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VLCAD deficiency: Pitfalls in newborn screening and confirmation of diagnosis by mutation analysis
Molecular Genetics and Metabolism 88 (2006) 166–170 www.elsevier.com/locate/ymgme
VLCAD deWciency: Pitfalls in newborn screening and conWrmation of diagnosis by mutation analysis A. Boneh a,d,¤, B.S. Andresen b,c, N. Gregersen b, M. Ibrahim a, N. Tzanakos a, H. Peters a,d, J. Yaplito-Lee a, J.J. Pitt a a
Metabolic Service and Newborn Screening Laboratory, Genetic Health Services Victoria, Melbourne, Australia b Research Unit for Molecular Medicine, Skejby Sygehus, Aarhus University, Aarhus, Denmark c Institute of Human Genetics, Aarhus University, Aarhus, Denmark d Department of Paediatrics, University of Melbourne, Australia Received 9 November 2005; received in revised form 21 December 2005; accepted 21 December 2005 Available online 20 February 2006
Abstract We diagnosed six newborn babies with very long-chain acyl-CoA dehydrogenase deWciency (VLCADD) through newborn screening in three years in Victoria (prevalence rate: 1:31,500). We identiWed seven known and two new mutations in our patients (2/6 homozygotes; 4/6 compound heterozygotes). Blood samples taken at age 48–72 h were diagnostic whereas repeat samples at an older age were normal in 4/6 babies. Urine analysis was normal in 5/5. We conclude that the timing of blood sampling for newborn screening is important and that it is important to perform mutation analysis to avoid false-negative diagnoses of VLCADD in asymptomatic newborn babies. In view of the emerging genotype–phenotype correlation in this disorder, the information derived from mutational analysis can be helpful in designing the appropriate follow-up and therapeutic regime for these patients. © 2005 Elsevier Inc. All rights reserved. Keywords: Fatty acid oxidation; Very long-chain fatty acids; VLCAD deWciency (VLCADD); Newborn screening; Mutation; Tandem mass spectrometry
Introduction Very long-chain acyl-CoA dehydrogenase deWciency (VLCADD, MIM 201475) is an autosomal recessive disorder of fatty acid oxidation. VLCAD (EC 1.3.99.13), which was identiWed in 1992 [1], is one of a family of acylCoA dehydrogenases that catalyse the dehydrogenation of straight-chain and branched-chain acyl-CoA esters. VLCAD is the Wrst enzyme in the spiral of oxidation of C10–C18 or longer straight-chain fatty acids. Essentially all patients who were historically thought to have longchain fatty acid dehydrogenase (LCAD) deWciency were found to actually have VLCADD. Patients diagnosed clinically can be grouped into three main groups [2,3].
*
Corresponding author. Fax: +61 3 8341 6390. E-mail address: [email protected] (A. Boneh).
1096-7192/$ - see front matter © 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.ymgme.2005.12.012
Patients may present early (possibly within days after birth) with a severe form, usually with cardiomyopathy. In a large series of babies diagnosed clinically, cardiomyopathy was the most common presenting symptom, leading to sudden death in some babies [4]. In the second group are patients who present later in childhood with less severe metabolic disturbances, yet with episodes of hypo-ketotic hypoglycaemia, and with no cardiomyopathy. The third group includes those who present after childhood with an exclusive muscular form and episodes of rhabdomyolysis. The exact prevalence of VLCADD is not known. Based on recent results of newborn screening programs the prevalence could vary between 1:42,500 and 1:125,000 births [5–7]. Herein, we describe our observations regarding the importance of the timing of newborn screening for this disorder, describe novel mutations, and summarise our experience in the management of VLCADD.
A. Boneh et al. / Molecular Genetics and Metabolism 88 (2006) 166–170
Methods Acylcarnitines in dried blood spot samples were measured using Xowinjection electrospray-tandem mass spectrometry. Newborn screening samples were normally collected at 48–72 h of age and extracted and butylated as previously described [8,9] and analysed using a Waters Quattro LC triple quadrupole mass spectrometer interfaced to a Gilson 215 autosampler and Agilent 1100 pumping system. Multiple reaction monitoring was used to measure the responses of various acylcarnitines and stable isotope internal standards. A standard of C14:1 carnitine was unavailable and the concentration of this metabolite was calculated by assuming the same response as C14 carnitine. Data were also expressed as standard deviation indices (SDI, deWned as the number of standard deviations the value deviates from the mean) relative to control infants. Mean and standard deviations for controls were calculated from log-transformed data for each combination of age (7 ranges), weight (6 ranges), and gestation (9 ranges). PCR ampliWcation of all exons, including part of the Xanking intron sequences, of the human VLCAD gene, was carried out as previously described [3] using a GeneAmp(r) PCR system 9700 (Applied Biosystems). Sequence analysis with M13 forward and reverse primers was performed using the BigDye(r) Terminator v1.1 Cycle Sequencing kit (Applied Biosystems) and an 3100-Avant genetic analyzer (Applied Biosystems). Sequence data were analysed with the Sequencer v3.1.1 software (Gene Codes Corporation).
Results Tandem mass spectrometry Six VLCADD subjects were detected by screening 189,000 infants between February 2002 and April 2005 (yielding a prevalence of 1:31,500 with a 95% conWdence interval of 1:15,300 to 1:90,900). A sibling (D1), born prior to the introduction of TMS newborn screening, was also diagnosed through family screening.
167
The results of acyl carnitine analyses in dried blood spot samples are illustrated in Fig. 1. All VLCADD subjects had signiWcantly increased levels of C14:1, C14, and C14:1/C10 ratio in the initial newborn screening sample. C14:1 levels in mol/L were higher than C14 levels in the Wrst 72 h (Figs. 1 A and B). The C14:1/C10 ratio was also diagnostic, extended up to 100 h after birth, and appeared to be somewhat more reliable in the second sample than the C14:1 value alone (e.g., babies C and F, Fig. 1C). C14, C14:1, and C14:1/C10 values generally declined in the second sample taken after 130 h (Fig. 1), consistent with previous observations that long-chain acylcarnitines such as C14 and C14:1 decrease signiWcantly in the immediate postnatal period in normal babies [10–12]. Thus, interpretation of these repeat samples requires age-matched reference ranges. We also used the standard deviation index (SDI) of C14:1, which takes into consideration age, weight, and gestation and hence is expected to have more diagnostic ability regardless of the age of sampling, as a convenient way to compensate for these changes. In general, SDIs greater than three are considered abnormal. As shown in Fig. 1D,14:1 level was still abnormal in most of the repeat samples when analysed in this fashion (generally >4). An exception to the Wnding of decreased C14:1 level in a repeat sample was patient B who had a higher level in the second sample, presumably because he was symptomatic at the time (with hypoglycaemia and cardiovascular collapse without cardiomyopathy on day four of life). All other patients have remained free of symptoms with normal urine organic acid and subsequent dried blood spot acylcarnitine proWles up to the age of six months. In particular,
Fig. 1. Dried blood spot metabolite levels in VLCADD patients as a function of age in hours. C14:1 and C14 values are in mol/L, shaded boxes represent neonatal reference ranges in two age ranges. (Values for D1 are approximate because sample was stored for four years.) (A) C14:1, (B) C14, (C) C14:1/C10 ratio, (D) C14:1 as the standard deviation index (SDI).
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secondary indicators of abnormal fatty acid oxidation such as urine dicarboxylic and 3-hydroxydicarboxylic acids were within normal limits. Mutation analysis To conWrm the diagnosis of VLCADD, we sequenced all exons comprising the entire coding region of the VLCAD gene from the identiWed newborns. In all babies, mutations were identiWed in both alleles of the gene. Two patients were homozygous. Baby A was homozygous for the most frequently found mutation in the VLCAD gene, namely c.848T>C, causing a change from valine to alanine at position 243 of the mature protein. Both parents were found to be heterozygous for this mutation. The c.848T>C mutation has been reported to be particularly frequent in newborns identiWed by tandem mass spectrometry-based screening [5]. It was initially identiWed as one of the mutations in compound heterozygous symptomatic patients [3,4,13]. However, two unrelated patients, who are homozygous for this mutation have been previously diagnosed (Andresen BS— Unpublished data). One of these patients presented with hypoglycemia at 48 h of age and experienced an episode of abnormal movements of the left hand and leg. After this he remained sleepy for several days. He has grown and developed normally, but started experiencing increasing troubles with rhabdomyolysis from the age of six years, indicating that the c.848T>C mutation may be disease causing also when present in homozygous form. Baby C was homozygous for a previously unreported mutation, c.1497-1499delCCT, which results in deletion of leucine at position 460 of the mature VLCAD protein. Both parents were found to be heterozygous for this mutation. The same genotype has been previously identiWed in a patient who was referred for mutation analysis because he had presented clinically with disease (Andresen BS— Unpublished data), indicating that this genotype may be disease causing. Baby B was compound-heterozygous for a c.1097G>A mutation (R326H) and for a c.1322G>A (G401D) mutation. His father was heterozygous for the c.1097G>A mutation and his mother was heterozygous for the c.1322G>A mutation. Both mutations have been reported in patients with clinical manifestation of VLCADD [3,4,13]. Baby D2 was compound-heterozygous for the c.848T>C mutation and a splice site mutation in intron 2 (c.7532A>C). The splice site mutation has been reported in several clinically aVected patients [3,4,13] and was found in four Italian patients [14]. Recently, another patient who has the same genotype has been identiWed (Andresen BS— Unpublished data). This patient presented with hypoglycaemia in association with a respiratory viral infection at 17 months of age and with seizures 10 months later. Thus, it seems that patients with this particular genotype are at risk of developing disease. Baby E was found to be compound-heterozygous for the c.1349G>A (R410H) mutation, which has been reported in
patients with clinically manifest VLCADD [3,15] and the novel mutation c.481G>A. His mother was heterozygous for the c.1349G>A mutation and his father was heterozygous for the c.481G>A mutation. The c.481G>A mutation causes a shift from alanine at position 121 of the mature VLCAD protein to a threonine (A121T). Alanine at this position is conserved in rat, bovine, mouse, and pig VLCAD. Human short-chain acyl-CoA dehydrogenase (SCAD) and human long-chain acyl-CoA dehydrogenase (LCAD) both have alanine at the homologous position, underscoring the importance of an alanine residue at this position for correct enzyme function. This mutation was predicted to be “benign” with a PSIC score diVerence of 1.079 by the PolyPhen prediction program (http:// tux.embl-heidelberg.de/ramensky/polyphen.cgi). Baby F was found to be compound-heterozygous for the c.1153C>T (R345W) mutation and the novel mutation, c.1117A>T, which causes a shift from isoleucine at position 333 of the mature VLCAD protein to a phenylalanine (I333F). The c.1153C>T mutation has been previously observed in a patient who presented with the muscular form (Muscular weakness, rhabdomyolysis, and elevated CK) of the disease at 19 years of age (Andresen BS— Unpublished data), indicating its disease causing potential. The c.1117A>T mutation is located on exon 11 and was not found in 100 control chromosomes [3], thus excluding the possibility of a common polymorphism. Isoleucine at this position is conserved in rat, bovine, mouse, and pig VLCAD. Human isovaleryl-CoA dehydrogenase also has isoleucine at the homologous position, whereas human SCAD, human medium-chain acyl-CoA dehydrogenase, human short/branched-chain acyl-CoA dehydrogenase, and human glutaryl-CoA dehydrogenase have a conservative substitution to leucine. However, this mutation was predicted to be “benign” with a PSIC score diVerence of 1.414 by the PolyPhen prediction program. It is therefore diYcult to estimate the disease causing potential of this mutation. Discussion In the period of February 2002 to April 2005, we detected six newborn babies with biochemical features of VLCADD through the newborn screening program in Victoria, using tandem mass spectrometry. There were no other clinically diagnosed patients with this disorder in the previous years, but we diagnosed a 4.5-year-old brother of one baby following cascade screening. Despite the relatively high rate of normal acylcarnitine proWles when babies were over four days of age, we pursued mutation analysis for all babies who were suspected of having VLCADD based on their Wrst newborn screening sample, taken at 48–72 h. A total of nine mutations were found in our six patients, which is in agreement with the reported large number of mutations found in previous studies [3,4]. Four of the newborns (A, B, C, and D2) had mutations that have previously been identiWed in symptomatic patients. Thus, our observation that
A. Boneh et al. / Molecular Genetics and Metabolism 88 (2006) 166–170
acylcarnitine proWle in the repeat samples was normal illustrates that newborns with genotypes that have previously been found in individuals with clinical manifestation of VLCADD may be missed by newborn screening if the second sample is drawn after day four of life. The two remaining newborns each had one known disease-associated mutation and one novel mutation. The possibility that these two newborn babies are carriers of rare polymorphisms is unlikely since C14:1 levels in their initial samples were similar to subjects with disease causing mutation on both alleles. Our results indicate a prevalence of 1:31,500 newborn babies, exceeding not only the reported prevalence as suggested by case-series of patients diagnosed clinically but also that based on current newborn screening reports [5–7]. Several reasons may be hypothesised for the higher prevalence of VLCADD in our population. DiVerent laboratories may deWne diVerent cut-oV values for acylcarnitines and guidelines and procedures for the conWrmation of diagnoses. In many instances further diagnostic tests are done if abnormal Wndings in the original blood spots are conWrmed in a repeat sample. We show that reliance on a repeat dried blood spot sample to conWrm the initial Wndings suggestive of VLCADD, when the baby is older than four days, may lead to a false-negative diagnosis (as mentioned above, we did not have false-positive diagnoses of VLCADD). Our results are in line with a recent report on the normalisation of the acylcarnitine proWle in conWrmatory plasma samples following newborn screening in one patient with trifunctional enzyme deWciency and one patient with VLCADD [11]. Indeed, the estimated VLCADD prevalence of 1:250,000 in one study was based on repeat tandem mass spectrometry analysis of blood spots at days 8–10 of life [5]. It is likely that newborn screening detects individuals with the milder forms of VLCADD (as in the case of medium-chain acyl-CoA dehydrogenase deWciency) and that this may explain some of the discrepancy between the prevalence suggested on the basis of MS/MS screening and that based on the number of clinical presentations in previous years. Clear correlation between genotype and phenotype has been demonstrated in VLCADD [3]. Individuals with two null mutations (like stop-, frameshift-, and severesplicing mutations) usually have a severe and early presentation, whereas individuals with at least one missense mutation (or deletion of an amino acid) have a milder and later presentation. Individuals who have their initial presentation in adulthood (with the muscular form of the disease) usually have missense mutations. This suggests that there is a correlation between the residual enzyme activity and the severity of disease. All newborns identiWed in the present study have at least one mutation that may lead to residual enzyme activity. In fact both the V243A and R410H mutations have been shown to result in residual enzyme activity by overexpression of recombinant protein in COS cells [3,15]. Some individuals with the milder form of VLCADD may have had unrecognized episodes and some may remain free of symptoms until adulthood or throughout life unless
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challenged by a metabolic stress. In line with this, Spiekerkoetter et al. [5] reported that an aVected older sibling of a newborn with the milder form of VLCADD, who was identiWed by MS/MS screening, had remained asymptomatic until diagnosis at three years of age. A high-MCT formula was initially prescribed to the Wrst three patients diagnosed, with carnitine supplementation at 100 mg/kg/day. However, this practice was changed to free breastfeeding, monitoring carnitine levels and supplementing as required, and a ‘sick-day’ regime similar to that used in MCAD deWciency. Older children are on a low fat diet. There have not been any episodes of metabolic decompensation noted under this regime, even when babies have been unwell. Growth and development are normal in all patients. We conclude that VLCADD can be reliably indicated by examination of an early blood sample but may be missed if the child is asymptomatic and older than four days. It is important to perform mutation analysis to obtain a correct diagnosis. The Wnding of potentially disease causing mutations in the newborns identiWed in the present study indicates that these newborns may be at risk of disease manifestation, but it is not possible at present to deWne how severe this risk is. Nevertheless, it should be stressed that even individuals with mild VLCADD may be at risk of disease manifestation, and that identiWcation of these individuals by screening before the disease has manifested clinically is important, so that preventive measures can be instituted. Regardless of the potential severity of the mutation, it is important to monitor all these patients and be proactive in management of intercurrent illnesses, as well as to detect possible future clinical manifestations as early as possible. Acknowledgment This work was supported by grants from the March of Dimes Foundation (Grant No.1-FY-2003-688). References [1] K. Izai, Y. Uchida, T. Orii, S. Yamamoto, T. Hashimoto, Novel fatty acid beta-oxidation enzymes in rat liver mitochondria. I. PuriWcation and properties of very-long-chain acyl-coenzyme A dehydrogenase, J. Biol. Chem. 267 (1992) 1027–1033. [2] C. Vianey-Saban, P. Divry, M. Brivet, M. Nada, M.T. Zabot, M. Mathieu, C. Roe, Mitochondrial very-long-chain acyl-coenzyme A dehydrogenase deWciency: clinical characteristics and diagnostic considerations in 30 patients, Clin. Chim. Acta 269 (1998) 43–62. [3] B.S. Andresen, S. Olpin, B.J. Poorthuis, H.R. Scholte, C. VianeySaban, R. Wanders, L. Ijlst, A. Morris, M. Pourfarzam, K. Bartlett, E.R. Baumgartner, J.B. deKlerk, L.D. Schroeder, T.J. Corydon, H. Lund, V. Winter, P. Bross, L. Bolund, N. Gregersen, Clear correlation of genotype with disease phenotype in very-long-chain acylCoA dehydrogenase deWciency, Am. J. Hum. Genet. 64 (1999) 479–494. [4] A. Mathur, H.F. Sims, D. Gopalakrishnan, B. Gibson, P. Rinaldo, J. Vockley, G. Hug, A.W. Strauss, Molecular heterogeneity in very-long-chain acyl-CoA dehydrogenase deWciency causing pediatric cardiomyopathy and sudden death, Circulation 99 (1999) 1337–1343.
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[5] U. Spiekerkoetter, B. Sun, T. Zytkovicz, R. Wanders, A.W. Strauss, U. Wendel, MS/MS-based newborn and family screening detects asymptomatic patients with very-long-chain acyl-CoA dehydrogenase deWciency, J. Pediatr. 143 (2003) 335–342. [6] U. Spiekerkoetter, C. Tokunaga, U. Wendel, E. Mayatepek, L. Ijlst, F.M. Vaz, N. van Vlies, H. Overmars, M. Duran, F.A. Wijburg, R.J. Wanders, A.W. Strauss, Tissue carnitine homeostasis in very-longchain acyl-CoA dehydrogenase-deWcient mice, Pediatr. Res. 57 (2005) 760–764, Epub 2005 Mar 2017. [7] B. Wilcken, V. Wiley, J. Hammond, K. Carpenter, Screening newborns for inborn errors of metabolism by tandem mass spectrometry, N. Engl. J. Med. 348 (2003) 2304–2312. [8] M.S. Rashed, P.T. Ozand, M.P. Bucknall, D. Little, Diagnosis of inborn errors of metabolism from blood spots by acylcarnitines and amino acids proWling using automated electrospray-tandem mass spectrometry, Pediatr. Res. 38 (1995) 324–331. [9] M.S. Rashed, M.P. Bucknall, D. Little, A. Awad, M. Jacob, M. Alamoudi, M. Alwattar, P.T. Ozand, Screening blood spots for inborn errors of metabolism by electrospray-tandem mass spectrometry with a microplate batch process and a computer algorithm for automated Xagging of abnormal proWles, Clin. Chem. 43 (1997) 1129–1141. [10] C.T. Cavedon, P. Bourdoux, K. Mertens, H.V. Van Thi, N. Herremans, C. de Laet, P. Goyens, Age-related variations in acylcarnitine and free carnitine concentrations measured by tandem mass spectrometry, Clin. Chem. 51 (2005) 745–752, Epub 2005 Feb 2011.
[11] M.F. Browning, C. Larson, A. Strauss, D.L. Marsden, Normal acylcarnitine levels during conWrmation of abnormal newborn screening in long-chain fatty acid oxidation defects, J. Inherit. Metab. Dis. 28 (2005) 545–550. [12] T.H. Zytkovicz, E.F. Fitzgerald, D. Marsden, C.A. Larson, V.E. Shih, D.M. Johnson, A.W. Strauss, A.M. Comeau, R.B. Eaton, G.F. Grady, Tandem mass spectrometric analysis for amino, organic, and fatty acid disorders in newborn dried blood spots: a two-year summary from the New England Newborn Screening Program, Clin. Chem. 47 (2001) 1945–1955. [13] B.S. Andresen, P. Bross, C. Vianey-Saban, P. Divry, M.T. Zabot, C.R. Roe, M.A. Nada, A. Byskov, T.A. Kruse, S. Neve, K. Kristiansen, I. Knudsen, M.J. Corydon, N. Gregersen, Cloning and characterization of human very-long-chain acyl-CoA dehydrogenase cDNA, chromosomal assignment of the gene, and identiWcation in four patients of nine diVerent mutations within the VLCAD gene, Hum. Mol. Genet. 5 (1996) 461–472. [14] R. Pons, P. Cavadini, S. Baratta, F. Invernizzi, E. Lamantea, B. Garavaglia, F. Taroni, Clinical and molecular heterogeneity in very-longchain acyl-coenzyme A dehydrogenase deWciency, Pediatr. Neurol. 22 (2000) 98–105. [15] T. Fukao, H. Watanabe, K. Orii, Y. Takahashi, A. Hirano, T. Kondo, S. Yamaguchi, T. Aoyama, N. Kondo, Myopathic form of very-longchain acyl-CoA dehydrogenase deWciency: evidence for temperaturesensitive mild mutations in both mutant alleles in a Japanese girl, Pediatr. Res. 49 (2001) 227–231.
VLCAD deWciency: Pitfalls in newborn screening and conWrmation of diagnosis by mutation analysis A. Boneh a,d,¤, B.S. Andresen b,c, N. Gregersen b, M. Ibrahim a, N. Tzanakos a, H. Peters a,d, J. Yaplito-Lee a, J.J. Pitt a a
Metabolic Service and Newborn Screening Laboratory, Genetic Health Services Victoria, Melbourne, Australia b Research Unit for Molecular Medicine, Skejby Sygehus, Aarhus University, Aarhus, Denmark c Institute of Human Genetics, Aarhus University, Aarhus, Denmark d Department of Paediatrics, University of Melbourne, Australia Received 9 November 2005; received in revised form 21 December 2005; accepted 21 December 2005 Available online 20 February 2006
Abstract We diagnosed six newborn babies with very long-chain acyl-CoA dehydrogenase deWciency (VLCADD) through newborn screening in three years in Victoria (prevalence rate: 1:31,500). We identiWed seven known and two new mutations in our patients (2/6 homozygotes; 4/6 compound heterozygotes). Blood samples taken at age 48–72 h were diagnostic whereas repeat samples at an older age were normal in 4/6 babies. Urine analysis was normal in 5/5. We conclude that the timing of blood sampling for newborn screening is important and that it is important to perform mutation analysis to avoid false-negative diagnoses of VLCADD in asymptomatic newborn babies. In view of the emerging genotype–phenotype correlation in this disorder, the information derived from mutational analysis can be helpful in designing the appropriate follow-up and therapeutic regime for these patients. © 2005 Elsevier Inc. All rights reserved. Keywords: Fatty acid oxidation; Very long-chain fatty acids; VLCAD deWciency (VLCADD); Newborn screening; Mutation; Tandem mass spectrometry
Introduction Very long-chain acyl-CoA dehydrogenase deWciency (VLCADD, MIM 201475) is an autosomal recessive disorder of fatty acid oxidation. VLCAD (EC 1.3.99.13), which was identiWed in 1992 [1], is one of a family of acylCoA dehydrogenases that catalyse the dehydrogenation of straight-chain and branched-chain acyl-CoA esters. VLCAD is the Wrst enzyme in the spiral of oxidation of C10–C18 or longer straight-chain fatty acids. Essentially all patients who were historically thought to have longchain fatty acid dehydrogenase (LCAD) deWciency were found to actually have VLCADD. Patients diagnosed clinically can be grouped into three main groups [2,3].
*
Corresponding author. Fax: +61 3 8341 6390. E-mail address: [email protected] (A. Boneh).
1096-7192/$ - see front matter © 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.ymgme.2005.12.012
Patients may present early (possibly within days after birth) with a severe form, usually with cardiomyopathy. In a large series of babies diagnosed clinically, cardiomyopathy was the most common presenting symptom, leading to sudden death in some babies [4]. In the second group are patients who present later in childhood with less severe metabolic disturbances, yet with episodes of hypo-ketotic hypoglycaemia, and with no cardiomyopathy. The third group includes those who present after childhood with an exclusive muscular form and episodes of rhabdomyolysis. The exact prevalence of VLCADD is not known. Based on recent results of newborn screening programs the prevalence could vary between 1:42,500 and 1:125,000 births [5–7]. Herein, we describe our observations regarding the importance of the timing of newborn screening for this disorder, describe novel mutations, and summarise our experience in the management of VLCADD.
A. Boneh et al. / Molecular Genetics and Metabolism 88 (2006) 166–170
Methods Acylcarnitines in dried blood spot samples were measured using Xowinjection electrospray-tandem mass spectrometry. Newborn screening samples were normally collected at 48–72 h of age and extracted and butylated as previously described [8,9] and analysed using a Waters Quattro LC triple quadrupole mass spectrometer interfaced to a Gilson 215 autosampler and Agilent 1100 pumping system. Multiple reaction monitoring was used to measure the responses of various acylcarnitines and stable isotope internal standards. A standard of C14:1 carnitine was unavailable and the concentration of this metabolite was calculated by assuming the same response as C14 carnitine. Data were also expressed as standard deviation indices (SDI, deWned as the number of standard deviations the value deviates from the mean) relative to control infants. Mean and standard deviations for controls were calculated from log-transformed data for each combination of age (7 ranges), weight (6 ranges), and gestation (9 ranges). PCR ampliWcation of all exons, including part of the Xanking intron sequences, of the human VLCAD gene, was carried out as previously described [3] using a GeneAmp(r) PCR system 9700 (Applied Biosystems). Sequence analysis with M13 forward and reverse primers was performed using the BigDye(r) Terminator v1.1 Cycle Sequencing kit (Applied Biosystems) and an 3100-Avant genetic analyzer (Applied Biosystems). Sequence data were analysed with the Sequencer v3.1.1 software (Gene Codes Corporation).
Results Tandem mass spectrometry Six VLCADD subjects were detected by screening 189,000 infants between February 2002 and April 2005 (yielding a prevalence of 1:31,500 with a 95% conWdence interval of 1:15,300 to 1:90,900). A sibling (D1), born prior to the introduction of TMS newborn screening, was also diagnosed through family screening.
167
The results of acyl carnitine analyses in dried blood spot samples are illustrated in Fig. 1. All VLCADD subjects had signiWcantly increased levels of C14:1, C14, and C14:1/C10 ratio in the initial newborn screening sample. C14:1 levels in mol/L were higher than C14 levels in the Wrst 72 h (Figs. 1 A and B). The C14:1/C10 ratio was also diagnostic, extended up to 100 h after birth, and appeared to be somewhat more reliable in the second sample than the C14:1 value alone (e.g., babies C and F, Fig. 1C). C14, C14:1, and C14:1/C10 values generally declined in the second sample taken after 130 h (Fig. 1), consistent with previous observations that long-chain acylcarnitines such as C14 and C14:1 decrease signiWcantly in the immediate postnatal period in normal babies [10–12]. Thus, interpretation of these repeat samples requires age-matched reference ranges. We also used the standard deviation index (SDI) of C14:1, which takes into consideration age, weight, and gestation and hence is expected to have more diagnostic ability regardless of the age of sampling, as a convenient way to compensate for these changes. In general, SDIs greater than three are considered abnormal. As shown in Fig. 1D,14:1 level was still abnormal in most of the repeat samples when analysed in this fashion (generally >4). An exception to the Wnding of decreased C14:1 level in a repeat sample was patient B who had a higher level in the second sample, presumably because he was symptomatic at the time (with hypoglycaemia and cardiovascular collapse without cardiomyopathy on day four of life). All other patients have remained free of symptoms with normal urine organic acid and subsequent dried blood spot acylcarnitine proWles up to the age of six months. In particular,
Fig. 1. Dried blood spot metabolite levels in VLCADD patients as a function of age in hours. C14:1 and C14 values are in mol/L, shaded boxes represent neonatal reference ranges in two age ranges. (Values for D1 are approximate because sample was stored for four years.) (A) C14:1, (B) C14, (C) C14:1/C10 ratio, (D) C14:1 as the standard deviation index (SDI).
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secondary indicators of abnormal fatty acid oxidation such as urine dicarboxylic and 3-hydroxydicarboxylic acids were within normal limits. Mutation analysis To conWrm the diagnosis of VLCADD, we sequenced all exons comprising the entire coding region of the VLCAD gene from the identiWed newborns. In all babies, mutations were identiWed in both alleles of the gene. Two patients were homozygous. Baby A was homozygous for the most frequently found mutation in the VLCAD gene, namely c.848T>C, causing a change from valine to alanine at position 243 of the mature protein. Both parents were found to be heterozygous for this mutation. The c.848T>C mutation has been reported to be particularly frequent in newborns identiWed by tandem mass spectrometry-based screening [5]. It was initially identiWed as one of the mutations in compound heterozygous symptomatic patients [3,4,13]. However, two unrelated patients, who are homozygous for this mutation have been previously diagnosed (Andresen BS— Unpublished data). One of these patients presented with hypoglycemia at 48 h of age and experienced an episode of abnormal movements of the left hand and leg. After this he remained sleepy for several days. He has grown and developed normally, but started experiencing increasing troubles with rhabdomyolysis from the age of six years, indicating that the c.848T>C mutation may be disease causing also when present in homozygous form. Baby C was homozygous for a previously unreported mutation, c.1497-1499delCCT, which results in deletion of leucine at position 460 of the mature VLCAD protein. Both parents were found to be heterozygous for this mutation. The same genotype has been previously identiWed in a patient who was referred for mutation analysis because he had presented clinically with disease (Andresen BS— Unpublished data), indicating that this genotype may be disease causing. Baby B was compound-heterozygous for a c.1097G>A mutation (R326H) and for a c.1322G>A (G401D) mutation. His father was heterozygous for the c.1097G>A mutation and his mother was heterozygous for the c.1322G>A mutation. Both mutations have been reported in patients with clinical manifestation of VLCADD [3,4,13]. Baby D2 was compound-heterozygous for the c.848T>C mutation and a splice site mutation in intron 2 (c.7532A>C). The splice site mutation has been reported in several clinically aVected patients [3,4,13] and was found in four Italian patients [14]. Recently, another patient who has the same genotype has been identiWed (Andresen BS— Unpublished data). This patient presented with hypoglycaemia in association with a respiratory viral infection at 17 months of age and with seizures 10 months later. Thus, it seems that patients with this particular genotype are at risk of developing disease. Baby E was found to be compound-heterozygous for the c.1349G>A (R410H) mutation, which has been reported in
patients with clinically manifest VLCADD [3,15] and the novel mutation c.481G>A. His mother was heterozygous for the c.1349G>A mutation and his father was heterozygous for the c.481G>A mutation. The c.481G>A mutation causes a shift from alanine at position 121 of the mature VLCAD protein to a threonine (A121T). Alanine at this position is conserved in rat, bovine, mouse, and pig VLCAD. Human short-chain acyl-CoA dehydrogenase (SCAD) and human long-chain acyl-CoA dehydrogenase (LCAD) both have alanine at the homologous position, underscoring the importance of an alanine residue at this position for correct enzyme function. This mutation was predicted to be “benign” with a PSIC score diVerence of 1.079 by the PolyPhen prediction program (http:// tux.embl-heidelberg.de/ramensky/polyphen.cgi). Baby F was found to be compound-heterozygous for the c.1153C>T (R345W) mutation and the novel mutation, c.1117A>T, which causes a shift from isoleucine at position 333 of the mature VLCAD protein to a phenylalanine (I333F). The c.1153C>T mutation has been previously observed in a patient who presented with the muscular form (Muscular weakness, rhabdomyolysis, and elevated CK) of the disease at 19 years of age (Andresen BS— Unpublished data), indicating its disease causing potential. The c.1117A>T mutation is located on exon 11 and was not found in 100 control chromosomes [3], thus excluding the possibility of a common polymorphism. Isoleucine at this position is conserved in rat, bovine, mouse, and pig VLCAD. Human isovaleryl-CoA dehydrogenase also has isoleucine at the homologous position, whereas human SCAD, human medium-chain acyl-CoA dehydrogenase, human short/branched-chain acyl-CoA dehydrogenase, and human glutaryl-CoA dehydrogenase have a conservative substitution to leucine. However, this mutation was predicted to be “benign” with a PSIC score diVerence of 1.414 by the PolyPhen prediction program. It is therefore diYcult to estimate the disease causing potential of this mutation. Discussion In the period of February 2002 to April 2005, we detected six newborn babies with biochemical features of VLCADD through the newborn screening program in Victoria, using tandem mass spectrometry. There were no other clinically diagnosed patients with this disorder in the previous years, but we diagnosed a 4.5-year-old brother of one baby following cascade screening. Despite the relatively high rate of normal acylcarnitine proWles when babies were over four days of age, we pursued mutation analysis for all babies who were suspected of having VLCADD based on their Wrst newborn screening sample, taken at 48–72 h. A total of nine mutations were found in our six patients, which is in agreement with the reported large number of mutations found in previous studies [3,4]. Four of the newborns (A, B, C, and D2) had mutations that have previously been identiWed in symptomatic patients. Thus, our observation that
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acylcarnitine proWle in the repeat samples was normal illustrates that newborns with genotypes that have previously been found in individuals with clinical manifestation of VLCADD may be missed by newborn screening if the second sample is drawn after day four of life. The two remaining newborns each had one known disease-associated mutation and one novel mutation. The possibility that these two newborn babies are carriers of rare polymorphisms is unlikely since C14:1 levels in their initial samples were similar to subjects with disease causing mutation on both alleles. Our results indicate a prevalence of 1:31,500 newborn babies, exceeding not only the reported prevalence as suggested by case-series of patients diagnosed clinically but also that based on current newborn screening reports [5–7]. Several reasons may be hypothesised for the higher prevalence of VLCADD in our population. DiVerent laboratories may deWne diVerent cut-oV values for acylcarnitines and guidelines and procedures for the conWrmation of diagnoses. In many instances further diagnostic tests are done if abnormal Wndings in the original blood spots are conWrmed in a repeat sample. We show that reliance on a repeat dried blood spot sample to conWrm the initial Wndings suggestive of VLCADD, when the baby is older than four days, may lead to a false-negative diagnosis (as mentioned above, we did not have false-positive diagnoses of VLCADD). Our results are in line with a recent report on the normalisation of the acylcarnitine proWle in conWrmatory plasma samples following newborn screening in one patient with trifunctional enzyme deWciency and one patient with VLCADD [11]. Indeed, the estimated VLCADD prevalence of 1:250,000 in one study was based on repeat tandem mass spectrometry analysis of blood spots at days 8–10 of life [5]. It is likely that newborn screening detects individuals with the milder forms of VLCADD (as in the case of medium-chain acyl-CoA dehydrogenase deWciency) and that this may explain some of the discrepancy between the prevalence suggested on the basis of MS/MS screening and that based on the number of clinical presentations in previous years. Clear correlation between genotype and phenotype has been demonstrated in VLCADD [3]. Individuals with two null mutations (like stop-, frameshift-, and severesplicing mutations) usually have a severe and early presentation, whereas individuals with at least one missense mutation (or deletion of an amino acid) have a milder and later presentation. Individuals who have their initial presentation in adulthood (with the muscular form of the disease) usually have missense mutations. This suggests that there is a correlation between the residual enzyme activity and the severity of disease. All newborns identiWed in the present study have at least one mutation that may lead to residual enzyme activity. In fact both the V243A and R410H mutations have been shown to result in residual enzyme activity by overexpression of recombinant protein in COS cells [3,15]. Some individuals with the milder form of VLCADD may have had unrecognized episodes and some may remain free of symptoms until adulthood or throughout life unless
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challenged by a metabolic stress. In line with this, Spiekerkoetter et al. [5] reported that an aVected older sibling of a newborn with the milder form of VLCADD, who was identiWed by MS/MS screening, had remained asymptomatic until diagnosis at three years of age. A high-MCT formula was initially prescribed to the Wrst three patients diagnosed, with carnitine supplementation at 100 mg/kg/day. However, this practice was changed to free breastfeeding, monitoring carnitine levels and supplementing as required, and a ‘sick-day’ regime similar to that used in MCAD deWciency. Older children are on a low fat diet. There have not been any episodes of metabolic decompensation noted under this regime, even when babies have been unwell. Growth and development are normal in all patients. We conclude that VLCADD can be reliably indicated by examination of an early blood sample but may be missed if the child is asymptomatic and older than four days. It is important to perform mutation analysis to obtain a correct diagnosis. The Wnding of potentially disease causing mutations in the newborns identiWed in the present study indicates that these newborns may be at risk of disease manifestation, but it is not possible at present to deWne how severe this risk is. Nevertheless, it should be stressed that even individuals with mild VLCADD may be at risk of disease manifestation, and that identiWcation of these individuals by screening before the disease has manifested clinically is important, so that preventive measures can be instituted. Regardless of the potential severity of the mutation, it is important to monitor all these patients and be proactive in management of intercurrent illnesses, as well as to detect possible future clinical manifestations as early as possible. Acknowledgment This work was supported by grants from the March of Dimes Foundation (Grant No.1-FY-2003-688). References [1] K. Izai, Y. Uchida, T. Orii, S. Yamamoto, T. Hashimoto, Novel fatty acid beta-oxidation enzymes in rat liver mitochondria. I. PuriWcation and properties of very-long-chain acyl-coenzyme A dehydrogenase, J. Biol. Chem. 267 (1992) 1027–1033. [2] C. Vianey-Saban, P. Divry, M. Brivet, M. Nada, M.T. Zabot, M. Mathieu, C. Roe, Mitochondrial very-long-chain acyl-coenzyme A dehydrogenase deWciency: clinical characteristics and diagnostic considerations in 30 patients, Clin. Chim. Acta 269 (1998) 43–62. [3] B.S. Andresen, S. Olpin, B.J. Poorthuis, H.R. Scholte, C. VianeySaban, R. Wanders, L. Ijlst, A. Morris, M. Pourfarzam, K. Bartlett, E.R. Baumgartner, J.B. deKlerk, L.D. Schroeder, T.J. Corydon, H. Lund, V. Winter, P. Bross, L. Bolund, N. Gregersen, Clear correlation of genotype with disease phenotype in very-long-chain acylCoA dehydrogenase deWciency, Am. J. Hum. Genet. 64 (1999) 479–494. [4] A. Mathur, H.F. Sims, D. Gopalakrishnan, B. Gibson, P. Rinaldo, J. Vockley, G. Hug, A.W. Strauss, Molecular heterogeneity in very-long-chain acyl-CoA dehydrogenase deWciency causing pediatric cardiomyopathy and sudden death, Circulation 99 (1999) 1337–1343.
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