1. MCADD, caused by mutation in the ACADM gene is the most common disorder of fatty acid  - oxidation. Treatment is very successfully if started before symptoms arise. Many countries offer newborn screening (NBS) for MCADD using tandem mass spectrometry (MS/MS) to detect raised octanoyl carnitine (C8) in dried blood spots (DBS). However NBS has led to the detection of individuals with previously unreported genotypes of uncertain clinical significance. Fat oxidation studies in cultured fibroblasts can assist in assessing the significance of previously unreported genotypes. The case presented here illustrates this and highlights the importance of assessing temperature sensitivity in determining pathogenicity. Thermolability of fat oxidation flux in a medium chain acyl-CoA dehdyrogenase deficient (MCADD) patient homozygous for the c.199T>C mutation Hulley S 1, Olpin SE 2, Clark S 2, Kirk R 3, Downing M 4, Sharrard MJ 1. Background Case History A healthy female infant was born at term to unrelated Caucasian parents. A half sibling was well and there was no family history of note. The infant was breast feeding well and underwent NBS on day 6. The initial dried blood spot (DBS) C8 was 0.56  mol/L (<0.5) with a C8:C10 ratio of 0.93 (<1.0). The infant was referred for further evaluation. At day 12 the infant remained well. Blood samples were taken for repeat DBS and plasma acyl carnitine analysis and ACADM genotyping, and urine for organic acid analysis by gas chromatography/mass spectrometry, and quantitation of hexanoylglycine. Pending investigation results, feeding advice and an emergency regimen were given. References: 1. Medium-chain acyl-CoA dehydrogenase (MCAD) mutations identified by MS/MS-based prospective screening of newborns differ from those observed in patients with clinical symptoms. Andresen BS et al. Am. J. Hum. Genet. 2001; 68: 1408 2. Protein misfolding is the molecular mechanism underlying MCADD identified in newborn screening. Maier EM et al. Human Molecular Genetics 2009; 18: 1612 3. Medium-chain acyl-CoA dehydrogenase deficiency: evaluation of genotype-phenotype correlation in patients detected by newborn screening. Gramer G et al. J Inherit Metab Dis 2015; 38: 780 4. The Y42H mutation in medium-chain acyl-CoA dehydrogenase, which is prevalent in babies identified by MS/MS-based newborn screening, is temperature sensitive.O’Reilly L et al. Eur. J. Biochem.2004: 271: 4053–4063 Investigations 1 Paediatric medicine, Sheffield Children’s NHS Trust, 2 Clinical Chemistry, Sheffield Children’s NHS Trust, 3 Sheffield Diagnostic Genetics Service, 4 Newborn Screening, Sheffield Children’s NHS Trust Acyl carnitine profiles are shown in figure 1a (DBS) and 1b (plasma). The DBS results indicates a C8 of 0.25 μmol/L ( 2.1). Sequencing of the ACADM gene detected homozygosity for the c.199T>C variant. Figure 3. Temperature effect on fat oxidation flux in MCADD 985A>G homozygotes, 985A>G / 199T>C compound heterozygotes and simple985A>G heterozygotes and Discussion The fat oxidation in the case presented here was normal at 37°C but reduced at 41°C to a level diagnostic for MCADD and is similar to that of the mean values seen in MCADD patients (figure 4). The infant has been managed with an emergency regimen and antipyretics and has not experienced any MCADD crises. The ACADM gene 199T>C mutation is present in about 1:500 individuals (1) and is found in 6% of MCADD genotypes in our NBS population. The resultant protein is a temperature sensitive misfolding variant (2), but expression studies have demonstrated that the mutant enzyme has Km and Vmax similar to the wild type. At 37°C, protein folding is sufficiently compromised reducing overall enzyme activity in the 985A>G/199T>C compound heterozygote to an MCADD level (figure 3), with further reduction at 41°C. Our experience together with that of Gramer (3) is that patients who are compound heterozygote for 199T>C and a ‘severe’ mutation may present with MCADD crises or episodes of lethargy (4). Fat oxidation in our homozygous 199T>C patient is normal at 37°C (although acylcarnitines are abnormal). Protein misfolding is sufficiently abnormal at 41°C to result in an MCADD biochemical phenotype. The patient has been successfully managed with an MCADD emergency regimen with particular attention to controlling fever. Conclusion 199T>C homozygosity in the ACADM gene results in a temperature sensitive form of MCADD Measuring fat oxidation flux at 41°C is important in assessing a genotype of uncertain significance, even if this is normal at 37°C Initial results Because the significance of this genotype was uncertain, tritium release fat oxidation flux studies on cultured fibroblasts were performed using four substrates, at 37°C and 41°C. Flux of less than 70% of simultaneous controls using myristate as a substrate, or less than 60% using octanoate is considered indicative of MCADD. Flux studies Figure 4. Tritium release fat oxidation flux studies in MCADD Discussion Figure 1a. MS/MS blood spot carnitine profile taken on day 12. C6 ↓ C8 ↓ C10 ↓ C16 ↓ Figure 1b. MS/MS plasma acyl carnitine profile taken on day 12. C6 ↓ C8 ↓ C10 ↓ Figure 2. GC/MS urine organic acid profile taken on day 12. Note insignificant hexanoylglycine peak *, with presence of adipate and suberate *. * * *