Clinical, biochemical, and genetic analysis of 28 Chinese patients with holocarboxylase synthetase deficiency.

BACKGROUND: This study aimed to describe the clinical, biochemical, and molecular characteristics of Chinese patients with holocarboxylase synthetase (HLCS) deficiency, and to investigate the mutation spectrum of HCLS deficiency as well as their potential correlation with phenotype. METHODS: A total of 28 patients with HLCS deficiency were enrolled between 2006 and 2021. Clinical and laboratory data were reviewed retrospectively from medical records. RESULTS: Among the 28 patients, six patients underwent newborn screening, of which only one was missed. Therefore, 23 patients were diagnosed because of disease onset. Among all the patients, 24 showed varying degrees of symptoms such as rash, vomiting, seizures, and drowsiness, while only four cases remained asymptomatic nowadays. The concentration of 3-hydroxyisovalerylcarnitine (C5-OH) in blood and pyruvate, 3-hydroxypropionate, methylcitric acid, 3-hydroxyvaleric acid, 3-methylcrotonylglycine in urine were increased greatly among affected individuals. After prompt supplement of biotin, both the clinical and biochemical symptoms were dramatically resolved and nearly all patients developed normal intelligence and physique on follow-up. DNA sequencing revealed 12 known and 6 novel variants in the HLCS gene of patients. Among them, the variant of c.1522C > T was the most common. CONCLUSIONS: Our findings expanded the spectrum of phenotypes and genotypes for HLCS deficiency in Chinese populations and suggested that with timely biotin therapy, patients with HLCS deficiency showed low mortality and optimistic prognosis. Newborn screening is crucial for early diagnosis, treatment, and long-term outcomes.

Successful treatment with secukinumab of psoriasis-like dermatitis in a patient with holocarboxylase synthetase deficiency.

Holocarboxylase synthetase deficiency (HSD) is a rare autosomal recessive disorder of biotin metabolism. Typical manifestations include irreversible metabolic disorders and erythroderma-like dermatitis. Most patients respond well to biotin supplementation. Psoriasis-like phenotype associated with this disease has been rarely reported in the literature and experiences with the use of biologics in patients with HSD are still lacking. We reported a rare case of recurrent psoriasis-like skin lesions in a 6-year-old child with HSD. The patient did not respond to initial therapy with high-dose oral biotin. Immunofluorescence staining showed an increased number of interleukin (IL)-17A+ cells in his skin lesions. Based on this finding, the patient was successfully treated with human anti-IL-17A monoclonal antibody (secukinumab). He did not report any side effects and remained healthy during the 2-year follow-up. We provide a comprehensive review of the reported cases of HSD with psoriasis-like dermatitis to date. The psoriasis-like phenotype of HSD is controversial in treatment and IL-17A inhibitor is an alternative therapeutic option.

Antenatal and postnatal radiologic diagnosis of holocarboxylase synthetase deficiency: a systematic review.

BACKGROUND: Holocarboxylase synthetase deficiency results in impaired activation of enzymes implicated in glucose, fatty acid and amino acid metabolism. Antenatal imaging and postnatal imaging are useful in making the diagnosis. Untreated holocarboxylase synthetase deficiency is fatal, while antenatal and postnatal biotin supplementation is associated with good clinical outcomes. Although biochemical assays are required for definitive diagnosis, certain radiologic features assist in the diagnosis of holocarboxylase synthetase deficiency. OBJECTIVE: To review evidence regarding radiologic diagnostic features of holocarboxylase synthetase deficiency in the antenatal and postnatal period. MATERIALS AND METHODS: A systematic review of all published cases of holocarboxylase synthetase deficiency identified by a search of Pubmed, Scopus and Web of Science. RESULTS: A total of 75 patients with holocarboxylase synthetase deficiency were identified from the systematic review, which screened 687 manuscripts. Most patients with imaging (19/22, 86%) had abnormal findings, the most common being subependymal cysts, ventriculomegaly and intraventricular hemorrhage. CONCLUSION: Although the radiologic features of subependymal cysts, ventriculomegaly, intraventricular hemorrhage and intrauterine growth restriction may be found in the setting of other pathologies, these findings should prompt consideration of holocarboxylase synthetase deficiency in at-risk children.

K16-biotinylated histone H4 is overrepresented in repeat regions and participates in the repression of transcriptionally competent genes in human Jurkat lymphoid cells.

Holocarboxylase synthetase (HCS) catalyzes the binding of biotin to lysine (K) residues in histones H3 and H4. Histone biotinylation marks are enriched in repressed loci, including retrotransposons. Preliminary studies suggested that K16 in histone H4 is a target for biotinylation by HCS. Here we tested the hypotheses that H4K16bio is a real histone mark in human chromatin and that H4K16bio is overrepresented in repressed gene loci and repeat regions. Polyclonal rabbit anti-human H4K16bio was generated and affinity purified. An extensive series of testing with synthetic and natural targets confirmed that this new antibody is specific for H4K16bio. Using anti-H4K16bio and chromatin immunoprecipitation assays, we demonstrated that H4K16bio is overrepresented in repeat regions [pericentromeric alpha satellite repeats and long terminal repeats (LTR)] compared with euchromatin promoters. H4K16bio was also enriched in the repressed interleukin-2 gene promoter in human lymphoid cells; transcriptional activation of the interleukin-2 gene by mitogens and phorbol esters coincided with a depletion of the H4K16bio mark at the gene promoter. The enrichment of H4K16bio depended on biotin supply; the enrichment at LTR22 and promoter 1 of the sodium-dependent multivitamin transporter (SMVT) was greater in biotin-supplemented cells compared with biotin-normal and biotin-deficient cells. The enrichment of H4K16bio at LTR15 and SMVT promoter 1 was significantly lower in fibroblasts from an HCS-deficient patient compared with an HCS wild-type control. We conclude that H4K16bio is a real phenomenon and that this mark, like other biotinylation marks, is overrepresented in repressed loci where it marks HCS docking sites.

Effects of single-nucleotide polymorphisms in the human holocarboxylase synthetase gene on enzyme catalysis.

Holocarboxylase synthetase (HLCS) is a biotin protein ligase, which has a pivotal role in biotin-dependent metabolic pathways and epigenetic phenomena in humans. Knockdown of HLCS produces phenotypes such as heat susceptibility and decreased life span in Drosophila melanogaster, whereas knockout of HLCS appears to be embryonic lethal. HLCS comprises 726 amino acids in four domains. More than 2500 single-nucleotide polymorphisms (SNPs) have been identified in human HLCS. Here, we tested the hypotheses that HLCS SNPs impair enzyme activity, and that biotin supplementation restores the activities of HLCS variants to wild-type levels. We used an in silico approach to identify five SNPs that alter the amino acid sequence in the N-terminal, central, and C-terminal domains in human HLCS. Recombinant HLCS was used for enzyme kinetics analyses of HLCS variants, wild-type HLCS, and the L216R mutant, which has a biotin ligase activity near zero. The biotin affinity of variant Q699R is lower than that of the wild-type control, but the maximal activity was restored to that of wild-type HLCS when assay mixtures were supplemented with biotin. In contrast, the biotin affinities of HLCS variants V96F and G510R are not significantly different from the wild-type control, but their maximal activities remained moderately lower than that of wild-type HLCS even when assay mixtures were supplemented with biotin. The V96 L SNP did not alter enzyme kinetics. Our findings suggest that individuals with HLCS SNPs may benefit from supplemental biotin, yet to different extents depending on the genotype.

A novel molecular mechanism to explain biotin-unresponsive holocarboxylase synthetase deficiency.

Biotin (vitamins H and B7) is an important micronutrient as defects in its availability, metabolism or adsorption can cause serious illnesses, especially in the young. A key molecule in the biotin cycle is holocarboxylase synthetase (HLCS), which attaches biotin onto the biotin-dependent enzymes. Patients with congenital HLCS deficiency are prescribed oral biotin supplements that, in most cases, reverse the clinical symptoms. However, some patients respond poorly to biotin therapy and have an extremely poor long-term prognosis. Whilst a small number of mutations in the HLCS gene have been implicated, the molecular mechanisms that lead to the biotin-unresponsive phenotype are not understood. To improve our understanding of HLCS, limited proteolysis was performed together with yeast two-hybrid analysis. A structured domain within the N-terminal region that contained two missense mutations was identified in patients who were refractory to biotin therapy, namely p.L216R and p.L237P. Genetic studies demonstrated that the interaction between the enzyme and the protein substrate was disrupted by mutation. Further dissection of the binding mechanism using surface plasmon resonance demonstrated that the mutations reduced affinity for the substrate through a >15-fold increase in dissociation rate. Together, these data provide the first molecular explanation for HLCS-deficient patients that do not respond to biotin therapy.

Holocarboxylase synthetase deficiency: novel clinical and molecular findings.

Multiple carboxylase deficiency (MCD) is an autosomal recessive metabolic disorder caused by defective activity of biotinidase or holocarboxylase synthetase (HLCS) in the biotin cycle. Clinical symptoms include skin lesions and severe metabolic acidosis. Here, we reported four unrelated Thai patients with MCD, diagnosed by urine organic acid analysis. Unlike Caucasians, which biotinidase deficiency has been found to be more common, all of our four Thai patients were affected by HLCS deficiency. Instead of the generally recommended high dose of biotin, our patients were given biotin at 1.2 mg/day. This low-dose biotin significantly improved their clinical symptoms and stabilized the metabolic state on long-term follow-up. Mutation analysis by polymerase chain reaction-sequencing of the entire coding region of the HLCS gene revealed the c.1522C>T (p.R508W) mutation in six of the eight mutant alleles. This suggests it as the most common mutation in the Thai population, which paves the way for a rapid and unsophisticated diagnostic method for the ethnic Thai. Haplotype analysis revealed that the c.1522C>T was on three different haplotypes suggesting that it was recurrent, not caused by a founder effect. In addition, a novel mutation, c.1513G>C (p.G505R), was identified, expanding the mutational spectrum of this gene.

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.

Carnitine transporter and holocarboxylase synthetase deficiencies in The Faroe Islands.

Carnitine transporter deficiency (CTD) and holocarboxylase synthetase deficiency (HLCSD) are frequent in The Faroe Islands compared to other areas, and treatment is available for both disorders. In order to evaluate the feasibility of neonatal screening in The Faroe Islands we studied detection in the neonatal period by tandem mass spectrometry, carrier frequencies, clinical manifestations, and effect of treatment of CTD and HLCSD. We found 11 patients with CTD from five families and 8 patients with HLCSD from five families. The natural history of both disorders varied extensively among patients, ranging from patients who presumably had died from their disease to asymptomatic individuals. All symptomatic patients responded favourably to supplementation with L: -carnitine (in case of CTD) or biotin (in case of HLCSD), but only if treated early. Estimates of carrier frequency of about 1:20 for both disorders indicate that some enzyme-deficient individuals remain undiagnosed. Prospective and retrospective tandem mass spectrometry (MS/MS) analyses of carnitines from neonatally obtained filter-paper dried blood-spot samples (DBSS) uncovered 8 of 10 individuals with CTD when using both C(0) and C(2) as markers (current algorithm) and 10 of 10 when using only C(0) as marker. MS/MS analysis uncovered 5 of 6 patient with HLCSD. This is the first study to report successful neonatal MS/MS analysis for the diagnosis of HLCSD. We conclude that CTD and HLCSD are relatively frequent in The Faroe Islands and are associated with variable clinical manifestations, and that diagnosis by neonatal screening followed by early therapy will secure a good outcome.

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.

Partial response to biotin therapy in a patient with holocarboxylase synthetase deficiency: clinical, biochemical, and molecular genetic aspects.

We report the clinical course and biochemical findings of a 10-year-old, mentally retarded girl with late-onset holocarboxylase synthetase (HCS, gene symbol HLCS) deficiency and only partial response to biotin. On treatment, even with an unusually high dose of 200mg/day, activities of the biotin-dependent mitochondrial carboxylases in lymphocytes remained below 50% of the mean control values. Not only urinary 3-hydroxyisovaleric acid excretion has been persistently elevated, but also plasma and, with even higher concentrations, cerebrospinal fluid 3-hydroxyisovaleric acid have not normalized. The unusual and insufficient response of this patient to biotin treatment can be explained by the effect of the combination of the common HLCS allele IVS10 +5 g>a on one chromosome and a truncating mutation on the other. This case illustrates mechanisms involved in the genotype-phenotype correlation that unequivocally exists in HCS deficiency.

Clinical findings and biochemical and molecular analysis of four patients with holocarboxylase synthetase deficiency.

Holocarboxylase synthetase (HLCS) deficiency (HLCSD) is a rare autosomal recessive disorder of biotin metabolism. HLCS catalyzes the biotinylation of the four human biotin-dependent carboxylases. Using the newly available human genomic sequence, we report the map of HLCS genomic structure and the predicted exon/intron boundaries. Moreover, the molecular studies of four patients (two Italians, one Iranian, and one Australian) affected by HLCS deficiency are here reported. The clinical findings, the age of onset, and response to biotin treatment differed between our patients. The diagnosis was made by organic acid analysis and confirmed by enzymatic analysis in three patients. Six mutations in the HLCS gene were identified, including two novel (N511K and G582R) and four known missense mutations (L216R, R508W, V550M, and G581S). Five of the mutations are localized within the HLCS biotin-binding domain, whereas the L216R amino acid change is located in the N-terminal region outside of the putative biotin-binding domain. This mutation, previously reported in a heterozygous state, was detected for the first time in a patient with homozygous status. The patient's severe clinical phenotype and partial responsiveness to biotin support a genotype-phenotype correlation through the involvement of residues of the N-terminal region in a substrate specificity recognition or regulation of the HLCS enzyme.