©American College of Medical Genetics and Genomics GENETEST REVIEW Biotinidase deficiency: “if you have to have an inherited metabolic disease, this is the one to have” Barry Wolf, MD, PhD1,2 Biotinidase recycles the vitamin biotin. Biotinidase deficiency is an about the natural history of the disorder. Regardless, the disorder is autosomal recessively inherited neurocutaneous disorder. The symp- an ideal example of an inherited metabolic disorder that if untreated toms of the disorder can be successfully treated or prevented by can result in major disabilities, but if identified early can be readily administering pharmacological doses of biotin. The biotinidase gene treated by the oral administration of a vitamin. (BTD) has been cloned and sequenced; its genomic organization has Genet Med 2012:14(6):565–575 been determined and more than 150 mutations have been identified. The disorder meets the major criteria for newborn screening and is Key Words: biotin; biotinidase; biotinidase deficiency; carboxylase being universally adopted in the United States and in many countries deficiency; mutation analysis; newborn screening around the world. Newborn screening will limit our understanding INTRODUCTION purified to homogeneity from plasma.3,10,11 Normal serum Biotinidase (E.C. 3.5.1.12) is the enzyme that recycles the vita- or plasma biotinidase is a monomeric sialylated glycopro- min biotin.1,2 As a vitamin, biotin comes from the diet either in tein with a molecular mass of 76–77 kDa. The enzyme has at the free, non-protein-bound form or as small biotinylated pep- least nine isoforms (four major and five minor) between pH tides bound to protein.3 Biotin in the free form can directly enter 4.15 and 4.35 as observed by isoelectric focusing.12 It has six the biotin pool and be used by the four carboxylases that use it putative N-linked glycosylation sites (arginine-X-threonine/ to convert them from the inactive to the active forms.4 The car- serine). Glycosylation of the protein could increase its mass boxylases propionyl-CoA carboxylase and β-methylcrotonyl- by 13 to 19 kDa; the molecular mass of the glycosylated CoA carboxylase are important for protein catabolism, ­pyruvate enzyme is estimated to be between 70 and 76 kDa, which carboxylase is essential for gluconeogeneis, and acetyl CoA car- is consistent with that of the reported glycosylated serum boxylase is the first step in fatty acid synthesis.5 Protein-bound enzyme.3,10,11,13 Most of the microheterogeneity observed on biotin must be proteolytically degraded to release biocytin isoelectric focusing results from differences in the degree of (biotinyl-ε-lysine) and/or small biotinyl-peptides that can be sialylation. further cleaved by biotinidase, releasing the free biotin to enter Biotinidase is a thiol enzyme that migrates to the α1-region on the pool.3 A major function of biotinidase is to recycle biotin agarose electrophoresis. The serum enzyme has a pH optimum liberated from biocytin and/or small biotinyl-peptides from of 5–6 when biocytin or biotinyl-p-aminobenzoate (artificial degraded holocarboxylases. substrate) is the substrate.1,3,11 Biocytin is cleaved more readily Biotinidase deficiency (OMIM# 253260 and 609019), the than larger biotinyl-peptides.3 Biotinidase apparently does not major cause of late-onset biotin-responsive multiple carboxylase cleave biotin from intact holocarboxylases. The biotinyl-binding deficiency, is an autosomal recessively inherited neurocutane- site of biotinidase is specific for the ureido group of the biotinyl ous disorder.2,6,7 The symptoms of the disorder can be success- moiety of various substrates.11,14 Biotinidase plays a role in the fully treated or prevented by administering pharmacological processing of dietary protein-bound biotin7,15 and has been doses of biotin. Newborn screening of the disorder is performed shown to transfer biotin from biocytin to nucleophiles, such as in most states and many countries. Frequently asked questions histones16; the physiologic significance of the latter activity is by parents and health-care providers and various issues about not known. biotinidase deficiency have been addressed in a recent paper.8 The three-dimensional structure of the majority of the enzyme has been predicted through its homology with nitrilases and THE ENZYME amidases from microorganisms.17 The active site of the enzyme Biotinidase has been shown to have biotinyl-hydrolase and involved in the cleavage of the amide bond of biocytin is simi- biotinyl-transferase activities.9 Human biotinidase has been lar in all the enzymes. The portion of the enzyme that is not 1Department of Medical Genetics, Henry Ford Hospital, Detroit, Michigan, USA; 2Center for Molecular Medicine and Genetics, Wayne State University, Detroit, Michigan, USA. Correspondence: Barry Wolf ([email protected]) Submitted 1 August 2011; accepted 6 September 2011; advance online publication 5 January 2012. doi:10.1038/gim.2011.6 GENETICS in MEDICINE | Volume 14 | Number 6 | June 2012 565 GENETEST REVIEW WOLF | Biotinidase deficiency predicted by the modeling is likely important for binding the which contains the N-terminal methionine of the mature biotinyl moiety of biocytin. enzyme. The presence of an intron between the two possible initiation codons could allow for alternative splicing, which Biotinyl-hydrolase activity could produce transcripts encoding a protein with a 41- or a Polyclonal and monoclonal antibodies prepared against puri- 21-residue signal peptide.23 fied human biotinidase react on immunoblot with biotinidase The nucleotide sequence upstream of exons 1 and 2 has been in extracts of normal fibroblasts and lymphoblasts.9 These examined for putative promoter elements. Promoter features antibodies react with normal serum biotinidase that has been identified from −600 to +400 are consistent with the ubiqui- desialylated by treatment with neuraminidase. Individuals with tous expression of biotinidase with characteristics of a CpG profound biotinidase deficiency can be classified into at least island, lack of a TATA element, six consensus methylation sites, nine distinct biochemical phenotypes on the basis of the pres- and three initiator (Inr) sequences, which are thought to be ence or absence of cross-reacting material (CRM) to biotini- important in transcription initiation of TATA-less promoters. dase, the number of isoforms, and the distribution frequency of A consensus sequence for the liver-specific transcription factor the isoforms.18 The isoform patterns observed in the individu- HNF-5 is present at −352. The nucleotide sequence 5′ of exon 2, als with partial biotinidase deficiency were not different from which contains the second putative ATG initiation codon, has those of individuals with profound biotinidase deficiency who features associated with housekeeping genes but does contain had CRM. a consensus sequence for the liver-specific transcription factor C/EBP within 300 bp of the 5′ end of exon 2. Biotinyl-transferase activity More than 100 children with profound biotinidase deficiency CLINICAL FEATURES OF BIOTINIDASE have been assessed for biotinyl-transferase activity and the pres- DEFICIENCY ence of CRM to antibodies prepared against purified serum bio- Clinically, children with profound biotinidase deficiency, if tinidase.16 Sera from all the symptomatic individuals studied— untreated, usually exhibit one or more of the following symp- both CRM-negative and CRM-positive—had no biotinyl- toms: hypotonia; seizures; eczematous skin rash; alopecia; respi- transferase activity. Sera from children detected by newborn ratory problems, such as hyperventilation, laryngeal stridor, and screening who were CRM-negative had no biotinyl-transferase apnea; conjunctivitis, candidiasis; ataxia; developmental delay; activity, whereas a considerable number of the CRM-positive hearing loss; and vision problems, such as optic atrophy.2,24 children had varying degrees of transferase activity. There was a Metabolically, most untreated individuals with biotinidase statistically significant difference in biotinyl-transferase activity deficiency will have one or more of the following: ketolactic aci- between the population of symptomatic enzyme-deficient chil- dosis, organic aciduria, and mild hyperammonemia.24,25 How- dren and those children who were identified by newborn screen- ever, the absence of organic aciduria or metabolic ketoacidosis ing. There was also a difference in the biotinyl-hydrolase activity does not exclude the diagnosis of biotinidase deficiency in a between the symptomatic and newborn screening group.19 symptomatic child. Symptoms of untreated profound biotinidase deficiency usu- MOLECULAR ORGANIZATION AND ally appear between the ages of 1 week and 10 years, with a mean CHARACTERIZATION OF THE BIOTINIDASE GENE age of 3.5 months.25 Some children with biotinidase deficiency The gene for human biotinidase (BTD) is located on chromo- exhibit only a single symptom, whereas others have multiple some 3q25.20 The complementary DNA (cDNA) for human neurological, cutaneous, or biochemical findings. biotinidase from a human cDNA hepatic library has two The most common neurologic features of individuals with putative ATG initiation codons and an open reading frame of untreated, profound biotinidase deficiency are seizures and 1,629 bp, relative to the first ATG codon.21 The cDNA encodes hypotonia.2,6,25–27 The seizures are usually myoclonic, but may for a mature protein of 543 amino acids with a molecular mass be grand mal and focal; some children