Acta Neuropathol (1992) 83:190-195
Acta Heuropathologica ~) Springer-Verlag 1992
Autopsy findings in two siblings with infantile Refsum disease C.W. Chow 1, A. Poulos 2, A. J. Feilenberg 2, J. Christodoulou 3, and D. M. Danks 3 1 Department of Anatomical Pathology, Royal Children's Hospital, Parkville, Melbourne,Victoria 3052, Australia 2 Department of Chemical Pathology, Adelaide Children's Hospital 3 Murdoch Institute, Royal Children's Hospital, Melbourne Received May 17, 1991/Accepted August 6, 1991
Summary. Recognition of adrenal atrophy during a review of autopsy findings in two sisters who died at 8 months and 3 1/2 years prompted estimation of very long chain fatty acids, phytanic acid and pristanic acid on wet liver fixed in formalin for 12 years. These were shown to be markedly increased and defects in multiple peroxisoreal functions and decrease in particulate catalase were shown in cultured fibroblasts, confirming an abnormality of peroxisomal biogenesis. The patients had presented with failure to thrive, recurrent diarrohea and vomiting, poor mental development, retinal pigmentation, blindness and in the older patient deafness, with only mild dysmorphic features. Autopsy in the older patient showed adrenal atrophy, cirrhosis, and foamy histiocytes in multiple organs. The brain showed no demyelination, little cytoarchitectural abnormality, occasional perivascular histiocytes in the grey matter and meninges and prominent Purkinje cells in the molecular layer of the cerebellum. In the younger patient the changes were very subtle in spite of the marked clinical similarity. Despite the young age at death the clinicopathological features are most suggestive of infantile Refsum disease. In many situations anatomical pathology can be very useful in the recognition and study of peroxisomal disorders. Key words: Sisters - Adrenal atrophy - Cirrhosis Ectopic Purkinje cells - Young age at death
radiology, biochemistry and morphology, the last including both ultrastructure of peroxisomes and general autopsy findings. Broadly the disorders have been divided into those associated with defects of biogenesis of peroxisomes, those with abnormalities of several peroxisomal functions, and those in which only one single enzyme is at fault [17, 27]. The first group is represented by Zellweger syndrome (ZS), neonatal adrenoleukodystrophy (NALD) and infantile Refsum disease(IRD), with hyperpipecolic acidaemia considered by some not to be a separate entity [10]. Although there are many autopsy reports on ZS [4, 5, 24] and N A L D [2, 8, 9], there has been only one on IRD [22]. Two sisters were seen at the Royal Children's Hospital (RCH) and died in the 1960's and 1970's. At that stage no obvious diagnosis was made. Review of the autopsy prompted biochemical investigations on material kept at the time of the post-mortem examination. This confirmed an abnormality of peroxisomal biogenesis. Correlation with clinical findings suggests IRD as the most likely diagnosis. The autopsy findings in these two cases are presented, together with a discussion on the difficulties in an exact classification in some cases and the role of morphology in the investigation of this group of diseases.
Case reports Patient 1
Since the absence of peroxisomes was noted in Zellweger syndrome by Goldfischer et al. [6], the list of diseases associated with abnormal peroxisomal structure and/or function has greatly expanded. Classification of many patients, however, remains arbitrary as the enzyme defect and molecular biology remain unknown, and is frequently based on a combination of clinical features,
Offprint request to: C.W. Chow (address see above)
This girl born in 1964 weighing only 2.15 kg at full term was first seen at RCH at the age of 5 weeks for an inspiratory stridor. Failure to thrive was a problem from the first weeks, and episodes of respiratory infection and of diarrohea and vomiting occurred repeatedly. Apart from three wide fontanelles and open sutures, there were no significant dysmorphic features. Stool examination showed no fat globules and sweat test was negative. Blood examination showed occasional Burr cells and acanthocytes. Chromosomes were normal. X-raysshowed no abnormalities in the epiphyses. Addison disease was considered but electrolytes were normal and 24-h urine showed 17-ketosteroid of 0.6 mg and
191 17-ketogenic steroid of 0.3 mg. At 6 months she was noticed to have a roving nystagmus. Ophthalmological consultation showed that there was no pupillary reaction to light, and fundal examination showed coarse brown-black pigmentation in the peripheral fields. Her conditions remained poor with no obvious neurological development and she died at 8 months following a cerebral haemorrhage.
Patient 2 This girl was born at full term in 1973 after a normal pregnancy.The birth weight was 2.61 kg. She was seen at 6 months because of failure to thrive. There were no craniofacial dysmorphic features.
Fig. 1 a,b. Adrenal showing marked cortical atrophy in older sibling (a) and preserved architecture in younger (b). H & E , • 40 Fig. 2. Liver in older sibling showing well-established cirrhosis. Masson's trichrome, x 48
Stool examination showed some fat globules. The sweat test was negative and a duodenal biopsy was normal. Her motor milestones were markedly delayed. She did not follow objects with eyes but did respond to aural stimuli by turning her head. Fundal examination showed peripheral equatorial clumps of pigment. Her liver was enlarged to 2-3 cm below the costal margin. Liver biopsy showed moderate fibrosis and occasional PAS-positive histiocytes (PASH) in the portal tracts. No tissue was processed for electron microscopy. X-rays showed moderate changes of rickets but no obvious abnormalities in the epiphyses. The serum calcium concentration was 6.4 mg%, and phosphate 3.0 mg%. In view of the family history she was considered to have a severe neurodegenerative disorder although the nature of this was unclear. Over the next 3 years she was admitted on multiple occasions for vomiting,
Fig. 3. Inferior olive showing focal concentration of neurons at the periphery of the nucleus. H & E , • 40 Fig. 4 a,b, Cerebellum showing marked displacement of Purkinje cells into molecular layer in older sibling (~), and normal location in younger (b). H & E , x 64
192 diarrhoea and dehydration. It became obvious that she was severely mentally retarded. At 3 years her head control was very poor. Her fontanelle was still open and she had developed cataracts and become deaf. At 3 1/2 years she was again admitted with dehydration. In spite of intravenous fluid, her serum sodium varied from 111 to 120 mEq/1. Her conditions gradually deteriorated and she died. The parents were unrelated. There were three other girls who were alive and healthy.
Biochemical findings Tissue was only available from patient 2. Firstly wet tissue of the liver, kept in formalin for 12 years, was assessed for the concentration of very long chain fatty acids (VLCFA), PA, and PTA. The results are shown in Table 1. As the concentrations of VLCFA, PA and PTA were strongly suggestive of a disorder of peroxisomal function, other tests were conducted on the cultured fibroblasts to characterise the disease further. Deficiencies in a number of enzyme activities including phytanic acid oxidase, particulate catalase, DHAP-AT and alkyl DHAP synthase were observed.
Biochemical methods Hexacosanoic acid/docosanoic acid (26:0/22:0) and lignoceric acid/docosanoic acid (24:0/22:0) ratios, and phytanic (PA) and pristanic acid (PTA) levels were measured as described earlier [14, 15]. Dihydroxyacetonephosphate acyltransferase (DHAP-AT) and alkyl dihydroxyacetonephosphate synthase (alkyl DHAP synthase) activities were assayed as described by van Crugten et al. [23] and Singh et al. [20], respectively. Phytanic acid oxidation was measured as described by Poulos et al. [13].
Autopsy findings Patient 2 As it was the autopsy findings in this patient which prompted the review and further investigations, the findings in this 3 1/2 year old girl will be presented first. The major abnormalities were seen in the adrenals, the liver and the brain. The adrenals were markedly atrophied, with the cortex replaced by scattered cells with foamy and striate cytoplasm and an occasional nodule of cortical cells (Fig. la). The medulla was normal. The liver showed wellestablished cirrhosis (Fig. 2). There was no significant haemosiderosis. Prominent PASH were present in the liver as well as in the lymph nodes, spleen and bone marrow in moderate numbers. The kidneys were normal, specifically showing no cortical cysts. In spite of the severe neurological deficit, the brain was on the whole remarkably well preserved. The cerebrum did not show any cytoarchitectural abnormality. The inferior olives retained the normal wavy outlines. There was focal concentration of neurons along the periphery of the nucleus (Fig. 3), but nothing like the prominent peripheral palisading in ZS [4]. Demyelination was not seen. Occasional small perivascular clusters of PASH were seen in the pontine nuclei, molecular layer of the cerebellum and the meninges. The cerebellar cortex showed moderate decrease in granular cells. The Purkinje cells were extensively displayced into the molecular layer which also showed mild gliosis (Fig. 4a). Due to the adrenal atrophy a peroxisomal disorder was suspected and biochemistry was requested on the material kept at the time of autopsy.
Patient 1 In this 8-month-old baby girl the adrenals were moderately decreased in size weighing 1 g each. The cortical architecture was, however, normal (Fig. lb). Careful searching with special staining showed occasional PASH between the columns of the zona fasciculata. The liver showed no significant fibrosis. Only an occasional PASH was seen in the liver, but not in the spleen, lymph nodes, or bone marrow. The kidney showed only a few small cortical cysts. The brain showed a large haemorrhage in the left frontal lobe. There was no cytoarchitectural abnormality, demyelination or gliosis. PASH were not seen. In the cerebellar cortex, Purkinje cells were normally situated at the junction of the molecular and granular layers (Fig. 4b).
Discussion O n c e a p e r o x i s o m a l disorder was suspected, the first screening test was an estimation of the c o n c e n t r a t i o n o f V L C F A [1]. S e r u m or fresh tissue was n o t available, b u t it was t h o u g h t that p e r h a p s formalin-fixed liver might still be suitable as the fatty acids are saturated and, therefore, stable. C o m p a r e d with control specimens, s o m e of w h i c h h a d b e e n k e p t in a similar state for c o m p a r a b l e periods, V L C F A , P A and P T A were m a r k e d l y elevated, strongly suggesting t h a t a disturb a n c e of p e r o x i s o m a l f u n c t i o n was i n d e e d involved.This highlights the i m p o r t a n c e of k e e p i n g material at the time of a u t o p s y in patients w h e n the n a t u r e of a suspected g e n e t i c / m e t a b o l i c disease is unclear. Fresh tissues f r o m multiple organs and fibroblast cultures are obviously ideal. Paraffin blocks can be suitable sources of D N A , and as s h o w n here, fixed wet tissue can occassionally also be e x t r e m e l y useful, s o m e t i m e s m a n y years after the d e a t h o f the patients. C o n f i r m a t i o n of the disturbance in p e r o x i s o m a l function was o b t a i n e d by d e m o n s t r a t i n g deficiencies in the activity of a n u m b e r of e n z y m e s , including p h y t a n i c acid oxidase, D H A P - A T , alkyl D H A P synthase, and particulate catalase activity. E a c h of these activities has b e e n r e p o r t e d to be associated p r e d o m i n a n t l y with peroxisomes in rat liver and h u m a n skin fibroblast [18, 19, 25]. D u e to the unavailability of suitable tissues for ultrastructural analysis we were n o t able to c o n f i r m m o r p h o logically an a b n o r m a l i t y in p e r o x i s o m a l biogenesis, a l t h o u g h the e n z y m o l o g i c a l data s h o w n in Table 1 are consistent with this view. T h e next p r o b l e m was to d e t e r m i n e w h e t h e r the features fit b e t t e r with ZS, N A L D or I R D . S o m e selected clinicopathological features of these three entities c o n s i d e r e d o f discriminatory value are c o m p a r e d with the index patients in Table 2. T h e classical Z S patients present in the n e o n a t a l p e r i o d with typical facies, severe h y p o t o n i a , seizures, c h o n d r o d y s p l a s i a p u n c t a t a , die within w e e k s or m o n t h s , and at a u t o p s y s h o w p r o m i n e n t cortical cysts in the kidneys, h a e m o s i d e r o s i s and a variable d e g r e e o f fibrosis of the liver, and m a r k e d migration abnormalities in t h e brain, involving m o s t conspicuously the inferior olives, the c e r e b r u m and the cerebellum [4, 5, 24]. N A L D patients m a y also present in the perinatal p e r i o d with h y p o t o n i a , seizure and d y s m o r p h i c feature, but generally w i t h o u t t h e typical facies of ZS or c h o n d r o d y s p l a s i a p u n c t a t a . T h e y t e n d to die later,
193 Table 1. Biochemical changes in patient 2
Patient 2
Normal
Phytanic acid (~g/g wet weight) Liver 1910 Pristanic acid (~tg/g wet weight) Liver 252 C26:0/C22:0fatty acid ratio Liver 1.380 Fibroblasts 1.538 C~4:0/C22:0fatty acid ratio 2 . 6 6 0 Liver Phytanic acid oxidase (pmol/h per mg protein) Fibroblasts 0.07 DHAP-AT (nmol/h per mg protein) Fibroblasts 0.21 Alkyl DHAP synthase (nmol/h per mg protein) Fibroblasts 0.44 Catalase (% sedimentable) Fibroblasts 10
7.2-29.1 (n = 5)
0.57 (n > 50)
1.2-8.0 (n > 50)
>1.09 (n > 10)
>46 (n > 50)
DHAP-AT= Dihydroxyacetonephosphate acyltransferase usually aged several months to a few years and at autopsy the most prominent changes are severe atrophy of the adrenals, extensive demyelination of the cerebrum and cerebellum, and prominent PASH in multiple organs, while renal cortical cysts are not a feature [2, 8,
9]. IRD patients may also show mild dysmorphic features and hypotonia which are somewhat variable, but often present some time after the neonatal period, with failure to thrive, hepatomegaly, developmental delay and problems of vision and hearing. Seizures are not prominent features although they have occasionally been reported in association with cerebral haemorrhage, They tend to survive for many years, into the teens
[3, 10, 12, 15]. In the single autopsy report available, on a 12-year-old, the prominent features were cirrhosis of the liver, PASH in lymph nodes, spleen and perivascular spaces in the brain, lack of active demyelination, and prominent displacement of Purkinje cells into the molecular layer [22]. In the present siblings, the presentation was outside the neonatal period, like IRD; and hypotonia and seizures were not noted. The age of death was, however, very young. The lack of typical facies, severe hypotonia, chondrodysplasia punctata, renal cortical cysts in one sibling, and conspicuous migration abnormalities in the brain are strongly against the diagnosis of ZS. Although most of the clinical and pathological feature are consistent with N A L D as well as IRD, the lack of demyelination, the hallmark of NALD, makes this diagnosis difficult to support. The marked changes in the liver, mild changes in the brain, and prominent Purkinje cells in the molecular layer are similar to the previous report on IRD, although in that report, the adrenal did not show atrophic changes which were well developed in the present older patient. Taken together, there seems to be more similarity to IRD than NALD, although there was some difficulty in assigning a specific diagnosis. As pointed out by many, there is considerable overlap between the main phenotypes associated with defects of peroxisome biogenesis and differentiation in some patients may not be possible [10, 26]. One might argue whether there is any purpose in trying to divide them in view of complementation experiments showing that all three phenotypes can be associated with the same complementation group, while the ZS phenotype is associated with at least five different groups [16, 21]. However, until the basic enzyme defects and molecular biology of these conditions are clearly defined, it is probably appropriate to split than to lump, as there are as yet few reports on different phenotypes within the same family, comparable to those concerning X-linked adrenoleucodystrophy and adrenomyeloneuropathy [11]. With the rapid advancement in knowledge and technology in the biochemistry of this group of diseases, it is worthwhile defining exactly what role anatomical
Table 2. Comparison between Zellweger syndrome (ZS), neonatal adrenoleukodystrophy (NALD), infantile Refsum disease (IRD) and index patients
Clinical Age at presentation Survival Craniofacial dysmorphia Severe hypotonia Seizures Chondrodysplasia punctata Autopsy Adrenal Liver fibrosis Renal cortical cysts Neuronal migration defect Demyelination
ZS
NALD
IRD
Patient 1
Patient 2
Neonatal Months Marked Prominent Prominent Common
Neonatal Months to years Mild Prominent Prominent Absent
Weeks to months Years to teens Mild Variable Usually absent Absent
5 weeks 8 months Mild Absent Absent Absent
6 month 3 1/2 years Mild Absent Absent Absent
Hypoplasia Nil to severe Prominent Severe Nil
Atrophy Nil to severe Nil Mild to severe Severe
Hypoplasia Severe Nil Mild Nil
Hypoplasia Nil Occasional Nil Nil
Atrophy Severe Nil Mild Nil
194 pathology has to play in the investigation of patients with perixosomal disorders. On reviewing the biopsy and autopsy files at R C H from 1961 to 1990, there are 3 patients with X-linked adrenoleukodystrophy, 13 patients with ZS, 3 patients with IRD (including the present cases), 1 patient with rhizomelic chondrodysdysplasia punctata, 2 patients with Pseudozellweger syndrome, 1 patient with a peroxisomal disorder which remains unclassified, and 2 patients with possible peroxisomal disorders suggested by morphology, but could not be confirmed by biochemistry. This is not a complete list of patients with peroxisomal disorders seen at R C H as not all patients had a biopsy or an autopsy. A m o n g the patients with X-linked adrenoleukodystrophy, one patient, a 3-year-old boy, died rapidly after a short period of diarrhoea and dehydration. Adrenal atrophy was noticed at autopsy. A n o t h e r patient, a 9-year-old boy, presented with dementia and had a brain biopsy because imaging suggested a diffuse astrocytoma. The biopsy showed demyelination, perivascular lymphocytic infiltration and many PASH which ultrastructurally contained many trilaminar profiles. The patients with IRD include the present siblings and another baby boy who presented at 16 months with hepatomegaly and p o o r vision and liver biopsy showed absence of peroxisomes and presence of membranebound collections of trilaminar profiles in a few histiocytes. In these patients, the diagnosis was unsuspected at the clinical level and made initially by tissue examination. With increasing awareness of the clinical spectrum of peroxisomal disorders and more liberal use of estimation of serum VLCFA as a screening test, this problem will no doubt decrease, but there will always be the odd cases who present unsuspected to the anatomical pathologists first, and it is important for them to recognise the diagnostic features. The ZS patients were all recognised at the clinical level, probably due to the long-standing interest of the D e p a r t m e n t of Genetics in this disease. In a few patients electron microscopy of the liver was requested to confirm or exclude the diagnosis. A n urgent assessment of peroxisomes can be provided overnight, information which can be useful for certain clinical decisions. In another group of patients, e.g. Pseudozellweger patients, electron microscopy of the liver and detailed autopsy findings may help in the delineation of this apparently extremely heterogeneous group of diseases [7, 27]. Lastly as in this pair of siblings, comparing the morphology at different ages may help in understanding the pathogenesis of the lesions. T h e marked prominence of Purkinje cells in the molecular layer in the older sister and the absence in the younger suggests this may be an acquired instead of a developmental lesion.
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Acta Heuropathologica ~) Springer-Verlag 1992
Autopsy findings in two siblings with infantile Refsum disease C.W. Chow 1, A. Poulos 2, A. J. Feilenberg 2, J. Christodoulou 3, and D. M. Danks 3 1 Department of Anatomical Pathology, Royal Children's Hospital, Parkville, Melbourne,Victoria 3052, Australia 2 Department of Chemical Pathology, Adelaide Children's Hospital 3 Murdoch Institute, Royal Children's Hospital, Melbourne Received May 17, 1991/Accepted August 6, 1991
Summary. Recognition of adrenal atrophy during a review of autopsy findings in two sisters who died at 8 months and 3 1/2 years prompted estimation of very long chain fatty acids, phytanic acid and pristanic acid on wet liver fixed in formalin for 12 years. These were shown to be markedly increased and defects in multiple peroxisoreal functions and decrease in particulate catalase were shown in cultured fibroblasts, confirming an abnormality of peroxisomal biogenesis. The patients had presented with failure to thrive, recurrent diarrohea and vomiting, poor mental development, retinal pigmentation, blindness and in the older patient deafness, with only mild dysmorphic features. Autopsy in the older patient showed adrenal atrophy, cirrhosis, and foamy histiocytes in multiple organs. The brain showed no demyelination, little cytoarchitectural abnormality, occasional perivascular histiocytes in the grey matter and meninges and prominent Purkinje cells in the molecular layer of the cerebellum. In the younger patient the changes were very subtle in spite of the marked clinical similarity. Despite the young age at death the clinicopathological features are most suggestive of infantile Refsum disease. In many situations anatomical pathology can be very useful in the recognition and study of peroxisomal disorders. Key words: Sisters - Adrenal atrophy - Cirrhosis Ectopic Purkinje cells - Young age at death
radiology, biochemistry and morphology, the last including both ultrastructure of peroxisomes and general autopsy findings. Broadly the disorders have been divided into those associated with defects of biogenesis of peroxisomes, those with abnormalities of several peroxisomal functions, and those in which only one single enzyme is at fault [17, 27]. The first group is represented by Zellweger syndrome (ZS), neonatal adrenoleukodystrophy (NALD) and infantile Refsum disease(IRD), with hyperpipecolic acidaemia considered by some not to be a separate entity [10]. Although there are many autopsy reports on ZS [4, 5, 24] and N A L D [2, 8, 9], there has been only one on IRD [22]. Two sisters were seen at the Royal Children's Hospital (RCH) and died in the 1960's and 1970's. At that stage no obvious diagnosis was made. Review of the autopsy prompted biochemical investigations on material kept at the time of the post-mortem examination. This confirmed an abnormality of peroxisomal biogenesis. Correlation with clinical findings suggests IRD as the most likely diagnosis. The autopsy findings in these two cases are presented, together with a discussion on the difficulties in an exact classification in some cases and the role of morphology in the investigation of this group of diseases.
Case reports Patient 1
Since the absence of peroxisomes was noted in Zellweger syndrome by Goldfischer et al. [6], the list of diseases associated with abnormal peroxisomal structure and/or function has greatly expanded. Classification of many patients, however, remains arbitrary as the enzyme defect and molecular biology remain unknown, and is frequently based on a combination of clinical features,
Offprint request to: C.W. Chow (address see above)
This girl born in 1964 weighing only 2.15 kg at full term was first seen at RCH at the age of 5 weeks for an inspiratory stridor. Failure to thrive was a problem from the first weeks, and episodes of respiratory infection and of diarrohea and vomiting occurred repeatedly. Apart from three wide fontanelles and open sutures, there were no significant dysmorphic features. Stool examination showed no fat globules and sweat test was negative. Blood examination showed occasional Burr cells and acanthocytes. Chromosomes were normal. X-raysshowed no abnormalities in the epiphyses. Addison disease was considered but electrolytes were normal and 24-h urine showed 17-ketosteroid of 0.6 mg and
191 17-ketogenic steroid of 0.3 mg. At 6 months she was noticed to have a roving nystagmus. Ophthalmological consultation showed that there was no pupillary reaction to light, and fundal examination showed coarse brown-black pigmentation in the peripheral fields. Her conditions remained poor with no obvious neurological development and she died at 8 months following a cerebral haemorrhage.
Patient 2 This girl was born at full term in 1973 after a normal pregnancy.The birth weight was 2.61 kg. She was seen at 6 months because of failure to thrive. There were no craniofacial dysmorphic features.
Fig. 1 a,b. Adrenal showing marked cortical atrophy in older sibling (a) and preserved architecture in younger (b). H & E , • 40 Fig. 2. Liver in older sibling showing well-established cirrhosis. Masson's trichrome, x 48
Stool examination showed some fat globules. The sweat test was negative and a duodenal biopsy was normal. Her motor milestones were markedly delayed. She did not follow objects with eyes but did respond to aural stimuli by turning her head. Fundal examination showed peripheral equatorial clumps of pigment. Her liver was enlarged to 2-3 cm below the costal margin. Liver biopsy showed moderate fibrosis and occasional PAS-positive histiocytes (PASH) in the portal tracts. No tissue was processed for electron microscopy. X-rays showed moderate changes of rickets but no obvious abnormalities in the epiphyses. The serum calcium concentration was 6.4 mg%, and phosphate 3.0 mg%. In view of the family history she was considered to have a severe neurodegenerative disorder although the nature of this was unclear. Over the next 3 years she was admitted on multiple occasions for vomiting,
Fig. 3. Inferior olive showing focal concentration of neurons at the periphery of the nucleus. H & E , • 40 Fig. 4 a,b, Cerebellum showing marked displacement of Purkinje cells into molecular layer in older sibling (~), and normal location in younger (b). H & E , x 64
192 diarrhoea and dehydration. It became obvious that she was severely mentally retarded. At 3 years her head control was very poor. Her fontanelle was still open and she had developed cataracts and become deaf. At 3 1/2 years she was again admitted with dehydration. In spite of intravenous fluid, her serum sodium varied from 111 to 120 mEq/1. Her conditions gradually deteriorated and she died. The parents were unrelated. There were three other girls who were alive and healthy.
Biochemical findings Tissue was only available from patient 2. Firstly wet tissue of the liver, kept in formalin for 12 years, was assessed for the concentration of very long chain fatty acids (VLCFA), PA, and PTA. The results are shown in Table 1. As the concentrations of VLCFA, PA and PTA were strongly suggestive of a disorder of peroxisomal function, other tests were conducted on the cultured fibroblasts to characterise the disease further. Deficiencies in a number of enzyme activities including phytanic acid oxidase, particulate catalase, DHAP-AT and alkyl DHAP synthase were observed.
Biochemical methods Hexacosanoic acid/docosanoic acid (26:0/22:0) and lignoceric acid/docosanoic acid (24:0/22:0) ratios, and phytanic (PA) and pristanic acid (PTA) levels were measured as described earlier [14, 15]. Dihydroxyacetonephosphate acyltransferase (DHAP-AT) and alkyl dihydroxyacetonephosphate synthase (alkyl DHAP synthase) activities were assayed as described by van Crugten et al. [23] and Singh et al. [20], respectively. Phytanic acid oxidation was measured as described by Poulos et al. [13].
Autopsy findings Patient 2 As it was the autopsy findings in this patient which prompted the review and further investigations, the findings in this 3 1/2 year old girl will be presented first. The major abnormalities were seen in the adrenals, the liver and the brain. The adrenals were markedly atrophied, with the cortex replaced by scattered cells with foamy and striate cytoplasm and an occasional nodule of cortical cells (Fig. la). The medulla was normal. The liver showed wellestablished cirrhosis (Fig. 2). There was no significant haemosiderosis. Prominent PASH were present in the liver as well as in the lymph nodes, spleen and bone marrow in moderate numbers. The kidneys were normal, specifically showing no cortical cysts. In spite of the severe neurological deficit, the brain was on the whole remarkably well preserved. The cerebrum did not show any cytoarchitectural abnormality. The inferior olives retained the normal wavy outlines. There was focal concentration of neurons along the periphery of the nucleus (Fig. 3), but nothing like the prominent peripheral palisading in ZS [4]. Demyelination was not seen. Occasional small perivascular clusters of PASH were seen in the pontine nuclei, molecular layer of the cerebellum and the meninges. The cerebellar cortex showed moderate decrease in granular cells. The Purkinje cells were extensively displayced into the molecular layer which also showed mild gliosis (Fig. 4a). Due to the adrenal atrophy a peroxisomal disorder was suspected and biochemistry was requested on the material kept at the time of autopsy.
Patient 1 In this 8-month-old baby girl the adrenals were moderately decreased in size weighing 1 g each. The cortical architecture was, however, normal (Fig. lb). Careful searching with special staining showed occasional PASH between the columns of the zona fasciculata. The liver showed no significant fibrosis. Only an occasional PASH was seen in the liver, but not in the spleen, lymph nodes, or bone marrow. The kidney showed only a few small cortical cysts. The brain showed a large haemorrhage in the left frontal lobe. There was no cytoarchitectural abnormality, demyelination or gliosis. PASH were not seen. In the cerebellar cortex, Purkinje cells were normally situated at the junction of the molecular and granular layers (Fig. 4b).
Discussion O n c e a p e r o x i s o m a l disorder was suspected, the first screening test was an estimation of the c o n c e n t r a t i o n o f V L C F A [1]. S e r u m or fresh tissue was n o t available, b u t it was t h o u g h t that p e r h a p s formalin-fixed liver might still be suitable as the fatty acids are saturated and, therefore, stable. C o m p a r e d with control specimens, s o m e of w h i c h h a d b e e n k e p t in a similar state for c o m p a r a b l e periods, V L C F A , P A and P T A were m a r k e d l y elevated, strongly suggesting t h a t a disturb a n c e of p e r o x i s o m a l f u n c t i o n was i n d e e d involved.This highlights the i m p o r t a n c e of k e e p i n g material at the time of a u t o p s y in patients w h e n the n a t u r e of a suspected g e n e t i c / m e t a b o l i c disease is unclear. Fresh tissues f r o m multiple organs and fibroblast cultures are obviously ideal. Paraffin blocks can be suitable sources of D N A , and as s h o w n here, fixed wet tissue can occassionally also be e x t r e m e l y useful, s o m e t i m e s m a n y years after the d e a t h o f the patients. C o n f i r m a t i o n of the disturbance in p e r o x i s o m a l function was o b t a i n e d by d e m o n s t r a t i n g deficiencies in the activity of a n u m b e r of e n z y m e s , including p h y t a n i c acid oxidase, D H A P - A T , alkyl D H A P synthase, and particulate catalase activity. E a c h of these activities has b e e n r e p o r t e d to be associated p r e d o m i n a n t l y with peroxisomes in rat liver and h u m a n skin fibroblast [18, 19, 25]. D u e to the unavailability of suitable tissues for ultrastructural analysis we were n o t able to c o n f i r m m o r p h o logically an a b n o r m a l i t y in p e r o x i s o m a l biogenesis, a l t h o u g h the e n z y m o l o g i c a l data s h o w n in Table 1 are consistent with this view. T h e next p r o b l e m was to d e t e r m i n e w h e t h e r the features fit b e t t e r with ZS, N A L D or I R D . S o m e selected clinicopathological features of these three entities c o n s i d e r e d o f discriminatory value are c o m p a r e d with the index patients in Table 2. T h e classical Z S patients present in the n e o n a t a l p e r i o d with typical facies, severe h y p o t o n i a , seizures, c h o n d r o d y s p l a s i a p u n c t a t a , die within w e e k s or m o n t h s , and at a u t o p s y s h o w p r o m i n e n t cortical cysts in the kidneys, h a e m o s i d e r o s i s and a variable d e g r e e o f fibrosis of the liver, and m a r k e d migration abnormalities in t h e brain, involving m o s t conspicuously the inferior olives, the c e r e b r u m and the cerebellum [4, 5, 24]. N A L D patients m a y also present in the perinatal p e r i o d with h y p o t o n i a , seizure and d y s m o r p h i c feature, but generally w i t h o u t t h e typical facies of ZS or c h o n d r o d y s p l a s i a p u n c t a t a . T h e y t e n d to die later,
193 Table 1. Biochemical changes in patient 2
Patient 2
Normal
Phytanic acid (~g/g wet weight) Liver 1910 Pristanic acid (~tg/g wet weight) Liver 252 C26:0/C22:0fatty acid ratio Liver 1.380 Fibroblasts 1.538 C~4:0/C22:0fatty acid ratio 2 . 6 6 0 Liver Phytanic acid oxidase (pmol/h per mg protein) Fibroblasts 0.07 DHAP-AT (nmol/h per mg protein) Fibroblasts 0.21 Alkyl DHAP synthase (nmol/h per mg protein) Fibroblasts 0.44 Catalase (% sedimentable) Fibroblasts 10
7.2-29.1 (n = 5)
0.57 (n > 50)
1.2-8.0 (n > 50)
>1.09 (n > 10)
>46 (n > 50)
DHAP-AT= Dihydroxyacetonephosphate acyltransferase usually aged several months to a few years and at autopsy the most prominent changes are severe atrophy of the adrenals, extensive demyelination of the cerebrum and cerebellum, and prominent PASH in multiple organs, while renal cortical cysts are not a feature [2, 8,
9]. IRD patients may also show mild dysmorphic features and hypotonia which are somewhat variable, but often present some time after the neonatal period, with failure to thrive, hepatomegaly, developmental delay and problems of vision and hearing. Seizures are not prominent features although they have occasionally been reported in association with cerebral haemorrhage, They tend to survive for many years, into the teens
[3, 10, 12, 15]. In the single autopsy report available, on a 12-year-old, the prominent features were cirrhosis of the liver, PASH in lymph nodes, spleen and perivascular spaces in the brain, lack of active demyelination, and prominent displacement of Purkinje cells into the molecular layer [22]. In the present siblings, the presentation was outside the neonatal period, like IRD; and hypotonia and seizures were not noted. The age of death was, however, very young. The lack of typical facies, severe hypotonia, chondrodysplasia punctata, renal cortical cysts in one sibling, and conspicuous migration abnormalities in the brain are strongly against the diagnosis of ZS. Although most of the clinical and pathological feature are consistent with N A L D as well as IRD, the lack of demyelination, the hallmark of NALD, makes this diagnosis difficult to support. The marked changes in the liver, mild changes in the brain, and prominent Purkinje cells in the molecular layer are similar to the previous report on IRD, although in that report, the adrenal did not show atrophic changes which were well developed in the present older patient. Taken together, there seems to be more similarity to IRD than NALD, although there was some difficulty in assigning a specific diagnosis. As pointed out by many, there is considerable overlap between the main phenotypes associated with defects of peroxisome biogenesis and differentiation in some patients may not be possible [10, 26]. One might argue whether there is any purpose in trying to divide them in view of complementation experiments showing that all three phenotypes can be associated with the same complementation group, while the ZS phenotype is associated with at least five different groups [16, 21]. However, until the basic enzyme defects and molecular biology of these conditions are clearly defined, it is probably appropriate to split than to lump, as there are as yet few reports on different phenotypes within the same family, comparable to those concerning X-linked adrenoleucodystrophy and adrenomyeloneuropathy [11]. With the rapid advancement in knowledge and technology in the biochemistry of this group of diseases, it is worthwhile defining exactly what role anatomical
Table 2. Comparison between Zellweger syndrome (ZS), neonatal adrenoleukodystrophy (NALD), infantile Refsum disease (IRD) and index patients
Clinical Age at presentation Survival Craniofacial dysmorphia Severe hypotonia Seizures Chondrodysplasia punctata Autopsy Adrenal Liver fibrosis Renal cortical cysts Neuronal migration defect Demyelination
ZS
NALD
IRD
Patient 1
Patient 2
Neonatal Months Marked Prominent Prominent Common
Neonatal Months to years Mild Prominent Prominent Absent
Weeks to months Years to teens Mild Variable Usually absent Absent
5 weeks 8 months Mild Absent Absent Absent
6 month 3 1/2 years Mild Absent Absent Absent
Hypoplasia Nil to severe Prominent Severe Nil
Atrophy Nil to severe Nil Mild to severe Severe
Hypoplasia Severe Nil Mild Nil
Hypoplasia Nil Occasional Nil Nil
Atrophy Severe Nil Mild Nil
194 pathology has to play in the investigation of patients with perixosomal disorders. On reviewing the biopsy and autopsy files at R C H from 1961 to 1990, there are 3 patients with X-linked adrenoleukodystrophy, 13 patients with ZS, 3 patients with IRD (including the present cases), 1 patient with rhizomelic chondrodysdysplasia punctata, 2 patients with Pseudozellweger syndrome, 1 patient with a peroxisomal disorder which remains unclassified, and 2 patients with possible peroxisomal disorders suggested by morphology, but could not be confirmed by biochemistry. This is not a complete list of patients with peroxisomal disorders seen at R C H as not all patients had a biopsy or an autopsy. A m o n g the patients with X-linked adrenoleukodystrophy, one patient, a 3-year-old boy, died rapidly after a short period of diarrhoea and dehydration. Adrenal atrophy was noticed at autopsy. A n o t h e r patient, a 9-year-old boy, presented with dementia and had a brain biopsy because imaging suggested a diffuse astrocytoma. The biopsy showed demyelination, perivascular lymphocytic infiltration and many PASH which ultrastructurally contained many trilaminar profiles. The patients with IRD include the present siblings and another baby boy who presented at 16 months with hepatomegaly and p o o r vision and liver biopsy showed absence of peroxisomes and presence of membranebound collections of trilaminar profiles in a few histiocytes. In these patients, the diagnosis was unsuspected at the clinical level and made initially by tissue examination. With increasing awareness of the clinical spectrum of peroxisomal disorders and more liberal use of estimation of serum VLCFA as a screening test, this problem will no doubt decrease, but there will always be the odd cases who present unsuspected to the anatomical pathologists first, and it is important for them to recognise the diagnostic features. The ZS patients were all recognised at the clinical level, probably due to the long-standing interest of the D e p a r t m e n t of Genetics in this disease. In a few patients electron microscopy of the liver was requested to confirm or exclude the diagnosis. A n urgent assessment of peroxisomes can be provided overnight, information which can be useful for certain clinical decisions. In another group of patients, e.g. Pseudozellweger patients, electron microscopy of the liver and detailed autopsy findings may help in the delineation of this apparently extremely heterogeneous group of diseases [7, 27]. Lastly as in this pair of siblings, comparing the morphology at different ages may help in understanding the pathogenesis of the lesions. T h e marked prominence of Purkinje cells in the molecular layer in the older sister and the absence in the younger suggests this may be an acquired instead of a developmental lesion.
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