Hum Genet (1992) 89:439-444

9 Springer-Verlag 1992

Genetic and biochemical heterogeneity in patients with the rhizomelic form of chondrodysplasia punctata a complementation study Judith C. Heikoop 1, Ronald J. A. Wanders 2, Anneke Strijland 1, Rally Purvis 2, Ruud B. H. Schutgens 2, and Joseph M. Tager 1. 1E. C. Slater Institute for Biochemical Research, University of Amsterdam, Academic Medical Centre, Meibergdreef 15, NL-1105 AZ Amsterdam, The Netherlands 2Department of Pediatrics, University Hospital Amsterdam, Academic Medical Centre, Meibergdreef 9, NL-1105 AZ Amsterdam, The Netherlands Received August 1, 1991 / Revised November 25, 1991

Summary. The genetic relationship between 10 patients with clinical manifestations of rhizomelic chondrodysplasia punctata (RCDP) was studied by complementation analysis after somatic cell fusion. Biochemically, 9 out of the 10 patients were characterized by a partial deficiency of acyl-CoA: dihydroxyacetone phosphate acyltransferase (DHAP-AT) and an impairment of plasmalogen biosynthesis, phytanate catabolism and the maturation of peroxisomal 3-oxoacyl-CoA thiolase; 3oxoacyl-CoA thiolase was strongly reduced in the peroxisomes of these patients. Fusion of fibroblasts from these 9 patients with Zellweger fibroblasts resulted in complementation as indicated by the restoration of DHAP-AT activity, plasmalogen biosynthesis, and punctate fluorescence after staining with a monoclonal antibody to peroxisomal thiolase. No complementation was observed after fusion of different combinations of the 9 RCDP cell lines, suggesting that they belong to a single complementation group. The tenth patient was characterized biochemically by a deficiency of DHAP-AT and an impairment of plasmalogen biosynthesis. However, maturation and localization of peroxisomal thiolase were normal. Fusion of fibroblasts from this patient with fibroblasts from the other 9 patients resulted in complementation as indicated by the restoration of plasmalogen biosynthesis. We conclude that mutations in at least two different genes can lead to the clinical phenotype of RCDP.

Introduction Chondrodysplasia punctata is a heterogeneous group of bone dysplasias whose common characteristic is the * Present address: Institute of Medical Biochemistry, University of Bari, Piazza G. Cesare, 1-70124 Bari, Italy Correspondence to: J. C. Heikoop, c/o Ms. G. E. E. van Noppen, Publications Secretary, E. C. Slater Institute for Biochemical Research, University of Amsterdam, Meibergdreef 15, NL-1105 AZ Amsterdam, The Netherlands

radiological finding of punctate epiphyseal and extraepiphyseal calcifications. Chondrodysplasia punctata has been reported in several distinct genetic forms. The two major types are the autosomal recessive rhizomelic form (RCDP) and the Conradi-Hfinermann type with autosomal-dominant inheritance (Spranger et al. 1971). In 1984, Heymans et al. reported several biochemical abnormalities in RCDP, including an impairment in the ability to synthesize plasmalogens. RCDP was therefore assigned to the newly recognized group of peroxisomal disorders. Additional studies on a liver biopsy from an RCDP patient revealed that peroxisomes were undetectable in some hepatocytes, whereas other liver cells displayed an increased number of exceptionally large and irregularly shaped peroxisomes (Heymans et al. 1986). Nevertheless, in cultured skin fibroblasts from RCDP patients, catalase is present in particles with the same density (Balfe et al. 1990; Heikoop et al. 1990; Singh et al. 1991) and morphology (Heikoop et al. 1991) as peroxisomes from control fibroblasts, in contrast to the situation in fibroblasts from patients with Zellweger syndrome and related disorders with a generalized impairment of peroxisomal functions (reviewed in Wanders et al. 1988; Lazarow and Moser 1989). Further studies have shown that the two peroxisomal enzymes essential for de novo plasmalogen biosynthesis, viz. acyl-CoA: dihydroxyacetone phosphate acyltransferase (DHAP-AT) and alkyldihydroxyacetone phosphate synthase (alkylD H A P synthase), are (partially) deficient in fibroblasts from RCDP patients (Heymans et al. 1986; Poulos et al. 1988; Schutgens et al. 1988). Furthermore, in liver (Hoefler et al. 1988) and in fibroblasts (Balfe et al. 1990; Heikoop et al. 1990; Singh et al. 1991) from these patients, 3-oxoacyl-CoA thiolase is present in the unprocessed precursor form. In contrast to most peroxisomal proteins, 3-oxoacyl-CoA thiolase is normally synthesized as a larger precursor with an amino-terminal peptide extension that is cleaved upon maturation of the enzyme (Furuta et al. 1982; Fujiki et al. 1985). Recent fractionation (Balfe et al. 1990; Heikoop et al. 1990;

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Singh et al. 1991) and i m m u n o e l e c t r o n - m i c r o s c o p y studies ( H e i k o o p et al. 1991) have revealed that only a very small a m o u n t of the 3 - o x o a c y l - C o A thiolase is present in the catalase-containing peroxisomes of R C D P fibroblasts, suggesting that there is an impairment of the uptake of the e n z y m e into the peroxisomes. Finally, phytanic acid levels have been f o u n d to be strongly elevated in R C D P patients ( H e y m a n s et al. 1986; H o e f l e r et al. 1988; Poulos et al. 1988; Schutgens et al. 1988). R C D P is thus a peroxisomal disorder characterized by impairment of some, but not all, peroxisomal functions. In other forms of chondrodysplasia punctata, peroxisomal functions are not impaired (Schutgens et al. 1988). The aim of the study described in this paper was to determine the genetic relationship between different R C D P patients, by using c o m p l e m e n t a t i o n analysis after somatic cell fusion of cultured skin fibroblasts.

Materials and methods

Cell lines and culture conditions Fibroblast cell lines RCDP 1-9 were derived from 9 unrelated patients with the clinical and biochemical features characteristic of the rhizomelic form of chondrodysplasia punctata (see Schutgens et al. 1988; Heikoop et al. 1990, 1991). The code numbers of these cell lines are MCHE85AD, BALC88AD, GJOLD90AD, VERS90AD, VRI87AD, KHJE90AD, RJAN90AD, ACLA90AD and ALL90AD, respectively. Furthermore, fibroblasts were studied from an additional patient (code number REKEL91AD), to be described in detail elsewhere (R. J. A. Wanders et al., submitted for publication). Briefly, this patient was born after an uneventful pregnancy, at 37 weeks, from consanguineous parents: birthweight, 2720g; length 46cm. At birth, dysmorphic features were noticed, including a high forehead, large fontanelles and a low/broad nasal bridge. Furthermore, severe hypotonia and cataracts were observed. Radiological studies revealed the stigmata characteristic of RCDP, including severe metaphyseal dysplasia and shortening of the humeri and femora (Spranger et al. 1971). There was failure to thrive and the patient suffered from frequent infections that eventually caused her death at 6 months of age. The biochemical abnormalities in fibroblasts from this patient were restricted to deficiency of DHAP-AT and impairment of de novo plasmalogen biosynthesis. Finally, cells from a control subject and two cell lines from patients with Zellweger syndrome [code numbers W78/515 and GOM85AD (see Wiemer et al. 1989 for details)] were used in these studies. The cells were cultured in a 1 : 1 mixture of Ham F10 (Gibco, Glasgow, UK) and Dulbecco's modified Eagle medium (Gibco) supplemented with 10% (by vol) fetal calf serum (Gibco), under 5% CO2.

Biochemical assays De novo plasmalogen biosynthesis in fibroblasts was measured as described by Schrakamp et al. (1988). The activity of DHAP-AT was measured in fibroblasts according to Schutgens et al. (1986).

Immunoblot analysis Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate was carried out essentially as described by Laemmli (1970). Immunoblot analyses and visualization of the antigen-antibody complexes were performed as described previously (Heikoop et al. 1991). A monoclonal antibody against 3oxoacyl-CoA thiolase (code number 3G4) was generated as described (Heikoop et al. 1991).

Immunofluorescence microscopy Cells were seeded on glass cover-slips and cultured as described above. At near-confluency, the cells were fixed, permeabilized and immunolabelled with the monoclonal antibody against 3-oxoacyl-CoA thiolase as described (Heikoop et al. 1991). The fluorescence staining pattern was viewed in an Olympus IMT2 (RFL) fluorescence microscope, using an IMT2-DMB filter for excitation.

Cell fusion and assessment of complementation Fusion mediated by polyethylene glycol (tool. wt. 1000: Merck, Darmstadt, FRG) and co-cultivation of the respective cell lines were carried out as described by Brul et al. (1988a). The fusion efficiency, defined as the percentage of total nuclei in multinucleate cells, was always in the range of 60%-90%. The occurrence of complementation was tested in three ways. First, heterokaryons were immunolabelled with a monoclonal anti-(3-oxoacyl-CoA thiolase) antibody at 24, 48 or 72 h after fusion as described above. Secondly, DHAP-AT activity was measured in the heterokaryons 24, 48 or 72 h after fusion, as described by Schutgens et al. (1986). Thirdly, the de novo synthesis of plasmalogens was measured 48 h after fusion, as described by Schrakamp et al. (1988). Protein was determined according to Smith et al. (1985).

Results

Biochemical and morphological parameters in cultured skin fibroblasts D e novo plasmalogen biosynthesis was impaired in R C D P cell lines, as described earlier ( H e y m a n s et al. 1984, 1986; H o e f l e r et al. 1988; Schutgens et al. 1988), and also in cell line R E K E L 9 1 A D (see below). Furtherm o r e , in cell lines R C D P 1 - R C D P 9, the activities of a l k y l - D H A P synthase and phytanic acid oxidase were impaired (Hoefler et al. 1988; Poulos et al. 1988; Schutgens et al. 1988). The activity of D H A P - A T was only partially deficient (range: 1.4-6.2 n m o l / 2 h . m g protein) in these cell lines when c o m p a r e d with the values for the control fibroblasts (mean + SD: 7.8 + 2.0 n m o l / 2 h . m g protein, n = 59). The residual activity of D H A P - A T in fibroblasts f r o m R C D P 1 - R C D P 9 was significantly higher than in fibroblasts from patients with Zellweger s y n d r o m e ( m e a n + SD: 0.7_+ 0.5 n m o l / 2 h • protein, n = 1 0 ) . In contrast, in cell line R E K E L 9 1 A D , the activities of a l k y l - D H A P synthase and phytanic acid oxidase were c o m p a r a b l e to values in control fibroblasts. H o w e v e r , the D H A P - A T activity was below the limit of detection. I m m u n o b l o t t i n g experiments revealed that, in cell lines R C D P I - R C D P 9, peroxisomal 3 - o x o a c y l - C o A thiolase was present in the 44 k D a precursor form, as described earlier (Balfe et al. 1990; H e i k o o p et al. 1990; Singh et al. 1991). With respect to this p a r a m e t e r , cell lines R C D P I - R C D P 9 also differed from cell line R E K E L 9 1 A D , in which the 3o x o a c y l - C o A thiolase was present in the 41 k D a m a t u r e f o r m as in control fibroblasts (not shown). Incubation of control cells with a m o n o c l o n a l antib o d y directed against peroxisomal 3 - o x o a c y l - C o A thiolase gave rise to a punctate i m m u n o f l u o r e s c e n c e pattern, consistent with the presence of 3 - o x o a c y l - C o A thiolase in peroxisomes (Fig. 1A). Only a diffuse green fluores-

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Fig. 1A,-D. Pattern of immunofluorescence observed after staining of fixed fibroblasts with a monoclonal anti-(3-oxoacyl-CoA thiolase) antibody. Fibroblasts were grown to near-confluency, fixed and permeabilized. The 3-oxoacyl-CoA thiolase protein was detected by incubation first with the monoclonal antibody 3G4, then with a biotin-conjugated anti-(mouse Ig) serum, and finally with strepavidin-labelled with fluorescein isothiocyanate as fluorescent marker. A Control cells; B Zellweger syndrome (cell line GOM85AD); C cell line RCDP 1; D cell line REKEL91AD

Complementation analysis of cell lines RCDP 1 - R C D P 9

fluorescence using antibodies to a peroxisomal enzyme, such as catalase (Wiemer et al. 1989). This morphological criterion for the presence of peroxisomes has been used by Brul et al. (1988b) in complementation analysis of cell lines from patients with the Zellweger syndrome and related disorders. The results described in the previous section prompted us to use the immunofluorescence pattern with the monoclonal anti-(3-oxoacyl-CoA thiolase) antibody to assess complementation. When fibroblasts from a patient with Zellweger syndrome were fused with fibroblasts from different R C D P cell lines ( R C D P 1 - R C D P 9), a punctate fluorescence labelling pattern emerged in the heterokaryons. Figure 2A shows the result for the cell line R C D P 1 after fusion with Zellweger syndrome fibroblasts (cell line G O M 8 5 A D ) . The results for the 8 other R C D P cell lines were comparable (not shown). Cell line R E K E L 9 1 A D was not included in these experiments because of the presence of punctate fluorescence when stained for 3-oxoacyl-CoA thiolase. In contrast to the fusions with Zellweger syndrome fibroblasts, no punctate fluorescence could be observed in heterokaryons derived from the fusion of different combinations of cell lines R C D P 1 - R C D P 9. The result for cell lines R C D P 1 and R C D P 2 obtained 3 days after somatic cell fusion is shown in Fig. 2B.

Morphological assessment. The presence of peroxisomes in fibroblasts can be visualized by indirect immuno-

Biochemical assessment. As shown by Brul et al. (1988a), the activity of D H A P - A T , which is deficient in fibro-

cence was observed with a Zellweger syndrome cell line (Fig. 1B) and cell line R C D P 1 (Fig. 1C) when stained for 3-oxoacyl-CoA thiolase. Similar results were obtained with cell lines R C D P 2 - R C D P 9 (not shown), in agreement with earlier results (Heikoop et al. 1991). However, when cell line R E K E L 9 1 A D was stained for 3-oxoacyl-CoA thiolase, a punctate fluorescence pattern was observed (Fig. 1D), and the distribution and size of the spots was comparable with those of control fibroblasts. This result suggests that 3-oxoacyl-CoA thiolase is associated with peroxisomes in fibroblasts from patient R E K E L 9 1 A D , in contrast to the situation in all other R C D P cell lines studied.

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Fig. 2A, B. Pattern of immunofluorescence observed with a monoclonal antibody directed against 3-oxoacyl-CoA thiolase, after fusion of fibroblasts from patients with RCDP and Zellweger syndrome. Fibroblasts were fused using polyethylene glycol. The fibroblasts were fixed and permeabilized 3 days after fusion. The

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fixed multinucleate cells were processed further for immunofluorescence microscopy using a monoclonal anti-(3-oxoacyl-CoA thiolase) antibody. A RCDP 1 x Zellweger syndrome (cell line GOM85AD); B RCDP 1 x RCDP 2

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Fig. 3 A - E . D H A P - A T activity in heterokaryons obtained after fusion of different cell lines. D H A P - A T activity was measured at intervals after fusion (Q) or co-cultivation (9 of Zellweger syndrome (ZS) and RCDP fibroblasts. The cells were fused using polyethylene glycol. A RCDP 1 • ZS (cell line W78/515): B RCDP 2 x ZS (cell line W78/515), C R E K E L 9 1 A D x ZS (cell line W78/ 515); D R E K E L 9 1 A D x RCDP 1; E R E K E L 9 1 A D x RCDP 2

Days after fusion

blasts from patients with Zellweger syndrome and related disorders, can be used as an index of complementation in these disorders. However, the residual activity of D H A P - A T in fibroblasts from patients R C D P 1 - R C D P 9 is significantly higher than in fibroblasts from patients with Zellweger syndrome and related disorders. For this reason, assessment of complementation on the basis of D H A P - A T activity alone is not sensitive enough for use in the study of the genetic relationship between R C D P patients. D H A P - A T activity could, however, be used to assess complementation between cell line R E K E L 9 1 A D and cell lines R C D P 1 - R C D P 9. The results of representative fusion experiments involving cell line REKEL91AD and cell lines R C D P 1, R C D P 2 and a Zellweger syndrome cell line (W781515) are shown in Fig. 3. In these

combinations, there was a time-dependent 2-3 fold increase in D H A P - A T activity after fusion. In the co-cultivation controls, little, if any, increase was observed. These cell lines clearly complement each other. Another way of assessing complementation of cell lines derived from patients with disorders of peroxisomal biogenesis is to measure the restoration of the capacity for synthesizing plasmalogens after fusion of different cell lines, as demonstrated by Roscher et al. (1989). De novo plasmalogen biosynthesis is strongly impaired in all the RCDP fibroblasts studied (Schutgens et al. 1988, see also Table 1), despite the high residual activity of D H A P - A T in cell lines R C D P 1 - R C D P 9. The results of representative fusion experiments involving the cell lines R C D P 1, R C D P 2, R E K E L 9 1 A D

443 Table 1. Complementation analysis of fibroblasts from patients

with RCDP and Zellweger syndrome (ZS). The cells were fused using polyethyleneglycol. Complementation was assessed by measuring de novo plasmalogen biosynthesis in heterokaryons Fibroblast cell lines

48 h after fusion of the cells. As a control, cells were co-cultivated for 48 h. PE, Phosphatidylethanolamine; PC, phosphatidylcholine; pPE, plasmalogen phosphatidylethanolamine; pPC, plasmalogen phosphatidylcholine

Values in co-cultivated cells Ratio (3I-I]14C) % pPE % pPC in alkenyl chains of in PE in PC pPE pPC

Control ZS (W78/515) RCDP 1 RCDP 2 REKEL91AD

78.4 17.7 11.9 2.3 5.1

2.1 0.4 0.4 0.3 0.3

1.2 53.4 63.8 349.1 49.9

0.9 9.1 6.9 13.2 5.2

ZS and RCDP 1 ZS and RCDP 2 ZS and REKEL91AD RCDP 1 and RCDP 2 RCDP I and REKEL91AD RCDP 2 and REKEL91AD

13.5 21.5 18.1 14.2 9.8 1.4

0.5 0.4 0.3 0.3 0.5 0.6

71.5 31.3 29.6 33.2 41.3 246.1

6.9 7.6 10.1 7.1 9.3 11.5

and a Zellweger syndrome cell line (W78/515) are shown in Table 1. For each complementation assay, plasmalogen synthesis capacity was measured in the co-cultivation controls and after fusion of two cell lines. A substantial increase in plasmalogen synthesis after fusion compared with that after co-cultivation was taken as evidence of complementation. Fusion of R C D P fibroblasts with fibroblasts from a patient with Zellweger syndrome (cell line W78/515) resulted in an increase in de novo plasmalogen biosynthesis, as expected. In the combinations in which cell line R E K E L 9 1 A D was fused with R C D P cell lines R C D P 1 and R C D P 2, a clear increase in de novo plasmalogen biosynthesis was also observed. However, when R C D P 1 fibroblasts were fused with R C D P 2 cells, no complementation was observed. Selffusion of R C D P fibroblasts did not affect the rate of de novo plasmalogen synthesis.

Discussion

Our complementation studies clearly show that the 9 unrelated patients who exhibit the rhizomelic form of chondrodysplasia punctata and who are characterized biochemically by a (partial) deficiency of D H A P - A T and alkyl-DHAP synthase, impairment of the oxidation of phytanic acid and impairment of the maturation of peroxisomal 3-oxoacyl-CoA thiolase belong to the same complementation group. Morphological and fractionation studies have revealed that the amount of 3-oxoacylCoA thiolase is strongly reduced in the peroxisomes of these R C D P patients (Balfe et al. 1990; Heikoop et al. 1990, 1991; Singh et al. 1991), indicating that the mutation may involve a gene encoding a receptor or another component of the peroxisomal import machinery specific for the uptake of the affected proteins. The enzyme 3oxoacyl-CoA thiolase is a peroxisomal protein that does not contain the carboxyterminal tripeptide-serine/leucine/

Values in fused cells % pPE % pPC in PE in PC

27.1 45.1 53.3 7.7 37.1 29.2

0.4 0.6 0.9 0.3 0.9 0.8

Ratio (3I-I/14C) in alkenyl chains of pPE

pPC

13.3 6.4 4.2 85.4 7.7 6.4

4.5 2.9 2.1 10.3 4.1 3.6

Complementation

+ + + + +

lycine (SKL) targeting signal, identified by Gould et al. (1988) as being required for the uptake of a number of peroxisomal proteins into peroxisomes (Hijikata et al. 1987; Bout et al. 1988; Fairbairn and Tanner 1989). Recently, Swinkels et al. (1990) have presented evidence for the presence of a peroxisomal targeting signal in the amino-terminal pre-piece of peroxisomal 3-oxoacyl-CoA thiolase. Thus, at least one component of the import machinery used by 3-oxoacyl-CoA thiolase differs from those used by SKL-containing proteins. The gene encoding such a component could be mutated in the 9 classic R C D P patients. The fact that, in R C D P fibroblasts, the ultrastructural appearance of peroxisomes and the distribution of catalase and the 69 kDa integral membrane protein are indistinguishable from the situation in control fibroblasts suggests that the primary lesion in R C D P is an impairment in the post-translational uptake of only a few newly made proteins into peroxisomes (Balfe et al. 1990; Heikoop et al. 1990, 1991; Singh et al. 1991). In Zellweger syndrome and related disorders with a generalized impairment of peroxisomal functions, the uptake defect involves a variety of peroxisomal proteins, suggesting that, in these disorders, a more universal component of the import machinery is affected (Wanders et al. 1988; Lazarow and Moser 1989). Our complementation analysis studies show that the 9 classic R C D P patients studied belong to a single complementation group. We have recently identified a patient (to be described in detail elsewhere) who has the clinical characteristics of RCDP, but whose biochemical abnormalities are limited to a deficiency of D H A P - A T and impairment of de novo plasmologen biosynthesis. The mutation in this R C D P patient seems to have no effect on peroxisomal 3-oxoacyl-CoA thiolase. Complementation occurred after somatic cell fusion of cultured skin fibroblasts from this patient with those of patients with the classic form of RCDP. Therefore, we

444 c o n c l u d e t h a t m u t a t i o n s in at least two d i f f e r e n t genes l e a d to the clinical p h e n o t y p e of R C D P . A deficiency o f D H A P - A T a n d h e n c e an i m p a i r m e n t in p l a s m a l o g e n b i o s y n t h e s i s are the o n l y b i o c h e m i c a l abn o r m a l i t i e s that h a v e b e e n d e t e c t e d so far in p a t i e n t R E K E L 9 1 A D , T h e p r i m a r y defect in the 9 classic R C D P p a t i e n t s affects n o t o n l y p l a s m a l o g e n b i o s y n t h e s i s b u t also o t h e r b i o c h e m i c a l p a r a m e t e r s . H o w e v e r , c h a n g e s in these p a r a m e t e r s d o n o t a p p e a r to b e r e l e v a n t for the clinical p h e n o t y p e of R C D P . W e t h e r e f o r e c o n c l u d e that an i m p a i r m e n t in the de n o v o synthesis of p l a s m a logens ( a n d o t h e r e t h e r p h o s p h o l i p i d s ) m a y p l a y a k e y role in the p a t h o g e n e s i s o f the clinical p h e n o t y p e of R C D P . F u r t h e r studies a r e n e e d e d in o r d e r to i d e n t i f y the exact m e c h a n i s m s i n v o l v e d in the p a t h o g e n e s i s of the clinical signs a n d s y m p t o m s of R C D P .

Acknowledgements. The authors are grateful to Wendy van Noppen and Els Vlugt-Van Daalen for their help in the preparation of the manuscript. This work was supported by a Programme Grant from the Netherlands Organization for Scientific Research (NWO), under the auspices of the Section for Medical Sciences, and by a grant from the Prinses Beatrix Fonds.

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