Brown-Vialetto-Van Laere and Fazio Londe syndrome is associated with a riboflavin transporter defect mimicking mild MADD: a new inborn error of metabolism with potential treatment

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Brown-Vialetto-Van Laere and Fazio Londe syndrome is associated with a riboflavin transporter defect mimicking mild MADD: a new inborn error of metabolism with potential treatment
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  RAPID COMMUNICATION Brown-Vialetto-Van Laere and Fazio Londe syndromeis associated with a riboflavin transporter defect mimickingmild MADD: a new inborn error of metabolismwith potential treatment Annet M. Bosch  &  Nico G. G. M. Abeling  &  Lodewijk IJlst  &  Hennie Knoester  & W. Ludo van der Pol  &  Alida E. M. Stroomer  &  Ronald J. Wanders  &  Gepke Visser  & Frits A. Wijburg  &  Marinus Duran  &  Hans R. Waterham Received: 13 September 2010 /Revised: 20 October 2010 /Accepted: 26 October 2010 /Published online: 26 November 2010 # The Author(s) 2010. This article is published with open access at Abstract  We report on three patients (two siblings and oneunrelated) presenting in infancy with progressive muscleweakness and paralysis of the diaphragm. Metabolic studiesrevealed a profile of plasma acylcarnitines and urine organicacids suggestive of a mild form of the multiple acyl-CoAdehydrogenation defect (MADD, ethylmalonic/adipic acidsyndrome). Subsequently, a profound flavin deficiency inspite of a normal dietary riboflavin intake was established inthe plasma of all three children, suggesting a riboflavintransporter defect. Genetic analysis of these patients demon-strated mutations in the C20orf54 gene which encodes thehuman homolog of a rat riboflavin transporter. This gene wasrecently implicated in the Brown-Vialetto-Van Laere syn-drome, a rare neurological disorder which may either present in infancy with neurological deterioration with hypotonia,respiratory insufficiency and early death, or later in life withdeafness and progressive ponto-bulbar palsy. Supplementa-tion of riboflavin rapidly improved the clinical symptoms aswell as the biochemical abnormalities in our patients,demonstrating that high dose riboflavin is a potentialtreatment for the Brown-Vialetto-Van Laere syndrome aswell as for the Fazio Londe syndrome which is considered to be the same disease entity without the deafness. Introduction Brown-Vialetto-van Laere syndrome (MIM 211530), is arare neurological disorder which may present in infancy witha devastating neurological deterioration with hypotonia,respiratory insufficiency and early death, or later in life withdeafness and progressive ponto-bulbar palsy (Green et al.2010; Sathasivam 2008). The same clinical presentation without deafness is also known as Fazio Londe disease(MIM 211500). Both are now considered to be a singledisease entity (Dipti et al. 2005).The multiple acyl-CoAdehydrogenation defect (MADD) isan inherited defect of mitochondrial fatty acid beta-oxidation Communicated by: K. Michael GibsonMarinus Duran and Hans R. Waterham contributed equallyCompeting interests: None declared.A. M. Bosch : H. Knoester  :  F. A. WijburgDepartment of Pediatrics, Academic Medical Center,University of Amsterdam,Amsterdam, The Netherlands N. G. G. M. Abeling : L. IJlst  : A. E. M. Stroomer  : R. J. Wanders : M. Duran : H. R. WaterhamLaboratory Genetic Metabolic Diseases,Academic Medical Center, University of Amsterdam,Amsterdam, The NetherlandsW. L. van der PolRudolf Magnus Institute of Neuroscience, Department of Neurologyand Pediatric Neuromuscular Center   ‘ Spieren Voor Spieren ’ ,University Medical Center Utrecht,Utrecht, The NetherlandsG. Visser Department of Metabolic Diseases,Wilhelmina Children ’ s Hospital,Utrecht, The NetherlandsA. M. Bosch ( * )Department of Pediatrics, Academic Medical Center (H7 270),University Hospital of Amsterdam,Meibergdreef 9 1105 AZ,Amsterdam, The Netherlandse-mail: Inherit Metab Dis (2011) 34:159  –  164DOI 10.1007/s10545-010-9242-z  and branched-chain amino acid catabolism (MIM231680).Thisdisorderischaracterizedbythemalfunctioningofanarrayof dehydrogenation reactions caused by the absence of either electron transfer flavoprotein (ETF) or electron transfer flavoprotein oxidoreductase (ETFDH). The electrons whichare liberated in the dehydrogenation reactions are primarilycaptured byflavin adeninedinucleotide(FAD),a metaboliteof thevitaminriboflavin.MADDmaypresentatanyage,varyingfroma lethal neonatal form andan infantile form characterized by hazardous hypoketotic hypoglycaemia (Rhead et al. 1987) to relatively mild adult lipid storage myopathy (Antozzi et al.1994). Riboflavin responsive forms of MADD are usually theconsequence of a defective ETFDH (Olsen et al. 2007). We present two siblings and one unrelated patient, presenting in infancy with progressive muscle weaknessand paralysis of the diaphragm, later recognized as FazioLonde and Brown-Vialetto-van Laere syndrome. Our  patients demonstrated abnormalities on metabolic evalua-tion mimicking mild MADD and were found to be severelyflavin deficient. Riboflavin therapy resulted in a rapidclinical and biochemical improvement. Patients and methods Patient 1A6montholdboy,the firstchildofhealthyparents,presentedwith a short history of progressive muscle weakness followed by life threatening apnoeic spells necessitating artificialventilation. Neurological examination revealed generalizedmuscle weakness with a severe head lag. A completediaphragmatic paralysis was detected on ultrasound. Spinalmuscular atrophy (SMA) and SMA with respiratory distress(SMARD) were excluded by genetic testing. Muscle histol-ogy at the age of 7 months demonstrated unclassifiedmyopathic abnormalities. Biochemical analysis of the mito-chondrial respiratory chain in fresh muscle tissue gaveinconclusive results. Metabolic studies revealed abnormalconcentrations of short- and medium-chain plasma acylcarni-tines and a urine organic acid profile was suggestive of MADD (Tables 1 and 2). Because of the possibility of  riboflavin-responsiveness (Olsen et al. 2007), the patient was treated with high dose oral riboflavin (vitamin B2, 10 mg/kg body weight/day). The MADD associated metabolic abnor-malities disappeared within days. Cessation of riboflavinsupplementation resulted in a recurrence of the abnormalmetabolic profile in spite of a normal dietary riboflavinintake, and riboflavin medication was restarted. ClassicalMADD was ruled out by sequence analysis of the  ETFDH  ,  ETFA  and  ETFB  genes. The biochemical profile, absence of urine thiosulfate and sulfo(thio)cysteine (Duran et al. 1997)argued against an ethylmalonic syndrome (ETHE) (Tiranti et al. 2009). Therefore we hypothesized a decreased availabilityof flavin adenine dinucleotide (FAD), the cofactor of MADD, due to defective uptake, synthesis or transport, to be the underlying cause of the disease.A tracheotomy was placed and the boy ’ s muscle toneslowly improved over the next month. He was able to walk independently at the age of 22 months. As the diaphrag-matic paralysis persisted, he needed nightly ventilation untilthe age of 41 months. Recently, the clinical diagnosis of Fazio Londe Syndrome was made. He is currently46 months old, his cognitive development is fully normaland he demonstrates no further cranial nerve palsy.Patient 2Recently, the 3 months-old sister of patient 1 presented withfailure to thrive and generalized axial muscle weakness.Based on the results of the additional studies in her brother,flavin deficiency was suspected, subsequently confirmed by Table 1  Plasma acylcarnitines ( μ  mol/L) in patient, 2, and 3 compared to a MADD patient, a patient with the ethylmalonic acid syndrome, andhealthy controls. Bold values are outside normal rangePat 1 before riboflavin Pat 2 before riboflavin Pat 3 at start of treatment MADD ETHE ControlsC0  18.3  35.1 37.6  4.2  35.4 22  –  55C2  3.2  4.0  0.74 1.9  5.1 3.4  –  13C4  0.65 2.97  0.43  8.55 2.07  0.07  –  0.58C5  0.29 1.99 0.91 0.31 0.32  0.04  –  0.22C6  0.27 0.87  0.02  0.14  0.05 0.02  –  0.12C8 0.18  0.70 0.53 0.25  0.04 0.04  –  0.22C10 0.25  0.86 0.77 0.49  0.06 0.04  –  0.30C12  0.15 0.44 0.64 0.49  0.05 0.04-0.12C14  0.14 0.23 0.46 0.39  <0.08C16 0.19 0.19  0.32 0.37  0.09 0.06  –  0.24C18:1 0.16 0.13  0.42 0.53  0.16 0.06  –  0.28160 J Inherit Metab Dis (2011) 34:159  –  164  the analysis of plasma flavins and acylcarnitines, andtreatment with riboflavin (10 mg/kg body weight /day) wasstarted. This resulted in normalization of her muscle tonewithin 7 days and a rapid catch-up growth. Sensorineuralhearing loss was excluded by brainstem evoked responseaudiometry. Therefore, the clinical diagnosis of Fazio LondeSyndrome was made. After 3 months of riboflavin supple-mentation her growth and development are normal.Patient 3A now 7 year old girl, second child of healthy parents,demonstrated feeding problems and a slow motor develop-ment since birth. At the age of 5 months she had generalizedweakness, more pronounced in distal than proximal musclegroups, and developed respiratory insufficiency necessitatingartificial ventilation due to diaphragmatic paralysis. Further-more, she was found to have a severe sensorineural hearingloss. SMA and SMARD were excluded by genetic testing.Muscle histology at the age of one year showed slight  predominance of type 1 fibers without type grouping.Biochemical analysis of the mitochondrial respiratory chainin muscle tissue was inconclusive.At the age of 6 months, metabolic studies revealed anacylcarnitine profile suggestive of MADD (Table 1). A fat restricted diet and supplementation with carnitine (50 mg/ kg / day), riboflavin (10 mg/kg /day) and glycine and 3-hydroxybutyrate according to the publication by Van Hoveet al. (2003) were started. Her muscle strength improvedand from the age of 2 years artificial ventilation was onlynecessary during sleep. Cognitive development was normal.Classical MADD was ruled out by enzymatic testing infibroblasts and mutation analysis of the ETFDH, ETFA andETFB genes. Following the normalization of the acylcarnitine profiles, the fat restriction was gradually discontinued and theglycine, 3-hydroxybutyrate and carnitine supplementationswere stopped without problems. However, withdrawal of riboflavin at the age of 4 years resulted in a rapid clinicaldeterioration with vomiting, progressive fatigue, and eleva-tions of lactate, liver enzymes and CK. The acylcarnitine profile became abnormal again. Reintroduction of riboflavin(50 mg b.i.d.) resulted in clinical improvement and normal-izationofthebiochemicalabnormalities.Sequenceanalysisof the mitochondrial FAD transporter  (Spaan et al. 2005) was  performed on suspicion of a possible mitochondrial ribofla-vin transporter defect, but showed no abnormalities.After reintroduction of riboflavin supplementation the patient had a stable clinical course. She walked with a foot drop and attended a school for hearing impaired children. The parents gradually decreased the dose of riboflavin to 10 mg b.i.d. because they suspected gastrointestinal side effects of thehighdosesofriboflavin.However,after5yearsshedeveloped palsies of the 7th and 12 th cranial nerves, and becamewheelchair bound. Furthermore, following a lower respira-tory tract infection at the age of 6.5 years she became onceagain completely ventilator dependent, and her musclestrength slowly deteriorated. Only recently, the clinicaldiagnosis of Brown-Violetto-Van Laere syndrome wasconfirmed. The riboflavin dose was increased to 50 mgt.i.d.,thus far without improvement of her symptoms.MethodsSelective screening for inborn errors of metabolism was performedbystandardtechniquesincludinganalysisoforganicacids in urine and acylcarnitines in plasma. Plasma riboflavin,flavin mononucleotide (FMN) and FAD concentrations weremeasured by high-performance liquid chromatography usingfluorescence detection of the analytes according to themethod of Capo-chichi et al. (2000) with minor modifications.Because plasma flavins were deficient in all three patientsdespite a normal intake, we suspected a defect in intestinalriboflavin uptake and we therefore performed mutationanalysis of the  C20orf54  gene. The protein encoded by thisgene is highly similar to the rat rRFT2 protein, which has been shown to be the transporter of riboflavin in the smallintestine (Yamamoto et al. 2009). Mutation analysis of thegene was performed by sequencing all exons plus flankingintronic sequences amplified from genomic DNA extractedfrom the patients ’  cells. Confirmation of the mutation in the parental genes was performed in genomic DNA extractedfrom blood cells.Fibroblast fatty acid oxidation studies were carried out in patients 1 and 3 by incubating the cells with U- 13 C palmitate Pat 1 before riboflavin MADD ETHE ControlsEMA 62 2648 48 <15Glutarate 1 19986 1 <15Adipate 70 5207 2 2  –  262-OH-glutarate 106 832 19 11  –  51Suberate 26 764 1 <15Hexanoylglycine + ++  –  Isovalerylglycine + ++ + Table 2  Urine organic acids(mmol/mol creat) in patient 1compared to a MADD patient, a patient with the ethylmalonicacid syndrome, and healthycontrolsJ Inherit Metab Dis (2011) 34:159  –  164 161  for 96 hours followed by the analysis of acylcarnitines inthe supernatant (Ventura et al. 1999).After the diagnosis had been established, the acylcarnitine profiles of the newborn screening bloodspots of patients 1and 2 were evaluated by standard ESI-tandem massspectrometric analysis. The newborn sample of patient 3was no longer available. Results The initial diagnostic work-up of patient 1 included ananalysis of plasma acylcarnitines and urine organic acids(Tables 1 and 2). A moderate accumulation of short and medium-chain acylcarnitines was observed. The long-chainacylcarnitines were normal. The profiles observed in patients 2 and 3 were similar but differed somewhat in thelevels of the long-chain acylcarnitines, demonstrating the biochemical variability of this defect. The acylcarnitine profiles of the newborn screening bloodspots of patients 1and 2 were normal (not shown), which demonstrates that newborn screening for a riboflavin transporter by thismethod is not feasible.For comparison, plasma of a patient with an ETHE defect was also investigated, demonstrating an accumulation of short-chain acylcarnitines only (Table 1). Finally, plasma of an infantile patient with hypoketotic hypoglycaemic MADDresulting from a homozygous mutation of the ETFDH geneshowed increased levels of all characteristic short-, medium-,and long-chain acylcarnitines as well as glutarylcarnitine.Glutarylcarnitine was normal in both the riboflavin trans- porter patients and the ETHE patient (Table 1). The C4carnitine consisted mainly of the isobutyrylcarnitine, point-ing to a considerable vulnerability of Acad 8 in all con-ditions described here.Organic acid analysis of the urine of patient 1 demon-strated a pattern resembling that of mild MADD (Table 2).Short and branched chain acylglycines were clearlyelevated as was the D-2-hydroxyglutaric acid. The ETHE patient had a comparable excretion pattern, althoughhexanoyl-glycine appeared to be normal. Patient 2 and 3were not investigated. Neither the riboflavin transporter  patient nor the ETHE patient had an increased urine glutaricacid, in contrast to the ETFDH patient (Table 3).The fibroblast fatty acid oxidation studies in patients 1and 3 revealed a normal profile of acylcarnitines, in sharpcontrast to the results obtained in the ETFDH patient,showing accumulation of all C4-C16 intermediates.Concentrations of riboflavin, FMN and FAD in plasma before treatment revealed a deficiency of all flavins in patients 1 and 2 (Table 3) whereas patient 3 had markedlydecreased levels of FMN and FAD. Riboflavin levelsnormalized within weeks after the start of riboflavinsupplementation. Cessation of supplementation in patients1 and 3 resulted in rapid recurrence of the deficient state.Plasmaflavinsof the mother of patients 1and2were normal.Patient 1 and 2 were found to be homozygous for a pathogenicspliceacceptorsitemutationin C20orf54 : c.1198-2A>C which is most probably pathogenic as it concerns amutation of an invariant nucleotide of a splice acceptor siteand thus will lead to incorrect mRNA splicing. The parents,second cousins, were heterozygous for this mutation.Patient 3 was found to be heterozygous for two mutationsin the C20orf54 gene: c.49T>C (p.W17R) and c.639C>G(p.Y213X). The latter mutation was also identified byGreen et al. (2010). The c.49T>C mutation affects atryptophan at position 17, which is highly conserved inorthologs of different species. Discussion Riboflavin is the precursor of FAD, which acts as an electronacceptor in a number of acyl-CoA dehydrogenation reactionsinvolved in mitochondrial fatty acid oxidation and branched-chain amino acid catabolism (Gregersen et al. 2008). An inherited or acquired deficiency of riboflavin will thereforemimic the biochemical presentation observed in classicalMADD, which was indeed observed in a riboflavin-deficient rat (Goodman 1981).Thebiochemicalabnormalitiesobservedin our patients are reminiscent of the mild forms of MADD,srcinally describedas ethylmalonic/adipic aciduria (Tanakaet  Table 3  Plasma flavin values in patients 1, 2,and 3 before and after the start of riboflavin therapyRiboflavin (nmol/l) FMN (nmol/l) FAD (nmol/l)Controls (mean +/- 2 SD) (n=43, ages 0  –  35 y) 3.9  –  49 2.8  –  11.4 46  –  114Patient 1 before treatment 1.4 1.7 31Patient 1 2 weeks after start therapy 18.5 2.1 100Patient 2 before treatment <1 <1 30Patient 2 4 weeks after start treatment 46.0 4.0 70Patient 3 at start of treatment 7.6 <1 24.5Patient 3 with supplementation 12.3 8.3 105162 J Inherit Metab Dis (2011) 34:159  –  164  al. 1977). Our data suggest that the short-chain and medium-chain acyl-CoA dehydrogenases are more vulnerable to theshortage of the physiological electron acceptor than the long-chain analogues. Similarly, glutaryl-CoA dehydrogenase wasonly mildly affected, in contrast to the D-2 hydroxyglutaratedehydrogenase.Based on the profiles of acylcarnitines and organic acidsit is difficult to make the distinction between the riboflavintransporter defect and the ETHE defect. Both conditions,however, have their own diagnostic analytes, i.e., riboflavinin the riboflavin transporter defect (Brown-Vialetto-VanLaere / Fazio Londe syndrome) and thiosulfate and sulfo(thio)cysteine in ETHE (Duran et al. 1997). Metabolicstudies in intact fibroblasts were normal in the transporter defect cells, probably because the riboflavin in the cellculture medium corrected the defect resulting in a normalfatty acid oxidation. Likewise, maternal riboflavin supply isthe probable cause of the normal clinical condition of the patients at birth and the fact that the acylcarnitines werenormal in the newborn blood spots of patients 1 and 2.Riboflavin deficiency usually presents with different clinical symptoms (Powers 2003), but neurological symp- toms resolving with riboflavin supplementation have been previously reported in a child with moderate riboflavindeficiency (Leshner  1981). Although further pathophysio-logical studies are needed to explain the clinical symptoms,we demonstrate that the riboflavin deficiency in our patientsresulted from a defect in the riboflavin transporter, encoded by the  C20orf54  gene.Remarkably, at the same time that we discovered themolecular defects in our patients, causing a defectiveintestinal riboflavin transport, Green et al. (2010) reportedthe identification of mutations in the  C20orf54  gene as thecause of the Brown-Vialetto-Van Laere Syndrome (MIM211530). Indeed, the clinical presentation of all three patients is compatible with the diagnosis of Brown-Vialetto-van Laere (patient 3) or the Fazio Londe (patients1 and 2) syndrome. This implies that at least part of theclinical signs and symptoms observed in these syndromesare caused by a deficiency of riboflavin and subsequentlyof FAD and FMN. The striking clinical and biochemicalimprovement on riboflavin supplementation seen in our  patients strongly supports this hypothesis. We therefore presume that riboflavin may be an effective therapy in theBrown-Vialetto-Van Laere syndrome, at least in young patients. Early treatment appears to be crucial as diaphrag-matic paralysis may be irreversible. Furthermore, in spite of the riboflavin supplementation since the age of 6 months, patient three now, at age 7, demonstrates the neurologicaldeterioration frequently observed in untreated Brown-Vialetto-Van Laere patients. This can be due to damagewhich already occurred during the extended periods of lowFAD values probably present during the first 6 months of life as well as around the age of four, or to periods of limited riboflavin availability during intercurrent illnesses,as she demonstrated clinical deterioration mostly during periods of viral illness. On the other hand, it is possible that riboflavin supplementation only shifts the clinical course of the Brown-Vialetto-Van Laere / Fazio Londe syndrome to alater presentation of the clinical symptoms. A long termfollow up of a cohort of early treated children, and moreinsight in the pathophysiology is warranted.Finally, our results demonstrate that selective metabolicscreening, including acylcarnitine profiling and organicacid analysis of the urine, is warranted in all patients withunexplained hypotonia and that plasma flavins should bemeasured in patients with riboflavin responsive MADD inwhom sequencing of the relevant genes fails to detected amutation. Acknowledgements  The authors wish to thank M. Turkenburg for technical assistance, and F.M. Vaz and R.C.M. Hennekam for valuableinput and discussions. Open Access  This article is distributed under the terms of theCreative Commons Attribution Noncommercial License which per-mits any noncommercial use, distribution, and reproduction in anymedium, provided the srcinal author(s) and source are credited. References Antozzi C, Garavaglia B, Mora M et al. (1994) Late-onset riboflavin-responsive myopathy with combined multiple acyl coenzyme Adehydrogenase and respiratory chain deficiency. Neurology44:2153  –  2158Capo-chichi CD, Guenat JL, Feillet F, Namour F, Vidailhet M (2000)Analysis of riboflavin and riboflavin cofactor levels in plasma byHPLC. J Chrom B 739:219  –  224Dipti S, Childs A, Livingston JH, Aggarwal AK, Miller M, Williams C,Crow YJ (2005) Brown-Vialetto-Van Laere syndrome; variabilityinageatonsetand diseaseprogression highlightingthephenotypicoverlap with Fazio-Londe disease. Brain Dev 27:443  –  446Duran M, Dorland L, Van den Berg IETet al. (1997) The ethylmalonicacid syndrome is associated with deranged sulfur aminoacidmetabolism leading to urinary excretion of thiosulfate andsulfothiocysteine. 8th Int Congress IEM,Vienna, Abstract 048Goodman SI (1981) Organic aciduria in the riboflavin-deficient rat.Am J Clin Nutr 34(11):2434  –  2437Green P, Wiseman M, Crow YJ, et al. (2010) Brown-Vialetto-VanLaere syndrome, a pontobulbar palsy with deafness, is caused bymutations in c20orf54. Am J Hum Genet 12;86(3):485-489Gregersen N, Andresen BS, Pedersen CB, Olsen RK, Corydon TJ,Bross P (2008) Mitochondrial fatty acid oxidation defects  –  remaining challenges. J Inherit Metab Dis 31(5):643  –  657Leshner RT (1981) Riboflavin deficiency-a reversible neurodegenera-tive disease. Ann Neurol 10:294  –  295Olsen RK, Olpin SE, Andresen BS et al (2007) ETFDH mutations as amajor cause of riboflavin-responsive multiple acyl-CoA dehy-drogenation deficiency. Brain 130(8):2045  –  2054Powers HJ (2003) Riboflavin (vitamin B2) and health. Am J Clin Nutr 77:1352  –  1360J Inherit Metab Dis (2011) 34:159  –  164 163
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