[Gene variant analysis of a patient with multiple carboxylase deficiency].

OBJECTIVE: To explore the genetic basis for a patient featuring multiple carboxylase deficiency (MCD). METHODS: PCR and Sanger sequencing were used to detect variant in the coding region of BT and HLCS genes in the patient. Suspected variants were verified in her parents and 80 unrelated healthy controls by a PCR-restriction fragment length polymorphism (PCR-RFLP) method. RESULTS: The patient was found to carry compound heterozygous variants of the HLCS gene, namely c.286delG (p.Val96Leufs*162) and c.1648G>A (p.Val550Met). The c.286delG (p.Val96Leufs*162) was verified to be novel variant based on the result of PCR-RFLP analysis. No variant was found in the coding regions of BT gene in the patient. CONCLUSION: The compound c.286delG (p.Val96Leufs*162) and c.1648G>A (p.Val550Met) variants probably underlie the MCD disorder in this patient. Above results have enriched the variant spectrum of MCA.

Genetic mimics of cerebral palsy.

The term "cerebral palsy mimic" is used to describe a number of neurogenetic disorders that may present with motor symptoms in early childhood, resulting in a misdiagnosis of cerebral palsy. Cerebral palsy describes a heterogeneous group of neurodevelopmental disorders characterized by onset in infancy or early childhood of motor symptoms (including hypotonia, spasticity, dystonia, and chorea), often accompanied by developmental delay. The primary etiology of a cerebral palsy syndrome should always be identified if possible. This is particularly important in the case of genetic or metabolic disorders that have specific disease-modifying treatment. In this article, we discuss clinical features that should alert the clinician to the possibility of a cerebral palsy mimic, provide a practical framework for selecting and interpreting neuroimaging, biochemical, and genetic investigations, and highlight selected conditions that may present with predominant spasticity, dystonia/chorea, and ataxia. Making a precise diagnosis of a genetic disorder has important implications for treatment, and for advising the family regarding prognosis and genetic counseling. © 2019 International Parkinson and Movement Disorder Society.

Biotin in metabolism, gene expression, and human disease.

Biotin is a water-soluble vitamin that belongs to the vitamin B complex and which is an essential nutrient of all living organisms from bacteria to man. In eukaryotic cells biotin functions as a prosthetic group of enzymes, collectively known as biotin-dependent carboxylases that catalyze key reactions in gluconeogenesis, fatty acid synthesis, and amino acid catabolism. Enzyme-bound biotin acts as a vector to transfer a carboxyl group between donor and acceptor molecules during carboxylation reactions. In recent years, evidence has mounted that biotin also regulates gene expression through a mechanism beyond its role as a prosthetic group of carboxylases. These activities may offer a mechanistic background to a developing literature on the action of biotin in neurological disorders. This review summarizes the role of biotin in activating carboxylases and proposed mechanisms associated with a role in gene expression and in ameliorating neurological disease.

Biochemical signatures mimicking multiple carboxylase deficiency in children with mutations in MT-ATP6.

Elevations of specific acylcarnitines in blood reflect carboxylase deficiencies, and have utility in newborn screening for life-threatening organic acidemias and other inherited metabolic diseases. In this report, we describe a newly-identified association of biochemical features of multiple carboxylase deficiency in individuals harboring mitochondrial DNA (mtDNA) mutations in MT-ATP6 and in whom organic acidemias and multiple carboxylase deficiencies were excluded. Using retrospective chart review, we identified eleven individuals with abnormally elevated propionylcarnitine (C3) or hydroxyisovalerylcarnitine (C5OH) with mutations in MT-ATP6, most commonly m.8993T>G in high heteroplasmy or homoplasmy. Most patients were ascertained on newborn screening; most had normal enzymatic or molecular genetic testing to exclude biotinidase and holocarboxylase synthetase deficiencies. MT-ATP6 is associated with some cases of Leigh disease; clinical outcomes in our cohort ranged from death from neurodegenerative disease in early childhood to clinically and developmentally normal after several years of follow-up. These cases expand the biochemical phenotype associated with MT-ATP6 mutations, especially m.8993T>G, to include acylcarnitine abnormalities mimicking carboxylase deficiency states. Clinicians should be aware of this association and its implications for newborn screening, and consider mtDNA sequencing in patients exhibiting similar acylcarnitine abnormalities that are biotin-unresponsive and in whom other enzymatic deficiencies have been excluded.

[Gene mutation analyses in Chinese children with multiple carboxylase deficiency].

OBJECTIVE: To confirm the diagnosis of multiple carboxylase deficiency (MCD) on the gene level and explore the mutations in Chinese children with MCD. METHODS: Biotinidase (BT) and holocarboxylase synthetase (HLCS) genes were analyzed by PCR and direct sequencing for the 4 BT deficiency patients and 8 HLCS deficiency patients, respectively. The identified mutations in the parents of the patients and 50 normal controls were screened by PCR-restriction fragment length polymorphism and direct DNA sequencing. RESULTS: Total detection rate of gene mutation is 100% in the 12 children with MCD. Six mutations were detected in the 4 children with BT deficiency, they were c. 98-104del7ins3, c. 1369G>A (V457M), c. 1157G>A(W386X), c. 1284C>A(Y428X), c. 1384delA and c. 1493_1494insT. The last four were novel mutations. Four mutations were found in the 8 children with HLCS deficiency. They were c. 126G>T (E42D), c. 1994G>C (R665P), c. 1088T>A (V363D) and c. 1522C>T (R508W). The last two were hot-spot mutations [75%(12/16)], and c. 1994G>C (R665P) was a novel mutation. CONCLUSION: This study confirmed the diagnosis of 12 patients with MCD on the gene level. Six mutations were found in the BT gene and 4 in the HLCS gene, including 5 novel mutations. Two mutations of the HLCS gene are probably hot-spot mutations in Chinese children with HLCS deficiency.

A case of holocarboxylase synthetase deficiency with insufficient response to prenatal biotin therapy.

Holocarboxylase synthetase (HCS) deficiency is an inborn error of biotin metabolism, leading to a multiple carboxylases deficiency. As the affected fetus sometimes presents with enlargement of the cerebral ventricles and intrauterine growth retardation (IUGR), prenatal administration of biotin has been attempted in some pregnancies. We present herein the case of a Japanese neonate with HCS deficiency who received maternal administration of biotin (10mg/day) from 33 weeks' gestation. After biotin administration, the fetal body weight increased and gestation was continued to full term. However, lactic acidemia and metabolic acidosis were observed after birth. To evaluate the effects of prenatal therapy, we collected serum samples and measured the acylcarnitine profiles using high-performance liquid chromatography electrospray ionization tandem mass spectrometry. At birth, levels of propionylcarnitine and 3-hydroxyisovalerylcarnitine had already increased. At 2h after birth, these levels of acylcarnitines were further increased. At 3.5h after the start of biotin, these chemical findings were slightly improved. In conclusion, we considered that prenatal biotin therapy at 10mg/day may have been inadequate to avoid neonatal acidotic crisis in this case.

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.

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.

Biotin in metabolism and its relationship to human disease.

Biotin, a water-soluble vitamin, is used as cofactor of enzymes involved in carboxylation reactions. In humans, there are five biotin-dependent carboxylases: propionyl-CoA carboxylase; methylcrotonyl-CoA carboxylase; pyruvate carboxylase, and two forms of acetyl-CoA carboxylase. These enzymes catalyze key reactions in gluconeogenesis, fatty acid metabolism, and amino acid catabolism; thus, biotin plays an essential role in maintaining metabolic homeostasis. In recent years, biotin has been associated with several diseases in humans. Some are related to enzyme deficiencies involved in biotin metabolism. However, not all biotin-responsive disorders can be explained based on the classical role of the vitamin in cell metabolism. Several groups have suggested that biotin may be involved in regulating transcription or protein expression of different proteins. Biotinylation of histones and triggering of transduction signaling cascades have been suggested as underlying mechanisms behind these non-classical biotin-deficiency manifestation in humans.

[Importance of biotin metabolism]. / Importancia del metabolismo de la biotina.

Biotin is a water soluble enzyme cofactor that belongs to the vitamin B complex. In humans, biotin is involved in important metabolic pathways such as gluconeogenesis, fatty acid synthesis, and amino acid catabolism by acting a as prosthetic group for pyruvate carboxylase, propionyl-CoA carboxylase, beta-methylcrotinyl-CoA carboxylase, and acetyl-CoA carboxylase. Carboxylases are synthesized as apo-carboxylases without biotin and the active form is produced by their covalent binding of biotin to the epsilon-amino group of a lysine residue of the apocarboxylases. This reaction is catalyzed by the holo-carboxylase synthetase. The last step in the degradation of carboxylases, the cleavage of the biotinyl moiety from the epsilon-amino group lysine residues, is catalyzed by biotinidase and results in the release of free biotin, which can be recycled. Biotin regulates the catabolic enzyme propionyl-CoA carboxylase at the posttranscriptional level whereas the holo-carboxylase synthetase is regulated at the transcriptional level. Aside from its role in the regulation of gene expression of carboxylases, biotin has been implicated in the induction of the receptor for the asialoglycoprotein, glycolytic enzymes and of egg yolk biotin binding proteins. Biotin deficiency in humans is extremely rare and is generally associated with prolonged parenteral nutrition, the consumption of large quantities of avidin, usually in the form of raw eggs, severe malnutrition and, inherited metabolic disorders. In humans, there are autosomal recessive disorders of biotin metabolism that result from the disruption of the activity of biotinidase or holo-carboxylase synthetase.

Mutations causing profound biotinidase deficiency in children ascertained by newborn screening in the United States occur at different frequencies than in symptomatic children.

Biotinidase deficiency is an autosomal recessive disorder of biotin metabolism that can lead to varying degrees of neurologic and cutaneous symptoms when untreated. Because this disorder meets the criteria for newborn screening, many states and countries perform this testing. Because newborn screening should result in complete ascertainment of mutations causing profound biotinidase deficiency (less than 10% of mean normal serum activity), we compared the mutations in a group of 59 children with profound biotinidase deficiency who were identified by newborn screening in the United States with 33 children ascertained by exhibiting symptoms. Of the 40 total mutations identified among the two populations, four mutations comprise 59% of the disease alleles studied. Two of these mutations occur in both populations, but in the symptomatic group at a significantly greater frequency. The other two common mutations occur only in the newborn screening group. Because two common mutations do not occur in the symptomatic population, it is possible that individuals with these mutations either develop mild or no symptoms if left untreated. However, inasmuch as biotin treatment is inexpensive and innocuous, it is still recommended that all children with profound biotinidase deficiency be treated.

Mechanism of biotin responsiveness in biotin-responsive multiple carboxylase deficiency.

Holocarboxylase synthetase (HCS) catalyses the biotinylation of the four biotin-dependent carboxylases found in humans. A deficiency in HCS results in biotin-responsive multiple carboxylase deficiency. We have evaluated the biotin responsiveness associated with six missense mutations previously identified in affected patients by expression of plasmids containing the mutated HCS in an Escherichia coli strain mutated in the corresponding BirA gene. We demonstrate that the mutations identified in the MCD patients are indeed responsible for their reduced HCS activity. Four of the mutations, clustering in the putative biotin binding domain as deduced from the structure of the E. coli enzyme, are consistent with an explanation for biotin responsiveness based on altered affinity for biotin. The remaining mutations, located outside the biotin binding region, were associated with a more limited biotin responsiveness that may be explained by the degree of residual enzyme activity present. The data suggest that the concentration of circulating biotin is as low as 100 times below the Km of the enzyme, so that any increase in biotin concentration through dietary supplementation would result in saturation of the available mutant enzyme. We suggest that these alternative explanations are sufficient to account for the apparent universality of biotin responsiveness in biotin responsive multiple carboxylase deficiency.

Profound biotinidase deficiency caused by a point mutation that creates a downstream cryptic 3' splice acceptor site within an exon of the human biotinidase gene.

Biotinidase recycles the vitamin biotin from biocytin upon the degradation of the biotin-dependent carboxylases. We have identified a novel point mutation within the biotinidase gene that encodes the signal peptide in two unrelated individuals with profound biotinidase deficiency. Sequence analysis of genomic DNA from these individuals revealed a G to A transition (G100-->A) located 57 bases downstream of the authentic splice acceptor site in exon B. Although this mutation predicts a G34S substitution, it also generates a 3' splice acceptor site. Sequence of the PCR-amplified cDNA from the homozygous child revealed that all the product was shorter than that of normal individuals and was the result of aberrant splicing. The aberrantly spliced transcript lacked 57 bases, including a second in-frame ATG, that encode most of the putative signal peptide and results in an in-frame deletion of 19 amino acids. The mutation results in failure to secrete the aberrant protein into the blood. This is the first reported example in which a point mutation creates a cryptic 3' splice acceptor site motif that is used preferentially over the upstream authentic splice site. The preferential usage of the downstream splice site is not consistent with the 5'-3' scanning model, but is consistent with the exon definition model of RNA splicing.

Biotinidase deficiency: result of treatment with biotin from age 12 years.

A boy with severe symptoms of biotinidase deficiency diagnosed at the age of 12 years showed a remarkable improvement of his neurological picture and normalization of brain magnetic resonance imaging abnormalities when prescribed oral biotin.

Clustering of mutations in the biotin-binding region of holocarboxylase synthetase in biotin-responsive multiple carboxylase deficiency.

Holocarboxylase synthetase (HCS) catalyses the biotinylation of the four biotin-dependent carboxylases found in humans. A deficiency in HCS results in biotin-responsive multiple carboxylase deficiency (MCD). We have identified six different point mutations in the HCS gene in nine patients with MCD. Two of the mutations are frequent among the MCD patients analyzed. Four of the mutations cluster in the putative biotin-binding domain as deduced from the corresponding Escherichia coli enzyme and consistent with an explanation for biotin-responsiveness based on altered affinity for biotin. The two others may define an additional domain involved in biotin-binding or biotin-mediated stabilization of the protein.

Combined pedigree and twin family study to determine the sources of variation in serum biotinidase activity: the usefulness of multiple study designs.

Biotinidase, the enzyme responsible for recycling the vitamin biotin, is deficient in most individuals with late-onset multiple carboxylase deficiency. Based on clinical criteria, biotinidase deficiency appears to be inherited as an autosomal recessive trait; however, the inheritance of biotinidase serum activity as a quantitative trait has not been studied previously. In this study, both segregation analysis of proband families and the analysis of twin family data were used to determine the relative contributions of a major gene, polygenes and environment to the variation in serum biotinidase activity. Segregation analysis of 24 families of biotinidase-deficient individuals indicated that serum biotinidase activity is determined by the segregation of a single codominant major gene with the variability about the mean of each major genotype attributable to environmental effects. Significant polygenic effects could not be detected by this analysis. Variance component analysis of 128 twin families, which included the twins, their spouses, and their offspring, indicated that 70% of total variance in biotinidase activity is attributable to additive genetic effects, 22% to individual environmental effects, and 8% to shared environmental effects. The model also included an age effect for females. A portion (27%) of the estimated additive variance may be attributed to the segregation of the major gene. This study emphasizes the usefulness of studying multiple data sets representing different types of family relationships.