Human Molecular Genetics, 2001, Vol. 10, No. 17 1753-1760
© 2001 Oxford University Press
Molecular scanning of the human sorbin and SH3-domain-containing-1 (SORBS1) gene: positive association of the T228A polymorphism with obesity and type 2 diabetes
1Department of Internal Medicine and 2Graduate Institute of Clinical Medicine, National Taiwan University Hospital, 7 Chung Shan South Road, Taipei, Taiwan and 3Division of Endocrinology, Diabetes and Hypertension, Department of Medicine, University of California, Los Angeles, School of Medicine, Los Angeles, CA 90095, USA
Received March 9, 2001; Revised and Accepted June 13, 2001.
DDBJ/EMBL/GenBank accession nos+.
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ABSTRACT |
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In the mouse, the SH3P12 or the c-Cbl-associated protein (CAP) has been shown as an important signaling molecule in insulin-stimulated glucose uptake. The human homolog for the sorbin and SH3-domain-containing-1 gene, termed SORBS1, might play a role in human disorders with insulin resistance. To explore the genetic role of SORBS1 in human obesity and type 2 diabetes, we investigated the nucleotide polymorphisms in the SORBS1 gene with molecular scanning. After scanning for a total of 13 136 bp in each of 40 chromosomes, we have identified 14 single nucleotide polymorphisms (SNPs) in the human SORBS1 gene. Among them, two SNPs affected amino acid coding (R74W and T228A), four occurred within exons but did not affect amino acid coding, and the remaining eight occurred within introns, which were located outside of the consensus region of the splicing mechanism. Further studies in 202 non-obese, 113 obese and 455 subjects with type 2 diabetes revealed that the A-allele of the T228A polymorphism in exon 7 exerted a protective role for both obesity [relative risk 0.466; 95% confidence interval (95% CI) 0.265–0.821] and diabetes (relative risk 0.668; 95% CI 0.472–0.945). Neither allele of the R74W polymorphism was associated with either obesity or diabetes. In conclusion, our results suggest that the A228 allele of the T228A polymorphism of the SORBS1 gene is a protective factor for both obesity and diabetes, and also imply that the SORBS1 gene plays an important role in the pathogenesis of human disorders with insulin resistance.
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INTRODUCTION |
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Insulin resistance is one of the key features in various disorders, such as obesity, type 2 diabetes, hypertension and atherosclerosis (1,2). Although substantial progress has been made in understanding the pathogenesis of insulin resistance (3), there are still areas that are not fully explored in adipose tissue. In differentiated mouse adipocytes (3T3-L1 adipocytes), insulin stimulates glucose uptake and storage of glucose as glycogen and lipid much better than those in 3T3-L1 fibroblasts (4–7). Furthermore, fat transplantation improves glycemia and insulin sensitivity in lipodystrophic mice (8). Therefore, adipose tissue plays an important role in glucose homeostasis and insulin resistance. To investigate the role of adipose tissue in insulin sensitivity and to explore the molecular basis of insulin resistance in adipocytes, we studied mRNA expression in 3T3-L1 cells during adipogenesis using differential display (9). Among numerous genes affected in the process of differentiation of 3T3-L1 cells, SH3P12 [also known as c-Cbl-associated protein (CAP)] was up regulated. The expression of SH3P12 could be further enhanced by the treatment of BRL 49653, a thiazolidinedione (TZD) (9), which is a new class of anti-diabetic agents with a primary effect in improving insulin resistance via binding to the nuclear peroxisome proliferator-activated receptor
(PPAR) (10,11). The mouse SH3P12/CAP belongs to a growing family of proteins containing a sorbin homology domain and it has three Src homology 3 (SH3) domains in the C-terminal region. This gene was first cloned by screening a mouse cDNA expression library with a SH3 binding ligand (12), and subsequently isolated by a yeast two-hybrid system using c-Cbl as bait and a blot overlay method with the labeled 1-afadin (13,14), and therefore denoted as CAP or ponsin because of its interaction with both 1-afadin and vinculin in a competitive binding manner (13). Moreover, SH3P12/CAP has been found to interact with insulin receptor, Sos, flotillin and focal adhesion kinase (14–17), indicating that SH3P12/CAP might be involved in the signaling pathways to the reorganization of the cytoskeleton and insulin-stimulated glucose uptake process in adipocytes. More interestingly, TZD can increase the expression of SH3P12/CAP in adipose tissues, indicating that SH3P12/CAP is a candidate for insulin resistance (16,18,19).
The human homolog of mouse SH3P12/CAP, the sorbin and SH3-domain-containing-1 gene (formally designated as SORBS1), has been cloned and mapped to chromosome 10q23.3–24.1 (20). Sequence comparison revealed an 88% homology in amino acid sequences between the two species. To test the hypothesis that a genetic variation in SORBS1 will affect the development of obesity and type 2 diabetes, we further delineated the genomic organization and screened for the molecular variants of this gene. We found an amino acid polymorphism, T228A in the exon 7 of SORBS1, that showed a significant association with human obesity and type 2 diabetes.
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RESULTS |
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Genomic organization of the human SORBS1 gene
We had cloned and sequenced several cDNAs that contained the various alternatively spliced exons (GenBank accession nos AF136380, AF136381, AF356525–AF356527). The intron/exon boundaries and the sizes of introns of the SORBS1 gene were then determined by the alignment with the genomic sequences (AL158165 and AL160288) obtained from NCBI (Table 1). Based on the homology and alignment search, the coding region of this gene was encoded in 34 exons, separated by 33 introns that spanned in a region of >120 kb (Fig. 1). There were four gaps in the genomic sequences as indicated. The sizes of introns 4, 6, 8 and 18 were estimated by long-range PCR (Table 1).
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Molecular scanning of SORBS1
To screen for molecular variants (or nucleotide polymorphisms) of SORBS1, all exons and the flanking introns of 50–150 bp were amplified and sequenced in 40 chromosomes from 20 type 2 diabetic subjects who were non-obese (n = 10) and obese (n = 10) with primers as described in Table 2. After sequencing a region of 13 136 bp in 40 chromosomes, 14 single nucleotide polymorphisms (SNPs) were identified (Table 3). Among them, two SNPs affected amino acid coding (R74W and T228A), four occurred within exons without change in amino acid coding, and the remaining eight occurred within introns, which were located outside of the consensus region of the splicing mechanism. The molecular variant R74W in exon 4 was a C
T change, which led to a substitution of CGG (Arg) with TGG (Trp). A PCR-based denaturing high-performance liquid chromatography (DHPLC) assay using WAVE Nucleic Acid Fragment Analysis System (Transgenomic, Inc., San Jose, CA) was developed. The frequencies of R74W heterozygotes were not different among three groups of subjects (i.e. 15/184 = 8.2% for non-diabetic non-obese control subjects; 11/183 = 6.0% for diabetic subjects; and 4/113 = 3.5% for obese subjects). No homozygous variant was detected. The second missense variation, T228A, occurred in exon 7, substituting ACG (Thr) with GCG (Ala). The allelic frequency for the variant Ala allele was 0.075 in 40 chromosomes.
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Phenotypes of T228A of SORBS1
To characterize the phenotypes which could be associated with T228A, we genotyped 315 non-diabetic subjects [169 female; age 43 ± 1 years; body mass index (BMI) 29.6 ± 0.5 kg/m2, mean ± SEM] and 455 subjects with type 2 diabetes as defined by the 1998 WHO criteria (21). Since diabetes can lead to weight loss and alterations in glucose/insulin processing, only non-diabetic subjects were used in analyses of BMI and metabolic measures. Their ß cell function (%B) and insulin resistance (IR) were estimated using the homeostasis model assessment (HOMA) based on the fasting plasma glucose and insulin concentrations. We identified 243 TT, 67 TA and 5 AA subjects with an allelic frequency of 12.2% for the A allele. The observed genotypic frequencies are in compliance with the Hardy–Weinberg equilibrium (P = 0.994). Since only five AA subjects were identified, they were pooled with the TA subjects as the TA/AA group during analysis.
As compared with the TT group, the TA/AA groups had a lower BMI (30.1 ± 0.6 versus 27.6 ± 1.0 kg/m2, P = 0.041; Fig. 2A), a lower fasting plasma insulin concentration (11 ± 1 versus 8 ± 1 mU/l, P = 0.025), 2 h plasma insulin concentration after oral glucose challenge (51 ± 5 versus 32 ± 6 mU/l, P = 0.039) and a lower IR (2.77 ± 0.18 versus 1.96 ± 0.24, P = 0.022; Fig. 2B). However, no difference was noted in fasting plasma glucose concentration (94 ± 1 versus 93 ± 1 mg/dl, P = 0.238), 2 h plasma glucose concentration after oral glucose challenge (113 ± 2 versus 107 ± 4 mg/dl, P = 0.189) and %B (133 ± 8 versus 108 ± 14%, P = 0.146). Since obesity (a higher BMI) is associated with insulin resistance, we re-examined the impact of this polymorphism by entering BMI as a covariate and we found that this polymorphism was no longer an independent determinant of IR (P = 0.271). Therefore, we concluded that this polymorphism had a primary impact on BMI and in turn affected IR in this population.
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Association studies of the T228A polymorphism with obesity and type 2 diabetes
We divided a sample of 315 normal glucose-tolerant subjects identified through routine yearly exams at National Taiwan University Hospital (NTUH) into two groups based on their BMI. Obesity was defined as BMI
30 kg/m2. There were 202 non-obese and 113 obese subjects with their genotypic and allelic frequencies of the T228A polymorphism shown in Table 4, and we also genotyped 455 patients with type 2 diabetes. Again, their observed genotypic frequencies are in compliance with the Hardy–Weinberg equilibrium (P = 0.978, 0.708 and 0.939, respectively). The frequencies for those with hetero- or homozygous variants (T/A or A/A genotype) of the T228A polymorphism were significantly lower in the obese (15%, P = 0.02) and diabetes (19.2%, P = 0.05) groups as compared to those in the normal controls (27.2%) (Table 4). Non-obese group had almost twice the frequency of the variant A allele as compared to obese group (14.9 versus 7.5%, P = 0.007). Also, a difference was noted in the distribution of the allelic frequencies between diabetes and non-obese groups (P = 0.039). Therefore, these results indicated that the A-allele of the T228A polymorphism of the SORBS1 gene is a protective marker for both obesity [relative risk 0.466; 95% confidence interval (95% CI) 0.265–0.821] and diabetes (relative risk 0.668; 95% CI 0.472–0.945).
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DISCUSSION |
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The importance of adipose tissue in pathogenesis of insulin resistance stems from the discovery of TZD as an anti-diabetic agent. We previously reported the expression of mouse SH3P12 (also known as CAP, or named SORBS1 for the human homolog in this paper) was activated during the adipogenesis and was also stimulated by TZD (9). Therefore, it is a candidate gene for the pathogenesis of insulin resistance/obesity. To evaluate its role, we cloned and characterized the human SORBS1 (20). Molecular analysis revealed two missense variants. Among them, only T228A meets the criteria for polymorphism as a potential candidate for the polygenic diseases (22). Analysis of this polymorphism in non-diabetic subjects revealed that the T228A polymorphism exerted its influence on insulin resistance through obesity. Population association studies confirmed that this polymorphism was associated not only with obesity, but also with type 2 diabetes.
SORBS1/SH3P12/CAP contains a region showing significant sequence similarity with the peptide hormone sorbin and three adjacent SH3 domains in the C-terminus (23). CAP interacts a novel phosphatidylinositol 3-kinase-independent pathway in adipocytes to participate in insulin signaling (16). Insulin initiates its actions by binding to its tyrosine kinase receptor, leading to the phosphorylation of intracellular substrates, such as c-Cbl, which interacts with the adaptor protein CAP, through one of three adjacent SH3 domains in its C-terminus. Upon phosphorylation of c-Cbl, the c-Cbl–CAP complex dissociates from the insulin receptor and moves to a caveolin-enriched triton-insoluble membrane fraction (24). Flotillin forms a ternary complex with c-Cbl and CAP, directing the localization of the c-Cbl–CAP complex to a lipid raft subdomain of the plasma membrane (16). Thus, localization of the c-Cbl–CAP complex to lipid rafts generates a pathway that is crucial in the regulation of glucose uptake. These events lead to stimulation of the transport of glucose into adipocytes. As a result, CAP could play an important role in insulin sensitivity. Furthermore, both CAP mRNA and proteins are expressed predominantly in 3T3-L1 adipocytes and not in 3T3-L1 fibroblasts (14). Therefore, SORBS1/SH3P12/CAP is essential for adipose tissue and a defect in SORBS1/SH3P12/CAP could interfere with insulin’s signaling pathway and in turn affect the development of obesity.
The entire coding sequences of the human SORBS1 gene including exon–intron boundaries and the adjacent introns were examined at single nucleotide level by PCR and then direct sequencing reaction from 40 chromosomes. Although two amino acid substitutions were identified in the human SORBS1 gene, only T228A was found to be pathogenic. As SORBS1 plays a key role in adipogenesis, a defect in SORBS1, such as T228A, could affect insulin sensitivity, the development of obesity and the pathogenesis of type 2 diabetes. Furthermore, exon 7, which contained this polymorphism, is detected in liver and muscle in addition to adipose tissue (20), suggesting that this polymorphism could affect insulin sensitivity. Phenotypic analysis revealed that the variant Ala allele was associated with a leaner body build than those without this polymorphism. Furthermore, the A-allele of this polymorphism was also associated with a lower IR than the T-allele. In agreement with these observations, we also found that the A-allele of this polymorphism was protective for both obesity and diabetes. Obesity is a well-known risk factor for diabetes; however, not all obese subjects develop diabetes. In the association study, we found that the frequency of the variant Ala allele was highest in the non-diabetic lean group (14.9%), followed by diabetic group (10.4%) and lowest in non-diabetic obese group (7.5%). These results suggest that the primary influence of this polymorphism is the protective effect of the development of obesity. The primary influence on obesity of this polymorphism was also implied from the phenotypic analysis, which revealed that this polymorphism had no impact on insulin sensitivity after adjustment of BMI. These observations were in agreement with the hypothesis that a molecular variant of SORBS1 could affect the development of obesity and diabetes. How this change in amino acid at codon 228 might affect the protein function remains to be elucidated. Interestingly, the threonine at codon 228 was predicted as a MAP kinase phosphorylation site (within the context of PISQTPPSF) that might be important for regulation of this protein’s function. Further studies of this amino acid change is required to confirm its biological consequence observed in human subjects.
An issue which remains to be resolved is the possibility of type I error, which could occur in any genetic studies for various reasons (25,26). One of the illustrations of this possibility in type 2 diabetes is the genetic study of the insulin gene. Although the role of the insulin gene in type 1 diabetes is well-established (27), its role in type 2 diabetes is controversial. In spite of the original positive association of the insulin gene with type 2 diabetes (28), the majority of subsequent studies failed to confirm this relationship (29–32), including the genetic studies (33) and functional study (34) from the original group. The possibility of type I error of the original study was entertained, even by the original group (33), until a highly convincing positive report which appeared 17 years later (35). Therefore, we cannot exclude the possibility of type I error at this moment. Nevertheless in any population study, spurious associations may arise when case and control subjects are not recruited in the same way, such that the only difference between groups is disease status. In the present study, we only enrolled one single ethnic group from the same hospital. Furthermore, quantitative study also confirmed the association of this polymorphism with insulin resistance and obesity, which are the risk factors for type 2 diabetes. However, additional studies of this polymorphism are essential to confirm the role of this polymorphism in the Taiwanese population as well as other ethnic groups.
In conclusion, we identified a polymorphic T228A amino acid substitution in the human SORBS1 gene, which was associated with a lean body build and a reduced insulin resistance. The subjects with at least one Ala allele were at a reduced risk for both obesity (relative risk 0.466; 95% CI 0.265–0.821) and diabetes (relative risk 0.668; 95% CI 0.472–0.945), as compared to those with two Thr alleles. These observations suggest that the SORBS1 gene plays a role in the pathogenesis of insulin resistance, obesity and type 2 diabetes.
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MATERIALS AND METHODS |
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Subjects
315 ostensibly normal glucose-tolerant subjects were recruited during their admission at NTUH for a routine yearly physical check-up. 455 type 2 diabetic subjects, according to the WHO criteria 1998 (21), were recruited from the Metabolic Clinic of the NTUH. Informed consent was obtained. This study was approved by the Institutional Review Board of NTUH. To identify SNPs with >5% frequency, we screened 10 type 2 diabetic patients (seven female; age 63.7 ± 3.2 years; BMI 23.5 ± 0.9 kg/m2; duration of diabetes 12.1 ± 3.1 years) and 10 severe obese patients who were just diagnosed as type 2 diabetes after an oral glucose tolerance test (five female; age 39.9 ± 2.7 years; BMI 38.2 ± 0.6 kg/m2) for the molecular variants in the SORBS1 gene.
Laboratory and phenotypic characterization of the subjects
A 75 g oral glucose tolerance test was given to non-diabetic individuals. The concentrations of plasma glucose, total cholesterol and triglyceride were measured in fasting samples by an autoanalyzer (Hitachi 7250 special, Tokyo, Japan). A fasting plasma glucose level of 126 mg/dl and/or a 2 h post-load glucose >200 mg/dl was diagnosed as diabetes. Serum insulin levels were determined by a microparticle enzyme immunoassay (MEIA) using AxSYM system from Abbott Diagnostics (Abbott Laboratories, Dainabot Co. Ltd, Japan). The HOMA is applied to estimate the degree of insulin resistance [HOMA IR = (insulin x glucose)/22.5] and ß cell function [HOMA ß = 20 x insulin/(glucose – 3.5)], where ‘insulin’ refers to concentration in µU/ml and ‘glucose’ refers to concentration in mM (36,37).
Screening strategy
To enhance the chance of detecting a polymorphism in the SORBS1 gene and to improve the power of detecting a modest effect, we used the following strategy: first, we screened 40 diseased chromosomes from 20 diabetic patients for molecular variants to identify a polymorphism (the frequency of the common allele <95%) (22). By examining the disease chromosomes for molecular variants, the chance of detecting a mutation should be enhanced. Then, we focused on those polymorphisms that could affect amino acid coding or peptide structure and have an allelic frequency of <95% for the common allele. For two polymorphisms of interest (R74W and T228A), we examined their role in subsets of subjects (184 non-obese/non-diabetic subjects, 113 obese/non-diabetic subjects and 183 patients with type 2 diabetes). Since the T228A polymorphism gave positive results, we genotyped this polymorphism in additional subjects, which were recruited later using the same procedures as described above.
Extraction of genomic DNA and mutation screening
Searching the human genome sequence in NCBI database with human SORBS1 cDNA sequence (GenBank accession nos AF136380, AF136381, AF356525–AF356527 and BF959891), we found three BACs, i.e. AL158165, AL160288 and AL157890, that contained SORBS1 cDNA sequence. Based on the homology search and alignment study, we characterized the exon/intron junctions and designed the primers located in flanking introns for screening of nucleotide variations in the exons and the flanking intronic sequences. PCR was set up under standard conditions to amplify genomic DNA from 20 (10 non-obese and 10 severe obese type 2 diabetic) subjects with the appropriate primers (Table 2) in a total reaction volume of 30 µl containing 50 mM Tris–HCl pH 9.1, 16 mM ammonium sulfate, 3.5 mM MgCl2, 4.5 µg BSA, 6 pmol of each primer and 0.2 mM dNTP including 20 ng genomic DNA and 1.2 U KlenTaq DNA polymerase. PCR were performed with initial denaturation at 96°C for 2 min, and then with 35 cycles of 1 min at 94°C, 55 s at 65°C and 1 min at 72°C and a final 10 min extension at 72°C. PCR products were purified with GFX PCR DNA purification kit (Amersham Pharmacia, Piscataway, NJ) and then sequenced with PCR primers (footnote a in Table 2) using Big Dye Terminator chemistry, except for exons 15, 16, 25 and 32 which were sequenced with the primers exon156P (5'-TTGGT TCTCT TACCT TAGCC AG-3'), exon25P (5'-GGAGG CAAAT AGGCA AATAT TGC-3') and exon32P (5'-GCTGA TGCAG AACAT GGCCT TC-3'). Variants were identified with the programs in Biology WorkBench 3.2 (http://workbench.sdsc.edu). Total genomic DNA was purified from peripheral blood leukocytes using DNA extraction kit of Puregene (Minneapolis, MN).
PCR–RFLP analysis of T228A variants
For PCR–RFLP analysis of the T228A variants resulting in a change of nucleotide 682 in exon 7 [ACG (Thr228) to GCG (Ala228)], PCR reaction was carried out in a total volume of 15 µl containing 10 ng human genomic DNA, 1.5 mM MgCl2, 50 mM KCl, 10 mM Tris–HCl pH 8.3, 0.2 mM dNTP, 0.2 µM of each exon7f (5'-TACCT CACTG CATGC CCACT CTC-3') and exon7r (5'-GACTG CTGGG AGGAG ACATT CAGAA-3') primers and 0.6 U of KlenTaq polymerase (Ab Peptides, St Louis, MO).The PCR mixture were denatured at 96°C for 2 min, and then with 35 cycles of 30 s at 94°C, 30 s at 65°C and 30 s at 72°C and a final 10 min extension at 72°C. PCR products (size of 518 bp) were then digested with KasI restriction enzyme for genotyping. Digested DNA fragments were separated on 1.5% agarose gel and analyzed on ultraviolet transilluminator. Presence of 518, 360 and 158 bp fragments indicated a genotype of T/A, while a single uncut fragment indicated T/T, and 360 and 158 bp indicated A/A genotypes, respectively.
DHPLC analysis for R74W variants
PCR amplified with primers exon4f (5'-AGACT TCGCA TGGCT GTAAC CAG-3') and exon4wr (5'-CAAGA GGTAT CTGAT GAACT CACAC-3') were performed in a total of 25 µl reaction volume containing 50 mM Tris–HCl pH 9.1, 16 mM ammonium sulfate, 3.5 mM MgCl2, 4.5 µg BSA, 5 pmol of each primer and 0.2 mM dNTP, 150 ng genomic DNA and 1.0 U KlenTaq DNA polymerase. PCR products were denatured for 10 min at 96°C and then reannealed by gradually cooling the samples from 96 to 25°C for 30 min. The crude PCR product (6–8 µl) was then loaded and separated through 55–59% acetonitrile gradient at 63°C using WAVE Nucleic Acid Fragment Analysis System (Transgenomic, Inc., San Jose, CA).
Statistical analyses
Data were presented as means ± SD, unless otherwise specified. Statistical analyses, including two-sample t-test, analysis of variance, 
2 tests, correlation analysis and multivariate linear regression were performed by using the PC version of the Statistical Analysis System (SAS, 6.12 edn, SAS Institute Inc., Cary, NC). For comparing the genotype frequencies in different degrees of obesity, we used a cut-off value of BMI 30 kg/m2 for obesity in our analyses. A P-value <0.05 was considered statistically significant.
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ACKNOWLEDGEMENTS |
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This work was supported by the grants from the National Science Council NSC 89-2314-B-002-065 (L.-M.C.), the Department of Education 89-B-FA01-1-4 (L.-M.C.), National Taiwan University Hospital 89M-014 (L.-M.C.), Taipei, Taiwan, NIH/NIDDK RO1DK52337 (K.C.C.), and American Diabetes Association (K.C.C.).
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FOOTNOTES |
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+ To whom correspondence should be addressed. Tel: +886 2 23123456; Fax: +886 2 23938859; Email: leeming@ha.mc.ntu.edu.tw +AF136380, AF136381, AF356525–AF356527
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