Possible misdiagnosis of Krabbe disease

76

Brief clinical and laboratory observations

Possible misdiagnosis of Krabbe disease David A. Wenger* and Vincent M. Riccardi, Denver, Colo.

KRABBE DISEASE or globoid cell leukodystrophy is a severe genetic disease of children with onset of symptoms at about 4 to 6 months of age, although forms with later onset have been reported. Symptoms include tonic seizures, optic atrophy, convulsions, and deafness with a rapid course ending in death between 1 1/2 and 2 years of age. All cases appear to be inherited in an autosomal recessive manner? The primary defect is reported to be a deficiency of the enzyme galactosyl ceramide beta-galactosidase (galactocerebrosidase) which is necessary for the degradation of galactosyl ceramide, a major component of myelin. -~ The amount of gal-cer storage in Krabbe disease is not large when compared to Gu2-ganglioside storage in Tay-Sachs diseasel apparently due to the diminished quantity of myelin formed in these children:~; for some reason myelination appears to be shut off early. This suggests that all of the symptoms and pathologic observations cannot be explained simply on the basis of a cerebrosidase defciency. Because o f this, we looked for other natural substrates in brain that could be acted on by the same enzyme. At about that time Miyatake and SuzukP reported that the same enzyme could act on psychosine, the deacylated derivative of cerebroside which is cytotoxic, owing to the free amine group. We then reported that the same enzyme could not degrade another myelin-specific compound, monogalactosyl diglyceride? Patients with Krabbe disease could not degrade this compound nor lactosyl ceramide, a glycolipid that also has a beta-linked galactosyl residue?. ~ Measurement of the degradation of lac-cer has provided a more sensitive diagnostic test for Krabbe disease, in which From the B. F Stolinsky Laboratories, Departments of Pediatrics and Neurology, and the Genetics Unit, Department of Biophysics and Genetics, University of Colorado Medical Center. Supported in part by a Basil O'Connor Starter Research Grantfrom the National Foundation-March of Dimes, National Institutes of Health grants HD 07773 and HD 08315 (D.A.W.) and by the National ~bundation-March of Dimes, The Kaiser Family Foundation, and The Colorado-Wyoming Regional Medical Program (Grant 5 GO3 RM 0000-40), Division of Regional Medical Programs DHE W (V.M.R.). *Reprint address: Department of Pediatrics. University of Colorado Medical Center, Denver; Colo. 80220.

The Journal of Pediatrics January 1976

leukocytes, fibroblasts, and amniotic fluid cell cultures are utilizeds 8 In these cell types, patients with Krabbe disease have 0-10% of normal activity for this enzyme, whereas carriers ordinarily have about 50% of normal activity. 7 Using our recently developed assay for beta galactosidase activitS we have demonstrated a new mutant allele for this enzyme which has activity in the heterozygote that can overlap with the values typical of patients with homozygous Krabbe disease. We have evaluated two unrelated families with this distinctive mutation. Its importance includes the possibility of a false positive diagnosis of Krabbe disease both in infants and in fetuses at amniocentesis. See related article, p. 175. Abbreviations used gal-cer: galactosyl ceramide lac-cer: lactosyl' ceramide TECHNIQUES Biopsied skin was cultured, and leukocytes were prepared according to our previous reporU The fibroblast pellet and the frozen leukocyte pellet were homogenized in distilled water, and the protein concentration was determined as described previously. 7 Assays for gal-cer and lac-cer beta galactosidase as well as for control lysosomal enzymes were performed according to earlier reports X::7 It is of interest that the use o f pure sodium taurocholate is essential when lac-cer is used as a substrate. FAMILY STUDIES Family 1 was discovered when the proband, a public health nurse, served as a control during evaluation of an unrelated family with classical Krabbe disease. Her personal health history is unremarkable, and her intellectual capacity is normal. She has no neurologic problems. Likewise, among other family members there are no relevant neurologic abnormalities or retardation. In multiple leukocyte preparations Proband 1 had galcer and lac-cer beta galactosidase activity consistently below 10% of our control values (Table I, Fig. 1). Her cultured skin fibroblasts had 19% of normal activity for gal-cer beta galactosidase and 14% of normal for lac-cer beta galactosidase (Table II). Five additional blood relatives and the spouse have thus far been studied (Table I). The proband's son has normal galactolipid beta galactosidase activity, but her daughter has levels at about 50% of normal. Moreover, one sister had leukocyte enzyme levels at less than 10% of normal, while another sister and their mother had normal values. These data indicate that our proband, with extremely low gal-cer and lac-cer beta

Volume 88 Number 1

Brief clinical and laboratory observations

77

32 LEIJKOCYTES 28

r-]Lactosyl

24

~Galactosyl

2o

ceramide

ceramide

12

8

CONTROLS

m

OBLIGATE #1

OBLIGATE #2

PROBAND I

PROBAND 1

repeat

hUSBAND of

PEOBAND

HIGHEST AFFECTED I

Fig. 1. Comparison of leukocyte homogenate beta galaetosidase activities using lac-cer or gal-cer as substrate. Proband I is easily distinguished from normals and two unrelated obligate heterozygotes for typical Krabbe disease.

galactosidase activity, is a heterozygote for Krabbe disease. The mutation in this family apparently involves the gene for the specific gal-cer or lac-cer beta galactosidase. The measured activities of other lysosomal enzymes, including synthetic substrate beta galactosidase, were normal. Mixing of homogenates of the proband's leukocytes or fibroblasts with leukocytes and fibroblast preparations from controls resulted in the expected decrease in control activity; there was no evidence of a soluble inhibitor in the proband's cells. In Family 2 the proband and his sister have typical Krabbe disease. He has only traces of gal-cer and lac-cer beta galactosidase in peripheral leukocytes. His father, an obligate heterozygote without symptoms, has only 10% of normal activity for leukocyte gal-cer beta gatactosidase and 6% of normal for leukocyte lac-cer beta galactosidase. A second leukocyte sample from a child with an undiagnosed degenerative disease accompanied the samples of Family 2, from Belgium, and showed normal results for gal-cer and lac-cer beta galactosidase. A third sample, from an unrelated female obligate heterozygote, was received at the same time; values for her gal-cer and laccer beta galactosidase were in the usual carrier range (1.45

and 11.6 nanamoles cleaved/mg protein/hour at 37 ~ C, respectively). Fibroblast cultures from Proband 2 and his parents gave the following mean values for gal-cer beta galactosidase activity (nanamoles cleaved/rag protein/ hour at 37 ~ C: proband -0.13, father- 1.48, mother-2.08) (see Table II, for control values), DISCUSSION The assay for the enzyme deficient in Krabbe disease is sensitive and specific. In a recent report we presented our conditions for the identification of patients and carriers of Krabbe disease/ Using peripheral blood leukocytes, all our known obligate carriers for Krabbe disease had galcer and lac-cer beta galactosidase activities between 4060% of our controls/Using cultured skin fibroblasts some obligate carriers have had as little as 20% of control values/ All of the affected (homozygous) children had enzyme activities of less than 10% of the controls. The heterozygous proband and one sister in Family 1 and the proband's heterozygous father in Family 2 had leukocyte values of both gal-cer and lac-cer beta galactosidase activity clearly under 10% of our control values. This was demonstrated on multiple blood samples. Other lysosomal enzymes, including 4-methylumbelliferyl-beta galac-

78

Brief clinical and laboratory observations

The Journal of Pediatrics January 1976

Table I. Leukocyte galactosyl ceramide and lactosyl ceramide beta galactosidase activity in Family 1

Substrate (nmoles/mg prot/ hr) Subjects Proband Proband's husband Proband's son Proband's daughter Proband's sister I Proband's sister II Proband's mother Controls (avg) SD

Gal-eer 0.45* 4.58 4.87 1.93" 4.32 0.18" 3.33 4.69 0.88

[ Lac-cer 2.40* 28.00 23.90 12.60" 21.30 2.23* 17.20 30.19 8.3

Enzyme activities in leukocyte homogenates expressed as nanamoles galactose released per mg proteinper hour at 37~ Heterozygotes and normalscan easilybe distinguishedper the gal-cerand lac-ceractivities; all showed normal activitywith 4-methylumbelliferyl-beta-galactosidase as substrate, All samples were processed in duplicate or triplicate, and printed valuesrepresentmeans. *Heterozygotes. Table II. Fibroblast galactosyl ceramide and lactosyl ceramide beta galactosidase activity

Substrate (nmoles/mg prot/hr) Subjects

Gal-cer

[ Lae-eer

Proband (Family 1) Highest affected Controls (avg) SD

0.85 0.64 4.48 _+ 1.60

2.29 2.26 16.22 _+ 5.65

Enzyme activities in fibroblast homogenates expressed as nanamoles galactose released per mg protein per hour at 37~ Proband 1 shows activityin the same range as that of our highest affected patient. tosidase, had normal activities. When a leukocyte preparation from each of these patients was mixed with an equal amount of control leukocyte preparation, the resultant gal-cer and lac-cer beta galactosidase activities were at about half-normal levels. Mixing of their respective leukocyte preparations with leukocyte preparations from patients with Krabbe disease resulted in little if any measurable activity. We conclude from these data that some heterozygotes for the Krabbe disease mutation have gal-cer and lac-cer beta galactosidase activities which overlap with those of affected homozygotes, and that the observed heterozygote deficiency is not due to a soluble inhibitor. It is not clear why Proband 1 and her daughter have such different heterozygote enzyme levels. It is unlikely that the daughter has a different mutant allele, since this

would entail her mother (the proband) being a compound heterozygote, which is incompatible with her son's normal enzyme levels. Consideration of all other possible explanations is beyond the scope of this paper, which is intended as a preliminary report demonstrating the need for caution in the interpretation of enzyme data for the diagnosis of Krabbe disease. Skin fibroblasts from the proband in Family 1 were cultured to demonstrate whether the low activity for gal-cer and lac-cer beta gatactosidase was restricted to the leukocytes. Her fibroblast gal-cer beta galactosidase activity was 19% of control activity (Table II). Two unrelated obligate carriers for Krabbe disease also had fibroblast gal-cer beta galactosidase activities which were 20% of those of our controls. 7 Although fibroblasts from patients with Krabbe disease have beta galaetosidase activities with both substrates under 10% of controls, differentiation of homozygotes and heterozygotes can still be difficult (cf. Proband 1 and Highest affected values, Table II). In Family 2 the proband is affected with Krabbe disease. His mother represents heterozygosity for the usual mutant allele, while his father is heterozygous for a different mutation, similar to that seen in Family 1. Thus, Family 2 again demonstrates that very low gal-cer and lac-cer beta galactosidase levels do not always indicate homozygosity. In addition this second family establishes a definite relationship of such a mutation to Krabbe disease. The most important immediate consequence of these considerations is the need for caution in situations in which enzyme activity would serve as the sole criterion for Krabbe disease, as in amniocentesis specimens2 The prenatal diagnosis of Krabbe disease reported in our previous publication was based on less than 2% activity compared to control cultured amniotic fluid cells} In cultured amniotic cells a level of activity for this enzyme at 10-15% of controls would present a problem, if one of the heterozygous parents were known to exhibit the very low activity we have demonstrated for the heterozygotes in the present two families. It thus becomes imperative to know the relative gal-cer and lac-cer beta galactosidase levels of the heterozygous parents before conclusions are drawn from the levels in cultured amniotic fluid cells. The same reasoning would apply to a famiIy with a child whose clinical course suggested Krabbe disease and whose leukocyte gal-cer beta galactosidase levels were found to be 10-15% of normal. Before it could be concluded that the child had Krabbe disease, the parents would have to be checked for their status in respect to their activities of gal-cer and lac-cer beta galactosidases, and the carrier status for each of them thereby documented.

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Brief clinical and laboratory observations

Similar concerns have been noted previously for a different disease and distinct enzyme deficiency. In 1973 Vidgoff and associates 1~reported a family which included a woman with a marked deficiency of hexosominidase A in all tissues studied. She actually represents the carrier state for Tay-Sachs disease, though her hexosominidase A level taken alone would have suggested that she was an affected homozygote. There are thus at least two heritable disorders for which there is overlap between the carrier and the affected person in terms o f enzyme activity. Based on these findings, and considering the greater availability and less strict indications for prenatal diagnostic amniocentesis, it is suggested that great care be exercised in the diagnosis of these diseases, particularly in the interpretation of data for intrauterine diagnosis. The data and conclusions discussed here were presented in preliminary form to the Society for Pediatric Research, Denver, April 18, 1975.11 The typing skills of Cheryl Jirschele and the excellent technical assistance of Martha Sattler and Cameron Clark are gratefully acknowledged. We also thank Dr. Jules Leroy for the samples from Family 2 and their controls. REFERENCES 1. Wilson J, Lake BD, and Dunn HG: Krabbe's leukodystrophy, some clinical, genetic and pathogenic considerations, J Neurol Sci. 10:563, 1970.

2. Suzuki K, and Suzuki Y: Globoid cell leukodystrophy (Krabbe's disease): Deficiency of galactocerebroside betagalactosidase, Proc Natl Acad Sci USA 66:302, 1970. 3. Eto Y, Suzuki K, and Suzuki K: Globoid cell leukodystrophy (Krabbe's disease): isolation of myelin with normal glycolipid composition, J Lipid Res 11:473, 1970. 4. Miyatake T, and Suzuki K: Globoid cell leukodystrophy: Additional deficiency of psychosine galactosidase, Biochem Biophys Res Commun 48:538, 1972. 5. Wenger DA, Sattler M, and Markey SP: Deficiency of monogalactosyl diglyceride beta-galactosidase activity in Krabbe's disease, Biochem Biophys Res Commun 53:680, 1973. 6. Wenger DA, Sattler M, and Hiatt W: Globoid cell leukodystrophy: deficiency of lactosyl ceramide beta-galactosidase, Proc Nail Acad Sci USA 71:854, 1974. 7. Wenger DA, Sattler M, Clark C, and McKelvey H: An improved method for the identification of patients and carriers of Krabbe's disease, Clin Chim Acta 56:199, 1974. 8. Suzuki Y, and Suzuki K: Krabbe's globoid cell leukodystrophy: deficiency of galactocerebrosidase in serum, leukocytes and fibroblasts, Science 171:73, 1971. 9. Atkins L, and Milunsky A: Prenatal diagnosis of genetic disorders, in Milunsky A, editor, The prevention of genetic disease and mental retardation, Philadelphia, 1975, WB Saunders Company, p 250. 10. VidgoffJ, Buist NRM, and O'Brien JS: Absence of beta-Nacetyl-D-hexosaminidase A activity in a healthy woman, Am J Hum Genet 25:372, 1973. 11. Wenger DA, and Riccardi VM: The possible false diagnosis of Krabbe disease, Pediatr Res 9:319, 1975 (abstr).

tioned? More recently N-acetyl-D, L-penicillamine has been shown to be an effective agent in mercury poisoningr All these chelating agents, however, are ultimately dependent upon renal excretion of the mercury chelate complex to reduce the mercury blood level. Therefore peritoneal dialysis is indicated to remove this complex when the patient is anuric. ~

Peritoneal dialysis in mercurial diuretic intoxication Jean E. Robillard, M.D.,* Linda K. Rames, M.D., Robert L. Jensen, A.B., and Robert J. Roberts, M.D., Ph.D., Iowa City, Iowa ALTHOUGH

THE A P P R O P R I A T E

TREATMENT

Abbreviation used BAL: dimercaprol pro-

c e d u r e s for mercury intoxication are still debated, dimer-

caprol has been employed successfully in cases of acute poisoning by mercury salts? Calcium ethylene diamine tretra-acetate has also been used for treatment of acute mercury intoxication'-", however, its efficacy was ques-

From the Department of Pediatrics, University Hospitals. *Reprint address: Department of Pediatrics, Division of Nephrology, Ste-dustine Hospital, 3175 Chemin SainteCatherine, Montrkal, P.Q., Canada H3T 1C5.

79

This case report involves a child with congenital heart disease who was admitted in acute renal failure following intoxication with a mercurial diuretic (mercaptomerin). Blood levels and peritoneal clearance of mercury together with tissue levels of mercury from autopsy material are reported. CASE REPORT Patient T. M., a 3-month-old white female who had, during the neonatal period, a Rashkind procedure for transposition of the