American J o u r n a l of Medical Genetics 37:489-503 (1990)
Clinico-Pathological Report Progressive Neurologic Deterioration in a Nine-YearOld White Male ~
Lewis A. Barness, Sunita Chandra, Pamela Kling, Ftenata Laxova, David B. Allen, and Enid Gilbert-Barness Departments of Pediatrics (L.A.B., P.K., R.L., D.B.A., E.G.B.), Pathology (S.C., E.G.B.), and Medical Genetics (R.L.), University of Wisconsin Medical School, Madison, Wisconsin
KEY WORDS: Schilder’s disease, long chain fatty acids, ALD, genetic disorders CLINICAL PRESENTATION Dr. Pamela Kling: Chief Resident in Pediatrics CHIEF COMPLAINT The patient was a 7%-year-old white boy, admitted for evaluation of neurologic deterioration. HISTORY OF PRESENT ILLNESS Five months before admission, his mother noted that his left eye “would wander” and that he occasionally bumped into things. He was seen by a n ophthalmologist 2V2 months before admission who thought he had left exotropia and mild hyperopia. For several months, his teacher noted he did not appear interested or listening in class, with significant deterioration in his grades. A local audiologic evaluation showed decreased hearing bilaterally. He also appeared to have a personality change, with disinterest in the activities he once enjoyed. He often showed “dulled” responses to situations and clearly defective memory. At other times, he appeared emotionally labile and became extremely excited with laughing fits, usually inappropriate. He seemed to be preoccupied with the illness and death of his grandmother and his own death. He showed decreased coordination with worsening motor skills, appeared ataxic, and got into accidents easily. His mother felt it was “like having a two year old again.” PAST MEDICAL HISTORY His birth weight was 3,010 g a t term. He was born to a primigravida with no complications during pregnancy, Received for publication May 29, 1989; revision received April 18, 1990. Address reprint requests to Lewis A. Barness, M.D., H41434 Clinical Science Center, University of Wisconsin Medical School, Madison, WI 53792.
0 1990 Wiley-Liss, Inc.
labor, delivery, or neonatal course. He was a healthy child with no hospitalizations, operations, or chronic illnesses. He had received immunizations without reactions and had no allergies to medications.
DEVELOPMENT He lifted his head a t 3 weeks, rolled over a t 3 months, sat with support a t 5 months, and stood with support a t 6 months. He walked by 10 months, talked at ll/z years, was toilet trained a t 2% years, and rode a tricycle a t 3 years. FAMILY HISTORY [See Dr. Laxova’s text.] PHYSICAL EXAMINATION The patient was a 71/z-year-old white boy who was alert, talkative, but occasionally confused. Pulse was 92 beatslmin, respirations 32lmin, and blood pressure 114176 mmiHg. He was afebrile. His height was 128 cm and weight 25.3 kg (both a t the 75th centile). He was normocephalic, with left exotropia and apparently normal optic discs. Ear exam showed decreased hearing bilaterally. He had generous tonsils, lung and heart sounds were normal on auscultation. The spine was straight. Abdomen and bowel sounds were normal. Genitalia were normal for age (Tanner I). Skin was normal in pigmentation and texture. The boy was alert, but could not remember day of week or city. He had poor judgment, poor short-term memory, and could follow only a series of 2 instructions. There was some echolalia and excessive laughter. He spoke very loudly with poor enunciation.His math skills seemed appropriate for age. He had a n ataxic gait, was unable to skip or walk heel-to-toe. He had past-pointing on finger-to-nose testing and had poor rapid alternating movements. The Romberg sign was absent. His deep tendon reflexes were 2+14+ and symmetric, no Babinski’s reflex. Tone was normal and strength was 5 + I5 + . Gross sensation and sharplsoft discrimination were intact. Other than the previously noted findings, the cranial nerves were intact.
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EVALUATION Audiologic evaluation showed a 75-80 dB sensorineural hearing loss. Ophthalmologic exam showed some pallor of the optic nerves, suggestive of optic atrophy. On admission he had a hematocrit of 3996, hemoglobin of 13.0 g, and white blood cell count of 4,900/mm3 with 3% band and 42% segmented neutrophils, 28%, lymphocytes, 11%atypical lymphocytes, and 6%monocytes. Results of urinalysis and chemical survey were normal. Serum sodium was 138 mmoliliter, potassium, 4.1 mmoliliter, chloride, 103 mmol/liter, and carbon dioxide, 28 mmoliliter. CSF protein was 19 m g 8 and glucose, 66 mg8. CSF albumin was 62.796, CSF gamma globulin, 11.4%,with albumin: globulin ratio of 1.68. There was one mononuclear cell and no red blood cells. A CT scan showed symmetric areas of abnormality in the septum semiovale, posteriorly. Following contrast, there was diffuse enhancement. The EEG was abnormal and showed widespread symmetrical disturbance of cerebral function characterized by slowing in the parietal and occupital regions. Additional laboratory studies showed a n ACTH level of 198 pgiml (normal in our lab is less than 80 pg/ml). A morning cortisol level was 12.3 +g% which remained essentially unchanged (12.2 kg%) after injection of 0.01 mgikg IM synthetic ACTH. CLINICAL COURSE Over the next 2 years the patient suffered a progressive downhill course with emotional outbursts, blindness, deafness, inability to walk, difficulty swallowing, then decreased responsiveness. He required institutionalization and died with massive bilateral pneumonia.
DISCUSSION Dr. Lewis A. Barness: Professor of Pediatrics One approach to the diagnosis of a child such a s this is to attempt to categorize the illness. This child obviously had a progressive neurodegenerative disease. In contrast to nonprogressive disorders which are usually due to genetic or anatomic abnormalities or trauma or other structural abnormalities, progressive neurodegenerative diseases fall into 2 categories: metabolic and unknown (like President Grant who said he knew two songs: one was “Yankee Doodle” and the other wasn’t). Next, one tries to localize the lesion: gray matter lesions usually start with dementia and seizures and include the storage diseases; the history of this child is not consistent with such a disorder, White matter lesions usually start with motor abnormalities such a s spasticity, hypotonia, and ataxia and include the leukodystrophies or the kind of demyelinating lesions which must have been present in this child. A third category includes the systemic lesions such as spinocerebellar as in (F’riedreich) ataxia, ataxia telangiectasia, Bassen-Kornzweig, and Refsum syndromes and basal ganglia including the dystonias, Hallervorden-Spatz, and Huntington chorea. At first, the boy developed a n ocular palsy. Later he
became deaf after 7 years of apparent good health. This was quickly followed by a personality change, disinterest, and liability. With the laughing fits, I thought of Tourette syndrome, a catch-all disease, until the destructive process proceeded even more quickly. Subacute sclerosing panencephalitis should have presented greater evidence off cortical involvement, particularly seizures, at this point. Were there any similar diseases in the family? Dr. Pamela Kling: The grandfather is said to have had multiple sclerosis. Three cousins died with neurologic disorders. Dr. Lewis A. Barness: The family history is interesting. It is now recognized that the diagnosis of multiple sclerosis has also been given to individuals with such entities a s amyotrophic lateral sclerosis, juvenile adrenoleukodystrophy, as well as those with multiple sclerosis. Later it is noted the patient had optic atrophy, a sign of multiple sclerosis. What was the sex of the cousins with progressive neurologic disorders? Dr. Pamela Kling: They were boys. Dr. Lewis A. Barness: In contrast to muscle diseases or amino acid disorders, few progressive neurologic disorders are X-linked. If this is truly X-linked, the differential diagnosis becomes relatively limited. That the father works in sanitation may suggest toxins. However, no other relative was apparently ill. Physical findings of interest other than the exotropia were the large tonsils. Tangiers disease, analphalipoproteinemia with neurological manifestations generally presents a t a much younger age and the tonsils usually yellow. The neurological findings were consistent with diffuse neurological involvement. Dr. David Allen: What was the skin color? Dr. Kling: The skin was normal in color. Results of the CBC, urine, chemical survey, and lumbar puncture were normal. Were the serum sodium, potassium and chloride levels normal? Dr. Kling: Yes. Dr. Barness: For the diagnosis most likely, the sodium, and chloride levels would be expected to be low with a n elevated potassium. The history and diffuse brain abnormalities seen are consistent with a number of CNS degenerative lesions. In concluding the diagnostic approach, gray matter diseases are unlikely for reasons already stated. There is no evidence of storage disease such as large liver or spleen. There is no evidence of cerebromacular degeneration. The age-of-onset argues against aminoacidopathies. Of the leukodystrophies, Krabbie cerebrosidosis, Canavan disease, and metachromatic leukodystrophy generally start earlier and usually are associated with cerebrospinal fluid abnormalities. Of the demyelinating diseases, neuromyelitis optica appears consistent with much of the course. Children with multiple sclerosis do not usually develop signs of dementia so early. The course followed by this child is characteristic of juvenile adrenoleukodystrophy [Moser, 19891.This peroxisomal disorder is X-linked. Symptoms usually begin a t age 4-10 most often a s hyperactivity or attention deficit disorders followed by emotional changes, disor-
Progressive Neurologic Deterioration ders of speech and writing, and auditory defects. Later, dementia, seizures, and optic nerve involvement occur and death occurs in 1-2 years. Adrenal insufficiency occurs in about 30% of boys. This child had adrenal insufficiency, though asymptomatic, since he was shown to be nonresponsive to his own ACTH or to administered ACTH.
ADDENDUM After coming to this diagnosis, the following findings were entered into the Meditel computer assisted diagnosis system: confusion, mental deficiencies, hearing loss, ocular palsy, ataxia, and happy affect. The first diagnosis that was retrieved was “Schilder’s disease.” “Schilder’s disease” is now recognized as a group of diseases including X-linked adrenoleukodystrophy, multiple sclerosis, Pelizaeus-Merzbacher disease, metachromatic leukodystrophy, and ceroid lipofucscinosis. PATHOLOGICAL FINDINGS Sunita Chandra, M.B.B.S.: Associate Professor of Pathology Gross Examination At autopsy the body was a well-nourished 9-year-old white boy. He weighed 25.3 kg and was 128 cm long. Scalp hair was dark brown, and the body hair was light brown. The skin and conjunctivae were nonicteric. Pupils were dilated, fixed, and equal in size. Nose, ears, and teeth were unremarkable. No malformations of the face were noted. The chest was normal externally, and the abdomen was slightly distended. The external genitalia were normal. The upper limbs were unremarkable. The lower limbs were thin and there was a bilateral talipes equinovarus deformity. The pleural and pericardial cavities were unremarkable. Abdominal distension was caused by distended bowel. The peritoneal surface of the abdominal wall was tan and smooth. The mucosa of the stomach and small and large intestines was smooth and unremarkable. The heart was normal and weighed 120 g. The heart valves, right and left ventricular walls, and myocardium were unremarkable. The mucosa of the bronchial tree was slightly congested. No evidence of obstruction was present. Both lungs were heavy and together weighed 500 g. On the cut surface, patchy areas of firmness were evident. Microscopic sections showed bronchopneumonia. The liver was slightly enlarged and weighed 900 g (normal 800 g), The external surface was tan and smooth and mildly congested. Microscopic sections showed mild congestion and absence of periportal fibrosis (Fig. 1). The spleen was moderately enlarged and weighed 120 g and was soft and congested. The adrenal glands were small. The right weighed 1.2 g and the left 0.8 g (normal 3.5-4 g) (Fig. 2). The cut surfaces were very thin. The cortex was pale, and the medulla was unremarkable. Microscopic sections of the adrenal glands showed a n intact zona glomerulosa. The zona fasciculata and zona reticularis contained aggregates and nodules of ballooned cells (Fig. 3 ) and many multinucleated giant cells. The ballooned cells contained pink granular cytoplasm and few striations (Fig.
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4). Perivascular and interstitial lymphocytic infiltration was noted. Ultrastructural studies of the adrenal cortex showed linear bilamellar cytoplasmic inclusions (Fig. 5). Biochemical studies of adrenal gland and brain tissue were performed by Dr. Hugo Moser (see genetics discussion by Dr. Renata Laxova). The brain weighed 1,350 g. It was bilaterally symmetrical, normal in size and gyral pattern (Fig. 6 ) . The olfactory bulbs and tracts and optic chiasm were intact. The pons, medulla, and upper cervical cord were grossly unremarkable. Horizontal section of the brain showed tannish-brown areas involving all the lobes of the brain (frontal, parietal, temporal, and occipital) and periventricular areas. These soft areas were sunken from the brain surface (Fig. 7A). These soft areas in the white matter picked up the Oil Red 0 stain, indicating breakdown of myelin and engulfment by macrophages (Fig. 7B). Sections from the pons, medulla, and cerebellum showed similar very tan, soft areas involving the white matter (Fig. 8A, B). Microscopic sections from the cerebral cortex showed a normal cortex. However, sections from the white matter showed severe demyelination, gliosis, perivascular accumulation of lymphocytes, and macrophages (Fig. 9A, B). The macrophages were more concentrated a t the edges of the lesions (Fig. 9C). The cytoplasm of these macrophages was weakly Oil Red 0 positive and many macrophages contained PAS positive material in their cytoplasm (Fig. 10A, B). Metachromatic stains were done and showed intact axis cylinders a t the progressive edges; however, in more severely involved areas axis cylinders were destroyed (Fig. 11). Ultrastructure of brain tissue from affected areas showed similar bilamellar enlarged intracytoplasmic inclusions. After the death of this child, the mother became pregnant again. The fetus was shown to be affected by prenatal diagnosis. Pregnancy was terminated a t 211/2 weeks of gestation. No obvious external abnormalities were seen in this fetus. The internal organs were macerated but normal. The liver was normal. The combined weight of both adrenals was 1.2 g. Microscopic sections of the adrenals showed a thin zona glomerulosa. Ballooning and occasional presence of giant cells was seen in the zona fasciculata and zona reticularis. PAS stains were mildly reactive. Ultrastructure examination of the adrenal cortex showed randomly oriented intracytoplasmic linear inclusions consisting of two 25 wide electron dense bands separated by a clear zone.
PATHOLOGICAL DISCUSSION Dr. Sunita Chandra: Associate Professor of Pathology Adrenoleukodystrophy is a disorder of defective peroxisomal oxidation of very long chain fatty acids. It is associated with progressive central nervous system demyelination, adrenal atrophy, and rarely adrenal insufficiency. Two distinct types of adrenoleukodystrophy (ALD) that differ in their age-of-onset, clinical course, and
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Fig. 1. Microscopic appearance of liver showing mild congestion and absence of periportal fibrosis. Hematoxylin and eosin (H&E), x 100.
Fig. 2. Gross appearance of urinary system with adrenal glands which are small.
Fig. 3. Microscopic appearance of adrenal gland showing nodules of ballooned cells in zona fasciculata and reticularis. H&E, x 25.
Fig. 5 . Electron micrograph of adrenal cortex showing linear bilaminar intracytoplasmic inclusions (arrow).Uranyl acetate and citrate, x 8,000. Fig. 4. Microscopic appearance of adrenal nodules composed of ballooned cells, containing pink granular cytoplasm and few striations.
H&E. x400.
Progressive Neurologic Deterioration mode of inheritance have been identified [Goldfischeret al., 19851. The X-linked form of ALD usually has its onset in prepubertal boys. It is characterized by progressive destruction of cerebral white matter and adrenal cortex. Death usually occurs in adolescence. Neonatal ALD appears in the neonatal period. The affected chil-
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dren suffer from severe hypotonia, seizures, and usually die by 6 years. In both types of ALD, there is accumulation of very long chain fatty acids (CZ6and C2J. Fibroblasts from these patients are deficient in their ability to oxidize long chain fatty acids [Goldfischer et al., 1985; Moser et al., 1980, 1984, 19891. Table I indicates the differences between the 2 types of adrenoleukodystrophy. The basic defect in ALD appears to be a deficiency of peroxisomal oxidation of fatty acids, and subsequent accumulation of very long chain fatty acids in the tissues, adrenal glands, and central nervous system in the X-linked form of ALD, and also in the liver and reticuloendothelial system in the neonatal form of ALD [Kelley et al., 19861. The pathological characteristics of both forms of ALD are shown in Table 11.
Peroxisomes and Their Function The peroxisomes are cytoplasmic organelles with a single membrane wall and electron-dense matrix [De Duve, 19831.The peroxisomes are present in all animal and plant cells, including cultured skin fibroblasts. Lazarow and Duve 119761 showed that rat liver peroxisomes catalyze the P-oxidation of fatty acids. At least 40 enzymes now have been shown to have peroxisomal localization [Tolbert, 19811.In recent years, it has become clear that in mammals peroxisomes fulfill a number of essential functions:
Fig. 6. Gross appearance ofthe brain showing normal gyral pattern.
1. Catabolism of very long chain fatty acids; peroxisomes seem specially equipped to bring about shortening of very long chain (> C ~ Zfatty ) acids LBremer,
Fig. 7. (A)Horizontal section of the brain showing tan-brown, soft areas involving white matter of all lobes of the brain and periventricular areas. ( B )Horizontal section of the brain showing soft. sunken areas in the white matter. Oil Red 0 stain.
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Fig. 8. (A) Section of the cerebrum showing normal-appearing cerebral cortex (dark area) and a pale, degenerated white matter. H&E, x 25. (B) Section of the cerebellum showing pale, degenerated white matter. Dentate nucleus cannot be identified. H&E, x 25.
TABLE I. Adrenoleukodystrophy: Clinical Differences Between X-Linked Adrenoleukodystrophy and Neonatal Adrenoleukodystrophy Characteristics --__
X-linked adrenoleukodystrophy
Age-of-onset Clinical course Storage material Pathology: Organs involved Biochemical abnormalities: (1) i. Plasma concentration of VLCFA" (2) ii. Ratio of C26:O :C22 :0 (3) iii. Hepatic catalase activity (4) iv. Total serum bile acids-trihydroxycoprostanic acid (THEA) (normally absent)
Neonatal adrenoleukodystrophy _ _
~
Prepubertal boys Slowly progressive, death in adolescence Very long chain fatty acids (VLCFA) (C26-C24) in the tissues
Neonatal Death, by 6 years Same
Brain, adrenals
Brain, adrenals, liver, macrophages
Increased
Increased
Abnormal Normal 50% is sedimented (10.3 nmoliml) Normal
Abnormal Abnormal, increase 12% is sedimented (8inmoliml) Normal
Absent
Serum bile acids
~
"VLCFA, Very long chain fattyacids.
TABLE 11. Adrenoleukodystrophy: Pathologic Changes Characteristics
X-linked adrenoleukodystrophy ~
Brain
Adrenal glands
-
Demyelination (centripetally spreading) Perivascular inflammatory infiltrates follow the margin of demyelination Diffuse gliosis Relative neuronal and axonal preservation Normal cytoarchitecture of cortex
Ultrastructure Liver
Atrophy of zona fasciculata and reticularis Ballooned adrenocortical cells with or without striations Bilamellar and laminar lipid inclusions Normal
Ultrastructure Deroxisomes
Peroxisomes identified
adrenoleukodystrophy _ _ _Neonatal - - ~
~~
-
Diffuse demyelination Perivasculara inflammation not intense
Mild primary maldevelopment of cerebral cortex Same Usually not present Same Periportal fibrosis, macrophages PAS positive material Reduced
.
Progressive Neurologic Deterioration
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malogen and alkyl-glycerophospholipids). It has been shown in rodent liver and brain that 2 of the enzymes required for the introduction of ether bands in the biosynthesis of ether phospholipids acyl CoA :dihydroxyacetone phosphate acyltransferase and acyldyhydroxyacetone phosphate synthase are located predominantly in peroxisomes [Hajra et al., 1979, 19821. 3. The biosynthesis of bile acids [Hagey et al., 1982; Kase et al., 19831. 4. The catabolism of pipecolic acid, an intermediate in the degradation of L-lysine [Trijbels et al., 19791. 5. The catabolism of dicarboxylic acid; the p-oxidation of C12 dicarboxylic acid to C6-C10 dicarboxylic acids is a peroxisomal process [Mortensen et al., 19831. Glutaryl-CoA can also be catalyzed by peroxisomes [Vamecq et al., 19841. 6. The peroxisomes play a n essential role in the catabolism of phytanic acid, although some reports indicate that phytanic oxidase is a mitochondria1 enzyme [Tsai et al., 19691. 7. The oxidation of polyamines in rat liver [Holtta, 19771.
Fig. 9. (A)Microscopic section of the cerebellum taken from the soft areas showing markedly demyelinated white matter. H&E, x 100. (B) Microscopic section of cerebral white matter showing demyelination, gliosis, perivascular accumulation of lymphocytes, and macrophages. H&E, x 250. ( C )Microscopic appearance of cerebral white matter from the degenerated area showing collection of macrophages a t the edge of the lesion. H&E, x 100.
1977; Mannaerts et al., 1979; Osmundsen et al., 1980; Singh et al., 19841. The reaction products are subsequently metabolized further by the mitochondria. 2. The biosynthesis of ether-phospholipids (plas-
It is obvious that any abnormality of the peroxisomes such as a decrease in the number or abnormality in function that may occur may lead to a n inherited disorder with serious clinical consequences. Moser has classified the peroxisomal disorders into 3 groups (Table IIIa). In group 1, activities of multiple peroxisomal enzymes are deficient and the number of peroxisomes is reduced. It is hypothesized that when peroxisomal structure is absent or reduced certain peroxisomal enzymes are degraded abnormally rapidly and this accounts for the multiple enzyme defects [Rachubinski et al., 1984; Moser, 19891. In group 2, activities of multiple peroxisomal enzymes are diminished, but the number of peroxisomes is normal. Moser thinks that diminished activity of peroxisoma1 enzymes in group 2 is due to the impaired peroxisoma1 utilization of flavin adenine-dinucleotide [Goldfischer et al., 19861. In group 3, peroxisomal structure is normal but the activity of a single peroxisomal enzyme is reduced. Activity of other peroxisomal enzymes is normal [Goldfischer et al., 1985; Moser et al., 19841. Biochemical abnormalities in Zellweger syndrome (ZS), infantile Refsum disease (IRD), and neonatal adrenoleukodystrophy (NALD) involve the metabolism of very long chain fatty acids (VLCFA),bile acids, phytanic acids, pipecolic acid, and plasmogen [Goldfischer et al., 1985; Hanson et al., 1979; Heymans et al., 1985; Kelley e t al., 1986; Moser et al., 1984; Poll-The, 1985; Schutgens et al., 1986; Trijbels et al., 19791. There are some differences in these disorders with respect to age of onset, severity of nervous system dysfunction, and duration of survival (Table IV). The difference between ZS, NALD, and IRD could be the result of different alleles or a nonallelic gene mutation or may be due to phenotypic variation of the same mutation.
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Fig. 10. (A) Microscopic appearance of white matter of the brain from demyelinated area showing macrophages with weak Oil RedO reaction ofthe cytoplasm (arrow).Oil RedO stain, x 100. (B)Microscopic appearance ofdemyelinated white matter of the brain showing PAS reactivity ofmacrophages (arrow).PAS stain, ~ 2 5 0 .
TABLE IIIb. Classification of Peroxisomal Diseases*
Fig, 11. Microscopic appearance of brain showing intact axis cylinders a t the progressive edge. Metachromatic stain, X 100.
I. Disorders of peroxisomal biogenesis a. Zellweger syndrome b. Hyperpipecolic acidemia c. Infantile Refsum syndrome d. Neonatal adrenoleukodystrophy 11. Peroxisomes intact: enzymes abnormal a. Rhizomelic chondrodysplasia punctata b. Conradi-Hunnerman chondrodysplasia (?) 11. Pseudo-Zellweger syndromes a. Single enzyme deficiencies 1. Thiolase deficiency 2. Acyl-CoA-oxidase deficiency 3. Acatalasemia b. Defective transport-rapid degradation 1. Pseudo-Zellweger @-oxidation of very long chain fatty acids) IV. Single peroxisomal function deficiency a. X-linked adrenoleukodystrophy b. Adult Refsum c. Hyperoxaluria type-I *Modified from Zellweger, H. (1989):Perorsisomopathies: New developments. Dev Med Child Neurol 31:264-266.
TABLE IIIa. Classification Peroxisomal Disorders* Group 1: Activities of multiple peroxisomal enzymes are deficient and number of peroxisomes is reduced a. Zellweger cerebrohepatorenal syndrome b. Neonatal adrenoleukodystrophy c. Hyperpipecolic acidemia (peroxisomal structure may be normal in hyperpipecolic acidemia) d. Infantile Refsum syndrome e. Rhizomelic chondrodysplasia punctata Group 2: Activitiese of multiple peroxisomal enzyme is reduced, and number of peroxisomes is normal a. X-linked adrenoleukodystrophy b. Acatalasemia (peroxisomes have been shown to present or even increased in the mouse with acatalasemia) *Courtesy of Dr. Hugo W. Moser, Professor Neurology and Pediatrics, Johns Hopkins liniversity.
The prenatal diagnosis of ZS, NALD, and IRD is based on the same criteria. All 3 disorders can be identified by the finding of a n accumulation of VLCFA in cultured amniotic fluid or chorionic villus fibroblasts [Hajra et al., 1985; Moser et al., 1982; Schutgens e t al., 19861. The prenatal diagnosis of IRD can be made by the detection of deficient phytanic acid oxidase in cultured amniotic fluid cells. (Poll-The et al., 1983). Diagnosis of ZS and IRD early in the first trimester can be made by demonstrating deficient activity of dihydroxyacetone phosphate acyltransferase in chorionic villi or fibroblasts, and impaired de novo plasmalogen biosynthesis in chorionic villus fibroblasts or a n abnormal intracellu-
Progressive Neurologic Deterioration lar localization of catalase [Jajra et al., 1985; Poll-The et al., 1985; Schutgens et al., 1986; Wanders et al., 19861.A precise diagnosis of the above diseases can help in genetic counseling, as the treatment of these conditions is not successful. Dr. Enid F. Gilbert-Barness: Professor of Pediatrics and Pathology Zellweger [1989] noted the rapid increase in studies on peroxisomal diseases. Cell fusion techniques, complementation studies of heterokarya, immunoblot studies of the enzymes of p-oxidation of very long chain fatty acids (VLCFA) led him to a modified taxonomy (Table IIIb). Moser [ 19891 summarizes the differential diagnosis of these by measuring VLCFA, pipecolic acid, phytanic acid, and bile acids in plasma, plasmalogens, and catalase in red blood cells, plasmalogen synthesis, catalase subcellular localization, phytanic oxidation, and VLCFA in fibroblasts. If the peroxisomes are present but VLCFA are increased immunoblot for acyl-CoA oxidase, bifunctional enzyme, and thiolase are needed.
GENETIC COMMENTS Dr. Renata Laxova:Professor of Pediatrics and Medical Genetics Genetic Aspects In considering the genetics of the disorder in this family, several noteworthy issues come to mind. They include (1)the variable clinical course and age-of-onset in several presumed affected individuals, (2) the reproductive options currently available to heterozygous females, and (3)the implications of the assignment of the adrenoleukodystrophy (ALD) gene to Xq28. Clinical Manifestations It appears that in this family, ALD is transmitted (as in most families) through a gene on the X chromosome. However, the variable manifestations and ages of onset are not usual for most X linked recessive disorders. The propositus, IV-3 in Figure 12 had a relatively typical, quite rapid clinical course of his disease, with onset of symptoms around age 6-7 and death a t age 9 % years. If the disease in the propositus and that in his male first cousins once removed (Fig. 12) is caused by the same abnormal X linked gene, it must have been transmitted from the mother of the patient’s maternal grandfather, in generation 1in the pedigree. No information is available about her, but her daughter’s sons are affected and her son’s daughters are proven carriers of the disease. This leads to the strong suspicion that the affected males in the family arelwere suffering from the same disorder. A review of available records was undertaken in an attempt to characterize more accurately the clinical course of the disease in the patient’s relatives, i.e., 11-6, 111-4, 111-5, and 111-7 (Fig. 12). The patient‘s maternal grandfather, 11-6, died in 1965 a t age 42 years. The principal anatomical diagnosis was “disseminated sclerosis, central nervous system.” Secondary diagnoses included aspiration pneumonia, emphysema, cachexia, atrophy of muscles of limbs with
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flexion deformities of lower limbs. He had had a diagnosis of “multiple sclerosis” for 13 years, established at about age 29. On autopsy his adrenal glands weighed 5 g each, and “no histologic diagnostic abnormalities were noted.” Little information exists on patient 111-4, the patient’s mother’s paternal first cousin. He died a t age 16 years in 1972. The only records available to us indicated a “leukodystrophy well worked up previously.” His final diagnosis was Schilder disease. No autopsy was available. Patient 111-5, brother of 111-4, died in 1971, at age 14 years. He had a 5 year history of progressive “mental retardation, optic atrophy, long tract signs, and seizures.” The family was told he had a “brain tumor.” On admission before death, the patient was described as severely demented and obtunded, in acute distress; limbs were held in quadriflexion secondary to the multiple contractures, reflexes were absent. He died of respiratory arrest, secondary to what-according to the records-was thought to be a progressive “dumping effect secondary to tube feedings and inappropriate ADH secretion.’’ His principal anatomic diagnosis was Schilder disease. Secondary diagnoses included acute and chronic mucopurulent tracheobronchitis, bilateral moderate bronchopneumonia. The adrenal glands weighed 4.1 g each. On section, the cortex was less than 1 mm, appeared light brown with a gray-tan, relatively prominent medulla. Several 0.2 cm gelatinous, well-circumscribed nodules were present in the cortical region of the left adrenal gland. 111-7, brother of 111-4 and 111-5, was hospitalized in 1978 a t age 20 with discharge diagnosis of “Addison’s disease, adrenal insufficiency, and headaches believed to be on a psychogenic basis.” Oral cortisone acetate 37.5 mg/day was prescribed. He was alive in 1986 a t the time of prenatal testing of the patient’s maternal aunt. No additional information is available about him currently. This is unfortunate, because familial X linked Addison disease as a n expression of ALD has been found to be associated with elevated C26 fatty acids [O’Neill et al., 19821. Such testing, if permitted by the patient, could help to delineate his diagnosis more accurately. In summary, the ages of onset of symptoms in this family (assuming the same disease in all affected individuals) ranged from around 7 years (in the propositus) to about 29 years in his grandfather. The duration of the disease ranged from 2-3 years in the propositus to 13 years in his grandfather; it is unknown in 111-7, the youngest (and presumably least severely affected) of the 3 brothers. A close relationship has been suggested between ALD and adrenomyeloneuropathy (AMN). The latter, although thought to present in the third decade with a milder and longer clinical course, has been described in a member of a t least one family in which 4 others had ALD [Davis et al., 19791. In addition, Griffin et al. [19771 and Schaumburg et al. 119771 considered AMN to be a probable variant of ALD. It is possible, then, that in the family presented here, the disease manifestations, while caused by the same mutant gene, confirm the existence of a wide range of variable expressions. This is a n unusual phenomenon for X-linked recessive
Liver Fibrosis and micronodular cirrhosis Hepatic siderosis
Retinal degeneration Oral involvement Pathologic changes Adrenals
Eye abnormalities Cataracts
Minor facial anomalies Renal cysts Patellar calcification
CharacteristicsInheritance Sex Survival
-
Absent
Absent
Not involved
-
Retinal degeneration
Absent -
Absent
AR M & F Adult
Refsum’s disease
Absent
Atrophy
-
Absent
X-linked Male Variabl+ usu. adolescent Absent
X-linked ALD (adult form)
Absent
Not involved
I
Absent
-
Absent
AR M&F Adult
Actalasemia
+
++
Not involved
-
+
Cataract present
Present Present
Present
AR M&F Usu 1 yr
Zell weger syndrome disease
Absent
Periportal fibrosis (mild)
Atrophy
-
Absent -
Absent
AR M&F 1 to 6-8 yr
Neonatal ALD
Absent
Absent
Not involved
+
Minor anomalies -
Male -
Infantile Refsum
TABLE IV. General Characteristics and Specific Biochemical Findings
+
Absent
Not involved
-
+
Cataracts
-
Typical facial appearance
AR M & F Usually 2 yr
Chondrodysplasia punctata
Absent
++
Not involved
-
Cataracts
Calcific stippling t
Present
AR Male Mostly 2 yr
Hyperpipecolic acidemia
Brain demyelination Neuronal heterotopia Ultrastructure lamellar inclusions Peroxisomes Liver Biochemical abnormalities (body fluids) Very long chain fatty acids ((2261 C22) Pipecolic acid Phytanic acid Intermediates of bile acid synthesis Plasminogen contents in tissues De novo synthesis Enzyme activity Dehydroxyacetone acyl transferase Alkyl dihydroxyacetone synthetase Elevated Elevated Elevated
Normal Elevated Elevated Normal
Elevated Normal Normal
Normal
Deficient
Deficient
Deficient
Deficient
Normal
Deficient
Decreased Decreased
Elevated
Elevated Elevated Elevated
Absent
Decreased Decreased
Elevated
Elevated Elevated Elevated
Decreased
++
-
+
Normal Normal
Elevated
Absent
Normal
-
++
in braid adrenals
-
+
Decreased
Deficient
-
Deficient
Decreased -
Elevated Elevated Elevated
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Barness et al.
I
II
111
IV
Fig. 12. Family history of propositus (indicated by arrow); affected males are indicated by shading, female carriers have dots. The diagnoses in quotation marks are those which the relatives were given by physicians caring for individual patients. 11-6, “multiple sclerosis”; 111-4, “neurological disorder”; 111-5, “brain tumor”; 111-7, “Addison disease”; IV-3, propositus, pregnancy terminated, infertile.
conditions. Furthermore, the situation presented here demonstrates one of the most important principles of clinical genetics, namely the need to direct attention to the whole family as well a s to the individual patient. Had this occurred, the confusing and inaccurate diagnoses in individual affected patients could have been avoided, and possibly much additional anguish prevented in this and many other families with a n incidence of genetic neurodegenerative disease. Carrier testing on cultured fibroblasts, of patient’s mother, 111-9, and her sister, 111-12, was performed before the patient’s death, by Drs. Ann and Hugo Moser a t the J.F. Kennedy Institute of Johns Hopkins University, Baltimore, Maryland. Results are given in Table V. Comparison of the ratios of C26 to C22 in these 2 women with those of ALD patients, obligate heterozygotes, and control individuals (Fig. 13) was clearly indicative of unequivocal carrier status in both patient’s mother and her sister. (This was not surprising since their father presumably had a n X-linked disease. The patient’s maternal aunt, 111-12, conceived in 1986. An amniocentesis for sex determination showed a male fetus whose amniocytes were kindly evaluated by Drs. Ann and Hugo Moser. The result, compared with those from control individuals and affected patients, shown in Table VI, indicated a n affected fetus. The parents elected to terminate the pregnancy and gave permission for a n autopsy (see above). The values of very
Adrenoleukodystrophy (ALD)-Adrenomyeloneuropathy (AMN) Ratio of C26.0 to C22.0Total Lipid Fatty Acids in Cultured Skin Fibroblasts April 1960 ALD-AMN patients diagnosis eslablished 0 or possible0
-
0.65 -
0.75 -
0.80
-
0.70
0.65 -
00
.p .4 : 0..
0.
.
0.
a mean
:j
060 0.55
c
0 706 f 0 236
I
f 0.202
oI
SD
: :
0.25
0.20
Patient’s maternal aunt Patient’s mother
Total”
C26.0iMg
C22.0iMg
Ratiob ~
15.49
0.204
0.638
0.320
51.87
0.244
0.799
0.305
Saturated very long fatty acidslmg protein (fibroblasts). bC26.0/C22.0 = within range of known heterozygotes for ALD.
a
0.05
Clinico-Pathological Report Progressive Neurologic Deterioration in a Nine-YearOld White Male ~
Lewis A. Barness, Sunita Chandra, Pamela Kling, Ftenata Laxova, David B. Allen, and Enid Gilbert-Barness Departments of Pediatrics (L.A.B., P.K., R.L., D.B.A., E.G.B.), Pathology (S.C., E.G.B.), and Medical Genetics (R.L.), University of Wisconsin Medical School, Madison, Wisconsin
KEY WORDS: Schilder’s disease, long chain fatty acids, ALD, genetic disorders CLINICAL PRESENTATION Dr. Pamela Kling: Chief Resident in Pediatrics CHIEF COMPLAINT The patient was a 7%-year-old white boy, admitted for evaluation of neurologic deterioration. HISTORY OF PRESENT ILLNESS Five months before admission, his mother noted that his left eye “would wander” and that he occasionally bumped into things. He was seen by a n ophthalmologist 2V2 months before admission who thought he had left exotropia and mild hyperopia. For several months, his teacher noted he did not appear interested or listening in class, with significant deterioration in his grades. A local audiologic evaluation showed decreased hearing bilaterally. He also appeared to have a personality change, with disinterest in the activities he once enjoyed. He often showed “dulled” responses to situations and clearly defective memory. At other times, he appeared emotionally labile and became extremely excited with laughing fits, usually inappropriate. He seemed to be preoccupied with the illness and death of his grandmother and his own death. He showed decreased coordination with worsening motor skills, appeared ataxic, and got into accidents easily. His mother felt it was “like having a two year old again.” PAST MEDICAL HISTORY His birth weight was 3,010 g a t term. He was born to a primigravida with no complications during pregnancy, Received for publication May 29, 1989; revision received April 18, 1990. Address reprint requests to Lewis A. Barness, M.D., H41434 Clinical Science Center, University of Wisconsin Medical School, Madison, WI 53792.
0 1990 Wiley-Liss, Inc.
labor, delivery, or neonatal course. He was a healthy child with no hospitalizations, operations, or chronic illnesses. He had received immunizations without reactions and had no allergies to medications.
DEVELOPMENT He lifted his head a t 3 weeks, rolled over a t 3 months, sat with support a t 5 months, and stood with support a t 6 months. He walked by 10 months, talked at ll/z years, was toilet trained a t 2% years, and rode a tricycle a t 3 years. FAMILY HISTORY [See Dr. Laxova’s text.] PHYSICAL EXAMINATION The patient was a 71/z-year-old white boy who was alert, talkative, but occasionally confused. Pulse was 92 beatslmin, respirations 32lmin, and blood pressure 114176 mmiHg. He was afebrile. His height was 128 cm and weight 25.3 kg (both a t the 75th centile). He was normocephalic, with left exotropia and apparently normal optic discs. Ear exam showed decreased hearing bilaterally. He had generous tonsils, lung and heart sounds were normal on auscultation. The spine was straight. Abdomen and bowel sounds were normal. Genitalia were normal for age (Tanner I). Skin was normal in pigmentation and texture. The boy was alert, but could not remember day of week or city. He had poor judgment, poor short-term memory, and could follow only a series of 2 instructions. There was some echolalia and excessive laughter. He spoke very loudly with poor enunciation.His math skills seemed appropriate for age. He had a n ataxic gait, was unable to skip or walk heel-to-toe. He had past-pointing on finger-to-nose testing and had poor rapid alternating movements. The Romberg sign was absent. His deep tendon reflexes were 2+14+ and symmetric, no Babinski’s reflex. Tone was normal and strength was 5 + I5 + . Gross sensation and sharplsoft discrimination were intact. Other than the previously noted findings, the cranial nerves were intact.
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EVALUATION Audiologic evaluation showed a 75-80 dB sensorineural hearing loss. Ophthalmologic exam showed some pallor of the optic nerves, suggestive of optic atrophy. On admission he had a hematocrit of 3996, hemoglobin of 13.0 g, and white blood cell count of 4,900/mm3 with 3% band and 42% segmented neutrophils, 28%, lymphocytes, 11%atypical lymphocytes, and 6%monocytes. Results of urinalysis and chemical survey were normal. Serum sodium was 138 mmoliliter, potassium, 4.1 mmoliliter, chloride, 103 mmol/liter, and carbon dioxide, 28 mmoliliter. CSF protein was 19 m g 8 and glucose, 66 mg8. CSF albumin was 62.796, CSF gamma globulin, 11.4%,with albumin: globulin ratio of 1.68. There was one mononuclear cell and no red blood cells. A CT scan showed symmetric areas of abnormality in the septum semiovale, posteriorly. Following contrast, there was diffuse enhancement. The EEG was abnormal and showed widespread symmetrical disturbance of cerebral function characterized by slowing in the parietal and occupital regions. Additional laboratory studies showed a n ACTH level of 198 pgiml (normal in our lab is less than 80 pg/ml). A morning cortisol level was 12.3 +g% which remained essentially unchanged (12.2 kg%) after injection of 0.01 mgikg IM synthetic ACTH. CLINICAL COURSE Over the next 2 years the patient suffered a progressive downhill course with emotional outbursts, blindness, deafness, inability to walk, difficulty swallowing, then decreased responsiveness. He required institutionalization and died with massive bilateral pneumonia.
DISCUSSION Dr. Lewis A. Barness: Professor of Pediatrics One approach to the diagnosis of a child such a s this is to attempt to categorize the illness. This child obviously had a progressive neurodegenerative disease. In contrast to nonprogressive disorders which are usually due to genetic or anatomic abnormalities or trauma or other structural abnormalities, progressive neurodegenerative diseases fall into 2 categories: metabolic and unknown (like President Grant who said he knew two songs: one was “Yankee Doodle” and the other wasn’t). Next, one tries to localize the lesion: gray matter lesions usually start with dementia and seizures and include the storage diseases; the history of this child is not consistent with such a disorder, White matter lesions usually start with motor abnormalities such a s spasticity, hypotonia, and ataxia and include the leukodystrophies or the kind of demyelinating lesions which must have been present in this child. A third category includes the systemic lesions such as spinocerebellar as in (F’riedreich) ataxia, ataxia telangiectasia, Bassen-Kornzweig, and Refsum syndromes and basal ganglia including the dystonias, Hallervorden-Spatz, and Huntington chorea. At first, the boy developed a n ocular palsy. Later he
became deaf after 7 years of apparent good health. This was quickly followed by a personality change, disinterest, and liability. With the laughing fits, I thought of Tourette syndrome, a catch-all disease, until the destructive process proceeded even more quickly. Subacute sclerosing panencephalitis should have presented greater evidence off cortical involvement, particularly seizures, at this point. Were there any similar diseases in the family? Dr. Pamela Kling: The grandfather is said to have had multiple sclerosis. Three cousins died with neurologic disorders. Dr. Lewis A. Barness: The family history is interesting. It is now recognized that the diagnosis of multiple sclerosis has also been given to individuals with such entities a s amyotrophic lateral sclerosis, juvenile adrenoleukodystrophy, as well as those with multiple sclerosis. Later it is noted the patient had optic atrophy, a sign of multiple sclerosis. What was the sex of the cousins with progressive neurologic disorders? Dr. Pamela Kling: They were boys. Dr. Lewis A. Barness: In contrast to muscle diseases or amino acid disorders, few progressive neurologic disorders are X-linked. If this is truly X-linked, the differential diagnosis becomes relatively limited. That the father works in sanitation may suggest toxins. However, no other relative was apparently ill. Physical findings of interest other than the exotropia were the large tonsils. Tangiers disease, analphalipoproteinemia with neurological manifestations generally presents a t a much younger age and the tonsils usually yellow. The neurological findings were consistent with diffuse neurological involvement. Dr. David Allen: What was the skin color? Dr. Kling: The skin was normal in color. Results of the CBC, urine, chemical survey, and lumbar puncture were normal. Were the serum sodium, potassium and chloride levels normal? Dr. Kling: Yes. Dr. Barness: For the diagnosis most likely, the sodium, and chloride levels would be expected to be low with a n elevated potassium. The history and diffuse brain abnormalities seen are consistent with a number of CNS degenerative lesions. In concluding the diagnostic approach, gray matter diseases are unlikely for reasons already stated. There is no evidence of storage disease such as large liver or spleen. There is no evidence of cerebromacular degeneration. The age-of-onset argues against aminoacidopathies. Of the leukodystrophies, Krabbie cerebrosidosis, Canavan disease, and metachromatic leukodystrophy generally start earlier and usually are associated with cerebrospinal fluid abnormalities. Of the demyelinating diseases, neuromyelitis optica appears consistent with much of the course. Children with multiple sclerosis do not usually develop signs of dementia so early. The course followed by this child is characteristic of juvenile adrenoleukodystrophy [Moser, 19891.This peroxisomal disorder is X-linked. Symptoms usually begin a t age 4-10 most often a s hyperactivity or attention deficit disorders followed by emotional changes, disor-
Progressive Neurologic Deterioration ders of speech and writing, and auditory defects. Later, dementia, seizures, and optic nerve involvement occur and death occurs in 1-2 years. Adrenal insufficiency occurs in about 30% of boys. This child had adrenal insufficiency, though asymptomatic, since he was shown to be nonresponsive to his own ACTH or to administered ACTH.
ADDENDUM After coming to this diagnosis, the following findings were entered into the Meditel computer assisted diagnosis system: confusion, mental deficiencies, hearing loss, ocular palsy, ataxia, and happy affect. The first diagnosis that was retrieved was “Schilder’s disease.” “Schilder’s disease” is now recognized as a group of diseases including X-linked adrenoleukodystrophy, multiple sclerosis, Pelizaeus-Merzbacher disease, metachromatic leukodystrophy, and ceroid lipofucscinosis. PATHOLOGICAL FINDINGS Sunita Chandra, M.B.B.S.: Associate Professor of Pathology Gross Examination At autopsy the body was a well-nourished 9-year-old white boy. He weighed 25.3 kg and was 128 cm long. Scalp hair was dark brown, and the body hair was light brown. The skin and conjunctivae were nonicteric. Pupils were dilated, fixed, and equal in size. Nose, ears, and teeth were unremarkable. No malformations of the face were noted. The chest was normal externally, and the abdomen was slightly distended. The external genitalia were normal. The upper limbs were unremarkable. The lower limbs were thin and there was a bilateral talipes equinovarus deformity. The pleural and pericardial cavities were unremarkable. Abdominal distension was caused by distended bowel. The peritoneal surface of the abdominal wall was tan and smooth. The mucosa of the stomach and small and large intestines was smooth and unremarkable. The heart was normal and weighed 120 g. The heart valves, right and left ventricular walls, and myocardium were unremarkable. The mucosa of the bronchial tree was slightly congested. No evidence of obstruction was present. Both lungs were heavy and together weighed 500 g. On the cut surface, patchy areas of firmness were evident. Microscopic sections showed bronchopneumonia. The liver was slightly enlarged and weighed 900 g (normal 800 g), The external surface was tan and smooth and mildly congested. Microscopic sections showed mild congestion and absence of periportal fibrosis (Fig. 1). The spleen was moderately enlarged and weighed 120 g and was soft and congested. The adrenal glands were small. The right weighed 1.2 g and the left 0.8 g (normal 3.5-4 g) (Fig. 2). The cut surfaces were very thin. The cortex was pale, and the medulla was unremarkable. Microscopic sections of the adrenal glands showed a n intact zona glomerulosa. The zona fasciculata and zona reticularis contained aggregates and nodules of ballooned cells (Fig. 3 ) and many multinucleated giant cells. The ballooned cells contained pink granular cytoplasm and few striations (Fig.
491
4). Perivascular and interstitial lymphocytic infiltration was noted. Ultrastructural studies of the adrenal cortex showed linear bilamellar cytoplasmic inclusions (Fig. 5). Biochemical studies of adrenal gland and brain tissue were performed by Dr. Hugo Moser (see genetics discussion by Dr. Renata Laxova). The brain weighed 1,350 g. It was bilaterally symmetrical, normal in size and gyral pattern (Fig. 6 ) . The olfactory bulbs and tracts and optic chiasm were intact. The pons, medulla, and upper cervical cord were grossly unremarkable. Horizontal section of the brain showed tannish-brown areas involving all the lobes of the brain (frontal, parietal, temporal, and occipital) and periventricular areas. These soft areas were sunken from the brain surface (Fig. 7A). These soft areas in the white matter picked up the Oil Red 0 stain, indicating breakdown of myelin and engulfment by macrophages (Fig. 7B). Sections from the pons, medulla, and cerebellum showed similar very tan, soft areas involving the white matter (Fig. 8A, B). Microscopic sections from the cerebral cortex showed a normal cortex. However, sections from the white matter showed severe demyelination, gliosis, perivascular accumulation of lymphocytes, and macrophages (Fig. 9A, B). The macrophages were more concentrated a t the edges of the lesions (Fig. 9C). The cytoplasm of these macrophages was weakly Oil Red 0 positive and many macrophages contained PAS positive material in their cytoplasm (Fig. 10A, B). Metachromatic stains were done and showed intact axis cylinders a t the progressive edges; however, in more severely involved areas axis cylinders were destroyed (Fig. 11). Ultrastructure of brain tissue from affected areas showed similar bilamellar enlarged intracytoplasmic inclusions. After the death of this child, the mother became pregnant again. The fetus was shown to be affected by prenatal diagnosis. Pregnancy was terminated a t 211/2 weeks of gestation. No obvious external abnormalities were seen in this fetus. The internal organs were macerated but normal. The liver was normal. The combined weight of both adrenals was 1.2 g. Microscopic sections of the adrenals showed a thin zona glomerulosa. Ballooning and occasional presence of giant cells was seen in the zona fasciculata and zona reticularis. PAS stains were mildly reactive. Ultrastructure examination of the adrenal cortex showed randomly oriented intracytoplasmic linear inclusions consisting of two 25 wide electron dense bands separated by a clear zone.
PATHOLOGICAL DISCUSSION Dr. Sunita Chandra: Associate Professor of Pathology Adrenoleukodystrophy is a disorder of defective peroxisomal oxidation of very long chain fatty acids. It is associated with progressive central nervous system demyelination, adrenal atrophy, and rarely adrenal insufficiency. Two distinct types of adrenoleukodystrophy (ALD) that differ in their age-of-onset, clinical course, and
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Fig. 1. Microscopic appearance of liver showing mild congestion and absence of periportal fibrosis. Hematoxylin and eosin (H&E), x 100.
Fig. 2. Gross appearance of urinary system with adrenal glands which are small.
Fig. 3. Microscopic appearance of adrenal gland showing nodules of ballooned cells in zona fasciculata and reticularis. H&E, x 25.
Fig. 5 . Electron micrograph of adrenal cortex showing linear bilaminar intracytoplasmic inclusions (arrow).Uranyl acetate and citrate, x 8,000. Fig. 4. Microscopic appearance of adrenal nodules composed of ballooned cells, containing pink granular cytoplasm and few striations.
H&E. x400.
Progressive Neurologic Deterioration mode of inheritance have been identified [Goldfischeret al., 19851. The X-linked form of ALD usually has its onset in prepubertal boys. It is characterized by progressive destruction of cerebral white matter and adrenal cortex. Death usually occurs in adolescence. Neonatal ALD appears in the neonatal period. The affected chil-
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dren suffer from severe hypotonia, seizures, and usually die by 6 years. In both types of ALD, there is accumulation of very long chain fatty acids (CZ6and C2J. Fibroblasts from these patients are deficient in their ability to oxidize long chain fatty acids [Goldfischer et al., 1985; Moser et al., 1980, 1984, 19891. Table I indicates the differences between the 2 types of adrenoleukodystrophy. The basic defect in ALD appears to be a deficiency of peroxisomal oxidation of fatty acids, and subsequent accumulation of very long chain fatty acids in the tissues, adrenal glands, and central nervous system in the X-linked form of ALD, and also in the liver and reticuloendothelial system in the neonatal form of ALD [Kelley et al., 19861. The pathological characteristics of both forms of ALD are shown in Table 11.
Peroxisomes and Their Function The peroxisomes are cytoplasmic organelles with a single membrane wall and electron-dense matrix [De Duve, 19831.The peroxisomes are present in all animal and plant cells, including cultured skin fibroblasts. Lazarow and Duve 119761 showed that rat liver peroxisomes catalyze the P-oxidation of fatty acids. At least 40 enzymes now have been shown to have peroxisomal localization [Tolbert, 19811.In recent years, it has become clear that in mammals peroxisomes fulfill a number of essential functions:
Fig. 6. Gross appearance ofthe brain showing normal gyral pattern.
1. Catabolism of very long chain fatty acids; peroxisomes seem specially equipped to bring about shortening of very long chain (> C ~ Zfatty ) acids LBremer,
Fig. 7. (A)Horizontal section of the brain showing tan-brown, soft areas involving white matter of all lobes of the brain and periventricular areas. ( B )Horizontal section of the brain showing soft. sunken areas in the white matter. Oil Red 0 stain.
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Fig. 8. (A) Section of the cerebrum showing normal-appearing cerebral cortex (dark area) and a pale, degenerated white matter. H&E, x 25. (B) Section of the cerebellum showing pale, degenerated white matter. Dentate nucleus cannot be identified. H&E, x 25.
TABLE I. Adrenoleukodystrophy: Clinical Differences Between X-Linked Adrenoleukodystrophy and Neonatal Adrenoleukodystrophy Characteristics --__
X-linked adrenoleukodystrophy
Age-of-onset Clinical course Storage material Pathology: Organs involved Biochemical abnormalities: (1) i. Plasma concentration of VLCFA" (2) ii. Ratio of C26:O :C22 :0 (3) iii. Hepatic catalase activity (4) iv. Total serum bile acids-trihydroxycoprostanic acid (THEA) (normally absent)
Neonatal adrenoleukodystrophy _ _
~
Prepubertal boys Slowly progressive, death in adolescence Very long chain fatty acids (VLCFA) (C26-C24) in the tissues
Neonatal Death, by 6 years Same
Brain, adrenals
Brain, adrenals, liver, macrophages
Increased
Increased
Abnormal Normal 50% is sedimented (10.3 nmoliml) Normal
Abnormal Abnormal, increase 12% is sedimented (8inmoliml) Normal
Absent
Serum bile acids
~
"VLCFA, Very long chain fattyacids.
TABLE 11. Adrenoleukodystrophy: Pathologic Changes Characteristics
X-linked adrenoleukodystrophy ~
Brain
Adrenal glands
-
Demyelination (centripetally spreading) Perivascular inflammatory infiltrates follow the margin of demyelination Diffuse gliosis Relative neuronal and axonal preservation Normal cytoarchitecture of cortex
Ultrastructure Liver
Atrophy of zona fasciculata and reticularis Ballooned adrenocortical cells with or without striations Bilamellar and laminar lipid inclusions Normal
Ultrastructure Deroxisomes
Peroxisomes identified
adrenoleukodystrophy _ _ _Neonatal - - ~
~~
-
Diffuse demyelination Perivasculara inflammation not intense
Mild primary maldevelopment of cerebral cortex Same Usually not present Same Periportal fibrosis, macrophages PAS positive material Reduced
.
Progressive Neurologic Deterioration
495
malogen and alkyl-glycerophospholipids). It has been shown in rodent liver and brain that 2 of the enzymes required for the introduction of ether bands in the biosynthesis of ether phospholipids acyl CoA :dihydroxyacetone phosphate acyltransferase and acyldyhydroxyacetone phosphate synthase are located predominantly in peroxisomes [Hajra et al., 1979, 19821. 3. The biosynthesis of bile acids [Hagey et al., 1982; Kase et al., 19831. 4. The catabolism of pipecolic acid, an intermediate in the degradation of L-lysine [Trijbels et al., 19791. 5. The catabolism of dicarboxylic acid; the p-oxidation of C12 dicarboxylic acid to C6-C10 dicarboxylic acids is a peroxisomal process [Mortensen et al., 19831. Glutaryl-CoA can also be catalyzed by peroxisomes [Vamecq et al., 19841. 6. The peroxisomes play a n essential role in the catabolism of phytanic acid, although some reports indicate that phytanic oxidase is a mitochondria1 enzyme [Tsai et al., 19691. 7. The oxidation of polyamines in rat liver [Holtta, 19771.
Fig. 9. (A)Microscopic section of the cerebellum taken from the soft areas showing markedly demyelinated white matter. H&E, x 100. (B) Microscopic section of cerebral white matter showing demyelination, gliosis, perivascular accumulation of lymphocytes, and macrophages. H&E, x 250. ( C )Microscopic appearance of cerebral white matter from the degenerated area showing collection of macrophages a t the edge of the lesion. H&E, x 100.
1977; Mannaerts et al., 1979; Osmundsen et al., 1980; Singh et al., 19841. The reaction products are subsequently metabolized further by the mitochondria. 2. The biosynthesis of ether-phospholipids (plas-
It is obvious that any abnormality of the peroxisomes such as a decrease in the number or abnormality in function that may occur may lead to a n inherited disorder with serious clinical consequences. Moser has classified the peroxisomal disorders into 3 groups (Table IIIa). In group 1, activities of multiple peroxisomal enzymes are deficient and the number of peroxisomes is reduced. It is hypothesized that when peroxisomal structure is absent or reduced certain peroxisomal enzymes are degraded abnormally rapidly and this accounts for the multiple enzyme defects [Rachubinski et al., 1984; Moser, 19891. In group 2, activities of multiple peroxisomal enzymes are diminished, but the number of peroxisomes is normal. Moser thinks that diminished activity of peroxisoma1 enzymes in group 2 is due to the impaired peroxisoma1 utilization of flavin adenine-dinucleotide [Goldfischer et al., 19861. In group 3, peroxisomal structure is normal but the activity of a single peroxisomal enzyme is reduced. Activity of other peroxisomal enzymes is normal [Goldfischer et al., 1985; Moser et al., 19841. Biochemical abnormalities in Zellweger syndrome (ZS), infantile Refsum disease (IRD), and neonatal adrenoleukodystrophy (NALD) involve the metabolism of very long chain fatty acids (VLCFA),bile acids, phytanic acids, pipecolic acid, and plasmogen [Goldfischer et al., 1985; Hanson et al., 1979; Heymans et al., 1985; Kelley e t al., 1986; Moser et al., 1984; Poll-The, 1985; Schutgens et al., 1986; Trijbels et al., 19791. There are some differences in these disorders with respect to age of onset, severity of nervous system dysfunction, and duration of survival (Table IV). The difference between ZS, NALD, and IRD could be the result of different alleles or a nonallelic gene mutation or may be due to phenotypic variation of the same mutation.
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Fig. 10. (A) Microscopic appearance of white matter of the brain from demyelinated area showing macrophages with weak Oil RedO reaction ofthe cytoplasm (arrow).Oil RedO stain, x 100. (B)Microscopic appearance ofdemyelinated white matter of the brain showing PAS reactivity ofmacrophages (arrow).PAS stain, ~ 2 5 0 .
TABLE IIIb. Classification of Peroxisomal Diseases*
Fig, 11. Microscopic appearance of brain showing intact axis cylinders a t the progressive edge. Metachromatic stain, X 100.
I. Disorders of peroxisomal biogenesis a. Zellweger syndrome b. Hyperpipecolic acidemia c. Infantile Refsum syndrome d. Neonatal adrenoleukodystrophy 11. Peroxisomes intact: enzymes abnormal a. Rhizomelic chondrodysplasia punctata b. Conradi-Hunnerman chondrodysplasia (?) 11. Pseudo-Zellweger syndromes a. Single enzyme deficiencies 1. Thiolase deficiency 2. Acyl-CoA-oxidase deficiency 3. Acatalasemia b. Defective transport-rapid degradation 1. Pseudo-Zellweger @-oxidation of very long chain fatty acids) IV. Single peroxisomal function deficiency a. X-linked adrenoleukodystrophy b. Adult Refsum c. Hyperoxaluria type-I *Modified from Zellweger, H. (1989):Perorsisomopathies: New developments. Dev Med Child Neurol 31:264-266.
TABLE IIIa. Classification Peroxisomal Disorders* Group 1: Activities of multiple peroxisomal enzymes are deficient and number of peroxisomes is reduced a. Zellweger cerebrohepatorenal syndrome b. Neonatal adrenoleukodystrophy c. Hyperpipecolic acidemia (peroxisomal structure may be normal in hyperpipecolic acidemia) d. Infantile Refsum syndrome e. Rhizomelic chondrodysplasia punctata Group 2: Activitiese of multiple peroxisomal enzyme is reduced, and number of peroxisomes is normal a. X-linked adrenoleukodystrophy b. Acatalasemia (peroxisomes have been shown to present or even increased in the mouse with acatalasemia) *Courtesy of Dr. Hugo W. Moser, Professor Neurology and Pediatrics, Johns Hopkins liniversity.
The prenatal diagnosis of ZS, NALD, and IRD is based on the same criteria. All 3 disorders can be identified by the finding of a n accumulation of VLCFA in cultured amniotic fluid or chorionic villus fibroblasts [Hajra et al., 1985; Moser et al., 1982; Schutgens e t al., 19861. The prenatal diagnosis of IRD can be made by the detection of deficient phytanic acid oxidase in cultured amniotic fluid cells. (Poll-The et al., 1983). Diagnosis of ZS and IRD early in the first trimester can be made by demonstrating deficient activity of dihydroxyacetone phosphate acyltransferase in chorionic villi or fibroblasts, and impaired de novo plasmalogen biosynthesis in chorionic villus fibroblasts or a n abnormal intracellu-
Progressive Neurologic Deterioration lar localization of catalase [Jajra et al., 1985; Poll-The et al., 1985; Schutgens et al., 1986; Wanders et al., 19861.A precise diagnosis of the above diseases can help in genetic counseling, as the treatment of these conditions is not successful. Dr. Enid F. Gilbert-Barness: Professor of Pediatrics and Pathology Zellweger [1989] noted the rapid increase in studies on peroxisomal diseases. Cell fusion techniques, complementation studies of heterokarya, immunoblot studies of the enzymes of p-oxidation of very long chain fatty acids (VLCFA) led him to a modified taxonomy (Table IIIb). Moser [ 19891 summarizes the differential diagnosis of these by measuring VLCFA, pipecolic acid, phytanic acid, and bile acids in plasma, plasmalogens, and catalase in red blood cells, plasmalogen synthesis, catalase subcellular localization, phytanic oxidation, and VLCFA in fibroblasts. If the peroxisomes are present but VLCFA are increased immunoblot for acyl-CoA oxidase, bifunctional enzyme, and thiolase are needed.
GENETIC COMMENTS Dr. Renata Laxova:Professor of Pediatrics and Medical Genetics Genetic Aspects In considering the genetics of the disorder in this family, several noteworthy issues come to mind. They include (1)the variable clinical course and age-of-onset in several presumed affected individuals, (2) the reproductive options currently available to heterozygous females, and (3)the implications of the assignment of the adrenoleukodystrophy (ALD) gene to Xq28. Clinical Manifestations It appears that in this family, ALD is transmitted (as in most families) through a gene on the X chromosome. However, the variable manifestations and ages of onset are not usual for most X linked recessive disorders. The propositus, IV-3 in Figure 12 had a relatively typical, quite rapid clinical course of his disease, with onset of symptoms around age 6-7 and death a t age 9 % years. If the disease in the propositus and that in his male first cousins once removed (Fig. 12) is caused by the same abnormal X linked gene, it must have been transmitted from the mother of the patient’s maternal grandfather, in generation 1in the pedigree. No information is available about her, but her daughter’s sons are affected and her son’s daughters are proven carriers of the disease. This leads to the strong suspicion that the affected males in the family arelwere suffering from the same disorder. A review of available records was undertaken in an attempt to characterize more accurately the clinical course of the disease in the patient’s relatives, i.e., 11-6, 111-4, 111-5, and 111-7 (Fig. 12). The patient‘s maternal grandfather, 11-6, died in 1965 a t age 42 years. The principal anatomical diagnosis was “disseminated sclerosis, central nervous system.” Secondary diagnoses included aspiration pneumonia, emphysema, cachexia, atrophy of muscles of limbs with
497
flexion deformities of lower limbs. He had had a diagnosis of “multiple sclerosis” for 13 years, established at about age 29. On autopsy his adrenal glands weighed 5 g each, and “no histologic diagnostic abnormalities were noted.” Little information exists on patient 111-4, the patient’s mother’s paternal first cousin. He died a t age 16 years in 1972. The only records available to us indicated a “leukodystrophy well worked up previously.” His final diagnosis was Schilder disease. No autopsy was available. Patient 111-5, brother of 111-4, died in 1971, at age 14 years. He had a 5 year history of progressive “mental retardation, optic atrophy, long tract signs, and seizures.” The family was told he had a “brain tumor.” On admission before death, the patient was described as severely demented and obtunded, in acute distress; limbs were held in quadriflexion secondary to the multiple contractures, reflexes were absent. He died of respiratory arrest, secondary to what-according to the records-was thought to be a progressive “dumping effect secondary to tube feedings and inappropriate ADH secretion.’’ His principal anatomic diagnosis was Schilder disease. Secondary diagnoses included acute and chronic mucopurulent tracheobronchitis, bilateral moderate bronchopneumonia. The adrenal glands weighed 4.1 g each. On section, the cortex was less than 1 mm, appeared light brown with a gray-tan, relatively prominent medulla. Several 0.2 cm gelatinous, well-circumscribed nodules were present in the cortical region of the left adrenal gland. 111-7, brother of 111-4 and 111-5, was hospitalized in 1978 a t age 20 with discharge diagnosis of “Addison’s disease, adrenal insufficiency, and headaches believed to be on a psychogenic basis.” Oral cortisone acetate 37.5 mg/day was prescribed. He was alive in 1986 a t the time of prenatal testing of the patient’s maternal aunt. No additional information is available about him currently. This is unfortunate, because familial X linked Addison disease as a n expression of ALD has been found to be associated with elevated C26 fatty acids [O’Neill et al., 19821. Such testing, if permitted by the patient, could help to delineate his diagnosis more accurately. In summary, the ages of onset of symptoms in this family (assuming the same disease in all affected individuals) ranged from around 7 years (in the propositus) to about 29 years in his grandfather. The duration of the disease ranged from 2-3 years in the propositus to 13 years in his grandfather; it is unknown in 111-7, the youngest (and presumably least severely affected) of the 3 brothers. A close relationship has been suggested between ALD and adrenomyeloneuropathy (AMN). The latter, although thought to present in the third decade with a milder and longer clinical course, has been described in a member of a t least one family in which 4 others had ALD [Davis et al., 19791. In addition, Griffin et al. [19771 and Schaumburg et al. 119771 considered AMN to be a probable variant of ALD. It is possible, then, that in the family presented here, the disease manifestations, while caused by the same mutant gene, confirm the existence of a wide range of variable expressions. This is a n unusual phenomenon for X-linked recessive
Liver Fibrosis and micronodular cirrhosis Hepatic siderosis
Retinal degeneration Oral involvement Pathologic changes Adrenals
Eye abnormalities Cataracts
Minor facial anomalies Renal cysts Patellar calcification
CharacteristicsInheritance Sex Survival
-
Absent
Absent
Not involved
-
Retinal degeneration
Absent -
Absent
AR M & F Adult
Refsum’s disease
Absent
Atrophy
-
Absent
X-linked Male Variabl+ usu. adolescent Absent
X-linked ALD (adult form)
Absent
Not involved
I
Absent
-
Absent
AR M&F Adult
Actalasemia
+
++
Not involved
-
+
Cataract present
Present Present
Present
AR M&F Usu 1 yr
Zell weger syndrome disease
Absent
Periportal fibrosis (mild)
Atrophy
-
Absent -
Absent
AR M&F 1 to 6-8 yr
Neonatal ALD
Absent
Absent
Not involved
+
Minor anomalies -
Male -
Infantile Refsum
TABLE IV. General Characteristics and Specific Biochemical Findings
+
Absent
Not involved
-
+
Cataracts
-
Typical facial appearance
AR M & F Usually 2 yr
Chondrodysplasia punctata
Absent
++
Not involved
-
Cataracts
Calcific stippling t
Present
AR Male Mostly 2 yr
Hyperpipecolic acidemia
Brain demyelination Neuronal heterotopia Ultrastructure lamellar inclusions Peroxisomes Liver Biochemical abnormalities (body fluids) Very long chain fatty acids ((2261 C22) Pipecolic acid Phytanic acid Intermediates of bile acid synthesis Plasminogen contents in tissues De novo synthesis Enzyme activity Dehydroxyacetone acyl transferase Alkyl dihydroxyacetone synthetase Elevated Elevated Elevated
Normal Elevated Elevated Normal
Elevated Normal Normal
Normal
Deficient
Deficient
Deficient
Deficient
Normal
Deficient
Decreased Decreased
Elevated
Elevated Elevated Elevated
Absent
Decreased Decreased
Elevated
Elevated Elevated Elevated
Decreased
++
-
+
Normal Normal
Elevated
Absent
Normal
-
++
in braid adrenals
-
+
Decreased
Deficient
-
Deficient
Decreased -
Elevated Elevated Elevated
500
Barness et al.
I
II
111
IV
Fig. 12. Family history of propositus (indicated by arrow); affected males are indicated by shading, female carriers have dots. The diagnoses in quotation marks are those which the relatives were given by physicians caring for individual patients. 11-6, “multiple sclerosis”; 111-4, “neurological disorder”; 111-5, “brain tumor”; 111-7, “Addison disease”; IV-3, propositus, pregnancy terminated, infertile.
conditions. Furthermore, the situation presented here demonstrates one of the most important principles of clinical genetics, namely the need to direct attention to the whole family as well a s to the individual patient. Had this occurred, the confusing and inaccurate diagnoses in individual affected patients could have been avoided, and possibly much additional anguish prevented in this and many other families with a n incidence of genetic neurodegenerative disease. Carrier testing on cultured fibroblasts, of patient’s mother, 111-9, and her sister, 111-12, was performed before the patient’s death, by Drs. Ann and Hugo Moser a t the J.F. Kennedy Institute of Johns Hopkins University, Baltimore, Maryland. Results are given in Table V. Comparison of the ratios of C26 to C22 in these 2 women with those of ALD patients, obligate heterozygotes, and control individuals (Fig. 13) was clearly indicative of unequivocal carrier status in both patient’s mother and her sister. (This was not surprising since their father presumably had a n X-linked disease. The patient’s maternal aunt, 111-12, conceived in 1986. An amniocentesis for sex determination showed a male fetus whose amniocytes were kindly evaluated by Drs. Ann and Hugo Moser. The result, compared with those from control individuals and affected patients, shown in Table VI, indicated a n affected fetus. The parents elected to terminate the pregnancy and gave permission for a n autopsy (see above). The values of very
Adrenoleukodystrophy (ALD)-Adrenomyeloneuropathy (AMN) Ratio of C26.0 to C22.0Total Lipid Fatty Acids in Cultured Skin Fibroblasts April 1960 ALD-AMN patients diagnosis eslablished 0 or possible0
-
0.65 -
0.75 -
0.80
-
0.70
0.65 -
00
.p .4 : 0..
0.
.
0.
a mean
:j
060 0.55
c
0 706 f 0 236
I
f 0.202
oI
SD
: :
0.25
0.20
Patient’s maternal aunt Patient’s mother
Total”
C26.0iMg
C22.0iMg
Ratiob ~
15.49
0.204
0.638
0.320
51.87
0.244
0.799
0.305
Saturated very long fatty acidslmg protein (fibroblasts). bC26.0/C22.0 = within range of known heterozygotes for ALD.
a
0.05
