Gregory S. Heard

Biotinidase Deficiency: A Novel Vitamin Recycling Defect

The recent finding that biotinidase deficiency is the primary biochemical defect in late-onset multiple carboxylase deficiency has stimulated new interest in the inherited disorders of biotin-dependent carboxylases. The clinical and biochemical features of biotinidase deficiency are discussed. We also speculate about two exciting areas currently being investigated: the localization of action of biotinidase, and the possible role of the enzyme as a binding or carrier protein for biotin.

Clinical Findings in Four Children with Biotinidase Deficiency Detected through a Statewide Neonatal Screening Program

Four children with biotinidase deficiency were identified during the first year of a neonatal screening program for this disease in the Commonwealth of Virginia. Two unrelated probands were identified among the 81,243 newborn infants who were screened. In addition, two siblings of one of these infants were found to be affected. Both probands had mild neurologic symptoms at two and four months, respectively, and the two older children had more severe neurologic abnormalities, cutaneous findings, and developmental delay at two and three years of age. However, none of the affected children had acute metabolic decompensation. Previous studies have shown that the administration of biotin to affected children can be a lifesaving procedure that can reverse acute symptoms and prevent irreversible neurologic damage. Our findings demonstrate that subtle neurologic abnormalities may appear as early as at two months of age and that developmental abnormalities may occur even in the absence of episodes of overt metabolic decompensation. Since screening and treatment are both inexpensive and effective and the incidence of the disease is well within the range of that of other metabolic diseases for which screening is performed, biotinidase deficiency should be added to the group of metabolic diseases for which screening is done in the neonatal period.

Biotinidase Deficiency: The Possible Role of Biotinidase in the Processing of Dietary Protein-bound Biotin

Biotinidase (EC 3.5.1.12) cleaves covalently-bound biotin from partially degraded carboxylases and from biocytin, thereby recycling biotin. We have recently demonstrated that the activity of biotinidase is deficient in serum from children with late-onset multiple carboxylase deficiency (Wolf et al., 1983a, b). Using a newly developed, sensitive radioassay, we have shown that biotinidase activity is also deficient in peripheral blood leukocytes and fibroblasts of affected patients (Wolf and McVoy, 1984). The enzyme activities in the parents of these children are intermediate between deficient and normal values. These findings indicate that the primary enzyme defect in late-onset multiple carboxylase deficiency is in biotinidase activity and that the disorder is inherited as an autosomal recessive trait. Children lacking biotinidase activity are unable to recycle biotin and depend on exogeneous biotin to prevent the clinical and biochemical features of biotin deficiency. Although previous reports have described’ impaired’ intestinal absorption of free biotin in patients with late-onset multiple carboxylase deficiency, the absorption of free biotin was found to be normal in one of these patients when loading studies were repeated while her tissues were not biotin-depleted. Moreover, this patient was found to be biotinidase deficient (Thoene and Wolf, 1983). Because there is considerable clinical variability in this disorder (Wolf et al., 1983c) and the concentrations of free and protein-bound biotin in foods are variable, biotinidase may also play a critical role in the processing of dietary protein-bound biotin.

Neonatal screening for biotinidase deficiency: Results of a 1-year pilot study

We screened 81,243 infants born in Virginia during the 1-year period beginning Jan. 24, 1984, for deficiency of the enzyme biotinidase. A simple colorimetric screening procedure was used to detect the presence or absence of biotinidase activity on the same blood-soaked filter paper cards that are currently used in most neonatal metabolic screening programs. Two newborn infants with biotinidase deficiency were identified during the 12-month pilot study. In addition, two affected siblings of one of the newborn infants were detected through secondary family screening. On the basis of these results, the disorder appears to be at least as frequent as several others for which newborn screening is currently conducted. There were no known false-negative test results, and only 0.09% false-positive results that necessitated requests for second blood samples. False-positive test results can be readily identified by the use of a quantitative assay, which can also be used to confirm the diagnosis and to detect heterozygous family members in the case of true positives. On the basis of currently recognized criteria, biotinidase deficiency should be considered for inclusion among the metabolic disorders for which screening is performed in the neonatal period.

Biotinidase deficiency: Accumulation of lactate in the brain and response to physiologic doses of biotin

Biotinidase deficiency is the most common cause of late onset, biotin-responsive multiple carboxylase deficiency (MCD). We studied the two oldest known boys with this disorder who had high CSF content of lactate that could have contributed to the clinical disorder. The symptoms of these patients implied that near physiologic, rather than pharmacologic, doses of biotin may be sufficient for treatment.

PANCREATIC BIOTINIDASE ACTIVITY: THE POTENTIAL FOR INTESTINAL PROCESSING OF DIETARY PROTEIN-BOUND BIOTIN

Biotinidase cleaves biotin from biocytin (ε-N-biotinyllysine) and biotinyl-peptides resulting from the degradation of carboxylases and, consequently, is important in recycling the vitamin. Some of the variability in the clinical features and age of onset of biotinidase deficiency (ranges from 3 weeks to several years) may be a consequence of differences not only in the total dietary biotin consumption but also in the form of the vitamin in the diet. Biotinidase-deficient children whose diets contain foods rich in free (as opposed to protein-bound) biotin may take longer to become biotin-deficient and, hence, to manifest the signs and symptoms of biotinidase deficiency. We have shown that biotinidase activity in rats is not enriched in intestinal brush border membranes but it is present in mucosa from all sections of the small intestine, and it is also in pancreatic homogenates (24.0±5.5 pmol/min/mg protein), isolated secretory granules (6.3 pmol/min/mg) and pancreatic juice from cannulated ducts (316±189 pmol/min/ml or 7.0±4.2 pmol/min/mg). The apparent Km of N-biotinyl-p-aminobenzoate (an artificial substrate) for biotinidase was 10 μM and broad pH optima of 4 to 8 were observed for enzymes from both rat serum and pancreatic juice. These findings suggest that biotinidase has an important role in increasing the bioavailability of dietary biotin.

Neonatal Screening for Biotinidase Deficiency: An Update

Biotinidase (EC 3.5.1.12) hydrolyses biotin from small biotinyl peptides and biocytin that result from the proteolytic degradation of biotin-dependent holocarboxylases (Pispa, 1965; Craft et al., 1985). The released biotin can then be reutilized by the body. Biotinidase also appears to play an important role in the processing of biotin from dietary protein-bound sources (Wolf et al, 1984). We have shown that most individuals with late-onset multiple carboxylase deficiency have a primary defect in biotinidase activity (Wolf et al, 1983a). Children with this autosomal recessively inherited disorder may exhibit seizures, hypotonia, ataxia, alopecia, skin rashes, hearing loss and developmental delay, which may ultimately result in coma or death (Wolf et al, 1983b). Although most affected individuals have had metabolic ketoacidosis and organic aciduria, some have not. All children who have been diagnosed early have improved markedly after treatment with pharmacologic doses of biotin. Others who are diagnosed late often sustain neurologic abnormalities even after biotin therapy. Therefore, biotinidase deficiency qualifies for inclusion in a newborn screening programme for inherited metabolic disorders by satisfying three major criteria. First, symptoms of the disease do not appear at birth but usually occur at several months of age. Second, affected individuals may manifest serious physical and mental disability. Third, the disorder can be treated easily and effectively with vitamin supplementation. In order to determine the feasibility of screening and the incidence of biotinidase deficiency, we have conducted a pilot neonatal screening programme in the Commonwealth of Virginia.

EFFECTS OF AGE AND BIOTIN STATUS ON POSTNATAL DEVELOPMENT OF PLASMA BIOTINIDASE ACTIVITY IN RATS

Biotinidase activity was measured in plasmas of 1-, 7-, 14-, and 21-day-old rats from control dams and dams that had been fed a biotin-depleting diet from Day 15 of gestation. Biotinidase activity increased significantly in the plasma of rats from control and depleted mothers until Postnatal Day 14, after which there was a small but significant decline at Day 21. Differences between the mean activities of the two groups of pups on each sampling day were not significant and there were no significant differences in activity levels attributable to sex. Plasma albumin concentrations increased from birth until Day 21, and plasma biotinidase activity and albumin concentration were significantly correlated (r = +/- 0.43). We suggested that these two proteins may be controlled by a common mechanism in the early postnatal period, and that biotin deficiency does not affect the development of biotinidase activity. Because biotin-depleted neonatal pups show developmental changes in biotinidase activity similar to those of human newborns, and they can be produced reliably by depleting dams from Day 15 of gestation, they may be useful models for studying the developmental abnormalities associated with human biotinidase deficiency.

875 BIOTINIDASE DEFICIENCY DETECTED BY A STATEWIDE NEONATAL SCREENING PROGRAM

Biotinidase deficiency is an autosomal recessively inherited disorder that usually manifests during infancy or early childhood with seizures, skin rash, alopecia, hearing loss, developmental delay and metabolic compromise, and occasionally results in coma and death. If diagnosed early the disorder can be treated effectively with biotin. To estimate the incidence of biotinidase deficiency and the potential cost-effectiveness of neonatal screening we conducted a pilot program in the Commonwealth of Virginia. After screening 77,145 newborns in ten months we have confirmed the diagnosis in two infants: a female and a male ascertained at 4 mos. and 2 mos. of age, respectively. Each infant had normal EEGs and developmental milestones, and neither had cutaneous or metabolic abnormalities. However, both were slightly hypertonic with brisk reflexes, and the female had abnormal brainstem auditory evoked potentials. Treatment with biotin (10 mg/d) was initiated within 3 days of confirming the diagnosis. Our preliminary estimate of the incidence of the disorder is 1 in 38,500 (95% confidence limits: 1 in 11,700 and 1 in 228,000). Affected infants are not symptomatic at birth, but may develop physical and neurologic abnormalities if untreated. The qualitative screening test for biotinidase activity is simple and inexpensive. Therefore, we recommend that testing for biotinidase deficiency be incorporated into all newborn screening programs.