Syndrome of encephalopathy, petechiae, and ethylmalonic aciduria

ELSEVIER

Syndrome of Encephalopathy, Petechiae, and Ethylmalonic Aciduria M. T e r e s a Garcia-Silva, M D * , A n t o n i a Ribes, PhD*, Y o | a n d a C a m p o s , MSc*, B a r b a r a G a r a v a g l i a , M S §, and J o a q u i n A r e n a s , P h D *

We report a boy 20 months of age with encephalopathy, petechiae, and ethylmalonic aciduria (EPEMA). Other clinical features were severe hypotonia, orthostatic acrocyanosis, and chronic diarrhea. Magnetic resonance imaging (MRI) of the brain demonstrated bilateral lesions in the lenticular and caudate nuclei, periaqueductal region, subcortical areas, white matter, and brainstem. Short and medium chain Acyi-CoA dehydrogenase and cytochrome c oxidase (COX) activities in fibroblasts were normal. Muscle histochemistry disclosed diffuse COX deficiency, and respiratory chain activities in muscle disclosed severe COX deficiency. Twelve other patients with similar clinical features have been reported. Muscle COX activity, studied only in four, demonstrated a clear-cut defect. © 1997 by Elsevier Science Inc. All rights reserved. G a r c f a - S i l v a M T , Ribes A, C a m p o s Y, G a r a v a g l i a B, Arenas J. S y n d r o m e o f e n c e p h a l o p a t h y , petechiae, and e t h y l m a l o n i c aciduria. Pediatr N e u r o l 1997; 17:165-170.

From the Departments of *Pediatrics; Mitochondrial Diseases Unit; and *Biochemistry; Hospital 12 Octubre; Madrid; *Institut Bioquimica Clinica; Corporaci6 Sanitaria and CSIC; Barcelona, Spain; and ~Divisione di Biochimica e Genetica; Istituto Neurologico "C.Besta"; Milan, Italy.

© 1997 by Elsevier Science Inc. All rights reserved. PlI S0887-8994(97)00048-9 • 0887-8994/97/$17.00

Introduction Burlina et al. [1,2] described a new s y n d r o m e characterized by the association of encephalopathy, relapsing petechiae, and e t h y l m a l o n i c ( E M A ) aciduria ( E P E M A ) [1,2]. This p r o g r e s s i v e encephalopathy: mental retardation, p y r a m i d a l signs, and bilateral lesions in striatum, r e s e m b l e s L e i g h S y n d r o m e (LS: necrotizing subacute e n c e p h a l o m y e l o p a t h y ) . Other clinical features are orthostatic acrocyanosis and chronic m u c o i d diarrhea. E P E M A is a fatal disorder, with onset usually in infancy and with early death. Apparently it is transmitted in an autosomalr e c e s s i v e inheritance pattern. Patients with E P E M A s y n d r o m e exhibit normal mitochondrial fatty acid oxidation in fibroblasts. The disease has b e e n related to a defect o f isoleucine m e t a b o l i s m [1], although this association has not been firmly established. C y t o c h r o m e c oxidase ( C O X ) activity in fibroblasts measured in s o m e patients did not demonstrate a clear defect [2-5]. In others, C O X activity m e a s u r e d in m u s c l e tissue d e m o n s t r a t e d a m o r e m a r k e d defect [3,4,6]. W e report a patient with E P E M A s y n d r o m e with normal respiratory chain in fibroblasts and dramatic decrease in C O X activity in muscle. W e also report the clinical history of his sister, w h o probably manifested the s a m e disease.

Case Report Patient. The child was the third born to healthy unrelated parents. He had two sisters; one was healthy; the other is described herein. At 5 months of pregnancy, there was a threat of abortion. Birth and neonatal period were uneventful. The infant was breast-fed, with supplement of formula, from birth. At 2 months of age, he manifested irritability and feeding difficulties. After experiencing diarrhea resulting from CampyIobacter, he presented with psychomotor regression. Diarrhea did not resolve after routine treatment. Later, he presented with recurrent perianal dermatitis with occasional mucoid stools and failure to thrive. He developed progressive hypotonia, lying in a frog position: lost social smile; was hyporeactive; and manifested episodes of hyperventilation. Results of ophthalmologic examination were normal. Laboratory investigations revealed metabolic acidosis and hyperlactatemia (4.7 to 9.5 raM, normal up to 2.2 mM). Magnetic resonance imaging (MRI) of the brain disclosed lesions characterized by prolongation of the relaxation times involving many structures, including both lenticular and caudate nuclei, periaqueductal region, bilateral scattered subcortical areas, hemispheric white matter foci, and brainstem. Patchy areas of hyperintensity were visible in caudate and lenticular nuclei. Electromyography (EMG) exhibited a myopathic pattern. Muscle biopsy displayed lipid droplets. At 7 months of age, he was admitted to our hospital. His weight was

Communications should be addressed to: Dr. Garcfa Silva; Department of Pediatrics; Mitochondrial Diseases Unit; Hospital 12 Octubre; Carretera de Andalucla Km 5,4:28041 Madrid, Spain. Received October 29, 1996; accepted December 6, 1996.

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Table 1.

Plasma and urine metabolites

Patient At diagnosis (7 mo) At 9 mo t Antemortem (20 mo) + Controls, n = 20

Organic Acids* EMA 2-MeS 196 343 1,185 9.3 ± 6.2

32 38 146 5.4 -+ 3.8

Acylglycipes* BG IVG 7.0 8.1 20.0 ~<0.1

6.9 ~0.1 10.5 -<0.3

Plasma carnitine (lttM) Total AcyUFree -

Free 15 39 ND 47 ± 9

22 54 ND 38 + 8

-

0.47 0.36 ND 0.26 -+ 0.14

Urine carnitine* Free Acyl ND 85 95 18 _± 18

ND 160 288 19 -+ 3

* Values are expressed in millimoles per mole creatinine. ~ Carnitine therapy. Abbreviations: 2-MeS = 2-Methylsucinate BG = Butyrylglycine EMA = Ethylmalonate IVG = Isovalerylglycine ND = Not determined

6,500 kg (less than 3rd percentile), length was 68 cm (25th percentile), and head circumference was 42 cm (less than 3rd percentile). Mild hypertelorism and low nasal bridge were apparent. He was lethargic and severely hypotonic, with absence of head control. He could not grasp objects, had neither social smile, ocular pursuit, nor auditory interest; he manifested pyramidal signs in the lower extremities. Plasma lactic acid levels were 2.3 to 2.6 mM. Amino acids, ammonia, pyruvate, ketone bodies, and [3-OHbutyrate/acetoacetate ratio were normal. Plasma carnitine level was low, and results of organic acid analysis were abnormal (Table 1). A second muscle biopsy was performed. Histochemical studies revealed diffuse COX deficiency, with no evidence of mitochondrial proliferation or increased number of lipid droplets. Electron microscopy demonstrated an increased number of mitochondria, with variability in their size. He was treated with carnitine 100 mg/kg/day, riboflavin 100 rag/day, thiamine 100 mg/day, and vitamin C 900 mg/day. Initially, the child improved in muscle tone and motility, holding his head up. He tried to roll over, made his first sounds, and displayed social smile. At 14 months of age, muscle tone and social contact worsened. He could not sit up and was irritable. We added coenzyme Q~o (50 rag/day) to his treatment regimen. Although the child showed slight improvement in tone and contact, his marked irritability was unchanged. At 16 months of age, petechiae of the trunk and "cutis marmorata" of the extremities were first observed (results of coagulation studies were normal). The distal portions of the legs were cold and sweating and smelled strongly of "feet sweat." Edema of the dorsum of the feet and distal orthostatic acrocyanosis were evident. At times, he had mueoid stools for 5 to 6 days (average of 2 per day). He had mild hepatomegaly, with normal results of liver function tests. Cardiac and renal function were normal. At 20 months of age, he weighed 7.9 kg and measured 77 cm; head circumference was 44 cm. He was severely retarded, did not develop language, and presented dystonic movements when he grasped objects. He was unable to sit, having axial hypotonia; deep tendon reflexes in the lower extremities were increased, and he manifested bilateral sustained ankle clonus. He had flexion spasms, and tonic and gelastic seizures. Results of laboratory studies demonstrated slight metabolic acidosis; plasma lactate levels were normal, but organic acid excretion remained severely abnormal (Table 1). A second MRI demonstrated similar lesions in the basal ganglia and brainstem (Fig 1). White matter and subcortical lesions had disappeared. He was discharged and died unexpectedly several days later. Postmortem examination was not authorized. Sister of the Patient. The sister's clinical and laboratory data were obtained from her parents and from clinical records of different hospitals. She was born at 35 weeks gestation, with birth weight of 2,300 gm, length of 45 cm, and head circumference of 30 cm. She presented with septicemia secondary to urinary infection by Escherichia coli on the first day of life. She was breast feeding, supplemented by commercial infant

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formula. Later she presented with feeding difficulties, vomiting, failure to thrive (weight and length under the 3rd percentile), rickets, hepatomegaly, hypotonia, and severe developmental delay. In addition, urinary infection was diagnosed. Laboratory studies demonstrated metabolic acidosis, glycosuria, leukocyturia, and microhematuria. Levels of transaminases were normal. At 7 months of age, she manifested hyperchloremic metabolic acidosis, nephrotic syndrome, and hypogammaglobulinemia. Tubular renal function, blood lactate, and ammonia levels were normal. Intestinal biopsy revealed partial mucosal atropy in moderate degree. EMG, performed because of the severe hypotonia and developmental delay,

Figure 1. Brain magnetic resonance imaging axial T2-weighted scan at the level of basal ganglia demonstrates bilateral patchy areas of hyperintensity in caudate and lenticular nuclei.

Table 2. Respiratory chain enzyme activities in muscle homogenates and fibroblasts and mitochondrial IS-oxidation rate activities in fibroblasts

Parameter

Patient

Muscle Controls (n = 30) (range)

NADH dehydrogenase Succinate dehydrogenase NADH cytochrome c reductase Succinate cytochrome c reductase COX CS SCAD MCAD

780 3.4 55 8.5 2.8 160

550-900 3.5-15 20-34 3.4-15 21-75 75-210

Patient

Fibroblasts Controls (range)

ND ND ND ND 14.7 28.4 0,76 2,54

ND ND ND ND 10-51 (n = 15M4 (n = 0.85-1 6 (n 2.22M.3 (n -

12) 8) 18) 181

Enzyme activities in muscle calculated as nanomoles per minute per milligram protein, referred to the specific activity of CS. Enzyme activities in fibroblasts calculated as nanomoles per minute per milligram protein. Abbreviations: CS = Citrate synthase MCAD = Medium chain acylCoA dehydrogenase NADH - Reduced nicotinamide adenine dinucleotide ND = Not determined SCAD = Short chain acylCoA dehydrogenase

demonstrated no abnormalities. Muscle biopsy morphologic analysis was normal (COX staining was not performed). Her death, at 7.5 months of age, was attributed to pneumonia and septicemia. Postmortem examination demonstrated bronchopneumonia, pleural effusion, and indications of disseminated intravascular coagulation. The brain evidenced edema and disseminated thrombosis. Liver examination showed mild steatosis. Peripheral nerves studied were normal. Electron microscopy showed no mitochondrial abnormalities in liver, kidney, or muscle.

Methods Organic Acids" and Carnitine. Organic acids were analyzed as trimethylsilyl derivatives in a HP5989A gas chromatography mass spectrometry system (Hewlett-Packard, Palo Alto, CA) as previously described [7]. However, the specific acylglycines were quantified by selected ion monitoring with heptanoylglycine as the internal standard (a gift from Dr. N. Gregersen, University Hospital, Aarhus, Denmark). Plasma and urine free and total carnitine were determined by a radiochemical procedure [8]. Fibroblasts. Fibroblasts were grown from explants of forearm skin biopsy tissues in a 5% COa atmosphere. Culture medium was Eagle's modified essential medium with 5.5 mM glucose, 20 mM HEPES, and 10% newborn calf serum (NCS). The activities of COX and citrate synthase were determined in fibroblasts by previously described spectrophotometric methods [9,10]. Protein levels were determined according to the method of Lowry et al. I I 11. The activity of short and medium-chain acylCoA dehydrogenases was measured according to the method of Frerman and Goodman [12]. En=ymes in Muscle. The activities of rotenone-sensitive NADH cytochrome c reductase, succinate dehydrogenase, succinate cytochrome c reductase, COX, and citrate synthase (CS) were measured in muscle homogenates after centrifugation at 800 g for 10 min, as described previously [ 131. The activities of each enzyme were referred to that of CS to correct for mitochondrial w)lume. Molecular Genetics, Total DNA was isolated from muscle. For Southern blot analysis, DNA was digested with PvuII or BamHI and subjected to electrophoresis on 0.8% agarose gel. After blotting the specimens onto nitrocellulose membranes, hybridization to two digoxigenin-labeled probes, one for entire mtDNA and the other for the nuclear-encoded 18S rRNA, was performed [14]. To detect point mutations at positions 8344 and 3243 of the mtDNA, we used previously described methods [ 15~161.

Results

Biochemical Studies. Urine organic acid analyses showed a prominent increase in EMA, 2-methylsuccinate (2-MeS), butyrylglycine (BG), and isovalerylglycine (IVG), but the excretion of other dicarboxylic acids was consistently normal. At diagnosis, both total and free plasma carnitine levels were low and acyl/free carnitine ratio was in the upper normal range (Table 1). Therefore, carnitine treatment was instituted. Plasma carnitine normalized, although the acyl/free ratio remained in the upper normal range, and the degree of excretion of carnitine esters was high during treatment (Table 1). Mitochondrial ~-oxidation studies in cultured fibroblasts revealed normal octanoate but slightly decreased butyrate oxidation rate (Table 2). COX activity in fibroblasts was normal, but its activity in muscle homogenates was 7% of control value. Other respiratory chain complexes in muscle were normal (Table 2). Molecular Genetics of mtDNA. Southern blot analysis revealed no significant rearrangement of mtDNA. Moreover, the ratio of mtDNA to nuclear 18S rRNA was normal, indicating no mtDNA depletion. Point mutations at positions 8344 and 3243 of mtDNA were not detected.

Discussion At onset, the patient presented clinical features resembling those of LS [17,18], i.e,, neurologic deterioration with bilateral symmetric lesions in striatum and lactic acidosis. The spectrum of image abnormalities in LS varies widely depending on the intensity of metabolic brain defect and the age of the patient, as well as on the evolution of the disease, It ranges from selective involvement of the putamen to involvement of diffuse brain

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Table 3. Antecedents and first clinical features of patients with EPEMA syndrome Reported Patients

A g e at onset Positive family history Sex (M/F) A g e o f survival Infants deceased First s y m p t o m s Diarrhea Hypotonia Petechiae Failure to thrive F e e d i n g difficulties Developmental delay Irritability Lactic acidosis Distal a c r o c y a n o s i s N e u r o l o g i c deterioration Seizures

(n = 12)*

Present Case

<6 mo 6/12 9/3 6 mo to 7 yr 10/12

2 mo + M 20 m o +

7/12 4/12 4/12 4/12 2/12 4/12 2/12 1/l 2 1/12 1/ 12 I/12

+ + + + -

* Data f r o m references 2, 4-6, 21. Abbreviations: E P E M A = E n c e p h a l o p a t h y , petechiae, a n d e t h y l m a l o n i c aciduria + = Present = Absent

structures [19]. In the present case, lesions not only involved the classic structures such as putamen, caudate nucleus, and periaqueductal region, but also extended to striatrum and brainstem. Therefore, the extended brain damage was not restricted to vascular areas. White matter and subcortical lesions originally observed in his first study disappeared over the time, probably related to brain maturation, which initially was disturbed. In the present case, as in the previous reported patients with EPEMA syndrome, postmortem examination was not performed and information about the characteristic neuropathologic changes necessary for diagnosis of LS was lacking [20]. The clinical features of the patient, the presence of lipid droplets in the first muscle biopsy, together with significant excretion of EMA and hypocarnitinemia, prompted us to study both [3-oxidation and respiratory chain enzymes. However, biochemical studies in fibroblasts failed to disclose any abnormality. A second muscle biopsy disclosed no lipid droplets, but biochemical and histochemical studies, not performed in the first biopsy, revealed severe COX deficiency. The patient subsequently developed a complex clinical syndrome: encephalopathy, petechiae, orthostatic acrocyanosis, chronic diarrhea, and EMA aciduria. This phenotype was virtually identical to that previously documented by Burlina et al. [1,2], but their patients were not investigated for mitochondrial dysfunction in muscle. At present, 12 other patients with EPEMA syndrome have been reported by other investigators [ 1-6,21 ]. Their clinical data are compared with those of the present case and are reviewed in Tables 3 and 4.

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In EPEMA syndrome, clinical features usually have onset before 6 months of life. In most patients, outcome is fatal, since 11 of the 13 patients have died, usually at less than 2 years of age. Death may occur suddenly, owing to metabolic decompensation or respiratory insufficiency due to infectious illness. Ozand et al. [5] reported that before death some patients with no evidence of infectious disease manifested lung opacities on roentgenograms. Ozand et al. [5] considered this phenomenon, as well as the petechiae, hemorrhages, and microhematuria, to be secondary to vasculopathy. Petechiae, distal swelling, and orthostatic acrocyanosis have been studied by different investigators, who ruled out peripheral dysautonomia, autoimmune disorder, coagulopathy, or platelet dysfunction [2,4]. Skin biopsy of the petechiae failed to disclose any specific change in vessels or nerves [6]. Petechiae, distal swelling, and orthostatic acrocyanoses may be attributed to possible vasculopathy [5]. Neurologic signs and diarrhea are frequent findings at onset of EPEMA syndrome. However, during its evolution, diarrhea is inconsistently observed or is absent in some patients. The etiology of the diarrhea is unknown. Intestinal biopsy may disclose mild mucosal atrophy in patients with normal results of malabsorption intestinal functional test 12]. In addition to increased excretion of EMA, laboratory investigations in these patients usually demonstrate metabolic acidosis and variable hyperlacticacidemia (at times, levels are normal). Plasma levels of free and total carnitine

Table 4.

Clinical course of patients with EPEMA syndrome Reported Patients Features

Neurologic Mental retardation Pyramidal dysfunction Hypotonia Microcephaly Seizures Episodes o f c o m a Muscle wasting Dystonia Other Petechiae Failure to thrive Orthostatic a c r o c y a n o s i s Metabolic decompensation Chronic diarrhea Respiratory failure Mild facial d y s m o r p h i a Liver involvement

(n = 12)*

Present Case

12/12 11/12 10/12 6/12 5/12 4/12 2/12 1/12

+ + + + + + +

12/12 8/12 7/12 7/12

+ + + +

6/12 4/12 3/12 2/12

+ + + -

* D a t a from references 2, 4-6, 21. Abbreviations: E P E M A = E n c e p h a l o p a t h y , petechiae, and ethylmalonic aciduria + = Present = Absent

may be normal or decreased. Muscle carnitine level can be either normal or decreased [4]. As in the patients of Burlina et al. [1], the concentration of specific metabolites in the present patient did not correlate well with changes in clinical conditions, but an antemortem urine sample revealed increased excretion of organic acids, acylglycines, and acylcarnitines (Table 1). EMA aciduria is a typical finding in multiple acyl CoA dehydrogenase disorders and in short chain Acyl-CoA dehydrogenase (SCAD) deficiency, but there is no obvious connection between the accumulation of EMA and the COX defect. Consistent with our findings, increased EMA with normal [3-oxidation in fibroblasts has been described in some respiratory chain disorders [23], usually associated with slight lactic acidosis and neurological abnormalities. Lehnert and Ruitenbeck [23] reported that EMA is a sensitive indicator of progressive neuromuscular disorders, including mitochondrial diseases. A patient with late-onset riboflavin-responsive myopathy, combined multiple acyl-CoA dehydrogenase, and respiratory chain deficiency has been reported [24]. In this patient, the enzymatic defect is still obscure and the excretion of EMA is probably due to secondary inhibition of SCAD by some yet unknown mechanism. On the other hand, impaired butyrate oxidation has been demonstrated in ulcerative colitis [25], probably due to inhibition of SCAD by persulfides produced by the human colon [26]. A similar mechanism might be responsible for EMA excretion in patients with some type of intestinal disorder, such as patients with EPEMA syndrome with chronic diarrhea, since many other patients with muscular COX deficiency do not present this peculiar urinary organic acid profile. Normal [3-oxidation and increased EMA excretion have been reported in patients with muscle COX deficiency [23]. Lehnert et al. [23] reported three patients with EMA and muscle COX deficiency. They did not report vascular involvement in their patients. Two had later onset and less severe encephalopathy. The third patient appeared to manifest features similar to those of EPEMA syndrome, but chronic diarrhea, petechiae, distal acrocyanosis, and swelling were not reported. This patient had microhematuria, glycosuria, and enteral symptoms, subsequently undergoing neurologic regression. Ozand et al. [5] attributed the microhematuria of their patients to vasculopathy. Patients with EPEMA syndrome with the same underlying genetic defect may exhibit variable clinical expression. The sister of our patient might have had the same disease. She manifested some clinical features common in patients with respiratory chain deficiencies: severe developmental delay, hypotonia, liver involvement, intestinal mucosal atrophy, and nephrotic syndrome [27]. On the other hand, she had some features common in the EPEMA syndrome: positive family history, hypotonia, developmental delay, failure to thrive, and gastrointestinal and liver involvement. No information derived from neuroradiologic studies of the girl before her death is available. Postmortem examination demonstrated only edema and diseminated

thrombus; no evidence of LS disease was detected. Because levels of urinary organic acids and respiratory chain in muscle were not determined, we did not reach a definitive diagnosis. Patients with rapid fatal evolution may be overlooked if metabolic studies, including determination of urinary organic acids levels and morphologic and biochemical analyses of muscle are not performed. Clinical manifestations in our patient were not consistent with the more typical phenotypes of COX deficiency, but there were some similarities with mtDNA depletion with variable tissue expression, LS, or mitochondrial neurogastrointestinal encephalomyopathy (MNGIE). In particular, patients with mtDNA depletion manifest good correlation between degree of depletion and COX deficiency. However, Southern blot analysis with nuclear 18S ribosomal DNA as normalizing nuclear probe failed to demonstrate mtDNA depletion. With regard to LS, some patients usually exhibit COX deficiency in fibroblasts and a COX defect in muscle that is less marked than that of our patient [13]. COX is a complex enzyme composed of 3 mtDNA-encoded subunits and 10 nuclear-encoded subunits. Some of the latter have tissue-specific isoforms. Therefore, patients with EPEMA and COX deficiency may have alterations in one or more nuclear-encoded subunits shared by affected tissues such as muscle, brain, and blood vessels. Our data suggest that in a group of patients, previously reported to manifest Leigh's disease and presenting with distinctive brain MRI abnormalities and muscle COX deficiency, short-chain organic acids are abnormal. However, there is no obvious association between COX defect and increased excretion of EMA.

The authors thank Paz Briones, PhD, who performed carnitine in plasma and urine and respiratory chain activities in fibroblasts (lnstituto de Bioqufmica Clfnica, Barcelona, Spain); Aria Cabello, MD, who studied the muscle histochemistry and Alberto Mufioz, MD, who performed brain MR (Hospital 12 Octubre, Madrid, Spain) and Jos6 Santos Borbujo, MD, who was the neurologist who assessed the patient (Hospital Virgen de la Concha, Salamanca, Spain).

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