ADULT-ONSET FORM IN VLCAD DEFICIENCY: SEVEN CASES Sousa C. 1 , Marcão A. 1 , Nogueira C. 1 , Fonseca H. 1 , Rocha H. 1 , Silva C. 2 ,Moniz M. 3 , Evangelista T. 4 , Guimas A. 5 , Maré R. 6 and Laura Vilarinho 1 1 Unidade de Rastreio Neonatal, Metabolismo e Genética, Departamento de Genética Humana. Instituto Nacional de Saúde Doutor Ricardo Jorge 2 Serviço de pediatria, Centro Hospitalar Póvoa Varzim 3 UCIEP, Hospitalar Professor Doutor Fernando Fonseca 4 Serviço de Neurologia, Centro Hospitalar de Lisboa Norte 5 Serviço de Medicina Interna, Centro Hospitalar do Porto 6 Serviço de Neurologia, Hospital São Marcos INTRODUCTION Very long chain acyl-CoA dehydrogenase deficiency (VLCADD, MIM 201475) is an autosomal recessive disorder characterized by impaired mitochondrial β- oxidation of fatty acids with a chain length between 14 and 18 carbons. The prevalence of VLCAD deficiency in Portugal is 1/101,613. VLCADD has three forms of clinical presentation: severe early-onset; intermediate with childhood onset and adult-onset, of mild severity, characterized by exercise intolerance, myalgia and recurrent episodes of rhabdomyolysis (1). The VLCAD gene (ACADVL) contains 20 exons that encode a 655-amino-acid protein and it is located on chromosome 17p13. More than 116 mutations have been identified in the literature (HGMD). The development of electrospray ionization tandem mass spectrometry (MS/MS) has allowed beyond the screening of neonatal forms a marked improvement on diagnosis of the adult onset form, through the analysis of acylcarnitine profiles from blood spots, using C14:1 as primary marker. The molecular study of ACADVL gene allowed the confirmation of the patients. Table 1- Clinical classification of the seven VLCADD patients. Family A-patients 1 and 2 are brothers; Family C-patients 4 and 5 are brothers. Family A Family B Family C Family D Family E Patient 1 Patient 2 Patient 3 Patient 4 Patient 5 Patient 6 Patient 7 Age/sex 13 Years Female 19 Years Male 51 Years Male 11 Years Male 20 Years Male 14 Years Female 63 Years Female Clinical phenotype -Severe generalized muscle pain -Dark urine after prolonged physical effort -CPK↑ -mioglobin↑ -mioglubinuria -Episodes of muscle pain -Dark urine after physical effort of moderate intensity since the age 17 -At age 18 hospitalization due to not elucidated rhabdomyolysis - Experienced the first of several recurrent episodes of myalgia at 20 years -Muscle weakness -Rhabdomyolysis following prolonged exercice -CPK↑ -Exercise intolerance -Dark urine -Myalgia -Rhabdomyolysis - Rhabdomyolysis -Rhabdomyolysis -Myalgia PATIENTS AND METHODS The authors report seven individuals from five families with clinical symptoms and ages between 11 y-63y (Table 1). Blood spot samples are collected in Whatman 903 filter paper. Acylcarnitines were analysed as butyl esters on an API 2000 triple quadrupole tandem mass spectrometer (AppliedBiosystems, Sciex) with an ion spray device, as previously described with minor modifications (2). Genomic DNA was extracted from blood of patients and their parents by standard methods. All 20 exons of the ACADVL gene (3), and respective flanking regions, were amplified by PCR, using newly designed primers and was performed on an automatic sequencer (ABI Prism 3130XL). Table 2- Biochemical and molecular data from the seven VLCADD patients. DISCUSSION / CONCLUSION In spite of the heterogeneity of genotype usually associated with VLCADD, in these cases we found the same mutation (p.L500del) in 10/14 allels and another mutation p.R366H in 3/4 reaming allels. This fact may indicate that this mutations are commonly associated with mild VLCADD form in the Portuguese population. When rhabdomyolysis is present in a patient, and after differential diagnosis exclusion, it is important to consider the possibility of a VLCAD deficiency. However late-onset forms may be undetectable by acylcarnitine profile in asymptomatic period, and whenever possible samples should be taken in in crisis period. If VLCADD is considered suspicious the molecular analysis of ACADVL should be performed even in the presence of a normal acilcarnitines profile , to avoid a late diagnosis. REFERENCES (1) Pons R, Cavadini P, et al (2000) Clinical and molecular heterogeneity in very-long-chain acyl-coenzyme A dehydrogenase deficiency. Pediatr Neurol. 22(2):98-105. (2) Rashed MS, Ozand PT, Bucknall MP, Little D. Diagnosis of inborn errors of metabolism from blood spots by acylcarnitinesand amino acids profiling using automated electrospray tandem mass spectrometry. Pediatr Res. 1995;38:324–331 (3) Andresen BS, Bross P, et al (1996) Cloning and characterization of human very-long-chain acyl-CoA dehydrogenase cDNA, chromosomal assignment of the gene and identification in four patients of nine different mutations within the VLCAD gene. Hum Mol Genet. 5(4):461-72. RESULTS The analysis by tandem mass spectrometry of the acylcarnitines profile in seven individuals with clinical symptoms revealed accumulation of tetradecenoyl carnitine (C14:1), suggesting the diagnosis of VLCADD. The molecular characterization allowed the identification of mutations in all cases (Table 2), thus confirming this diagnosis. C14:1 µM C14:2 µM C14:1/C16 C14:1/C12:1 Genotype Deduced effect Ref. value <0.18 <0.08 <0.19 <2.60 Patient 1 0.35 0.23 0.30 4.38 c.[1500 _1502delCCT]+ [1500 _1502delCCT] p.[L500del]+[L500del] Patient 2 0.25 0.01 0.10 1.25 c.[1500 _1502delCCT]+ [1500 _1502delCCT] p.[L500del]+[L500del] Patient 3 7.17 0.67 2.43 15.42 c.[1097G>A]+[1097G>A] p.[R366H]+[R366H] Patient 4 0.47 0.17 0.46 5.88 c.[1500 _1502delCCT]+ [1500 _1502delCCT] p.[L500del]+[L500del] Patient 5 0.22 0.10 0.16 1.70 c.[1500 _1502delCCT]+ [1500 _1502delCCT] p.[L500del]+[L500del] Patient 6 1.27 0.30 1.72 9.77 c.[187_192insA]+[1097G>A] p.[P65Tfs*7]+[R366H] Patient 7 0.25 0.09 0.52 3.64 c.[1500 _1502delCCT]+ [1500 _1502delCCT] p.[L500del]+[L500del]