Fatal familial infantile glycogen storage disease: Multisystem phosphofructokinase deficiency

An infant girl of consanguinous Bedouin parents suffered from fatal early onset of progressive generalized muscle weakness. Her older brother suffered from similar weakness and cardiomyopathy, which led to his death at the age of 21 months. A muscle biopsy performed on the propositus at the age of 9 months was PAS-negative, and showed nonspecific myopathic changes. A second muscle biopsy, performed at 21 months of age, a few days before her death, and postmortem study of heart and liver, disclosed excessive extralysosomal glycogen storage and reduced phosphofructokinase-1 (PFK-1) activity. Because the genes encoded for PFK-1 in liver and muscle are located on separate chromosomes, the reduced enzyme activity in both tissues could not be related to a single mutation for this enzyme. Activity of 6-phosphofructose-2-kinase(PFK-2), a recently discovered physiological activator to all PFK-1 isozymes, was normal in the liver. The possibility that this multisystem PFK-1 deficiency may be related to the absence of a yet unknown activator, common to all PFK-1 isozymes, is discussed. Key words: infantile glycogen storage phosphofructokinase MUSCLE 81 NERVE 15:455-458 1992

FATAL FAMILIAL INFANTILE GLYCOGEN STORAGE DISEASE: MULTISYSTEM PHOSPHOFRUCTOKb NASE DEFICIENCY RAM1 AMIT, MD, NAVA BASHAN, PhD, JACOB M. ABARBANEL, MD, YEHUDA SHAPIRA, MD, SHAUL SOFER, MO, and SHIMON MOSES, MD

Muscle phosphofructokinase- 1 (PFK-1) deficiency is a rare glycogen storage disorder (Tarui's disease, glycogenosis type VII) characterized by childhood or adult onset of exercise intolerance, muscle cramps, and myoglobinuria. 1,8212 Biochemically, there is mild accumulation of normally structured glycogen in the subsarcolemmal space. The infantile form of this enzymatic defect is

From the Departments of Pediatrics (Dr. Amit. Dr Bashan. Dr Sofer, and Dr. Moses) and Neurology (Dr. Abarbanel), Soroka Medical Center Faculty of Health Sciences, Ben-Gurion University, Beer-Sheva. Department of Pediatrics (Dr Shapira), Hadassah University Hospital, Mt Scopus. Jerusalem, Israel. Presented as a poster at the 19th American Chlld Neurology Society Meeting, Atlanta, Georgia, October 1990 Acknowledgments: We thank Profs. H. G. Hers and E Van Schaftingen of the International Institute of Cellular and Molecular Pathology in Brussels for their contribution to the metabolic work up in measuring the PFK-2 activity and fructose-2,6-biphosphate content We are grateful to Prof A. Gutman of the Department of Clinical Biochemistry at Hadassah University Hospital, Jerusalem, for her most constructive comments Address reprint requests to Rami Amit. MD, Division of Neurology. Children's Hospital of Pittsburgh, 3705 Fifth Avenue at DeSoto, Pittsburgh, PA 15213. Accepted for publication March 26, 1991 CCC 0148-639x1921040455-04$04.00 0 1992 John Wiley & Sons, Inc.

Infantile Phosphofructokinase Deficiency

characterized by fatal progressive muscle weakness. Human PFK-1 is under the control of 3 structural loci that code for specific subunits, represented in muscle (M), liver (L), and platelet (P) types.'"14 A tissue may comprise 1, 2, or 3 subunits, which will randomly be expressed in tetramers of different isozymes. Human muscle and liver express homotetramers, i.e., M4 and L4, respectively; whereas red blood cells (RBC) contain 5 isozymes composed of M and L subunits. The M subtype constitutes the major part of the heart isozyme. In patients with type I (classic Tarui's disease), which is the most common form of PFK-I deficiency, a total lack of the muscle PFK-1 and partial reduction of RBC PFK-1 activity were found. In these patients, M-type subunits were totally missing. T h e nature of the molecular defect in the infantile type is unknown. Only 4 cases (2 of them siblings) of infantile PFK-1 deficiency with an isolated muscle enzymatic defect have been reported to date.3.53'0We now present 2 siblings of consanguinous parents with fatal progressive weakness. Biochemical analysis performed in 1 patient showed reduced PFK-1 activity in skeletal muscle and liver.

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CASE HISTORIES

I. The first patient was an 18-month-old female, the product of a normal full-term pregnancy. The delivery and postnatal period were uneventful. The parents are healthy second cousins of Bedouin origin. At 5 months, mild generalized hypotonia and decreased deep tendon reflexes were found. Mental and social development was intact. CPK was 350 IU (normal < 100). Fasting and nonfasting levels of serum glucose, carnitine, lactate, pyruvate, and ketone bodies were normal. A closedneedle muscle biopsy showed marked differences in fiber size, central nuclei, and infiltration of connective tissue among the fibers. ATPase (pH 9.4) staining showed only dark (type 11) fibers. PAS staining was negative for glycogen. T h e sample taken for EM did not contain muscle fibers. T h e infant suffered from recurrent bouts of pneumonia requiring frequent ad prolonged hospitalizations. At 18 months, her mental status and social adaptation were age appropriate. She was extremely hypotonic, weak, and areflexic. There were no joint contractures. Cranial nerves and fundi were intact. Laboratory findings (during the course of acute bacterial pneumonia): CBCmarked leukocytosis with shift to the left, urine analysis was normal; SGOT- 175 U, SGPT-72 U, CPK- 172 U. Chest X-ray showed bilateral atelectasis and infiltration, but no cardiomegaly. EKG was normal. A few days before she died of severe respiratory failure, an open muscle biopsy that was obtained from the right quadriceps showed marked differences in fiber size, central nuclei and large vacuoles filled with PAS-positive and diastasedigestible granular material. An EM study showed extralysosomal accumulation of dense granular material (Fig. 1). Postmortem biochemical studies of heart muscle and liver tissues were performed (Tables 1 and 2).

Patient

This patient was patient 1’s older brother. He suffered from progressive generalized muscle weakness, which had its onset at early infancy. CPK was mildly elevated, but no further neuromuscular investigations were performed. Radiographic and electocardiographic studies showed increased heart size, compatible with cardiomyopathy. He died at the age of 21 months of severe respiratory failure. Patient 2.

Glycogen content was determined by enzymatic hy-

Methods and Results of Biochemical Studies.

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FIGURE 1. Electromicrograph illustrating increased subsarcolemmal accumulation of glycogen (~7600).

drolysis and enzymatic analysis of glucose as described by Johnson et al.7 For assays of activities of glycogenolytic or glycolytic enzymes, specimens were homogenized in a glass homogenizer with 10 volumes of 0.05 mol/L Tris buffer, 5 mol/L EDTA at pH 7.5. Protein concentration was measured accordig to Lowry et al.’ Lactate dehydrogenase, phosphorylase, and phosphofructokinase activities were assayed by conventional spectrophotometric methods.‘,’ l~~~ Alpha glucosidase activity was measured by a fluorometric method.+’ PFK-2 activity and fructose-2,6-hiphosphate (F26P2) content were assayed at the International Institute of Cellular and Molecular Pathology in Brussels (courtesy of Profs. H. G. Hers and E. Van Schaftingen).

Table 1. Enzyme activities and glycogen concentration in skeletal muscle Control biopsies were obtained from pediatric orthopedic patients Muscle a-Glucosidase (pH 4 0) Phosphofructokinase-1 a-Glucosidase (pH 7 0) Phosphorylase (+AMP) Phosphorylase (-AMP) Lactate dehydrogenase Glycogen concentrations (mgig wet weight)

Patient 0 10 k 0 02 0-0 08 0 27 t 0 06 0 3 008 0 15 ? 0 05 21 ?05 81

Controls ( n = 6) 0 07 ? 0 099 0 38 0 27 0 18 19 6k05

0 25

*

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Table 2. PFK-1 and PFK-2 activities, glycogen and fructose-2,6-biphosphate content in liver, heart, and muscle. Patient

PFK-1 (pnol/min/mg protein) F26P2 kmol/g PFK-2 mU/g Glycogen (mgig wet weight)

Controls

Heart

Liver

Muscle

Heart

Liver

0

0

-

0.286

0.013

-

2.8 0.7

1.4 -

-

-

-

-

168

185

-

7.3

0.8 2 0.1 (n = 10) 25

Glycogen concentration was greatly elevated in muscle, heart, and liver. Acid maltase and glycogen phosphorylase activities were normal. Activity of PFK-1 ranged between 0% and 30% of normal controls. F26P2 was normal both in liver and muscle. PFK-2 activity in liver was similar to that of controls and, in muscle, was undetectable, most probably due to the thawing of the sample during air transportation. DISCUSSION

Patient 1 died before her second birthday of respiratory failure resulting from progressive muscle weakness. Biochemical analysis of skeletal muscle revealed a significant decrease of PFK activity. It is conceivable that patient 2 suffered from the same enzymatic defect. In his case, the clinical picture of progressive muscle weakness was accompanied by cardiomyopathy. The consanguinity of the parents is compatible with autosomal recessive transmission. T o the best of our knowledge, these are the fifth and sixth cases and the second family with the infantile form of PFK-1 deficiency to be reported. The neuromuscular involvement in our patients resembles that of other previously reported cases, i.e., congenital or early infantile onset of symptoms with progression to death in respiratory failure.3..5,1 0 Clinical manifestations of cardiomyopathy were not reported previously. Patient 2 presented clinical, radiological, and electrocardiographic manifestations of cardiomyopathy. Patient 1 had no clinical manifestations of heart involvement, but postmortem examination of the heart revealed a massive increase of glycogen concentration and absence of PFK-1 activity. Involvement of the liver in this patient was documented by similar biochemical findings. Multiple tissue involvement

Infantile Phosphofructokinase Deficiency

of this enzymatic defect has not been reported previously in either adult or the infantile form. The biochemical analysis of the skeletal muscles in the cases reported to date are characterized by mild accumulation of normally structured glycogen in subsarcolemmal spaces. An important finding in patient 1 was the negative PAS-staining on the first muscle biopsy (performed at 6 months) and the massive accumulation of glycogen (81 mg/g wet weight) in a second biopsy that was obtained at 18 months. In all previously reported cases, muscle biopsy that was performed before 6 months of age showed only a mild increase in glycogen concentration. “Inadequate sampling” of the first closed-needle biopsy in patient l cannot be ruled out as the cause of the difference in glycogen concentrations. However, it is a reasonable hypothesis that glycogen storage increases with the progression of the disease. This may be related to the ability of the patient to use a fetal functional form of the enzyme in early infancy. As the child matures and loses the fetal enzyme, glycogen content is increased because of the presence of an adult, deficient form of the enzyme. Thus, glycogen storage may be missed in a biopsy obtained too early in the course of infantile PFK- 1 deficiency. The involvement of the liver in patient 1 cannot be explained on the basis of a deficiency of the M-type isozyme. It is also unlikely that 2 different isozymes (M and L), which are under different genetic control, are involved. It is possible, however, that the reduced PFK activity is secondary to a deficiency of an activator or to the presence of an inhibitor common to different types of PFK-1 isozymes. This assumption led us to investigate PFK-2 activity. This recently discovered enzyme converts fructose-6-phosphate to fructose-2,6-biphosphate (F26P2), which is not a metabolic intermediate, but rather the most prominent intracellular activator of‘ mammalian PFK-1 studied to date.” Because only one PFK-2 isozyme common to all tissue is known, its normal activity in this patient’s liver, and the presence of normal levels of F26P2 in muscle and liver, rules out the possibility of PFK-2 deficiency. Such an enzymatic defect has not yet been described. Nevertheless, the multisystem involvement in this patient theoretically may be related to a single, yet unknown activator or inhibitor common to all PFK-1 subtypes. From the paucity of cases of infantile PFK deficiency published to date, it is apparent that it is not compatible with life beyond late infancy or early childhood. It is also apparent that this form

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is clinically and biochemically different from the adult type. It is yet not clear whether all the infantile cases reported to date have a common metabolic origin. Although we did not indentify the exact enzymatic defect, the unusual biochemical findings in our patients may contribute to a better

understanding of this metabolic error. Based on our findings, it seems justified to include the measurements of glycogen concentration and of glycogenolytic enzymes in the work-up of cases with congenital or early infantile onset of progressive muscle weakness,

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