Identification of two novel amino acid polymorphisms in beta-cell/liver (GLUT2) glucose transporter in Japanese subjects

Diabetologia, Feb 1995

The beta-cell/liver glucose transporter (GLUT2) gene was screened for mutations using single-strand conformation polymorphism analysis (SSCP) in 30 Japanese subjects with non-insulin dependent diabetes mellitus (NIDDM). Analysis of all exons and adjacent intron regions identified six SSCP polymorphisms, three of which resulted in amino acid substitutions: V101I, T110I and G519E. The V101I and G519E substitutions represent new polymorphisms in this gene. The six polymorphisms were observed in both NIDDM and control groups and there were no significant differences in allele frequencies between groups. A portion of the insulin receptor substrate 1 gene in 30 NIDDM subjects and in normal control subjects was also screened for mutations. Two SSCP variants that change the sequence of the protein, δS686/687 (deletion of the codons for serine-686 and 687) and G972R, were identified in two different NIDDM subjects, both whom were also heterozygous for the V101I polymorphism in GLUT2. The GLUT2 and IRS1 amino acid polymorphisms did not show a simple pattern of co-inheritance with NIDDM in the families of these subjects suggesting that neither polymorphism is sufficient to cause NIDDM but may increase diabetes-susceptibility through their interaction with other loci and environmental factors.

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Identification of two novel amino acid polymorphisms in beta-cell/liver (GLUT2) glucose transporter in Japanese subjects

Diabetologia Identification of two novel amino acid polymorphisms in beta-cell/ liver (GLUT2) glucose transporter in Japanese subjects F. Shimada 0 1 H. Makino 0 1 H. Iwaoka 1 S. Miyamoto 1 N. Hashimoto 0 1 A. Kanatsuka 0 1 G.I. Bell 1 S. Yoshida 0 1 0 Inohana Chuo-ku , Chiba 260, Japan Abbreviations: NIDDM, Non-insulin-dependent diabetes mellitus; GLUT2, glucose transporter 2; IRS1, insulin receptor substrate-1; SSCR single-stranded conformation polymorphism; PCR, polymerase chain reaction; Thr, threonine; Phe, phenylalanine; Ala, alanine; Ser, serine; Ile, isoleucine; Val, valine; Gly,glycine 1 1Second Department of Internal Medicine, Chiba University School of Medicine, Chiba, Japan 2Narita Red Cross Hospital, Narita, Japan 3Division of Endocrinology,Chiba Children's Hospital , Chiba , Japan 4Howard Hughes Medical Institute and Departments of Biochemistry and Molecular Biology and Medicine, University of Chicago , Chicago,Illinois , USA Summary The beta-cell/liver glucose transporter (GLUT2) gene was screened for mutations using single-strand conformation polymorphism analysis (SSCP) in 30 Japanese subjects with non-insulin dependent diabetes mellitus (NIDDM). Analysis of all exons and adjacent intron regions identified six SSCP polymorphisms, three of which resulted in amino acid substitutions: V101I, T l l 0 I and G519E. The V101I and G519E substitutions represent new polymorphisms in this gene. The six polymorphisms were observed in both N I D D M and control groups and there were no significant differences in allele frequencies between groups. A portion of the insulin receptor substrate i gene in 30 N I D D M subjects and in normal control subjects was also screened for mutations. Two SSCP variants that change the sequence of the protein, AS686/687 (deletion of the codons for serine-686 and 687) and G972R, were identified in two different N I D D M subjects, both whom were also heterozygous for the V101I polymorphism in GLUT2. The G L U T 2 and IRS1 amino acid polymorphisms did not show a simple pattern of co-inheritance with N I D D M in the families of these subjects suggesting that neither polymorphism is sufficient to cause N I D D M but may increase diabetes-susceptibility through their interaction with other loci and environmental factors. [Diabetologia (1995) 38: 211-215] Diabetes; glucose transporter; insulin receptor substrate-1; insulin; genetics - 9 Springer-Verlag1995 Genetic factors play an important role in the development of non-insulin dependent diabetes mellitus (NIDDM), a heterogeneous disorder characterized by defects in insulin secretion as well as insulin action [ 1 ]. Mutations associated with N I D D M have been identified in the insulin [ 2 ], insulin receptor [ 3, 4 ], glucokinase [ 5-7 ] and mitochondrial genes [ 8, 9 ]. This suggests that direct screening of other candidate genes for mutations in a cohort of well-characterized subjects may lead to the identification of other diabetes-susceptibility loci. G L U T 2 is the glucose transporter isoform responsible for the transport of glucose across the plasma membrane of the beta cell and hepatocyte [ 10 ]. Decreased expression of this glucose transporter has been associated with defects in glucose-stimulated insulin secretion in animal models of N I D D M and I D D M [ 11, 12 ]. The results of association studies to assess the contribution of G L U T 2 to the development of N I D D M have been inconsistent [ 13, 14 ]. However, recent studies in Pima Indians have shown evidence of linkage between GLUT2 and acute insulin response. These studies also identified an amino acid polymorphism in GLUT2, Tl10I, although this polymorphism could not account for the linkage between G L U T 2 and acute insulin response [ 15 ]. Direct screening of G L U T 2 for mutations in a cohort of African-American women who had gestational Materials and methods Subjects. Thirty subjects with N I D D M and 48 normal control subjects were studied (Table 1). The control subjects had no family history of diabetes and normal glucose tolerance on administration of a 75-g oral glucose tolerance test. The diagnosis of N I D D M was based on World Health Organisation criteria [ 18 ], All patients were informed of the purpose of study and gave their informed consent. These studies were approved by the ethics committee of Chiba University Hospital. Screening for nucleotide substitutions in GLUT2 and insulin receptor substrate-1. D N A was isolated from human lymphocytes. The 11 exons and adjacent introns of GLUT2 [!9] were amplified using polymerase chains reaction (PCR) and primers specific for each exon. A region of IRS1 [ 20 ] representing amino acids 445 to 1028 [ 21 ] was also screened for mutations by SSCR (The sequences of the primers used to amplify GLUT2 and IRS1 are available from the authors on request). Standard PCR conditions were used with 3~P-labelled primers. The PCR products were diluted 15-fold with formamide buffer (95 % formamide, 0.05 % bromphenol blue and 0.05 % xylene cyanol), heated at 95 ~ for 3 rain and 1.5 gl of each sample was loaded onto a 5 % non-denaturing polyacrylamide gel (30 40 x 0.03; acrylamide:N,N'-methylene-bis-acrylamide = 49:1). Each sample was run on four gels containing 0 or 10 % glycerol at room temperature or 4 ~ The gels were then transferred to paper, dried and exposed to film (XAR-5; Kodak, Rochester, N.Y., USA) with an intensifying screen for ~[2h at -70~ The sequences of novel bands observed by SSCP were determined directly and also after subcloning the amplified DNA into M13. Statistical analysis Data are given as mean + SEM. The association of polymorphisms with NIDDM was analysed by 2 x 2 contingency tables and the significance of difference was tested by a Fisher's exact test. Results P o l y m o r p h i s m s in G L U T 2 . Six S S C P v a r i a n t s w e r e i d e n t i f i e d o n s c r e e n i n g t h e e n t i r e p r o t e i n c o d i n g reg i o n o f G L U T 2 t o g e t h e r w i t h a d j a c e n t i n t r o n segm e n t s in 30 J a p a n e s e s u b j e c t s w i t h N I D D M (Table 2). T h e T l l 0 I p o l y m o r p h i s m , t h e silent s u b s t i t u tions in t h e c o d o n s f o r Thr-198 a n d P h e - 4 7 9 , a n d t h e C / T p o l y m o r p h i s m 15 b p b e f o r e t h e s p l i c e - a c c e p t o r site o f e x o n 5 h a v e b e e n d e s c r i b e d previously. T h e s u b s t i t u t i o n s V 1 0 1 I a n d G 5 1 9 E r e p r e s e n t n e w p o l y morphisms/variants [ 16 ]. T h e V 1 0 1 I w a s m o s t easily i d e n t i f i e d b y S S C P in a 5 % p o l y a c r y l a m i d e gel w i t h GLUT2 IRS-1 Location Exon3 Exon3 c Exonl0 Exon4B c Intron5 ~ Exonl0 Codon 686/687 Codon 972 Codon 805 Nucleotide change Amino acid Allele frequency (%) NIDDM a Controlb Codon 101 Codon 110 Codon 519 Codon 198 Codon 479 GTA --~ ATA ACT -4 ATT G G A -~ G A A ACG -4 ACA C --~ T TTC -4 TTT AAGCIAGC G G G -~ A G G GCA -4 GCG Val -~ Ile Thr -4 Ile Gly ~ Glu Thr -4 Thr Phe -4 Phe ASer/Ser Gly --+ Arg Ala -4 Ala 7.3 4.2 1.0 4.2 4.2 22.9 1.0 1.0a 39.6 412 3.1 1.0 3.1 3.1 27.0 0 0 37.5 an = 96 (48 individuals, including 30subjects screened by SSCP and 18 others); b n = 96 (48 individuals); c These polymorphisms were found in the same subjects and thus may be in linkage disequilibrium; d this substitution was found only in members of the FA family , F A GLUT2v1~176 IRS-1 G972/I~972 GLUT2V101G519/il~ IRS*I G972/G972 GLUT2 IRS-1 $6 10 % glycerol at room temperature and G519E with a 5 % polyacrylamide gel without glycerol at 4 ~ We also tested 18 additional subjects with N I D D M and 48 non-diabetic subjects for these polymorphisms. There was no difference in frequency of any of these polymorphisms between N I D D M and non-diabetic groups (Table 2). The G519E variant was identified in an N I D D M subject who was also heterozygous for the V101I polymorphism (Fig. 1). Thus, this subject (FA II-1) was a c o m p o u n d heterozygote. Characterization of the pattern of segregation of these two polymorphisms in the subject's family indicated that the allele haplotypes are ll~ 519 and Vl~ the native haplotype is v l ~ 519. Polymorphisms in IRS1. Almid et al. [ 22 ] have reported two amino acid variants/polymorphisms in IRS1, A513P and G972R, and suggested that amino acid polymorphisms in IRS1 may be involved in the development of NIDDM. We screened the region of amino acids 445 to 1028 of IRS1 for mutations in 30 Japanese subjects with NIDDM. Three SSCP variants were noted (Table 2): a silent substitution in the codon for Ala-805 (GCA/GCG), the G972R polymorphism, and a six nucleotide deletion resulting in the deletion of the codons for Ser-686 and Set 687 (AS686/687). We also tested 18 additional subjects with N I D D M and 48 non-diabetic subjects for G972R and AS686/687. These variants were found in one N I D D M subject each and in none of the non-diabetic subjects (Table 2). Presence o f GLUT2 and IRS1 amino acid polymorphisms in the same subject. The two IRS1 amino acid polymorphisms were present in subjects with the V101I polymorphism in GLUT2, one of whom was also a compound heterozygote of GLUT2 (Fig.l). Analysis of the co-segregation of the G L U T 2 and IRS1 variants with N I D D M in these families did not show a simple pattern of inheritance. Discussion GLUT2 is the major glucose transporter expressed in beta cells and hepatocytes and plays a central role in the regulation of glucose metabolism in these cells. Diabetes is characterized in part by the inability of the beta cell to secrete insulin in response to glucose whereas the response to non-glucose secretagogues is retained, and it has been proposed that decreased expression of beta-cell G L U T 2 is responsible for the loss of the glucose-stimulated insulin secretory response [ 23 ]. The decreased levels of GLUT2 may impair the glucose-stimulated insulin secretory response by reducing high K mglucose transport. They may also affect glucose responsiveness directly by uncoupling G L U T 2 and cellular proteins and/or structures involved in glucose signalling such as glucokinase. In this regard Hughes et al., [ 24 ] have shown that the heterologous expression of G L U T 2 confers glucose responsiveness to non-beta cells. If decreased G L U T 2 expression impairs glucose sensing, mutations in G L U T 2 may have a similar effect. Four amino acid polymorphisms V10I, T l l 0 I , V197I and G519E have now been described in GLUT2 (Fig. 2). They are located in transmembrane segments 2 (V101 and T l l 0 ) and 5 (V197), and the intracellular COOH-terminal domain (G519). The T l l 0 I and V197I mutations have been expressed in vitro. The Ile-110 change had no effect on 2-deoxyglucose uptake whereas the Ile-197 change abolished transport activity [ 17 ]. The effect of substitutions at Val-101 and Gly-519 on G L U T 2 transport activity have not been determined. The residue Val-101 is invariant in sequences of the mammalian facilitative glucose/fructose transporter family [ 25, 26 ], and thus its mutation may affect transporter activity or function. However, the V101I substitution was present in both N I D D M and non-diabetic subjects suggesting that its presence per se is not sufficient to cause N I D D M although it may contribute to the development of N I D D M in the presence of other genetic or acquired % ~6 NH2| 9 810 | Oooo+o%0~ .oCDC~. % CHO d e f e c t s in b e t a - c e l l f u n c t i o n a n d insulin a c t i o n or b o t h . T h e r e s i d u e G l y - 5 1 9 in G L U T 2 is n o t c o n s e r v e d a n d is G l y in t h e h u m a n a n d r a t a n d A l a in m o u s e [ 26 ], a n d its e f f e c t o n b e t a - c e l l f u n c t i o n is u n k n o w n . D u r i n g t h e c o u r s e o f this study, t w o f a m i l i e s w e r e i d e n t i f i e d t h a t w e r e s e g r e g a t i n g a m i n o acid p o l y m o r p h i s m s in b o t h G L U T 2 a n d I R S 1 t o g e t h e r w i t h N I D D M . This m a y b e c o i n c i d e n t a l o r m a y s u g g e s t t h a t b o t h loci c o n t r i b u t e to t h e d e v e l o p m e n t o f N I D D M . H o w e v e r , t h e p a t t e r n o f i n h e r i t a n c e o f t h e G L U T 2 a n d I R S 1 p o l y m o r p h i s m s w i t h N I D D M was n o t c o n s i s t e n t w i t h a s i m p l e m o d e l f o r g e n e t i c liability. T h e i d e n t i f i c a t i o n o f a d d i t i o n a l f a m i l i e s in w h i c h t h e s e p o l y m o r p h i s m s a r e s e g r e g a t i n g m a y r e s o l v e t h e i r r e l a t i v e c o n t r i b u t i o n to t h e d e v e l o p m e n t o f gluc o s e i n t o l e r a n c e a n d d i a b e t e s . Acknowledgements. We thank Drs. S. Seino (Chiba University) and Y. Tokuyama (University of Chicago) for their helpful advice. This research was supported by grants from the Ministry of Education, Science and Culture, Japan (No. 05 670840), Otsuka Pharmaceutial Co. 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F. Shimada, Dr. H. Makino, H. Iwaoka, S. Miyamoto, N. Hashimoto, A. Kanatsuka, G. I. Bell, S. Yoshida. Identification of two novel amino acid polymorphisms in beta-cell/liver (GLUT2) glucose transporter in Japanese subjects, Diabetologia, 1995, 211-215, DOI: 10.1007/BF00400096