Development and characterization of a mouse with profound biotinidase deficiency: A biotin-responsive neurocutaneous disorder

doi:10.1016/j.ymgme.2010.10.005

Molecular Genetics and Metabolism

Volume 102, Issue 2, February 2011, Pages 161-169

https://doi.org/10.1016/j.ymgme.2010.10.005 Get rights and content

Abstract

Biotinidase deficiency is the primary enzymatic defect in biotin-responsive, late-onset multiple carboxylase deficiency. Untreated children with profound biotinidase deficiency usually exhibit neurological symptoms including lethargy, hypotonia, seizures, developmental delay, sensorineural hearing loss and optic atrophy; and cutaneous symptoms including skin rash, conjunctivitis and alopecia. Although the clinical features of the disorder markedly improve or are prevented with biotin supplementation, some symptoms, once they occur, such as developmental delay, hearing loss and optic atrophy, are usually irreversible. To prevent development of symptoms, the disorder is screened for in the newborn period in essentially all states and in many countries. In order to better understand many aspects of the pathophysiology of the disorder, we have developed a transgenic biotinidase-deficient mouse. The mouse has a null mutation that results in no detectable serum biotinidase activity or cross-reacting material to antibody prepared against biotinidase. When fed a biotin-deficient diet these mice develop neurological and cutaneous symptoms, carboxylase deficiency, mild hyperammonemia, and exhibit increased urinary excretion of 3-hydroxyisovaleric acid and biotin and biotin metabolites. The clinical features are reversed with biotin supplementation. This biotinidase-deficient animal can be used to study systematically many aspects of the disorder and the role of biotinidase, biotin and biocytin in normal and in enzyme-deficient states.

Introduction

Biotin is an essential vitamin required for the activation of four biotin-dependent carboxylases. In humans, holocarboxylase synthetase (HCS) converts inactive apocarboxylases into functionally active holocarboxylases by covalently attaching biotin to ε-amino groups of specific lysyl-residues of each of the four biotin-dependent carboxylases (propionyl CoA carboxylase (PCC), ß-methylcrotonyl CoA carboxylase (MCC), pyruvate carboxylase (PC), and acetyl CoA carboxylase (ACC)) [1]. These holocarboxylases have important roles in gluconeogenesis, fatty acid synthesis and the catabolism of several branch-chain amino acids and odd-carbon fatty acids. The holocarboxylases are proteolytically degraded to biocytin (biotinyl-ε-N-lysine) and biotinylated-peptides. Biocytin is the product of the proteolytic degradation of biotin-dependent carboxylases [1]. Similarly proteolysis of dietary proteins generates biotinylated-peptides and biocytin. Biotinidase (EC 3.5.1.12) is an amido-hydrolase that hydrolyzes biocytin (biotinyl-ε-lysine) and small biotinyl-peptides thereby recycling biotin [2], [3]. In addition, biotinidase has biotinyl-transferase activity capable of transferring biotin to nucleophilic acceptor molecules, such as histones [4].

Biotinidase deficiency (OMIM 253260) is a biotin-responsive, autosomal recessively inherited metabolic disorder [5]. If untreated, children with profound biotinidase deficiency (less than 10% of mean normal serum activity) can exhibit neurological features such as hypotonia, seizures, developmental delay, sensorineural hearing loss and optic atrophy; and cutaneous abnormalities, such as skin rash and alopecia [6]. Biochemically, these individuals may exhibit metabolic acidosis, mild hyperammonemia and/or organic acidemia/uria. The symptoms improve or can be prevented if affected children are treated with pharmacological doses of biotin [7]. However, the developmental delay, hearing loss, and visual abnormalities are usually irreversible if they occur prior to initiation of biotin therapy [8]. Currently, most states in the United States and many countries screen their newborns for biotinidase deficiency [9].

With the success of newborn screening for biotinidase deficiency, we are losing the window-of-opportunity to learn about the natural history and the pathophysiology of symptomatic, untreated biotinidase-deficient individuals. Therefore, we developed a transgenic mouse with biotinidase deficiency by knocking-out the biotinidase gene (BTD). Biotinidase-deficient (BTD^−/−) mice have no detectable biotinidase activity and develop neurocutaneous symptoms when placed on a biotin-deficient diet. In addition, symptoms can be reversed in BTD^−/− mice treated with pharmacological doses of biotin. This biotinidase-deficient mouse model will allow us to gain a better understanding of the natural history of biotinidase deficiency, the pathophysiology of the disorder, and the role biotinidase and biotin deficiency play in normal and biotinidase-deficient states.

Section snippets

Generation of vector constructs

Construction of the target vector pL253-ΔBtdNeo with the disrupted BTD gene sequence required multi-step recombinant engineering to generate multiple vectors. BTD homologous sequence (from BAC-Btd ) was cloned into pL253 in a series of recombination steps involving PCR amplifications, restriction digestions, gel purification of correct fragments, ligation, colony screening, and plasmid preparation. We adopted a highly efficient recombination based method as described [10]. This method takes

Resultant transgenic knockout mice

The resulting F1 mice were back-crossed to C57BL/6 background for ten generations to produce a congenic animal line. Currently, we have bred the genetically engineered mice for 20 generations without any breeding complications.

Mice were genotyped using the three-primer-two-allele PCR methodology. They were assigned to one of three genotypic groups: BTD^+/+ or wildtype, BTD^+/− or heterozygous, and BTD^−/− or deficient. A representative genotyping agarose gel is shown in Fig. 1C. The ratio of BTD^+/+

Discussion

Our laboratory first demonstrated that late-onset multiple carboxylase deficiency was due to a deficiency of biotinidase activity [5], characterized the variability in the clinical expression of the biotinidase deficiency [19], [20], [21], developed a method to screen newborns for biotinidase deficiency using blood-soaked filter paper spots [22], and piloted the first newborn screening program for the disorder [23]. We subsequently isolated and cloned the human biotinidase gene [24]

Acknowledgments

This work was funded by the Safra Research Foundation (B.W.) and National Institutes of Health grants R37 DK36823 (DMM) and R37 DK36823-26S1 (DMM). We thank Dr. Kenneth Barton from the Department of Radiation Oncology at Henry Ford Hospital for helping us with the Computer Tomography studies and Dr. Jieli Chen from the Department of Neurology at Henry Ford Hospital for helping us with Motor-neuronal assessment studies.

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In this scenario, the KO animals at day 8 are markedly symptomatic, whereas the KO mice at day 1 are asymptomatic. Under these conditions, the major differences in gene expression are most likely to be the direct effect of the enzyme deficiency and the resultant biotin deficiency [6]. Most of the significantly affected genes are up-regulated (Fig. 1D).
Biotinidase deficiency is an autosomal recessively inherited disorder characterized by neurological and cutaneous abnormalities. Untreated individuals with biotinidase deficiency cannot recycle biotin from biocytin (N-biotinyl-ϵ-lysine), the proteolytic digestion product of protein-bound biotin. Biotin therapy can markedly resolve symptoms, or can prevent the development of symptoms if initiated early. To understand better the pathogenesis of the neurological problems in the disorder in humans, we have compared gene transcription changes during the first week post-birth in the brains of biotinidase-deficient, transgenic, knock-out mice at days 1 and 8 and compared to changes in wildtype mice at the same times. The knockout pups that were not supplemented with unconjugated biotin became symptomatic by day 8 and exhibiting failure to thrive. Wildtype pups remained asymptomatic under the same experimental conditions. We compared all four possible combinations and noted the most significant up- and down-regulated genes in the knockout animals at Day 8 compared to those at Day 1, reflecting the changes in gene expression over the first week of development. These alterations involved neurological development and immunological function pathways and provide some clues to avenues for further research. At this time, these preliminary analyses provide us with limited, but new information. However, with the development of new algorithms and programs examining various mechanisms and pathways in the central nervous system, these analyses may help us to understand better the role of biotinidase and the pathogenesis of biotinidase deficiency.
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Biotinidase is the enzyme that is necessary for the recycling of the vitamin, biotin. Biotinidase deficiency is an autosomal recessively inherited metabolic disorder. If untreated, individuals with biotinidase deficiency usually develop neurological and cutaneous symptoms that can result in coma or death. Symptomatic individuals can be markedly improved by treating them with pharmacological doses of biotin; however, some clinical features may be irreversible. Fortunately, essentially all symptoms can be prevented if treatment is initiated at birth or before the symptoms develop. Because of this, the disorder is currently screened for in newborns in all states in the United States and in many countries around the world. This is the story of one laboratory's work in bringing basic science research from the discovery of the disorder to its translation into clinical medicine and its impact on the individuals with the disorder and their families.
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Certain inborn errors of metabolism result from deficiencies in biotin containing enzymes. These disorders are mimicked by dietary absence or insufficiency of biotin, ATP deficit being a major effect, whose responsible mechanisms have not been thoroughly studied. Here we show that in rats and cultured cells it is the result of reduced TCA cycle flow, partly due to deficient anaplerotic biotin-dependent pyruvate carboxylase. This is accompanied by diminished flow through the electron transport chain, augmented by deficient cytochrome c oxidase (complex IV) activity with decreased cytochromes and reduced oxidative phosphorylation. There was also severe mitochondrial damage accompanied by decrease of mitochondria, associated with toxic levels of propionyl CoA as shown by carnitine supplementation studies, which explains the apparently paradoxical mitochondrial diminution in the face of the energy sensor AMPK activation, known to induce mitochondria biogenesis. This idea was supported by experiments on AMPK knockout mouse embryonic fibroblasts (MEFs). The multifactorial ATP deficit also provides a plausible basis for the cardiomyopathy in patients with propionic acidemia, and other diseases. Additionally, systemic inflammation concomitant to the toxic state might explain our findings of enhanced IL-6, STAT3 and HIF-1α, associated with an increase of mitophagic BNIP3 and PINK proteins, which may further increase mitophagy. Together our results imply core mechanisms of energy deficit in several inherited metabolic disorders.
Characterization and functional analysis of cellular immunity in mice with biotinidase deficiency
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BD mice used in this study develop clinical symptoms and exhibit biochemical profile of biotinidase deficiency [10]. BD mice when deprived of biotin by feeding them a biotin-deficient diet for two weeks become symptomatic and exhibit clinical features of the disorder [10]. This is the first study focusing on immunological aspects of biotinidase and biotin deficiency to evaluate immune organs and assess cellular immunological function using BD mice.
Biotinidase deficiency is an autosomal recessively inherited metabolic disorder that can be easily and effectively treated with pharmacological doses of the vitamin, biotin. Untreated children with profound biotinidase deficiency may exhibit neurological, cutaneous and cellular immunological abnormalities, specifically candida infections. To better understand the immunological dysfunction in some symptomatic individuals with biotinidase deficiency, we studied various aspects of immunological function in a genetically engineered knock-out mouse with biotinidase deficiency. The mouse has no detectable biotinidase activity and develops neurological and cutaneous symptoms similar to those seen in symptomatic children with the disorder. Mice with profound biotinidase deficiency on a biotin-restricted diet had smaller thymuses and spleens than identical mice fed a biotin-replete diet or wildtype mice on either diet; however, the organ to body weight ratios were not significantly different. Thymus histology was normal. Splenocyte subpopulation study showed a significant increase in CD4 positive cells. In addition, in vitro lymphocyte proliferation assays consistently showed diminished proliferation in response to various immunological stimuli. Not all symptomatic individuals with profound biotinidase deficiency develop immunological dysfunction; however, our results do show significant alterations in cellular immunological function that may contribute and/or provide a mechanism(s) for the cellular immunity abnormalities in individuals with biotinidase deficiency.
Biotinidase knockout mice show cellular energy deficit and altered carbon metabolism gene expression similar to that of nutritional biotin deprivation: Clues for the pathogenesis in the human inherited disorder
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We have previously demonstrated the importance of studying the early specific molecular and metabolic effects of biotin deficiency before other non-specific effects, such as malnutrition. In these experiments we used mice of C57BL/6 background that are homozygous for the knocked-out biotinidase BTD gene (KO) [2]. Wild-type (WT) mice were of the same strain and homozygous for the normal BTD gene.
Biotin is the prosthetic group of carboxylases that have important roles in the metabolism of glucose, fatty acids and amino acids. Biotinidase has a key role in the reutilization of the biotin, catalyzing the hydrolysis of biocytin (ε-N-biotinyl-l-lysine) and biocytin-containing peptides derived from carboxylase turnover, thus contributing substantially to the bioavailability of this vitamin. Deficient activity of biotinidase causes late-onset multiple carboxylase in humans, whose pathogenic mechanisms are poorly understood. Here we show that a knock-out biotinidase-deficient mouse from a C57BL/6 background that was fed a low biotin diet develops severe ATP deficit with activation of the energy sensor adenosine monophosphate (AMP)-activated protein kinase (AMPK), inhibition of the signaling protein mTOR, driver of protein synthesis and growth, and affecting the expression of central-carbon metabolism genes. In addition, sensitivity to insulin is augmented. These changes are similar to those observed in nutritionally biotin-starved rats. These findings further our understanding of the pathogenesis of human biotinidase deficiency.
Temporal development of genetic and metabolic effects of biotin deprivation. A search for the optimum time to study a vitamin deficiency
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Biotin deficiency (Bt-D) is usually studied at the point at which the animal model exhibits the signs of full-blown deficiency symptoms; in rats, this typically occurs at 6–8 weeks of feeding a deficient diet. To differentiate specific deficiency effects from those of undernutrition, biotin sufficient and deficient rats were studied at 2, 3, 4, and 5 weeks on the deficiency diet, before the onset of weight loss and deficiency signs. The deficiency state was confirmed by biochemical and molecular analyses. Blood and liver metabolites were determined and western blots of signaling proteins, and qRT-PCR gene expression studies. The main effects of Bt-D were already well established by the fourth week on the diet; thus, we consider the fourth week as the optimum time to study the consequences of biotin depletion. Early effects, which were already apparent at week 2, included cellular energy deficit (as assessed by increased AMP/ATP ratio), activation of the AMPK energy sensor, and changes of carbon metabolism gene transcripts (e.g., phosphoenolpyruvate carboxykinase, carnitine palmitoyl transferase 1, liver glucokinase and fatty acid synthetase). Reduced post-prandial blood concentrations of glucose were also observed early; we speculate that these are attributable to augmented sensitivity to insulin and increased glucose utilization, a likely effect of AMPK induction of translocation of glucose transporter GLUT4 to the cell membranes and increased hexokinase expression. Other late-onset changes (week 4) included increased serum concentrations of lactate and free fatty acids and decreased liver glycogen and serum concentrations of triglycerides and total cholesterol. The identification of the early specific molecular and metabolic disturbances of biotin deficiency might be useful in identifying individuals with marginal deficiency of this vitamin, which appears to be common in normal human pregnancy. The study of time-course of other vitamin deficiencies, such as this one, might help to better understand and cope with their effects.

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Development and characterization of a mouse with profound biotinidase deficiency: A biotin-responsive neurocutaneous disorder

Abstract

Introduction

Section snippets

Generation of vector constructs

Resultant transgenic knockout mice

Discussion

Acknowledgments

J. Biol. Chem.

Clin. Chim. Acta

Clin. Chim. Acta

Methods Enzymol.

J. Nutr.

J. Pediatr.

J. Pediatr.

J. Pediatr.

Am. J. Clin. Nutr.

J. Nutr.

Am. J. Clin. Nutr.

J. Nutr.

The biotin-dependent enzymes

Adv. Enzymol.

Biocytinase, an enzyme concerned with hydrolytic cleavage of biocytin

Proc. Soc. Exp. Biol. Med.

Biotinidase deficiency: the possible role of biotinidase in the processing of dietary protein-bound biotin

J. Inherit. Metab. Dis.

Disorders of biotin metabolism

Biotinidase deficiency: a novel vitamin recycling defect

J. Inherit. Metab. Dis.

Disorders of biotin metabolism: treatable neurological syndromes