Abstract Holocarboxylase synthetase (HLCS) is an en- zyme that catalyzes the incorporation of biotin into apo- carboxylases, and its deficiency causes biotin-responsive multiple carboxylase deficiency. The reported sequences of cDNA for human HLCS from liver, lymphocyte, and KG-1 myeloid cell lines differ at their 5′ regions. To elu- cidate variations of the human HLCS mRNA and longer 5′ cDNA ends, we performed screening of the human liver cDNA library and rapid amplification of the cDNA ends (RACE). Our results suggest the existence of three types of HLCS mRNA that start at different exons. The first type starts at exon 1, and the second type starts at exon 3, and both are found in various human tissues. The third type, corresponding to the cDNA from the KG-1 cell, starts at exon 2 of the HLCS gene. Various splicing patterns from exons 3–6 were also observed. None of the variations of cDNA found created a new initiation codon. Mutation screening from exons 6–14, therefore, was suf- ficient to detect amino acid changes in HLCS in patients. Our direct sequencing strategy for screening mutations in the HLCS gene revealed mutations in five Japanese pa- tients and seven non-Japanese patients. Our analyses in- volving 12 Japanese and 13 non-Japanese patients and studies by others indicate that (1) there is no panethnically prevalent mutation; (2) the Arg508Trp, Gly581Ser, and Val550Met mutations are found in both Japanese and non- Japanese populations; (3) the IVS10+5G→A mutation is predominant and probably a founder mutation in Euro- pean patients; (4) the 655–656insA, Leu237Pro, and 780delG mutations are unique in Japanese patients; (5) the spectrum of the mutations in the HLCS gene may vary substantially among different ethnic groups. Introduction Holocarboxylase synthetase (HLCS: EC 6.3.1.10) is an enzyme that catalyzes biotin incorporation into carbox- ylases (Achuta Murthy and Mistry 1972; Wolf 2001). In mammalian cells, four carboxylases that require biotin as a prosthetic group are known: acetyl-CoA carboxylase (EC 6.4.1.2), a rate-limiting enzyme in fatty acid synthe- sis; pyruvate carboxylase (EC 6.4.1.1), a key enzyme of gluconeogenesis; propionyl-CoA carboxylase (EC 6.4.1.3) and 3-methylcrotonyl-CoA carboxylase (EC 6.4.1.4), en- zymes involved in amino acid catabolism (Wood and Bar- den 1977). Biotinylation of these carboxylases is essential for their enzymatic activities. Any defect in HLCS results in the decreased activity of many carboxylases, thereby affecting various metabolic processes in cells. HLCS defi- ciency (McKusick 253270) is therefore also known as bi- otin-responsive multiple carboxylase deficiency (MCD; Wolf 2001). We previously isolated human cDNA for HLCS from liver (Suzuki et al. 1994). The isolated cDNA encoded Xue Yang · Yoko Aoki · Xue Li · Osamu Sakamoto · Masahiro Hiratsuka · Shigeo Kure · Sepidh Taheri · Ernst Christensen · Koji Inui · Mitsuru Kubota · Miki Ohira · Misao Ohki · Jun Kudoh · Kazuhiko Kawasaki · Kazunori Shibuya · Ai Shintani · Shuichi Asakawa · Shinsei Minoshima · Nobuyoshi Shimizu · Kuniaki Narisawa · Yoichi Matsubara · Yoichi Suzuki Structure of human holocarboxylase synthetase gene and mutation spectrum of holocarboxylase synthetase deficiency Hum Genet (2001) 109 : 526–534 DOI 10.1007/s004390100603 Received: 2 July 2001 / Accepted: 22 August 2001 / Published online: 5 October 2001 ORIGINAL INVESTIGATION X. Yang · Y. Aoki · X. Li · O. Sakamoto · M. Hiratsuka · S. Kure · K. Narisawa · Y. Matsubara · Y. Suzuki (✉) Department of Medical Genetics, Tohoku University School of Medicine, 1-1 Seiryo-machi, Aoba-ku, Sendai 980-8574, Japan e-mail: ysuzuki@mail.cc.tohoku.ac.jp, Tel.: +81-22-7178140, Fax: +81-22-7178142 S. Taheri Department of Pediatrics, National Hospital, Torshavn, Faroe Islands E. Christensen Department of Clinical Genetics, Juliane Marie Centre, Rigshospitalet, Copenhagen, Denmark K. Inui Department of Pediatrics, Faculty of Medicine, Osaka University, Osaka, Japan M. Kubota Pediatrics and Developmental Medicine, Hokkaido University Graduate School of Medicine, Sapporo, Japan M. Ohira · M. Ohki Laboratory of Gene Structure 1, Kazusa DNA Research Institute, Kisarazu, Chiba, Japan J. Kudoh · K. Kawasaki · K. Shibuya · A. Shintani · S. Asakawa · S. Minoshima · N. Shimizu Department of Molecular Biology, Keio University School of Medicine, Tokyo, Japan © Springer-Verlag 2001