American Journal of Medical Genetics 102:183± 187 (2001)
Antenatal Presentation of Carnitine Palmitoyltransferase II De®ciency Orly N. Elpeleg,1* Cathy Hammerman,2 Ann Saada,1 Avraham Shaag,1 Elena Golzand,2 Drorith Hochner-Celnikier,3 Itai Berger,1 and Michel Nadjari3 1
Metabolic Disease Unit, Shaare-Zedek Medical Center, Jerusalem, Israel Neonatal Intensive Care Unit, Shaare-Zedek Medical Center, Jerusalem, Israel 3 Department of Obstetrics and Gynecology, Hadassah Medical Centers, Jerusalem, Israel 2
Carnitine palmitoyl transferase (CPT) II de®ciency is usually manifested around puberty by exercise induced myoglobinuria. Two Ashkenazi Jewish sibs with the rare antenatal form of CPTII de®ciency are reported. On the 5th gestational month periventricular calci®cations and markedly enlarged kidneys were found in both of them. The activity of CPTII in lymphocytes was undetectable and both sibs were homozygous for the 1237delAG mutation. Because of the serious consequences of homozygosity for this mutation, genotype determination of all Ashkenazi patients with the adolescent form of CPTII de®ciency is warranted. ß 2001 Wiley-Liss, Inc. KEY WORDS: long chain fatty acid oxidation; fetal malformation syndrome INTRODUCTION Long chain fatty acids (LCFA) are imported into the mitochondria for chain shortening and acetyl-CoA production by b-oxidation. This process is carnitinedependent and is regulated by two carnitine palmitoyltransferases (CPT) and by carnitine:acylcarnitine translocase. The outer membrane CPT I is involved in the esteri®cation of palmitoyl-CoA to yield palmitoylcarnitine. After translocation into the mitochondrial inner membrane, the palmitoylcarnitine molecule is converted into carnitine and palmitoyl-CoA by the activity of CPT II [McGarry and Foster, 1980]. De®ciency of CPT II is typically manifested around puberty with exercise intolerance and myoglobinuria [DiMauro and DiMauro, 1973]. Presentation in infancy is less common; it is characterized by recurrent
*Correspondence to: Orly N. Elpeleg, M.D., Metabolic Disease Unit, Shaare-Zedek Medical Center, Jerusalem 91031, Israel. E-mail:
[email protected] or
[email protected] Received 19 July 2000; Accepted 19 February 2001
ß 2001 Wiley-Liss, Inc.
episodes of encephalopathy, cardiomegaly, rhabdomyolysis and hepatomegaly and is commonly associated with hypoglycemia and long chain acylcarnitines accumulation in tissues [Demaupsie et al., 1991; Hug et al., 1991; Elpeleg et al., 1993]. Rarely, presentation is antenatal with cerebral periventricular cysts and cystic dysplastic kidneys [Witt et al., 1991; Zinn et al., 1991; Taroni et al., 1994; North et al., 1995; Pierce et al., 1999]. This clinical variability is likely attributed to the variable residual enzymatic activity. We report the clinical, biochemical and molecular ®ndings in two patients of Ashkenazi Jewish origin who presented antenatally with CPT II de®ciency. CASE REPORTS Patient 1 was a 2,325 gram male infant who was born at 36 weeks of gestation to a 24-year-old G3P3 healthy mother. His Ashkenazi Jewish parents were unrelated and their two previous children were healthy. Fetal screening at 23 weeks revealed an absence of the corpus callosum, ventriculomegaly and intracerebral periventricular calci®cations; markedly enlarged polycystic kidneys and cardiomegaly with a thickened myocardium (Fig. 1a±c). Repeated examination at 29 weeks was unchanged apart from the ®nding of normal heart size with normal myocardial walls. The patient was born by spontaneous vaginal delivery. Apgar scores were 9 and 8 at 1 and 5 min, respectively. Initial physical examination revealed a crying infant with respirations of 71/min, a pulse of 130/ min and blood pressure of 86/55. Physical features were not abnormal. Head circumference was 32 cm with normal fontanelle and sutures. There was a high arched and narrow palate. The heart showed a regular sinus rhythm with no murmur. Abdominal examination revealed easily palpated, enlarged and irregular kidneys. No organomegaly was present at this point. Neurologic examination was remarkable for exaggerated head lag, mild generalized hypotonia and somewhat decreased activity and responsiveness. Brain CT scan demonstrated mild enlargement of the lateral ventricles, hypodense areas surrounding the anterior horns with subependymal calci®cations (Fig. 3a±c). Echocardiogram showed a small tumor attached to the
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Fig. 1. a±c: Ultrasound performed on the 23rd gestational week of Patient 1. a: enlarged kidneys; b: cardiomegaly, 4 chamber view; c: markedly enlarged occipital horn.
right side of the septum. Renal ultrasound showed enlarged cystic kidneys. On the second day, renal insuf®ciency was noted with serum creatinine 2.6 mg% and potassium 9 mM. Therapy with kayexalate, calcium, bicarbonate, glucose and insulin was initiated and renal function slowly improved. By the end of the ®rst week the abdomen became distended and the liver enlarged. Over the next month the patient became progressively more obtunded, stopped tolerating feeds, and had frequent regurgitation. His condition deteriorated with compensated metabolic acidosis and increasing hepatomegaly. Repeated echocardiogram revealed massive myocardial thickening with normal ejection fraction. Peak liver functions were AST 495 u/L, ALT 197 u/L, direct bilirubin 9.7 mg% and INR 4.2. Liver biopsy revealed diffuse macrovesicular fatty changes. Creatine kinase, initially normal, increased to 1,200 u/L. Free carnitine in plasma was 2.3 mM (N > 23 mM). Urinary organic acid analysis disclosed severe non-ketotic dicarboxylic aciduria. Despite aggressive medical therapy the infant succumbed to his disease at the age of 43 days. Patient 2 was the sibling of Patient 1, a product of the fourth pregnancy. Fetal screening at 22 weeks revealed a female with markedly enlarged kidneys, brain calci®cations and suspected cerebral hemorrhage (Fig. 2a±c). The pregnancy was terminated on Week 23. The placenta was of normal appearance and its weight was appropriate for age; fat stain was negative. Muscle histology was unremarkable. In the liver, fat
droplets of variable size were present in the cytoplasm of the hepatocytes. METHODS The oxidation of [9,10]-3H palmitic acid in fresh lymphocytes and cultured ®broblasts was determined as previously described [Manning et al., 1990]. The activities of CPT I and II in lymphocytes were determined by the stable isotope exchange method with measurement of the CoA dependent exchange of labeled carnitine with palmitoylcarnitine [Zierz and Engel, 1985]. The assay was performed at 2 concentrations of DL-palmitoylcarnitine, 0.1 mM and 1.0 mM, because the higher concentration inhibits CPT I activity. Both activities were also assayed by measuring the formation of palmitoylcarnitine from palmitoyl-CoA and labeled carnitine, in the baseline state and after the addition of Triton X-100 that abolishes CPT I activity [Zierz and Engel, 1985]. Genomic DNA was isolated from fresh blood of both patients and their parents. The 5 exons of the CPT II gene and the exon-intron junctions were ampli®ed, using oligonucleotide primers as previously described [Verderio et al., 1995]. Two additional primers within exon 4, CPT5 50 -TGAGGAGAGCCTGAGGAAAG-0 3 and CPT6 50 -AATGGGGAAGTCATCTAGGC-30 were used for sequence analysis. Direct sequencing was performed on an automatic sequencer (ABI Prism 377, Perkin-Elmer, Norwalk, CT) using Dye Terminator
Fig. 2. a±c: Ultrasound performed on the 22nd gestational week of Patient 2. a: Enlarged ventricles with periventricular hemorrhage, F frontal horn, O occipital horn, H hemorrhage; b: cerebral calci®cations, E an orbit, C a calci®cation; c: enlarged kidney.
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Fig. 3. A±C: Brain CT scan of Patient 1 disclosing mild enlargement of the lateral ventricles, hypodense areas surrounding the anterior horns and subependymal calci®cations.
Cycle Sequencing Core Kit (Perkin-Elmer) according to the manufacturer's instructions. RESULTS The oxidation of [9,10-3H] palmitic acid by lymphocytes of Patient 1 and 2 was 2.8 and 3.2 pmol/min/mg protein, respectively (control 129 59 pmol/min/mg protein). The total CPT activity in lymphocytes of
Patient 1 was 0.52 nmol/min/mg protein (control 0.89 0.26 nmol/min/mg protein) in the presence of 0.1 mM of DL-palmitoylcarnitine and was below detection limit when CPT I activity was inhibited with 1.0 mM DL-palmitoylcarnitine. The activity of CPT using the forward assay was 0.42 and 0.64 nmol/min/ mg protein in Patient 1 and 2 respectively (control 1.23 0.44 nmol/min/mg protein) and was undetectable in the presence of Triton X-100 in both patients (control
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1.20 0.43 nmol/min/mg protein). Because of the small blood volume available from Patient 2, the total CPT activity was not determined in his lymphocytes. The determination of the sequence of the 5 exons of the CPT II gene of Patient 1 disclosed homozygosity for 2 mutations in exon 4, a frame-shift mutation, 1237delAG and a missense mutation, Phe448Leu (nucleotides and amino acids are counted from the ®rst ATG to include the leader sequence). Homozygosity for these mutations was also found in genomic DNA of Patient 2. Both parents were heterozygous for the mutations. DISCUSSION CPT II de®ciency is manifested by three phenotypes and the clinical severity is directly related to the residual enzymatic activity. The classical form has its onset in adolescence and is characterized by muscle pain and myoglobinuria after strenuous exercise [DiMauro and DiMauro, 1973]. In ®broblasts of these patients residual enzymatic activity is 15±26% of the control. The impact of the enzymatic de®ciency on global LCFA oxidation in lymphocytes is usually minimal as evidenced by [9,10-3H] myristate oxidation > 50% of the control [Bonnefont et al., 1996]. The clinical outcome is favorable though fatalities have been reported [Kelly et al., 1989]. Because this form is considered benign, mutation analysis is rarely performed. The infantile phenotype is characterized by recurrent metabolic decompensation with hepatomegaly, hypertrophic cardiomyopathy, rhabdomyolysis and sudden death [Demaugre et al., 1991; Hug et al., 1991; Taroni et al., 1992; Elpeleg et al., 1993; Elpeleg et al., 1994; Ross et al., 1996; Yamamoto et al., 1996]. Residual enzymatic activity in ®broblasts of these patients is 4±10% of the control and LCFA oxidation, re¯ected by [9,10-3H] myristate oxidation is
