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

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.

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.

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 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).

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 HbA_1c 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.

Data on the study groups were compared using χ² 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 was performed with an α (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 Δ 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 Δs of 0.1 and 0.15 in allele frequency was 70 and 95%, respectively.

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.