account in the evaluation of disease-related missense mutations in patients in the future.

A missense variant remote from the active site impairs stability of human phosphoglucomutase 1.

Missense variants of human phosphoglucomutase 1 (PGM1) cause the inherited metabolic disease known as PGM1 deficiency. This condition is categorised as both a glycogen storage disease and a congenital disorder of glycosylation. Approximately 20 missense variants of PGM1 are linked to PGM1 deficiency, and biochemical studies have suggested that they fall into two general categories: those affecting the active site and catalytic efficiency, and those that appear to impair protein folding and/or stability. In this study, we characterise a novel variant of Arg422, a residue distal from the active site of PGM1 and the site of a previously identified disease-related variant (Arg422Trp). In prior studies, the R422W variant was found to produce insoluble protein in a recombinant expression system, precluding further in vitro characterisation. Here we investigate an alternative variant of this residue, Arg422Gln, which is amenable to experimental characterisation presumably due to its more conservative physicochemical substitution. Biochemical, crystallographic, and computational studies of R422Q establish that this variant causes only minor changes in catalytic efficiency and 3D structure, but is nonetheless dramatically reduced in stability. Unexpectedly, binding of a substrate analog is found to further destabilise the protein, in contrast to its stabilising effect on wild-type PGM1 and several other missense variants. This work establishes Arg422 as a lynchpin residue for the stability of PGM1 and supports the impairment of protein stability as a pathomechanism for variants that cause PGM1 deficiency. SYNOPSIS: Biochemical and structural studies of a missense variant far from the active site of human PGM1 identify a residue with a key role in enzyme stability.

A Hotspot for Disease-Associated Variants of Human PGM1 Is Associated with Impaired Ligand Binding and Loop Dynamics

Human phosphoglucomutase 1 (PGM1) plays a central role in cellular glucose homeostasis, catalyzing the conversion of glucose 1-phosphate and glucose 6-phosphate. Recently, missense variants of this enzyme were identified as causing an inborn error of metabolism, PGM1 deficiency, with features of aglycogen storage disease and a congenital disorder of glycosylation. Previous studies of selected PGM1 variants have revealed various mechanisms for enzyme dysfunction, including regions of structural disorder and side-chain rearrangements within the active site. Here, we examine variants within a substrate-binding loop in domain 4 (D4) of PGM1 that cause extreme impairment of activity. Biochemical, structural, and computational studies demonstrate multiple detrimental impacts resulting from these variants, including loss of conserved ligand-binding interactions and reduced mobility of the D4loop, due to perturbation of its conformational ensemble. These potentially synergistic effects make this conserved ligand-binding loop a hotspot for disease-related variants in PGM1 and related enzymes.

Clinical and molecular genetic characterization of two patients with mutations in the phosphoglucomutase 1 (PGM1) gene

Background The phosphoglucomutase 1 (PGM1) enzyme plays a central role in glucose homeostasis by catalyzing the inter-conversion of glucose 1-phosphate and glucose 6-phosphate. Recently, PGM1 deficiency has been recognized as a cause of the congenital disorders of glycosylation (CDGs). Methods Two Chinese Han pediatric patients with recurrent hypoglycemia, hepatopathy and growth retardation are described in this study. Targeted gene sequencing (TGS) was performed to screen for causal genetic variants in the genome of the patients and their parents to determine the genetic basis of the phenotype. Results DNA sequencing identified three variations of the PGM1 gene (NM_002633.2). Patient 1 had a novel homozygous mutation (c.119delT, p.Ile40Thrfs*28). In patient 2, we found a compound heterozygous mutation of c.1172G>T(p.Gly391Val) (novel) and c.1507C>T(p.Arg503*) (known pathogenic). Conclusions This report deepens our understanding of the clinical features of PGM1 mutation. The early molecular genetic analysis and multisystem assessment were here found to be essential to the diagnosis of PGM1-CDG and the provision of timely and proper treatment.

Disruption of the Responsible Gene in a Phosphoglucomutase 1 Deficiency Patient by Homozygous Chromosomal Inversion.

Phosphoglucomutase 1 (PGM1) deficiency is a recently defined disease characterized by glycogenosis and a congenital glycosylation disorder caused by recessive mutations in the PGM1 gene. We report a case of a 12-year-old boy with first-cousin parents who was diagnosed with a PGM1 deficiency due to significantly decreased PGM1 activity in his muscle. However, Sanger sequencing revealed no pathogenic mutation in the PGM1 gene in this patient. As this case presented with a cleft palate in addition to hypoglycemia and elevated transaminases and creatine kinase, karyotyping was performed and identified homozygous inv(1)(p31.1p32.3). Based on the chromosomal location of the PGM1 gene at 1p31, we analyzed the breakpoint of the inversion. Fluorescence in situ hybridization (FISH) combined with long PCR analysis revealed that the inversion disrupts the PGM1 gene within intron 1. Since the initiation codon in the PGM1 gene is located within exon 1, we speculated that this inversion inactivates the PGM1 gene and was therefore responsible for the patient's phenotype. When standard molecular testing fails to reveal a mutation despite a positive clinical and biochemical diagnosis, the presence of a gross structural variant that requires karyotypic examination must be considered.

Three patients with glycosylation deficiencies, chronically elevated transaminases, and low serum ceruloplasmin and copper, caused by mutations in the gene encoding the transmembrane protein TMEM199

Background: A decade ago we reported on four children with glycosylation deficiencies and a liver disease mimicking Wilson-disease, but without known genetic cause. Two (patients #1 and #2) were later diagnosed with phosphoglucomutase-1 deficiency.

Identification of a Novel Pathomechanism Underlying a Congenital Disorder of Glycosylation: Crosstalk between Hexosamine Biosynthetic Pathway, PI3K/AKT Signaling, and Protein Glycosylation

Phosphoglucomutase 1 (PGM1) deficiency is a recently identified subtype of congenital disorder of glycosylation that can be treated by dietary modification. PGM1 is required for the interconversion of glucose 1‐phosphate and glucose 6‐phosphate, and is a key enzyme in glycogen metabolism. Although the exact mechanism remains unclear, PGM1 deficiency has been associated with abnormal intracellular glucose and galactose metabolites, as well as reduced N‐linked glycosylation in the endoplasmic reticulum and Golgi. These metabolic disturbances are thought to be responsible for both missing and truncated glycans that have been detected in patient serum transferrin. A recent landmark study demonstrated that oral D‐galactose supplementation improved cellular glycosylation in six patients and resolved hypogonadotropic hypogonadism in two patients. While the clinical benefits of galactose supplementation in PGM1 deficiency are clear, its biological effect on the biochemical and cellular level has not been characterized in detail. It was assumed that dietary galactose supplementation improves galactosylation by increasing intracellular UDP‐galactose relative to UDP‐glucose; however, this has not been confirmed experimentally, nor has an alternative mechanism of action been explored. Here, we report no difference in the ratio of intracellular UDP‐glucose to UDP‐galactose between patient and control cells, despite significant improvement in N‐linked and O‐linked glycosylation in patient cells following galactose supplementation. We found no evidence to support galactose acting as a pharmacological chaperone for PGM1 missense variants. Surprisingly, we discovered disruptions in the hexosamine biosynthetic pathway (HBP) at the metabolic and transcriptional level, as well as downregulation of PI3K/AKT signaling in PGM1 deficient patient cells. Interestingly, galactose supplementation appears to rescue HBP and PI3K/AKT signaling. Collectively, our preliminary studies suggest a hitherto unexplored crosstalk between HBP, PI3K/AKT signaling, and protein glycosylation.Support or Funding InformationThis study is supported in part by the Hayward Foundation and by 1 U54 GM104940 from the National Institute of General Medical Sciences of the National Institutes of Health, which funds the Louisiana Clinical and Translational Science Center.

Asp263 missense variants perturb the active site of human phosphoglucomutase 1.

Structural data are available in the PDB under the accession numbers 5JN5 and 5TR2.

False-Positive Newborn Screen Using the Beutler Spot Assay for Galactosemia in Glucose-6-Phosphate Dehydrogenase Deficiency.

Classical galactosemia is detected through newborn screening by measuring galactose-1-phosphate uridylyltransferase (GALT) in the USA primarily via the Beutler spot assay. We report on an 18-month-old patient with glucose-6-phosphate dehydrogenase (G6PD) deficiency that was originally diagnosed with classical galactosemia. The patient presented with elevated liver function enzymes and bilirubinemia and was immediately treated with soy-based formula. Confirmatory tests revealed deficiency of the GALT enzyme, however, full-sequencing of GALT was normal, suggestive of a different ideology. The Beutler spot assay uses three other enzymatic steps in addition to GALT. A deficiency in either of these enzymes can result in suspected decreased GALT activity when using the Beutler assay. Congenital Disorders of Glycosylation screening for phosphoglucomutase-1 deficiency was negative. Quantitative analysis of G6PD enzyme in red blood cells showed a severe deficiency and a deletion in G6PD. Soy-formula, the standard treatment for galactosemia, has been reported to trigger hemolysis in G6PD deficient patients. G6PD and phosphoglucomutase-1 deficiencies should be considered when confirmatory tests are negative for pathogenic variants in GALT and galactose-1-phosphate level is normal.

PGM1 deficiency diagnosed during an endocrine work-up of low IGF-1 mediated growth failure

Objective and importance: Phosphoglucomutase 1 (PGM1) deficiency, first described as a glycogenosis (type XIV) is also a congenital disorder of glycosylation (CDG). We want to illustrate the wide clinical spectrum of PGM1 deficiency and in particular the associated disturbance in glucose metabolism and the endocrine dysfunction. Treatment with d-galactose is experimental. Case presentation: PGM1 deficiency was diagnosed in an 8-year-old boy, who was referred because of an unexplained complex syndrome, including recurrent hypoglycaemia and low IGF-1 mediated growth failure. Conclusion: The timely diagnosis of this disorder is particularly important, because d-galactose treatment can improve the latter symptoms.

Defining the Phenotype and Assessing Severity in Phosphoglucomutase-1 Deficiency

To define phenotypic groups and identify predictors of disease severity in patients with phosphoglucomutase-1 deficiency (PGM1-CDG). We evaluated 27 patients with PGM1-CDG who were divided into 3 phenotypic groups, and group assignment was validated by a scoring system, the Tulane PGM1-CDG Rating Scale (TPCRS). This scale evaluates measurable clinical features of PGM1-CDG. We examined the relationship between genotype, enzyme activity, and TPCRS score by using regression analysis. Associations between the most common clinical features and disease severity were evaluated by principal component analysis. We found a statistically significant stratification of the TPCRS scores among the phenotypic groups (P<.001). Regression analysis showed that there is no significant correlation between genotype, enzyme activity, and TPCRS score. Principal component analysis identified 5 variables that contributed to 54% variance in the cohort and are predictive of disease severity: congenital malformation, cardiac involvement, endocrine deficiency, myopathy, and growth. We established a scoring algorithm to reliably evaluate disease severity in patients with PGM1-CDG on the basis of their clinical history and presentation. We also identified 5 clinical features that are predictors of disease severity; 2 of these features can be evaluated by physical examination, without the need for specific diagnostic testing and thus allow for rapid assessment and initiation of therapy.

Induced Structural Disorder as a Molecular Mechanism for Enzyme Dysfunction in Phosphoglucomutase 1 Deficiency

Human phosphoglucomutase 1 (PGM1) plays a central role in cellular glucose homeostasis, mediating the switch between glycolysis and gluconeogenesis through the conversion of glucose 1-phosphate and glucose 6-phosphate. Recent clinical studies have identified mutations in this enzyme as the cause of PGM1 deficiency, an inborn error of metabolism classified as both a glycogen storage disease and a congenital disorder of glycosylation. Reported here are the first crystal structures of two disease-related missense variants of PGM1, along with the structure of the wild-type enzyme. Two independent glycine-to-arginine substitutions (G121R and G291R), both affecting key active site loops of PGM1, are found to induce regions of structural disorder, as evidenced by a nearly complete loss of electron density for as many as 23 aa. The disordered regions are not contiguous in sequence to the site of mutation, and even cross domain boundaries. Other structural rearrangements include changes in the conformations of loops and side chains, some of which occur nearly 20Å away from the site of mutation. The induced structural disorder is correlated with increased sensitivity to proteolysis and lower-resolution diffraction, particularly for the G291R variant. Examination of the multi-domain effects of these G➔R mutations establishes a correlation between interdomain interfaces of the enzyme and missense variants of PGM1 associated with disease. These crystal structures provide the first insights into the structural basis of enzyme dysfunction in PGM1 deficiency and highlight a growing role for biophysical characterization of proteins in the field of precision medicine.

Isoelectric Focusing of Serum Apolipoprotein C-III as a Sensitive Screening Method for the Detection of O-glycosylation Disturbances.

Apolipoprotein C-III (ApoC-III) is a glycoprotein carrying the most common O-linked glycan structure and is abundantly present in serum, what renders it a suitable marker for analysis of O-glycosylation abnormalities. Isoelectric focusing followed by a Western blot of ApoC-III, using PhastSystem™ Electrophoresis System (GE Healthcare), was introduced as a rather simple and rapid method for screening of certain subtypes of inherited glycosylation disorders. The study's aim was to establish this method in our laboratory, what included performing the analysis in a group of 170 healthy individuals to set the reference range of detected relative amounts of sialylated ApoC-III isoforms and to evaluate the gender- and age-dependent differences. A significant relative increase of asialo-ApoC-III with growing age was found. Secondly, we examined serum from patients with selected metabolic disorders and detected minor O-glycosylation changes in diseases such as Prader-Willi syndrome, PGM1 (phosphoglucomutase 1) or MAN1B (class 1B alpha-1,2-mannosidase) deficiency. Our results show that this method allows for a sensitive detection of ApoC-III O-glycosylation status, however this might be modulated by several factors (i.e. nutrition, medication) whose exact role remains to be determined.

Compromised Catalysis and Potential Folding Defects in in Vitro Studies of Missense Mutants Associated with Hereditary Phosphoglucomutase 1 Deficiency

Recent studies have identified phosphoglucomutase 1 (PGM1) deficiency as an inherited metabolic disorder in humans. Affected patients show multiple disease phenotypes, including dilated cardiomyopathy, exercise intolerance, and hepatopathy, reflecting the central role of the enzyme in glucose metabolism. We present here the first in vitro biochemical characterization of 13 missense mutations involved in PGM1 deficiency. The biochemical phenotypes of the PGM1 mutants cluster into two groups: those with compromised catalysis and those with possible folding defects. Relative to the recombinant wild-type enzyme, certain missense mutants show greatly decreased expression of soluble protein and/or increased aggregation. In contrast, other missense variants are well behaved in solution, but show dramatic reductions in enzyme activity, with kcat/Km often <1.5% of wild-type. Modest changes in protein conformation and flexibility are also apparent in some of the catalytically impaired variants. In the case of the G291R mutant, severely compromised activity is linked to the inability of a key active site serine to be phosphorylated, a prerequisite for catalysis. Our results complement previous in vivo studies, which suggest that both protein misfolding and catalytic impairment may play a role in PGM1 deficiency.

Mutations in hereditary phosphoglucomutase 1 deficiency map to key regions of enzyme structure and function.

Recent studies have identified phosphoglucomutase 1 (PGM1) deficiency as an inherited metabolic disorder in humans. PGM1 deficiency is classified as both a muscle glycogenosis (type XIV) and a congenital disorder of glycosylation of types I and II. Affected patients show multiple disease phenotypes, reflecting the central role of the enzyme in glucose homeostasis, where it catalyzes the interconversion of glucose 1-phosphate and glucose 6-phosphate. The influence of PGM1 deficiency on protein glycosylation patterns is also widespread, affecting both biosynthesis and processing of glycans and their precursors. To date, 21 different mutations involved in PGM1 deficiency have been identified, including 13 missense mutations resulting in single amino acid changes. Growing clinical interest in PGM1 deficiency prompts a review of the molecular context of these mutations in the three-dimensional structure of the protein. Here the known crystal structure of PGM from rabbit (97 % sequence identity to human) is used to analyze the mutations associated with disease and find that many map to regions with clear significance to enzyme function. In particular, amino acids in and around the active site cleft are frequently involved, including regions responsible for catalysis, binding of the metal ion required for activity, and interactions with the phosphosugar substrate. Several of the known mutations, however, are distant from the active site and appear to manifest their effects indirectly. An understanding of how the different mutations that cause PGM1 deficiency affect enzyme structure and function is foundational to providing clinical prognosis and the development of effective treatment strategies.

Molecular and cytological investigations of phosphoglucomutase (PGM1) in the K562 cell line

Phosphoglucomutase 1 (PGM1) deficiency is a stable characteristic of the erythroleukaemic cell line, K562, whereas the activity of the isozymes of the other two PGM loci (PGM2 and PGM3) is slightly elevated. In this study the molecular basis of PGM1 deficiency was investigated by a combined approach utilising protein electrophoresis, immunodetection, cytogenetic techniques, and DNA and RNA analysis. Isoelectric focusing and activity staining confirmed that K562 has no detectable PGM1 activity. Immunoblot analysis of extracts, separated by isoelectric focusing, starch gel and SDS gel electrophoresis, using monospecific anti-PGM1 antibodies showed that K562 contained no detectable immunoreactive material. Karyotype analysis revealed the presence of two intact chromosomes 1 and a derivative chromosome 1, der(1)t(1; 11), each of which carried a copy of the PGM1 gene as demonstrated by fluorescence in situhybridization using a PGM1 cosmid as probe. Southern blot analysis using a PGM1 cDNA clone as probe suggested that the PGM1 genes had not been subject to any gross structural rearrangements. We were also able to determine that K562 is type PGM1 2+1+ by restriction endonuclease analysis of genomic DNA. Very low levels of PGM1 mRNA which appeared to be full length transcripts were detected in K562 using a reverse transcriptase PCR technique. We conclude that the most likely cause of PGM1 enzyme deficiency in K562 is abnormal regulation of transcription.

Molecular and cytological investigations of phosphoglucomutase (PGM1) in the K562 cell line.

Phosphoglucomutase 1 (PGM1) deficiency is a stable characteristic of the erythroleukaemic cell line, K562, whereas the activity of the isozymes of the other two PGM loci (PGM2 and PGM3) is slightly elevated. In this study the molecular basis of PGM1 deficiency was investigated by a combined approach utilising protein electrophoresis, immunodetection, cytogenetic techniques, and DNA and RNA analysis. Isoelectric focusing and activity staining confirmed that K562 has no detectable PGM1 activity. Immunoblot analysis of extracts, separated by isoelectric focusing, starch gel and SDS gel electrophoresis, using monospecific anti-PGM1 antibodies showed that K562 contained no detectable immunoreactive material. Karyotype analysis revealed the presence of two intact chromosomes 1 and a derivative chromosome 1, der(1)t(1;11), each of which carried a copy of the PGM1 gene as demonstrated by fluorescence in situ hybridization using a PGM1 cosmid as probe. Southern blot analysis using a PGM1 cDNA clone as probe suggested that the PGM1 genes had not been subject to any gross structural rearrangements. We were also able to determine that K562 is type PGM1 2+1+ by restriction endonuclease analysis of genomic DNA. Very low levels of PGM1 mRNA which appeared to be full length transcripts were detected in K562 using a reverse transcriptase PCR technique. We conclude that the most likely cause of PGM1 enzyme deficiency in K562 is abnormal regulation of transcription.