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BRIEF REPORT |
Division of Endocrinology and Metabolism, Departments of Medicine (N.P.), Immunology and Immunology Graduate Program (N.P., N.S., W.T., W.B., N.B.), Physiology (S.K.), and Research and Development (N.S., P.Y.), Faculty of Medicine, Siriraj Hospital, Mahidol University, Bangkok, 10700 Thailand; The First Department of Medicine (A.Doi, H.F., M.N., K.N.), Wakayama Medical University, Wakayama 641-8509, Japan; and Section on Genetics and Epidemiology (A.Dor.), Joslin Diabetes Center, Harvard Medical School, Boston, Massachusetts 02215
Address all correspondence and requests for reprints to: Nattachet Plengvidhya, M.D., Division of Endocrinology and Metabolism, Department of Medicine, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700, Thailand. E-mail: sinpv.natpl{at}gmail.com.
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Abstract |
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Objective: The aim of this study was to investigate whether or not genetic variants in PAX4, a crucial transcription factor in ß-cell development, contribute to MODY in Thais.
Design and Methods: We screened PAX4 coding sequences in 46 MODY probands without mutation in known MODY genes and in 74 nondiabetic controls using PCR-single-stranded conformational polymorphism analysis followed by direct sequencing. Genotyping of variants identified was done by PCR-restriction fragment length polymorphism analysis.
Results: Eight sequence differences were identified. Two novel variations (R164W and IVS7–1G>A) were found in two different probands. Neither was found in the 74 nondiabetic controls and additional 270 healthy subjects of Thai origin. R164W segregated with diabetes in the family of the proband and in vitro studies showed that it impairs the repressor activity of PAX4 on the insulin and glucagon promoters. The remaining six variants were previously described and observed in both groups. One of them, R192H, was three times more frequent in MODY probands than in 342 nondiabetic controls (minor allele frequency = 0.196 vs. 0.064; P < 0.00001). The same variant was associated with a younger age at diagnosis among 254 Thai subjects with adult-onset type 2 diabetes (44.6 ± 15 vs. 49.7 ± 11 yr; P = 0.048).
Conclusions: We have identified two possible pathogenic mutations of PAX4, R164W, and IVS7–1G>A. For one of these, we have shown evidence of segregation with diabetes and a functional impact on PAX4 activity. Single-nucleotide polymorphism R192H might influence the age at onset of diabetes.
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Introduction |
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PAX4, a paired-homeodomain transcription factor, functions as a transcription repressor through a pair homeobox and homeodomain (12, 13). Such action plays a critical role in pancreatic ß-cell development and function (14). PAX4 first appears in the endocrine progenitor cells at embryonic d 9.5 and is later selectively expressed in ß-cells (13), where it is required to maintain the expression of Pdx1 and Nkx 6.1, two essential modulators of pancreatic ß-cell development (14). Heterozygous PAX4 knockout (KO) mice have few mature ß- and 
-cells, and numerous, abnormally clustered 
-cells, suggesting that PAX4 is a critical regulator of the commitment of progenitor cells to the different islet cell lineages (15). Remarkably, the abnormalities of PAX4 KO mice resemble those of mice with a targeted disruption of insulin-promoter-factor 1, a known MODY gene (16). Pax4 also appears to be important for the regeneration of ß-cell in adult life, as suggested by the finding that PAX4 mutations impair the ability of ß-cells to proliferate (17). On this basis, we investigated whether sequence variants in PAX4 contribute to MODY in the Thai population or not.
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Subjects and Methods |
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The study included 46 diabetic probands of MODY families recruited at the Diabetic Clinic, Siriraj Hospital, Bangkok, Thailand. The inclusion criteria were: 1) the proband and at least one first-degree relative diagnosed with type 2 diabetes before age 35 yr, 2) two or more generations affected by diabetes, 3) diabetes treatment with diet and/or oral agents, 4) no history of diabetic ketoacidosis, and 5) absence of glutamic acid decarboxylase antibody. Mutations in any of the six known MODY genes were excluded by PCR-single-stranded conformational polymorphism (SSCP) analysis followed by direct sequencing. Nondiabetic subjects were 74 healthy staff members in the Department of Immunology and Department of Research and Development at Mahidol University, Bangkok, Thailand. All of them had fasting plasma glucose levels less than 100 mg/dl and had no family history of diabetes in first-degree relatives. A total of 270 additional nondiabetic subjects were healthy blood donors without a history of diabetes. The study was approved by the Faculty of Medicine Siriraj Hospital Research Ethics Committee. All study subjects signed a consent form before their enrollment.
Mutation screening and sequence analysis of PAX4
PAX4 exons and exon-intron boundaries were screened for nucleotide variants in the 46 MODY probands and 74 nondiabetic subjects by PCR followed by SSCP analysis. PCR products showing a mobility shift were directly sequenced by the ABI Prism BigDye 228 Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems, Foster City, CA). Fluorescent signals were detected with the ABI Collection software and analyzed by the Sequencer Navigator software and Chromas program version 1.4.4 (Conor McCarthy, Griffith University, Queensland, Australia).
Genotyping of PAX4 variants
The R164W, IVS7–1G>A, and R192H variants were genotyped in an additional 270 nondiabetic subjects by PCR-restriction fragment length polymorphism analysis using the enzymes HaeIII, BsrI, and MseI (Fermentas Inc., Hanover, MD), respectively. DNA fragments were separated on 12% polyacrylamide gels or 2% agarose gels, and genotype and allele frequencies compared between MODY probands and nondiabetic subjects by 
2 tests with Yates’ correction for continuity or Fisher exact tests (Statistical Package for the Social Sciences; SPSS, Inc., Chicago, IL). P values < 0.05 were considered significant.
Functional study of PAX4 variant
Full-length human wild-type PAX4 cDNA was amplified from PCR Ready First Strand cDNA of normal human placenta (BioChain Institute, Inc., PSA Vista, Singapore) by PCR using platinum Pfx DNA polymerase (Invitrogen, Leek, The Netherlands) and subcloned into a pcDNA 3.1 expression vector. The R164W mutation was introduced by site-directed mutagenesis (QuikChange Mutagenesis Kit; Stratagene, La Jolla, CA) to generate pcDNA3.1-PAX4-R164W. Human insulin and glucagons promoters were isolated by PCR using Pfu DNA polymerase (Stratagene) and separately subcloned into pGL3 reporter vectors to generate human insulin and glucagon promoter-firefly luciferase reporters. PAX4 wild-type and mutant constructs (500 ng) were transfected into MIN6 or TC-1.6 cells using the FUGENE 6 transfection reagent (Roche Diagnostics, Roche Applied Science, Indianapolis, IN) along with 100 ng pGL3-human insulin promoter, and 10 ng pRL-SV40 (to control for the transfection efficiency). After 24 h, the transactivation activity of the normal and mutant PAX4 proteins was measured by the Dual-Luciferase Reporter Assay System (Promega Corp., Madison, WI). The significance of differences among constructs was tested by one-way ANOVA followed by Scheffe’s post hoc test. A P value < 0.05 was considered significant.
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Results and Discussion |
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TopAbstractIntroductionSubjects and MethodsResults and DiscussionReferences |
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). Two were novel variants that were found in heterozygosity with the wild type in two different probands. One was a C to T substitution at codon 164 (CGG>TGG), resulting in the replacement of arginine with tryptophan (R164W). The other was a G to A substitution at the splice acceptor site of intron 7 (IVS7–1G>A) (Fig. 1A). Neither mutation was found in the 344 nondiabetic subjects of Thai origin. The R164W mutation segregated with diabetes, being present in the proband’s 52-yr-old father, who was diagnosed as having diabetes at age 50 yr, and 29-yr-old sister, who both had type 2 diabetes, as well as in her 14-yr-old brother, who had impaired glucose tolerance (Fig. 1B). However, two sisters (28 and 22 yr old), who also had impaired glucose tolerance, did not carry the mutation and were probably phenocopies as observed in the case of other MODY genes (4, 7). No relatives were available for the segregation analysis of the IVS7–1G>A mutation with diabetes, but evaluation of the pedigree (data not show) revealed that two older sisters and one older brother had diabetes and early-onset renal failure (40 yr old), and all died at age 52–53 yr of end-stage renal disease. Her youngest sister was diagnosed as having diabetes at age 30 yr, was still alive, and already has diabetic retinopathy and nephropathy at age 40 yr.
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). The R192H variant was three times more frequent in the 46 MODY probands than in 342 nondiabetic controls (minor allele frequency = 0.196 vs. 0.064; P < 0.00001). We further genotyped the R192H in 254 Thai adult onset type 2 diabetes but found no significant difference in allele frequencies between patients and nondiabetic controls (Table 2). However, the mean age at diagnosis of heterozygotes was significantly lower than among major allele homozygotes (44.6 ± 15.0 vs. 49.7 ± 10.7 yr; P = 0.048).
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). By contrast, the R164W mutant repressed the promoter by only 35% (P < 0.01 for mutant vs. wild-type). Similar results were obtained with a human glucagon promoter reporter system in -TC1.6 cells (Fig. 2C). The PAX4 wild type repressed the promoter activity by 57%, whereas the R164W repressed it by only 35% (P < 0.01 for mutant vs. wild-type). These differences between wild type and mutant were not due to differences in transfection efficiencies or in the expression of the transfected constructs (data not shown).
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) responsible for PAX4 binding to target DNA sequences; 4) it concerns an amino acid residue conserved among species; 5) the mutation is rather severe, replacing a polar with a nonpolar amino acid; and 6) this amino acid substitution impairs the repressor activity of PAX4 on the insulin and glucagon promoters.
Given that the impairment of PAX4 repressor activity caused by the mutation is relatively small, we hypothesize that such abnormality causes diabetes through the interaction of multiple mechanisms, each of which is not sufficient by itself to lead to hyperglycemia. One of these may be the disruption of ß-cell development in the embryo, resulting into reduced ß-cell mass. Another mechanism may involve decreases insulin secretion in response to glucose in adult ß-cell. Because heterozygous PAX4 KO mice have numerous, abnormally clustered -cells (15), this mutation may similarly perturb the islet architecture in humans and affect the insulin to glucagon ratio, which is a critical determinant of gluconeogenesis and glycogenolysis.
The evidence supporting a pathogenetic role for the IVS7–1G>A variant is not as definitive because we did not have access to the proband’s relatives to determine its segregation with diabetes and could not directly study the impact of this mutation because this would require access to islet mRNA from the proband. On the other hand, this mutation appears to be especially severe, abolishing the acceptor splice site of intron 7 and potentially leading to exon skipping, intron retention, or usage of another acceptor splice site. It would seem unlikely that a mutation having such predicted effects was silent. Indeed, the characteristic of diabetes in the pedigree suggests that this mutation may determine an especially severe form of diabetes characterized by an early onset of renal complications.
We also found that a relatively common polymorphism (R192H) was overrepresented in MODY probands compared with nondiabetic controls. Although highly significant, this result should be taken with caution because it concerns only 46 MODY cases. Furthermore, we failed to demonstrate an association between this polymorphism and later-onset type 2 diabetes, as previously shown in Japanese individuals (18). However, our data indicate that this variant may influence the age at onset rather than the overall risk of type 2 diabetes.
In conclusion, we have identified two possible pathogenic mutations of PAX4, R164W and IVS7–1G>A. For one of these, we have shown evidence of segregation with diabetes and a functional impact on PAX4 activity. We have also found that SNP R192H might also influence the risk of diabetes development, in particular for those forms characterized by an early onset.
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Acknowledgments |
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Footnotes |
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Author Disclosure Statement: The authors have nothing to disclose.
First Published Online April 10, 2007
Abbreviations: KO, Knockout; MODY, maturity onset diabetes of the young; SNP, single-nucleotide polymorphism; SSCP, single-stranded conformational polymorphism.
Received September 1, 2006.
Accepted April 3, 2007.
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References |
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TopAbstractIntroductionSubjects and MethodsResults and DiscussionReferences |
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gene in maturity-onset diabetes of the young (MODY1). Nature 384:458–460[CrossRef][Medline] gene in maturity-onset diabetes of the young (MODY3). Nature 384:455–458[CrossRef][Medline] genes in patients with early-onset type 2 diabetes mellitus/MODY. Diabet Med 16:956–963[CrossRef][Medline] gene (MODY3) are not a major cause of early-onset non-insulin-dependent (type 2) diabetes mellitus in Japanese. J Hum Genet 43:107–110[CrossRef][Medline]This article has been cited by other articles:
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  T. Brun and B. R Gauthier A focus on the role of Pax4 in mature pancreatic islet {beta}-cell expansion and survival in health and disease J. Mol. Endocrinol., February 1, 2008; 40(2): 37 - 45. [Abstract] [Full Text] [PDF]
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and 
, Normal fasting glucose; shaded circle and shaded square, impaired glucose tolerance; • and 
, diabetes; 