Clinical manifestations

Presentation and course

More than 75 children with holocarboxylase synthetase deficiency have been described (54; 56; 07; 19; 30; 52; 88; 47; 62; 72; 09; 21; 15; 16; 37; 23; 65; 66; 44; 14; 33). These children usually develop symptoms in the newborn period, but some have become symptomatic at several months of age; one patient presented at 20 months of age (77). They often exhibit respiratory abnormalities, such as tachypnea, hyperventilation, or apnea, as well as feeding difficulties, hypotonia or hypertonia, seizures, lethargy, and irritability. The disorder can ultimately result in developmental delay and coma. Many children develop rashes and several have had alopecia. One child exhibited dermatological features of ichthyosis (03). A child with holocarboxylase synthetase deficiency had a psoriasis-like dermatitis that markedly improved with the anti-IL-17A monoclonal antibody (secukinumab) (36). A few patients have had ataxia, tremors, hypothermia, and hyporeflexia or hyperreflexia. Abnormal urine odor was noted in several patients. One affected child had a history of bacteremia. In a retrospective review of 75 children with holocarboxylase deficiency, subependymal cysts, ventriculomegaly, intrauterine growth retardation, and intraventricular hemorrhage were seen in antenatal and postnatal radiograms (05). The disorder should be considered in the differential diagnosis of children with one or more of these features.

Siblings of several of the children, who undoubtedly had the same disorder, had become comatose and died before a diagnosis was made or appropriate treatment started. Holocarboxylase synthetase deficiency should be considered in children presenting with seizures in the newborn period or early infancy and failing to respond to routine anticonvulsive therapies.

An adult with holocarboxylase synthetase deficiency exhibited a psoriasis-like dermatitis (81).

Prognosis and complications

Children with holocarboxylase synthetase deficiency who have been diagnosed early and have had an early start on biotin therapy have done well. The oldest patients with this disorder, who were diagnosed soon after birth, are now almost teenagers. Patients who have had multiple episodes of metabolic compromise prior to diagnosis and treatment have frequently suffered permanent neurologic damage. Compliance with biotin therapy has been a problem in one patient.

Clinical vignette

A 1-week-old female was brought to the emergency department with a new onset of myoclonic seizures. She had been experiencing vomiting and lethargy for one day. The child had metabolic acidosis with an arterial blood pH of 7.20, a bicarbonate concentration of 12 meq/L, and a base excess of 23. She also had mild hyperammonemia, increased glycine concentration on amino acid analysis, and ketones in the urine. Urinary organic acids showed elevated concentrations of lactate, beta-hydroxyisovalerate, methylcitrate, and beta-methylcrotonylglycine, consistent with the diagnosis of multiple carboxylase deficiency. She was treated with 10 mg of biotin, and the seizures stopped within two hours. Within six hours she became more alert and active. Serum biotinidase activity was normal. The activities of the mitochondrial carboxylase, propionyl-CoA carboxylase, pyruvate carboxylase, and beta-methylcrotonyl-CoA carboxylase were all deficient in cultured skin fibroblast incubated in a medium containing low concentrations of biotin. The carboxylase activities became normal when the fibroblasts were incubated in a medium containing increased concentrations of biotin. Specific assay of biotin holocarboxylase synthetase activity confirmed the deficiency. The child was continued on the biotin and thrived without any further episodes of neurologic or biochemical compromise.

Biological basis

Etiology and pathogenesis

Biotin, a water-soluble B-complex vitamin, is the coenzyme for four carboxylases in humans that have important roles in gluconeogenesis, fatty acid synthesis, and the catabolism of several branched-chain amino acids. Biotin is covalently attached to the various apocarboxylases by the same enzyme biotin holocarboxylase synthetase (45). The carboxyl group of biotin is linked by an amide bond to an e-amino group of a specific lysine residue of the apoenzyme. This reaction occurs through two partial reactions. The first is the adenosine triphosphate-requiring phosphorylation of biotin resulting in biotinyl 5-adenosine monophosphate. In the second partial reaction this intermediate product reacts with apocarboxylases to give biotinylated holoenzyme. There is evidence that biotin plays a regulatory role in controlling the transcription of the synthetase through a signaling mechanism that requires guanylate cyclase and cGMP-dependent protein kinase (64). Some studies indicate that, in addition to the mitochondrial and cytosolic localization of holocarboxylase synthetase, the enzyme localizes to the nucleus where it likely biotinylates histones (46). This is supported by decreased biotinylation of histones in fibroblasts from individuals with holocarboxylase synthetase deficiency. In addition to its role in intermediary metabolism, holocarboxylase synthetase has been shown to be involved in gene regulation, possibly as a nuclear receptor repressor (26; 35; 95). Studies indicate that biotin holocarboxylase synthetase has a potential role in histone biotinylation and is involved in chromatic dynamics as a nuclear factor (32). The authors suggest that these nuclear roles are separate functions from the enzyme’s biotin-dependent cytosolic metabolic roles.

The primary enzyme defect, biotin holocarboxylase synthetase deficiency, was demonstrated by several laboratories at about the same time (10; 22; 61). Studies of a group of patients with holocarboxylase synthetase deficiency revealed that they have all had elevated Michaelis constant values of biotin, ranging from 3 to 70 times that of normal, and that the age of onset of symptoms in the patients correlated negatively with the Michaelis constant values (11). These results suggested that the kinetic properties of the synthetase in vitro correlated with the degree of increase in carboxylase activity. In one report, however, a patient with the highest Michaelis constant of biotin for the enzyme had a mild clinical course. Some differences were noted in the stability of the mutant enzymes, suggesting biochemical heterogeneity in the disorder. Of the patients studied thus far, all have had residual synthetase activity in their tissues, a finding that suggests that total deficiency of the enzyme may be lethal prenatally.

Holocarboxylase synthetase deficiency is inherited as an autosomal recessive trait. Both males and females have been affected. Children in the same family have died of symptoms similar to those of their siblings, in whom the diagnosis has been confirmed. Consanguinity has been reported in one family. Evaluation of parents of patients with holocarboxylase synthetase deficiency has not yielded activities in the range intermediate between that of deficient and normal individuals in leukocytes or fibroblasts. This is probably because the enzymatic assay is not sufficiently sensitive to discriminate between these two groups (11). Therefore, there is no available enzymatic testing for heterozygosity. Bovine holocarboxylase synthetase has been purified to homogeneity (13). The cDNA for human holocarboxylase synthetase has been cloned and sequenced (68; 31), and the gene has been localized to chromosome 21q22.1 (68). The first molecular defects identified that cause holocarboxylase synthetase deficiency are a single base deletion and a nonsense mutation (68). Other mutations have also been identified, especially in the biotin-binding region of the enzyme (02; 18; 48; 57; 58; 28; 59; 93; 44; 74). Mutations outside the biotin-binding region of the synthetase result in enzymes with normal Km values for biotin but low Vmax values (58). Haplotype analyses in Japanese children with the disorder have revealed two mutations that appear to be founder mutations (92). Children with these latter mutations usually respond poorly to biotin treatment. A child homozygous for the L216R mutation, which is usually associated with a poor outcome even with biotin supplementation, can do better clinically if diagnosed early and treated with a daily dose of as much as 1.2 mg of biotin (63). Mutational analysis of seven children with holocarboxylase synthetase deficiency from Europe and the Middle East revealed seven different novel mutations (01). One child with an abnormal gene splicing mutation resulting in a moderate decrease in normal synthetase mRNA did not exhibit symptoms until he was eight years old (59). Mutation analysis may be helpful in predicting the clinical phenotype of the disorder (58). A child was reported with partial response to biotin at a dose of 200 mg/day (60). Even with this extremely high dose of biotin, her carboxylase activities increased to less than 50% of mean normal activity. She also continued to have elevated organic acid metabolites in her urine, plasma, and cerebrospinal fluid. These findings are similar to those of another child reported previously (88). Failure of improvement or limited responsiveness to biotin therapy may be due to mutations in the enzyme that reduce the affinity of the substrate to interact, particularly in a structured domain within the N-terminal region of the enzyme (40). A case of holocarboxylase synthetase deficiency with normal pyruvate carboxylase activity in the lymphocytes was reported in an 8-year-old girl with clinical toxicity without the classic dermatological involvement (79).

Multiple variants that cause holocarboxylase synthetase deficiency have been described (70; 80; 27; 91). Mutations are located across the gene. Several mutations that occur in individuals from the Faroe Islands (prevalence is 10 times that in the rest of the world) and from Japan are likely due to founder effects. Clinical biotin-responsiveness correlates positively with residual holocarboxylase activity. Children with holocarboxylase synthetase deficiency from Samoa have a form of the disorder that is only partially responsive to biotin, and these children usually have a poor outcome (83). In a study of an affected child with incomplete responsiveness to biotin therapy, the turnover rate of the aberrant enzyme was shown to be twice that of normal and offers an explanation for the incomplete responsiveness to biotin (04). In addition, an individual with holocarboxylase synthetase deficiency was found to have a paracentric inversion of chromosome 21 involving a deletion in the holocarboxylase synthetase gene on one allele and a missense mutation in the gene on the other allele (53). There have been multiple reports of Chinese children with novel pathogenic variants in the HCS gene (90; 89; 96). One of these children also had hearing loss, which appears to be rare in this disorder (96). However, hearing evaluation should be considered in these individuals. In a study of 28 children found to have holocarboxylase synthetase deficiency in China, most were identified symptomatically with metabolic compromise, whereas only five were ascertained by newborn screening (34). The clinical and biochemical symptoms resolved with biotin therapy in almost individuals.

Deficient holocarboxylase synthetase activity results in failure to biotinylate the various biotin-dependent carboxylases, so that an affected individual develops multiple carboxylase deficiency. Subsequently, the results are the accumulation of organic acid metabolites that are commonly seen in each of the isolated carboxylase deficiencies. Patients with holocarboxylase synthetase deficiency are not biotin deficient. Biotin holocarboxylase synthetase is involved in the regulation of its own mRNA concentrations (50). In a biotin-deficient state, the synthetase is down-regulated in the liver, whereas it is constitutively expressed in the brain. This mechanism may spare functions of biotin in the brain compared to those in other organs, such as liver and kidney. In holocarboxylase synthetase deficiency, some of the symptoms of the disorder may be due to this down-regulation in liver, and biotin supplementation may be important to overcoming this effect.

Children with holocarboxylase synthetase deficiency have all exhibited metabolic ketoacidosis and organic aciduria. Most of those who were evaluated had hyperammonemia. The metabolic acidosis is usually accompanied by an increased anion gap and elevated lactate. The ketosis is due to the accumulation of abnormal organic acid metabolites, beta-hydroxypropionate, methylcitrate, beta-hydroxyisovaleric acid, beta-methylcrotonylglycine, and beta-hydroxybutyrate in urine (72). Hyperammonemia plays a major role in causing the lethargy, somnolence, and coma seen in the disorder. The hyperammonemia is due to the secondary inhibition of N-acetylglutamate synthetase, which is the producer of N-acetylglutamate, the activator of carbamyl phosphate synthetase in the urea cycle.

Immunological studies in one patient prior to biotin treatment revealed the absence of lymphocytic response to phytohemagglutinin in vitro. The response was restored after biotin was added to the culture medium. Abnormalities in immunological function were attributed to the effects of accumulations of abnormal metabolites.

EEG studies of three patients were all diffusely abnormal, with one patient showing burst-suppression activity that improved markedly on biotin therapy (84). CT scan of the head has been reported in two children: one was normal, and the other revealed low-density changes scattered throughout the white matter, with loss of demarcation between the white and gray matter, mild to moderate ventricular dilation, and cerebral atrophy (84).

Epidemiology

Fourteen children with holocarboxylase synthetase deficiency have been reported, and 20 patients are known. Because only a small number of patients have been reported, racial and ethnic distributions are not known.

Prevention

Early initiation of biotin therapy has prevented or ameliorated clinical symptoms and metabolic compromise in all patients.

Prenatal diagnosis of holocarboxylase synthetase deficiency has been performed by demonstrating deficient activities of the mitochondrial carboxylases in cultured amniocytes (29). When these cells were cultured in medium supplemented with biotin, the carboxylase activities increased to near-normal or normal. In addition, elevations of beta-hydroxyisovalerate or methylcitrate have been demonstrated in the amniotic fluid of affected fetuses using stable isotope dilution (29). Although this latter method is a simpler and more rapid method for prenatal diagnosis, both diagnostic procedures can be performed to confirm the diagnosis using the same sample of amniotic fluid. Prenatal diagnosis was successfully performed by measuring holocarboxylase synthetase activity in chorionic villi (75).

Prenatal treatment of holocarboxylase synthetase deficiency has been performed in four separate at-risk pregnancies (51; 55; 75; 76). The mothers were treated with 10 mg of biotin orally, one from the 34th week of gestation, one from the 23rd week of gestation, and two throughout pregnancy. In all cases, the children were asymptomatic at birth, although in one pregnancy the child became symptomatic after biotin was withheld for a short period of time. The child's condition returned to normal once biotin treatment was restarted. The diagnosis was confirmed enzymatically in all children. Molecular prenatal diagnosis using a chorionic villus sample from an at-risk pregnancy has been performed (39). This method is more rapid and can be performed earlier than other prenatal diagnostic techniques. All children have remained asymptomatic on biotin therapy (42; 75; 76). Because biotin therapy initiated at birth apparently prevents the development of symptoms in affected newborns, it is unclear if prenatal treatment of affected children is necessary. A mother pregnant with a child with holocarboxylase synthetase deficiency was treated with 10 mg of biotin per day from 33 weeks gestation (94). However, the baby had metabolic lactic acidosis at birth, with elevated 3-hydroxyisovalerylcarnitine; the latter increased after several hours. The authors concluded that prenatal biotin therapy at 10 mg/day may not be adequate in all cases to prevent metabolic compromise in an affected infant soon after birth. Elevated hydroxyisovalerylcarnitine was found on newborn screening and on repeat testing of an unaffected infant born to a woman with holocarboxylase synthetase deficiency who was treated with biotin (49). Urinary organic acid analysis was normal at 39 days of age. Such a scenario can yield a false-positive result.

Most, but not all, children with holocarboxylase synthetase deficiency can be identified by newborn screening using tandem mass spectroscopy (38). However, it is possible that an affected individual with the deficiency can have a normal newborn screen (17).

Differential diagnosis

Nonspecific clinical symptoms include vomiting, hypotonia, and seizures, and are often characteristic of treatable disorders, such as sepsis, gastrointestinal obstruction, and cardiorespiratory problems. Concomitantly, after exclusion of these conditions, or when these findings are accompanied by metabolic ketoacidosis or hyperammonemia, the presence of an inborn error of metabolism should be considered. Both holocarboxylase synthetase deficiency and biotinidase deficiency may present initially with these clinical features, and both have been misdiagnosed as other disorders before they were correctly identified (67; 08).

Other symptoms characteristic of biotin-responsive multiple carboxylase deficiencies, such as rash or alopecia, can occur in children with zinc deficiency or essential fatty acid deficiency. Frequent viral, bacterial, or fungal infections due to immunological dysfunction may occur in multiple carboxylase deficiency. Children with holocarboxylase synthetase deficiency may have metabolic acidosis and large anion gaps with elevated concentrations of lactate in the serum and urine. An amino acid analysis may reveal hyperglycinemia, which is also found in other organic acidemias.

Holocarboxylase synthetase deficiency must be differentiated from the isolated carboxylase deficiencies. Urinary organic acid analysis is useful for differentiating isolated carboxylase deficiencies from the biotin-responsive multiple carboxylase deficiencies (08). Beta-hydroxyisovalerate is the most common urinary metabolite observed in holocarboxylase synthetase deficiency, biotinidase deficiency, and acquired biotin deficiency, but it is also seen in isolated beta-methylcrotonyl-coenzyme A carboxylase deficiency. Elevated concentrations of urinary lactate, methylcitrate and beta-hydroxypropionate, in addition to beta-hydroxyisovalerate and beta-methylcrotonylglycine, are indicative of multiple carboxylase deficiency. The isolated carboxylases are not biotin-responsive, whereas the multiple carboxylase deficiencies are. A trial of biotin is useful in discriminating the disorders. Isolated carboxylase deficiencies can be excluded by demonstrating deficient enzyme activity of one of the three mitochondrial carboxylases in peripheral blood leukocytes (prior to biotin therapy), or in cultured fibroblasts, whereas the activities of the other two carboxylases are normal.

Holocarboxylase synthetase deficiency must be differentiated from biotinidase deficiency (08). The symptoms of holocarboxylase synthetase deficiency and biotinidase deficiency are similar, and, thus, clinical differentiation may be difficult; however, the age of onset of symptoms can be useful in discriminating between these two disorders. Holocarboxylase synthetase deficiency usually manifests before three months of age, whereas biotinidase deficiency usually manifests after three months of age. There are exceptions for both disorders, and age of onset alone is not reliable. Both multiple carboxylase deficiencies are characterized by deficient activities of the mitochondrial carboxylases in peripheral blood leukocytes prior to biotin administration. These activities increase to near-normal or normal after biotin treatment (67). Patients with holocarboxylase synthetase deficiency have deficient carboxylase activities in fibroblasts incubated in medium containing only the biotin contributed by fetal calf serum (low biotin), whereas fibroblasts from patients with biotinidase deficiency have activities within the normal range. The activities of the carboxylases in holocarboxylase synthetase deficiency become near-normal to normal when they are cultured in medium supplemented with biotin (high biotin). Holocarboxylase synthetase deficiency and biotinidase deficiency can be definitively diagnosed by direct enzymatic assay.

Diagnostic workup

Holocarboxylase synthetase deficiency is usually suspected when high concentrations of the metabolites, beta-hydroxyisovalerate, beta-methylcrotonylglycine, beta-hydroxypropionate, methylcitrate, or lactate are found in the urine of a child exhibiting some or all of the above clinical findings. In at least one child, severe ketosis masked the diagnostic metabolites commonly seen in urine from individuals with holocarboxylase synthetase deficiency (12). Prior to biotin treatment, the activities of all three mitochondrial carboxylases are deficient in extracts of the peripheral blood leukocytes. Within days after starting biotin therapy, the carboxylase activities increase to near-normal or normal in these cells. Skin fibroblasts from these patients are deficient in all of the mitochondrial carboxylase activities when the cells are cultured in medium containing low concentrations of biotin. On incubation of these cells in high concentrations of biotin, the carboxylase activities increase to near-normal or normal.

Definitive diagnosis requires the demonstration of deficient activity of holocarboxylase synthetase in peripheral blood leukocytes or skin fibroblasts (10). These assays are complicated and require that appropriate tissues be sent to specific laboratories that have experience measuring this enzyme activity. A simple assay of the activity of the first partial reaction in the holocarboxylase synthetase reaction has been developed. This activity was deficient in all of those patients previously shown to have synthetase deficiency (43), so that this assay allows more rapid confirmation of the diagnosis of this disorder. In addition, the diagnosis can be made using a sensitive method based on the incorporation of tritiated biotin into the apocarboxyl carrier protein, a subunit of acetyl CoA carboxylase from E coli (69).

Management

Immediate treatment of infants with holocarboxylase synthetase deficiency should consist of adequate hydration, especially if dehydration is evident. Large quantities of parenteral fluids facilitate the excretion of the abnormal organic acids. Adequate nutrition is essential. In addition, protein may have to be restricted because the specific branched-chain amino acids are a source of organic acids; the protein is a source of nitrogen, and this will contribute to hyperammonemia. Protein restriction is not necessary once the child is stable and is on biotin therapy. Because inadequate caloric intake can result in tissue breakdown and endogenous protein degradation, it is imperative to supply sufficient calories in the form of parenteral glucose or oral polysaccharides. Severe acidosis may require bicarbonate supplementation in addition to hydration.

The mainstay of treatment for holocarboxylase synthetase deficiency is oral biotin supplementation. The clinical and biochemical abnormalities have improved in most patients following oral administration of 10 mg of biotin per day. However, one child has required 40 mg of biotin a day; a second child with one of the highest Michaelis constant values of biotin continued to excrete abnormal organic acids even when treated with 60 to 80 mg of biotin a day (09). Biotin treatment is lifelong. Several children in Thailand were successfully treated with only 1.2 mg of biotin a day (73). Effective low-dose therapy may depend on the individual’s specific genotype. To determine the biotin requirement of the patient, both the urinary excretion of abnormal organic acids and the degree of normalization of carboxylase activities in peripheral blood leukocytes should be monitored. Enzyme activities should achieve near-normal or normal levels when biotin therapy is appropriate. Because of differences in the Michaelis constant values of biotin for the synthetase in these patients, the quantity of biotin required must be determined on an individual basis. If biotin treatment is delayed, many of the neurologic abnormalities fail to improve.

A child with holocarboxylase synthetase deficiency with a nonsense mutation and a missense mutation at the 3’ end of the enzyme, the region that interacts with biotin, was treated with 100 mg of biotin per day (78). The child’s metabolic abnormalities corrected in blood and in the cerebral spinal fluid, as did the carboxylase activities. The biotin concentration in plasma was above that of the Km of the aberrant enzyme, and the concentration in the cerebrospinal fluid was half of that in plasma. The authors suggest that determining the mutation(s) and monitoring carboxylase activities and plasma and cerebrospinal fluid biotin concentrations following treatment based on the molecular characteristics of the aberrant enzyme are useful in optimizing the degree of biotin supplementation required.

It has been shown that high doses of biotin, such as that used to treat holocarboxylase synthetase deficiency, can interfere with immunoassays that use biotin-strept(avidin) technologies. Therefore, it is important that health professionals are aware of this when individuals with the disorder are having laboratory tests performed (85).

Special considerations

Pregnancy

A biotin-compliant woman with holocarboxylase synthetase deficiency, treated with 100 mg of biotin/day, had an uncomplicated pregnancy and delivery. There were no metabolic derangements during the pregnancy, and there were no deleterious effects in the newborn using this dosage of biotin (41).