Diabetes
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Online Appendix
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Ng, D. P.K.
Right arrow Articles by Krolewski, A. S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Ng, D. P.K.
Right arrow Articles by Krolewski, A. S.
Social Bookmarking
Add to CiteULike   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

Diabetes 53:865-869, 2004
© 2004 by the American Diabetes Association, Inc.

Brief Genetics Report

Scrutiny of the Glutamine-Fructose-6-Phosphate Transaminase 1 (GFPT1) Locus Reveals Conserved Haplotype Block Structure not Associated With Diabetic Nephropathy

Daniel P.K. Ng1,2,3, William H. Walker1, Kee-Seng Chia3, Serena Choo3, James H. Warram1, and Andrzej S. Krolewski1,2

1 Section on Genetics and Epidemiology, Joslin Diabetes Center, Boston, Massachusetts
2 Department of Medicine, Harvard Medical School, Boston, Massachusetts
3 Community, Occupational and Family Medicine, National University of Singapore, Singapore, Singapore


   ABSTRACT
TOP
 ABSTRACT
RESEARCH DESIGN AND METHODS
 REFERENCES
 
Glutamine-fructose-6-phosphate transaminase 1 (GFAT) is the rate-limiting enzyme of the hexosamine pathway that has been implicated in the pathogenesis of diabetic nephropathy. As such, we hypothesized that GFPT1, which encodes for GFAT, may confer genetic susceptibility to this complication among Caucasians. Screening of all known functional regions of GFPT1 revealed six single nucleotide polymorphisms (SNPs) that were located in the promoter, introns, and 3' untranslated region. The ~60 kb GFPT1 locus was encompassed in a single conserved haplotype block, and two tagging SNPs were sufficient to capture >90% of the haplotype diversity. Analysis of these SNPs in a case-control study made up of type 1 diabetic subjects (324 case subjects with diabetic nephropathy and 289 control subjects with normoalbuminuria despite >15 years of diabetes) revealed no significant association even after stratification by sex, diabetes duration, glucose control, and blood pressure. Similar results were obtained among type 2 diabetic subjects (202 case and 114 control subjects). Genetic variation in GFPT1 is thus unlikely to have a major impact on susceptibility to diabetic nephropathy.

The hexosamine biosynthetic pathway is an alternative pathway of glucose metabolism that has recently been implicated in the pathogenesis of diabetic nephropathy (1). Glutamine-fructose-6-phosphate transaminase 1 (GFAT) is the rate-limiting enzyme of this pathway and converts fructose 6-phospate to glucosamine 6-phosphate. Glucosamine 6-phosphate is subsequently converted to uridine diphosphate N-acetylglucosamine, which is used for the O-glycosylation of intracellular proteins, including transcription factors. Concerning diabetic nephropathy, the hexosamine pathway mediates hyperglycemia-induced transforming growth factor-ß1 production by glomerular mesangial cells, an effect that could be prevented by GFAT inhibition (2). Overexpression of GFAT in rat mesangial cells increases the expression of transforming growth factor-ß1 and its receptors, even under normoglycemic conditions (3). A similar phenomenon has been reported for NIH-3T3 fibroblasts overexpressing GFAT (4). Gene expression of plasminogen activator inhibitor-1 is also dependent on the hexosamine pathway as mediated by protein kinase C isoforms and activation of the transcription factor Sp1 by O-glycosylation (3,5,6).

With mounting evidence implicating GFAT in diabetic nephropathy, we hypothesized that GFPT1 (OMIM: 138292, human chromosome 2p13) may be an important susceptibility gene for diabetic nephropathy because it encodes for this enzyme. In this first report to examine the issue, we screened the GFPT1 locus for common DNA polymorphisms by sequencing all known functional regions, including the promoter, all exons, and exon/intron junctions. In total, 17% of the ~60 kb locus was screened. Six polymorphisms were detected, all of which were single nucleotide polymorphisms (SNPs) (Table 1). One SNP was located ~1.1 kb upstream of the start adenine thymine guanine codon (denoted g.-1093A>G), another in the 3' untranslated region (denoted c.2846T>G), and the remainder in intronic regions (denoted IVS1 + 36C>T, IVS5 + 25T>C, IVS5 + 102G>T, and IVS11 + 7G>A). All SNPs were common with minor allele frequency ranging from 0.39 to 0.47. Five of the six SNPs were found in dbSNP and one was absent (Table 1).


View this table:
[in this window]
[in a new window]
  		TABLE 1 Polymorphisms detected in GFPT1

 
As recent reports have suggested that chromosomal regions contain SNPs that could be partitioned into "haplotype blocks" within which there is substantial linkage disequilibrium (7,8), we calculated the linkage disequilibrium between the six SNPs using Lewontin’s D', a commonly used measure of linkage disequilibrium (9). All SNPs were in strong linkage disequilibrium with each other (range of D' = 0.88 to 1.00) (data not shown). The frequencies of six SNP haplotypes were estimated from genotype data from 200 randomly selected Caucasian type 1 diabetic individuals (100 case and 100 control subjects). Three haplotypes (denoted A, B, C) predominated, each with an estimated frequency >0.10. Together, these accounted for >90% of the haplotype diversity at this locus (Fig. 1). A conserved haplotype block thus spans the GFPT1 locus and two tagging SNPs (tagSNPs) (IVS5 + 25T>C and IVS5 + 102G>T) were sufficient to define haplotypes A, B, and C (Fig. 1).



View larger version (18K):
[in this window]
[in a new window]
  		FIG. 1. Genomic structure of the ~60 kb GFPT1 locus and its associated haplotypes. Exons (black vertical blocks) and location of the six SNPs (vertical arrows) are shown; major alleles are in shaded boxes. *TagSNPs that in combination captured 92% of all possible six-SNP haplotypes. {dagger}Remaining 8% of haplotypes had an estimated frequency of <0.03 each.

 
These tagSNPs were tested for association with diabetic nephropathy using a population-based case-control study design. Among both type 1 and 2 diabetic patients, case subjects were defined as having advanced diabetic nephropathy indicated by persistent proteinuria, chronic renal failure, or end-stage renal disease. Control subjects with type 1 diabetes had normoalbuminuria despite a diabetes duration >=15 years. Control subjects with type 2 diabetes had normoalbuminuria despite a known diabetes duration >=6 years, allowing for the fact that the diagnosis of type 2 diabetes is generally established many years after the onset of hyperglycemia. Clinical characteristics of the study groups are shown in Table 2.


View this table:
[in this window]
[in a new window]
  		TABLE 2 Clinical characteristics of study subjects with type 1 and type 2 diabetes

 
No significant difference in allele or genotype distributions was observed for either IVS5 + 25T>C or IVS5 + 102G>T between case and control subjects with type 1 diabetes; estimated haplotype frequencies were also similar between these two groups of patients (Table 3). Stratification according to sex, median values for diabetes duration (26 years), HbA1c (8.5%), and systolic (127 mmHg) or diastolic (77 mmHg) blood pressure revealed no significant association between these tagSNPs and diabetic nephropathy (data not shown). Similarly, among the type 2 diabetic subjects, allele and genotype distributions of the tagSNPs were comparable between case and control subjects, and haplotype frequencies were also similar in the two groups (Table 3). No significant association was detected after stratifying according to sex, median values for HbA1c (8.2%), duration of diabetes (12 years), and systolic (135 mmHg) or diastolic (78 mmHg) blood pressure (data not shown).


View this table:
[in this window]
[in a new window]
  		TABLE 3 Allele, genotype, and haplotype distributions of tagSNPs in case and control subjects

 
Our current findings, therefore, do not support GFPT1 as being an important susceptibility gene for diabetic nephropathy among Caucasians with type 1 or 2 diabetes. This was the case for genotypes at individual SNPs as well as haplotypes formed from the two tagSNPs. This combination of tagSNPs is capable of capturing >90% of the haplotype diversity at this gene locus in our Caucasian sample, although its performance in other human populations (e.g., Chinese and African populations) should be assessed empirically. Besides reducing the genotyping workload by utilizing tagSNPs, employment of both geno- and haplotype analyses also permitted us to evaluate the contribution of common SNPs and currently unidentified, rarer polymorphisms to diabetic nephropathy. Although examination of individual SNPs tested whether their genotypes were common susceptibility determinants of diabetic nephropathy, haplotype analysis addressed the possibility that a putative rarer polymorphism, residing on a haplotype, may also contribute to this genetic risk. Although our study largely excludes a major susceptibility role for GFPT1 for diabetic nephropathy among Caucasians, a minor effect cannot be easily ruled out, and the question as to whether this gene confers susceptibility in other human populations remains open. Genome scans recently conducted at the Joslin Diabetes Center in Caucasian families with type 1 and 2 diabetes have provided suggestive evidence for linkage between diabetic nephropathy and a large region on chromosome 2p that includes GFPT1 (A.S.K., unpublished data). Our current findings, however, do not support this gene locus as being a contributor to these linkage results. Since additional genes are likely related to the hexosamine pathway and its regulation, these may form the focus of future investigations (10).


   RESEARCH DESIGN AND METHODS
TOP
 ABSTRACT
 RESEARCH DESIGN AND METHODS
REFERENCES
 
Since 1991, individuals with type 1 diabetes have been recruited for studies of the genetics of nephropathy from among patients attending the Joslin Clinic. Diabetes has been classified as type 1 if it was diagnosed at <30 years of age and continuous treatment with insulin began within 1 year of diagnosis. As of 2001, 352 case subjects with diabetic nephropathy and 307 control subjects with normoalbuminuria had been enrolled in the study. Details of the procedures for recruiting these patients were described previously (11). Since 1998, individuals with type 2 diabetes have also been recruited for studies of the genetics of nephropathy from among patients attending the Joslin Clinic. Diabetes has been classified as type 2 if it was diagnosed between ages 30 and 64 years and was treated for at least 2 years with diet or oral hypoglycemic agents. Only patients <75 years of age at enrollment are included in the study. As of 2002, 303 case subjects with diabetic nephropathy and 168 control subjects with normoalbuminuria had been enrolled into the study.

Diagnosis of diabetic nephropathy.
The Joslin Clinic provides care for ~4,000 patients with type 1 diabetes and 12,000 patients with type 2 diabetes. The majority of these patients are Massachusetts residents referred to the clinic within 5 years of the diagnosis of diabetes. A large proportion of them remain under the care of the clinic for life. Certain demographic and clinical information and most laboratory results are computerized and available for research purposes. The computer databases were used to identify patients eligible for our genetic studies. Diabetic nephropathy was determined on the basis of the medical records of the Joslin Clinic (supplemented with records of other physicians if necessary) and results of routine urinary analyses, including measurements of the albumin-to-creatinine ratio (11).

Patients were classified as control subjects if they had type 1 diabetes with a duration >=15 years or type 2 diabetes with a duration >=6 years and their albumin-to-creatinine ratio was <17 mg/g for men or <25 mg/g for women in at least two of the last three urine specimens spanning at least a 2-year interval. Patients with microalbuminuria or intermittent proteinuria were not included in this study. Patients were considered case subjects if they had persistent proteinuria or if they had end-stage renal disease due to diabetic nephropathy. Persistent proteinuria was defined as two of three successive urinalyses positive by either reagent strip (>=2+ on Multistix; Bayer, Elkhart, IN) or an albumin-to-creatinine ratio >=250 mg/g for men or >=355 mg/g for women. Patients with persistent proteinuria and serum creatinine >=2.0 mg/dl were considered case subjects with chronic renal failure.

Examination of study participants.
All patients selected for the genetic studies were examined at the clinic or at their homes. After consenting to participate in the study, each subject had a standardized physical examination and provided a diabetes history regarding diagnosis, treatment, and complications. Each individual provided a blood sample for biochemical measurements and DNA extraction. Patient medical records were thoroughly reviewed to minimize the possibility of the presence of nondiabetic kidney disease, and patients were also directly questioned whether they were ever diagnosed for nondiabetic kidney disease by MDs. The Committee on Human Subjects of the Joslin Diabetes Center approved the protocols and informed consent procedures for our studies.

GFPT1 DNA polymorphisms screening.
GFPT1 consists of 20 exons, including a recently described alternative exon (12,13). To identify DNA polymorphisms in GFPT1, all exons (with exon/intron boundaries) and 1.5 kb of the promoter region were amplified from the genomic DNA of eight individuals (four case and four control subjects) using PCR (online appendix 1 [available at http://diabetes.diabetesjournals.org]). PCR (25 µl reaction volume) was typically performed on 20 ng of genomic DNA using 0.6 units of Taq polymerase (PGC Scientific) in the presence of 1.5 mmol/l MgCl2 for 40 cycles. Cycling parameters were denaturation at 95°C for 45 s, annealing at product-specific temperatures (Table 1) for 45 s, and extension at 72°C for 60 s with final extension at 72°C for 10 min. Primer sequences were designed based on human GFPT1 genomic DNA sequence (GenBank accession number NT_022184). All PCR products were sequenced using an ABI 377 automated DNA sequencer in conjunction with dye terminator chemistry in accordance with the manufacturer’s protocol (Perkin Elmer). DNA sequence chromatograms were manually inspected to ensure proper base calling by the sequencing analysis software. As the PCR products were 500 bp in length on average, sequencing them from both forward and reverse directions resulted in considerable sequence overlap. This allowed us to double check the detection of DNA polymorphisms. Polymorphisms were annotated according to the nomenclature advocated by Antonarakis and the Nomenclature Working Group (14).

Genotyping.
When this study was performed, genomic DNA was available for genotyping from 289 (94%) and 324 (92%) type 1 diabetic control and case subjects, respectively, as well as 114 (68%) and 202 (67%) type 2 diabetic control and case subjects, respectively. Almost one-third of the patients with type 2 diabetes did not have DNA extracted as of the time of this study. Those with DNA available were not different from those without DNA with regard to sex, age at examination, duration of diabetes, or clinical characteristics such as distribution of HbA1c or blood pressure.

Genotyping of GFPT1 polymorphisms was performed by DNA hybridization using allele-specific oligonucleotides probes, as previously described (15). DNA sequences of the allele-specific oligonucleotide probes and PCR primers are detailed in Table 1. The two tagSNPs, IVS5 + 25T>C and IVS5 + 102G>T, were also genotyped as restriction fragment length polymorphisms. IVS5 + 25T>C was genotyped using PshAI (New England Biolabs, Beverly, MA) so that when the C allele was present, the restriction site was absent and the 581-bp PCR product migrated intact as a single band. When the T allele was present, two bands of sizes 302 and 279 bp were seen after digestion. For IVS5 + 102G>T, genotyping was performed using AseI (New England Biolabs). When the G allele was present, the restriction site was absent, but when the T allele was present, digestion occurred and two fragments of 383 and 198 bp were seen. Genotypes of 80 individuals obtained using allele-specific oligonucleotides and restriction fragment length polymorphism methods were in strong agreement for both IVS5 + 25T>C (99% concordant) and IVS5 + 102G>T (100% concordant), consistent with the absence of any significant genotyping error.

Statistical analysis.
Data on the study groups were compared using {chi}2 and Student’s t tests for categorical and continuous variables, respectively (SAS system for Windows version 6.12; SAS Institute, Cary, NC). Identification of haplotype blocks was performed according to Gabriel et al. (8) with D' >=0.8 as evidence of strong linkage disequilibrium. Haplotype frequencies were estimated using the EM algorithm in SAS. Within each haplotype block, tagSNPs were identified that best captured the diversity of common haplotypes present (i.e., those with frequencies >=5%).

Power analysis.
Power analysis was performed with an {alpha} (type 1 error) set at 5%. Based on sample sizes of 289 control subjects and 324 case subjects for patients with type 1 diabetes, power to detect a {Delta} of 0.1 in allele frequency of the tagSNPs was >90%. For sample sizes, 114 control subjects and 202 case subjects for patients with type 2 diabetes, power to detect {Delta}s of 0.1 and 0.15 in allele frequency was 70 and 95%, respectively.


   ACKNOWLEDGMENTS
 
This research was supported by National Institutes of Health Grants DK041526, DK053534, and DK058549.

We thank Adam Smiles of the Joslin Diabetes Center for development and maintenance of genetic and phenotypic databases.


   FOOTNOTES
 
Additional information for this article can be found in an online appendix at http://diabetes.diabetesjournals.org.

Address correspondence and reprint requests to Andrzej S. Krolewski, MD, PhD, Section on Genetics and Epidemiology, Joslin Diabetes Center, One Joslin Pl., Boston, MA 02215. E-mail: andrzej.krolewski{at}joslin.harvard.edu

Received for publication July 25, 2003 and accepted in revised form December 5, 2003

Abbreviations: GFAT, glutamine-fructose-6-phosphate transaminase; SNP, single nucleotide polymorphism; tagSNP, tagging SNP


   REFERENCES
TOP
 ABSTRACT
 RESEARCH DESIGN AND METHODS
 REFERENCES
 

  1. Schleicher ED, Weigert C: Role of the hexosamine biosynthetic pathway in diabetic nephropathy. Kidney Int77 (Suppl.) :S13 –S18,2000
  2. Kolm-Litty V, Sauer U, Nerlich A, Lehmann R, Schleicher ED: High glucose-induced transforming growth factor beta1 production is mediated by the hexosamine pathway in porcine glomerular mesangial cells. J Clin Invest101 :160 –169,1998[Medline]
  3. James LR, Fantus IG, Goldberg H, Ly H, Scholey JW: Overexpression of GFAT activates PAI-1 promoter in mesangial cells. Am J Physiol Renal Physiol279 :F718 –F727,2000[Abstract/Free Full Text]
  4. Weigert C, Brodbeck K, Lehmann R, Haring HU, Schleicher ED: Overexpression of glutamine:fructose-6-phosphate-amidotransferase induces transforming growth factor-beta1 synthesis in NIH-3T3 fibroblasts. FEBS Lett488 :95 –99,2001[Medline]
  5. Du XL, Edelstein D, Rossetti L, Fantus IG, Goldberg H, Ziyadeh F, Wu J, Brownlee M: Hyperglycemia-induced mitochondrial superoxide overproduction activates the hexosamine pathway and induces plasminogen activator inhibitor-1 expression by increasing Sp1 glycosylation. Proc Natl Acad Sci U S A97 :12222 –12226,2000[Abstract/Free Full Text]
  6. Goldberg HJ, Whiteside CI, Fantus IG: The hexosamine pathway regulates the plasminogen activator inhibitor-1 gene promoter and Sp1 transcriptional activation through protein kinase C-beta I and -delta. J Biol Chem277 :33833 –33841,2002[Abstract/Free Full Text]
  7. Daly MJ, Rioux JD, Schaffner SF, Hudson TJ, Lander ES: High-resolution haplotype structure in the human genome. Nat Genet29 :229 –232,2001[Medline]
  8. Gabriel SB, Schaffner SF, Nguyen H, Moore JM, Roy J, Blumenstiel B, Higgins J, DeFelice M, Lochner A, Faggart M, Liu-Cordero SN, Rotimi C, Adeyemo A, Cooper R, Ward R, Lander ES, Daly MJ, Altshuler D: The structure of haplotype blocks in the human genome. Science296 :2225 –2229,2002[Abstract/Free Full Text]
  9. Lewontin RC: The interaction of selection and linkage. I: general considerations; heterotic models. Genetics49 :49 –67,1964[Free Full Text]
  10. Oki T, Yamazaki K, Kuromitsu J, Okada M, Tanaka I: cDNA cloning and mapping of a novel subtype of glutamine:fructose-6-phosphate amidotransferase (GFAT2) in human and mouse. Genomics57 :227 –234,1999[Medline]
  11. Warram JH, Gearin G, Laffel L, Krolewski AS: Effect of duration of type I diabetes on the prevalence of stages of diabetic nephropathy defined by urinary albumin/creatinine ratio. J Am Soc Nephrol7 :930 –937,1996[Abstract]
  12. DeHaven JE, Robinson KA, Nelson BA, Buse M: A novel variant of glutamine: fructose-6-phosphate amidotransferase-1 (GFAT1) mRNA is selectively expressed in striated muscle. Diabetes50 :2419 –2424,2001[Abstract/Free Full Text]
  13. Niimi M, Ogawara T, Yamashita T, Yamamoto Y, Ueyama A, Kambe T, Okamoto T, Ban T, Tamanoi H, Ozaki K, Fujiwara T, Fukui H, Takahashi EI, Kyushiki H, Tanigami A: Identification of GFAT1-L, a novel splice variant of human glutamine: fructose-6-phosphate amidotransferase (GFAT1) that is expressed abundantly in skeletal muscle. J Hum Genet46 :566 –571,2001[Medline]
  14. Antonarakis SE, the Nomenclature Working Group: Recommendations for a nomenclature system for human gene mutations. Hum Mutat11 :1 –3,1998[Medline]
  15. Araki S, Ng DP, Krolewski B, Wyrwicz L, Rogus JJ, Canani L, Makita Y, Haneda M, Warram JH, Krolewski AS: Identification of a common risk haplotype for diabetic nephropathy at the protein kinase C-beta1 (PRKCB1) gene locus. J Am Soc Nephrol14 :2015 –2024,2003[Abstract/Free Full Text]

Add to CiteULike CiteULike   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 DiabetesHome page
K. E. North, N. Franceschini, I. B. Borecki, C. C. Gu, G. Heiss, M. A. Province, D. K. Arnett, C. E. Lewis, M. B. Miller, R. H. Myers, et al.
Genotype-by-Sex Interaction on Fasting Insulin Concentration: The HyperGEN Study
Diabetes, January 1, 2007; 56(1): 137 - 142.
[Abstract] [Full Text] [PDF]

Home page
 DiabetesHome page
L. H. Canani, C. Capp, D. P.K. Ng, S. G.L. Choo, A. L. Maia, G. B. Nabinger, K. Santos, D. Crispim, I. Roisemberg, A. S. Krolewski, et al.
The Fatty Acid-Binding Protein-2 A54T Polymorphism Is Associated With Renal Disease in Patients With Type 2 Diabetes
Diabetes, November 1, 2005; 54(11): 3326 - 3330.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Online Appendix
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Ng, D. P.K.
Right arrow Articles by Krolewski, A. S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Ng, D. P.K.
Right arrow Articles by Krolewski, A. S.
Social Bookmarking
Add to CiteULike   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Diabetes Diabetes Care Clinical Diabetes Diabetes Spectrum