Multiple Carboxylase Deficiency Research Articles

Selective screening for inborn errors of metabolism and secondary methylmalonic aciduria in pregnancy at high risk district of neural tube defects: A human metabolome study by GC-MS in China

Objective Urease pretreatment-gas chromatography-mass spectrometry (UP-GC-MS) has become a valuable tool in the field of metabolome research, including analysis of inborn errors of metabolism (IEMs) and acquired metabolic disturbances secondary to nutrition or drugs. This research aims to screen IEMs in Chinese patients and to explore the cause of neural tube defects (NTDs), a congenital malformation very common in North China. Design and methods Urine samples from 618 patients at high risk of IEMs in China were collected, and UP-GC-MS was performed in the selective screening. Urinary methylmalonate (MMA) levels in pregnancy with and without NTDs fetus, respectively, at Luliang district, a countryside region with NTDs incidence 227/10 000, Shanxi Province, North China, were analyzed by GC-MS-selective ion monitoring, and compared with that from control region. Results Among the 618 patients, 22 kinds and 59 cases of IEM were found. Methylmalonic aciduria (MMAuria) is on top of the list, followed by neonatal intrahepatic cholestasis caused by citrin deficiency (NICCD), phenylketonuria (PKU), multiple carboxylase deficiency (MCD), etc. Satisfactory therapeutic effects have been achieved in patients such as NICCD, MCD, and galactosemia. At Luliang district, urinary MMA levels in pregnancy, no matter NTDs-affected or unaffected, are both significantly ( P < 0.01) higher than that in normal control, while serum B 12 levels in NTDs-affected pregnancy are significantly lower than that both in NTDs-unaffected group ( P < 0.01) and in normal control ( P < 0.01). Furthermore, B 12 < 52.5 pmol/L is associated with a 7.78-fold increased NTDs risk ( P < 0.01) at Luliang district. Conclusions Selective screening for IEMs by UP-GC-MS provides valuable evidences for the diagnosis of IEMs. MMAuria secondary to B 12 deficiency is quite common at Luliang district, suggesting B 12 deficiency is involved in the development of NTDs in the specific population. This metabolome research by UP-GC-MS provides valuable epidemiological information that helps to understand the prevalence and the possible intervention strategy of NTDs and IEMs, especially in Chinese population.

Reduced half-life of holocarboxylase synthetase from patients with severe Multiple Carboxylase Deficiency

Multiple carboxylase deficiency is a clinical condition caused by defects in the enzymes involved in biotin metabolism, holocarboxylase synthetase (HLCS) or biotinidase. HLCS deficiency is a potentially fatal condition if left untreated, although the majority of patients respond to oral supplementation of 10-20 mg/day of biotin. Patients who display incomplete responsiveness to this therapy have a poor long-term prognosis. Here we investigated cell lines from two such HLCS-deficient patients homozygous for the c.647T>G p.L216R allele. Growth of the patients' fibroblasts was compromised compared with normal fibroblasts. Also the patient cells were not sensitive to biotin-depletion from the media, and growth rates could not be restored by re-administration of biotin. The molecular basis for the HLCS deficiency was further investigated by characterisation of the p.L216R protein. The HLCS mRNA was detected in MCD and normal cell lines. However, protein and enzyme activity could not be detected in the patients' cells. In vitro kinetic analysis revealed that enzyme activity was severely compromised for recombinantly expressed p.L216R and could not be increased by additional biotin. Furthermore, the turn-over rate for the mutant protein was double that of wildtype HLCS. These results help provide a molecular explanation for the incomplete biotin-responsiveness of this p.L216R form of HLCS.

Determination of 3-hydroxyisovalerylcarnitine and other acylcarnitine levels using liquid chromatography–tandem mass spectrometry in serum and urine of a patient with multiple carboxylase deficiency

Due to its increased concentration in blood, 3-hydroxyisovalerylcarnitine (C5OH-I) is an important indicator for the diagnosis of organic acidemias in newborns. However, C5OH-I has not been used as a standard in tandem mass spectrometric (MS/MS) assays because its isolation is difficult. We developed a new synthesis of C5OH-I and investigated its behavior by MS/MS. A method using the multiple reaction monitoring (MRM) mode of MS/MS with HPLC was developed which provides high accuracy, precision and reproducibility. Acylcarnitine profiles in the serum and urine of a patient with multiple carboxylase deficiency (MCD) showed increased levels compared to a healthy patient.

1H NMR spectra of methylcitric acid in urine

Methylcitric acid (3-carboxy-3-hydroxy-2-methyl-pentanedioic acid; MCA) is elevated in body fluids of patients with propionic acidaemia (PA; McKusick: 232000, 232050), methylmalonic aciduria (MMA; McKusick: 251000, 251120) and multiple carboxylase deficiency (McKusick: 253260, 253270). MCA molecule has two stereogenic centers so that it can occur in the form of four stereoisomers. Only two stereoisomers of MCA, (2S, 3S) and (2R, 3S), were found in human urine. They have different spectra and can be easily distinguished. We performed measurements of 1H, 13C NMR and gs-HMQC (gradient-selected Heteronuclear Multiple Quantum Coherence) on the samples of unprocessed urine of 6 patients suffering from propionic acidaemia and methylmalonic aciduria and identified the protons of MCA diastereoisomers, 3-hydroxypropionic acid and methylmalonic acid. Having used NMR spectroscopy to identify stereoisomers of MCA in urine, we can conclude that this technique gives the possibility of analysing MCA diastereoisomers in body fluids.

From an inborn error patient to a search for regulatory meaning: A biotin conducted voyage

This article summarizes some findings of a research that I have pursued for the past 25 years, whose roots are immersed in the field of inherited metabolic disorders, and deal with different aspects of the vitamin biotin, starting with a patient with multiple carboxylase deficiency (MCD). Several of MCD clinical manifestations resemble those of infant malnutrition; we demonstrated that about one-third of infants with this common nutritional disorder were indeed biotin-deficient, and that this deficiency is metabolically significant, by studying urine instead of blood, studying urinary organic acids by gas chromatography–mass spectrometry. Remarkably, the metabolic abnormalities became apparent only after protein feeding was started, suggesting that this phenomenon may contribute to the worsening of malnourished individuals when they are abruptly fed. Afterwards, we studied biotin deficiency at the tissue level. Carboxylase activities and masses were significantly reduced in liver, kidney, muscle, adipose tissue, intestine, and spleen, but brain and heart were spared; their mRNAs remained unchanged. On the other hand, holocarboxylase synthetase (HCS) mRNA levels were markedly low in the deficient animals, and increased upon biotin injection. Over 2000 human genes have been identified that depend on biotin for expression. To probe into the “logic” of this enigma, we have started comparative studies among evolutionarily distant organisms, such as mouse and Saccharomyces cerevisiae, and we are now looking for biotin effects on specific genes and proteins, such as HCS and hexokinases, and on their proteomes.

Molecular genetics of biotin metabolism: old vitamin, new science

Biotin is a water-soluble vitamin that participates as a cofactor in gluconeogenesis, fatty acid synthesis and branched chain amino acid catabolism. It functions as the carboxyl carrier for biotin-dependent carboxylases. Its covalent attachment to carboxylases is catalyzed by holocarboxylase synthetase. Our interest in biotin has been through the genetic disease, “biotin-responsive multiple carboxylase deficiency,” caused by deficient activity of holocarboxylase synthetase. As part of these studies, we made the unexpected findings that the enzyme also targets to the nucleus and that it catalyzes the attachment of biotin to histones. We found that patients with holocarboxylase synthetase deficiency have a much reduced level of biotinylated histones, yet the importance of this process is unknown. The dual nature of biotin, as the carboxyl-carrier cofactor of carboxylases and as a ligand of unknown function attached to histones, is an enigma that suggests a much more involved role for biotin than anticipated. It may change our outlook on the optimal nutritional intake of biotin and its importance in biological processes such as development, cellular homeostasis and regulation.

Nuclear matrix proteins and hereditary diseases

The review summarizes literature data on alterations of structure or expression of different nuclear matrix proteins in hereditary syndromes. From the point of view of involvement of nuclear matrix proteins in etiology and pathogenesis of the disease hereditary pathologies can be classified in pathologies with pathogenesis associated with defects of nuclear matrix proteins and pathologies associated to changes of the nuclear matrix protein spectrum. The first group includes laminopathies, hereditary diseases with abnormal nuclear-matrix associated proteins and triplet extension diseases associated with accumulation of abnormal proteins in the nuclear matrix. Laminopathies are hereditary diseases coupled to structural defects of the nuclear lamina. These diseases include Emery-Dreifuss muscular dystrophy, limb girdle muscular dystrophy, dilated cardiomyopathy (DCM) with conduction system disease, familial partial lipodystrophy (FPLD), autosomal recessive axonal neuropathy (Charcot-Marie-Tooth disorder type 2, CMT2), mandibuloacral dysplasia (MAD), Hutchison Gilford Progeria syndrome (HGS), Greenberg Skeletal Dysplasia, and Pelger-Huet anomaly (PHA). Most of them are due to mutations in the lamin A/C gene, one - to mutations in emerin gene, some are associated with mutations in Lamin B receptor gene. In Werner's, Bloom's, Cockayne's syndromes, Fanconi anemia, multiple carboxylase deficiency mutations in nuclear matrix protein or enzyme gene lead to deficient DNA repair, abnormal regulation of cell growth and differentiation or other specific metabolic functions. Proteins with a long polyglutamic tract synthesized in the cells of patients with dentato-rubral and pallido-luysian atrophy, myotonic dystrophy and Huntington disease interfere with transcription on the nuclear matrix. Down's syndrome is a representative of the group of diseases with altered nuclear matrix protein spectrum.

Diagnosis of Methylmalonic Acidemia from Dried Blood Spots by HPLC and Intramolecular-Excimer Fluorescence Derivatization

Methylmalonic acidemias, a group of heterogeneous disorders, are characterized by accumulation of methylmalonic acid (MMA) and its byproducts in biological fluids (1)(2). Methylmalonic acidemia is now included in all tandem mass spectrometry (MS/MS)-based newborn screening programs (3)(4)(5). Detection is based on the finding of increased propionylcarnitine and/or increased propionylcarnitine-to-acetylcarnitine ratio in dried blood spots (DBS) by MS/MS. These markers, however, are not specific because they are increased in propionic acidemia and, possibly, in multiple carboxylase deficiency (3). In most programs, newborns or patients with initial positive results are recalled for a second blood spot, and a urine sample is collected for organic acid analysis to differentiate among the three disorders. In the present study, we used the intramolecular-excimer fluorescence derivatization approach of Nohta and coworkers (6)(7) to form a fluorescent derivative of MMA. This would allow the detection of MMA in DBS samples from affected neonates, leading to a conclusive diagnosis with the remains of the DBS within a short time, often the same working day. From a DBS, four 3.2-mm discs were punched and extracted into 250 μL of methanol containing 20 μmol/L malonic acid (MA) as internal calibrator by vortex-mixing for 30 s and standing at room temperature for 1 h. After evaporation and reconstitution of the residue …

Paradoxical Regulation of Biotin Utilization in Brain and Liver and Implications for Inherited Multiple Carboxylase Deficiency

Holocarboxylase synthetase (HCS) catalyzes the biotinylation of five carboxylases in human cells, and mutations of HCS cause multiple carboxylase deficiency (MCD). Although HCS also participates in the regulation of its own mRNA levels, the relevance of this mechanism to normal metabolism or to the MCD phenotype is not known. In this study, we show that mRNA levels of enzymes involved in biotin utilization, including HCS, are down-regulated during biotin deficiency in liver while remaining constitutively expressed in brain. We propose that this mechanism of regulation is aimed at sparing the essential function of biotin in the brain at the expense of organs such as liver and kidney during biotin deprivation. In MCD, it is possible that some of the manifestations of the disease may be associated with down-regulation of biotin utilization in liver because of the impaired activity of HCS and that high dose biotin therapy may in part be important to overcoming the adverse regulatory impact in such organs.

Clinical onset and prognosis of Asian children with organic acidemias, as detected by analysis of urinary organic acids using GC/MS, instead of mass screening

Organic acidemias (OAs) have been detected worldwide in symptomatic patients using gas chromatography mass spectrometry. We diagnosed 188 Asian cases of OAs by analysis of urinary organic acids and investigated their clinical onset and outcome. Methylmalonic acidemia (MMA) was most common (74 cases), followed by propionic acidemia (23 cases), ornitine transcarbamylase deficiency (22 cases), and multiple carboxylase deficiency (15 cases). For these 188 patients, onset was most frequent in the neonatal period or early infancy. Approximately 30% of the patients had a family history of similar symptoms or diseases. Although the outcome of OA patients varied, patients with early onset generally had poor outcomes despite early detection. Of the 45 MMA patients whose clinical data were available, 25 were clinically vitamin B12-responsive, while the remaining 20 were non-responsive. A favorable outcome was obtained in 7 of the 25 B12-responsive patients, and in only 3 of the 20 B12-nonresponsive patients. It was suggested that even in B12-responsive MMA cases, earlier detection and B12 therapy were needed to improve the prognosis. We concluded that detection of such patients at the presymptomatic stages using newborn mass screening is essential for prognosis improvement with OAs.

Dermatologic Signs of Biotin Deficiency Leading to the Diagnosis of Multiple Carboxylase Deficiency

The biotin-responsive, multiple carboxylase deficiencies are autosomal recessively inherited disorders of metabolism in which biotin-dependent carboxylases show diminished activity. This results in an accumulation of organic acids in the urine. The clinical picture involves the nervous system, skin, respiratory system, digestive system, and immune system. The disorder has a good prognosis if biotin therapy is introduced early. If not, it can result in irreversible damage to the central nervous system and early death from metabolic acidosis. We report a 4-year-old girl with unexplained seizures that did not respond well to anticonvulsants. The development of skin problems, which histologically could match the diagnosis of a nutritional dermatitis, together with the fact that the child was constantly eating without gaining weight, led us to the diagnosis of a metabolic disorder. The accumulation of organic acids in the urine suggested the possibility of a biotin deficiency. With biotin therapy the skin problems resolved completely. The seizures also diminished. This case shows that in young children with unexplained seizures that do not respond well to classic anticonvulsant therapy, the possibility of biotin deficiency should always be considered. This article also includes a thorough review of the skin manifestations and other problems caused by biotin deficiency.

Biotinidase Deficiency: New Directions and Practical Concerns.

Biotinidase deficiency is a readily treatable inherited disorder. Discovery of the enzyme deficiency as the cause for late-onset multiple carboxylase deficiency initially seemed to answer almost all of the questions about the disorder. However, as is the case for most inborn errors of metabolism, finding the enzyme that causes the disorder, cloning the gene, and determining the spectrum of clinical features of the disease only opens a Pandora's box. As researchers have found, there are still many important and interesting questions about this disorder that must be addressed and answered. However, when compared with other inherited metabolic diseases, biotinidase deficiency is still one of the most readily treatable. If a child must have an inborn error of metabolism, let it be biotinidase deficiency and let it be identified by newborn screening.

A genomic approach to mutation analysis of holocarboxylase synthetase gene in three Chinese patients with late-onset holocarboxylase synthetase deficiency

Objective Multiple carboxylase deficiency (MCD, MIM:253270) is a common organic aciduria and caused by deficiency of either biotinidase or holocarboxylase synthetase (HLCS; EC 6.3.4.10). Patients commonly present during early infancy with acute metabolic derangements and severe metabolic acidosis. Recently, a late onset form of HLCS deficiency was also described. The different phenotypes (early and late presenting) may be related to a spectrum of mutations in HLCS gene. Applications of mutation analysis in HLCS had been limited previously by the requirement of cDNA from living tissue for study. We described here a genomic approach for molecular diagnosis of HLCS deficiency which we have used to detect mutations in Chinese patients who had the late-onset form of HLCS deficiency. In addition, a fibroblast cell line with MCD from Coriell Cell repositories was also studied. Design and methods Three Chinese patients with late onset HLCS deficiency were studied. The genomic sequence of HLCS was retrieved and newly designed primers were used to cover all coding sequences of the gene. PCR products were analyzed by direct sequencing. Population allelic frequencies of mutations detected were determined by genotyping of control samples by restriction fragment length polymorphism. Results We found a recurrent mutation, R508W, in the three unrelated Chinese patients. Two were homozygous for this mutation. The other patient was a compound heterozygote of R508W and a novel mutation, D634N. The results suggest that R508W may be an important and relatively prevalent disease-causing mutation in Chinese MCD patients. A fibroblast cell-line from an African patient revealed an additional novel mutation, R565X and a known mutation, V550M. Conclusion R508W is a recurrent mutation in Chinese MCD patients which is associated with the late onset phenotype. This new genomic approach for mutation analysis of HLCS gene provides new opportunities in studies of MCD.

Usage of derivatives of D-glucosamine in emotional hypertension due to stress

Biotin (vitamin B7, hexahydro-2-oxo-1H-thieno[3,4-d]imidazole-4-pentanoic acid) is not synthesized by mammals: dietary sources include plant based foods, yeast, liver, kidneys, heart, pancreas, egg yolk, poultry, and cow’s milk. Synthesis via the gut bacteria may also play a role. Recommended daily allowances are based on normal intake; EU RDA is 50 μg/day, the US adequate intake (AI) for adults is 30 μg/day.Biotin is required for the function of four carboxylases: pyruvate carboxylase, propionyl CoA carboxylase, multiple CoA carboxylase, and acetylCoA carboxylase I and II. Holocarboxylase synthase is required to attach biotin to their active sites. Biotin also modulates the expression of up to 2000 genes, and has effects on immune function, largely through biotinylation of histones and other proteins.Biotinidase, the enzyme which splits biotin from biotinyl lysine (biocytin), and from peptides containing biocytin, recycles biotin from endogenous turnover and facilitates absorption from exogenous sources, together with the sodium-dependent multivitamin transporter.Nutritional deficiency of biotin is rare and most cases relate to inborn errors of biotin metabolism and transport, long-term use of parenteral nutrition, chronic anticonvulsant therapy, or avidin binding of dietary biotin due to excessive ingestion of egg whites.Excessive ingestion of biotin from supplements is increasingly common. Biotin has no direct toxicity, but high circulating levels can interfere with many commercially available immunoassays which use the interaction between biotin and streptavidin. This can result in a spurious but plausible biochemical picture of disease supported by two or more independent immunoassay based tests.The laboratory detection of functional biotin deficiency relies upon detection of multiple carboxylase deficiency, identified and followed by measurement of urinary 3-hydroxyisovaleric acid (3HIVA, ideally using isotope dilution GC-MS, or isotope dilution-LC-MSMS (ID-LC-MSMS)) or plasma 3-hydroxyisovaleryl carnitine (3HIVc, measured using ID-LC-MSMS). These methods are reviewed, together with methods for assessing biotinidase activity.This chapter also describes methods for the direct measurement of biotin in foods, blood, serum/plasma, and urine. These relied for many years on microbiological assays. These were followed by binding assays using avidin with radioactive biotin or enzyme linked biotin as tracers, with or without chromatographic separation to distinguish between biotin, metabolites of biotin, and biotinylated peptides. Latterly, the introduction of specific assays using ID-LC-MSMS should allow traceable and harmonized assay of plasma/serum biotin. These could be particularly helpful to detect excessive ingestion of biotin supplements.We conclude that there is a need for laboratory methods with adequate sensitivity and specificity to reliably diagnose biotin deficiency and monitor biotin levels in patients receiving supplements. There are a limited number of reliable markers of biotin status and no single marker can reliably distinguish moderately deficient biotin from sufficient and supplemented. Measurement of 3HIVA and 3HIVc, the functional markers of biotin status, is currently the recommended approach.

Holocarboxylase synthetase is an obligate participant in biotin-mediated regulation of its own expression and of biotin-dependent carboxylases mRNA levels in human cells.

Holocarboxylase synthetase (HCS) catalyzes the covalent attachment of biotin to five biotin-dependent carboxylases in human cells. Multiple carboxylase deficiency (MCD) is a life-threatening disease characterized by the lack of carboxylase activities because of deficiency of HCS activity. Here, we report the obligatory participation of HCS in the biotin-dependent stimulation of the level of HCS mRNA and those of acetyl-CoA carboxylase and the alpha subunit of propionyl-CoA carboxylase in human cells. Fibroblasts from patients with MCD are unable to increase HCS mRNA in response to biotin unless the vitamin concentration is raised 100-fold, in keeping with mutations that cause a reduced affinity for biotin by the mutant enzyme. The outcome is deficient synthesis of biotinyl-5'-AMP, the active form of the vitamin in the biotinylation reaction. HCS and carboxylase mRNA levels in normal and MCD fibroblasts and HepG2 cells can be restored by the addition of the cGMP analogue, 8-Br-cGMP, and can be abolished by the addition of inhibitors of the soluble form of guanylate cyclase. We propose a regulatory role for biotin in the control of HCS and carboxylase mRNA levels through a signaling cascade that requires HCS, guanylate cyclase, and cGMP-dependent protein kinase.

Structure of human holocarboxylase synthetase gene and mutation spectrum of holocarboxylase synthetase deficiency.

Holocarboxylase synthetase (HLCS) is an enzyme that catalyzes the incorporation of biotin into apo-carboxylases, and its deficiency causes biotin-responsive multiple carboxylase deficiency. The reported sequences of cDNA for human HLCS from liver, lymphocyte, and KG-1 myeloid cell lines differ at their 5' regions. To elucidate variations of the human HLCS mRNA and longer 5' cDNA ends, we performed screening of the human liver cDNA library and rapid amplification of the cDNA ends (RACE). Our results suggest the existence of three types of HLCS mRNA that start at different exons. The first type starts at exon 1, and the second type starts at exon 3, and both are found in various human tissues. The third type, corresponding to the cDNA from the KG-1 cell, starts at exon 2 of the HLCS gene. Various splicing patterns from exons 3-6 were also observed. None of the variations of cDNA found created a new initiation codon. Mutation screening from exons 6-14, therefore, was sufficient to detect amino acid changes in HLCS in patients. Our direct sequencing strategy for screening mutations in the HLCS gene revealed mutations in five Japanese patients and seven non-Japanese patients. Our analyses involving 12 Japanese and 13 non-Japanese patients and studies by others indicate that (1) there is no panethnically prevalent mutation; (2) the Arg508Trp, Gly581Ser, and Val550Met mutations are found in both Japanese and non-Japanese populations; (3) the IVS10+5G-->A mutation is predominant and probably a founder mutation in European patients; (4) the 655-656insA, Leu237Pro, and 780delG mutations are unique in Japanese patients; (5) the spectrum of the mutations in the HLCS gene may vary substantially among different ethnic groups.

Overview of common inherited metabolic diseases in a Southern Chinese population of Hong Kong

Background: The Joint metabolic clinic at the Prince of Wales Hospital was established in January 1997 to provide a comprehensive multi-disciplinary care to patients with inherited metabolic diseases (IMDs). Patients are referred from both within and outside our hospital. Until July, 2000, more than 40 patients and families with 20 different biochemical diagnoses attend the clinic for regular follow up. A pattern of more common IMDs among Hong Kong Chinese emerged from our case registry. In order to advance the understanding of Chinese metabolic diseases, we examined the molecular basis of those diseases with unique features in Chinese or were locally prevalent. Mutations were found in patients with primary carnitine deficiency, ornithine transcarbamylase deficiency, X-linked adrenoleukodystrophy, glutaric aciduria type I, and galactosemia. We also analyzed the mutations in multiple carboxylase deficiency and Niemann-pick type C on four families. Conclusions: Although IMDs are a significant cause of mortality and morbidity among pediatric patients, with a better understanding of the molecular genetics of these diseases, prenatal diagnosis of these common IMDs will be facilitated, which is currently the most effective way of controlling IMDs.

Evaluation of Urinary Acylglycines by Electrospray Tandem Mass Spectrometry in Mitochondrial Energy Metabolism Defects and Organic Acidurias

We analyzed the urinary acylglycine excretion in 26 patients with mitochondrial energy metabolism disorders and in 55 patients with organic acidurias by electrospray tandem mass spectrometry (ESI-MS/MS), monitoring precursor ions of m/z 90. Urinary concentrations of the different acylglycines were quantified using deuterated internal standards. Normal values for the most important acylglycines were established. In MCAD and MAD (neonatal form) deficiencies, typical excretion patterns of urinary acylglycines were found in all the samples. In isovaleric aciduria, propionic aciduria, and 3-methylcrotonylglycinuria typical glycine conjugates were always found. Methylmalonic aciduria (mutase deficiency), multiple carboxylase deficiency, and 3-hydroxy-3-methylglutaric aciduria revealed pathological acylglycine profiles, even if not specific for the disease. In all these diseases acylglycine excretion seems to be less influenced by the clinical status than organic acid excretion. This method is a useful diagnostic tool for these metabolic disorders, complementary to organic acids and acylcarnitine profiles.

Relationship between kinetic properties of mutant enzyme and biochemical and clinical responsiveness to biotin in holocarboxylase synthetase deficiency.

Holocarboxylase synthetase (HCS) deficiency is a metabolic disorder that causes a biotin-responsive multiple carboxylase deficiency. We analyzed the kinetic properties of seven mutant HCS proteins. Two of these enzymes harbored mutations within the putative biotin-binding region of HCS and showed elevated Km values for biotin compared with that of the wild-type form (Km mutant; Gly581Ser: 45 times, delThr610: 3 times). The remaining five mutations (Arg183Pro, Leu216Arg, Leu237Pro, Val333Glu, and Val363Asp) were located outside the biotin-binding region. The enzymes containing these mutations showed normal or low Km values for biotin (non-Km mutant). Symptoms of patients who have the non-Km, mutants, as well as those of patients who have the Km, mutants, responded to biotin therapy. This is probably because the Km value for biotin of normal HCS is higher than the physiologic concentration of biotin in human cells. The Vmax values of all mutant HCS proteins were considerably decreased, but to a variable degree. The responsiveness to biotin supplementation of propionyl-CoA carboxylase activity in cultured cells bearing the mutations correlated well with the degree of reduction in the Vmax of HCS. Patients who have mutant HCS proteins with lower Vmax showed poorer clinical and biochemical responses to biotin therapy. These observations suggest that the reduction of Vmax is an essential factor for pathophysiology and prognosis of HCS deficiency under treatment with large amounts of biotin. The determination of HCS genotype can be valuable for characterizing the clinical phenotype in HCS deficient patients.

Direct and transplacental intrauterine fetal therapy

Several approaches for an intrauterine fetal therapy have been reported in recent years for different congenital diseases. This article concentrates especially on the treatment of the congenital adrenal hyperplasia and other rare enzymatic defects, on abnormal fetal thyroid function and fetal tachyarrhythmia. Results of intrauterine treatment of congenital adrenal hyperplasia are optimal, when maternal dexamethason therapy is started very early in pregnancy, preferable at 6 weeks of gestation. Other rare congenital defects are methylmalonic acidemia, a functional vitamin B12 deficiency, and biotin-responsive multiple carboxylase deficiency. Transplacentar treatment was reported by administration of cyanocobalamin and biotin, respectively. Sonographic prenatal diagnosis of a goiter in fetuses with intrauterine hypothyreosis has been described previously. Treatment by injection of thyroxine into the amniotic fluid was successful and size of the fetal thyroid decreased. Fetuses are at risk of hyperthyreosis, if maternal thyroid stimulating antibodies cross the placenta. Presenting symptoms might be fetal tachycardia, general hydrops or even intrauterine fetal death. Initiation of prenatal therapy with propylthiouracil is recommended in these cases. Successful intrauterine therapy of fetal tachyarrhythmia has been reported using digoxin alone or in combination with flecainide. In most cases fetal hydrops resolved and therapy via cordocentesis was not necessary. However in fetuses with complete AV-block and general hydrops there is no standardised protocol for the intrauterine treatment in the literature.