TY - JOUR
T1 - Biallelic GALM pathogenic variants cause a novel type of galactosemia
AU - Wada, Yoichi
AU - Kikuchi, Atsuo
AU - Arai-Ichinoi, Natsuko
AU - Sakamoto, Osamu
AU - Takezawa, Yusuke
AU - Iwasawa, Shinya
AU - Niihori, Tetsuya
AU - Nyuzuki, Hiromi
AU - Nakajima, Yoko
AU - Ogawa, Erika
AU - Ishige, Mika
AU - Hirai, Hiroki
AU - Sasai, Hideo
AU - Fujiki, Ryoji
AU - Shirota, Matsuyuki
AU - Funayama, Ryo
AU - Yamamoto, Masayuki
AU - Ito, Tetsuya
AU - Ohara, Osamu
AU - Nakayama, Keiko
AU - Aoki, Yoko
AU - Koshiba, Seizo
AU - Fukao, Toshiyuki
AU - Kure, Shigeo
N1 - Funding Information:
The authors would like to thank the patients and families who participated in this study. We thank Yoko Chiba, Kumi Ito, Miyuki Tsuda, Mami Kikuchi, Makiko Nakagawa, Yoko Tateda and Kiyotaka Kuroda for providing technical assistance. We also acknowledge the support from the Biomedical Research Core of the Tohoku University Graduate School of Medicine and the Biomedical Research Unit of Tohoku University Hospital. This research was supported by the Japanese Agency for Medical Research and Development (AMED) through grant numbers JP17ek0109151h0003 (Initiative on Rare and Undiagnosed Diseases [IRUD] to S. Kure), JP17ek0109276 (to T.F.), and JP17km0405001 (the Program for Promotion of Genome Medicine to S. Koshiba). This work was also supported by grants from the Project for Promoting Public Utilization of Advanced Research Infrastructure (MEXT) (to S. Koshiba) and the Takeda Science Foundation (to A.K.).
Funding Information:
The GALM complementary DNA (cDNA) (IRAK015M11) was provided by the RIKEN Bioresource Center (BRC) through the National Bio-Resource Project of the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan,22–25 and was subcloned into pCMV6-AN-DDK (PS100014, OriGene, Rockville, MD, USA) or pET21d(+) (69743, Novagen, Merck Millipore, Darmstadt, Germany) with a His6 tag at the N-terminus using the In-Fusion HD Cloning Kit (TaKaRa, Shiga, Japan). Then, 293FT cells (R700-07, Invitrogen™) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing high glucose supplemented with 10% fetal bovine serum, and 1% penicillin and streptomycin (100 U/ml and 100 μg/ml, respectively) at 37 °C in a 5% CO2 incubator. Epstein–Barr virus transformed lymphoblastoid cell lines (EBV-LCLs) were established from patients 2, 3, 8, and healthy controls using previously described methods.26 All cell lines were tested for mycoplasma
Publisher Copyright:
© 2018, American College of Medical Genetics and Genomics.
PY - 2019/6/1
Y1 - 2019/6/1
N2 - Purpose: Galactosemia is caused by metabolic disturbances at various stages of galactose metabolism, including deficiencies in enzymes involved in the Leloir pathway (GALT, GALK1, and GALE). Nevertheless, the etiology of galactosemia has not been identified in a subset of patients. This study aimed to explore the causes of unexplained galactosemia. Methods: Trio-based exome sequencing and/or Sanger sequencing was performed in eight patients with unexplained congenital galactosemia. In vitro enzymatic assays and immunoblot assays were performed to confirm the pathogenicity of the variants. Results: The highest blood galactose levels observed in each patient were 17.3–41.9 mg/dl. Bilateral cataracts were observed in two patients. In all eight patients, we identified biallelic variants (p.Arg82*, p.Ile99Leufs*46, p.Gly142Arg, p.Arg267Gly, and p.Trp311*) in the GALM encoding galactose mutarotase, which catalyzes epimerization between β- and α-D-galactose in the first step of the Leloir pathway. GALM enzyme activities were undetectable in lymphoblastoid cell lines established from two patients. Immunoblot analysis showed the absence of the GALM protein in the patients’ peripheral blood mononuclear cells. In vitro GALM expression and protein stability assays revealed altered stabilities of the variant GALM proteins. Conclusion: Biallelic GALM pathogenic variants cause galactosemia, suggesting the existence of type IV galactosemia.
AB - Purpose: Galactosemia is caused by metabolic disturbances at various stages of galactose metabolism, including deficiencies in enzymes involved in the Leloir pathway (GALT, GALK1, and GALE). Nevertheless, the etiology of galactosemia has not been identified in a subset of patients. This study aimed to explore the causes of unexplained galactosemia. Methods: Trio-based exome sequencing and/or Sanger sequencing was performed in eight patients with unexplained congenital galactosemia. In vitro enzymatic assays and immunoblot assays were performed to confirm the pathogenicity of the variants. Results: The highest blood galactose levels observed in each patient were 17.3–41.9 mg/dl. Bilateral cataracts were observed in two patients. In all eight patients, we identified biallelic variants (p.Arg82*, p.Ile99Leufs*46, p.Gly142Arg, p.Arg267Gly, and p.Trp311*) in the GALM encoding galactose mutarotase, which catalyzes epimerization between β- and α-D-galactose in the first step of the Leloir pathway. GALM enzyme activities were undetectable in lymphoblastoid cell lines established from two patients. Immunoblot analysis showed the absence of the GALM protein in the patients’ peripheral blood mononuclear cells. In vitro GALM expression and protein stability assays revealed altered stabilities of the variant GALM proteins. Conclusion: Biallelic GALM pathogenic variants cause galactosemia, suggesting the existence of type IV galactosemia.
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U2 - 10.1038/s41436-018-0340-x
DO - 10.1038/s41436-018-0340-x
M3 - Article
C2 - 30451973
AN - SCOPUS:85055249201
VL - 21
SP - 1286
EP - 1294
JO - Genetics in Medicine
JF - Genetics in Medicine
SN - 1098-3600
IS - 6
ER -