Biochemical and Molecular Study of CLINICAL PRESENTATION Carnitine-Acylcarnitine Translocase Deficiency in a Neonate with Hyperammonaemic Encephalopathy Leong HY1,5, Ngu LH1, Abd Azize NA2, Yakob Y2, Habib A3, Md Yunus Z3, Catchpole A4, Yap S5, Olpin S4 1Genetic Dept, Hosp Kuala Lumpur, 2Molecular Diagnostic & Protein Unit, and 3Biochemistry Unit, Institute of Medical Research, Kuala Lumpur, Malaysia, 4Dept of Clinical Chemistry, and 5Inherited Metabolic Disease Dept, Sheffield Children’s Hospital, Sheffield, United Kingdom. INTRODUCTION Carnitine-acylcarnitine translocase (CACT) deficiency is a disorder of long chain fatty acid transport across the mitochondria inner membrane. Long chain free fatty acids enter mitochondria via the carnitine shuttle: Carnitine transported across the plasma membrane. Acyl group transferred from acyl CoA to carnitine through carnitine palmitoyltransferase I (CPT1). Acylcarnitine transported across the inner mitochondrial membrane via CACT. Acyl groups transferred from acylcarnitines to CoA inside the mitochondria through carnitine palmitoyltransferase II (CPTII). Transporter Long-chain fatty acids Acyl-CoA CPT-1 Acylcarnitine Carnitine Medium-chain CPT 2 b-oxidation Plasma membrane Outer mitochondrial membrane Inner mitochondrial membrane CACT 1 CLINICAL PRESENTATION Family history of infantile deaths (figure 1) ~ 3rd child: ?CPT II / CACT deficiency (↑ C16, C18, C18:1) Consanguineous parents (indigenous Bajau ethnic group) Girl, born term (birth weight 2.75 kg) Encephalopathic, hypothermic & fitted at day 3 Hyperammonaemia (342 to 858 µmol/L) Treated with: ~ peritoneal dialysis ~ anti-ammonia scavengers (sodium benzoate, sodium phenylbutyrate and arginine) ~ low fat, high carbohydrate diet Plasma ammonia normalised 1 month old - Another hyperammonaemia (1582 µmol/L) Died 2½ months old - cause of death unknown patient Onset Day 2 Day 1 Symptoms Apnoea Hypoglc Encepha Apnoea Apnoea Hypoglyc Seizure Apnoea Hypoglyc Seizure Encepha Blood tests ↓ pH ND  NH3 NH3 normal Acylcarnitine  C16, C18 Normal  C16 Died Day 64 Day 3 Day 12 Day 20 2½ mths Diagnosis Unknown ?CPT II/ CACT def CACT def Fig 1. Clinical features of the siblings & the patient’s for comparison. Hypoglyc, hypoglycaemia; Encepha, encephalopathy; ↓ pH, metabolic acidosis; NH3, hyperammonaemia; ND, not done. RESULTS Plasma ammonia: 342 µmol/L. Urine organic acid: Moderate peaks of adipate & suberate. Large lactate peaks. No orotic acids. Blood spot acylcarnitines: ↑ C16 - 6.91 μmol/L (ref. 0.2–5.5) ↑ C16+C18/C2 ratio - 1.36 (ref. 0.36–0.59) Normal C18:1 - 1.2 µmol/L (ref. 0.2–2.45) Normal free carnitine - 13.71 µmol/L (ref. 10–89) CPT II assay in fibroblast sonicates is by coversion of [14C]palmitoly-carnitine to [14C]palmitoyl-CoA with inhibition of CPT I activity4: Normal activity (table 2) CACT assay5,6: [2-14C]pyruvate is oxidised to [14C]acetyl-CoA -- [14C]acetylcarniitne Very low activity (table 2) CACT Enzyme Assay Patient Positive control Normal control CPT II nmol/mg prot /min 6.5 0.6 6.9  1.3 (n=6) CACT nmol/mg prot/min 0.011 0.012 0.485  0.097 (n=4) Tritium release assay of -oxidation of fatty acids in skin fibroblasts1,2,3 (table 1): Very low activity in long chain substrates. Increased octanoate oxidation indicating compensatory activity towards medium chain substrates that enter the mitochondria directly. Table 2: CPT II & CACT enzyme assays in fibroblasts SLC25A20 gene sequencing Homozygous splice site mutation c.199-10T>G in intron 2 (fig 2). Both parents heterozygous carriers. Intron 2 [9,103H] myristate [9,103H] palmitate [9,103H] oleate [2,2,3,33H] octanoate % of simultaneous controls 6% 5% 208% Fig 2. DNA chromatogram: homozygous c.199-10T>G Table 1: Fatty acid oxidation flux studies in fibroblasts DISCUSSION Our patient developed severe hyperammonaemic encephalopathy at day 3. Vitoria et al7 reported that 24 out of 34 (70.6%) patients were symptomatic in the first week with hypoketotic hypoglycaemia (68%), hyperammonaemia (54%), hepatomegaly (34%), arrthymia and/or bradycardia (32%) and respiratory distress (30%). Biochemical findings of CACT deficiency include high plasma long-chain acylcarnitines (C16, C18, C18:1 and C18:2) and low carnitine. Our patient showed only a mild increase C16 with normal C18 & C18:1. The splicing mutation 261-10T>G has been reported in patients from China, Japan and Vietnam and is associated with a severe clinical phenotype. It leads to skipping of either exon 3, or both exons 3 and 4 resulting in a truncated gene product.8 In homozygous state, it led to a severe impairment of mitochondrial fatty acid -oxidation and CACT enzyme activity in our patient. Vitoria et al1 found that clinical outcomes correlate with the fatty acid oxidation rate in cultured fibroblasts. REFERENCES 1. Manning et al. A comparison of [9,10-3H]myristic acids for the detection of fatty acid oxidation defects in intact cultured fibroblasts. JIMD 1990;13:58-68. 2. Olpin et al. Differential diagnosis of hydroxydicarboxylic aciduria based on release of 3H2O from [9,10-3H]myristic and [9,10-3H]palmitic acids by intact cultured fibroblasts. JIMD 1992;15:883-890. 3. Olpin et al. [9,10-3H]Oleic acid- for the improved detection of long chain fatty acid oxidation defects in intact cells. JIMD 1997;20:415-419. 4. Olpin et al. Mutation and biochemical analysis in CPT II deficiency. JIMD 2003;26:543-557. 5. Pande et al. Carnitine-acylcarnitine translocase deficiency with severe hypoglycemia and auriculo ventricular block. Translocase assay in permeabilized fibroblasts. J Clin Invest 1993;91:1247. 6. Olpin et al. Carnitine-acylcarnitine trasnlocase deficiency-a mild phenotype. JIMD 1997;20:714-715. 7. Vitoria I et al. Carnitine-acylcarnitine translocase deficiency: experience with four cases in Spain and review of the literature. JIMD Rep. 2015;20:11-20. 8. Ogawa A et al. Identification of two novel mutations of the carnitine/acylcarnitine translocase (CACT) gene in a patient with CACT deficiency. J Hum Genet 2000;45:52–55. ACKNOWLEDGEMENTS We would like to thank the healthcare providers at the Queen Elizabeth Hospital, Kota Kinabalu, Malaysia for their input with the patient. CORRESPONDENCE hueyyinleong@yahoo.com