First prenatal diagnosis of acyl-CoA oxidase deficiency

J. Inher. Metab. Dis. 13 (1990) 371-374 © SSIEM and KluwerAcademicPublishers.Printed in the Netherlands

Short Communication

First Prenatal Diagnosis of Acyl-CoA Oxidase Deficiency R. J. A. WANDERS1, A. SCHELEN1, N. FELLER1, R. B. H. SCHUTGENS1, F. STELLAARD2, C. JAKOBS2, B. MITULLA3 and G. SEIDLITZ4 1Department of Paediatrics, University Hospital Amsterdam, Meibergdreef 9, 1105 A Z Amsterdam, The Netherlands; 2Department of Paediatrics, Free University of Amsterdam, De Boelelaan II 17, 1007 MB Amsterdam, The Netherlands; 3Bezirkkrankenhaus Suhl, Suhl, German Democratic Republic; 4Ernst-Moritz-Arndt Universitgtt, Greifswald, German Democratic Republic

In 1976 Lazarow and de Duve (1976) reported that peroxisomes from rat liver are capable of oxidizing fatty acids. The functional significance of a second fi-oxidation system next to that in mitochondria remained obscure for some time. In recent years, however, it has become clear that the peroxisomat system plays an indispensable role in fatty acid fl-oxidation, being involved in the fl-oxidation of a distinct set of compounds. Indeed, the first step(s) in the fl-oxidation of very long chain fatty acids (VLCFAs) take place virtually exclusively in peroxisomes. Furthermore, the floxidative chain-shortening of di- and trihydroxycholestanoyl-CoA, intermediates in the formation of chenodeoxycholic acid and cholic acid, respectively, occurs solely in peroxisomes (see Wanders et al., 1990 for review). The importance of the peroxisomal fl-oxidation system is stressed by tho identification of a growing number of inherited diseases in man in which peroxisomal floxidation is impaired. Apart from the peroxisome deficiency disorders (ZeUweger syndrome, neonatal adrenoteukodystrophy (ALD), infantile Refsum disease) in which peroxisomal fl-oxidation is impaired due to the absence of peroxisomes, these include: Zellweger-like syndrome (combined oxidase-bifunctional protein-thiolase and dihydroxyacetone phosphate acyltransferase deficiency), X-linked adrenoleukodystrophy (peroxisomal VLCFA-CoA synthetase deficiency), pseudoneonatal ALD (acylCoA oxidase deficiency), bifunctional protein deficiency and pseudo-Zellweger syndrome (peroxisomal thiolase deficiency). All these disorders are devastating diseases with severe neurological involvement and death within the first decade of life, which warrants prenatal diagnosis. In this report we describe the first successful prenatal diagnosis in a case of pseudoneonatal ALD in which plasmalogen biosynthesis was normal but peroxisomal fl-oxidation was impaired due to a deficiency of acyl-CoA oxidase. 371

372

Wanders et at.

MATERIALS AND METHODS

Analytical techniques De novo ptasmalogen biosynthesis, very long chain fatty acid levels and very long chain fatty acid fl-oxidation were all measured according to published procedures (Schutgens et al., 1989; Wanders et al., 1990). Very long chain fatty acids and the bile acids trihydroxycholestanoic acid and cholic acid were measured in amniotic fluid samples using stable isotope dilution methods (Stellaard et al., 1988; Jakobs et al., 1989). Immunoblotting was performed according to standard procedures using antisera raised against the peroxisomal r-oxidation enzyme proteins (acyl-CoA oxidase, bifunctional protein and 3-ketoacyt-CoA thiolase) as purified from rat liver (Wanders et al., 1990).

Case report Amniotic fluid was taken at the 20th week of gestation from a 28-year-old gravida. She had previously given birth to a child who died at the age of 3 years and 11 months with the presumed diagnosis of neonatal adrenoleukodystrophy. The parents were consanguineous. When examined at the age of 3 years and t0 months the patient showed generalized hypotonia, failure to thrive, deafness, hepatomegaly, psychomotor retardation, absent reflexes and frequent convulsions. At autopsy a number of abnormalities were found including atrophy of skeletal muscles, brainstem ganglions and cerebral nerves. Furthermore, in the brain and adrenals large PASpositive, sudanophilic astrocytes and macrophages were found. The liver was fibrotic. Based on these results the patient was presumed to suffer from neonatal ALD, although no biochemical investigations were carried out.

RESULTS AND DISCUSSIONS Prenatal diagnosis was requested by a family in which a previous child died at the age of 3 years and 11 months from neonatal adrenoleukodystrophy. Diagnosis, however, was based only on post m o r t e m autopsy findings (see case report) and was not confirmed biochemically. Neonatal ALD has been described in patients in whom peroxisomes are (virtually) absent, leading to the generalized loss of peroxisomal functions (Kelley et aI., 1986). However, a similar phenotype has recently been described in two patients in whom the defect was at the level of acyl-CoA oxidase, the first enzyme in peroxisomal r-oxidation. In these patients all other peroxisomal functions were found to be normal (Poll-The et al., 1988). For this reason we decided to study both plasmalogen biosynthesis and peroxisomal r-oxidation in this at risk pregnancy. The results summarized in Table 1 show that plasmalogen biosynthesis was normal in amniocytes. However, gas chromatographic analysis of VLCFAs revealed strongly elevated values. Furthermore, C26:0 r-oxidation activity was strongly deficient indicating a defect in peroxisomal r-oxidation. Using antibodies J. Inher. Metab. Dis. 13 (1990)

Prenatal Diagnosis of Acyl CoA Oxidase Deficiency

373

Table 1 Results of analyses in amnioeytes, amniotic fluid, cultured fetal fibroblasts and fetal liver in a patient at risk for (pseudo)neonatal adrenoleukodystrophy

Parameter Amniocytes Plasmalogen biosynthesis pPE in PE (%) pPC in PC (%) [3H]/[14C] ratio in: Alkenyl PE Alkenyl PC C26:0/C22:0 ratio C24:0/C22:0 ratio C26:0//-oxidation activity (pmol min- 1mg- 1) Immunoblotting Acyl-CoA oxidase Bifunctional protein Peroxisomal thiolase Amniotic fluid Very long chain fatty acids C26:0 level (nmol/L) C26:1 level (nmol/L) C26:0/C22:0 ratio C26:1/(222:0 ratio Bile acids Trihydroxycholestanoic acid (nmol/L) Cholic acid (nmol/L) Cultured (fetal) fibroblasts Ptasmalogen biosynthesis pPE in PE (%) pPC in PC (%) [3H]/[14C] in alkenyl PE [3H]/[14C] in alkenyl PC C26:0/C22:0 ratio C24:0/C22:0 ratio C26:0 fl-oxidation activity Fetal liver material Immunoblotting Acyl-CoA oxidase Bifunctional protein Peroxisomal thiolase

Controls

Zellweger patients

Index case

89.8 -+ 6.8 (21) 19.8 -+ 8.8 (22)

11.9-16.3 (3) 1.2-2.6 (3)

78,1 5.6

1.0 _+ 0.3 (16) 0.6 -+ 0.3 (16) 0.10 -+ 0.06 (8) 1.50 _+ 0.32 (16)

Not measured Not measured 0.53-0.94 (3) 2.76-3.23 (3)

1.0 0.6 1.06 3.16

1.8-3.8 (5)

0.05-0.30 (3)

0.13

+ + +

+ +

m

40-141 (20) 0-31 (20) 0.054-0.247 (20) 0~).077

112 134 0.181 ~216

330 275 0.42 0.34

0-0.80 (10) 170-600 (10)

69 130

0.36 100

85.0 _+4.2 (38) 7.3 5:2.6 (38) 1.5 _+0.9 (38) 1.1 _+ 0.7 (38) 0.01-0.09 (50) 1.30-2.09 (50) 3.0-7.5 (16)

21.0 _ 12,9 (19) 1.0 _ 0.4 (19) 75 _+98 (19) 7.5 +__5.3 (19) 0.68-2.09 (22) 1.66-3.03 (22) 0.01-0.38 (8)

88.3 4.5 0.4 0.4 0.35 2.05 0.46

+ + +

m

m

+ +

For experimentaldetails see Materials and Methods section directed against the three peroxisomal fl-oxidation enzyme proteins, it was subsequently found on immunoblotting that both the 72 kDa, 52 kDa and 20 kDa components of acyl-CoA oxidase were absent, whereas the 78 kDa bifunctional protein and the 41 kDa, mature form of peroxisomal thiolase were present normally. Interestingly, C26:0 and C26:1 levels were also strongly elevated in amniotic fluid of the at risk fetus. THCA-levels, however, were normal, in line with the current view that acylCoA oxidase is not involved in THCA fl-oxidation (Wanders et al., 1990).

J. lnher. Metab. Dis. 13 (1990)

374

Wanders et at.

The diagnosis of an isolated acyl-CoA oxidase deficiency was subsequently confirmed after abortion via analyses of fetal skin fibroblasts and fetal liver material (by immunoblotting) (see Table 1). In conclusion, this paper describes the first prenatal diagnosis of acyl-CoA oxidase deficiency in a pregnancy at risk for neonatal ALD. The results obtained stress the importance of doing multiple analyses in chorionic villi, chorionic villous fibroblasts or amniocytes, i.e. plasmalogen biosynthesis and peroxisomal fl-oxidation (Wanders et al., 1990).

REFERENCES

Jakobs, C., ten Brink, H., Kok, R. M., Stellaard, F, Kleijer, W. J., Wanders, R. J. A. and Schutgens, R. B. H. Very long chain fatty acids in amniotic fluid from a fetus affected with Zellweger syndrome. Eur. J. Pediatr. 148 (1989) 581 Kelley, R. I., Datta, N. S, Dobijns, W. S., Hajra, A. K., Moser, A. B., Noetzel, M.J., Zackai, E. H. and Moser, H. W. Neonatal adrenoleukodystrophy: new cases, biochemical studies and differentiation from Zellweger, and related polydystrophy syndromes. Am. J. Med. Genet. 23 (1986) 869-901 Lazarow, P. B. and de Duve, C. A fatty acyl-CoA oxidizing system in rat liver peroxisomes: enhancement by clofibrate, a hypolipidemic drug. Proc. Natl. Acad. Sci. USA 73 (1976) 2043-2046 Poll-The, B. T., Roels, F., Ogier, H., Scotto, J., Vamecq, J., Schutgens, R. B. H., Wanders, R. J. A., van Roermund, C. W. T., van Wijland, M. J. A., Schram, A. W., Tager, J. M. and Saudubray, J. M. A new peroxisomal disorder with enlarged peroxisomes and a specific deficiency of acyl-CoA oxidase (pseudo-neonatal adrenoleukodystrophy). Am. J. Hum. Genet. 42 (1988) 422-434 Schutgens, R. B. H., Schrakamp, G., Wanders, R. J. A., Heymans, H. S. A., Tager, J. M. and van den Bosch, H. Prenatal and perinatal diagnosis of peroxisomal disorders. J. Inher. Metab. Dis. 12 Suppl. 1 (1989) 118-134 Stellaard, F., Langelaar, S. A., Kok, R. M., Kleijer, W. J., Schutgens, R. B. H. and Jakobs, C. Prenatal diagnosis of Zellweger syndrome by determination of trihydroxychotestanoic acid in amniotic fluid. Eur. J. Pediatr. 148 (1988) 175 Wanders, R. J. A., van Roermund, C. W. T., Schutgens, R. B. H., Barth, P. G., Heymans, H. S. A., van den Bosch, H. and Tager, J. M. The inborn errors of peroxisomal fl-oxidation. J. Inher. Metab. Dis 13 (1990) 4-36

J. Inher. Metab, Dis. 13 (1990)