Lethal neonatal deficiency of carnitine palmitoyltransferase II associated with dysgenesis of the brain and kidneys

Lethal neonatal deficiency of carnitine palmitoyltransferase II associated with dysge n esls ' of the brain and kidneys Kathryn N. North, MD, Charles L. Hoppel, MP, Umberto De Girolami, MD, Harry P. W. Kozakewich, MD, a n d Mark S. Korson, MD From the Departments of Medicine and Pathology, Children's Hospital, Boston, Massachusetts, and the Department of Pharmacology and Medicine, Case Western ReserveUniversity, Veterans Administration Medical Center, Cleveland, Ohio

We describe neonatal onset of a lethal multiorgan deficiency of carnitine palmitoyltransferase II (CPT II) associated with dysmorphic features, cardiomyopathy, and cystic dysplasia of the brain and kidneys. Concentrations of Iong-chain acylcarnitines were elevated in blood and multiple tissues, diffuse lipid accumulation was present at autopsy, and a profound deficiency of CPT II activity was evident in heart, liver, muscle, and kidney tissue. This disorder constitutes another recognizable malformation syndrome with a metabolic basis. Deficiency of CPT II should be included in the differential diagnosis of patients with cystic renal dysplasia, dysmorphism, central nervous system malformations, and early death, along with glutaric acidemia type II, Zellweger syndrome, and other disorders in which peroxisomal l~-oxidation is impaired. The clinicopathologic similarities among these disorders raise the possibility that a common biochemical mechanism, namely the disruption of l~-oxidation of fatty acids, is responsible for the abnormal organogenesis. (J PEDIATR1995; 127:414-20) Camitine palmitoyltransferase II is involved in the transport of long-chain fatty acids from the cytosol to the mitochondrial matrix, where fatty acid 13-oxidation occurs. After traversing the outer mitochondrial membrane, camitine is conjugated with palmitoyl--coenzyme A by CPT I to yield free CoA and palmitoylcamitine, which is transferred across the inner mitochondrial membrane by camitine acylcarnitine translocase. CPT II then cleaves palmitoylcamitine to form free carnitine and palmitoyl CoA, which undergoes 13-oxidation. 1 "Classic" CPT II deficiency is manifested in adulthood with recurrent exercise intolerance and myoglobinuria. 1 Childhood and neonatal onset forms of the disorder have recently been recognized and are characterized by hypoketotic hypoglycemia, hepatopathy, cardiomyopathy, and sudden Submitted for publication Feb. 7, 1995; accepted April 24, 1995. Reprint requests: Kathryn N. North, MD, Department of Nettrology, Children's Hospital, Bridge Road, Camperdown, New South Wales 2050, Australia. 9/20/65858

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death. 26 The neonatal lethal form of the disorder is associated with minimal residual enzyme activity and a marked accumulation of long-chain acylcamitines in multiple tis-

sues.2 We describe a patient with neonatal lethal multiorgan CPT II deficiency associated with dysmorphic features, cardioCPT CoA ETF ETF-QO

Camitine palmitoyltransferase Coenzyme A Electron transfer flavoprotein ETF-ubiquinone oxidoreductase

myopathy, cystic dysplasia of the brain and kidneys, and fatty infilträtion of multiple organs. This disorder constitutes another recognizable malformation syndrome with a metabolic basis. CASE REPORT

A term female infant was referred on day 4 of life because of hyperammonemia and seizures. The family history was noncontributory. The pregnancy was complicated by marked oligohydramnios, and respiratory distress developed soon after delivery. Coagulase-

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Fig. 1. Large cytoplasmic lipid vacuoles present in hepatocytes (A) and ventricular myocardial cells (B). (Hematoxylineosin stain; x400.) negative staphylococcus was cultured in blood, and the infant was treated with intravenous administration of 10% dextrose and antibiotics. An abdominal ultrasound study on day 1 demonstrated bilaterally enlarged kidneys with cortical cysts and a normal liver. Cranial ultrasonography showed a left pefiventricular cyst and scattered areas of increased echogenicity throughout the brain and basal ganglia. On day 3 of life, seizures with prolonged apnea developed and tracheal intubation was required. Serum bicarbonate, glucose, calcium, magnesium, lactate, creatinine, and urea concentrations were normal; urinary ketones were not present, the total serum bilirubin concentration was elevated (191.5 ~unol/L [11.2 mg/dl], direct 77 pmol/L), and liver aminotransferase values were mildly increased. The serum ammonia concentration was 1811 pmol/L (normal range 10 to 47) (3084 pg/dl). At this fime the infant had no response to stimulation, the pupils were fixed and dilated, the fundi were normal, and the comeas were cloudy. Dysmorphic features included microcephaly (head circumference 30.5 cm); a high, sloping forehead; tat occiput; overfolded helices; long, tapering fingers and toes; extra digital creases on fmgers 2 to 4 bilaterally; widely spaced nipples; contractures of the knees, elbows, and small joints of the hands; and hypoplastic toenails. A soft pansystofic murmur was andible at the left stemal edge, and firm renal masses were palpable bilaterally. Hemodialysis was performed for a 72-hour period, and the clinical status of the infant improved slightly; the pupils became reactive, and the serum ammonia concentration subsequently remained within the normal range. The liver progressively increased in size and renal function deteriorated. On day 8 of life, worsening bradycardia developed, with widening of the QRS complexes, hypotension, and oliguria. The infant died at 10 days of age. Invesfigations. Serum titers for congenital infections showed negative findings; plasma amino acid values were normal; urinary amino acids included marked elevations of lysine, omithine, and arginine; and orotic acid was not detectable by high-performance liquid chromatography. Urinary organic acids demonstrated no di-

agnostic pattem; in particular, no glutaric acid was identified, but small amounts of 5-hydroxyhexanoic, suberic, and sebacic acids were present. Analysis of very long chain fatty acids showed a slight increase in the C26/C22 ratio; the phytanic acid level was within the normal range, and the pipecolic acid level was significantly elevated (12.7 gmol/L; normal range 0.8 to 5.3), consistent with changes resulting from hepatocellular disease (A. Moser, R. Kelley: personal communication, 1994). The urinary acylglycine profile showed increased excretion of long-chaln dicarboxylic acids (12- to 16-carbon chains). The blood acylcarnitine profle demonstrated a major elevation of long-chain acylcamitines (16- to 18-carbon chains), and very little acetylcarnitine. Autopsy findings. Autopsy was performed 3 hours after death. Macroscopic examination of the internal organs revealed a markedly fatty liver, and the cardiac left ventricular free wall and sepmm were hypertrophied. The kidneys were markedly enlarged (48.2 gm; expected weight 30 gm) and had poorly defined corticomedullary differentiation with increased connective tissue, small papillae and calices, and scattered cortical and medullary cysts up to 0.3 cm in diameter. Light microscopy showed striking vacuolar changes in the cytoplasm of the hepatocytes (Fig. 1, A), in the ventricular myocytes (Fig. 1, B), and to a lesser extent in the epithelium of the renal~ proximäl convoluted tubules, the provisional adrenal cortex, skeletal muscle, and pancreatic exocrine cells. The vacuoles consisted of lipid (oll red O staining of formalin-fixed cryostat sections). The kidneys were markedly dysplastic, with cystic change evident in the nephron mlit and particularly the collecting ducts, in both the cortex and the medulla (Fig. 2). The medullary collecting ducts were frequently associated with a connective tissue collar, and there was dismption of the usually well defined lateral borders of the medullae. Perilobar and intralobar nephrogenic nests were present. Tissue for electron microscopy from the heart, liver, diaphragm, adrenal glands, and kidneys was immediately fixed in 2% glutaraldehyde, postfixed in osmium tetroxide, and stained with uranylacetate and lead citrate. Ulkastructurally the lipid accumnlation

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[:ig. 2. A, Coronal whole mount of kidney, showing poorly formed medullae and papillae, and cystic dysplasia affecting the cortex and medulla. (Hematoxylin-eosin stain; x 1.) B, Renal parenchyma with focal tubular ectasia of collecting ducts and nephron tubule. Note the disorganization of the medulla (right-hand side ofphotograph). (Hematoxylin-eosin stain; x25.)

[:ig. 3. Electron micrograph of myocardial myocytes with lipid droplets (L) and scrolls of osmiophilic membranes (M). Note mitochondrial inclusions of glycogen (solid arrow), lipid (curved arrow), and fing-shaped bodies (open arrow). (x4689.)

appeared as lightly osmiophilic droplets consistent with neutral fat. At the periphery of some of these droplets, and admixed with them, were thin, irregular membranous profiles of uncertain origin (Fig. 3). Scrolls of osmiophific membranes (lipofuscin-like) were present in the ventricular myocytes and renal proximal tubular epithelium. A few mitochondria in the ventricular myocytes and diaphragmatic muscle cells contained lipid droplets as well as ring-shaped inclusions of uncertain origin, ventricular myocytes also had rare mitochondria with glycogen inclusions (Fig. 3, B). There was mitochondrial enlargement in some skeletal muscle fibers, and frequent

giant mitochondria were seem in the provisional adrenal cortex. Peroxisomes wem present in hepatocytes and renal proximal convoluted tubular epithelium, and appeared normal in size and number. The brain was moderately reduced in weight (265 gm; expected weight 335 gm). Although there was no definite evidence of gyral anomalies over the convexities, the cingulate gyms was abnormal, with focal irregularities and interruptions, particularly in the posterior region. A large cystic cavity was noted next to the anterior horn of the lateral ventricle. Microscopically, the cyst was lined by dense

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glial tissue with foci of hemosiderin-laden macrophages and free pigment, and surrounded by multiple small cystic spaces lined by ependyma (Fig. 4). There was extensive reactive gliosis, characterized by an increase in the number of astrocytes and a marked increase in the stainable cytoplasm around the nucleus (gemistocytic shapes). There were scattered Alzheimer type 2 glia in the frontal and temporal cortex. Glial heterotopias were noted in the subarachnoid space at the base of the brain and adjacent to the splenium and the corpus callosum. There was focal disruption of cortical lamination in the posterior portion of the cingulate gyrus (Fig. 5). METHODS Analysis of carnitine and acylcarnitine was pefformed in heart muscle, skeletal muscle, and liver and kidney tissue by means of high-performance liquid chromatography of 4'bromophenacyl derivatives.7 Activity of CPT was assayed in cardiac muscle, skeletal muscle, and liver and kidney tissue with three complementary assays. Total CPT activity was measured in both the forward direction (assay I) and the backward or reverse direction (assay II) by methods described previously.8 CPT assay t was modified to include a pretreatment of the fissue homogenate with Lubrol P×, 10 mmol/L, for 10 minutes on ice. CPT assay III was a modified forward reaction to measure malonyl-CoA-sensitive activity. The incubation medium contained 80 mmol potassium chloride per liter; 3-(N-morpholino)propanesulfonic acid, 50 mmol/L (pH 7.2); ethylene glycol-bis(~-aminoethyl ether)N,NJV',N'-tetraacetic acid, 2 mmol/L; palmitoylCoA, 40 pmol/L; L-carnitine, 0.5 mmol/L; and bovine serum albumin, 4 mg/tal. The reaction was performed at 37 ° C for 3 minutes; total assay III activity was measured in the absence of malonyl-CoA, whereas malonyl-CoA-insensitive assay III contained 200 pmol malonyl-CoA per liter. The difference between the two measurements reflects malonylCoA-sensifive activity. The assays were performed on two different days with lx and 2x protein content with similar results. Activity of citrate synthase, a mitochondrial enzyme, was also measured as a fissue control. RESULTS Long-chain acylcarrdtines were markedly increased in all tissues (Table I). The concentration of total carnitine was within the normal range in skeletal and heart muscle and was mildly increased in liver and kidney. Free camitine levels were decreased in skeletal and heart muscle, normal in liver, and mildly elevated in kidney. In all tissues the total CPT activity measured in both the forward direction and the backward, or reverse, reaction was markedly decreased relative to control values (Table II). The malonyl-CoA sensitivity of the forward reaction (reflecting CPT I activity) was normal in skeletal muscle and increased in heart muscle and in liver and kidney tissue relative to control values. These results indicate a marked decrease or absence of CPT II ac-

Fig. 4. Paraventricular cysts showing dense gfiosis lining large cyst- and ependyma-lined cystic spaces. (Luxol fast blue-hematoxylin-eosin stain; x58.) tivity, with normal or increased activity of CPT I in all tissues measured. DISCUSSION Examination of out patient confirmed a profound deficiency of CPT II inmultiple organs; excessive long-chain acylcarnitine values and a small acetylcarnitine peak were present in the blood, indicating diminished flux through mitochondrial [3-oxidation. However, additional biochemical abnormalities differed considerably from what is usually seen in CPT II deficiency. Although mild to moderate hyperammonemia can occur in [3-oxidation defects, the marked hyperammonemia was atypical. Concurrent staphylococcal sepsis may have contributed to its severity. The absence of ketosis, acidosis, or hypoglycemia may be related to the fact that the patient received intravenous infusions of fluid with 10% dextrose continuously from birth. Dicarboxylic aciduria has not been described previously in association with CPT II deficiency, although the results of urinary organic acid analyses have not been reported for previous neonatal

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Fig. 5. Focal disruption of cortical lamination (arrow) in posterior portion of cingulate gyrus. Note lack of normal layering and irregular clustering of neurons. (Cresyl violet stain; x58.)

Table I. Tissue concentrations of carnitine in a newbom with CPT II deficiency and normal subjects Carnitine/acylcarnitine Total carnitine Patient Control subjects* % Normal mean Free camitine Patient Control subjects* % Normal mean Long-chain acylcamitines Patient Control subjects* % Normal mean

Liver

Kidney

Skeletal muscle

Heart muscle

1.23 0.69 + 0.26 178

2.38 0.52 + 0.15 457

1.92 3.52 + 1.61 55

2.33 0.63 -+ 0.21 370

0.71 0.63 + 0.23 113

0.98 0.37 + 0.14 264

0.44 2.53 + 1.21 17

0.80 0.50 + 0.14 160

0.58 0.03 _+0~0t 1933

0.69 0.02 _+0.01 3450

0.60 0.09 + 0.05 667

0.21 0.02 _+0.01 1050

All measurements are expressed in micromolesper gram of wet weight. Long-chain acylcamitines were markedly increased in all tissues. Total camitine concentrafion was within the normal range in skeletalmuscle and was mildly increasedin heart, liver, and kidney fissue.Free carnitinelevels were decreasedin skeletal muscle, normal in liver tissue, mad mildly elevated in kidney and heart muscle. *Control subjects: Liver, muscle (psoas), heart, and kidney controI autopsy data obtained from five infants and three adults who died of pulmonary, congenital heart, and infectious diseases. (Kindly provided by Dr. Douglas Kerr.) Values are mean + SD.

lethal cases. The absence of dicarboxylic aciduria in milder phenotypes is thought to result from partial peroxisomal oxidation of long-chain fatty acids, followed by complete mitochondrial oxidation of the medium-chain intermediates. 1 Dicarboxylic aciduria in out patient may reflect not only the severity of the enzyme defect but also inadequate compensatory mechanisms, perhaps because of a secondary toxic inhibition of mitochondrial [3-oxidation. The associafion of mulfiple malformations with neonatal lethal CPT II deficiency has been documented previously in two families. 9,10 C o m m o n findings at autopsy included diffuse lipid accumulation, cardiomegaly, dysplasfic renal pa-

renchyma, and neuronal migration defects. In our patient, there were focal disruptions of cortical lamination and gfial heterotopias. The paraventricular cysts were immediately adjacent to the ventricular system and lined by glia, which suggests cerebral dysgenesis, although a destmctive lesion could not be excluded. Severe white matter reactive gfiosis was evident, along with multifocal Alzheimer type 2 glia, characteristic of hyperammonemic encephalopathy. However, diffuse white mauer echogenicity was evident on cranial ultrasonography pefformed on day 1, suggesting a prenatal onset to some of the white matter changes. Although differences in residual enzyme activity may ex-

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Table II. Results of carnitine palmitoyltransferase assays of tissue from a neonate with CPT II deficiency and normal subjects Carnitine palmitolyl transferase

Liver tissue

Kidney tissue

Assay I (forward reaction) Patient 547 Control values* 2850 + 180 % Normal mean 19 Assay II (reverse reaction) Patient 783 Control values* 10260 + 1710 % Normal mean 8 Assay lIl (modified forward reaction) Total Patient 378 Control values* 326 + 66 Malonyl-CoA insensitive (CPT II) Patient 49 Control values* 218 + 67 % Normal mean 22 Malonyl-CoA sensitive (CPT I) Patient 329 Control values* j " 107 + 35 % Normal mean 307 Citrate synthase Patient 3.1 Control values* 6.2 + 1.8 % Normal mean 50

Skeletal muscle

Heart muscle

52 2707 2

13 594 + 184 2

43 2035 + 1009 4

346 8352 4

0 2503 + 625 0

240 6024 + 2897 4

205 404

77 187 + 69

371 491 + 80

8 376 2

0 115 + 58 0

8 405 + 95 2

197 29 679

77 72 + 28 107

363 86 + 23 422

14.7 19 + 5 77

32.9 62 + 8 53

5.7 13.1 44

The total CPT activity,measuredin nanomoles per minute per milligramof wet weight tissue, in both the forward reaction (assay I) and the reversereaction (assay Il), was markedly decreased relative to prior and concurrent control values. The malonyl CoA sensitivityof the forward reaction (reflectingCPT I activity) was increased relativeto prior and concurrent controls in liver, kidney, and heart tissue. These results indicateeitlaera marked decrease or an absence of CPT Il activity in all tissue samples, with normal or increased CPT I activity. *Control values are derived from autopsy specimens (as in Table I) and are recorded as mean + SD. Liver, n = 5; kidney, n = 1; skeletalmuscle, n = 6; and heart; n = 3. Concurrent assays of control specimens were also performed for all tissues.

plain the age at onset and severity of symptoms in CPT II deficiency, 3 there is no ready explanation for the pathogenesis of the congenital malformations. The initial differential diagnosis in our patient included glutaric acidemia type II and Zellweger syndrome because of the presence of renal cysts and central nervous system abnormalities in association with liver dysfunction and other dysmorphic features. Both of these disorders are associated with derangement of fatty acid oxidation and dicarboxylic aciduria. The biochemical similarities between these disorders and neonatal lethal CPT II deficiency may provide some insight into the mechanism of abnormal organogenesis in all three diseases. In glutaric acidemia type II, there is a deficiency of electron transfer flavoprotein or ETF-ubiquinone oxidoreductase, resulting in impaired mitochondrial oxidation of fatty acids and certain amino acids. 11 A subset of patients have congenital malformations, including cystic renal dysplasia, central nervous system abnormalities (neuronal migration defects, degenerative lesions, and cerebral gliosis), facial dysmorphism, and diffuse lipid accumulation. 11-14 The

pathologic abnormalities in these patients are very similar to those observed in out patient and are restricted to patients with severe deficiency of ETF-QO. Compared with those with ETF-QO deficiency, patients with deficiency of ETF have some residual oxidation of fatty acids in mutant cells, which implicates this pathway in the pathogenesis of anomalles in the kidney and brain. Zellweger syndrome is also characterized by cystic renal dysplasia, facial dysmorphism, focal neuronal migration defects, liver dysfuncUon, and lipid accumulation.15 Compared with the kidneys in glutaric acidemia and neonatal lethal CPT II deficiency, the kidneys are not as markedly dysplastic, 16 and disordered neuronal migration, although focal, tends to be more severe, x7 Zellweger syndrome is caused by a defect in peroxisomal biogenesis, which results in multiple biochemical abnormalities. However, 5% of patients with a phenotype indistinguishable from Zellweger syndrome have defects of single or multiple peroxisomal 13-oxidation enzymes only (e.g., deficiency of bifunctional protein or 3-oxoacyl C o A thiolase), again implicating a primary role

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for fatty acid oxidaüon in normal brain and kidney formation. TM Severe impalrment of the oxidafion of long-chain fatty acids and decreased flux through the Ô-oxidafion päthway may result in abnormal organogenesis through a variety of mechanisms. The altered ratio of acylcarnitine to acyl CoA would result in decreased avallabiüty of acyl CoA for cell membrane phosphoüpid synthesis and other important biosynthetic pathways. Accumulation of intermediary metabolites may exert a toxic effect on mitochondfial funcfion in utero, 12 resulfing in decreased avallability of adenosine triphosphate, glucose, or both during cmcial times in development. The ultrastructural abnormalities of the mitochondria in out patient (lipid and glycogen inclusions, giant mitochondfia, and osmiophiüc membranes that may represent degenerafing mitochondria) provide direct evidence of a dismption of cellular energy metabotism.

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4. 5.

6. 7.

8.

9.

CONCLUSIONS The present case, in combination with those reported by Zinn et al.9 and Witt et al., 1° expands the phenotype of neonatal lethal CPT I1 deficiency. This disorder should be included, along with glutaric acidemia type II, Zellweger syndrome, and other disorders of peroxisomal [3-oxidation, in the differential diagnosis for patients with cystic renal dysplasia, dysmorphism, centräl nervous system malformations, and early death. Whether the final common pathway causing abnormal organogenesis in these disorders is "energy deficiency" at the cellular level; lack of substrate for normal biosynthesis, or a toxic effect of accumulated metabolites remains to be elucidated. We are indebted to Dr. Antonio Perez-Atayde for interpretation of the eleclIon photomicrographs, and to Howard Mulhem for technical assistance. We also thank Joseph Volpe, Richard Kelley, Arm Moser, Charles Roe, and Stephen Kahler for their helpful discussions in interpreting the results in this patient, and the implications for pathogenesis of the other disorders. REFERENCES

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