- Email: [email protected]
Studies in glycogen storage disease
214
February, 1968 T h e J o u r n a l ,~/ P E I ) I A T R
I(:S
Studies in glycogen storage disease III. Limit dextrinosis: A genetic study
A case of type 1II glycogen storage disease is reported in uhich amylo-1,6-glucosidase activity was markedly reduced in liver and muscle. The leukocyte debranching enzyme activity was higher than that observed in previous cases and was more in the range thought to be characteristic o[ heterozygotes. Although both parents had some reduction in the activity of leukocyte debranching enzyme, their muscle enzyme activity was normal. The father's hepatic amyIo-l,6-glucosidase activity was normal, while the mother's was probably reduced. These results indicate tissue variability in the expression of both the homozygotic state in the patient and the heterozygotic state in the parents. The reasons for such findings are not apparent. Both the maternal and paternal grandfathers had significant reduction o[ arrzylo-l,6-gtueosidase activity consistent with the heterozygous state, while both grandmothers had normal enzyme levels. The~e results and those of leukocyte debranching enzyme assays in eight other relatives would be most consistent with a simple Mendelian recessive mode of inheritance of this type of glycogen storage disease in this patient.
Christine Williams, M.D., and James B. Field, M.D. PITT
SB URGt'[
~
PA,
T H E M o D E 0 IV inheritance of type I I I glycogen storage disease has not been completely elucidated. An autosomal recessive inheritance was suggested by Sidbuvy's report that 4 patients with this disease have married normal individuals and produced 6 children, all of whom are unaffected by the disease. 1 Furthermore, this mode of inheritance is supported by his studies mea-
From the Clinical Research Unit and the Department o[ Medicine, University o[ Pittsburgh School of Medicine. Supported by United States Public Health Service Grant AM-08333 and General Clinical Research Center Grant~" FR-56 and FR-84 from the National Institutes of Health. Vol. 72, No. 2, pp. 214-221
suring erythrocyte glycogen in family members of patients with this type of glycogen storage disease. 2 Williams and associates a reported reductions in leukocyte debranching enzyme activity (amylo-1, 6-glueosidase) in both parents of a patient with type I I I glycogen storage disease compatible with autosomal recessive transmission of the disease. Although Brandt and De Luca 4 reported type I I I glycogen storage disease in a mother and 3 children, suggesting a dominant mode of inheritance, these authors do not exclude the possibility of autosomal recessive inheritance since the parents were tirst cousins. Studies of families of patients with type
Volume 72 Number 2
I I I glycogen storage disease have been complicated by the demonstration of tissue variability of the enzyme deficiency, depending on whether the assay measured liberation of glucose from a phosphorylase limit dextrin or incorporation of 1+C glucose into glycogen? Furthermore, although it has not been possible to separate amylo-1, 6glucosidase activity from oligo- 1,4--+ 1,4 glucantransferase activity, patients have been described in w h o m one but not the other activity has been absent from a specific tissue. Based on these various considerations, Hers ~ has subdivided type I I I glycogen storage disease into 4 types. Patients with type A lack both enzyme activities in liver and muscle regardless of the method used for assay, while type B patients are deficient of both enzymatic activities in liver. Although a homogenate of their muscle can incorporate 1"C glucose into glycogen, it cannot liberate glucose from a limit dextrin and has no oligo-1,4-->1,4 glucantransferase activity. Amylo-l,6-glucosidase is absent from muscle by both assays in type C glycogen storage disease. Activity is present in liver when incorporation of z~C glucose into glycogen is measured, but not when liberation of glucose from limit dextrin is the basis for the assay. Oligo-1,4--+1,4 glucantransferase activity is present in both liver and muscle of such patients. Patients with type D have amylo-l,6-glucosidase deficiency in both organs when assayed by liberation of glucose from limit dextrin, but both tissues have activity when the assay involves incorporation of ~4C glucose into glycogen. Both liver and muscle were devoid of oligo1,4--+i,4 glucantransferase activity. Brandt and De L u c a 4 suggested that in addition to that in muscle and liver, enzyme activity in leukocytes m a y be independently genetically expressed, and that this variable m a y therefore also have to be incorporated into the subgrouping. T h e following is a case report of a patient with type I I I glycogen storage disease who had markedly reduced debrancher enzyme activity in muscle and liver. However, his leukocyte amylo-l,6-glucosidase activity
Genetic study in glycogen storage disease
2 15
was higher than that reported in other cases of this diseas&' ';' ~ but in the range previously assigned to presumed heterozygotes? In addition, leukocyte debranching enzyme activity was assayed in m a n y other family members in an attempt to elucidate the mode of inheritance of the disease. CASE REPORT
The patient, N. D., was the product of a 37week gestational period which was complicated by mild pre-eclampsia. Delivery was normal. His parents, ages 22 and 23 respectively, gave no history of consanguinity. There are no other siblings. At birth he weighed 1.9 kilograms and at one minute had an Apgar score of 9. During the two weeks he remained in the nursery, he gained weight steadily, did not become jaundiced, and had no apparent symptoms of hypoglycemia. No hepatomegaly was present. Rapid respirations, frequently above 60 per minute but without cyanosis or retractions, were present during the first 3 days of life and were attributed only to the infant's prematurity. Although at home he had frequent vomiting and changes of formula, he continued to gain weight slowly. At age 4 months hepatomegaly was noted by the family pediatrician, and one month later the infant was admitted to the Children's Hospital of Pittsburgh for evaluation of hepatomegaly and failure to thrive. Upon admission he was described as a "frail, irritable, pot-bellied Caucasian male infant with elfin facies." The child's mother denied that he was irritable at home although she had noted excessive sweating on occasion when waking him for night feedings. Height, weight (4.4 kilograms), head and chest measurements were all below the third percentile for his age. Upon physical examination his anterior fontanel was large, measuring 5 by 6 cm. Funduscopic examination was normal. A healing papular rash was present over the upper third of the chest and a generalized decrease of subcutaneous tissue was noted. A grade I I / V I systolic ejection murmur was heard over the left precordium; no thrill or cardiomegaly was noted. The liver edge was firm and non-nodular, extending 6 cm. below the right costal margin. The spleen and kidneys were not palpable. Muscle tone and head control were poor, but deep tendon reflexes were normal. Urinalysis was normal and acetonuria was not present. The hematocrit varied from 30 to 37 per cent. The white blood cell count ranged
2 16
Williams and Field
The Journal of Pediatrics February 1968
from 7,500 to 11,900 cells per cubic millimeter with 17 to 49 per cent polymorphonuclear forms and the rest lymphocytes. Bleeding and coagulation times were normal. Fasting blood sugar levels varied from 30 to 75 rag. per cent. Blood-urea-nitrogen was 13 mg. per cent; calcium, 10.4 mg. per cent; phosphorus, 5.6 mg. per cent; sodium, 137 to 145 mEq. per liter; chloride, 102 to 113 mEq. per liter; carbon dioxide combining power, 15.4 mEq. per liter; and pH, 7.22. Serum cholesterol was 176 mg. per cent; serum glutamic oxaloacetie transaminase, 975; alkaline phosphatase, 8.9 Bessy-Lowry units, and thymoI turbidity, 0.8 units. There were 3.3 Gin. of albumin and 2.0 Gin. of globulin. T A globulin was absent while both y G and V M globulins were decreased. The protein-bound iodine and butanol extractable iodine were normal. An oral glucose tolerance test was performed with the following results: 0 minutes--35 mg. per cent; 30 minutes--67; 60 minutes--142; 120 minutes--147; 180 minu t e s - - l l 7 ; 4 hours--95; and 5 hours--94 mg. per cent. A "double barrel" glucagon tolerance test was performed according to the method of Hug. s Glucagon (1 rag. intravenously) given after a 14 hour fast produced no hyperglycemic response, whereas postprandially there was a norreal increase in blood glucose (Fig. 1). A presumptive diagnosis of type I I I glycogen storage disease was made on the basis of the clinical symptoms, the positive Hug test and the elevated serum glutamic oxaloacetic transaminase (with normal liver function studies). Bone marrow, excretory urogram, bone age, and electrocardiographic examinations were within normal limits. An open biopsy of the liver was performed. Grossly, apart from its increased size, the liver
200
,~
150
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I00
09J
/
.,,"""*~"'' ' *
POST
PRANDIAL
50
"i
o
30
io
"-FASTING
i
8
90
,2o
TIME ( M I N I
Fig. 1. "Double barrel" glucagon tolerance test (1 mg. intravenously).
,;o
appeared normal. Microscopically, however, the hepatocytes were large, swollen, and contained pale, clear cytoplasm. Large amounts of glycogen were present in the cytoplasm by periodic acid Schiff and periodic acid-Schiff-diastase staining. The patient's hospital course was uneventful except for a postoperative urinary tract infection which responded to appropriate antibiotic therapy. At 9 months of age, the patient was admitted to the Clinical Research Unit of Children's Hospital for re-evaluation. His weight had increased to 6.84 kilograms. At this time his liver measured 11 cm. below the costal margin; the remainder of the physical examination was unchanged. Psychological evaluation was reported as "normal for age." Laboratory data were essentially the same as for his first admission. Serum gamma globulins remained at a low level of 300 mg. per cent; this was considered to be a physiological hypogammaglobulinemia. The child remained asymptomatic throughout his hospital course and has continued well to the present time. Treatment has consisted primarily of frequent feedings. METHODS T h e methods for assay of leukocyte "~ and h e p a t i c 9 d e b r a n c h i n g enzyme, leukocyte TM and hepatic ~ phosphorylase, a n d hepatic glueose-6-phosphatase 9 have been previously published. H e p a t i c , 9 muscle, 1. a n d e r y t h r o cyte a glycogen were m e a s u r e d as previously reported. Muscle d e b r a n c h i n g enzyme was assayed both b y i n c o r p o r a t i o n of glucose-14C into glycogen 12 ("reverse" reaction) and by release of glucose f r o m phosphorylase limit d e x t r i n 1~ ( " f o r w a r d " r e a c t i o n ) . T h e l a t t e r assay was modified so t h a t the reaction m i x t u r e consisted of 0.3 ml. of 0.02 M citrate-ethylenediamine tetraacetic acid buffer at p H 6.2, 0.1 ml. glass distilled water, 0.1 ml. of phosphorylase limit dextrin (10 mg. per milliliter), a n d 0.1 ml. muscle homogenized in the c i t r a t e - e t h y l e n e d i a m i n e t e t r a a c e t i c acid buffer. This a m o u n t of h o m o g e n a t e contained between 0.2 a n d 0.4 mg. muscle protein. T h e reaction was carried out at 37 ~ C. for 5 minutes, at which time it was stopped by a d d i t i o n of 0.2 ml. 0.3 N b a r i u m hydroxide a n d 0.2 ml. 5 p e r cent zinc sulfate. Free glucose released d u r i n g the i n c u b a t i o n was
Volume 72 Number 2
Genetic study in glycogen storage disease 2 1 7
marized in Table II. Although muscle debranching activity was markedly reduced in patient N. D., the activity as measured by both "forward" and "reverse" assays was normal in both parents. Hepatic debranching activity ("reverse" assay) was normal in the father but only 60 per cent of normal in the mother. Leukocyte debranching activity in both parents was significantly reduced from the normal mean. However, several of the 6 determinations were in the same range as found in normal patients, while the rest of the values were definitely lower than the normal range. Hence, the mean activity falls between these and is higher than the mean activity in other parents thought to be heterozygotes. 3 Liver, muscle, and erythrocyte glycogen was normal in both parents. White blood cell phosphorylase was also normal in both. Relatives of N. D. These results are summarized in Fig. 2. Of 12 additional relatives studied by means of leukocyte debranching assays, 6 had values consistent with a heterozygous state and 6 were normal. Both grandfathers of N. D. appeared to be carriers, while the grandmothers were normal. A maternal uncle, as well as a paternal uncle and aunt of the patient, were heterozygous for the trait. The final carrier detected was a brother of the paternal grandfather.
measured by the glucose oxidase method, (Glucostat). A control tube with boiled muscle homogenate was included in each assay. Under these conditions the assay was linear both with time and with different amounts of muscle homogenate. RESULTS
Patient N. D. The results establishing a diagnosis of type I I I glycogen storage disease are summarized in Table I. Amylo-l,6glucosidase activity was markedly decreased to 5 per cent and 10 per cent of normal in muscle and liver respectively. Furthermore, an homogenate of muscle failed to form significant amounts of glucose from a limit dextrin. In contrast to other patients studied, a, 6, 1, leukocyte debranching activity was approximately 40 per cent of normal. This has been the range previously presumed to represent the heterozygote. 3 Glycogen content was elevated in muscle and liver but not in erythrocytes. Polysaccharide isolated from a trichloroacetic acid extract of muscle had an end group of 11.8 per cent (normal 6 to 9 per cent) and was degraded to the extent of 5 per cent (normal 30 to 40 per cent) by phosphorylase free of the debrancher system. Types I and V I glycogen storage disease were excluded by the normal hepatic glucose-6-phosphatase and phosphorylase levels. Parents of N. D. These results are sum-
1
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[ ] HETEROZYGOTE PI.O, Iq~214 141
Fig. 2. "D" family pedigree.
U HOMOZYGOTE ( ).NO. D I E T E R I d I N A ~
2 18
Williamv a~zd Fidd
The Journal o[ Pediatrics February 1968
T a b l e I. Enzyme assays and special studies on N. D.
Tissue
I
Assay
I Results-+ S. E. I
Normals
Liver
Debranching (14C method)
(3)*
137,981 +60,000
1,123,000 -+435,000 e.p.m.'~ per gram protein
Muscle
Debranching ('*C method)
(2)
2:3,158+-14,214
1,307,000-+103,000 c.p.m, per gram protein
WBCt
Debranching (~4C method)
(4)
102 +- 14
303 -+ 13 c.p.m./10 ~ WBC
Liver
Phosphorylase
(1)
115.8(20 min.) 163.3 (30 rain.)
72-+14.7 (20 min. incubation) 81-+ 18.9 (30 min. incubation) mg. phosphorus per gram protein
WBC
Phosphorylase
( 1)
70.6
29.6 +- 1.2 #g phosphorus/10 r WBC
Liver
Glueose-6-phosphatase
(2)
41.8- 9.6
26.7 +- 5.1 rag. phosphorus per gram protein
RBCt
Glycogen
( 1)
127
59 -+9 gg glycogen per gram Hb. (21-135 range)
Liver
Glycogen
(3)
10.8%
5%
Muscle
Glycogen
( 1)
3.9%
1%
e'Number of determinations contained in parentheses. "~Abbrevlations: c.p.m., eoun|s per minute. WBC, white blood cells: RBC, ted blood cells.
T a b l e II. Enzyme assays and special studies in the parents of N. D.
Tissue
I
Assay
{
Mr. D.
I
Mrs. D.
1
Normals
Liver
Debranching ("~C)
863,195
614,298
t,123,000 -'2"435,000 c.p.m.f per grain protein
WBCt
Debranching ( 1~(1)
163-+39 (6)* Range 51-319
185-+33 (6)* Range 79-285
303-+13 e.p.m, per 107 WBC
Muscle
Debranching ('tO)
1,615,000
1,365,700
1,307,000 -+ 103,000 c.p.m, per gram protein
Muscle
Debranching (Limit dextrin
4.93-+1.29 (5)
4.44+-l).8l
WBC
Phosphorylase
38.1
34.8
29.6 -+ 1.2 ~g Phos. per 10r WBC
RBC }
Glycogen
23.6
27.5
59 -+ 9 #g glycogen per gram Hb. (21-135 range)
Muscle
Glycogen
0.87%
0.69%
1eA
(2)
5.43 -+ .52 mg. glueose per gram protein (Range 4.13 - 8.05)
*Number of detelminatlons contained in parentheses. t F m abbreviations, see Table I.
COMMENTS
T h e diagnosis of type I I I glycogen storage disease in our p a t i e n t was based on the clinical features, the m a r k e d reduction of activity of hepatic and muscle amylo-l,6glucosidase, the increased glycogen content
of liver and muscle, and its a b n o r m a l structure. T h e clinical features of this disease have been adequately reviewed elsewhere.l, 14, 1~ All of the symptoms found in patients with type I glycogen storage disease have been seen in patients with type
Volume 72 Number 2
I I I glycogenosis with the possible exception of xanthomata. 1 Furthermore, all of the laboratory abnormalities observed in patients with type I glycogen storage disease may be present in those with type I I I disease. However, in contrast to type I, ~6 patients with type I I I disease usually have markedly elevated serum glutamic oxalo acetic transaminase levels. 6, 14 Glucagon tolerance tests have also been employed to distinguish between these two forms of glycogen storage disease. Both types of patients have no rise in blood glucose following administration of glucagon during the fasting state. Patients with type I I I disease, however, frequently respond with a normal hyperglycemic response if tested postprandially, whereas type I patients are unresponsive, s, 17 Failure to respond to glucagon, both fasting and postprandialIy, has been reported in patients with a confirmed diagnosis of type I I I disease.4, 18 Patients with the type I I I defect demonstrate an elevation in blood glucose following intravenous administration of galactose while type I patients do not? 9 Definitive diagnosis of type I I I glycogen storage disease, however, is based on tissue analysis for amylo-l,6-glucosidase activity as well as on the content and nature of the stored glycogen. Enzyme activity can be assayed by measuring the release of free glucose from a phosphorylase limit dextrin. 13 Alternately, incorporation of 14C-glucose into glycogen has been used to assay debrancher activity? 2 In a series of 18 cases Illingworth 2~ reported liver glycogen contents varying between 8 and 26 per cent and muscle glycogen contents between 1 and 14 per cent. Erythrocyte 2, 21 and leukocyt& glycogen have been elevated in some but not all cases of this disease, a' 4, 22 Glycogen isolated from liver, muscle, and erythrocytes of patients with type I I I disease resembles a phosphorylase limit dextrin TM 24 with very short outer branches as determined by the per cent of glycogen digested by phosphorylase free of debranching enzyme. 2~ Williams and associates, 3 Field and Drash, 6 Steinitz and co-workers, ~ and Huijing 2s have all demonstrated marked reduction in leu-
Genetic study in glycogen storage disease
2 19
kocyte debranching enzyme in patients with type I I I glycogen storage disease. Our patient was unusual in that his leukocyte amylo-l,6-glucosidase activity was consistently higher than that of other patients..~, ~, ~, z5 This finding is similar to that reported by Brandt and De Luca 4 and indicates that leukocyte debranching enzyme assays may be misleading in patients with type I I I glycogen storage disease. On the basis of markedly reduced hepatic and muscle debranching enzyme activity, our patient would be classified according to Hers '~ subgrouping as an example of Group A. However, in view of the evidence found in this case and in those reported by Brandt and De Luca 4 that muscle and hepatic activity may be markedly reduced in association with low normal or normal leukocyte activity, it seems clear that the original subgrouping of limit dextrinosis into A, B, C, and D will have to be modified to incorporate leukocyte debrancher activity as well as that of liver and muscle. In order to investigate the mode of inheritance of limit dextrinosis, the parents and available relatives of N. D. were studied. Although leukocyte debranching activity in both parents was clearly below normal, the values were somewhat higher than those seen in the parents of the patient reported by Williams and colleagues? Actually, the mean activity of the parents' leukocyte assays consisted of some values which were in the expected heterozygote range and other values which were in the range found in normal subjects. Heterozygosity could not be detected when amylo-l,6-glucosidase was assayed in muscle obtained from the parents even though the patient himself had less than 5 per cent of normal muscle debrancher activity. This was true regardless of which assay for the enzyme was used. The father's hepatic amylo-l,6-glucosidase was normal while the mother's was lower than any of the 5 controls. The mother's value would be consistent with the heterozygous state, while the father's would not: Thus, in addition to the distributional anomalies of the enzymatic defect in the pa-
220
Williams and Field
tient, there is also tissue variability in terms of detection of the heterozygote. This suggests that the genetic control of the enzyme may differ from tissue to tissue, or that other factors are capable of modifying the genetic expression of the enzymatic defect. The problem of such distributional anomalies in inherited enzymatic defects has recently been considered in detail by Auerbach and DiGeorge 2G and a possible explanation based on a copy-choice mechanism for the transcription of genetic information to messenger ribonucleic acid proposed. Although this hypothesis could adequately explain the tissue variability of the enzyme defect in the homozygote, the results obtained in this report are unique, in that similar distributional anomalies were observed in the heterozygote. In contrast to the results obtained in families with type I I I glycogen storage disease, parents of patients with type I glycogenosis have consistently demonstrated heterozygous levels of gtucose-6-phosphatase activity in biopsies of small intestine, s, 9 In contrast to the somewhat confusing results obtained from study of both parents, assay of leukocyte debranching enzyme activity in 12 additional relatives of the patient provide data most consistent with an autosomal recessive type of inheritance in this patient. Both the paternal and maternal grandfathers were heterozygotes, while the paternal and maternal grandmothers were normal. In addition, a paternal aunt, uncle, and great uncle, and a maternal uncle were also heterozygotes. Sidbury~,Z, 21 has suggested that heterozygotes for type I I I glycogen storage disease may be detected by means of their elevated erythrocyte glycogen content, and in one family so studied a simple recessive type of inheritance was suggested. In the present case, however, our patient, as well as his parents, had normal erythroeyte glycogen content, hence this mode of investigating heterozygosity was not pursued. Detection of heterozygotes by leukocyte assay for debranching enzyme activity was possible in the case described by Williams and associates; s however, in a re-
The Journal of Pediatrics February 1968
cent case studied by Field and Drash s the mother was heterozygous as determined by leukocyte assay but the father's white blood cell debranching enzyme activity was normal. Brandt and De Luca's 4 report of a mother and her 3 children with limit dextrinosis was compatible with a dominant form of inheritance, although the authors favored a simple Mendelian recessive pattern of inheritance since the parents were first cousins. Previously, an autosomal recessive mode of inheritance for this disease has been preferred over the other possibilities in view of the high incidence of consanguinity among parents, the increased incidence of disease among siblings, and the fact that male and female subjects are affected with equal frequency, a, 27 The demonstration of several examples of heterozygosity in both the mother's and father's family in our own study would be most consistent with this mode of inheritance. The discrepant results of the parents' leukocyte debranching assays could be explained on the basis of other factors modifying the expression of the genetic defect. The authors are indebted to Dr. Barbara IIlingworth Brown, Washington University of St. Louis, who kindly performed the studies on the structure of muscle glycogen as well as the assay for muscle debranching enzyme using limit dextrin. Generous supplies of phosphorylase limit dextrin were kindly provided by Drs. Barbara Illingworth Brown, Washington University of St. Louis, and Joseph L. Larner, University of Minnesota, Minneapolis. REFERENCES
1. Sidbury, J. B., Jr.: The genetics of the glycogen storage diseases, in Steinberg, A. G., editor: Progress in medical genetics, New York, 1965, Grune and Stratton, Inc. 2. Sidbury, J. B., Jr., Cornblath, M., Fisher, J., and House, E.: Glycogen in erythrocytes of patients with glycogen storage disease, Pediatrics 27" 103, 1961. 3. Williams, H. E., Kendig, E. M., and Field, J. B.: Leukocyte debranching enzyme in glycogen storage disease, J. Clin. Invest. 42: 656, 1963. 4. Brandt, I. K., and De Luca, V. A., Jr." Type III glycogenosis: A family with an unusual tissue distribution of the enzyme lesion, Am. J. Med. 40: 779, 1966.
Volume 72 Number 2
5. Hers, H. G.: The mechanism of action of amylo-l,6-glucosidase, in Whelan, W. J., and Cameron, M. P., editors: Control of glycogen metabolism, Ciba Foundation Symposium, London, 1964, J. & A. Churchill, Ltd., pp. 151-175. 6. Field, J. B., and Drash, A. L.: Studies in glycogen storage disease. II. Heterogeneity in the inheritance of glycogen storage diseases, Tr. A. Am. Physiol. In press. 7. Steinitz, K., Bodur, H., and Arman, T.: Amylo- 1,6-glucosidase activity in leukocytes from patients with glycogen storage disease, Clin. & chim, acta 8: 807, 1963. 8. Hug, G.: Glucagon tolerance test in glycogen storage disease, J. PEDXAT. 60: 545, 1962. 9. Field, J. B., Epstein, S. M., and Egan, T.: Studies in glycogen storage disease. I. Intestinal glucose-6-phosphatase activity in patients with Von Gierke's disease and their parents, J. Clin. Invest. 44: 1240, 1965. 10. Williams, H. E., and Field, J. B.: Low leukocyte phosphorylase in hepatic phosphorylase deficient glycogen storage disease, J. Clin. Invest. 40: 1841, 1961. 11. Caroll, N. V., Longley, R. W., and Roe, J. H.: The determination of glycogen in liver and muscle by use of the anthrone reagent, J. Biol. Chem. 220: 583, 1956. 12. Hers, H. G.: Etudes enzymatiques sur fragments hepatiques, Rev. Intern. Hepatol. 12: 35, 1959. 13. Illingworth, B., Cori, G. T., and Cori, C. F.: Amylo-l,6-glucosidase in muscle tissue in generalized glycogen storage disease, J. Biol. Chem. 218: 123, 1956. 14. Van Creveld, S., and Huijing, F.: Glycogen storage disease: Biochemical and clinical data in sixteen cases, Am. J. Med. 38: 554, 1965. 15. FieId, R. A.: Glycogen deposition diseases, in Stanbury, J. B., Wyngaarden, J. B., and Fredrickson, D. S., editors: The metabolic basis of inherited disease, New York, 1966, McGraw-Hill Book Company, Inc. 16. Brante, G., Kaijser, K., and Ockerman, P. A.: Glycogenosis type I (lack of glucose-6-
Genetic study in glycogen storage disease
17.
18.
19. 20. 21.
22.
23.
24.
25.
26.
27.
221
phosphatase) in four siblings, Acta paediat. suppl. 157: 10, 1964. Hug, G., Krill, C. E., Perrin, E. V., and Guest, G. M.: Cori's disease (Amylo-l,6glucosidase deficiency): Report of a case in a Negro child, New England J. Med. 268: 113, 1963. Limbeck, G. A., and Kelley, V. C.: "Double barrel" glucagon test: Correlation with enzyme assays in limit dextrinosis, Am. J. Dis. Child. 109: 162, 1965. Schwartz, R., Ashmore, J., and Renold, A. E.: Galactose tolerance in glycogen storage disease, Pediatrics 19: 585, 1957. Illingworth, B.: Glycogen storage disease, Am. J. Clin. Nutrition 9: 683, 1961. Sidbury, J. B., Gitzelmann, R., and Fisher, J.: The glycogenoses. Further observations on glycogen in erythrocytes of patients with glycogenosis, Helvet. paediat, aeta 16: 506, 196l. Van Creveld, S., and Huijing, F.: Differential diagnosis of the type of glycogen storage disease in two adult patients with long history of glycogenosis, Metabolism 13: 191, 1964. Forbes, G. B.: Glycogen storage disease: Report of a case with abnormal glycogen structure in liver and skeletal muscle, J. PEDIAT. 42: 645, 1953. Illingworth, B., and Cori, G. T.: Structure of glycogens and amylopectins. III. Normal and abnormal human glycogen, J. Biol. Chem. 199: 653, 1952. Huijing, F.: Amylo-1, 6-glucosidase activity in normal leukocytes and in leukocytes of patients with glycogen storage disease, Clin. & chim. acta 9: 269, 1964. Auerbach, V. H., and DiGeorge, A. M.: Genetic mechanisms producing multiple enzyme defects. A review of unexplained eases and a new hypothesis, Am. J. M. Sc. 249: 718, 1965. Hers, H. G.: Glycogen storage disease, in Levine, R., and Luft, R., editors: Advances in metabolic disorders, New York, 1963, Academic Press, Inc.
February, 1968 T h e J o u r n a l ,~/ P E I ) I A T R
I(:S
Studies in glycogen storage disease III. Limit dextrinosis: A genetic study
A case of type 1II glycogen storage disease is reported in uhich amylo-1,6-glucosidase activity was markedly reduced in liver and muscle. The leukocyte debranching enzyme activity was higher than that observed in previous cases and was more in the range thought to be characteristic o[ heterozygotes. Although both parents had some reduction in the activity of leukocyte debranching enzyme, their muscle enzyme activity was normal. The father's hepatic amyIo-l,6-glucosidase activity was normal, while the mother's was probably reduced. These results indicate tissue variability in the expression of both the homozygotic state in the patient and the heterozygotic state in the parents. The reasons for such findings are not apparent. Both the maternal and paternal grandfathers had significant reduction o[ arrzylo-l,6-gtueosidase activity consistent with the heterozygous state, while both grandmothers had normal enzyme levels. The~e results and those of leukocyte debranching enzyme assays in eight other relatives would be most consistent with a simple Mendelian recessive mode of inheritance of this type of glycogen storage disease in this patient.
Christine Williams, M.D., and James B. Field, M.D. PITT
SB URGt'[
~
PA,
T H E M o D E 0 IV inheritance of type I I I glycogen storage disease has not been completely elucidated. An autosomal recessive inheritance was suggested by Sidbuvy's report that 4 patients with this disease have married normal individuals and produced 6 children, all of whom are unaffected by the disease. 1 Furthermore, this mode of inheritance is supported by his studies mea-
From the Clinical Research Unit and the Department o[ Medicine, University o[ Pittsburgh School of Medicine. Supported by United States Public Health Service Grant AM-08333 and General Clinical Research Center Grant~" FR-56 and FR-84 from the National Institutes of Health. Vol. 72, No. 2, pp. 214-221
suring erythrocyte glycogen in family members of patients with this type of glycogen storage disease. 2 Williams and associates a reported reductions in leukocyte debranching enzyme activity (amylo-1, 6-glueosidase) in both parents of a patient with type I I I glycogen storage disease compatible with autosomal recessive transmission of the disease. Although Brandt and De Luca 4 reported type I I I glycogen storage disease in a mother and 3 children, suggesting a dominant mode of inheritance, these authors do not exclude the possibility of autosomal recessive inheritance since the parents were tirst cousins. Studies of families of patients with type
Volume 72 Number 2
I I I glycogen storage disease have been complicated by the demonstration of tissue variability of the enzyme deficiency, depending on whether the assay measured liberation of glucose from a phosphorylase limit dextrin or incorporation of 1+C glucose into glycogen? Furthermore, although it has not been possible to separate amylo-1, 6glucosidase activity from oligo- 1,4--+ 1,4 glucantransferase activity, patients have been described in w h o m one but not the other activity has been absent from a specific tissue. Based on these various considerations, Hers ~ has subdivided type I I I glycogen storage disease into 4 types. Patients with type A lack both enzyme activities in liver and muscle regardless of the method used for assay, while type B patients are deficient of both enzymatic activities in liver. Although a homogenate of their muscle can incorporate 1"C glucose into glycogen, it cannot liberate glucose from a limit dextrin and has no oligo-1,4-->1,4 glucantransferase activity. Amylo-l,6-glucosidase is absent from muscle by both assays in type C glycogen storage disease. Activity is present in liver when incorporation of z~C glucose into glycogen is measured, but not when liberation of glucose from limit dextrin is the basis for the assay. Oligo-1,4--+1,4 glucantransferase activity is present in both liver and muscle of such patients. Patients with type D have amylo-l,6-glucosidase deficiency in both organs when assayed by liberation of glucose from limit dextrin, but both tissues have activity when the assay involves incorporation of ~4C glucose into glycogen. Both liver and muscle were devoid of oligo1,4--+i,4 glucantransferase activity. Brandt and De L u c a 4 suggested that in addition to that in muscle and liver, enzyme activity in leukocytes m a y be independently genetically expressed, and that this variable m a y therefore also have to be incorporated into the subgrouping. T h e following is a case report of a patient with type I I I glycogen storage disease who had markedly reduced debrancher enzyme activity in muscle and liver. However, his leukocyte amylo-l,6-glucosidase activity
Genetic study in glycogen storage disease
2 15
was higher than that reported in other cases of this diseas&' ';' ~ but in the range previously assigned to presumed heterozygotes? In addition, leukocyte debranching enzyme activity was assayed in m a n y other family members in an attempt to elucidate the mode of inheritance of the disease. CASE REPORT
The patient, N. D., was the product of a 37week gestational period which was complicated by mild pre-eclampsia. Delivery was normal. His parents, ages 22 and 23 respectively, gave no history of consanguinity. There are no other siblings. At birth he weighed 1.9 kilograms and at one minute had an Apgar score of 9. During the two weeks he remained in the nursery, he gained weight steadily, did not become jaundiced, and had no apparent symptoms of hypoglycemia. No hepatomegaly was present. Rapid respirations, frequently above 60 per minute but without cyanosis or retractions, were present during the first 3 days of life and were attributed only to the infant's prematurity. Although at home he had frequent vomiting and changes of formula, he continued to gain weight slowly. At age 4 months hepatomegaly was noted by the family pediatrician, and one month later the infant was admitted to the Children's Hospital of Pittsburgh for evaluation of hepatomegaly and failure to thrive. Upon admission he was described as a "frail, irritable, pot-bellied Caucasian male infant with elfin facies." The child's mother denied that he was irritable at home although she had noted excessive sweating on occasion when waking him for night feedings. Height, weight (4.4 kilograms), head and chest measurements were all below the third percentile for his age. Upon physical examination his anterior fontanel was large, measuring 5 by 6 cm. Funduscopic examination was normal. A healing papular rash was present over the upper third of the chest and a generalized decrease of subcutaneous tissue was noted. A grade I I / V I systolic ejection murmur was heard over the left precordium; no thrill or cardiomegaly was noted. The liver edge was firm and non-nodular, extending 6 cm. below the right costal margin. The spleen and kidneys were not palpable. Muscle tone and head control were poor, but deep tendon reflexes were normal. Urinalysis was normal and acetonuria was not present. The hematocrit varied from 30 to 37 per cent. The white blood cell count ranged
2 16
Williams and Field
The Journal of Pediatrics February 1968
from 7,500 to 11,900 cells per cubic millimeter with 17 to 49 per cent polymorphonuclear forms and the rest lymphocytes. Bleeding and coagulation times were normal. Fasting blood sugar levels varied from 30 to 75 rag. per cent. Blood-urea-nitrogen was 13 mg. per cent; calcium, 10.4 mg. per cent; phosphorus, 5.6 mg. per cent; sodium, 137 to 145 mEq. per liter; chloride, 102 to 113 mEq. per liter; carbon dioxide combining power, 15.4 mEq. per liter; and pH, 7.22. Serum cholesterol was 176 mg. per cent; serum glutamic oxaloacetie transaminase, 975; alkaline phosphatase, 8.9 Bessy-Lowry units, and thymoI turbidity, 0.8 units. There were 3.3 Gin. of albumin and 2.0 Gin. of globulin. T A globulin was absent while both y G and V M globulins were decreased. The protein-bound iodine and butanol extractable iodine were normal. An oral glucose tolerance test was performed with the following results: 0 minutes--35 mg. per cent; 30 minutes--67; 60 minutes--142; 120 minutes--147; 180 minu t e s - - l l 7 ; 4 hours--95; and 5 hours--94 mg. per cent. A "double barrel" glucagon tolerance test was performed according to the method of Hug. s Glucagon (1 rag. intravenously) given after a 14 hour fast produced no hyperglycemic response, whereas postprandially there was a norreal increase in blood glucose (Fig. 1). A presumptive diagnosis of type I I I glycogen storage disease was made on the basis of the clinical symptoms, the positive Hug test and the elevated serum glutamic oxaloacetic transaminase (with normal liver function studies). Bone marrow, excretory urogram, bone age, and electrocardiographic examinations were within normal limits. An open biopsy of the liver was performed. Grossly, apart from its increased size, the liver
200
,~
150
uJ. o3 O <,.)
I00
09J
/
.,,"""*~"'' ' *
POST
PRANDIAL
50
"i
o
30
io
"-FASTING
i
8
90
,2o
TIME ( M I N I
Fig. 1. "Double barrel" glucagon tolerance test (1 mg. intravenously).
,;o
appeared normal. Microscopically, however, the hepatocytes were large, swollen, and contained pale, clear cytoplasm. Large amounts of glycogen were present in the cytoplasm by periodic acid Schiff and periodic acid-Schiff-diastase staining. The patient's hospital course was uneventful except for a postoperative urinary tract infection which responded to appropriate antibiotic therapy. At 9 months of age, the patient was admitted to the Clinical Research Unit of Children's Hospital for re-evaluation. His weight had increased to 6.84 kilograms. At this time his liver measured 11 cm. below the costal margin; the remainder of the physical examination was unchanged. Psychological evaluation was reported as "normal for age." Laboratory data were essentially the same as for his first admission. Serum gamma globulins remained at a low level of 300 mg. per cent; this was considered to be a physiological hypogammaglobulinemia. The child remained asymptomatic throughout his hospital course and has continued well to the present time. Treatment has consisted primarily of frequent feedings. METHODS T h e methods for assay of leukocyte "~ and h e p a t i c 9 d e b r a n c h i n g enzyme, leukocyte TM and hepatic ~ phosphorylase, a n d hepatic glueose-6-phosphatase 9 have been previously published. H e p a t i c , 9 muscle, 1. a n d e r y t h r o cyte a glycogen were m e a s u r e d as previously reported. Muscle d e b r a n c h i n g enzyme was assayed both b y i n c o r p o r a t i o n of glucose-14C into glycogen 12 ("reverse" reaction) and by release of glucose f r o m phosphorylase limit d e x t r i n 1~ ( " f o r w a r d " r e a c t i o n ) . T h e l a t t e r assay was modified so t h a t the reaction m i x t u r e consisted of 0.3 ml. of 0.02 M citrate-ethylenediamine tetraacetic acid buffer at p H 6.2, 0.1 ml. glass distilled water, 0.1 ml. of phosphorylase limit dextrin (10 mg. per milliliter), a n d 0.1 ml. muscle homogenized in the c i t r a t e - e t h y l e n e d i a m i n e t e t r a a c e t i c acid buffer. This a m o u n t of h o m o g e n a t e contained between 0.2 a n d 0.4 mg. muscle protein. T h e reaction was carried out at 37 ~ C. for 5 minutes, at which time it was stopped by a d d i t i o n of 0.2 ml. 0.3 N b a r i u m hydroxide a n d 0.2 ml. 5 p e r cent zinc sulfate. Free glucose released d u r i n g the i n c u b a t i o n was
Volume 72 Number 2
Genetic study in glycogen storage disease 2 1 7
marized in Table II. Although muscle debranching activity was markedly reduced in patient N. D., the activity as measured by both "forward" and "reverse" assays was normal in both parents. Hepatic debranching activity ("reverse" assay) was normal in the father but only 60 per cent of normal in the mother. Leukocyte debranching activity in both parents was significantly reduced from the normal mean. However, several of the 6 determinations were in the same range as found in normal patients, while the rest of the values were definitely lower than the normal range. Hence, the mean activity falls between these and is higher than the mean activity in other parents thought to be heterozygotes. 3 Liver, muscle, and erythrocyte glycogen was normal in both parents. White blood cell phosphorylase was also normal in both. Relatives of N. D. These results are summarized in Fig. 2. Of 12 additional relatives studied by means of leukocyte debranching assays, 6 had values consistent with a heterozygous state and 6 were normal. Both grandfathers of N. D. appeared to be carriers, while the grandmothers were normal. A maternal uncle, as well as a paternal uncle and aunt of the patient, were heterozygous for the trait. The final carrier detected was a brother of the paternal grandfather.
measured by the glucose oxidase method, (Glucostat). A control tube with boiled muscle homogenate was included in each assay. Under these conditions the assay was linear both with time and with different amounts of muscle homogenate. RESULTS
Patient N. D. The results establishing a diagnosis of type I I I glycogen storage disease are summarized in Table I. Amylo-l,6glucosidase activity was markedly decreased to 5 per cent and 10 per cent of normal in muscle and liver respectively. Furthermore, an homogenate of muscle failed to form significant amounts of glucose from a limit dextrin. In contrast to other patients studied, a, 6, 1, leukocyte debranching activity was approximately 40 per cent of normal. This has been the range previously presumed to represent the heterozygote. 3 Glycogen content was elevated in muscle and liver but not in erythrocytes. Polysaccharide isolated from a trichloroacetic acid extract of muscle had an end group of 11.8 per cent (normal 6 to 9 per cent) and was degraded to the extent of 5 per cent (normal 30 to 40 per cent) by phosphorylase free of the debrancher system. Types I and V I glycogen storage disease were excluded by the normal hepatic glucose-6-phosphatase and phosphorylase levels. Parents of N. D. These results are sum-
1
f ll.M.
J.II.
Q,D
DD.T~ 2?3 20 g~
P~D,
I rig I04X$
A.D 01+7
M)
{|l
9 FELLY. Itl~tllO
~31t30
~1
{!)
"~"
~21
"~ 1~8t78
"*" 01~:~0
~ NOT TESTED
[ ] HETEROZYGOTE PI.O, Iq~214 141
Fig. 2. "D" family pedigree.
U HOMOZYGOTE ( ).NO. D I E T E R I d I N A ~
2 18
Williamv a~zd Fidd
The Journal o[ Pediatrics February 1968
T a b l e I. Enzyme assays and special studies on N. D.
Tissue
I
Assay
I Results-+ S. E. I
Normals
Liver
Debranching (14C method)
(3)*
137,981 +60,000
1,123,000 -+435,000 e.p.m.'~ per gram protein
Muscle
Debranching ('*C method)
(2)
2:3,158+-14,214
1,307,000-+103,000 c.p.m, per gram protein
WBCt
Debranching (~4C method)
(4)
102 +- 14
303 -+ 13 c.p.m./10 ~ WBC
Liver
Phosphorylase
(1)
115.8(20 min.) 163.3 (30 rain.)
72-+14.7 (20 min. incubation) 81-+ 18.9 (30 min. incubation) mg. phosphorus per gram protein
WBC
Phosphorylase
( 1)
70.6
29.6 +- 1.2 #g phosphorus/10 r WBC
Liver
Glueose-6-phosphatase
(2)
41.8- 9.6
26.7 +- 5.1 rag. phosphorus per gram protein
RBCt
Glycogen
( 1)
127
59 -+9 gg glycogen per gram Hb. (21-135 range)
Liver
Glycogen
(3)
10.8%
5%
Muscle
Glycogen
( 1)
3.9%
1%
e'Number of determinations contained in parentheses. "~Abbrevlations: c.p.m., eoun|s per minute. WBC, white blood cells: RBC, ted blood cells.
T a b l e II. Enzyme assays and special studies in the parents of N. D.
Tissue
I
Assay
{
Mr. D.
I
Mrs. D.
1
Normals
Liver
Debranching ("~C)
863,195
614,298
t,123,000 -'2"435,000 c.p.m.f per grain protein
WBCt
Debranching ( 1~(1)
163-+39 (6)* Range 51-319
185-+33 (6)* Range 79-285
303-+13 e.p.m, per 107 WBC
Muscle
Debranching ('tO)
1,615,000
1,365,700
1,307,000 -+ 103,000 c.p.m, per gram protein
Muscle
Debranching (Limit dextrin
4.93-+1.29 (5)
4.44+-l).8l
WBC
Phosphorylase
38.1
34.8
29.6 -+ 1.2 ~g Phos. per 10r WBC
RBC }
Glycogen
23.6
27.5
59 -+ 9 #g glycogen per gram Hb. (21-135 range)
Muscle
Glycogen
0.87%
0.69%
1eA
(2)
5.43 -+ .52 mg. glueose per gram protein (Range 4.13 - 8.05)
*Number of detelminatlons contained in parentheses. t F m abbreviations, see Table I.
COMMENTS
T h e diagnosis of type I I I glycogen storage disease in our p a t i e n t was based on the clinical features, the m a r k e d reduction of activity of hepatic and muscle amylo-l,6glucosidase, the increased glycogen content
of liver and muscle, and its a b n o r m a l structure. T h e clinical features of this disease have been adequately reviewed elsewhere.l, 14, 1~ All of the symptoms found in patients with type I glycogen storage disease have been seen in patients with type
Volume 72 Number 2
I I I glycogenosis with the possible exception of xanthomata. 1 Furthermore, all of the laboratory abnormalities observed in patients with type I glycogen storage disease may be present in those with type I I I disease. However, in contrast to type I, ~6 patients with type I I I disease usually have markedly elevated serum glutamic oxalo acetic transaminase levels. 6, 14 Glucagon tolerance tests have also been employed to distinguish between these two forms of glycogen storage disease. Both types of patients have no rise in blood glucose following administration of glucagon during the fasting state. Patients with type I I I disease, however, frequently respond with a normal hyperglycemic response if tested postprandially, whereas type I patients are unresponsive, s, 17 Failure to respond to glucagon, both fasting and postprandialIy, has been reported in patients with a confirmed diagnosis of type I I I disease.4, 18 Patients with the type I I I defect demonstrate an elevation in blood glucose following intravenous administration of galactose while type I patients do not? 9 Definitive diagnosis of type I I I glycogen storage disease, however, is based on tissue analysis for amylo-l,6-glucosidase activity as well as on the content and nature of the stored glycogen. Enzyme activity can be assayed by measuring the release of free glucose from a phosphorylase limit dextrin. 13 Alternately, incorporation of 14C-glucose into glycogen has been used to assay debrancher activity? 2 In a series of 18 cases Illingworth 2~ reported liver glycogen contents varying between 8 and 26 per cent and muscle glycogen contents between 1 and 14 per cent. Erythrocyte 2, 21 and leukocyt& glycogen have been elevated in some but not all cases of this disease, a' 4, 22 Glycogen isolated from liver, muscle, and erythrocytes of patients with type I I I disease resembles a phosphorylase limit dextrin TM 24 with very short outer branches as determined by the per cent of glycogen digested by phosphorylase free of debranching enzyme. 2~ Williams and associates, 3 Field and Drash, 6 Steinitz and co-workers, ~ and Huijing 2s have all demonstrated marked reduction in leu-
Genetic study in glycogen storage disease
2 19
kocyte debranching enzyme in patients with type I I I glycogen storage disease. Our patient was unusual in that his leukocyte amylo-l,6-glucosidase activity was consistently higher than that of other patients..~, ~, ~, z5 This finding is similar to that reported by Brandt and De Luca 4 and indicates that leukocyte debranching enzyme assays may be misleading in patients with type I I I glycogen storage disease. On the basis of markedly reduced hepatic and muscle debranching enzyme activity, our patient would be classified according to Hers '~ subgrouping as an example of Group A. However, in view of the evidence found in this case and in those reported by Brandt and De Luca 4 that muscle and hepatic activity may be markedly reduced in association with low normal or normal leukocyte activity, it seems clear that the original subgrouping of limit dextrinosis into A, B, C, and D will have to be modified to incorporate leukocyte debrancher activity as well as that of liver and muscle. In order to investigate the mode of inheritance of limit dextrinosis, the parents and available relatives of N. D. were studied. Although leukocyte debranching activity in both parents was clearly below normal, the values were somewhat higher than those seen in the parents of the patient reported by Williams and colleagues? Actually, the mean activity of the parents' leukocyte assays consisted of some values which were in the expected heterozygote range and other values which were in the range found in normal subjects. Heterozygosity could not be detected when amylo-l,6-glucosidase was assayed in muscle obtained from the parents even though the patient himself had less than 5 per cent of normal muscle debrancher activity. This was true regardless of which assay for the enzyme was used. The father's hepatic amylo-l,6-glucosidase was normal while the mother's was lower than any of the 5 controls. The mother's value would be consistent with the heterozygous state, while the father's would not: Thus, in addition to the distributional anomalies of the enzymatic defect in the pa-
220
Williams and Field
tient, there is also tissue variability in terms of detection of the heterozygote. This suggests that the genetic control of the enzyme may differ from tissue to tissue, or that other factors are capable of modifying the genetic expression of the enzymatic defect. The problem of such distributional anomalies in inherited enzymatic defects has recently been considered in detail by Auerbach and DiGeorge 2G and a possible explanation based on a copy-choice mechanism for the transcription of genetic information to messenger ribonucleic acid proposed. Although this hypothesis could adequately explain the tissue variability of the enzyme defect in the homozygote, the results obtained in this report are unique, in that similar distributional anomalies were observed in the heterozygote. In contrast to the results obtained in families with type I I I glycogen storage disease, parents of patients with type I glycogenosis have consistently demonstrated heterozygous levels of gtucose-6-phosphatase activity in biopsies of small intestine, s, 9 In contrast to the somewhat confusing results obtained from study of both parents, assay of leukocyte debranching enzyme activity in 12 additional relatives of the patient provide data most consistent with an autosomal recessive type of inheritance in this patient. Both the paternal and maternal grandfathers were heterozygotes, while the paternal and maternal grandmothers were normal. In addition, a paternal aunt, uncle, and great uncle, and a maternal uncle were also heterozygotes. Sidbury~,Z, 21 has suggested that heterozygotes for type I I I glycogen storage disease may be detected by means of their elevated erythrocyte glycogen content, and in one family so studied a simple recessive type of inheritance was suggested. In the present case, however, our patient, as well as his parents, had normal erythroeyte glycogen content, hence this mode of investigating heterozygosity was not pursued. Detection of heterozygotes by leukocyte assay for debranching enzyme activity was possible in the case described by Williams and associates; s however, in a re-
The Journal of Pediatrics February 1968
cent case studied by Field and Drash s the mother was heterozygous as determined by leukocyte assay but the father's white blood cell debranching enzyme activity was normal. Brandt and De Luca's 4 report of a mother and her 3 children with limit dextrinosis was compatible with a dominant form of inheritance, although the authors favored a simple Mendelian recessive pattern of inheritance since the parents were first cousins. Previously, an autosomal recessive mode of inheritance for this disease has been preferred over the other possibilities in view of the high incidence of consanguinity among parents, the increased incidence of disease among siblings, and the fact that male and female subjects are affected with equal frequency, a, 27 The demonstration of several examples of heterozygosity in both the mother's and father's family in our own study would be most consistent with this mode of inheritance. The discrepant results of the parents' leukocyte debranching assays could be explained on the basis of other factors modifying the expression of the genetic defect. The authors are indebted to Dr. Barbara IIlingworth Brown, Washington University of St. Louis, who kindly performed the studies on the structure of muscle glycogen as well as the assay for muscle debranching enzyme using limit dextrin. Generous supplies of phosphorylase limit dextrin were kindly provided by Drs. Barbara Illingworth Brown, Washington University of St. Louis, and Joseph L. Larner, University of Minnesota, Minneapolis. REFERENCES
1. Sidbury, J. B., Jr.: The genetics of the glycogen storage diseases, in Steinberg, A. G., editor: Progress in medical genetics, New York, 1965, Grune and Stratton, Inc. 2. Sidbury, J. B., Jr., Cornblath, M., Fisher, J., and House, E.: Glycogen in erythrocytes of patients with glycogen storage disease, Pediatrics 27" 103, 1961. 3. Williams, H. E., Kendig, E. M., and Field, J. B.: Leukocyte debranching enzyme in glycogen storage disease, J. Clin. Invest. 42: 656, 1963. 4. Brandt, I. K., and De Luca, V. A., Jr." Type III glycogenosis: A family with an unusual tissue distribution of the enzyme lesion, Am. J. Med. 40: 779, 1966.
Volume 72 Number 2
5. Hers, H. G.: The mechanism of action of amylo-l,6-glucosidase, in Whelan, W. J., and Cameron, M. P., editors: Control of glycogen metabolism, Ciba Foundation Symposium, London, 1964, J. & A. Churchill, Ltd., pp. 151-175. 6. Field, J. B., and Drash, A. L.: Studies in glycogen storage disease. II. Heterogeneity in the inheritance of glycogen storage diseases, Tr. A. Am. Physiol. In press. 7. Steinitz, K., Bodur, H., and Arman, T.: Amylo- 1,6-glucosidase activity in leukocytes from patients with glycogen storage disease, Clin. & chim, acta 8: 807, 1963. 8. Hug, G.: Glucagon tolerance test in glycogen storage disease, J. PEDXAT. 60: 545, 1962. 9. Field, J. B., Epstein, S. M., and Egan, T.: Studies in glycogen storage disease. I. Intestinal glucose-6-phosphatase activity in patients with Von Gierke's disease and their parents, J. Clin. Invest. 44: 1240, 1965. 10. Williams, H. E., and Field, J. B.: Low leukocyte phosphorylase in hepatic phosphorylase deficient glycogen storage disease, J. Clin. Invest. 40: 1841, 1961. 11. Caroll, N. V., Longley, R. W., and Roe, J. H.: The determination of glycogen in liver and muscle by use of the anthrone reagent, J. Biol. Chem. 220: 583, 1956. 12. Hers, H. G.: Etudes enzymatiques sur fragments hepatiques, Rev. Intern. Hepatol. 12: 35, 1959. 13. Illingworth, B., Cori, G. T., and Cori, C. F.: Amylo-l,6-glucosidase in muscle tissue in generalized glycogen storage disease, J. Biol. Chem. 218: 123, 1956. 14. Van Creveld, S., and Huijing, F.: Glycogen storage disease: Biochemical and clinical data in sixteen cases, Am. J. Med. 38: 554, 1965. 15. FieId, R. A.: Glycogen deposition diseases, in Stanbury, J. B., Wyngaarden, J. B., and Fredrickson, D. S., editors: The metabolic basis of inherited disease, New York, 1966, McGraw-Hill Book Company, Inc. 16. Brante, G., Kaijser, K., and Ockerman, P. A.: Glycogenosis type I (lack of glucose-6-
Genetic study in glycogen storage disease
17.
18.
19. 20. 21.
22.
23.
24.
25.
26.
27.
221
phosphatase) in four siblings, Acta paediat. suppl. 157: 10, 1964. Hug, G., Krill, C. E., Perrin, E. V., and Guest, G. M.: Cori's disease (Amylo-l,6glucosidase deficiency): Report of a case in a Negro child, New England J. Med. 268: 113, 1963. Limbeck, G. A., and Kelley, V. C.: "Double barrel" glucagon test: Correlation with enzyme assays in limit dextrinosis, Am. J. Dis. Child. 109: 162, 1965. Schwartz, R., Ashmore, J., and Renold, A. E.: Galactose tolerance in glycogen storage disease, Pediatrics 19: 585, 1957. Illingworth, B.: Glycogen storage disease, Am. J. Clin. Nutrition 9: 683, 1961. Sidbury, J. B., Gitzelmann, R., and Fisher, J.: The glycogenoses. Further observations on glycogen in erythrocytes of patients with glycogenosis, Helvet. paediat, aeta 16: 506, 196l. Van Creveld, S., and Huijing, F.: Differential diagnosis of the type of glycogen storage disease in two adult patients with long history of glycogenosis, Metabolism 13: 191, 1964. Forbes, G. B.: Glycogen storage disease: Report of a case with abnormal glycogen structure in liver and skeletal muscle, J. PEDIAT. 42: 645, 1953. Illingworth, B., and Cori, G. T.: Structure of glycogens and amylopectins. III. Normal and abnormal human glycogen, J. Biol. Chem. 199: 653, 1952. Huijing, F.: Amylo-1, 6-glucosidase activity in normal leukocytes and in leukocytes of patients with glycogen storage disease, Clin. & chim. acta 9: 269, 1964. Auerbach, V. H., and DiGeorge, A. M.: Genetic mechanisms producing multiple enzyme defects. A review of unexplained eases and a new hypothesis, Am. J. M. Sc. 249: 718, 1965. Hers, H. G.: Glycogen storage disease, in Levine, R., and Luft, R., editors: Advances in metabolic disorders, New York, 1963, Academic Press, Inc.