1084 Diabetes Volume 68, May 2019 Loss-of-Function Mutation in Thiamine Transporter 1 in a Family With Autosomal Dominant Diabetes Prapaporn Jungtrakoon,1,2,3 Jun Shirakawa,1,4 Patinut Buranasupkajorn,1,2,5 Manoj K. Gupta,1,4 Dario F. De Jesus,1,4 Marcus G. Pezzolesi,6 Aussara Panya,1,2,3 Timothy Hastings,2 Chutima Chanprasert,3 Christine Mendonca,2 Rohit N. Kulkarni,1,4 and Alessandro Doria1,2 Diabetes 2019;68:1084–1093 | https://doi.org/10.2337/db17-0821 Solute Carrier Family 19 Member 2 (SLC19A2) encodes cardiovascular, and neuroendocrine abnormalities such as thiamine transporter 1 (THTR1), which facilitates thia- optic atrophy (2), arrhythmia, heart defects (3,4), thyroid mine transport across the cell membrane. SLC19A2 function alterations (5), and seizures (6). homozygous mutations have been described as a cause Thiamine, also known as vitamin B1, acts as coenzyme of thiamine-responsive megaloblastic anemia (TRMA), of the cytosolic enzyme transketolase (TKT), which is part an autosomal recessive syndrome characterized by of the pentose phosphate pathway (PPP) responsible for megaloblastic anemia, diabetes, and sensorineural deaf- the generation of NADPH and ribose-5-phosphate— SLC19A2 ness. Here we describe a loss-of-function a precursor of high-energy ribonucleotides (e.g., ATP) mutation (c.A1063C: p.Lys355Gln) in a family with early- and nucleotides. Thiamine is also the coenzyme for several onset diabetes and mild TRMA traits transmitted in an SLC19A2 mitochondrial enzymes, including pyruvate dehydroge- autosomal dominant fashion. We show that - a a fi b nase (PDH), -ketoglutarate dehydrogenase ( -KGDH), de cient -cells are characterized by impaired thiamine a uptake, which is not rescued by overexpression of branched-chain -ketoglutarate dehydrogenase (BCKD), the p.Lys355Gln mutant protein. We further demonstrate and 2-hydroxyphytanoyl-CoA lyase. The megaloblastic ane- that SLC19A2 deficit causes impaired insulin secretion in mia associated with SLC19A2 mutations appears to be due conjunction with mitochondrial dysfunction, loss of pro- to impaired TKT activity, which, by reducing the availabil- tection against oxidative stress, and cell cycle arrest. ity of nucleotides, causes red cell precursors to continue to These findings link SLC19A2 mutations to autosomal grow in size without dividing. The coexistence of senso- dominant diabetes and suggest a role of SLC19A2 in rineural deafness and the other neurological and cardio- b-cell function and survival. vascular abnormalities found in the TRMA syndrome are suggestive of an underlying mitochondrial dysfunction, GENETICS/GENOMES/PROTEOMICS/METABOLOMICS which might be due, in part, to impaired PDH (7) and SLC19A2 encodes thiamine transporter 1 (THTR1), a trans- a-KGDH (8) activities causing defective energy production, membrane protein that facilitates the transport of thia- to which tissues with a high demand for energy, such as the mine by shielding its positive charge from the repulsive auditory, visual, cardiovascular, and central nervous sys- force of the membrane. Homozygous SLC19A2 mutations tems, are particularly sensitive (9–11). have been described as the cause of thiamine-responsive The form of diabetes that is part of the TRMA syndrome megaloblastic anemia (TRMA) (Online Mendelian Inheri- is nonautoimmune and is characterized by an insulin se- tance in Man [OMIM] #249270), a syndrome characterized cretion deficit and a time of onset ranging from birth to by megaloblastic anemia, diabetes, sensorineural deafness adolescence. Insulin treatment is usually needed, although (1), and the variable combination of other neurological, the insulin requirement may be reduced in some cases by 1Department of Medicine, Harvard Medical School, Boston, MA Corresponding author: Alessandro Doria, [email protected] 2 Section on Genetics and Epidemiology, Joslin Diabetes Center, Boston, MA Received 12 July 2017 and accepted 23 February 2019 3Section on Islet Cell and Regenerative Biology, Joslin Diabetes Center, Boston, MA This article contains Supplementary Data online at http://diabetes 4Division of Molecular Medicine, Research Department, Faculty of Medicine, Siriraj .diabetesjournals.org/lookup/suppl/doi:10.2337/db17-0821/-/DC1. Hospital, Mahidol University, Bangkok, Thailand 5Division of Hospital and Ambulatory Medicine, Department of Medicine, Faculty of © 2019 by the American Diabetes Association. Readers may use this article as Medicine, Chulalongkorn University, Bangkok, Thailand long as the work is properly cited, the use is educational and not for profit, and the 6Division of Nephrology and Hypertension, University of Utah School of Medicine, work is not altered. More information is available at http://www.diabetesjournals Salt Lake City, UT .org/content/license. diabetes.diabetesjournals.org Jungtrakoon and Associates 1085 high-dose thiamine supplementation (12,13). The TRMA approved by the Joslin Diabetes Center’s Committee on syndrome has been reported in .50 families worldwide Human Studies (CHS#5-05). Upon receipt, islets were (14). While in most cases heterozygous SLC19A2 mutation cultured overnight in Miami Media #1A (Cellgro). Mouse carriers have not been described as having diabetes, adult- islets were isolated from wild-type C57BL/6 mice. Islets onset glucose intolerance with or without a hearing defect were handpicked and cultured overnight in RPMI media was reported among these subjects in three TRMA kindreds containing 5 mmol/L glucose and 10% FBS. For immu- (15). The question as to whether a single allele defect of nostaining studies, islets were embedded in agarose and SLC19A2 may cause diabetes has never been addressed. In used. For insulin secretion studies, islets were starved in this study, we report a loss-of-function mutation in the Final Wash/Culture Media (Cellgro) for 5 h before being SLC19A2 gene linked, albeit with incomplete penetrance, stimulated with 10 nmol/L insulin. to autosomal dominant diabetes with mild TRMA clinical signs and show suggestive evidence for SLC19A2 deficiency Immunohistochemistry and Immunoblotting causing impaired insulin secretion and diabetes by de- Human and mouse islets were analyzed by immunostain- termining mitochondrial dysfunction, loss of protection ing using anti-THTR1 (sc-27656; Santa Cruz Biotechnol- against oxidative stress, and cell cycle arrest in b-cells. ogy) and anti-insulin (ab7842; Abcam). Images were taken with an LSM7 confocal laser scanning microscope (Carl RESEARCH DESIGN AND METHODS Zeiss). For immunoblotting, MIN6-m9 cells, mouse islets, Whole-Exome Sequencing or human islets were solubilized in M-PER lysis buffer Fifty-two extended families with autosomal dominant di- (Thermo Scientific) with protease inhibitors and phospha- abetes underwent whole-exome sequencing as reported by tase inhibitors (Sigma-Aldrich), and protein concentration Prudente et al. (16). Variants were filtered as previously was measured using a BCA protein assay kit (Pierce). described. Briefly, variants that were heterozygous, were Extracts were subjected to Western blotting with primary reported in public databases (dbSNP142, 1000 Genomes, antibodies overnight at 4°C. Mouse and human anti- and Exome Variant Server [EVS]) with frequency ,0.01, THTR1, anti–b-actin, and anti–a-tubulin antibodies were were shared by affected family members, and were poten- from Sigma-Aldrich (HPA016599), Cell Signaling Technol- tially deleterious as determined by a panel of prediction ogy (4970), and Abcam (ab7291), respectively. softwares were included in the analysis (16). In the former study, the focus was on variants that were located in genes Cellular Localization of SLC19A2-GFP Fusion Proteins with functional relevance to glucose metabolism as de- The coding sequence of the human SLC19A2 gene termined by an analysis with GeneDistiller (16). In the (NM_006996; OHu18269C [GenScript]) was subcloned current study, we reanalyzed those same exome sequenc- into the pAcGFP1-N1 vector (Addgene) at XhoI and AgeI ing data focusing on variants placed in a second set of sites. The SLC19A2-WT-GFP (GFP-tagged wild type [WT]) candidate genes, namely, genes that are mutated in rare construct was used as a template for generation of forms of syndromic diabetes. These included the WFS1, SLC19A2-K355Q-GFP (GFP-tagged mutant) by means of CISD2, ALMS1, and SLC19A2 genes, which are mutated in site-directed mutagenesis using the Q5 Site-Directed Mu- Wolfram type 1, Wolfram type 2, Alström, and TRMA tagenesis Kit (NEB). Primers for cloning and mutagenesis syndromes, respectively. Filtered variants were validated are provided in Supplementary Table 1. Plasmid con- and analyzed for segregation with diabetes in the families structs were transiently transfected into MIN6-m9 using by means of Sanger DNA sequencing. Variants were con- METAFECTENE PRO (Biontex). Twenty-four hours after sidered to segregate with diabetes if $80% of carriers .40 transfection, cells were trypsinized and transferred to years old had developed diabetes and there were no poly-L-lysine–coated glass-bottom dishes. Live cell imaging phenocopies whose phenotype was not consistent with was performed at 72 h after transfection with an LSM7 either type 1 or older-onset type 2 diabetes. confocal laser scanning microscope (Carl Zeiss). Cell Culture SLC19A2 Suppression and Quantitative RT-PCR The mouse insulinoma cell line MIN6-m9 (17) was kindly Analysis provided by Prof. S. Seino, Kobe University. Unless in- Lentiviral transduction particles (Sigma-Aldrich) carrying dicated otherwise,