Neonatal adrenoleukodystrophy

SUMMARY Nine cases of neonatal adrenoleukodystrophy are described. All patients had abnormal facial features, moderate to severe hypotonia, hepatomegaly, and retinitis pigmentosa. The clinical course was rapidly progressive in six cases and more protracted in three others. Biological signs of adrenal insufficiency were present in five cases. CT scan showed a demyelinating process in four patients. Trilamellar inclusions were found in the liver of four cases and dark and complex lipidic inclusions in three other cases. In the three necropsied patients there was severe alteration of the white matter involving particularly the cerebellum in two cases. Gyral and cytoarchitectonic disturbances were absent in all three cases. Increased plasma levels of very long chain fatty acids (8/8), phytanic acid (7/8) and bile fluid trihydroxycoprostanic acid (2/4) confirmed the deficiency of multiple peroxisomal enzymes. Clinical, histopathological and biochemical findings of these nine cases are compared to those reported in other neolatal adrenoleukodystrophy cases and to those of other neonatal peroxisomal disorders, that is cerebro-hepato-renal syndrome of Zellweger and infantile Refsum's disease.


78
showed some similarity to those of the patient described by Brown et al.'0 In all those three patients, psychomotor retardation became obvious only by the age of 4-7 months, and failure to thrive and/or jaundice were the most important initial clinical problems. Vision was moderately impaired up to 4-6 months. Psychomotor development stopped at 7 and 12 months respectively for patients 6 and 7, followed by a period of rapid decline.

Results
LABORATORY, RADIOLOGICAL AND NEUROPHYSIOLOGICAL STUDIES Table 2 summarises the main data in eight patients. Liver dysfunction was present in. three cases, mild adrenal insufficiency in five patients (1, 4, 5, 8,9) and CSF protein was increased in three. Elec- tromyography (EMG) showed polyphasic motor potentials in three patients (1, 2, 6) suggesting a neurogenic process but nerve conduction velocity (NCV) was decreased in two cases only (1, 2). In case 9, EMG showed decreased duration and amplitude of unit potentials suggestive of a myopathic process. CT scan showed a demyelinating process in five patients (1, 4,5,7,9).
As shown in table 3, the levels of plasma C26: 0 fatty acid were increased in eight patients, and C26: 1 in seven. All but one patient had increased C24:O/C22:0 ratios. C22:0 levels were decreased in four patients, whereas C24: 0 levels appeaied within the normal range in six. One patient (case 2) had only slightly increased levels of C26: 0. Phytanic acid levels were strongly increased in two patients (6,8) and slightly in five.
Bile acids analysis from samples of duodenal fluid aspirates were performed in two patients (6,8) and showed increase levels of trihydroxycoprostanic acid. Small traces of dihydroxycoprostanic and varanic acids were found. In two other patients (cases 2 and 7), serum bile acids concentrations were normal.  2). These jet-black structures were found to have irregular, angular outlines and a distinct limiting membrane with a cleft-like space separating them from the surrounding cytoplasm. These spaces were probably due to technical artefacts related to the hardness of their structure. The dark bodies were generally located in the hepatocytes near bile canaliculi but were also found in some duct and mesenchymal cells. Bile canaliculi with loss of mic-81 rovilli were sometimes dilated and their lumen was filled with deposits of loose membranous material. In the second liver sample of case 4, the homogeneous bodies and the dilatation of bile canaliculi could no longer be found but the steatosis was more marked. Lipid vacuoles have an unusual pattern and were divided in compartments by a ribbon-like material of interconnecting membranes (fig 3). In hepatocytes this material appeared to result from clustering of altered smooth membranes (fig 4). No trilamellar inclusions were found in those 4 cases.
In cases 6, 7, 8 and 9, mesenchymal cells contained ovoid or spindle-shaped inclusions with irregular outlines and some spike-like protusions. Their light or dense matrix was filled with globules of various densities and rectilinear lamellae showing a trilamellar structure (fig 5a). Hepatocytes also included heterogeneous dense bodies and trilamellar structures (fig 5b). Lipid vacuoles identical to those encountered in cases 1, 2, and 4 were scanty.
Peroxidatic activity of catalase in peroxisomes was studied in case 8: no activity could be detected. However, the hepatocytic cytoplasm of all the liver samples showed the presence of microbodies whose structure looked like peroxisomes. Their number and size were strikingly decreased (fewer than 50 microbodies per hepatocyte), and they were even absent in case 8. In all our cases, the mitochondria were morphologically normal.
Neuropathological study Brain. The brain weight was increased in two of -::  the three examined cases (1, 4). No significant abnormalities of cortical cytoarchitecture were found except in case 2 in which there was severe cortical atrophy. In all cases, there was a severe degeneration of the white matter involving both cerebral and cerebellar hemispheres while axons were preserved. In case 4, demyelination was striking in the cerebellar white matter and in the tegmentum of the brain stem ( fig 6). In cas!W 1, demyelination was also particularly important in the cerebellar white matter with many cuffs of mononuclear cells (fig 7). Heterotopic Purkinje cells were found to be aggregated in irregular clumps in the subcortical areas of the cerebellar cortex in cases 1 and 4 (fig 8).
No heterotopic neurons were observed in the corti- cal layers or in the subcortical white matter of the cerebral hemispheres. Each olivary nucleus was found to have normal morphology. Electron microscopic study was performed upon necropsy specimens. Ultrastructural preservation of tissue was poor. In addition to numerous lipid droplets, histiocytic cells and astrocytes contained dark inclusions with complex lamellar profiles and irregular electron dense bodies. Neither trilamellar inclusions nor mitochondrial abnormalities could be observed.
Sural nerve biopsy specimen (case 1) showed a decreased density of myelinated sheaths with preservation of Schwann cells and axons. No trilamellar inclusions were found in the Schwann cells.  Adrenal gland study (case 1) The adrenal glands were atrophic and weighed 2 g each. The main histological features were the presence of ballooned cortical cells in the zona fasciculata and reticularis, and the persistence of the fetal zone of the adrenal cortex. No striated cell was seen. Ultrastructural study was not performed.

Discussion
The criteria used for assignment of a patient to the neonatal adrenoleukodystrophy category have been the onset of disability in the neonatal period, involvement of adrenal cortex and cerebral white matter and increased very long chain fatty acids in plasma, cultured skin fibroblasts, brain and adrenal gland.5-'0-2 3 Clinical and biological data warrant the inclusion of our nine patients in this category. All patients had in common development delay from birth, craniofacial dysmorphism, moderate to severe hypotonia, hepatomegaly and retinitis pigmentosa. Pigmentary changes in the eyes were obvi-83 ous only after 4-6 months of age, but the electroretinogram was early extinguished. Elevated levels of plasma very long chain fatty acids confirmed the diagnosis. Pipecolic acid was found to be normal in the urines of three cases studied (1, 4,8) as in some other neonatal adrenoleukodystrophy cases.'2 Increased ACTI-f plasma levels suggesting an abnormal adrenal function were found in four cases, and low morning serum cortisol without rise after IV ACTH (case 1) in a fourth case. Adrenal glands were studied in one case showing atrophy and ballooned cortical cells without striated cells. The apparently normal adrenal function observed in two cases with normal ACTH levels is not incompatible with this diagnosis. Powers et a124 have shown that fetal adrenal glands from fetuses (childhood adrenoleukodystrophy) with abnormal fatty acids in cultured amniotic cells contain striations and lamellae pathognomonic of adrenoleukodystrophy. However, in this latter disorder, it is shown that signs of adrenal failure may be absent or of delayed onset.' 2 A demyelinating process was observed in three necropsied cases (1, 2, 4) and was demonstrated on CT scan in three other patients 3, 7, 9. The distinction between neonatal adrenoleukodystrophy and childhood adrenoleukodystrophy is clear but the situation in regard to cerebro-hepato-renal syndrome and neonatal adrenoleukodystrophy is less well defined.
The resemblance includes the onset of disability in the neonatal period, hypotonia with convulsions, retinal pigmentary degeneration with the presence of bileaflet inclusions and increased very long chain fatty acids in the retina,25 hepatomegaly with micronodular cirrhosis and trilamellar fatty acid inclu- 21 26 ad21an sions, striated adrenocortical cells'4 21 and increased very long chain fatty acids in plasma'' 21 27 and brain.'7 However, infants with cerebro-hepatorenal syndrome have a more severe neurologic deficit and rarely survive beyond the fifth month. The strikingly dysmorphic facial features are observed in all cerebro-hepato-renal syndrome patients, which is not the case in neonatal adrenoleukodystrophy.
Eventually, cerebral abnormalities observed in neonatal adrenoleukodystrophy as found in our cases are different from those encountered in cerebro-hepato-renal syndrome. 21 28 As in some neonatal adrenoleukodystrophy reports,5 7 0 we found no gyral and cytoarchitectonic disturbances in the cerebral hemispheres. Some Purkinje cells were found to be present in the subcortical white matter of the cerebellum but the inferior olivary nuclei showed a normal morphology.
Abnormalities of gyration with "micropolygyria" have been reported by some authors'69 but the highly characteristic disorder of neuronal migration Protected by copyright. http://jnnp.bmj.com/ Aubourg, Scotto, Rocchiccioli, Feldmann-Pautrat, Robain which seems to be constant in all cerebro-hepatorenal syndrome patients has never been found in neonatal adrenoleukodystrophy.
The situation in regard to neonatal adrenoleukodystrophy and infantile Refsum' s disease is more complex. The clinical features of the infantile Refsum' s disease include retinitis pigmentosa, deafness, facial dysmorphia, growth and/or mental retardation, peripheral neuropathy (inconstant) and hepatomegaly.'9 20 29-32 Liver biopsy shows mild to severe fibrosis and trilamellar inclusions in parenchymal and non parenchymal liver cells,'9 20 identical to those described in adrenoleukodystrophy, neonatal adrenoleukodystrophy and cerebro-hepato-renal syndrome. The clinical course of infantile Refsum' s disease is not as severe as in most patients with neonatal adrenoleukodystrophy and some patients survive beyond the tenth year. The patients described by Brown et al'0 and Noetzel et al'3 as neonatal adrenoleukodystrophy share the same clinical features as the three patients previously described by one of us,'9 and seem to have followed the same protracted course, these three children being still alive at 7, 8, and 11 years. Increased levels of plasma very long chain fatty acids2'33 and abnormal bile metabolites'832 have been documented in infantile Refsum' s disease cases. Similarly, plasma phytanic acid levels are increased in infantile Refsum's disease'920 as in cerebro-hepato-renal syndrome and neonatal adrenoleukodystrophy,2' reflecting a deficiency in phytanate oxidase'8 20 3' which is supposed to be located in part in peroxisome.'82' All those findings suggest that infantile Refsum's disease, cerebro-hepato-renal syndrome and neonatal adrenoleukodystrophy are closely related disorders involving multiple peroxisomal enzymes deficiency.
If neonatal adrenoleukodystrophy and infantile Revsum's disease are overlapping disorders, an attempt must be made to draw borderline between them. The severity of the disease in children with neonatal adrenoleukodystrophy or infantile Revsum's disease varies greatly and three different clinical pictures at least are encountered. In some patients (refs 4, 9, our cases 1 to 5 and 9), the neurological disability is as severe as in cerebrohepato-renal syndrome. Those children have no intellectual development and survive rarely beyond the second year of life. In other patients, the clinical course is more progressive. Neurological development is delayed from birth but these children may acquire some psychomotor development. Most of them can apparently see and hold the head following the first 6 months of life. Failure to thrive, moderate developmental delay, hepatomegaly, hypotonia, decreased hearing and impaired visual acuity call for attention between 3 and 6 months of life. For some patients, psychomotor development stops between 1 and 2 years and is followed by a rapid decline and death (refs 5-8, our cases 6 and 7). In the third group of patients, the course is more progressive. Some children can sit or walk alone by the age of five years and may be still alive after the age of ten years.'0 13 19 32 Absence of hepatic peroxisomes is considered to be an essential clue for the diagnosis of cerebrohepato-renal syndrome but has been reported also to occur in neonatal adrenoleukodystrophy'°12 Peroxisomes could not be identified after histochemical reaction in one of our cases. In the other cases, microbodies whose ultrastructural appearance was similar to peroxisome were found in all the liver biopsy samples but peroxidatic activity of catalase was not studied. Microbodies were detected in the liver of our cases, but their number and size were greatly decreased as also reported by Goldfischer et al in neonatal adrenoleukodystrophy. 34 Various types of inclusions have been described in the liver of neonatal adrenoleukodystrophy and infantile Refsum' s disease including especially trilamellar inclusions (refs 11, 19, 20, and four of our cases). Other types of hepatic inclusions have been reported as "arced lamellae in plump, spindle shaped cluster",5 "packed lamellar profiles"e. The dark inclusions observed in three of our cases (1, 2, and 4) seem to be peculiar on account of the homogeneous matrix, their irregular outlines and their hardness. Their ultrastructure resembled those observed in some cases of neonatal cholestasis35 but signs of cholestasis were mild or absent emphasising the pecularity of this ultrastructural pattern. These inclusions have also some similarity with those observed in cerebro-tendinous xanthomatosis,36 a possible peroxisomal disorder.'5 The second biopsy of case 4 shows that the ultrastructure may change in the course of time. Bile canaliculi dilatation and jet-back inclusions were less numerous in hepatocytes and were replaced by empty and lipid-filled septate vacuoles. Those complex lipidic inclusions have also been reported in lymph nodes' and thymus9 of neonatal adrenoleukodystrophy. If trilamellar inclusions are probably related to the accumulation of very long chain fatty acids, other lipidic inclusions probably contain more complex lipids. The various ultrastructural findings may also correspond to different phases in the evolution of the disease.
Cerebral pathology has not been studied in infantile Refsum' s disease, since most patients are still alive, whereas in neonatal adrenoleukodystrophy it is mainly a demyelinating process. In neonatal adrenoleukodystrophy the distribution of the lesions 84 Protected by copyright. Neonatal adrenoleukodystrophy is variable, and the cerebellum may be more involved than the cerebral hemispheres (refs 4, 7, and our cases). The severity of the myelin involvement is more severe than in cerebro-hepato-renal syndrome, including moderate to severe gliosis, and perivascular cuffs of mononuclear cells. This intense inflammatory cell response occuring within the demyelinated areas (as in childhood adrenoleukodystrophy) is obvious in neonatal adrenoleukodystrophy but absent in cerebrohepato-renal syndrome.2' Trilamellar inclusions are also found in brain macrophages478 1 1 but other complex lipid and membrane aggregate inclusions (refs 4, 9, our case 4) may be seen. As in the liver, the aspect of the lipid storage in the brain is variable and more data are required to determine whether the difference in the distribution of the lesions and in the morphological aspects of the inclusions are significant.
Biochemical abnormalities reflecting impairment of various peroxisomal enzymes have been documented in neonatal adrenoleukodystrophy and infantile Refsum' s disease as in cerebro-hepatorenal syndrome including impaired oxidation of very long chain fatty acids and phytanic acid (refs 10, 17, 18, 21, 33, and our cases), bile synthesis defect with elevated levels of certain intermediates (refs 18, 32, 37, and our cases 6 and 8) and increased plasma and urine pipecolic acid.'8 38 Recent studies have emphasised the role of peroxisome in the synthesis of glycerol-ether lipids39 and the presence of a deficiency of dihydroxyacetone phosphate (DHAP) acyltransferase and alkyl-DHAP synthetase404' in cerebro-hepato-renal syndrome. The presence of a deficiency in plasmalogens synthesis is under investigation in our patients but it must probably be assumed that neonatal adrenoleukodystrophy and infantile Refsum' s disease patients are also deficient in glycerol-ether lipids synthesis. In the absence of specific biochemical abnormalities, it must be determined whether variable deficiency in these peroxisomal enzymes or other biochemical abnormalities exist accounting for the wide clinical and histopathological spectrum encountered in these disorders.

Conclusions
Cerebro-hepato-renal syndrome, neonatal adrenoleukodystrophy and infantile Refsum' s disease are peroxisomal disorders that share many clinical, histopathological and biochemical features. In the absence of specific biochemical abnormalities one should determine if the large clinical and histopathological spectrum of these diseases is related to the variable deficiences in peroxisomal enzymes 85 or other biochemical disturbances. However, some major discrimination allows a temporary classification. Severe clinical outcome, and neuronal migration disturbances, are always encountered in cerebro-hepato-renal syndrome suggesting that this disease is the consequence of a specific gene mutation. At the present time, there is no definite distinction between neonatal adrenoleukodystrophy and infantile Refsum's disease but the different clinical courses observed in these diseases suggest that there may be at least three phenotypic variants of a specific enzyme deficiency or three different mutations. Liver and brain histopathological findings are to date unhelpful in establishing a nosological classification but may supply some clarification pending to a better understanding of peroxisomal function, specially outer-inner membrane transport of enzymes.