Adrenoleukodystrophy and variants

Journal of the Neurological Sciences, 1986, 72:103-112

103

Elsevier JNS 2598

Adrenoleukodystrophy and Variants Clinical, Neurophysiological and Biochemical Studies in Patients and Family Members Ikuo G o t o 1, T a k u r o K o b a y a s h i ~, Y a s u n o b u A n t o k u 2, Shojo T o b i m a t s u 1, and Yoshigoro K u r o i w a ~ 'Department of Neurology, Neurological Institute, Faculty of Medicine, Kyushu University60, Fukuoka and 2National Chikugo Hospital, Fukuoka Prefecture, Fukuoka (Japan)

(Received 13 May, 1985) (Received, revised 20 August, 1985) (Accepted 22 August, 1985)

SUMMARY Clinical, neurophysiological and biochemical studies were performed in patients with various forms of adrenoleukodystrophy (ALD) and their family members. The patients showed an abnormality in saturated very long chain fatty acids and in the somatosensory and brain stem auditory or visual evoked potentials. Female presumptive carriers without abnormal neurological manifestations also showed abnormality in the somatosensory or brain stem auditory evoked potentials and in saturated very long chain fatty acids. One ALD patient and his mother, a female carrier, had the decreased beta-galactosidase activity. The increase in saturated very long chain fatty acids was found, not only in sphingomyelin, but also in phosphatidylcholine and phosphatidylserine. Our results suggest that a generalized abnormal metabolism of VLFA and an abnormality in the central nervous system exist in our patients and female carriers.

Key words: Adrenoleukodystrophy- Multimodality evoked potentials - beta-galactosidase - Very long chain fatty acids

This study was partly supported by a Grant-in-Aidfrom the Ministryof Education, Science and Culture, SelectedIntractableNeurologicalDisorders, Japan, and Ministryof Health and Welfare,Japan. Correspondent: Ikuo Goto, Departmentof Neurology,NeurologicalInstitute,Facultyof Medicine, Kyushu University60, 3-1-1 Maldashi,Higashi-Ku,Fukuoka, Japan 812, 0022-510X/86/$03.50 © 1986 Elsevier Science Publishers B.V. (BiomedicalDivision)

104 INTRODUCTION Adrenoleukodystrophy (ALD) is usually an X-linked recessive disease that mainly involves the white matter of the nervous system and the adrenal cortex. Adrenoleukomyeloneuropathy (ALMN) and adrenomyeloneuropathy (AMN) have also been reported as variants of the disease (Schaumburg et al. 1975; Griffin et al. 1977; Marsden et al. 1982). The accumulation of saturated very long-chain fatty acids (VLFA) of cholesterol esters and sphingolipids was found in autopsied brain and adrenal glands of ALD (Igarashi et al. 1976a,b). Determination of saturated VLFA in plasma, erythrocyte membranes and skin fibroblasts provides reliable detection of affected persons and carriers (Kawamura et al. 1978; Moser et al. 1981; Tsuji et al. 1981; Molzer etal. 1982; Kobayashi etal. 1983). We have studied ALD and its variants. The purpose of this paper is to show the biochemical studies, particularly the saturated VLFA in phospholipid, as well as the neurophysiological and neurological studies in patients and female carriers. SUBJECTS Case I

An 8-year-old boy, the only child in this family, showed forced laughing, bilateral decreased vision, decorticate posturing and spastic quadriparesis with bilateral Babinski's signs. He was unable to swallow and speak. He had the clinical manifestations of leukodystrophy and bilateral diffuse low attenuation in the cerebral white matter, predominantly in the bilateral posterior horn areas on brain CT scan. He was finally diagnosed as having adrenoleukodystrophy. Case 2

A 21-year-old intelligent male showed forced laughing, nasal voice, spastic paraparesis with bilateral Babinski's signs and urinary disturbance. Motor conduction velocity of the tibial nerve was mildly decreased at 37.8 m/s and sural nerve biopsy showed slight loss of large myelinated fibers. This patient showed clinical manifestations of myeloneuropathy, and was diagnosed as having adrenomyeloneuropathy. His elder brother showed mental deterioration, concentric visual field defects, slurred speech, bilateral hearing loss, spastic quadriparesis with bilateral Babinski's signs and cerebellar ataxia at the age of 8 years. He was diagnosed having as Schilder's disease and died at the age of 12 years. Case 3

A 28-year-old intelligent male had forced laughing, marked scanning speech, irregular saccadic ocular movement, spastic quadriparesis with bilateral Babinski's signs, marked cerebellar ataxia and urinary disturbance. Brain CT scan showed atrophy of the ports and eerebeUum. Motor and sensory nerve conduction velocities in upper and lower extremities were normal. This patient had the clinical manifestations of

105 spinocerebellar degeneration, and was finally diagnosed as having adrenoleukomyelopathy presenting as spinocerebellar degeneration.

Examined family members A 28-year-old maternal male cousin of case 3 had scanning speech, marked cerebellar ataxia, spastic quadriparesis with bilateral Babinski's signs. Brain CT scan revealed atrophy of the pons and cerebellum. He showed clinical f'mdings of cerebellar degeneration. His final diagnosis was adrenoleukomyelopathy with spinocerebellar degeneration as the presenting symptoms. The 35-year-old mother and the 37-year-old father of case 1, the 51-year-old mother of case 2 and the 48- and 52-year-old maternal aunts of case 3 showed no neurological abnormalities. Subjects examined in this study The above-described 3 patients, 5 clinically healthy members in 3 families and a cousin of case 3 were investigated for endocrinological, neurophysiological and/or biochemical studies. Three to twenty-one subjects were also studied as controls for the neurophysiological and biochemical investigations. METHOD

Endocrinological studies Serum triiodothyronine (T3) and thyroxine (T4) were determined by a tube solid phase radioimmunoassay method (SPAC RIA kit, Daiichi Radioisotope, Japan); serum thyroid stimulating hormone (TSH) by a double antibody radioimmunoassay method (TSH kit, Daiichi Radioisotope, Japan); serum ACTH by a dextran coated charcoal radioimmunoassay method (ACTH RIA kit, CIS, France); plasma cortisol by a solid phase radioimmunoassay method (gamma-coatcortisol kit, Japan Travenol). The plasma cortisol response to ACTH loading was determined by the intravenous injection of ACTH, 0.25 mg. Urinary 17-ketosteroids and 17-hydroxycorticosteroids were determined by the method based on Zimmermann's reaction and the method based on the Porter-Silber reaction, respectively. Neurophysiological studies Recording electrodes were placed at the vertex (Cz) and referred to electrodes on the bilateral mastoid processes for brain stem auditory evoked potentials (BAEP). Electrodes at Erb's point, cervical spine and the contralateral somatosensory hand area of the stimulated side referred to an electrode at Fz for somatosensory evoked potentials (SEP); and at the mid-occipital area (Oz) referred to an electrode at Fz for visual evoked potentials (VEP). Clicks on BAEP were given binaurally at a rate of 8/s through a shielded earphone. The clicks were 100/as in duration with a 90 dB peak equivalent sound pressure level and a total summation of 1024 responses. Electrical stimulation on SEP consisted of square wave pulses of 0.1 ms in duration on the median nerve at the wrist at a rate of 5/s, with a total summation of 512 responses. A black-white

106 checkerboard pattern reversing at a rate of 1/s was monocularly given as the stimulus on pattem reversal VEP with a summation of 64 responses. Values greater than the mean + 2.5 SD were regarded as being prolonged.

Biochemical studies Venous blood samples were obtained with heparin-Na or EDTA-2Na as an anticoagulant from patients, family members and controls. Fibroblast culture was performed as described by Kobayashi et al. (1980). Lysosomal enzyme activities. Extracts of leukocytes were prepared by the method of Kampine et al. (1967). The activities oflysosomal enzymes in leukocytes and cultured fibroblasts were determined as described before (Goto et al. 1974, 1983). Fatty acids analysis. Erythrocyte membranes were prepared according to the modified method of Dodge et al. (1963). Lipids were extracted from plasma, erythrocyte membrane suspensions and cultured fibroblasts by the method of Folch et al. (1957). Sphingomyelin (SM), phosphatidylethanolamine (PE), phosphatidylcholine (PC) and phosphatidyl serine (PS) were separated by thin-layer chromatography. Fatty acids in SM, PE, PC and PS from plasma, erythrocyte membranes and cultured fibroblasts were analyzed by high performance liquid chromatography as described previously (Kobayashi et al. 1983; Antoku et al. 1984). Values greater than the mean + 2 SD were regarded as being abnormal. RESULTS

Endocrinological studies The urinary 17-ketosteroids and 17-hydroxycorticosteroids and the serum T3, T4, TSH, ACTH levels and plasma cortisol response after intravenous ACTH loading were normal in all 3 cases. Neurophysiological studies For SEP, case 2 showed a prolonged N 9 latency (11.36ms; control, 9.29 + 0.58 ms); case 3, a prolonged N9-N13 interval (5.38 ms; control, 3.38 + 0.40 ms); all 3 patients, a prolonged N13-N2o interval (8.78, 7.42 and 7.26 ms in cases 1, 2 and 3, respectively; control, 5.89 + 0.48 ms). Case 1 had a prolonged Plo0 latency on both sides for VEP (fight, 144.6 ms; left, 124.8 ms; control, fight 93,0 _+4.7 and left 94.0 + 4.9 ms). Cases 2 and 3 showed a prolonged I-III interval (2.67 and 3.07 ms, respectively; control 2.16 + 0.13) and a prolonged III-V interval (2.38 and 3.19 ms, respectively; control, 1.92 + 0,13 ms) for BAEP, although none of the cases revealed an abnormality of wave I latency. Tba'ee females examined in these 3 families had a prolonged III-V interval (2,24, 2.36 and 2.40 ms in ~ ¢ r s of cases 1 and2 and aunt of case 3, respectively) for BAEP, and a mother of case 2 showed a prolonged N 9 latency (10.88 ms) and N13-N2o interval (7.28 ms) for SEP in addition.

107

L ysosomal enzyme studies Case 1 and his mother had decreased beta-galactosidase activity in leukocytes, using p-nitrophenyl-beta-D-galactoside (22.9 and 28.9, respectively; normal, 51.0-92.0 nmol/mg of protein/h), 4-methylumbelliferyl-beta-D-galactoside (47.0 and 48.5, respectively; normal, 102.3-180.7 nmol/mg of protein/h) and GMl-ganglioside (14.6 and 16.7, respectively; normal 29.6-49.0 nmol/mg of protein/h) as substrates, and decreased beta-galactosidase activity in cultured fibroblasts, using p-nitrophenyl-betaD-galactosidase (104.3 and 146.7, respectively; normal, 217.4-463.5 nmol/mg of protein/h) and 4-methylumbelliferyl-beta-D-galactoside (196.5 and 321.0, respectively; normal, 372.0-1120.0 nmol/mg of protein/h). However, they had normal betagalactosidase activity in cultured fibroblasts, using Gr~,-ganglioside. Other lysosomal enzymes were normal. His father had normal lysosomal enzyme activities. Other patients had normal beta-galactosidase activities in leukocytes. Fatty acid analysis Fatty acid analysis was not done in an elder brother of case 2. Fatty acids in plasma and cultured fibroblasts (Table 1): Cases 1, 2 and 3 showed elevated ratios of C26: o and C24 :o to C22 : o in SM of plasma. Mothers of cases 1 and 2 and 2 maternal aunts and a male cousin of case 3 had elevated ratios of C26: o and C2a : o to C22 : o in SM of plasma. The ratios of C26 : o and C24: o to C22: o in SM of cultured fibroblasts were elevated in cases 1 and 2. Cases 3 and 4 female presumptive carriers were not examined. Fatty acids in erythrocyte membranes (Table 2) We were unable to evaluate the ratios of VLFA to long chain fatty acids (LFA), in PE because of a trace of VLFA. Higher ratios of VLFA to LFA in all 3 patients than TABLE 1 T H E R A T I O S OF VERY L O N G - C H A I N F A T T Y A C I D S TO L O N G - C H A I N F A T T Y A C I D S IN PATIENTS W I T H A L D A N D T H E V A R I A N T S OF A L D A N D T H E I R F A M I L Y M E M B E R S

C26:0/C22; 0 in

Case Case Case Case Case Case

1 (ALD) 2 (AMN) 3 (ALM, SCD) 1: Mother 2: Mother 3: Maternal aunt 1 Maternal aunt 2 Maternal cousin (son of aunt 2) Control

sphingomyelin

C24: o/C22: o in sphingomyelin

Plasma

Cultured fibroblasts

Plasma

Cultured fibroblasts

0.026 0.018 0.019 0.013 0.012 0.011 0.020 0.025

0.101 0.197

1.64 1.27 1.18 1.03 1.08 0.91 1.29 1.56

2.39 2.54

0.004 _+ 0.002 (n = 6)

0.017-0.025 (n = 3)

0.70 + 0.06 (n = 6)

1.82-2.43 (n = 3)

108 in controls were seen in C26 : 0 to C22: 0 in SM, PC and PS ; C26 : 0 to C:o: 0 in PS ; C26 : 0 to C18:0 in SM and PS; C26:0 to C~6:0 in PS; C24:0 to C22:0 and C24:0 to C~6:o in PC and PS. Significant elevations of the mean ratios in the patient group were found in C26:0 to C2z:o in SM, PC and PS; Cz6:0 to Cao:o and C26:0 to C~8:0 in SM and PS; C26:0 to C~6:0 in PC and PS; C24: 0 to Ce2 : 0 in PC and PS; C24:0 to Ceo : 0 in PS" and C24:0 to C~8:0 and Ca4:o to C16:0 in PC and PS. TABLE 2 THE AREA RATIOS OF VERY LONG-CHAIN FATTY ACIDS OR LONG-CHAIN FATTY ACIDS TO LONG-CHAIN FATTY ACIDS IN ERYTHROCYTE MEMBRANE PHOSPHOLIPIDS OF PATIENTS WITH VARIOUS FORMS OF ADRENOLEUKODYSTROPHY AND CONTROLS Ratios

Sphingomyelin Range

Mean

Phosphatidylcholine

Phosphatidylserine

Range

Mean

Range

Mean

C26:o / C22 : o

patient control

0.131-0.198 0.071-0.177

0.162" O.1O0

3.524-5.063 1.082-1.968

4.277** 1.585

0.242-0.466 0.071-0.115

0.329** 0.092

C26:o / C2o:o

patient control

0.420-0.920 0.170-0.420

0.727* 0.280

0.580-0.880 0.390-0.790

0.763 0.554

0.100-0.220 0.030-0.050

0.147"* 0.038

C26: o/ C18:o

patient control

O.150-0.360 0.040-0.130

0.257* 0.093

0.004-0.006 0.002-0.004

0.005 0.003

0.0006-0.0009 0.0001-0.0003

0.0007*** 0.0002

C26: o/ C~6:o

patient control

0.033-0.066 0.018-0.045

0.045 0.029

0.0012-0.0017 0.0007-0.0013

0.0015* 0.0009

0.0044-0.0089 0.0009-0.0019

0.0061"* 0.0013

C24:o / C22:o

patient control

2.270-2.760 1.610-2.470

2.580 2.222

2.880-5.240 0.740-2.170

4.387** 1.370

1.010-1.270 0.790-0.950

1.150"* 0.878

C24:o/ C2o:o

patient control

7.240-14.540 7.270-9.070

11.540 8.160

0.470-1.090 0.350-0.720

0.800 0.494

0.410-0.560 0.380-0.400

0.507* 0.354

C24:o/ C~s:o

patient control

2.560-5.030 1.430-3.710

4.043 2.394

0.0044-0.0061 0.0016-0.0039

0.0050* 0.0026

0.0023-0.0028 0.0015-0.0021

0.0025* 0.0018

C24:o / C16:o

patient control

0.560-0.920 0.280-1.240

0.697 0.658

0.0014-0.0016 0.0005-0.0012

0.0015"* 0.0008

0.018-0.023 0.008-0.016

0.021"** 0.012

C22:o/ C2o:o

patient control

3.190-5.370 1.610-3.990

4.407 3.150

0.160-0.210 0.230-0.500

0.177 0.388

0.410-0.480 0.300-0.460

0.443 0.406

C22:o/ Cl8:o

patient control

1.130-1.830 0.350-1.770

1.547 0.958

0.0009-0.0015 0.0013-0.0025

0,0012 0.0020

0.0020-0.0023 0.0017-0.0025

0.0022 0.0020

C22: o/ Cj6:o

patient control

0.230-0.330 0.170-0.590

0.270 0.292

0.0003-0.0005 0.0004-0.0007

0.0004 0.0006

0.017-0.019 0.009-0.019

0.018 0.014

C2o:o / Cl8:o

patient control

0.310-0.390 0.170-0.440

0.350 0.300

0.0042-0.0093 0.0046-0.0055

0.0070 0.0052

0.0041-0.0055 0.0042-0.0054

0.0049 0.0049

C2o: o/ C~6:o

patient control

0.042-0.078 0.046-0.150

0.064 0.096

0.0013-0.0030 0.0013-0.0017

0.0021 0.0015

0.040-0.045 0.026-0.040

0.042 0.033

C~s: o/ C~6 : o

patient control

0.140-0.220 0.110-0.330

0.180 0.244

0.260-0.320 0.270-0.310

0.300 0.292

7.790-9.790 5.290-7.510

8.537 6.620

* P < 0.05, ** P < 0.005, *** P < 0.001. Controls: Sphingomyelin, phosphatidylcholine and phosphatidylserine (n = 5).

109 DISCUSSION An increase in VLFA has been used to establish the diagnosis in presumptive ALD cases and to confirm the cartier state (Kawamura et al. 1978; Moser et al. 1981; Tsuji et al. 1981; Molzer et al. 1982; Kobayashi et al. 1983). Case 1 showed the characteristic clinical manifestations of leukodystrophy and an increase of VLFA. His mother also had an increase in VLFA, while his father showed no such biochemical abnormality. Case 2 showed the characteristic clinical manifestations with the increase of VLFA and can be diagnosed as having AMN. His brother was clinically diagnosed as having Schilder's disease. Their mother showed an increase in VLFA without neurological abnormalities. AMN and ALD coexist in this family, and are probably due to the same abnormal gene as suggested by Davies et al. (1979). Case 3 showed manifestations of spinocerebellar degeneration, and a maternal male cousin also had similar manifestations. These patients had an increase in VLFA. Two maternal aunts of case 3 also had the increase of VLFA without abnormal neurological fmdings. Our 3 patients and a cousin of case 3 can be diagnosed as having ALD or a variant, and their mothers and maternal aunts are thought to be female carders. An adrenal insufficiency has been described in some ALD patients before the appearance of neurological manifestations (Schaumburg et al. 1975). Our patients did not, however, demonstrate overt adrenal insufficiency at presentation, although they may show abnormal adrenal function at a later stage. A few authors reported that ALD patients and female carriers showed prolongation of the I-V interval of the BAEP, slowing of central conduction time of the SEP and delay in the major positive peak of the flash VEP (Markand et al. 1982; Garg et al. 1983). The abnormal prolongation of these peak and interpeak latencies indicates delayed conduction time in the respective neural pathways, probably due to demyelination. Case 1 had prolonged P~oolatency of the VEP which can be correlated with the clinical visual disturbance, possibly due to demyelination in the occipital visual area as revealed by the cranial CT scan. He also had a prolonged N~3-Nzo interval of the SEP, which suggests impairment of the central sensory pathways. Vercruyssen et al. (1982) reported prolonged I-V intervals of the BAEP and slowing of the central conduction time of the SEP have been reported in AMN patients. Case 2 had a delay in the N 9 and N13-N2o intervals of the SEP and in the I-III and III-V intervals of the BAEP suggesting impairment of the peripheral and central sensory pathways and the auditory pathways in the brain stem (Jones 1977, 1979; Chiappa 1980; Green et al. 1980; Kjaer 1980; Vercruyssen et ai. 1982; Garg et al. 1983). Case 3 revealed delayed N9-N13 and N13-N2o intervals of the SEP and I-III and III-V intervals of the BAEP which indicate impairment of the central sensory pathways and auditory pathways in the brain stem. The neurophysiological results in these 3 patients correlated well with the clinical neurological manifestations. Three presumptive female carriers without neurological abnormalities in these families showed prolonged III-V intervals of the BAEP and/or delayed N 9 and N~3-N2o intervals of the SEP. These results suggest that some abnormalities of the neural pathways exist even in the female carriers, as suggested by Garg et al. (1983). Our studies also suggest that the combination of the three different

110 evoked potential modalities provides further information concerning the functional state of the nervous system, particularly subclinical dysfunction, as suggested by several authors (Chiappa 1980; Green et al. 1980; Kjaer 1980; Khoshbin et al. 1981 ; Markand et al. 1982). Case 1 and his mother had decreased beta-galactosidase activity, while his father showed normal beta-galactosidase activity. The reason why this biochemical abnormality exists in our patient and his mother is not clear. An increase in VLFA in tissues has been reported in ALD (Igarashi 1976a,b). Recently, Tsuji et al. (1981) demonstrated the increased ratio of C26:o to C22:o in erythrocyte membrane" sphingomyelin of ALD patients. We also found a statistically significant increase in saturated VLFA in erythrocyte membrane sphingomyelin in patients with ALD and variant of ALD and carriers (Kobayashi et al. 1983). Erythrocyte membrane lipids are composed of phospholipids, free cholesterol and a small amount of glycolipids (Ways et al. 1964), and the erythrocyte phospholipids contain main 4 classes: SM, PE, PC and PS. No one has, however, referred to the VLFA in PE, PC and PS except for ours (Antoku et al. 1984). The increased VLFA in PC and PS, also found in our patients, revealed a more conspicuous difference than in SM, compared with control values. This accumulation of VLFA in tissues in ALD could be due to increased VLFA uptake, increased VLFA synthesis or decreased VLFA catabolism. The ratios ofC26 : o to other saturated even-chain fatty acids in our patients were definitely higher than those of controls. The ratios ofC22 : o to C2o: o, C~8: o or C~c,: ~ in PC and C22: o to C16: o in SM were decreased in our patients. The ratios of C2o: o to C18 : o or Ci6: o and C18 : o to C16: o did not, however, show a significant difference between our patients and the controls, and agrees with the results of Moser et al. (1981). These results suggest that our patients may have some defects in the degradation from saturated VLFA to saturated LFA, as suggested by Moser et al. (1980, 1981). Recent studies suggested an important roles of peroxisomal beta-oxidation of VLFA (Moser et al. 1984; Singh et al. 1984). The significance of and the metabolism of the increased saturated VLFA in our patients are, however, still obscure. Further investigations are necessary to elucidate the pathogenesis of ALD. ACKNOWLEDGEMENTS The authors would like to thank Drs. Satoshi Suzuki, Takeo Yoshimura and Hirotoshi Umezaki and Miss Nobue Shinno for their help. REFERENCES Antoku, Y., T. Sakai, I. Goto, H. Iwashita and Y. Kuroiwa(1984) Adrenoteukodystrophy-- Abnormality of very long-chainfatty acids in erythrocytemembrane phospholipids, Neurology, 34: 1499-1501. Chiappa, K.H. (1980) Pattern shift visual, brainstem auditory, and short-latencysomatosensoryevoked potentials in multiple sclerosis, Part 2, Neurology, 30:110-123. Davis, L.E., R.D. Snyder, D.N. Orth, W.E. Nicholson, M. Kornfeld and D.F. Seelinger (1979) Adrenoleukodystrophyand adrenomyeloneuropathyassociated with partial adrenal insufficiencyin three generations of a kindred, Amer. J. Med., 66: 342-347.

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