Neonatal screening for biotinidase deficiency in Hungary: Clinical, biochemical and molecular studies

From 1989 to 2001, 1,336,145 newborns were screened for biotinidase deficiency in Hungary. Fifty-eight children with the disorder were identified as enzyme-deficient. We have characterized the clinical and biochemical features and mutations of 20 of these children. Eleven children had profound biotinidase deficiency, 7 had partial biotinidase deficiency, and 2 were found to be heterozygous for profound deficiency by mutation analysis. Seventeen different mutations were identified in this population including seven novel mutations. Six of these new mutations are missense, 245C>A, 334G>A, 652G>C, 832C>G, 1253G>C, 1511T>A, and one is a unique allelic double mutation [212T>C;236G>A]. Of five Romanian Gypsies, four were homozygous for the 1595C>T mutation and one was heterozygous for this mutation. Most of the children with profound deficiency have been asymptomatic on therapy; however, four exhibited minimal brain abnormalities, motor delay and abnormal blood chemistries. Compliance with therapy must be questioned in these cases. Of clinical importance, all of the children with partial deficiency exhibited mild symptoms at the time of diagnosis, at several weeks to months of age. These symptoms resolved following biotin therapy. This is in contrast to the experience in the United States, where the children with partial deficiency have been asymptomatic at the time of diagnosis. This finding further indicates that children with partial deficiency should be treated. The incidence of biotinidase deficiency in Hungary is more than twice that observed in a worldwide survey. These results indicate that newborn screening in Hungary is effective and warranted.     

Worldwide survey of neonatal screening for biotinidase deficiency

Summary Neonatal screening for biotinidase deficiency has been conducted in 14 countries since 1984. To 31 December 1990, 8 532 617 newborns were screened. One hundred and forty-two infants with biotinidase deficiency were identified; 76 infants with profound deficiency (<10% of mean normal serum activity) and 66 infants with partial deficiency (10–30% of mean normal activity). The estimated incidence of profound biotinidase deficiency is 1:112 271 (1:85 000 to 1:145 000; 95% confidence limits) and the incidence of partial deficiency is 1:129 282 (1:112 700 to 1:177 000). The incidence of combined profound and partial deficiency is 1:60 089 newborns (1:49 500 to 1:73 100). The estimated frequency of the allele for biotinidase deficiency is 0.004 and an estimated 1 in 123 individuals is heterozygous for the disorder.     

Prospective ascertainment of complete and partial serum biotinidase deficiency in the newborn

Summary We screened 163 000 newborn filter-paper blood samples for serum biotinidase deficiency (McKusick 25326) and found 15 probands: three had complete deficiency (incidence 18.4 cases per million live births, 95% confidence interval 4–54 cases per million); the others had partial deficiency. The positive predictive value of the test for either form of biotinidase deficiency was 9.86%. We found seasonal variation in biotinidase activity in filter-paper blood samples. The cost per test was Can.$0.27 (1987 dollar value) and per case of complete deficiency ascertained, $15 500. Family studies indicated that complete serum biotinidase deficiency is a homozygous phenotype and partial deficiency is the heterozygous form. Homozygous cases were treated with biotin and have shown no clinical manifestations (55 patient-months of observation). None of the heterozygotes (n=42, age 3 months – 62 years) has clinical manifestations. The number of heterozygotes found by screening was much less than predicted probably because the screening test detects mainly the samples with very low (outlier) biotinidase activity. The variant allele(s) for biotinidase deficiency was more common in French Canadians than in other ethnic groups in Quebec; there was no evidence of regional clustering or founder effect.This is publication no. 89002 from the McGill University-Montreal Children's Hospital Research Institute.     

Neonatal screening for biotinidase deficiency in East-Hungary

Summary There are two types of multiple carboxylase deficiency, the neonatal form with holocarboxylase synthetase defect and the late-onset form with biotinidase deficiency. We report our preliminary experiences in screening for biotinidase deficiency. In total 43 493 infants were screened for the deficiency of the enzyme biotinidase; 0.14% false positive results that necessitated requests for second blood samples and two newborns with a biotinidase defect were identified during our pilot study. The definitive diagnosis required the demonstration of enzyme deficiency in serum. Both of the patients have residual biotinidase activity: 3.59% and 7.55%. These two newborns with biotinidase deficiency are treated with daily supplementation of free biotin. According to our preliminary results biotinidase deficiency satisfies all the criteria for incorporation into the national newborn mass screening.     

Comparison of patients with complete and partial biotinidase deficiency: Biochemical studies

Summary Seventeen partially biotinidase-deficient patients detected by neonatal screening or family studies were compared with four patients with classical biotinidase deficiency. Using a sensitive HPLC method for biotinidase in plasma (substrate: biocytin) the patients could be divided into two groups: one with residual biotinidase activity, and the second with undetectable biotinidase activity (0-activity). Biocytin excretion, characteristically elevated in 0-activity patients, decreased rapidly with increasing residual biotinidase activity and was almost normal when residual activity exceeded 2–3% of mean normal. In one patient with classical disease (0-activity) biotin deficiency, typical organic aciduria and multiple carboxylase deficiency were found as early as at the second week of life. In contrast, 13 infants with residual activities from 1.2% to 23% had no remarkable clinical or biochemical abnormalities. However, in three 5-, 14- and 15-year-old healthy siblings with residual biotinidase activities between 2.3% and 4.2%, biotin deficiency was proven by decreased activities of the mitochondrial carboxylases in lymphocytes (30–57% of mean normal) and, in the older siblings, also by subnormal plasma biotin concentrations. In biotinidase deficiency, biotin depletion presumably occurs earlier in the brain than in other tissues and may thus first affect the central nervous system. For this reason and because of discrete biochemical abnormalities found in a patient with residual biotinidase activity of 8%, we suggest that at least all patients with residual activities below 10% should be treated with biotin.Presented as poster and abstract (P145) at the 26th Annual Symposium of the Society for the Study of Inborn Errors of Metabolism in September, 1988 in Glasgow, Scotland.     

Markedly elevated serum biotinidase activity may indicate glycogen storage disease type Ia

We report two children who presented with symptoms suggestive of biotinidase deficiency. Rather than deficiency, markedly elevated serum biotinidase activities were found. Based upon literature reports of elevated biotinidase activities in children with glycogen storage disease (GSD) type Ia, we considered the latter in our differential diagnosis and subsequently confirmed GSD type Ia in both patients by enzymatic testing. GSD type Ia should be considered in children with markedly elevated serum biotinidase activity.     

Hyperchylomicronaemia due to lipoprotein lipase deficiency as a cause of false-positive newborn screening for biotinidase deficiency

Summary Two cases of molecular genetically proven lipoprotein lipase deficiency are reported. Both patients were detected owing to a false-positive neonatal screening test for biotinidase deficiency. We conclude that both the fluorimetric and the colorimetric screening tests for biotinidase deficiency used with dried blood samples are affected by severe hyperchylomicronaemia and that, most probably, severe plasma turbidity interferes with the assay.     

Biotinidase Activity in Patients with Liver Disease

To investigate whether biotinidase deficiency may occur in liver disease, we determined biotinidase activity, biotin levels, and organic acids in patients with liver disease. Serum biotinidase activity in patients with liver disease (2.63 1.40 nmol/min/ml) was significantly lower than in the control group (5.43 1.06 nmol/min/ml). Serum biotinidase activity in decompensated liver cirrhosis (LC) and hepatoma was significantly lower than in acute viral hepatitis (AVH), chronic viral hepatitis (CVH), and compensated LC. The mean serum level of biotin in decompensated LC (1.8 0.6 μg/ml) and hepatoma (1.7 0.8μg/ml) was significantly lower than in the control group (2.5 1.0 μg/ml), and urinary excretion of biotin was increased in patients with liver disease, particularly in decompensated LC. Biotinidase activity correlated positively with serum biotin level and correlated negatively with urinary biotin level. Moreover, in four of five patients with severe liver disease the excretion of propionate, lactate, and 3-hydroxybutyrate decreased after biotin supplementation. The data for patients with severe liver disease so resembled those for late-onset multiple carboxylase deficiency that biotinidase deficiency is likely in patients with severe liver disease.     

A partial deficiency of interleukin-7R alpha is sufficient to abrogate T-cell development and cause severe combined immunodeficiency

Both in vitro and in vivo studies established that interleukin 7 (IL-7) is essential for differentiation of immature T cells and B cells but not natural killer (NK) cells in the mouse. In humans, although both T-cell and B-cell progenitors express the functional IL-7 receptor that consists of IL-7R alpha and the gamma common (gamma c) chain, this lymphocyte receptor system is critical for T lineage but not for B lineage development. Indeed, complete gamma c deficiency like IL-7R alpha deficiency results in the arrest of T-cell but not B-cell development (T(-)B(+) SCID). However, partial deficiency of gamma c caused by missense mutations results in a T(+)B(+) phenotype and a delay of clinical presentation. It was therefore plausible to assume that partial deficiency of IL-7R alpha, like partial gamma c deficiency may lead to a milder clinical and immunologic phenotype. A P132S mutation in the IL-7R alpha was identified in 3 patients with severe combined immunodeficiency (SCID) within an extensively consanguineous family. Substitution of proline with serine in the extracellular portion of IL-7R alpha did not affect IL-7R alpha messenger RNA (mRNA) and protein expression, but severely compromised affinity to IL-7, resulting in defective signal transduction. In response to IL-7 stimulation, Jak-3 phosphorylation was markedly reduced in both patient cells as well as in COS cells reconstituted with mutant IL-7R alpha. Surprisingly, this partial deficiency of IL-7R alpha resulted in a severe phenotype, including markedly reduced circulating T cells while sparing B-cell numbers similar to gamma c chain deficiency. However, unlike the previously reported cases, serum immunoglobulins were virtually absent. Further, unlike gamma c deficiency, NK cell numbers and function was preserved. Despite the partial deficiency, clinical presentation was indistinguishable from a complete gamma c deficiency, including severe and persistent viral and protozoal infections and failure to thrive. Unlike partial gamma c deficiency, a partial deficiency of IL-7R alpha results in an arrest of T-cell development, leading to typical severe combined immunodeficiency. This underscores the critical role of IL-7R alpha chain in the differentiation of T cells. (Blood. 2000;96:2803-2807)     

Clinical evaluation of serum biotin levels and biotinidase activities in patients with various liver diseases

In order to evaluate the clinical significance of serum biotin and biotinidase in liver disease, serum biotin levels and biotinidase activities were determined in 83 patients with various liver diseases and 10 healthy controls. Serum biotin levels and biotinidase activities were determined by a simplified lactobacillus plantarum bioassay and liquid chromatography with fluorimetric detection respectively. Serum biotin levels in decompensated liver cirrhosis, hepatoma and fulminant hepatitis were found to be significant low compared with healthy controls, while it was significant high in autoimmune hepatitis. There was no significant difference between serum biotin levels in the other liver diseases and healthy controls. In various liver diseases except for both acute hepatitis and alcoholic liver disease biotinidase activities were significantly reduced than in healthy controls. Serum biotinidase activities were correlated with serum albumin, prothrombin time, ChE and total cholesterol respectively, suggesting that biotinidase activities may reflect the degree of liver damage. These results seem that biotin deficiency may occur in some cases of severe liver diseases.     

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.     

Short Report: Normalization of low biotinidase activity in a child with biotin deficiency after biotin supplementation

We report findings in a Japanese boy with severe skin rash attributable to biotin deficiency. The patient had an intracranial malformation and developed biotin deficiency due to tube feeding with a single formula for over one year. Results of urinary organic acid analysis were consistent with multiple carboxylase deficiency, and low biotinidase activity was also observed. After biotin supplementation, the skin rash improved and biotinidase activity normalized. We speculate that biotin is one regulating factor in the biosynthesis of biotinidase.     

Asymptomatic adults and older siblings with biotinidase deficiency ascertained by family studies of index cases

Summary We report 32 biotinidase-deficient patients detected by family studies in the index cases. The study group consisted of 10 mothers, 4 fathers and 18 siblings. There were 17 individuals (3 mothers, 4 fathers and 10 siblings) with profound biotinidase deficiency (BD) (< 10% of mean normal activity) and 15 (7 mothers and 8 siblings) with partial BD (10–30% of mean normal activity). In the profound BD group, only three siblings were symptomatic. Dermatitis, microcephaly, developmental delay and convulsions were observed. The patients with partial BD did not have any clinical symptoms except one sibling with borderline IQ score. None of the parents was symptomatic. Family investigation of patients with BD is very important for the detection of asymptomatic patients who are at risk of exhibiting symptoms at any age. Careful evaluation of these untreated individuals with BD is important to obtain additional information about the natural history of this disorder and may provide clues to phenotype–genotype relationships and treatment regimes.     

BiotinidaseK m-variants: detection and detailed biochemical investigations

Summary We describe a simple method for the detection of biotinidaseK _m-variants and detailed biochemical investigations in 5 such patient. They were detected among 103 patients with plasma biotinidase activity which ranged from undetectable to 30% of the mean normal value. Two different types of biotinidaseK _m-variants were found. (1) In 3 infants biotinidase had a single 105–430-fold elevatedK _m for biocytin. Biotinidase showed very low activities (0.2–4% of the mean normal value) in the routine colorimetric assay and was not functionalin vivo. Accordingly, these patients presented with classical clinical illness. (2) In two patients biotinidase showed biphasic kinetics indicating the presence of one component with a normalK _m and reducedV _max (1.7% and 12%), and another with 330- and 59-fold elevatedK _m, respectively. In these two patients, biotinidase proved to be at least partially functionalin vivo. However, the first patient developed severe symptoms and biotin deficiency late, at the age of 10–15 years, and the second had marginal biotin deficiency at the age of 2 years but no clinical symptoms. Comparative studies revealed that both patients had more severe biotin deficiency than age-matched patients with similar levels of residual biotinidase activity and a single normalK _m. Therefore, all patients with residual biotinidase activity should be evaluated for the presence of aK _m-mutation, since such patients should be treated with biotin. These can easily be detected by including a second substrate concentration (1.5 mmol/L) in the routine colorimetric biotinidase assay which is performed with 0.15 mmol/L biotin. Increased activity with the higher substrate concentration indicates the presence of aK _m-mutation. Detailed kinetic studies are needed to evaluate the distinct forms ofK _m-variants.     

Late-onset holocarboxylase synthetase deficiency

Summary We report a 21-month-old female patient whose urine organic acid profile suggested a biotin utilization abnormality consistent with multiple carboxylase deficiency. For most previously reported patients, holocarboxylase synthetase deficiency has correlated with the early-onset variant of multiple carboxylase deficiency; conversely, biotinidase deficiency has been characteristic of the late-onset form.In vitro enzyme studies revealed that our patient suffered from holocarboxylase synthetase deficiency. We suggest that holocarboxylase synthetase deficiency should be considered in the differential diagnosis of older patients in whom there is suspicion of a defect in biotin metabolism.     

Newborn screening compared to clinical identification of biochemical genetic disorders

A group of 28 patients with inherited metabolic disease (homocystinuria galactosaemia, maple syrup urine disease and biotinidase deficiency) diagnosed by screening were compared with a group of 17 similar patients identified clinically. The rate of hospitalization was similar for the two groups. The patients diagnosed clinically showed a higher incidence of mental retardation and their parents experienced greater stress and found greater difficulty in meeting their child's needs.     

Cerebral metabolic changes in biotinidase deficiency

Clinical and metabolic changes in the central nervous system are described in a patient with biotinidase deficiency before and after biotin treatment. Lactate, pyruvate and 3-hydroxyisovaleric acid as metabolic disease markers were measured in blood, cerebrospinal fluid and brain tissue by biochemical analyses or localized magnetic resonance proton spectroscopy. The patient improved markedly with biotin treatment. Nevertheless, neurological sequelae and abnormal intracerebral lactate concentrations persisted despite normalized metabolic disease markers in extracerebral fluids. Therefore, localized in vivo measurements of intracerebral metabolites may be a valuable tool for elucidating the pathogenesis of biotinidase deficiency.     

Reversible metabolic myopathy in biotinidase deficiency: its possible role in causing hypotonia

Summary A 5-year-old girl diagnosed with biotinidase deficiency at 9 months of age demonstrated limb and axial hypotonia which improved on biotin therapy. In this patient, electromyographic (EMG) studies prior to treatment were compatible with a mild myopathic process. Serial EMGs performed on biotin therapy demonstrated a gradual resolution of the myopathy. This is the first documented case of a reversible myopathy in a patient with biotinidase deficiency, which may contribute to the clinical finding of hypotonia.     

A new case of combined factor V and factor VIII deficiency further suggests that the LMAN1 M1T mutation is a frequent cause in Italian patients.

Combined factor V and factor VIII deficiency (F5F8D) is an extremely rare worldwide congenital hemorrhagic disorder that is more prevalent in the Mediterranean area. We report the clinical presentations and the identification of a LMAN1 mutation in a 3-year-old Italian boy who was diagnosed with F5F8D. The mutation identified (M1T) has already been found in several Italian patients. Since the LMAN1 M1T mutation has been identified in most patients with F5F8D, we suggest that the search for this mutation should be the first step in the molecular characterization of patients from an Italian ethnic background.     

Successful pregnancy in a treated patient with biotinidase deficiency

Summary We report a patient with biotinidase deficiency treated with biotin during pregnancy with favourable outcome.