Hidekazu Nishigori

Frameshift mutation, A263fsinsGG, in the hepatocyte nuclear factor-1beta gene associated with diabetes and renal dysfunction.

who had been diagnosed with diabetes at 19 years of age (BMI 18.3 kg/m 2 ) (Fig. LA). This mutation causes a frameshift and the generation of a mutant truncated protein of 264 amino acids that lacks the transcription activation domain. Screening other family members showed that the mutation was also present in the probands sister (IV-3), who had been diagnosed with glycosuria at 14 years of age during the course of an annual school physical examination. The mutation was also found in the probands father (III-2), who had been diagnosed with diabetes at 61 years of age. The two affected sisters (IV-2 and IV-3) are currently being treated with 10-20 U of insulin, and their father with diet. Medical records indicate that the paternal grandmother (D-2) had early-onset diabetes, and so also may have inherited the mutation. The frequency of HNF-1(3 gene mutations in our study population of Japanese subjects who have early-onset type 2 diabetes with a first-degree relative with diabetes is 2.5%. Together with the previous observation by Horikawa et al. (10), our results indicate that mutations in the HNF-ip gene are not a major cause of early-onset type 2 diabetes in Japanese, although the results apply only to the coding region of the HNF-1(3 gene. The proband and her sister had decreased renal function (creatinine clearance <50 ml/min) and multiple bilateral renal cysts with three to five cysts per kidney (0.3-1.2 cm in diameter) (Fig. IE). Their father also had chronic renal failure with renal cysts, which is now being treated by hemodialysis. While the two affected sisters showed no evidence of proteinuria, the father had persistent mild proteinuria (<300 mg/day), which was first noticed during the course of an annual health examination 20 years before the diagnosis of diabetes. The A263fsinsGG mutation was also found in the nondiabetic 2-year-old son (V-l) of the proband and the 5-year-old daughter (V-2) of the affected sister, who also had elevated levels of serum creatinine (1.3-1.8 mg/dl; normal 0.6-1.0 mg/dl) at birth. Moreover, subject V-l was found at birth to have bilateral renal cysts measuring several millimeters in diameter, and fetal ultrasound examination at 27 weeks of gestation revealed renal abnormalities, including bilateral enlarged kidney mass and multiple parenchymal hyperechoic nodules (Fig. LET). These observations suggest that the A263fsinsGG mutation in the HNF-ip gene might be associated with diabetes and renal dysfunction in this family. Renal dysfunction described in this report is defined broadly, including proteinuria, decreased renal function, and renal cysts. Moreover, these abnormalities appear to precede the onset of dia

Identification of mutations in the hepatocyte nuclear factor (HNF)-1 alpha gene in Japanese subjects with IDDM

One form of maturity-onset diabetes of the young, MODY3, is characterized by a severe insulin secretory defect, compared with MODY2, a glucokinase-deflcient diabetes. It has recently been shown that mutations of the gene encoding the transcription factor hepatocyte nuclear factor (HNF)-1α cause MODY3. Because of the rapid progress to overt diabetes and the high prevalence of required insulin treatment in patients with MODY3, we screened the HNF-1α gene for mutations in Japanese subjects with IDDM. Ten exons and flanking introns of the HNF-1α gene in these subjects were amplified by polymerase chain reaction and direct sequencing of the products. Mutations were identified in three (5.5%) of the 55 unrelated subjects with IDDM. A missense mutation of R272H (replacement of Arg by His in codon 272) in the DNA binding domain of HNF-1α was found in a subject who developed IDDM 1 year after diagnosis of NIDDM at 8 years of age. A frameshift mutation of P291fsinsC (insertion of a C in a polyC tract around codon 291 for Pro), which would generate a mutant truncated protein of 340 amino acids, was found in a subject who started insulin treatment when hyperglycemia and ketonuria were noticed at 13 years of age. A missense mutation of R583G (replacement of Arg by Gly in codon 583) in the transactivation domain of HNF-1α was found in a subject with sudden-onset IDDM at 20 years of age. None of these mutations were present in 100 nondiabetic subjects (200 normal chromosomes). These results indicate that the HNF-1α gene defects could lead to the development of not only early-onset NIDDM but also IDDM, implicating the importance of subclassification of HNF-1α-deficient IDDM from a classical type of autoimmune-based IDDM in Japanese.

Loss-of-function and Dominant-negative Mechanisms Associated with Hepatocyte Nuclear Factor-1β Mutations in Familial Type 2 Diabetes Mellitus

Hepatocyte nuclear factor (HNF)-1β, a homeodomain-containing transcription factor, regulates gene expression in a dimerized form in pancreas, liver, and some other tissues. Recent genetic studies have identified two HNF-1β mutations, R177X and A263fsinsGG, in subjects with a monogenic form of type 2 diabetes. Despite the defects being in the same gene, diverse severities of disease are observed in the affected subjects. To investigate the molecular mechanism by which mutations might cause various phenotypic features, wild type and mutant proteins were transiently expressed in insulin-producing (MIN6) and hepatic (HepG2) cells. Luciferase reporter assay showed that both mutations resulted in a marked reduction of transactivation activity. Because their dimerization activity was found to be intact by the yeast two-hybrid system, it was possible that they were dominant-negative to wild type activity. When co-expressed with wild type, both of the mutants significantly decreased wild type activity in HepG2 cells. In contrast, although A263fsinsGG functioned similarly in MIN6 cells, R177X failed to affect wild type activity in this cell line. Immunohistochemical analysis of the mutants suggests that this functional divergence might be generated by the modification of nuclear localization. These results suggest that HNF-1β mutations may impair pancreatic β-cell function by loss-of-function and dominant-negative mechanisms.

Mutations in the hepatocyte nuclear factor-1α gene (MODY3) are not a major cause of early-onset non-insulin-dependent (type 2) diabetes mellitus in Japanese

AbstractMaturity-onset diabetes of the young (MODY3), a monogenic subtype of non-insulin-dependent diabetes mellitus (NIDDM) with an early age of onset, is characterized by a primary defect in insulin secretion. Recently, it has been shown that mutations of the gene encoding the transcription factor hepatocyte nuclear factor-1α (HNF-1α) cause MODY3. Since NIDDM in Japanese is characterized by insulin secretory defects due to primary β-cell dysfunction, we screened 60 Japanese nonobese subjects with early-onset NIDDM for mutations in this gene, 45 of whom had a first-degree relative with NIDDM. Direct sequencing of the ten exons and flanking introns of the gene in these subjects identified eight nucleotide substitutions including two amino acid changes, Ile-27-Leu and Ser-487-Asn, the frequencies of which were not significantly different in subjects with early-onset NIDDM and nondiabetic subjects. These results suggest that mutations in the HNF-1α gene are not a major cause of early-onset NIDDM in Japanese.

Mutations in the Hepatocyte Nuclear Factor-1α Gene (MODY3) Are Not a Major Cause of Late-Onset NIDDM in Japanese Subjects

Maturity-onset diabetes of the young (MODY) is a monogenic form of NIDDM characterized by an early age of onset, often in childhood or adolescence and usually <25 years of age, and autosomal dominant inheritance. Clinical studies of subjects with one form of MODY (M0DY3), the gene for which has been localized to chromosome 12, have suggested that impaired insulin secretion is the primary defect responsible for hyperglycemia in these subjects (1,2). Recently, Mahtani et al. (3), studying Finnish families with late-onset NIDDM, has mapped a gene affecting the susceptibility to NIDDM associated with low insulin secretion, designated NIDDM2, to the region of the M0DY3 locus on chromosome 12 (3). They suggest that NIDDM2 and M0DY3 may be different alleles of the same gene, which with severe mutations causes MODY and with milder mutations causes late-onset NIDDM with low insulin secretion. Recently, it has been shown that mutations in the gene encoding hepatocyte nuclear factor-la (HNF-la), a homeodomain-containing transcription factor originally identified in liver, are the cause of M0DY3 (4), but the molecular mechanism by which mutations in this gene cause MODY is still unclear. The possibility that mutations in the HNF-la gene might also cause late-onset NIDDM prompted us to screen this gene for mutations in Japanese subjects with late-onset NIDDM, a group characterized by primary pancreatic (3-ceD dysfunction rather than insulin resistance (5). We studied 103 Japanese subjects with late-onset NIDDM (mean age at diagnosis, 62.0 ± 10.2 years) from two diabetes care centers in Hyogo and Gunma prefectures, 68 of which had at least one relative with NIDDM (48 in a first-degree relative). The subjects involved were not diabetic by annual health examination before 40 years of age. NIDDM was diag-

Cloning of cDNA and the gene encoding human hepatocyte nuclear factor (HNF)-3β and mutation screening in Japanese subjects with maturity-onset diabetes of the young

Aims/hypothesis. Molecular defects of the genes for transcription factors, hepatocyte nuclear factor (HNF)-4α, HNF-1α, HNF-1β and insulin promoter factor-1 cause maturity-onset diabetes of the young (MODY1, 3, 5, and 4, respectively). This suggests the HNF-related transcription cascade is important in insulin secretion which is induced by glucose. These genes and the gene encoding glycolytic enzyme glucokinase (MODY2) are, however, responsible for only 15–20 % of cases of MODY in the Japanese. Searching for a novel form of MODY in this population, we cloned a new candidate gene encoding human HNF-3β, a winged helix transcription factor, which also belongs to the same HNF-transcription cascade.¶Methods. The cDNA clone for human HNF-3β was isolated from a liver cDNA library. The gene was also cloned from a genomic library and its organization and chromosomal localization were determined. We screened 68 Japanese subjects with MODY/early-onset diabetes for mutations in this gene.¶Results. Human HNF-3β is composed of 457 amino acids. The human gene, which was mapped to the segment 30 cR from SHGC-37 039 on chromosome 20 p by radiation hybrid mapping, spans approximately 4.5 kb and consists of three exons. Direct sequencing of the exons and flanking regions identified one missense mutation A328 V and seven polymorphisms, although the functional significance of the mutation in the pathogenesis of diabetes is not known.¶Conclusion/interpretation. The characterization of the structure of the HNF-3β gene and its mapping in the framework of markers will be helpful in genetic studies of the various forms of diabetes mellitus. [Diabetologia (2000) 43: 121–124]