Main

Four biotin-dependent enzymes occur in man: PCC (EC 6.4.1.3), MCC (EC 6.4.1.4), PC (EC 6.4.1.1), and ACC (EC 6.4.1.2). The covalent binding of biotin to the inactive apocarboxylases is catalyzed by the enzyme HCS. Genetic defects of this enzyme lead to an inability to form active holocarboxylases resulting in MCD. HCS deficiency is a rare disorder, which in its classical form presents in the first days or weeks of life with severe life-threatening disease(1). Clinical symptoms are variable including feeding difficulties, vomiting, tachypnea, seizures, hyper- or hypotonia, and progressive loss of consciousness, which may lead to coma and death. Patients with a later onset form who presented with acute metabolic derangement at the age of 14-20 mo have also been reported(2–4). All patients suffered from metabolic acidosis, mild hyperammonemia, and massive organic aciduria at some time during the course of the disease. Oral biotin therapy with pharmacologic doses reversed most biochemical and clinical abnormalities; however, in some cases the response was only partial(5). In all well documented cases the primary defect was decreased affinity of HCS to biotin,i.e. a change in Km. The elevation in the Km value varied from patient to patient and seemed to be related to the severity of the illness(6).

We describe detailed biochemical studies in five patients with evidence of HCS deficiency. The efficacy of biotin therapy was evaluated by measuring organic acid concentrations in urine and biotin-dependent carboxylase activities in lymphocytes. The defect was further characterized by measurement of the level of holocarboxylase activities in cultured skin fibroblasts grown with variable biotin supply. This indirect assay, which has also been used by others(7, 8), provides a reliable alternative to the direct HCS assay, which requires isolation of an apocarboxylase as one of the substrates(9). An important aim of the study was to evaluate the correlation between findings in skin fibroblasts, clinical manifestations, and in vivo effectiveness of biotin supplementation.

CASE REPORTS

AD. Patient AD is the first child of healthy consanguineous Turkish parents who presented at the 2nd d of life with vomiting, excessive loss of weight, tachypnea, and muscular hypotonia. She responded promptly to biotin therapy introduced on the 3rd d of life. She is now 6.5 y old and so far shows no mental or physical impairment under biotin treatment. Details of this patient with a classical early onset form of HCS deficiency have been reported elsewhere(10).

SM. Patient SM is the first child of healthy nonconsanguineous German parents. At the age of 3 mo he developed feeding difficulties and tachypnea during a upper respiratory tract infection and became somnolent. He was subsequently hospitalized in a comatose state with severe metabolic acidosis. He responded well to biotin therapy, which was started at the age of 3.5 mo. He is now 3.5 y old and has developed normally so far.

KE. Patient KE is the first child of healthy nonconsanguineous French parents who presented at the age of 5 mo with seizures and hypotonia. When examined 3 mo later, alopecia and periorificial dermatitis were also present. She exhibited only a partial clinical and biochemical response to biotin therapy, although her skin rash resolved with a massive dose of biotin(4 × 25 mg/d). She is now 8 y old. Her psychomotor development corresponds to that of a 3.5-4-y-old child, and she attends a school for handicapped children.

TM. Patient TM is the first child of healthy nonconsanguineous German parents who suffered two life-threatening episodes of metabolic decompensation at the age of 13 and 17 mo. The first crisis followed orchidopexy, the second one was precipitated by an infection with mild fever. He showed feeding difficulties, vomiting, hyperventilation, and progressive loss of consciousness but recovered without biotin therapy and was apparently well when diagnosed at the age of 18 mo. Under biotin therapy no further episodes occurred. He is now 5 y old and has developed normally.

SY. Patient SY is the sixth child of healthy consanguineous Libyan parents who presented initially at 6 y of age with severe vomiting necessitating hospitalization and i.v. fluid treatment for further episodes over the next 2 y. Notable features were abdominal pains and violent vomiting with metabolic acidosis and ketonuria. The child was first seen in Germany at 8 y of age when he presented again with abdominal pains, vomiting, and tachypnea after orchidopexy. When seen again at 15 y of age the earlier suspected diagnosis of MCD was confirmed. Because he returned to Libya, further follow-up and introduction of biotin treatment has not been possible. The boy attended a normal school.

During metabolic crises each patient presented with severe metabolic acidosis, lactic acidemia, moderate hyperammonemia, and massive organic aciduria. Biotinidase activity in plasma of each patient was normal varying between 4.3 and 6.8 nmol/min/mL plasma (normal range: 4.0-8.0).

METHODS

Organic acids were determined in urine by gas chromatography/mass spectrometry(11). Biotin concentrations were determined in serum and plasma by a microbiological assay using Lactobacillus plantarum ATCC 8014(12). Lymphocytes were isolated from blood samples, and fibroblasts were cultured from skin biopsies as described previously(13). Informed consent was obtained from the parents of the patients. The study was approved by an ethical committee of University Children's Hospital, Basel.

Fibroblasts were routinely cultured in a standard medium containing 10% FCS(Life Technologies, Inc., Trading AG) with a final biotin concentration of 10 nmol/L contributed by the natural biotin content of FCS. Low biotin medium was prepared by replacing FCS by NBCS (Life Technologies, Inc. and BIOSPA GmbH, Germany), resulting in a final biotin concentration of 0.1 nmol/L. In this medium the growth rate of fibroblasts was similar to that in FCS-based medium. High biotin medium was prepared by supplementing either FCS- or NBCS-based medium with 10 000 nmol/L biotin. For the carboxylase reactivation studies, biotin depletion of fibroblasts was achieved by growing patient cells for one subculture in the NBCS-based medium and control cells for at least four subcultures in the NBCS-based medium or for at least two subcultures in a medium containing 10% biotin-free FCS. Biotin was removed by passing FCS twice through a column containing excess of avidin bound to Sepharose 4B (biotin concentration in medium <0.06 nmol/L). After cultures became confluent biotin was added to the medium according to the experimental plan with incubation for defined periods at 37 °C. Reactivation was terminated by removing the medium, washing the cell layer with PBS, and harvesting the cells immediately by trypsinization. For reactivation periods shorter than 1 h, previously harvested biotin-depleted cells were incubated in glass tubes with 2 mL of biotin-containing medium at 37 °C. The reactivation was stopped by adding 10 mL of ice-cold PBS and immediate centrifugation. Both procedures resulted in similar activities after incubation for 1 h. Cell pellets were stored at -70 °C until assayed for carboxylase activities. Basal activities, measured in cells grown in low biotin medium without addition of biotin, were used as blanks. Maximal activities were obtained in cells grown for one subculture in low biotin medium supplemented with biotin (10 000 nmol/L).

The activities of the biotin-dependent mitochondrial carboxylases (PCC, MCC, PC) were assayed in lymphocyte and fibroblast homogenates by measuring the incorporation of [14C]bicarbonate into acid-nonvolatile products with established methods(13, 14). Cytosolic ACC activity was assayed by measuring the citrate-activated incorporation of[14C]bicarbonate into malonyl-CoA as described previously(15, 16). Activity of glutamate dehydrogenase was determined as a mitochondrial biotin-independent control enzyme by NADH-linked assay(17). Protein concentration in homogenates was determined after trichloroacetic acid precipitation by a modified Lowry method(13).

RESULTS

Organic acids in urine. Before biotin therapy all patients showed a typical pattern of organic acids in urine suggesting MCD(Table 1). There was a markedly increased excretion of lactate, 3-HIVA, 3-MCG, and MeCit during metabolic crisis in four of the patients. Further typical metabolites such as 3-hydroxypropionate, tiglylglycine, and propionylglycine were also elevated (data not shown). Concentrations of the aforementioned metabolites were lower, and lactate excretion was not elevated in patient SY who had the latest onset of symptoms(6 y) and was evaluated during an episode of relative well-being.

Table 1 Concentrations of organic acids in urine(mmol/mol creatinine) of the patients
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Four patients (AD, SM, KE, TM) responded to oral biotin in varying doses with a rapid improvement of organic acids in urine (Table 1). In SM and TM 20 mg of biotin/d resulted in normal organic acid excretion, whereas organic acids, particularly 3-HIVA, remained elevated in AD and KE even with a higher biotin dose of 40 mg/d and with a massive dose of 100 mg/d in KE.

Carboxylase activities in lymphocytes. In three patients (KE, TM, SY) mitochondrial carboxylase activities, measured before the start of biotin therapy, were severely deficient in lymphocytes ranging from 1.1 to 23% of mean normal (Table 2). The activity of the cytosolic ACC was also deficient in each patient, however, to a lesser degree than the mitochondrial carboxylases. Plasma biotin concentration was within the normal range in each patient.

Table 2 Activities of the biotin-dependent carboxylases(PCC, MCC, PC, ACC), and a mitochondrial biotin-independent control enzyme, glutamate dehydrogenase (GLDH), in lymphocytes and plasma biotin concentrations of the patients before and/or during therapy with variable daily biotin doses
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Carboxylase activities were measured in lymphocytes of four patients during biotin therapy with increasing doses for at least a few weeks(Table 2). In KE and TM, for whom pretreatment values were obtained, there was a dramatic increase of activities. TM showed the best response, with carboxylase activities reaching the normal range with a biotin dose of 1.4 mg/kg of body weight/d. In two patients (AD, SM) mitochondrial carboxylase activities remained below the normal range with the lower biotin doses (<2.0 mg/kg of body weight/d) but became normal or remained only slightly below the normal range with the higher dose given (2.9 and 2.3 mg/kg of body weight/d, respectively). Patient KE showed the poorest response, and carboxylase activities remained below the normal range even with a massive biotin intake (up to 10 mg/kg of body weight/d). Also ACC activity remained below the normal range in this patient, whereas in the other three patients it was normal even with the lowest biotin dose given.

Carboxylase activities in fibroblasts. Activities of the mitochondrial carboxylases measured in fibroblasts grown for one subculture in media with three different biotin concentrations (Table 3) were severely deficient in the low biotin medium (0.1 nmol/L) but within the normal range in the high biotin medium (10 000 nmol/L) in all patients. Inconsistent results were obtained in fibroblasts grown in the standard FCS medium with a moderate biotin concentration of 10 nmol/L. Mitochondrial carboxylase activities in this medium were clearly deficient in only two patients (AD, SM), whereas they were normal in the others, with the exception of MCC activity in cells of SY. Also the activity of the cytosolic ACC showed variations being severely deficient in AD both in the low biotin and in the standard FCS medium but deficient only in the low biotin medium in cells of SM, TM, and SY. ACC activity was reproducibly normal in all three media for KE.

Table 3 Activities of the biotin-dependent carboxylases(PCC, MCC, PC, ACC), and a mitochondrial biotin-independent control enzyme, glutamate dehydrogenase (GLDH), in skin fibroblasts grown for 1 subculture in media with three different biotin concentrations
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Carboxylase reactivation studies in fibroblasts. The enzyme defect in each patient was further characterized by studying the kinetics of reactivation of apocarboxylases in intact biotin-depleted fibroblasts (Figs. 1 and 2). PCC activities were measured as representative of the mitochondrial carboxylases because of its high stability in enzyme homogenates and because preliminary experiments showed that the reactivation patterns were similar for all three mitochondrial carboxylases(results not shown).

Figure 1
figure 1

Time dependence of reactivation of deficient PCC activities in fibroblasts with 1 μmol/L biotin. PCC deficiency was induced in fibroblasts, and reactivation was performed as described under“Methods.” PCC activities are expressed as percent of maximal activities obtained in cells grown simultaneously for one passage in medium supplemented with 10 μmol/L biotin in which conditions PCC activity was within the normal range in all cell lines. Basal activities obtained in cells grown in biotin-deficient conditions without added biotin were used as blanks. Each symbol is the mean of duplicates of duplicate experiments. (A)▵, patient AD; ○, patient SM; •, patient TM; ▴, patient SY;▪, control cells (mean of values obtained in four different cell strains). (B) □, patient KE; ▪, control cells.

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Figure 2
figure 2

Dependence of reactivation of deficient PCC activities on biotin concentration using 24-h reactivation time. For details, see the legend to Figure 1 and “Methods.” Each symbol represents the mean of duplicates of duplicate experiments for the patients' cells and for the control cells mean of duplicates from four experiments with four different cell strains. Note the different biotin concentration scale at the x axis in A and B. (A) ▵, patient AD; ○, patient SM; •, patient TM. (B) □, patient KE; ▴, patient SY; ▪, control cells; the vertical lines show the lowest control value obtained.

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The rate with respect to time of reactivation of apocarboxylases showed a positive correlation with biotin concentration in all cell lines. However, in all patients it was considerably decreased compared with control cells, but to a different degree in each patient. With a biotin concentration of 1000 nmol/L (Fig. 1A), maximal activities were reached in cells of patient SM between 48 and 72 h, of TM between 30 and 40 h, and of SY between 16 and 24 h. In cells of AD 88% of maximal activities were reached after 96 h of incubation. In normal fibroblasts maximal activities were reached between 0.5 and 1 h. In fibroblasts of KE the activation profile appeared to be more complex (Fig. 1B). Initially there was a similarly rapid increase in carboxylase activities as seen in patient SY, reaching a plateau of 53-62% of maximal activities within 24-30 h. After 64-72 h, a further slower increase led to 87% of maximal activity after 8 d.

The concentration of biotin needed for restoration of carboxylase activities was higher in patient cells than in control cells and also depended on the preincubation time. The reactivation profiles using a 24-h incubation time are shown in Figure 2,A and B. In each cell line the activity reached a plateau with higher biotin concentrations. However, the level of the plateau of activity expressed as a percent of maximal activity(defined in “Methods”) in each cell line reflected the variable reactivation rate. The biotin concentration with which half of the plateau activity was reached was elevated in cells of each patient to a variable degree, being 4000 nmol/L in AD, 1000 nmol/L in SM, 300 nmol/L in TM (Fig. 2A), 34 nmol/L in SY, and 19 nmol/L in KE, whereas in normal cells it was 3-5 nmol/L (n = 4) (Fig. 2B). These findings are consistent with the decreased affinity of HCS for biotin in each patient. In this assay system PCC activity in fibroblasts of KE could be restored to only 55% without further increase, even when the biotin concentration was increased up to 100 000 nmol/L.

Effect of CHI on carboxylase reactivation. The possibility of decreased apocarboxylase synthesis as a consequence of insufficient biotin supply was investigated by measuring the reactivation of apo-MCC in intact biotin-depleted fibroblasts with an excess of biotin (10 μmol/L in patients' cells and 0.1 μmol/L in control cells) in the presence and absence of 20 μg/mL of the protein synthesis inhibitor CHI. At this concentration CHI effectively inhibited incorporation of[3H]phenylalanine into macromolecules in fibroblasts (results not shown). In this experiment activity of MCC rather than PCC was investigated because control fibroblasts became more readily deficient in MCC than in the other carboxylases under biotin deprivation. Appreciable reactivation of MCC was observed in the presence and absence of CHI in all cell lines. For example in KE MCC activity reached 40% of maximal activity after 8 h and 53% after 48 h in the presence of CHI compared with 33 and 62%, respectively, in the absence of CHI. In comparison MCC activity in control cells reached 69 and 68% after 8 and 24 h in the presence of CHI, and 82 and 73% without CHI. These results indicate that in these cell lines apocarboxylases are synthesized at a normal rate even under biotin-deficient conditions.

Turnover rate of holocarboxylases. The apparent turnover rate of holocarboxylases (Fig. 3) was studied in fibroblasts by growing them in the high biotin medium up to preconfluence, changing the medium to the NBCS-based low biotin medium, and following the decrease of carboxylase activities. In this low biotin medium, cells of all patients were unable to form new holocarboxylases. In all patients studied the rate of decrease was very similar, thus excluding an altered turnover rate of holocarboxylases as the cause of the clinical and biochemical differences between the patients. The half-life of holocarboxylases was estimated by treating the process mathematically as a first order decomposition. The half-life for PCC varied in the four patients studied (Fig. 3) between 3.1 and 3.6 d with a mean of 3.4 d, and for MCC it was slightly shorter, varying from 2.0 to 3.3 d with a mean of 2.5 d (results not shown; PC was not assayed).

Figure 3
figure 3

Decrease of PCC activity in fibroblasts of four patients after changing the culture medium from high biotin to low biotin medium. Fibroblasts were cultured in a high biotin medium (10 000 nmol/L) until preconfluence. Then the medium of part of the cultures was changed to low biotin medium (0.1 nmol/L). After selected periods, cells grown in both low and high biotin medium were harvested and assayed for carboxylase activities. The activities in cells grown in the low biotin medium are presented and expressed as percent of activities in cells grown in the high biotin medium. Each symbol represent the mean of duplicates of a single experiment. Symbols: ▵, patient AD; ○, patient SM; □, patient KE;•, patient TM.

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DISCUSSION

This study clearly indicates that indirect assays of HCS allow diagnosis of this heterogeneous disorder in patients with very variable clinical expression of the defect. Each showed biotin-responsive deficiency of all four carboxylases in lymphocytes and/or in cultured skin fibroblasts in the presence of normal plasma biotinidase activity.

The former notion of HCS deficiency as the “early onset MCD” has to be revised with increasing experience of this disease. In our patients the onset of clinical symptoms varied from the 2nd day of life (AD) to 6 y(SY) with considerable differences in the severity of disease. Although half of reported patients with HCS deficiency have presented with severe illness during the first 3 d of life(1), late onset patients have also been described who presented for the first time between 14 and 20 mo(2–4) and one as late as 8 y of age(18). Life-threatening episodes occurred both in neonatal and late onset patients during acute metabolic derangement.

The characteristic indication for MCD is elevated urinary excretion of 3-HIVA, 3-MCG, lactate, and MeCit. In contrast to biotinidase deficiency, where urinary metabolites may be normal, all patients with HCS deficiency excrete these metabolites, particularly 3-HIVA, in high concentration during acute episodes(1). However, the concentrations of the key metabolites in urine vary greatly depending on the clinical state(Table 1). A consistent biochemical abnormality in our patients when untreated was severe MCD in lymphocytes. This was found even in patients TM and SY during a phase of well-being in the presence of low organic acid excretion.

An important approach to study the enzyme defect in detail is the use of cultured fibroblasts. In the commonly used medium with 10% FCS (biotin concentration varying between 6 and 82 nmol/L) fibroblast carboxylase activities in 10 patients were reported to be clearly decreased to 0-30% of normal(6, 10, 19–21). However, in three of our patients (KE, TM, SY) these values were within the normal range in this medium. As also suggested by others(19, 22) there is a substantial risk of missing the correct diagnosis in such patients unless media with sufficiently low biotin concentrations are used. This can easily be achieved by replacing FCS with NBCS, which contains 100 times less biotin. Clearly deficient mitochondrial carboxylase activities (<24% of normal) were measured in each patient in the NBCS-based medium. Deficiency of the cytosolic carboxylase, ACC, was observed in our patients both in lymphocytes and/or in fibroblasts. In accordance with other reports(20, 22), its activity was influenced less than that of the mitochondrial carboxylases. Similar to findings in our patients, fibroblasts of most patients reported in the literature(3, 6, 8, 19) showed normal or at least significantly increased carboxylase activities when the culture medium was supplemented with 200-10 000 nmol/L biotin.

The evaluation of biotin treatment yielded important information on variations in dose response. In three of our patients (AD, SM, TM) clinical symptoms disappeared promptly with 10-20 mg/d, whereas in one patient (KE) the response was only partial. Her skin rash was cured only with the very high dose of 100 mg/d, but she has remained mentally retarded. Biochemical response evaluated by measuring activities of the mitochondrial carboxylases in lymphocytes showed further variation in dose response. In three patients normal or near to normal activities were obtained, in one (TM) with 1.4 mg/kg of body weight/d and in the other two (AD, SM) with 2-3 mg/kg of body weight/d. In contrast, in patient KE carboxylase activities remained below 50% of mean normal even with 100 mg biotin/d, i.e. 10 mg/kg of body weight/d. These variations in lymphocyte carboxylase activities also correlated well with the abnormalities in urinary 3-HIVA excretion, which remained clearly elevated only in KE.

Low carboxylase activities in white blood cells and elevated excretion of 3-HIVA during biotin therapy have been reported previously in patients with HCS deficiency(5, 20, 21, 23). Our study indicates that the cause is either insufficient biotin supplementation or a severe defect that does not allow complete biochemical normalization even with application of massive biotin doses.

Our findings show a wide range of biotin requirement in each patient. It is, however, not clear whether it is necessary to increase the daily biotin dose to normalize mild biochemical abnormalities observed in those of our patients (AD, SM, TM) with apparently normal clinical development. Most reported patients developed well on 20 mg or less of biotin/d, but they were observed clinically only for short periods. There is only one follow-up study of a 9-y-old child who received biotin therapy prenatally (after the death of a sibling) and 6 mg/d since the age of 3.5 mo. She developed normally with this low dose(24). It may be of importance that her daily biotin was divided into four portions. Several doses divided over the day may help to maintain biotin concentrations at a sufficiently high continuous level to compensate for the decreased affinity of HCS for biotin.

Our studies of carboxylase reactivation performed in intact fibroblasts showed that in each patient higher biotin concentrations and longer incubation times were needed for restoration of carboxylase activities than in biotin-depleted control fibroblasts. Similar abnormalities have also been described in cells of other patients with HCS deficiency(8, 25). The increased biotin requirement for carboxylase reactivation is in accordance with a Km change of HCS for biotin. However, in our indirect assay system it was not possible to measure the true Km value for biotin because intact cells and endogenous apocarboxylases were used. Similar indirect methods have been used by others in homogenates(2, 7) or in intact cell system(7, 8, 19), leading to similar results. Studies in which HCS activity was assayed directly with exogenous apoPCC as one of the substrates showed a good correlation between clinical presentation and the Km for biotin(6, 26). Our studies indicate that the same correlation holds true also with indirect measurement of HCS activity. In four patients (AD, SM, TM, SY) the variation of both the reactivation rate (Fig. 1) and the biotin requirement (Fig. 2) in fibroblasts correlated well with the time of onset of symptoms and with in vivo responsiveness to biotin therapy(see Tables 1 and 2). Ranging these four patients with respect to their reactivation patterns in fibroblasts reflects the degree of clinical severity of the disorder. For example patient AD who had the most abnormal reactivation patterns in fibroblasts presented at 2 d of age with severe clinical symptoms. In contrast patient SY in whose fibroblasts reactivation was closest to that of controls showed milder symptoms only at 6 y of age precipitated by intercurrent infection.

Patient KE was an exception because she showed a relatively late onset of symptoms (5 mo) and only a partial response to high dose biotin therapy. However, the reactivation profiles in her fibroblasts differed from those of the other patients, indicating only mildly decreased affinity of HCS for biotin but possibly reduced Vmax. Other possible primary defects, such as decreased apocarboxylase synthesis during biotin-deficiency or altered turnover rate of holocarboxylases, were ruled out. A defect in biotin transport was excluded by the studies of a similar rate of[3H]biotin transport into fibroblasts than found in the other patients and of controls (results not shown). A defect of biotin transport into mitochondria is very unlikely because the cytosolic ACC activity in lymphocytes was similarly affected as the mitochondrial carboxylases.

In patient KE there was a discrepancy between carboxylase activities in lymphocytes and fibroblasts, whereas in the other four patients the activity patterns correlated well between these two cell types. In lymphocytes of KE, carboxylase activities, including ACC, remained below 50% of mean control values despite highly elevated plasma biotin levels (up to 684 nmol/L;Table 2). In contrast, carboxylase activities were normal in fibroblasts grown in FCS medium (biotin concentration 10 nmol/L). In the only other well documented patient(5) with a partial clinical response to biotin therapy, the biochemical findings were different. In this patient carboxylase activities were similarly decreased in lymphocytes as in KE, but in fibroblasts PCC and MCC activities were also decreased (30 and 28% of normal, respectively) even when the cells were cultured with 8 mmol/L biotin(5). The reason for the discrepancy in cells of patient KE seems to be related to the unusual biphasic time dependence curve for carboxylase reactivation in fibroblasts (Fig. 1B). During an initial relatively fast phase carboxylases reached about 60% of maximal activities in fibroblasts,i.e. similar levels as obtained in lymphocytes during biotin therapy. However, further incubation of fibroblasts with biotin resulted in a second slower reactivation phase, which led to normalization of activities after 8 d. The normal carboxylase activities obtained in long-term cultures in the standard FCS medium (Table 3) are most probably caused by this phenomenon. One could speculate that in patient KE the mutation has two effects, one resulting in reduced sensitivity to reactivation of apocarboxylases by biotin, the other in an increase of the turnover rate of the HCS molecule itself, which is stabilized by high concentrations of biotin. A further possibility would be restoration of an abnormal three-dimensional structure of HCS by high biotin concentrations. Further clarification of the defect requires studies at the molecular level.

HCS deficiency is considered to be more rare than the other biotin-responsive disorder, biotinidase deficiency. Due to the great variability of presentation in HCS deficiency, the diagnosis based on clinical findings is difficult. Furthermore, diagnosis may be missed not only in patients with neonatal onset who may die before appropriate analyses have been performed, but also in late onset patients, e.g. when organic acid metabolites in urine show only mild abnormalities and carboxylase activities are normal in fibroblasts grown in FCS medium. Therefore, the incidence of this easily treatable disorder may be higher than anticipated.

Follow-up studies are necessary to know more about the long-term outcome of HCS deficiency. However, the prognosis in most patients seems to be good. Even with treatment a minority of patients may remain handicapped due to only partial responsiveness. Irreversible neurologic abnormalities as seen in biotinidase deficiency, i.e. neurosensory hearing loss and optic atrophy(1), have not been described. Our results confirm the extreme variability of both clinical and biochemical presentation and stress the importance of the assessment of the optimal daily biotin dose for each patient individually.