Identification of glucokinase mutation in subjects with post-renal transplantation diabetes mellitus

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Abstract

Mutations in the glucokinase (GCK) gene are considered to be a possible cause of maturity-onset diabetes of the young. The purpose of this study was to evaluate the contribution of this gene to the development of post-renal transplantation diabetes mellitus (PTDM). Identification of the GCK mutation was attempted in 58 selected renal allograft recipients with PTDM and 45 normal controls. The exons in the GCK gene were examined using polymerase chain reaction (PCR), followed by an analysis of single-stranded DNA conformational polymorphism (SSCP). The abnormal bands were then confirmed by DNA sequencing analysis. The family members of the patients affected with GCK mutation were also examined. Two of the 58 PTDM patients (3.4%) were found to have GCK mutations. One had the mutation on exon 5 and the other on intron 7. One control subject had the mutation on intron 9. The mutation on exon 5 was identified as a substitution of CCT (proline) for CTT (leucine) at codon 164, which has never been reported before. The family members of the PTDM patient with a mutation on exon 5 were analyzed by PCR, followed by SSCP, and two of them had the same mutation. The abnormal band seen on SSCP analysis of exon 7 was identified as the C→T substitution at the 39th nucleotide in intron 7. Two of the family members also displayed the same bands on the SSCP. One of the 45 normal controls had a known polymorphism located at the 8th nucleotide in intron 9. We found a GCK mutation on the exon in subjects with PTDM and we speculate that this mutation may be one of the possible contributing factors of PTDM, although variations of the GCK gene are not common causes of PTDM.

Introduction

Both genetic and non-genetic factors contribute to the development of non-insulin dependent diabetes mellitus (NIDDM). The mode of transmission of NIDDM has not been established, although a genetic predisposition is generally accepted [1], [2]. Although the primary defect which accounts for this prediposition is uncertain, both impaired insulin action (insulin resistance) and defective insulin secretion appear to characterize the diabetic state [3]. A large body of evidence suggests that genetic factors may be of crucial importance in the pathogenesis of NIDDM. Glucokinase (GCK) is among the few candidate genes which, if defective, might affect both pathways.

GCK is expressed in pancreatic beta cells, where it may play a key role in sensing plasma glucose to insulin release [4], [5]. GCK is also expressed in hepatocytes, where th phosphorylation of glucose by GCK may be essential to hepatic glucose disposal. The GCK gene has been characterized as containing 12 exons [6], [7], [8]. In a group of patients with GCK mutations, maturity onset diabetes of the young 2 (MODY2), a subtype of NIDDM, developed in patients <25 years of age and was inherited as an autosomal dominant trait [9]. Thereafter, several mutations responsible for MODY2 have been identified within the coding regions of the GCK gene [10]. Although most patients with mutations in the GCK gene identified so far have presented with MODY2, it may be difficult to make an accurate diagnosis of MODY2 without obtaining thorough clinical, laboratory and family histories because diabetes mellitus(DM) caused by mutations in the GCK gene is usually associated with mild symptoms. Therefore, it is reasonable to speculate that a considerable proportion of patients with the GCK gene mutation should be recognized as having secondary diabetes mellitus, which may be triggered by pregnancy, steroids, immunosuppressants or stress (e.g. surgery).

The exact mechanism of post-renal transplantation diabetes mellitus(PTDM) is still unclear. However, stress due to operation, immunosuppressants such as steroids and cyclosporine A[11], [12], a positive family history of DM [13], certain HLA phenotypes [14], or other genetic defects have been suggested as etiopathogenic factors of PTDM. This is supported by the facts that patients with kidney transplantation are known to have a much higher incidence of DM than normal populations and that PTDM occurs regardless of age. A genetic factor is assumed to be strongly associated with the development of PTDM. This is supported by the fact that PTDM develops independent of the dosage of steroid or CsA, but develops more frequently in those with a family history of DM [15], [16]. Patients with GCK mutations display impaired insulin secretion, and, therefore, are possibly at a high risk for developing diabetes mellitus by triggering factors, such as stress realted to surgery or medication such as steroid or CsA. DM in patients with GCK mutations who received renal transplantation, may be diagnosed as PTDM. In our study, we have tried to determine the relationship between the GCK gene mutations and PTDM.

Section snippets

Study population

The subject group consisted of 58 patients who were diagnosed with PTDM. They had a mean age of 43.5±5.3 years, ranging from 32 to 56 years of age. All patients were being followed at the Endocrinology Department of Yonsei University College of Medicine and all of their creatinine values were 2 mg/dl or less. No patients included in this study were diabetic before transplantation according to World Health Organization criteria (WHO) for DM on 75 g oral glucose tolerance test (OGTT) or two

Identification of nucleotide alterations in the GCK gene

All 12 exons of the GCK gene for the 58 PTDM patients were screened for mutations using SSCP. Abnormal bands were observed in exon 5 (Fig. 1a) and in exon 7 (Fig. 1b) in the PTDM patients (Table 2). We also screened 45 controls and the abnormal conformers were not seen in any of the controls with the exception of one mutant band in exon 9 (Fig. 1c), which is located at the 8th nucleotide in intron 9 (Table 2), and which was already reported as a common polymorphysm [6].

Identification of missense mutation in exon 5

Sequencing of the exon 5

Discussion

Mutations in the GCK gene have been found in ∼60% of French families with early-onset NIDDM or MODY [19], [20], [21], however the frequency of the GCK mutation in NIDDM is less than 1% [22], [23] and 2–6% of GDM subjects [24], [25]. So far, about 40 mutations in the GCK gene have been reported all over the world. Although mutations in the GCK gene have been identified in nine different exons, the missense mutations occurring in four exons (exon 5, 6, 7, and 8) appear to be important in

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