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Biochemical abnormalities in rhizomelic chondrodysplasia punctata
Biochemical abnormalities in rhizomelic chondrodysplasia punctata G e r a l d Hoefler, MD, Sigrid Hoefler, MD, Paul A. Watkins, MD, PhD, Winston W. C h e n , PhD, A n n Moser, BA, Virginia Baldwin, MD, B. McGillivary, MD, Joel C h a r r o w , MD, J. M. Friedman, MO, L a n e R u t l e d g e , MD, Takashi Hashimoto, PhD, a n d Hugo W. Moser, MD From The Kennedy Institute and Department of Neurology, Johns Hopkins University, Baltimore; British Columbia Children's Hospital, and Department of Pathology and Genetics, University of British Columbia, Vancouver; Division of Genetics, Children's Memorial Hospital, Department of Pediatrics, Northwestern University Medical School, Chicago; and Department of Biochemistry, Shinshu University School of Medicine, Asahi, Japan
Biochemical studies with emphasis on peroxisomal functions were conducted in six patients with well-documented rhizomelic chondrodysplasia punctata (RCDP) and c o m p a r e d with findings in patients with Zellweger syndrome and neonatal adrenoleukodystrophy (ALD). Patients with RCDP had three characteristic biochemical abnormalities: (I) profound defect in plasmalogen (ether lipid) synthesis, which is significantly greater than the analogous defect in Zellweger syndrome or neonatal ALD; (2) reduction of phytanic acid oxidation activity to I% to 5% of control, similar to that observed in Refsum disease, Zellweger syndrome, and neonatal ALD; (3) presence of the unprocessed form of peroxisomal 3-oxoacyl-coenzyme A thiolase in the postmortem liver of two patients. Other peroxisomal functions were normal, Including levels of very long chain fatty acids, pipecolic acid, and bile acid intermediates, and immunoblot studies of peroxlsomal acyl-CoA oxidase and bifunctional enzyme in postmortem liver. Unlike what is observed in Zellweger syndrome and neonatal ALD, catalase activity in cultured skin fibroblasts was sedimentable, indicating that peroxisome structure is not grossly deficient in RCDP. The biochemical abnormaltles in RCDP were consistent and set it apart from all the other known peroxisomal disorders. (J PEDIATR1988;112:726-33)
The term chondrodysplasia punctata is applied to puncrate epiphyseal and extraepiphyseal calcifications on roentgenograms of infants. First described by Conradi in 1914,1 the finding is nonspecific. It can result from maternal use of warfarin during pregnancy,2 and is observed in Zellweger cerebrohepatorenaI syndrome3 and other syndromes.
The majority of cases are genetically determined. These patients were originally referred to as having Conradi-
Supported in part by Grant HD 10981 from the U.S. Public Health Service. Dr. Gerald Hoefler supported by Fonds zur Foerderung der Wissenschaftlichen Forschung, Project No. J0145M. Dr. Sigrid Hoefler supported by a Fulbright Scholarship. Submitted for publication July 30, 1987; accepted Nov. 2, 1987. Reprint requests: Hugo W. Moser, MD, The Kennedy Institute, 707 N. Broadway, Baltimore, MD 21205.
Hunermann syndrome, but several distinct genetic disorders are now associated with chondrodysplasia punctata, and differ in respect to clinical manifestations and mode of inheritance. In 1971 Spranger et al." delineated the rhizomelic form of chondrodysplasia punctata. This form has an autosomal recessive mode of inheritance, associated with proximal shortening of the extremities, cataracts
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ALD CoA RCDP
Adrenoleukodystrophy CoenzymeA RhizomelicchondrodyspIasia punctata
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Table I. Biochemical findings in RCDP Patients with RCDP Control
Age at time of test Sex Fibroblast plasmalogen
1
2
3
4
5
6
2 mo M 36.5
12 mo F 29.2
10 days F 31.8
10 mo M 14.6
12 mo F 58.2
16 mo M 22.7
100 (10)
1.8
1.7
1.5
3.5
0.5
1.3
3 (50)
14.1
65.0
ND
41.3
24.0
4.0
90 _+ 3 (5)
91.0
81.0
97.0
83.0
87.0
95.0
100 (8)
102.0
152.0
73.0
130.0
54.0
118.0
0.62 _+ 0.16 (28)
synthesis (3H/146) Fibroblast phytanic acid oxidation (% control) Plasma phytanic acid (ug/mL) Catalase distribution (% sedimentable) Fibroblast VLCFA oxidation (% control) Fibroblast C26 :0 (#g/rag protein) Fibroblast C26:0/C22: 0 Plasma C26:0 (#g/mL) Plasma THCA (~mol/L) Plasma pipecolie acid (umol/L)
0.07 _+ 0.04 (525)
0.12
0.031
0.044
0.097
0.085
0.052
0.08 _+ 0.03 (525) 0.33 _+ 0.18 (1900) 0.1 (3) 4 (16)
0.089 0.18 0.1 2.3
0.092 0.23 ND 1
0.031 ND ND ND
0.24 0.36 0.1 1
0.096 0.29 ND 1.3
0.27 0.45 0.1 ND
Comparisonof biochemicalfindingsin six patientswithRCDPand controlvalues.Analysescarriedout as described(see Methods)and are givenas meanof two separate determinations;numberof controlsubjectsin parentheses. VLCFA, very longchainfatty acid; ND, not determined.
(72%), skin changes, and psychomotor retardation, and usually causes death before the end of the first year of life. The remaining patients assigned to the Conradi-Hunermann category are more mildly affected; often survive to adulthood, with normal or only mildly impaired mental function; and have a lower incidence of cataracts (17%). The mode of inheritance for these milder cases often is autosomal dominant.4 In 1979 Happle 5 reported an Xlinked dominant form of chondrodysplasia punctata, the severity of which is intermediate between the rhizomelic and autosomal dominant forms; cataracts and limb involvement are often asymmetric, and skin abnormalities show a linear and blotchy pattern, suggesting lyonization. In 1984 Curry et al. 6 reported two families in which chondrodysplasia punctata was associated with facial dysmorphism, short stature, and mild mental retardation; deletion of the terminal short arm of the X-chromosome; and deficient activity of steroid sulfatase. The pattern of inheritance in these families was X-linked recessive. Recent studies by Heymans et al. 7 have led to a significant advance in our understanding of RCDP, namely the assignment of this disorder to the peroxisomal disease category. These investigators had recognized points of resemblance between RCDP and Zellweger syndrome, and demonstrated deficient enzyme activity in plasmalogen synthesis and elevated plasma phytanic acid levels in RCDP. They concluded that the disorder involved the
peroxisome. 7 However, the number of peroxisomes does not appear to be diminished, and very long chain fatty acid metabolism is not impaired. Thus it is distinguishable from Zellweger syndrome, a conclusion confirmed by complementation studies? During the last 3 years a panel of biochemical assays has been developed that permits assessment of peroxisomal functionY We have applied these assays to the study of six patients with RCDP and compared them with alterations observed in other peroxisomal disorders. RCDP demonstrates consistent biochemical abnormalities that differ from those in all Of the other peroxisomal disorders. These characteristic abnormalities permit precise prenatal and postnatal diagnosis and provide new insights about pathogenic mechanisms. METHODS The six patients with RCDP were unrelated and had the generally accepted diagnostic criteria, 4 including proximal shortening of limbs, joint contractures, dysmorphic features, cataracts, and psychomotor retardation. R0entgenograms showedthe characteristic chondrodysplasia punctata lesions and coronal clefts of vertebral bodies. Patients 1, 2, and 3 died at 21/2 months, 14 months, and 2 weeks, respective!y, and the diagnosis was confirmed by portmortern examination. Patients 4, 5, and 6 were in stable condition at ages 9 to 16 months.
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Hoefler et al.
The Journal of Pediatrics May 1988
T a b l e II. Plasmalogen synthesis in cultured fibroblasts Cell line
No. of patients
3H/14C
SD
P"
Control Neonatal adrenoleukodystrophy Zellweger syndrome RCDP
28 10 6 6
0.62 4.66 10.40 32.19
0.16 1.63 2.85 21.31
0.0001 0.0001 0.003
Plasmalogen synthesis measurements carried out as described (see Methods). Values expressed as ratio of pH] to [~4C] incorporated into plasmalogens. *Values assessed using Student t test.
T a b l e III. Comparison of biochemical abnormalities in certain peroxisomal disorders
RCDP Fibroblast phytanic acid oxidation (% control) Plasma phytanic acid (patients with levels >4 /~g/mL) Catalase distribution (% sedimentable) Fibroblast VLCFA oxidation (% control) Fibroblast C26 : 0 (#g/rag protein) Fibroblast C26:0/C22:0 Plasma C26 : 0 (~g/mL) Plasma THCA (/zmol/L) Plasma pipecolic acid (#mol/L)
1.7 _+ 0.9 (6)
Adrenoleukodystrophy
Zellweger syndrome
Neonatal
1.4 +_+_1.3 (11)
4.6 __+4.8 (5)
X-linked
Adult Refsum disease
Control
98 _+ 13 (3) 0.5 _+ 0.5 (2)
3/3
12/26
11/18
0/10
89 +_ 6 (6)
10 +_ 7 (21)
13 +_ 8 (16)
91 +_ 5 (9)
105 _+ 33 (6)
10 (3)
4.6 -+ 1.6 (10)
16.0 _+ 4.4 (6)
ND
100 (8)
0.076 _+ 0.034 (5) 0.53 +_ 0.28 (37)
0.40 _+ 1.5 (26)
2.41 _+ 0.15 (28)
ND
0.07 + 0.04 (525)
1.05 _+ 0.46 (18)
0.69 + 0.19 (28)
0.07 (1)
0.08 + 0.03 (525)
0.320 _+ 0.112 (4) 3.06 _+ 1.23 (40) 2.14 + 0.71 (27)
1.62 +_ 0.87 (282)
0.15 +_ 0.09 (5)
1.34 + 0.72 (16)
0.1 (3)
11.4 + 5.2 (5)
10.9 _+ 14.6 (11)
1.4 _+ 0.54 (6)
80 _+ 79 (5)
132 +__77 (5)
4/4
100 (10)
89 +_ 2 (3)
0/50
90 + 3 (5)
0.2 (1) 0.33 +_ 0.18 (1900)
ND
ND
4 (5)
ND
0.1 4 (16)
Comparison of biochemical findings in patients with RCDP and other peroxisomal diseases. Values are given as mean _+SD; number of subjects in pa~rentheses. VLCFA, very long chain fatty'acid; ND, not determined.
Experimental procedures. [1-14C]phytanic acid (55 m C i / m m o l ) , [1-14C]hexadecan01 (11.4 m C i / m m o l ) , and [125I]i0dine (30 m C i / m L ) were purchased from Amersham Corp., Arlington Heights, Illinois; [ 1-14C]palmitic acid (58 m C i / m m o l ) from N e w England Nuclear, North Billerica, Massachusetts; and [ 1-~4C]lignoceric acid (47 mCi/rnmol) from Research Products International. [9',10'-3I-I]-sn hexadecylglycerol (5.5 m C i / m m o l ) was kindly provided by F. Paltauf, Technical University of Graz, Austria. Cell culture reagents were from Gibco Laboratories, Grand Island, New York. Nitrocellulose filters were from Schleicher & Schuell Inc., Keene, New Hampshire. L K 5 D Linear-K silica gel thin-layer chromatography plates (250 ~m thickness) were from W h a t m a n Chemical Separation
Inc., Clifton, New Jersey. All other reagents were of analytical grade and obtained from commercial sources. Protein A was labeled with lZSIaccording to the procedure of Greenwood et al. l~ Antibodies against purified rat liver peroxisomal acyl-CoA oxidase, bifunctiona! enzyme, and 3-0xoacyl-CoA thiolase were prepared as described previously? 1 Skin fibroblasts were maintained in culture as previously described? 2 C a t a l a s e activity was assayed by the method of Peters et al? s The subcellular distribution of catalase was measured as previously described? 4 Levels of very long chain fatty acids in fibroblasts and plasma were measured as previously described, 12,~5 trihydroxy coprostanic acid by the method of Bjorkhem and Falk, 16 and pipecolic acid as described by Van den Berg et al. 17Protein
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729
Fig. 1. !mmunoblot analysis of peroxisomal 3-oxoacyl-CoA thiolase. Total homogenates of postmortem liver samples were prepared, subjected to sodium dodecylsulfate/polyacrylamide gel electrophoresis, and blotted onto nitrocellulose membranes as described. After incubation with antibody specific for peroxisomal 3-oxoacyl-CoA thiolase, bound ~mmunoglobulins were detected by ~2~I-labeledprote!n A binding and autoradiography. Lane C, liver sample from control individual;Z, patient ~yith Ze!lweger syndrome; R1 and R2, RCDP; X, X-linked adrenoleukodystrophy. Amounts Of sample protein applied to gel are approximately 0. I mg per gel lane. Immunoreacti,)e band appearing at approximately 47 kilodaltons is nonspecific and thought to be unrelated to thiolase. Positions Of molecular size markers are indicated on left. Note that in RCDP and Zellweger syndrome, moleculhr mass of thiolase is 2 to 3 kilodaltons larger than in Control Or X-linked adren01eukodyst rophy.
Mol. Wt. .............
941(
--
67K
""
7~
' ? N
45 K 36K =zg
|
m
C
Z
RI
R2
X
~
C
Z
RI
R2
X
Fig. 2. Immunoblot'analysisof peroxisomal acyl-CoA oxidase (A) and bifunctional enzyme (B). Nitrocelluiose membranes were prepared as described (see legend, Fig. 1) and subsequently incubated with antibody either specific for peroxisomal acyl-CoA oxidase or bifunctional enzyme: detection of immunoreactive proteins was as in Fig, 1~Lane C, liver sample from control individual; Z, patient with Zellweger syndrome; R1 and R2, RCDP; X, X-linked adrenoleukod~,strophy.
concentration was determined by the method of Lowry et al. TM Immunnblot analysis. Liver samples, stored at - 7 0 ~ C, were allowed to thaw in 62.5 m m o l / L Tris-HC1 (pH 6.8), 1 m m o l / L phenazinemethylsulfonyl fluoride, 1% sodium dodecyl sulfate containing 50 m m o l / L dithiothreitol (50
mg tissue per milliliter), and then homogenized using a Ten Broeck tissue grinder followed by sonication (three pulses of 15 seconds each~at 50 W; (Heat Systems Ultrasonics Inc., Farmingdal~, N.Y.). After heating for 5 minutes at 70 ~ C, samples were centrifuged for 5 minutes in a Fisher microcentrifuge (Fisher Scientific Co., Pitts-
730
Hoefler et al.
The Journal of Pediatrics May 1988
Table IV. Summary of biochemical abnormalities in peroxisomal disorders that involve lipid metabolism
RCDP VLCFA levels Pipecolic ~tcid Bile acid intermediates Phytanic acid oxidation (% control) Plasmalogen biosynthesis Catalase activity (% sedimentable)
Normal Normal Normal 1%-5% Reduced 85%
Zeliweger syndrome
Neonatal
X-linked
Thiolase deficiency (pseudo-zellweger syndrome)*
+++ + § 1%-5% Reduced 10%
+++ + ++ 1%-5% Reduced 15%
++ Normal Normal Normal Normal 90%
+++ Normal ++ ND Normal ND
Adrenoleukodystrophy
+ to + + + , Degree to which metabolite levels are increased. VLCFA, very long chain fatty acid; ND, not determined. *Data from Schram AW, Goldfischer S, Van Roermond CWT, et al (Proc Natl Acad Sci USA 1987;84:2494-6) and Goldfischer SL, Collins J, Rapin I, et al (J PEDIATR 1986;108:25-32). ~Data from Poll-The BT, Ogier H, Saudubray JM, et al (Abstracts of the Society for the Study of Inborn Errors of Metabolism, Amersfoort, 1986:109).
burgh) and subjected to electrophoresis on 10% acrylamide gels according to the method of Laemmli? 9 Each gel lane contained protein corresponding to 1.25 mg (wet weight) liver. Immunoblot analyses were performed as described previously." Phytanic acid oxidation. Alpha-oxidation of [ l-~4C]phytanic acid by cultured skin fibroblasts was measured by ~4CO2 production. [1-~4C]phytanic acid (90 gCi) in 2 mL dry benzene was mixed with 500/xL 5 mmol/L NHaOH in ethanol, and the solution was incubated at 37 ~ C for 2 hours. The sample was evaporated to dryness by a stream of N2 gas and placed in an evacuated desiccator over PzO5 for 2 hours. Ammonium [~4C]phytanate was dissolved in 300 gL ethanol and mixed with 3 mL fetal bovine serum. When cultured fibroblasts reached confluence, the growth medium was replaced with 1 mL fresh growth medium containing 0.3 uCi ammonium [~4C] phytanate in 1% fetal bovine serum, and the cells were incubated at 37 ~ C for an additional 48 hours. Flasks were fitted with serum stoppers and center wells containing KOH-soaked glass fiber filter paper. After acidification of the medium with 0.5 mL 3N H2SO4, incubation was continued overnight at 37 ~ C, and i4coz trapped on filters was quantitated using a tolueneethanol scintillation fluid?~ Plasmalogen biosynthesis. Incorporation of [1-~4C]hexa decanol and [9",lO'-3H]-sn-hexadecylglycerol into the plasmalogen fraction of cultured human fibroblasts was determined according to the method of Roscher et al. 21 This method utilizes a double-label, double-substrate incubation. The peroxisomal component of plasmalogen biosynthesis is measured by the incorporation of [!-14C]hexa decanol into plasmalogens and compared with the microsomal component, assessed by the rate of incorporation of [9',10'-3H]hexadecylglycerol into plasma!ogens. Results
are expressed as the ratio of [3H] to [~4C] incorporation. An abnormally high ratio is observed when peroxisomal plasmalogen synthesis is deficient. The method was modified slightly: LK5D Linear-K silica gel thin-layer chromatography plates (Whatman) were used; they were developed twice with diethYlether/H20 (100:1 vol/vol), exposed to HC1 fumes, and then developed with light petroleum/diethylether (95:5 vol/vol) to a distance of 10 cm.
Fatty acid oxidation. Oxidation of [1-~4C]palmitic acid or [1-~4C]lignoceric acid to water-soluble products by cell suspensions was measured at pH 8.0 as described previously,~2 with the following modifications: After harvesting at confluence by trypsinization, cultured skin fibroblasts were suspended in 0.25 m o l / L sucrose containing 10 mmol/L Tris (C1-), pH 8.0, and 40 # g / m L digitonin. After incubation, reactions were terminated by the addition of 100 ~zL 1 N KOH, and the tubes were heated to 60 ~ C for1 hour. Ice-cold 18% HC104 (0.3 mL) was then added, and samples were kept at 4 ~ C for several hours. The precipitate was removed by centrifugation, and the supernatant was then partitioned By the method of Folch et al. z3 Radioactivity in the upper phase was determined by liquid scintillation spectrometry. The ratio of oxidation of lignoceric acid (C24:0) to palmitic acid (C16:0) provides a measure of very long chain fatty acid oxidation. RESULTS
Biochemical assays in plasma and cultured skin fibroblasts. Table I lists the results of the panel of biochemical assays that have been developed to assess peroxisomal structure and function. These assays measure biochemical reactions that are either carried out in the peroxisome or are affected in known peroxisomal disorders such as
Volume 112 Number 5
Rhizomelic chondrodysplasia punctata
Refsum disease
Acyl-CoA oxidase deficlencyt
Infantile
Adult
++ Normal Normal Normal Normal 90%
++ + ++ 1%-5% Reduced 10%
Normal Normal Normal 1%-5% Normal 85%
Zellweger syndrome? Tables II and III compare results in RCDP with those in other peroxisomal disorders. Plasmalogen synthesis. Peroxisomal plasmalogen synthesis was deficient in all patients with RCDP (Table I). The defect in RCDP was significantly more severe than in Zellweger syndrome or neonatal ALD (Table I1). Phytanie acid oxidation. As previously reported by Heymans et al., 7 phytanic acid levels were usually, but not invariably, elevated. Phytanic acid oxidation in cultured skin fibroblasts of patients with RCDP was reduced to the same extent as in adult Refsum disease, infantile Refsum disease, and Zellweger syndrome (Tables I and liD. Other measures of peroxisomal function and structure. Levels of very long chain fatty acids, pipecolic acid, and bile acid intermediates and very long chain fatty acid oxidation were normal in the patients with RCDP, indicating that the metabolism of these substances is not impaired in this disorder. The subcellular distribution of catalase in cultured skin fibroblasts was primarily sedimentable, as it is in normal cells, but differing sharply from the results in Zellweger syndrome and neonatal ALD. Immunoblot analysis of peroxisomal beta oxidation enzymes in postmortem liver tissue. Figs. 1 and 2 compare results in portmortem liver tissue of patients with RCDP with those in Zellweger syndrome and control specimens. The findings in RCDP are distinct and characteristic. The amount of protein reacting with the 3-oxoacyl-CoA thiolase antibody was present in normal quantity, but the molecular mass of this enzyme was apparently 2 to 3 kilodaltons larger than control values (Fig. 1). Unlike the findings in Zellweger syndrome and neonatal ALD, acylCoA oxidase and bifunctional enzyme were present in normal amounts (Fig. 2). DISCUSSION
Biochemical diagnosis of RCDP. There is increasing awareness that the phenotype of peroxisomal disorders covers a wide spectrum. Complementation studies indicate
731
that there are distinct genotypes, s The use of a panel of biochemical probes of peroxisomal function offers the opportunity to clarify the diagnosis and classifications of peroxisomal disorders. Application of these probes permits RCDP to be distinguished from all of the other known peroxisomal disorders (Table IV). These tests can be performed with accessible tissues, such as plasma and cultured skin fibroblasts, and are of general value for the diagnosis and categorization of patients who have or are suspected to have a peroxisomal disorder. These procedures have been used successfully for the prenatal diagnosis of peroxisomal disorders. 24 The profound defect of plasmalogen synthesis in RCDP should permit prenatal diagnosis utilizing either chorion villus samples or cultured amniocytes. We have monitored four pregnancies at risk for RCDP, and have identified one affected fetus by measuring plasmalogen synthesis and phytanic acid oxidase in a cultured chorion villus sample (Hoefler S, unpublished observation). Defect in plasmaiogen synthesis. The most striking biochemical defect in RCDP is the profound impairment of plasmalogen synthesis. Defective plasmalogen synthesis has been previously demonstrated in RCDP 25'26 and in Zellweger syndrome, 27,2s but the sensitive assay2~ used in our studies has permitted us to demonstrate for the first time that this defect is significantly more severe in RCDP. Webber et al. 29 have shown that the defective plasmalogen synthesis in Zellweger syndrome is related mainly to defective activity of DHAP acyltransferase, and that this in turn is related to low concentration of active enzyme molecules rather than a defect in the structure of the protein molecule itself. It is postulated that in the absence of peroxisomes the active enzyme is abnormally unstable. Analogous studies with RCDP will be of interest. Plasmalogens are constituents of virtually all membranes, 3~ but their biologic role has not been determined. Inasmuch as chondrodysplasia punctata is a feature in both RCDP and Zellweger syndrome, it seems plausible that the bone lesion is related to this biochemical defect. In neonatal ALD, plasmalogen synthesis is much less defective than in Zellwcger syndrome or RCDP, and chondrodysplasia punctata is not observed in neonatal A L D ) 1 RCDP as sole example of group 2 peroxisomal disorders. Previous classifications of peroxisomal disorders have subdivided them into three general categories. 32 In the group 1 disorders, which include Zellweger syndrome and neonatal ALD, the structure of the organelle is deficient or even lacking, and it is generally accepted that this is the cause of the multiple peroxisomal enzyme defects associated with these disorders. The group 3 disorders include X-linked ALD, hyperoxaluria type I, ~3 acyl-CoA oxidase deficiency, 34 3-oxoacyl-CoA thiolase deficiency,3~ and possibly
732
Hoefler et al.
adult Refsum disease. Peroxisome structure is normal in group 3 disorders, and it is presumed that they represent mutations that involve a single peroxisomal enzyme. The greatest conceptual difficulty has applied to the group 2 peroxisomal disorders, which are defined as disorders in which the activity of more than one peroxisomal enzyme is deficient but peroxisomal structure is intact22 To this category were assigned pseudo-Zellweger syndrome, a name coined by Goldfischer et al., 36 and RCDP. Pseudo-Zellweger syndrome is caused by 3-oxoacyl-coA thiolase deficiency, and this single enzyme defect may account for both the accumulation of very long chain fatty acids and bile acid intermediates25 This new knowledge obviates the need for the term "pseudo-Zellweger syndrome" and may reassign this disorder to category 3.
Defects in phytanie acid oxidation and processing of thiolase. A t this time, then, R C D P may be the only example of a group 2 peroxisomal disorder. There is convincing evidence for two enzyme defects, namely, peroxisomal plasmalogen synthesis and oxidation of phytanic acid. It is very unlikely that these two reactions can be traced to a single enzyme defect. The subcellular localization of phytanic acid oxidase is still uncertain. It has been postulated to be peroxisomal because defective phytanic acid oxidation is a constant feature in patients with group 1 peroxisomal disorders. In spite of this observation, the most recent subcellular fractionation studies suggest that phytanic acid oxidase is a mitochondrial enzyme. 37 The basis for the existence of these two enzymatic defects in R C D P requires additional study. The demonstration that the peroxisomal thiolase in the postmortem liver tissue of two R C D P patients existed in the unprocessed form 38 (Fig. 1) was unexpected. Up to now, this enzyme had been found to be present in this form only in the group 1 peroxisomal disorders, 11,39 It had been concluded that processing of the enzyme took place in the peroxisome and that this would fail to occur when the peroxisome structure was absent or deficient. This formulation does not appear applicable in R C D P , because of our observation that catalase in R C D P fibroblasts is in the particulate fraction (Table III), a finding that suggests that peroxisome structure is preserved, 4~ and the microscopic observation by Heymans et al. 26 that peroxisomes are present. The observation that very long chain fatty acids levels are normal suggests that the protein is catalytically active, at least to some extent. Heymans et al. reported the intriguing finding that in the liver tissue of one R C D P patient, "some hepatocytes lacked peroxisomes, while other cells displayed an increased number of exceptionally large and irregular shaped peroxisomes." Further study is required to unravel the pathogenesis of these apparently unrelated biochemical defects and their relationship to the phenotype of RCDP.
The Journal of Pediatrics May 1988 We thank Dr. Richard L. Kelley for performing the pipecolic acid assays, and Dr. Ingemar Bjorkhem for measuring levels of bile acid intermediates; and Dr. Fritz Paltauf for his generous gift. of the tritium labeled sn-hexadecylglycerol.
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28.
29.
measurement with the Folin phenol reagent. J Biol Chem 1951;193:265-75. Laemmli UK. Cleavage of structural protein during the assembly of the head of bacteriophage T4. Nature 1970; 227:680-5. Murad S, Kishimoto Y. Alpha hydroxylation of lignoceric acid to cerebronic acid during brain development. J Biol Chem 1975;250:5841-6. Roscher A, Molzer B, Bernheimer H, Stockier S, Mutz I, Paltauf F. The cerebro-hepato-renal (Zellweger) syndrome: an improved method for the biochemical diagnosis and its potential for prenatal diagnosis. Pediatr Res 1985;19:930-3. Singh I, Moser AB, Moser HW, Kishimoto Y. Adrenoleukodystrophy: impaired oxidation of very long chain fatty acids in white blood cells, cultured skin fibroblasts and amniocytes. Pediatr Res 1984;18:286-9. Folch J, Lees M, Sloane-Stanley GH. A simple method for the isolation and purification of total lipids from animal tissues. J Biol Chem 1957;226:457-509. Hajra AK, Datta NS, Jackson LG, et al. Prenatal diagnosis of Zellweger cerebro-hepato-renal syndrome. N Engl J Med 1985;312:445-6. Holmes RD, Wilson GN, Hajra AK. Peroxisomal enzyme deficiency in the Conradi-Hunerman form of chondrodysplasia punctata. N Engl J Med 1987;316:1608. Heymans HSA, Oorthuys JWE, Nelck G, Wanders RJA, Dingemans KP, Schutgens RBH. Peroxisomal abnormalities in rhizomelic chondrodysplasia punctata. J Inher Metab Dis 1986;9(Suppl 2):329-31. Datta NS, Wilson GN, Hajra AK. Deficiency of enzymes catalyzing the biosynthesis of glycerol-ether lipids in Zellweger syndrome: a new category of metabolic disease involving the absence of peroxisomes. N Engl J Med 1984;311: 1080-3. Schutgens RBH, Romeyn G J, Wanders RJA, Van den Bosch H, Schrakamp G, Heymans HSA. Deficiency of acyl-CoA: dihydroxyacetone phosphate acyltransferase in patients with Zellweger (cerebro-hepato-renal) syndrome. Biochem Biophys Res Commun 1984;120:179-84. Webber KO, Datta NS, Hajra AK. Properties of the enzymes
Rhizomelic chondrodysplasia punctata
30. 31.
32. 33.
34.
351
36.
37.
38.
39.
40.
733
catalyzing the biosynthesis of lysophosphatidate and its ether analog in cultured fibroblasts from Zellweger syndrome patients and normal controls. Arch Biochem Biophys 1987; 254:611-20. Mangold HK, Paltauf F. Ether lipids, biochemical and biomedical aspects. New York: Academic Press, 1983. Kelley RI, Datta NS, Dobyns WB, et al. Neonatal adrenoleukodystrophy: new cases, biochemical studies, and differentiation from Zellweger and related peroxisomal polydystrophy syndromes. Am J Med Genet 1986;23:869-901. Moser HW. Peroxisomal disorders [Editorial]. J PEDIATR 1986;108:89-91. Danpure C J, Jennings PR, Watts RWE. Enzymological diagnosis of primary byperoxaluria type I by measurement of hepatic alanine: glyoxylate aminotransferase activity. Lancet 1987;1:289-91. Poll-The BT, Ogier H, Saudubray JM, et al. A peculiar peroxisomal disorder: two siblings diagnosed previously as having neonatal adrenoleukodystrophy. Abstracts of the Society for the Study of Inborn errors of Metabolism, Amersfoort, 1986:109. Schram AW, Goldfischer S, Van Roermond CWT, et al. Human peroxisomal 3-oxoacyl-coenzyme A thiolase deficiency. Proc Natl Acad Sci USA 1987;84:2494-6. Goldfischer SL, Collins J, Rapin I, et al. Pseudo-Zellweger syndrome: deficiencies in several peroxisomal oxidative activities. J PEDIATR 1986;108:25-32. Skjeldal D, Stokke O. The subcellular localization of phytanic oxidase. 24th Annual Symposium Society for the Study of Inborn Errors of Metabolism, Amersfoort, 1986. Miura S, Mori M, Takiguchi M, et al. Biosynthesis and intracellular transport of enzymes of peroxisomal beta oxidation. J Biol Chem 1984;259:6397-6402. Tager JM, Ten Harmsen Van den Beck WA, Wanders RJA, et al. Peroxisomal beta-oxidation enzyme proteins in the Zellweger syndrome. Biochem Biophys Res Commun 1985; 126:1269-1275. Goldfischer S, Collins J, Rapin I, et al. Peroxisomal defects in neonatal-onset and X-linked adrenoleukodystrophy. Science 1985;227:67-70.
Biochemical studies with emphasis on peroxisomal functions were conducted in six patients with well-documented rhizomelic chondrodysplasia punctata (RCDP) and c o m p a r e d with findings in patients with Zellweger syndrome and neonatal adrenoleukodystrophy (ALD). Patients with RCDP had three characteristic biochemical abnormalities: (I) profound defect in plasmalogen (ether lipid) synthesis, which is significantly greater than the analogous defect in Zellweger syndrome or neonatal ALD; (2) reduction of phytanic acid oxidation activity to I% to 5% of control, similar to that observed in Refsum disease, Zellweger syndrome, and neonatal ALD; (3) presence of the unprocessed form of peroxisomal 3-oxoacyl-coenzyme A thiolase in the postmortem liver of two patients. Other peroxisomal functions were normal, Including levels of very long chain fatty acids, pipecolic acid, and bile acid intermediates, and immunoblot studies of peroxlsomal acyl-CoA oxidase and bifunctional enzyme in postmortem liver. Unlike what is observed in Zellweger syndrome and neonatal ALD, catalase activity in cultured skin fibroblasts was sedimentable, indicating that peroxisome structure is not grossly deficient in RCDP. The biochemical abnormaltles in RCDP were consistent and set it apart from all the other known peroxisomal disorders. (J PEDIATR1988;112:726-33)
The term chondrodysplasia punctata is applied to puncrate epiphyseal and extraepiphyseal calcifications on roentgenograms of infants. First described by Conradi in 1914,1 the finding is nonspecific. It can result from maternal use of warfarin during pregnancy,2 and is observed in Zellweger cerebrohepatorenaI syndrome3 and other syndromes.
The majority of cases are genetically determined. These patients were originally referred to as having Conradi-
Supported in part by Grant HD 10981 from the U.S. Public Health Service. Dr. Gerald Hoefler supported by Fonds zur Foerderung der Wissenschaftlichen Forschung, Project No. J0145M. Dr. Sigrid Hoefler supported by a Fulbright Scholarship. Submitted for publication July 30, 1987; accepted Nov. 2, 1987. Reprint requests: Hugo W. Moser, MD, The Kennedy Institute, 707 N. Broadway, Baltimore, MD 21205.
Hunermann syndrome, but several distinct genetic disorders are now associated with chondrodysplasia punctata, and differ in respect to clinical manifestations and mode of inheritance. In 1971 Spranger et al." delineated the rhizomelic form of chondrodysplasia punctata. This form has an autosomal recessive mode of inheritance, associated with proximal shortening of the extremities, cataracts
726
ALD CoA RCDP
Adrenoleukodystrophy CoenzymeA RhizomelicchondrodyspIasia punctata
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Rhizomelic chondrodysplasia punctata
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Table I. Biochemical findings in RCDP Patients with RCDP Control
Age at time of test Sex Fibroblast plasmalogen
1
2
3
4
5
6
2 mo M 36.5
12 mo F 29.2
10 days F 31.8
10 mo M 14.6
12 mo F 58.2
16 mo M 22.7
100 (10)
1.8
1.7
1.5
3.5
0.5
1.3
3 (50)
14.1
65.0
ND
41.3
24.0
4.0
90 _+ 3 (5)
91.0
81.0
97.0
83.0
87.0
95.0
100 (8)
102.0
152.0
73.0
130.0
54.0
118.0
0.62 _+ 0.16 (28)
synthesis (3H/146) Fibroblast phytanic acid oxidation (% control) Plasma phytanic acid (ug/mL) Catalase distribution (% sedimentable) Fibroblast VLCFA oxidation (% control) Fibroblast C26 :0 (#g/rag protein) Fibroblast C26:0/C22: 0 Plasma C26:0 (#g/mL) Plasma THCA (~mol/L) Plasma pipecolie acid (umol/L)
0.07 _+ 0.04 (525)
0.12
0.031
0.044
0.097
0.085
0.052
0.08 _+ 0.03 (525) 0.33 _+ 0.18 (1900) 0.1 (3) 4 (16)
0.089 0.18 0.1 2.3
0.092 0.23 ND 1
0.031 ND ND ND
0.24 0.36 0.1 1
0.096 0.29 ND 1.3
0.27 0.45 0.1 ND
Comparisonof biochemicalfindingsin six patientswithRCDPand controlvalues.Analysescarriedout as described(see Methods)and are givenas meanof two separate determinations;numberof controlsubjectsin parentheses. VLCFA, very longchainfatty acid; ND, not determined.
(72%), skin changes, and psychomotor retardation, and usually causes death before the end of the first year of life. The remaining patients assigned to the Conradi-Hunermann category are more mildly affected; often survive to adulthood, with normal or only mildly impaired mental function; and have a lower incidence of cataracts (17%). The mode of inheritance for these milder cases often is autosomal dominant.4 In 1979 Happle 5 reported an Xlinked dominant form of chondrodysplasia punctata, the severity of which is intermediate between the rhizomelic and autosomal dominant forms; cataracts and limb involvement are often asymmetric, and skin abnormalities show a linear and blotchy pattern, suggesting lyonization. In 1984 Curry et al. 6 reported two families in which chondrodysplasia punctata was associated with facial dysmorphism, short stature, and mild mental retardation; deletion of the terminal short arm of the X-chromosome; and deficient activity of steroid sulfatase. The pattern of inheritance in these families was X-linked recessive. Recent studies by Heymans et al. 7 have led to a significant advance in our understanding of RCDP, namely the assignment of this disorder to the peroxisomal disease category. These investigators had recognized points of resemblance between RCDP and Zellweger syndrome, and demonstrated deficient enzyme activity in plasmalogen synthesis and elevated plasma phytanic acid levels in RCDP. They concluded that the disorder involved the
peroxisome. 7 However, the number of peroxisomes does not appear to be diminished, and very long chain fatty acid metabolism is not impaired. Thus it is distinguishable from Zellweger syndrome, a conclusion confirmed by complementation studies? During the last 3 years a panel of biochemical assays has been developed that permits assessment of peroxisomal functionY We have applied these assays to the study of six patients with RCDP and compared them with alterations observed in other peroxisomal disorders. RCDP demonstrates consistent biochemical abnormalities that differ from those in all Of the other peroxisomal disorders. These characteristic abnormalities permit precise prenatal and postnatal diagnosis and provide new insights about pathogenic mechanisms. METHODS The six patients with RCDP were unrelated and had the generally accepted diagnostic criteria, 4 including proximal shortening of limbs, joint contractures, dysmorphic features, cataracts, and psychomotor retardation. R0entgenograms showedthe characteristic chondrodysplasia punctata lesions and coronal clefts of vertebral bodies. Patients 1, 2, and 3 died at 21/2 months, 14 months, and 2 weeks, respective!y, and the diagnosis was confirmed by portmortern examination. Patients 4, 5, and 6 were in stable condition at ages 9 to 16 months.
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Hoefler et al.
The Journal of Pediatrics May 1988
T a b l e II. Plasmalogen synthesis in cultured fibroblasts Cell line
No. of patients
3H/14C
SD
P"
Control Neonatal adrenoleukodystrophy Zellweger syndrome RCDP
28 10 6 6
0.62 4.66 10.40 32.19
0.16 1.63 2.85 21.31
0.0001 0.0001 0.003
Plasmalogen synthesis measurements carried out as described (see Methods). Values expressed as ratio of pH] to [~4C] incorporated into plasmalogens. *Values assessed using Student t test.
T a b l e III. Comparison of biochemical abnormalities in certain peroxisomal disorders
RCDP Fibroblast phytanic acid oxidation (% control) Plasma phytanic acid (patients with levels >4 /~g/mL) Catalase distribution (% sedimentable) Fibroblast VLCFA oxidation (% control) Fibroblast C26 : 0 (#g/rag protein) Fibroblast C26:0/C22:0 Plasma C26 : 0 (~g/mL) Plasma THCA (/zmol/L) Plasma pipecolic acid (#mol/L)
1.7 _+ 0.9 (6)
Adrenoleukodystrophy
Zellweger syndrome
Neonatal
1.4 +_+_1.3 (11)
4.6 __+4.8 (5)
X-linked
Adult Refsum disease
Control
98 _+ 13 (3) 0.5 _+ 0.5 (2)
3/3
12/26
11/18
0/10
89 +_ 6 (6)
10 +_ 7 (21)
13 +_ 8 (16)
91 +_ 5 (9)
105 _+ 33 (6)
10 (3)
4.6 -+ 1.6 (10)
16.0 _+ 4.4 (6)
ND
100 (8)
0.076 _+ 0.034 (5) 0.53 +_ 0.28 (37)
0.40 _+ 1.5 (26)
2.41 _+ 0.15 (28)
ND
0.07 + 0.04 (525)
1.05 _+ 0.46 (18)
0.69 + 0.19 (28)
0.07 (1)
0.08 + 0.03 (525)
0.320 _+ 0.112 (4) 3.06 _+ 1.23 (40) 2.14 + 0.71 (27)
1.62 +_ 0.87 (282)
0.15 +_ 0.09 (5)
1.34 + 0.72 (16)
0.1 (3)
11.4 + 5.2 (5)
10.9 _+ 14.6 (11)
1.4 _+ 0.54 (6)
80 _+ 79 (5)
132 +__77 (5)
4/4
100 (10)
89 +_ 2 (3)
0/50
90 + 3 (5)
0.2 (1) 0.33 +_ 0.18 (1900)
ND
ND
4 (5)
ND
0.1 4 (16)
Comparison of biochemical findings in patients with RCDP and other peroxisomal diseases. Values are given as mean _+SD; number of subjects in pa~rentheses. VLCFA, very long chain fatty'acid; ND, not determined.
Experimental procedures. [1-14C]phytanic acid (55 m C i / m m o l ) , [1-14C]hexadecan01 (11.4 m C i / m m o l ) , and [125I]i0dine (30 m C i / m L ) were purchased from Amersham Corp., Arlington Heights, Illinois; [ 1-14C]palmitic acid (58 m C i / m m o l ) from N e w England Nuclear, North Billerica, Massachusetts; and [ 1-~4C]lignoceric acid (47 mCi/rnmol) from Research Products International. [9',10'-3I-I]-sn hexadecylglycerol (5.5 m C i / m m o l ) was kindly provided by F. Paltauf, Technical University of Graz, Austria. Cell culture reagents were from Gibco Laboratories, Grand Island, New York. Nitrocellulose filters were from Schleicher & Schuell Inc., Keene, New Hampshire. L K 5 D Linear-K silica gel thin-layer chromatography plates (250 ~m thickness) were from W h a t m a n Chemical Separation
Inc., Clifton, New Jersey. All other reagents were of analytical grade and obtained from commercial sources. Protein A was labeled with lZSIaccording to the procedure of Greenwood et al. l~ Antibodies against purified rat liver peroxisomal acyl-CoA oxidase, bifunctiona! enzyme, and 3-0xoacyl-CoA thiolase were prepared as described previously? 1 Skin fibroblasts were maintained in culture as previously described? 2 C a t a l a s e activity was assayed by the method of Peters et al? s The subcellular distribution of catalase was measured as previously described? 4 Levels of very long chain fatty acids in fibroblasts and plasma were measured as previously described, 12,~5 trihydroxy coprostanic acid by the method of Bjorkhem and Falk, 16 and pipecolic acid as described by Van den Berg et al. 17Protein
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R h i z o m e l i c chondrodysplasia p u n c t a t a
729
Fig. 1. !mmunoblot analysis of peroxisomal 3-oxoacyl-CoA thiolase. Total homogenates of postmortem liver samples were prepared, subjected to sodium dodecylsulfate/polyacrylamide gel electrophoresis, and blotted onto nitrocellulose membranes as described. After incubation with antibody specific for peroxisomal 3-oxoacyl-CoA thiolase, bound ~mmunoglobulins were detected by ~2~I-labeledprote!n A binding and autoradiography. Lane C, liver sample from control individual;Z, patient ~yith Ze!lweger syndrome; R1 and R2, RCDP; X, X-linked adrenoleukodystrophy. Amounts Of sample protein applied to gel are approximately 0. I mg per gel lane. Immunoreacti,)e band appearing at approximately 47 kilodaltons is nonspecific and thought to be unrelated to thiolase. Positions Of molecular size markers are indicated on left. Note that in RCDP and Zellweger syndrome, moleculhr mass of thiolase is 2 to 3 kilodaltons larger than in Control Or X-linked adren01eukodyst rophy.
Mol. Wt. .............
941(
--
67K
""
7~
' ? N
45 K 36K =zg
|
m
C
Z
RI
R2
X
~
C
Z
RI
R2
X
Fig. 2. Immunoblot'analysisof peroxisomal acyl-CoA oxidase (A) and bifunctional enzyme (B). Nitrocelluiose membranes were prepared as described (see legend, Fig. 1) and subsequently incubated with antibody either specific for peroxisomal acyl-CoA oxidase or bifunctional enzyme: detection of immunoreactive proteins was as in Fig, 1~Lane C, liver sample from control individual; Z, patient with Zellweger syndrome; R1 and R2, RCDP; X, X-linked adrenoleukod~,strophy.
concentration was determined by the method of Lowry et al. TM Immunnblot analysis. Liver samples, stored at - 7 0 ~ C, were allowed to thaw in 62.5 m m o l / L Tris-HC1 (pH 6.8), 1 m m o l / L phenazinemethylsulfonyl fluoride, 1% sodium dodecyl sulfate containing 50 m m o l / L dithiothreitol (50
mg tissue per milliliter), and then homogenized using a Ten Broeck tissue grinder followed by sonication (three pulses of 15 seconds each~at 50 W; (Heat Systems Ultrasonics Inc., Farmingdal~, N.Y.). After heating for 5 minutes at 70 ~ C, samples were centrifuged for 5 minutes in a Fisher microcentrifuge (Fisher Scientific Co., Pitts-
730
Hoefler et al.
The Journal of Pediatrics May 1988
Table IV. Summary of biochemical abnormalities in peroxisomal disorders that involve lipid metabolism
RCDP VLCFA levels Pipecolic ~tcid Bile acid intermediates Phytanic acid oxidation (% control) Plasmalogen biosynthesis Catalase activity (% sedimentable)
Normal Normal Normal 1%-5% Reduced 85%
Zeliweger syndrome
Neonatal
X-linked
Thiolase deficiency (pseudo-zellweger syndrome)*
+++ + § 1%-5% Reduced 10%
+++ + ++ 1%-5% Reduced 15%
++ Normal Normal Normal Normal 90%
+++ Normal ++ ND Normal ND
Adrenoleukodystrophy
+ to + + + , Degree to which metabolite levels are increased. VLCFA, very long chain fatty acid; ND, not determined. *Data from Schram AW, Goldfischer S, Van Roermond CWT, et al (Proc Natl Acad Sci USA 1987;84:2494-6) and Goldfischer SL, Collins J, Rapin I, et al (J PEDIATR 1986;108:25-32). ~Data from Poll-The BT, Ogier H, Saudubray JM, et al (Abstracts of the Society for the Study of Inborn Errors of Metabolism, Amersfoort, 1986:109).
burgh) and subjected to electrophoresis on 10% acrylamide gels according to the method of Laemmli? 9 Each gel lane contained protein corresponding to 1.25 mg (wet weight) liver. Immunoblot analyses were performed as described previously." Phytanic acid oxidation. Alpha-oxidation of [ l-~4C]phytanic acid by cultured skin fibroblasts was measured by ~4CO2 production. [1-~4C]phytanic acid (90 gCi) in 2 mL dry benzene was mixed with 500/xL 5 mmol/L NHaOH in ethanol, and the solution was incubated at 37 ~ C for 2 hours. The sample was evaporated to dryness by a stream of N2 gas and placed in an evacuated desiccator over PzO5 for 2 hours. Ammonium [~4C]phytanate was dissolved in 300 gL ethanol and mixed with 3 mL fetal bovine serum. When cultured fibroblasts reached confluence, the growth medium was replaced with 1 mL fresh growth medium containing 0.3 uCi ammonium [~4C] phytanate in 1% fetal bovine serum, and the cells were incubated at 37 ~ C for an additional 48 hours. Flasks were fitted with serum stoppers and center wells containing KOH-soaked glass fiber filter paper. After acidification of the medium with 0.5 mL 3N H2SO4, incubation was continued overnight at 37 ~ C, and i4coz trapped on filters was quantitated using a tolueneethanol scintillation fluid?~ Plasmalogen biosynthesis. Incorporation of [1-~4C]hexa decanol and [9",lO'-3H]-sn-hexadecylglycerol into the plasmalogen fraction of cultured human fibroblasts was determined according to the method of Roscher et al. 21 This method utilizes a double-label, double-substrate incubation. The peroxisomal component of plasmalogen biosynthesis is measured by the incorporation of [!-14C]hexa decanol into plasmalogens and compared with the microsomal component, assessed by the rate of incorporation of [9',10'-3H]hexadecylglycerol into plasma!ogens. Results
are expressed as the ratio of [3H] to [~4C] incorporation. An abnormally high ratio is observed when peroxisomal plasmalogen synthesis is deficient. The method was modified slightly: LK5D Linear-K silica gel thin-layer chromatography plates (Whatman) were used; they were developed twice with diethYlether/H20 (100:1 vol/vol), exposed to HC1 fumes, and then developed with light petroleum/diethylether (95:5 vol/vol) to a distance of 10 cm.
Fatty acid oxidation. Oxidation of [1-~4C]palmitic acid or [1-~4C]lignoceric acid to water-soluble products by cell suspensions was measured at pH 8.0 as described previously,~2 with the following modifications: After harvesting at confluence by trypsinization, cultured skin fibroblasts were suspended in 0.25 m o l / L sucrose containing 10 mmol/L Tris (C1-), pH 8.0, and 40 # g / m L digitonin. After incubation, reactions were terminated by the addition of 100 ~zL 1 N KOH, and the tubes were heated to 60 ~ C for1 hour. Ice-cold 18% HC104 (0.3 mL) was then added, and samples were kept at 4 ~ C for several hours. The precipitate was removed by centrifugation, and the supernatant was then partitioned By the method of Folch et al. z3 Radioactivity in the upper phase was determined by liquid scintillation spectrometry. The ratio of oxidation of lignoceric acid (C24:0) to palmitic acid (C16:0) provides a measure of very long chain fatty acid oxidation. RESULTS
Biochemical assays in plasma and cultured skin fibroblasts. Table I lists the results of the panel of biochemical assays that have been developed to assess peroxisomal structure and function. These assays measure biochemical reactions that are either carried out in the peroxisome or are affected in known peroxisomal disorders such as
Volume 112 Number 5
Rhizomelic chondrodysplasia punctata
Refsum disease
Acyl-CoA oxidase deficlencyt
Infantile
Adult
++ Normal Normal Normal Normal 90%
++ + ++ 1%-5% Reduced 10%
Normal Normal Normal 1%-5% Normal 85%
Zellweger syndrome? Tables II and III compare results in RCDP with those in other peroxisomal disorders. Plasmalogen synthesis. Peroxisomal plasmalogen synthesis was deficient in all patients with RCDP (Table I). The defect in RCDP was significantly more severe than in Zellweger syndrome or neonatal ALD (Table I1). Phytanie acid oxidation. As previously reported by Heymans et al., 7 phytanic acid levels were usually, but not invariably, elevated. Phytanic acid oxidation in cultured skin fibroblasts of patients with RCDP was reduced to the same extent as in adult Refsum disease, infantile Refsum disease, and Zellweger syndrome (Tables I and liD. Other measures of peroxisomal function and structure. Levels of very long chain fatty acids, pipecolic acid, and bile acid intermediates and very long chain fatty acid oxidation were normal in the patients with RCDP, indicating that the metabolism of these substances is not impaired in this disorder. The subcellular distribution of catalase in cultured skin fibroblasts was primarily sedimentable, as it is in normal cells, but differing sharply from the results in Zellweger syndrome and neonatal ALD. Immunoblot analysis of peroxisomal beta oxidation enzymes in postmortem liver tissue. Figs. 1 and 2 compare results in portmortem liver tissue of patients with RCDP with those in Zellweger syndrome and control specimens. The findings in RCDP are distinct and characteristic. The amount of protein reacting with the 3-oxoacyl-CoA thiolase antibody was present in normal quantity, but the molecular mass of this enzyme was apparently 2 to 3 kilodaltons larger than control values (Fig. 1). Unlike the findings in Zellweger syndrome and neonatal ALD, acylCoA oxidase and bifunctional enzyme were present in normal amounts (Fig. 2). DISCUSSION
Biochemical diagnosis of RCDP. There is increasing awareness that the phenotype of peroxisomal disorders covers a wide spectrum. Complementation studies indicate
731
that there are distinct genotypes, s The use of a panel of biochemical probes of peroxisomal function offers the opportunity to clarify the diagnosis and classifications of peroxisomal disorders. Application of these probes permits RCDP to be distinguished from all of the other known peroxisomal disorders (Table IV). These tests can be performed with accessible tissues, such as plasma and cultured skin fibroblasts, and are of general value for the diagnosis and categorization of patients who have or are suspected to have a peroxisomal disorder. These procedures have been used successfully for the prenatal diagnosis of peroxisomal disorders. 24 The profound defect of plasmalogen synthesis in RCDP should permit prenatal diagnosis utilizing either chorion villus samples or cultured amniocytes. We have monitored four pregnancies at risk for RCDP, and have identified one affected fetus by measuring plasmalogen synthesis and phytanic acid oxidase in a cultured chorion villus sample (Hoefler S, unpublished observation). Defect in plasmaiogen synthesis. The most striking biochemical defect in RCDP is the profound impairment of plasmalogen synthesis. Defective plasmalogen synthesis has been previously demonstrated in RCDP 25'26 and in Zellweger syndrome, 27,2s but the sensitive assay2~ used in our studies has permitted us to demonstrate for the first time that this defect is significantly more severe in RCDP. Webber et al. 29 have shown that the defective plasmalogen synthesis in Zellweger syndrome is related mainly to defective activity of DHAP acyltransferase, and that this in turn is related to low concentration of active enzyme molecules rather than a defect in the structure of the protein molecule itself. It is postulated that in the absence of peroxisomes the active enzyme is abnormally unstable. Analogous studies with RCDP will be of interest. Plasmalogens are constituents of virtually all membranes, 3~ but their biologic role has not been determined. Inasmuch as chondrodysplasia punctata is a feature in both RCDP and Zellweger syndrome, it seems plausible that the bone lesion is related to this biochemical defect. In neonatal ALD, plasmalogen synthesis is much less defective than in Zellwcger syndrome or RCDP, and chondrodysplasia punctata is not observed in neonatal A L D ) 1 RCDP as sole example of group 2 peroxisomal disorders. Previous classifications of peroxisomal disorders have subdivided them into three general categories. 32 In the group 1 disorders, which include Zellweger syndrome and neonatal ALD, the structure of the organelle is deficient or even lacking, and it is generally accepted that this is the cause of the multiple peroxisomal enzyme defects associated with these disorders. The group 3 disorders include X-linked ALD, hyperoxaluria type I, ~3 acyl-CoA oxidase deficiency, 34 3-oxoacyl-CoA thiolase deficiency,3~ and possibly
732
Hoefler et al.
adult Refsum disease. Peroxisome structure is normal in group 3 disorders, and it is presumed that they represent mutations that involve a single peroxisomal enzyme. The greatest conceptual difficulty has applied to the group 2 peroxisomal disorders, which are defined as disorders in which the activity of more than one peroxisomal enzyme is deficient but peroxisomal structure is intact22 To this category were assigned pseudo-Zellweger syndrome, a name coined by Goldfischer et al., 36 and RCDP. Pseudo-Zellweger syndrome is caused by 3-oxoacyl-coA thiolase deficiency, and this single enzyme defect may account for both the accumulation of very long chain fatty acids and bile acid intermediates25 This new knowledge obviates the need for the term "pseudo-Zellweger syndrome" and may reassign this disorder to category 3.
Defects in phytanie acid oxidation and processing of thiolase. A t this time, then, R C D P may be the only example of a group 2 peroxisomal disorder. There is convincing evidence for two enzyme defects, namely, peroxisomal plasmalogen synthesis and oxidation of phytanic acid. It is very unlikely that these two reactions can be traced to a single enzyme defect. The subcellular localization of phytanic acid oxidase is still uncertain. It has been postulated to be peroxisomal because defective phytanic acid oxidation is a constant feature in patients with group 1 peroxisomal disorders. In spite of this observation, the most recent subcellular fractionation studies suggest that phytanic acid oxidase is a mitochondrial enzyme. 37 The basis for the existence of these two enzymatic defects in R C D P requires additional study. The demonstration that the peroxisomal thiolase in the postmortem liver tissue of two R C D P patients existed in the unprocessed form 38 (Fig. 1) was unexpected. Up to now, this enzyme had been found to be present in this form only in the group 1 peroxisomal disorders, 11,39 It had been concluded that processing of the enzyme took place in the peroxisome and that this would fail to occur when the peroxisome structure was absent or deficient. This formulation does not appear applicable in R C D P , because of our observation that catalase in R C D P fibroblasts is in the particulate fraction (Table III), a finding that suggests that peroxisome structure is preserved, 4~ and the microscopic observation by Heymans et al. 26 that peroxisomes are present. The observation that very long chain fatty acids levels are normal suggests that the protein is catalytically active, at least to some extent. Heymans et al. reported the intriguing finding that in the liver tissue of one R C D P patient, "some hepatocytes lacked peroxisomes, while other cells displayed an increased number of exceptionally large and irregular shaped peroxisomes." Further study is required to unravel the pathogenesis of these apparently unrelated biochemical defects and their relationship to the phenotype of RCDP.
The Journal of Pediatrics May 1988 We thank Dr. Richard L. Kelley for performing the pipecolic acid assays, and Dr. Ingemar Bjorkhem for measuring levels of bile acid intermediates; and Dr. Fritz Paltauf for his generous gift. of the tritium labeled sn-hexadecylglycerol.
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