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Archives ofDisease in Childhood 1992; 67: 115-119
A biotinidase Km variant causing late onset bilateraloptic
neuropathy
V Th Ramaekers, T M Suormala, M Brab, R Duran, G Heimann, E R
Baumgartner
AbstractA patient with a newly recognised variant ofbiotinidase
deficiency presented with acuteloss ofvision at the age of 10
years. Progressivebilateral optic neuropathy, spastic
paraparesis,and a predominantly motor type neuropathydeveloped over
the next five years. Metabolicinvestigations revealed biotin
depletioncausing multiple carboxylase deficiency. Thebasic defect
was a biotin recycling disorderdue to a mutant biotinidase with
residualactivity of 4-4% assayed routinely. Biocytinexcretion in
urine was only slightly increased.Further investigations on plasma
biotinidaserevealed biphasic kinetics with two differentreduced
values for maximum reaction velocity(Vmax) and two for the
Michaelis constant(Km), one being almost normal and the
otherconsiderably raised. In contrast to this patient,two age
matched children with partial bio-tinidase deficiency (2-8% and
2-9% of normal),but with a normal K. for biocytin,
remainedasymptomatic. After six months of oral substi-tution with
10 mg biotin per day the coeco-central and peripheral scotomata
regressed,the pyramidal signs in the lower limbs dis-appeared, and
further progression of themotor neuropathy arrested.We conclude
that the differential diagnosis
of unexplained bilateral optic neuropathy ofjuvenile onset,
particularly when associatedwith upper and lower motor neuron
disease,should include biotinidase deficiency.
Department ofPaediatrics andOphthalmology,Technical University
ofAachen, GermanyV Th RamaekersM BrabG HeimannMetabolic
Unit,University Children'sHospital, Basel,SwitzerlandT M SuormalaE
R BaumgartnerMetabolic Unit,University Children'sHospital 'het
WilhelminaKinderziekenhuis',Utrecht,The NetherlandsR
DuranCorrespondence to:Dr V Th Ramaekers,Department of
Paediatrics,Technical Universityof Aachen, Pauwelsstrasse 30,D-5100
Aachen,Federal Republic of Germany.Accepted 25 July 1991
Biotinidase deficiency represents an autosomalrecessive inborn
error of biotin metabolismcausing late onset, biotin responsive,
multiplecarboxylase deficiency. 1-3 Biotinidase acts byrecycling
biotin through liberation from bio-cytin (F-aminobiotinyl lysine)
or from shortbiotinyl peptides formed during proteolyticdegradation
of the biotin dependent carboxyl-ases: propionyl-CoA carboxylase
(PCC; EC6.4.1.3), 3-methylcrotonyl-CoA carboxylase(MCC; EC
6.4.1.4), pyruvate carboxylase (PC;EC 6.4.1.1), and acetyl-CoA
carboxylase (ACC;EC 6.4.1.2.). In biotinidase deficiency biocytinis
lost in urine4 leading to progressive biotindepletion and
subsequently to multiple car-boxylase deficiency.3
Biotinidase deficiency in its classical formusually presents
during the first year of life withvariable neurological signs
(hypotonia, seizures,ataxia), dermatitis, alopecia, and
recurrentinfections. Metabolic acidosis and organicaciduria due to
decreased activities of the biotindependent carboxylases are
inconsistently
found. Despite rapid clinical and biochemicalimprovement after
treatment with pharmaco-logical doses of biotin, neurological
damagesuch as sensorineural hearing loss, optic atrophy,ataxia, and
mental retardation has been reportedto persist.3 5 6 Since the
introduction of neonatalscreening a number of children with
partialbiotinidase deficiency have been detected ofwhom most have
remained clinically asympto-matic.7 8 In the present paper a new
variant ofbiotinidase deficiency with delayed onset
ofsymptomatology is described. In addition theeffect of biotin
treatment will be presented.
Case reportThe patient was the single child of healthy
non-consanguineous parents born in Poland after anuneventful
pregnancy and delivery; his birthweight was 3250 g.
Neurodevelopmentalprogress and growth in the first decade
werereported to have been completely normal. Atthe age of 10 years
the patient complained ofacute visual loss during the course of a
'flu'-likeillness. Bilateral optic atrophy was found andvisual
acuity was decreased to 20/100 withsmall bilateral coecocentral
scotomata (see fig 1,upper section). Computed tomography,
electro-encephalography, and routine laboratory in-vestigations
gave normal results. He was treatedempirically with parenteral
vitamin B-12 but hisvision continued to deteriorate. From the age
of14 he complained of fatigue, particularly afterexercise, with
weakness in the hands particularlywhen opening boxes or unscrewing
bottles.Upon referral at the age of 15 years visual
acuity was 20/200 on the right and 20/400 on theleft and a mixed
colour discrimination defect ofred-green and blue-yellow type was
found. Bothoptic discs showed appreciable temporal pallorwith
increased retinal transparency along thetemporal arcuate fibre
bundles. Coecocentral aswell as peripheral scotomata had become
en-larged (see fig 1, middle section). Other ocularfindings were
bilateral ptosis and mild chronicconjunctival injection. There was
intenseperspiration of the extremities but no signs oforgan
failure, alopecia, or dermatitis.
Psychometric assessment showed a full scaleintelligence quotient
of 90. There were nocranial nerve abnormalities apart from
mildptosis and lack of facial expression. In the upperlimbs
considerable thenar muscle wasting withfinger fasciculation was
observed and grip wasvery weak. In the lower limbs there was
distalmuscle wasting with bilateral pes cavus andhammer toes. His
gait was stiff and shufflingbut he was not ataxic. Tendon reflexes
in the
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Ramaekers, Suormala, Brab, Duran, Heimann, Baumgartner
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Figure I Visualfields. Top: initial examination at the age of
10years in Poland showed an enlarged blind spot extendinginto the
central visualfield. Middle: at the age of 15years the visualfields
demonstrated enlarged coecocentral scotomas withwedgelike
extensions into the temporal visualfield breaking through to the
periphery. Bottom: six months after treatment with10 mg biotinlday
the visualfields had improved with reduction ofdepth and extent
ofthe coecocentral scotomas and opening upofthe peripheral
visualfield borders. The roman numbersfrom 0 to IV represent the
identification bordersfor differently sizedobject, from the size of
1116 mm2 (0) to 16 mm2 (IV) respectively. Increasing light
intensity is indicated by arabic numbersfrom I to 4. The most
important scotomas have been shaded by a grey-white scale.
upper limbs were normal but in the legs theywere symmetrically
exaggerated with extensorplantar responses and ankle clonus. There
wasslight reduction of vibration sense distally in thelower
limbs.
Electroneuromyography in median, tibial,and common peroneal
nerves was consistentwith severe neuroaxonal denervation. Motorand
sensory conduction velocities were normal.Sensory nerve action
potentials derived fromthe sural and median nerve were
normal.Audiometry, brain stem and sensory evokedresponses were also
normal. The normalelectroretinogram excluded any defects at
thelevel of the retinal photoreceptor-ganglioncell unit. The
pattern visual evoked response(stimulus size=80 minutes of arc) was
extin-guished on the right and equivocal on the left.Brightness
stimuli showed plump responseswith diminished amplitudes and
prolongedlatencies. The findings on electroretinographyand from
evoked visual responses suggested
severe axonal fibre loss and conduction distur-bance.
In peripheral nerve, measurements of neuro-filament density
indicated shrinking of axons ofmyelinated nerve fibres but the
ultrastructure ofnon-myelinated nerve fibres appeared normal.A
muscle biopsy specimen showed light micro-scopic changes indicative
of a slowly progressiveneurogenic muscle atrophy.
Despite normal macroscopic appearance ofskin and hair,
microscopy of a skin biopsyspecimen showed chronic inflammatory
changescharacteristic of chronic dermatitis. On lightmicroscopy a
conjunctival biopsy specimenshowed extensive inflammatory changes
consis-tent with chronic conjunctivitis.Computed tomography,
magnetic resonance
imaging, electroencephalography, cardiac func-tion, analysis of
protein in the cerebrospinalfluid, and routine laboratory tests
gave normalresults. The first screening of urinary organicacid
excretion showed a moderately raised
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A biotinidase Km variant causing late onset bilateral optic
neuropathy
excretion of 3-OH-isovaleric acid and a trace of3-OH-propionate
(courtesy of Dr Lehnert,Freiburg). A colorimetric screening test
forbiotinidase was positive. Neurological assess-ment,
ophthalmological and neurophysiologicalexamination in both parents
gave normal results.Other family members were not available
forophthalmological studies.
EFFECT OF BIOTIN TREATMENTBefore detection of the metabolic
inborn errorthe patient's diet was normally balanced andwas lacking
food substances rich in biotin. Afterconfirmation of partial
biotinidase deficiencydaily substitution with 10 mg biotin was
started.After six months on biotin treatment the coeco-central
scotomata regressed and peripheralvisual fields showed recovery to
near normal(fig 1, bottom section). Colour discriminationhad also
improved. These clinical findingscould be substantiated by
examination of hisvisual evoked responses. While pattern
visualevoked response (representing the central 5-10°of arc)
remained unaltered, the brightnessstimuli demonstrated appreciable
recovery ofamplitudes and latencies. Subjective improve-ment with
less fatigue over the daytime andafter exercise was reported by the
patient. Afterbiotin supplementation further deterioration ofthe
motor neuropathy arrested. Eight monthsafter biotin supplementation
the pyramidalsigns in the lower limbs had disappeared.
Special biochemical studiesMETHODSBiochemical data obtained in
the patient werecompared with those of two age matched girlswho had
residual biotinidase activities of 2-8%and 2-9% of mean normal
(table 1). In contrastto the patient these 15 year old girls
appearedperfectly healthy and were neurologicallynormal.8
Informed consent was obtained from theparents and patient before
the samples of blood,cerebrospinal fluid, and urine were
collected.Organic acids were quantified using
gaschromatography/mass spectrometry. Biotinconcentrations were
determined in plasma andurine samples by a microbiological assay
usingLactobacillus plantarum-ATCC 8014.9 Biocytin
concentrations in urine were measured by amodified high
performance liquid chromato-graphy (HPLC).4 8 The activities of
biotindependent mitochondrial carboxylases (PCC,MCC, PC) in
lymphocytes were measuredbefore and after in vitro activation of
apo-carboxylases with excess of biotin as describedpreviously.'0
ACC activity was assayed inlymphocytes by measuring the citrate
activatedincorporation of ['4C]-bicarbonate into malonyl-CoA. "
Biotinidase activity in plasma wasmeasured colorimetrically using
0-15 mmol/lbiotinyl-p-aminobenzoate as substrate. 2 Kineticstudies
on biotinidase were performed by asensitive HPLC method that uses
the naturalsubstrate biocytin.8 Plasma from one of the girlswith
partial biotinidase deficiency was analysedin the same set of
assays as that of the patient.To measure the maximum reaction
velocity(Vmax) and Michaelis constant (Km) for bio-tinidase initial
velocities were measured usingseveral biocytin concentrations
varying from0-00125 to 2-0 mmol/l assay mixture. Theresults were
plotted using Eadie-Hofstee re-ciprocal plots, from which the
respective Vmaxand Km values were derived.
ResultsBefore biotin treatment organic acid analysisshowed only
moderate abnormalities in urinewith a slight rise of
3-OH-isovalerate of 30[tmol/mmol creatinine (normal 8±4
,tmol/mmol)and a trace of 3-OH-propionate. Plasma concen-trations
of 3-OH-isovalerate (9 ,tmol/l), 3-OH-propionate (12 [imol/1), and
3-OH-butyrate (44[imol/l) were also moderately increased. Thelatter
organic acids, except for 3-OH-butyrate,are not normally detectable
in plasma. However,in cerebrospinal fluid the concentration of
3-OH-isovalerate was 250 .tmol/l, that is, 27 timesthe
concentration in plasma. Plasma and cere-brospinal fluid lactate
and pyruvate wereall within the normal range.
Tiglylglycine,methylcitrate, and 3-methyl-crotonylglycinewere not
detected in any of the body fluidsinvestigated. Prompt response to
biotin supple-mentation was shown after 24 hours by nor-malisation
of 3-OH-isovalerate concentrationsin plasma (1 ,umol/l) and urine
(9-7 iimollmmolcreatinine). After four weeks a repeated
lumbarpuncture showed return of the 3-OH-isovalerate
Table I Biotinidase activity -in plasma and kinetic parameters
for plasma biotinidase calculated from Eadie-Hofsteereciprocal
plots presented in figure 2
Subject Activity Kinetic parameters` Factorst(nmollmin/ml
plasma)*
(nmol/minlml plasma) (x 10 6 molIl)
Patient 0 275 0-101-=V,,m, 5 56= KmI F=24952-38='Vmax..2
1000=Km2 p
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Ramaekers, Suormala, Brab, Duran, Heimann, Baumgartner
concentration in the cerebrospinal fluid to thenormal range.
Plasma biotin concentrationsbefore biotin treatment were
undetectable (table2). The mitochondrial carboxylase activities
inlymphocytes of the patient were appreciablydecreased (12-20% of
mean normal values), andACC activity was within the normal
range.Biotin responsiveness could be demonstrated invitro by the
normalisation of the carboxylaseactivities after preincubation with
excess biotin.In the two girls with partial biotinidase defic-iency
the mitochondrial carboxylases were onlymoderately decreased to
30-58% ofmean normalvalues.Compared with controls and the girls
with
partial biotinidase deficiency the urinary biocytinexcretion in
the patient before and during biotin
Table 2 Biotin concentrations in plasma and biotin and biocytin
concentrations in urine
Subject Biotin Plasma Urine (nmollmmol creatinine)treatment
biotin
(nmolll)* Biotin Biocytin
Patientt Nil
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A biotinidase Km variant causing late onset bilateral optic
neuropathy 119
organic aciduria and macroscopic skin or hairabnormalities were
absent. Another surprisingfinding was the absence of hearing
abnor-malities, whereas the majority of symptomaticpatients who
were diagnosed late suffered fromhearing loss. It should be
emphasised that thehistory of our patient did not show
any-evidenceof increased dietary intake of free biotin thatmight
explain the delayed onset of clinicalsymptoms. Biotin treatment in
our patient ledto considerable improvement of the optic
neuro-pathy, disappearance of pyramidal signs, andarrest of
progression of the neuropathy. How-ever the long term outcome is
not yet known. Inother patients with delayed onset of
biotinsupplementation there was prompt reversal ofthe metabolic
derangement and of most clinicalsymptoms with the exception of
optic atrophyand hearing abnormalities which persisted insome of
them. The selective vulnerability of thecentral nervous system in
biotinidase deficiencyhas been observed previously in a single
caseresembling Leigh's disease. This phenomenonwas attributed to
earlier depletion of biotinstores in the brain than in other
tissues,'6 whichwas confirmed by postmortem enzyme studiesof brain,
liver, and kidney." In the presentpatient the 27-fold higher
3-OH-isovalerateconcentration in cerebrospinal fluid comparedwith
plasma supports this hypothesis.The relationship between the
unusual clinical
course in our patient and the enzymatic findingsis not
understood. To our knowledge similarbiphasic kinetics have never
been observed inplasma of other patients with complete orpartial
biotinidase deficiency. In our patientbiocytin excretion was
significantly lower thanin patients with complete biotinidase
deficiencyindicating a considerable recycling of biotin. Onthe
other hand, biotin recycling in our patientwas more severely
compromised than in thegirls with similar residual biotinidase
deficiencybut a normal Km value. This is reflected by his2-4 fold
higher excretion of biocytin comparedwith the girls with partial
deficiency.8 Theabsence of symptoms in these two 15 year oldgirls
suggests that residual biotinidase activityas low as 2-3% with
normal Km maintainsbiotin concentrations high enough to result
insufficiently active carboxylases avoiding patho-genic
consequences.' However, in our patientsimilar residual activity in
combination with anabnormal Km does not allow sufficient
recyclingof biotin to prevent neurological abnormalities.
Further investigations are needed to clarifythe molecular basis
of the biphasic kinetics ofthis biotinidase Km variant. It is of
interest thatbiotinidase activity in the patient's parents
wasnormal in contrast to the intermediate activityobserved in
parents of patients with complete orpartial biotinidase
deficiency.7 8
In conclusion we recommend looking for Km
variants in biotinidase deficient patients with anunusual
clinical course. Disorders of biotinmetabolism should be included
in the differen-tial diagnosis of patients presenting with
un-explained sudden visual failure as early detectionand biotin
supplementation will prevent visualor neurological deterioration.
The basic screen-ing for disorders of the biotin cycle
shouldinclude both urinary organic acid analysis aswell as the
colorimetric biotinidase assay onplasma by using three different
substrate con-centrations. Testing of biotinidase activity
atdifferent substrate concentrations will roughlydetect biotinidase
Km variants.
Part of this work was supported by the Deutsche
ForschungsGemeinschaft (German Research Foundation). The
biochemicalpart of the work was supported by the Swiss National
ScienceFoundation grant No 3.871-0.88. We thank Ms J Engler and
hergroup (Department of Vitamin and Nutrition Research,
FHoffmann-La Roche and Co Ltd, Basel, Switzerland) for thebiotin
determinations in plasma and urine. Last but not least wearemost
grateful to DrJ Wilson (consultant paediatric neurologist,Hospital
for Sick Children, London), for his expert advice andhelp in
preparing the manuscript.
1 Sweetman L, Nyhan WL. Inheritable biotin-treatable dis-orders
and associated phenomena. Annu Rev Nutr 1986;6:317-43.
2 Wolf B, Grier RE, Allen RJ, Goodman SI, Kien CL.Biotinidase
deficiency: the enzymatic defect in late-onsetmultiple carboxylase
deficiency. Clin Chim Acta 1983;131:273-81.
3 Wolf B, Heard GS. Disorders of biotin metabolism. In:Scriver
CR, Beaudet AL, Sly WS, Valle D, eds. Themetabolic basis of
inherited disease. New York: McGraw-Hill, 1989:2083-103.
4 Suormala TM, Baumgartner ER, Bausch J, Holick W, WickH.
Quantitative determination of biocytin in urine ofpatients with
biotinidase deficiency using high-performanceliquid chromatography
(HPLC). Clin Chim Acta 1988;177:253-70.
5 Wolf B, Heard GS, Weissbecker KA, Secor McVoy JR, GrierRE,
Leshner RE. Biotinidase deficiency: initial clinicalfeatures and
rapid diagnosis. Ann Neurol 1985;18:614-7.
6 Wastell HJ, Bartlett K, Dale G, Shein A.
Biotinidasedeficiency: a survey of 10 cases. Arch Dis Child
1988;63:1244-9.
7 Secor McVoy JR, Levy HL, Lawler M, et al. Partialbiotinidase
deficiency: clinical and biochemical features.J Pediatr
1990;116:78-83.
8 Suormala TM, Baumgartner ER, Wick H, Scheibenreiter
S,Schweitzer S. Comparison of patients with complete andpartial
biotinidase deficiency: biochemical studies.J Inherited Metab Dis
1990;13:76-92.
9 Frigg M, Brubacher G. Biotin deficiency in chicks fed
awheat-based diet. IntJ Vitam Nutr Res 1976;46:314-21.
10 Suormala TM, Wick H, Bonjour JP, Baumgartner ER.
Rapiddifferential diagnosis of carboxylase deficiencies and
evalu-ation of biotin responsiveness in a single blood sample.
ClinChim Acta 1985;145:151-62.
11 Baumgartner ER, Suormala TS, Wick H, et al.
Biotinidasedeficiency: a cause of subacute necrotizing
encephalomye-lopathy (Leigh syndrome). Report of a case with
lethaloutcome. Pediatr Res 1989;26:260-6.
12 Knappe J, Brummer W, Biederbick K. Reinigung undEigenschaften
der Biotinidase aus Schweinenieren undLactobacillus casei.
Biochemische Zeitschrift 1963;338:599-613.
13 Ziegler K. Misuse of nonlinear Scatchard plots. TrendsBiochem
Sci 1989;14:314-7.
14 Singh G, Lott MT, Wallace DC. A mitochondrial DNAmutation as
a cause of Leber's hereditary optic neuropathy.N EnglJ' Med
1989;320:1300-5.
15 Wallace DC. Maternal genes: mitochondrial diseases.
In:McKusick VA, Roderick TH, Mori J, Paul NW, eds. Birthdefects
original article series. Medical and experimentalmammalian
genetics: a perspective. New York: Alan R Liss,1987;23:137-90.
16 Baker H, Frank 0, DeAngelis B, Feingold S. Vitamins inhuman
blood and cerebrospinal fluid after intramuscularadministration of
several B-vitamins. Nutrition ReportsInternational
1983;27:661-70.
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