PEDIATRICS Vol. 118 No. 3 September 2006, pp. 1065-1069 (doi:10.1542/peds.2006-0666)
ARTICLE |
Neonatal Screening for Very Long-Chain Acyl-CoA Dehydrogenase Deficiency: Enzymatic and Molecular Evaluation of Neonates With Elevated C14:1-Carnitine Levels
a Department of General Pediatrics, University Children's Hospital, Duesseldorf, Germany
b University of Amsterdam, Academic Medical Center, Departments of Pediatrics and Clinical Chemistry, Amsterdam, the Netherlands
c Department of Pediatrics and Vanderbilt Children's Hospital, Vanderbilt University, Nashville, Tennessee
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ABSTRACT |
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 TOP ABSTRACT METHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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OBJECTIVE. Neonatal screening programs for very long-chain acyl-coenzyme A dehydrogenase deficiency have been implemented recently in various countries. Mildly elevated C14:1-carnitine on day 3 of life strongly suggests very long-chain acyl-coenzyme A dehydrogenase deficiency.
DESIGN. We characterized 11 neonates with elevated C14:1-carnitine by enzyme and molecular analyses. Palmitoyl-coenzyme A oxidation was measured in lymphocytes. Sequencing of all 20 exons of the VLCAD gene was performed from genomic DNA.
RESULTS. Palmitoyl-coenzyme A oxidation revealed significantly decreased residual activities consistent with very long-chain acyl-coenzyme A dehydrogenase deficiency in 7 neonates. In 2 individuals, residual activities of 48% and 44%, respectively, suggested heterozygosity. Two disease-causing mutations were detected in 6 of 7 neonates with very long-chain acyl-coenzyme A dehydrogenase deficiency; in the remaining 1 patient, only 1 mutation was identified. Of 2 individuals with residual activities consistent with heterozygosity, 1 was heterozygous for a VLCAD mutation. The other child and both individuals with normal palmitoyl-coenzyme A oxidation had normal genotypes.
CONCLUSIONS. In 4 of 11 neonates identified with elevated C14:1-carnitine, very long-chain acyl-coenzyme A dehydrogenase deficiency was excluded. A C14:1-carnitine level >1 µmol/L strongly suggests very long-chain acyl-coenzyme A dehydrogenase deficiency, whereas concentrations 
1 µmol/L do not allow a clear discrimination among affected patients, carriers, and healthy individuals. Further diagnostic evaluation, including enzyme and molecular analyses, is essential to identify very long-chain acyl-coenzyme A dehydrogenase deficiency correctly.
Key Words: tetradecenoylcarnitine • very long-chain acyl-CoA dehydrogenase • fatty acid oxidation • tandem mass spectrometry
Abbreviations: VLCADD—very long-chain acyl-coenzyme A dehydrogenase deficiency • MS/MS—tandem mass spectrometry • CoA—coenzyme A • MCADD—medium-chain acyl-coenzyme A dehydrogenase deficiency
Fatty acid oxidation defects are a rare group of genetic disorders that may have serious clinical consequences for an affected neonate or young infant. If undiagnosed and untreated, these defects can cause severe cardiomyopathy and hepatopathy and may result in early death in 40% of symptomatic infants. Early detection and accurate diagnosis are, therefore, essential for achieving a favorable outcome. Neonatal screening programs for fatty acid oxidation defects, including very long-chain acyl-coenzyme A dehydrogenase deficiency (VLCADD), have been implemented recently in a number of European countries, as well as in Australia, and are currently required by law in 28 of the US states.1 This expansion of neonatal screening programs was feasible because of the development of electrospray ionization tandem mass spectrometry (MS/MS), a new diagnostic tool with high specificity and sensitivity.2
VLCAD catalyzes the initial step of mitochondrial ß-oxidation of long-chain fatty acids with a chain length of 14 to 18 carbons. In VLCADD, C14 to C18 acylcarnitines accumulate before the enzymatic block, with C14:1-carnitine as the disease-specific marker. Mildly elevated C14:1-carnitine in neonates on day 3 of life strongly suggests VLCADD.3 Since introduction of neonatal screening programs for VLCADD, the apparent incidence has increased4 and is now estimated at 1:50000 to 1:120000.3 VLCADD is, therefore, the most common defect in long-chain fatty acid oxidation.
The majority of children identified by neonatal screening was asymptomatic at time of diagnosis and remained asymptomatic during follow-up with preventive dietary measures, such as avoidance of fasting, long-chain fat restriction, and supplementation of medium-chain triglycerides.3 It is not yet apparent which patients diagnosed by neonatal screening would have become symptomatic if screening had not been performed.4 The clinical presentation of symptomatic VLCADD is quite heterogeneous with phenotypes of different severities. Early onset forms with cardiomyopathy, life-threatening arrhythmias,5 or Reye-like symptoms and later-onset milder phenotypes with hypoglycemia or myopathy have been reported.6 VLCADD is characterized by molecular heterogeneity.6,7 Despite this, genotype-phenotype correlation has been reported, with missense mutations resulting predominantly in milder phenotypes and null mutations associated with life-threatening phenotypes.8 To further characterize asymptomatic neonates identified with elevated C14:1-carnitine on neonatal screening suggestive for VLCADD, we performed enzyme and molecular analyses in a cohort of 11 MS/MS-positive neonates.
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METHODS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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Patients
Of an estimated 1000000 neonates screened for VLCADD, 11 presented with an elevated C14:1-carnitine. Because several laboratories are authorized to perform neonatal screening in Germany and the referring physician decides whether and where further workup should be performed, the cohort in our study may not be from the whole of Germany. C14:1-carnitine serves as disease-specific marker for VLCADD. The individuals identified were referred for further diagnostic workup.
Tandem Mass Spectrometry
Tandem mass spectrometry was performed as reported previously.2,9 Acylcarnitines were extracted from dried blood spots on neonatal screening cards. Analyses were performed on day 3 of life. According to neonatal screening procedures, an elevated C14:1-carnitine level required a second, "confirmatory" sample, which was usually obtained 5 to 7 days after birth. The most common cutoff in the different screening laboratories (Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, Oberschleissheim; Screening-Labor Hannover; Screening-Labor der Universitätskinderklinik Hamburg; Screening-Labor Becker und Olgemöller, München, Germany), who performed MS/MS studies on day 3 of life, is <0.25 µmol/L (ranging from <0.21 to 0.41 µmol/L). Recall rates in VLCADD were at 0.03%.
Isolation of Lymphocytes From Blood Samples
Lymphocytes from blood were isolated using Leucosep (Greiner bio-1, Frickenhausen, Germany) according to the manufacturer's protocol. Ficoll-Paque Plus solution (Amersham, Uppsala, Sweden) was added to the blood sample in a filter tube (Leucosep), and the sample was subjected to centrifugation at 2000 rpm for 10 minutes. The lymphocyte layer was transferred into a reaction tube with 10 mL of ammonium chloride buffer. After incubation for 10 minutes on ice, lymphocytes were pelleted (again at 2000 rpm for 10 minutes). The pellet was resuspended in 10 mL of 0.9% sodium chloride and centrifuged at 2000 rpm for 10 minutes. The resulting lymphocyte pellet was resuspended in 1 mL 0.9% sodium chloride solution. Finally, lymphocytes were spun down at 13000 rpm for 10 minutes. The resulting lymphocyte pellet was used for measurements of enzyme activity. Protein concentration was measured using the Bradford method.
Measurement of VLCAD Enzyme Activity
Palmitoyl-coenzyme A (CoA) oxidation was performed as reported previously.10 In short, lymphocytes were resuspended in a buffer solution at protein concentrations of 2 to 3 mg/mL. All of the samples were prepared in duplicate. The enzyme reaction was started by adding the specific substrate, palmitoyl-CoA. After 5 minutes, the reaction was stopped by adding HCl. Samples were incubated on ice for 5 minutes and afterward neutralized.
For HPLC measurements, acetonitrile-water (1:2) was added to each sample. After centrifugation, the supernatant was transferred to a glass flask and analyzed by HPLC for C16-carnitine, C16:1-carnitine, and C16:OH-carnitine.
Molecular Genetic Analysis
Genomic DNA from whole blood was isolated using DNA Mini kit (Qiagen, Hilden, Germany). All 20 of the VLCAD exons were amplified by polymerase chain reaction with the use of 21- to 29-bp-long intronic primer pairs.3 Exons 1 and 2, 3 and 4, 12 and 13, 14 and 15, 16 and 17 were amplified together. The amplified DNA-fragments were separated by electrophoresis and extracted (QIAquick Gel Extraction kit, Quiagen). PCR products were subjected to DNA sequence analysis using the Cycle sequencing kit, (Applied Biosystems, Weiterstadt, Germany). Analysis was performed using an Applied Biosystems Prism 310 Genetic Analyser.
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RESULTS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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C14:1-Carnitine and Free Carnitine
Neonatal screening on day 3 of life revealed elevated C14:1-carnitine in all 11 neonates, suggesting VLCADD (Fig 1). In 8, a confirmatory sample obtained between days 5 and 7 also revealed elevated C14:1, but in the remaining 3 neonates (patients 5, 8, and 9), the confirmatory sample on days 5 to 7 was normal. In our group of neonates, C14:1-carnitine was significantly increased (>1 µmol/L) in 5 subjects; in all 5, VLCADD was confirmed (Table 1). Free carnitine was within the reference ranges in all 11 neonates.
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Clinical Presentation and Follow-up
All of the neonates excluding patient 9 were asymptomatic at the time of diagnosis. Patient 9 suffered from a congenital heart disease, and surgical intervention was necessary within the first days of life. Patient 5 developed hypoketotic hypoglycemia associated with an infection at age 3 months.
VLCAD Enzyme Activity
In 6 neonates, palmitoyl-CoA oxidation in lymphocytes was below the defined limit of sensitivity of the assay (<0.6 nmol · [mg · min]–1; Table 1). In 1 neonate, low residual activity of 0.9 nmol · (mg · min)–1 was measured (Table 1). In comparison, healthy controls had activities of 7.5 ± 1.5 nmol · (mg · min)–1 (n = 19).
Decreased palmitoyl-CoA oxidation of 12% clearly indicates VLCADD in these 7 neonates (Table 1). In 2 individuals, we measured enzyme activities of 3.6 and 3.3 nmol · (mg · min)–1, respectively, corresponding with 48% and 44% residual activity, suggesting heterozygosity. Known heterozygous individuals composing a control group (n = 8) had residual activities of 3.7 ± 1.0 nmol · (mg · min)–1. In 2 of our 11 infants, activities were 
5 nmol · (mg · min)–1, indicating normal palmitoyl-CoA oxidation and excluding VLCADD.
VLCAD Sequence Analysis
In all 11 neonates, DNA sequence analysis of all 20 VLCAD exons and adjacent intronic splice consensus sequences was performed (Table 1). In 6 of 7 patients with significantly reduced palmitoyl-CoA oxidation suggesting VLCADD, 2 disease-causing mutations in the VLCAD gene were delineated; 1 patient was homozygous, and the other 5 were compound heterozygous, with 2 different mutations. In 1 infant with significantly reduced palmitoyl-CoA oxidation in lymphocytes (patient 7), only 1 single mutation was detected. One of 2 neonates with residual activities suggesting heterozygosity had a single VLCAD mutation (patient 8), and the other individual (patient 9) did not have a mutation identified. Both individuals with normal VLCAD activities showed a normal VLCAD genotype.
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DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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Our study demonstrates that an elevated C14:1-carnitine of >1 µmol/L on neonatal screening on day 3 of life correctly identifies VLCAD deficiency. Elevated C14:1-carnitine concentrations of
1 µmol/L do not allow sufficient discrimination between affected and healthy individuals. In 4 of 6 neonates with elevated C14:1-carnitine <1 µmol/L on day 3 of life, VLCADD could not be confirmed by enzyme or molecular analyses. C14:1-carnitine in confirmatory samples on days 5 to 7 of life was increased in 6 of 7 patients with VLCADD and in 2 of the remaining 4 healthy individuals. Normalization of C14:1-carnitine as caloric intake increases after birth, therefore, does occur in healthy individuals. However, this is also seen in patients with VLCADD and does not allow discrimination between affected and healthy children.11,12 An increase in long-chain acylcarnitines in healthy individuals with stresses and in neonates suggests induction of fatty acid oxidation during catabolism resulting in accumulation of metabolites that are potential substrates for the rate-limiting ß-oxidation enzyme, VLCAD.11 Previous studies in normal mice report that stress may induce long-chain acylcarnitine production.13 Moreover, acylcarnitine production in mice heterozygous for VLCADD is even more pronounced after stress.13 Because neonates are in a catabolic condition on day 3 of life, MS/MS screening cutoff values for long-chain acylcarnitines are significantly higher in the first 1–3 days of life as compared with values in well-nourished children.14 Despite these adjusted cutoff values, acylcarnitine analysis on day 3 of life does not correctly discriminate between affected subjects with VLCADD and healthy individuals. Therefore, we recommend that an elevated C14:1-carnitine on neonatal screening should always result in further diagnostic workup, including measurement of palmitoyl-CoA oxidation, as well as molecular genetic studies, even if the second, confirmatory plasma acylcarnitine sample is normal.11,12
In contrast, palmitoyl-CoA oxidation in lymphocytes does allow discrimination of healthy individuals and affected subjects. Our data show that palmitoyl-CoA oxidation in patients does not exceed 0.9 nmol ·(mg · min)–1, corresponding with 12% residual enzyme activity. However, clear discrimination of heterozygous individuals and neonates with a normal genotype is not possible using palmitoyl-CoA oxidation studies. Enzyme activities in the known VLCAD heterozygous individuals range from 2.1 to 5.0 nmol · (mg · min)–1 and, in the control group of individuals with a normal genotype, are from 5.5 to 9.0 nmol · (mg · min)–1. Molecular analysis may be required to give additional information about the carrier status. With respect to the tetradecenoylcarnitine concentrations at the time of neonatal screening, 2 groups of patients can be distinguished: those with a significant increase in C14:1-carnitine (>1 µmol/L) and those with a milder increase (1 µmol). There was no correlation between C14:1-carnitine levels and residual enzyme activity in our studies.
Molecular analysis revealed 2 disease-causing mutations in 6 neonates deficient of VLCAD by enzymatic analysis. In 2 individuals, only 1 mutation was found. In 1 of these neonates, enzyme analysis revealed significantly reduced activity, clearly suggesting VLCADD. It seems possible that the second mutation may be in a distant intronic regulatory control region not available for gene sequencing in the amplified products. One child with only a single mutation is clearly heterozygous for VLCADD according to enzyme analysis. In 1 neonate (patient 9) with reduced residual enzyme activity suggesting heterozygosity, no mutation in the VLCAD gene was found. In this case, it remains uncertain whether the child is a disease carrier or completely unaffected. All of the individuals with normal palmitoyl-CoA oxidation showed a normal genotype.
For medium-chain acyl-CoA dehydrogenase deficiency (MCADD), the most common fatty acid oxidation defect, only recently has a genotype-biochemical phenotype correlation been proposed.15 The common c.985A>G mutation that is homozygous in 80% of clinically presenting cases and heterozygous in additional 18% is associated with significantly higher octanoylcarnitine in the homozygous state than in compound heterozygosity for c.985A>G and a second mutation.15 Patients with homozygous c.199T>C presented with the lowest octanoylcarnitine levels.15 In contrast to MCADD, VLCADD is characterized by great molecular heterogeneity. In our cohort of 7 patients with VLCADD, the most common mutation, V243A, occurs on 3 of 13 alleles.3 This mutation in the homozygous state is associated with a residual VLCAD activity of 12%3 and with a milder tetradecenoylcarnitine increase on neonatal screening (0.58 µmol/L),3 suggesting milder functional effects on protein structure. Patient 5 with homozygosity for T220M presented with a more pronounced increase in tetradecenoylcarnitine (2.3 µmol/L), indicating that T220M might have a more severe effect on protein structure and function. Overall, because of compound heterozygosity in the majority of our patients, correlation of genotype with biochemical phenotype is not possible in our patient cohort.
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CONCLUSIONS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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Neonatal screening for VLCADD using tandem mass spectrometry does not allow discrimination between affected and healthy individuals, especially when the disease-specific marker C14:1-carnitine is above the cutoff value but
1 µmol/L. Neonates with an elevated C14:1-carnitine <1 µmol/L can be affected homozygotes, heterozygotes, or individuals with a normal genotype. There are other metabolites, such as C14 and C14:2, and analyte ratios, such as C14/C14:1, C14:1/C16, C14:1/C8, or C14:1/C4 that may help in discrimination among these groups. Whether a specificity of 100% can be reached using all of these metabolites and analyte ratios needs further investigation. Further enzymatic workup is essential to establish or exclude VLCADD and to avoid unnecessary treatment. Analysis in lymphocytes is feasible in the first days of life, and results are readily available within a few days. Despite the high rate of false-positive individuals for VLCADD in our cohort using C14:1-carnitine as the only disease-specific marker, neonatal screening using tandem mass spectrometry allows early detection of patients with VLCADD and, with appropriate treatment, will prevent morbidity and mortality. Furthermore, simulation modeling for MCADD, the most common fatty acid oxidation defect, predicts that almost all of the additional costs of screening would be offset by prevention of deleterious sequelae.16
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ACKNOWLEDGMENTS |
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This study was financially supported by a grant from the medical faculty of the Heinrich Heine University Düsseldorf, Düsseldorf, Germany.
We thank PD Dr Ina Knerr, Dr Uta Nennstiel-Ratzel, Dr Zoltan Lukacs, Prof Dr Bernhard Olgemöller, PD Dr Wulf Röschinger, Prof Dr Johannes Sander, Prof Dr René Santer, Prof Dr Wilfried Tillmann, and Dr A. Weise for providing neonatal screening results and clinical data from their patients.
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FOOTNOTES |
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Accepted May 3, 2006.
Address correspondence to Ute Spiekerkoetter, MD, Department of General Pediatrics, University Children's Hospital, Moorenstrasse 5, D-40225 Düsseldorf, Germany. E-mail: ute.spiekerkoetter{at}uni-duesseldorf.de
Michaela Liebig and Ina Schymik contributed equally to this study.
The authors have indicated they have no financial relationships relevant to this article to disclose.
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REFERENCES |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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- National Newborn Screening and Genetics Resource Center. US National Screening Status Report: MS/MS. Washington, DC: 2005. Available at: http://genes-r-us.uthscsa.edu/resources/newborn/msmstests.htm. Accessed August 26, 2005
- Zytkovicz TH, Fitzgerald EF, Marsden D, et al. Tandem mass spectrometric analysis for amino, organic and fatty acid oxidation disorders in newborn dried blood spots: two-year summary from the New England screening program.
Clin Chem. 2001;47
:1945
–1955
[Abstract/Free Full Text] - Spiekerkoetter U, Sun B, Zytkovicz T, Wanders R, Strauss AW, Wendel U. MS/MS-based newborn and family screening detects asymptomatic patients with very long-chain acyl-CoA dehydrogenase deficiency. J Pediatr. 2003;143 :335 –342[CrossRef][Web of Science][Medline]
- Wilcken B, Wiley V, Hammond J, Carpenter K. Screening newborns for inborn errors of metabolism by tandem mass spectrometry.
N Engl J Med. 2003;348
:2304
–2312
[Abstract/Free Full Text] - Bonnet D, Martin D, de Lonay P, et al. Arrhytmias and conduction defects as presenting symptoms of fatty acid oxidation disorders in children.
Circulation. 1999;100
:2248
–2253
[Abstract/Free Full Text] - Gregersen N, Andresen BS, Corydon MJ, et al. Mutation analysis in mitochondrial fatty acid oxidation defects: exemplified by acyl-CoA dehydrogenase deficiencies, with special focus on genotype-phenotype relationship. Hum Mutat. 2001;18 :169 –189[CrossRef][Web of Science][Medline]
- Strauss AW, Powell CK, Hale DE, et al. Molecular basis of human mitochondrial very long chain acyl-CoA dehydrogenase deficiency causing cardiomyopathy and sudden death in childhood.
Proc Natl Acad Sci USA. 1995;92
:10496
–10500
[Abstract/Free Full Text] - Andresen BS, Olpin S, Poorthuis BJ, et al. Clear correlation of genotype with disease phenotype in very long-chain acyl-CoA dehydrogenase deficiency. Am J Hum Genet. 1999;64 :479 –494[CrossRef][Web of Science][Medline]
- Roscher AA, Fingerhut R, Liebl B, et al. Expanded newborn screening using tandem mass spectrometry. Monatsschr Kinderh. 2001;149 :1297 –1303[CrossRef]
- Wanders RJA, Vreken P, Den Boer MEJ, et al. Disorders of mitochondrial fatty acyl-CoA b-oxidation. J Inherit Metab Dis. 1999;22 :442 –487[CrossRef][Web of Science][Medline]
- Schymik I, Liebig M, Mueller M, et al. Pitfalls of neonatal screening for very long-chain acyl-CoA dehydrogenase (VLCAD) deficiency using tandem mass spectrometry. J Pediatr. 2006; In press
- Browning MF, Larson C, Strauss A, et al. Normal acylcarnitine levels during confirmation of abnormal newborn screening in long-chain fatty acid oxidation defects. J Inherit Metab Dis. 2005;28 :545 –550[CrossRef][Web of Science][Medline]
- Spiekerkoetter U, Tokunaga C, Wendel U, et al. Changes in blood carnitine and acylcarnitine profiles of very long-chain acyl-CoA dehydrogenase-deficient mice subjected to stress. Eur J Clin Invest. 2004;34 :191 –196.[CrossRef][Web of Science][Medline]
- Cavedon CT, Bourdoux P, Mertens K, et al. Age-related variations in acylcarnitine and free carnitine concentrations measured by tandem mass spectrometry. Clin Chem. 2005;745 –752
- Waddell L, Wiley V, Carpenter K, et al. Medium-chain acyl-CoA dehydrogenase deficiency: Genotype-biochemical phenotype correlations. Mol Genet Metab. 2006; 87: 32–39[CrossRef][Web of Science][Medline]
- Venditti L, Venditti C, Berry G, et al. Newborn screening by tandem mass spectrometry for medium-chain acyl-CoA dehydrogenase deficiency: a cost effectiveness analysis.
Pediatrics. 2003;112
:1005
–1015
[Abstract/Free Full Text]
PEDIATRICS (ISSN 1098-4275). ©2006 by the American Academy of Pediatrics
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