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The Journal of Clinical Endocrinology & Metabolism Vol. 89, No. 2 971-978
Copyright © 2004 by The Endocrine Society

Insulin Promoter Factor-1 Mutations and Diabetes in Trinidad: Identification of a Novel Diabetes-Associated Mutation (E224K) in an Indo-Trinidadian Family

Brian N. Cockburn, Giovanna Bermano, Laura-Lee G. Boodram, Surujpal Teelucksingh, Takafumi Tsuchiya, Deepak Mahabir, Andrew B. Allan, Roland Stein, Kevin Docherty and Graeme I. Bell

Department of Life Sciences (B.N.C., L.G.B.), Faculty of Science and Agriculture, University of the West Indies, St. Augustine, Republic of Trinidad and Tobago; Department of Molecular and Cell Biology (G.B., A.B.A., K.D.), University of Aberdeen, Institute of Medical Sciences, Foresterhill, Aberdeen, AB25 2ZD, United Kingdom; Department of Biochemistry and Molecular Biology (B.N.C., L.G.B., T.T., G.I.B.), The University of Chicago, Chicago, Illinois 60637; Department of Clinical Medical Sciences (S.T.), Faculty of Medical Sciences, University of the West Indies, St. Augustine, Republic of Trinidad and Tobago; Nutrition and Metabolism Unit (D.M.), Ministry of Health, Republic of Trinidad and Tobago; and Department of Molecular Physiology and Biophysics (R.S.), Vanderbilt University Medical School, Nashville, Tennessee 37232

Address all correspondence and requests for reprints to: B. N. Cockburn, Ph.D., Department of Life Sciences, Faculty of Science and Agriculture, University of the West Indies, St. Augustine, Republic of Trinidad and Tobago. E-mail: bcockburn{at}fans.uwi.tt.


   Abstract
Top
 Abstract
Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
This study investigated the prevalence of insulin promoter factor-1(IPF-1) mutations in familial early-onset diabetes mellitus in Trinidad. We screened 264 unrelated subjects with type 2 diabetes diagnosed before 40 yr of age and a family history of diabetes for mutations in the minimal promoter and coding region of the IPF-1 gene (IPF1). This study population included 169 patients of East Indian descent (Indo-Trinidadians), 66 of African descent (Afro-Trinidadians), and 29 of mixed ancestry. We identified five IPF1 variants, including one new missense mutation E224K, the previously described diabetes-associated duplication P242 P243dupP, two silent mutations in the codons for Leu54 (c.162G>A) and Ala256 (c.768C>A), and a substitution in the 5'-untranslated region (c.-18C>T). The E224K mutation was found in two unrelated diabetic Indo-Trinidadians and 0 of 60 controls. It was present on the same haplotype in both patients suggesting a founder effect. The E224K mutation cosegregated with early-onset diabetes or impaired glucose tolerance in a large family, suggestive of the type 4 form of maturity-onset diabetes of the young rather than type 2 diabetes. Functional studies of E224K showed reduced transactivation activity. IPF1 mutations leading to synthesis of a mutant protein may contribute to the development of familial early-onset diabetes/maturity-onset diabetes of the young in Indo-Trinidadians.


   Introduction
Top
 Abstract
 Introduction
Subjects and Methods
 Results
 Discussion
 References
 
THE PREVALENCE OF diabetes is increasing worldwide, and this is also true of Latin America and the Caribbean (1). King et al. (1) estimated that in Trinidad and Tobago (a twin island republic located at the southernmost end of the Caribbean chain of islands) there were 39,000 people (age >= 20 yr) in yr 2000 with diabetes and predicted that this number would increase to 86,000 by yr 2025. The population of Trinidad is comprised of approximately 40% each of people of African and East Indian descent (2). Afro-Trinidadians are descendants of African slaves who were brought to the Caribbean from the early 1500s until slavery was abolished around 1834. The Indo-Trinidadians are descendants of indentured laborers who first started migrating from India to the island in 1845. They came primarily from the Gangetic Plain, from areas known today as Uttar Pradesh in the north, and the eastern region of Bihar. A smaller number also came from the northern region of Punjab, from Bengal in the east, and from Madras in the south. The remaining 20% of the population comprises individuals of mixed ethnicity (18%) with 2% being members of other ethnic groups including Caucasians (from Western European countries), Chinese, and Amerindians.

Genetic studies of diabetes are revealing the complex nature of this disease and providing new insight into the molecular mechanisms (3, 4). We have begun to study the genetics of diabetes in Trinidad with the goal of improving its diagnosis and treatment in this ethnically diverse population. As a first step, we are screening Indo- and Afro-Trinidadian patients with early-onset type 2 diabetes (diagnosed before 40 yr of age) and a family history of type 2 diabetes in at least two generations for mutations in genes associated with maturity-onset diabetes of the young (MODY) (3).

IPF-1, also known as pancreatic duodenal homeobox-1 (PDX-1), plays an important role in regulating the development and normal function of the pancreatic ß-cell (5). Complete deficiency of IPF-1 results in pancreatic agenesis and permanent neonatal diabetes, whereas mutations in the heterozygous state have been associated with both the type 4 form of MODY4 and type 2 diabetes (6, 7, 8). We screened 264 diabetic subjects for mutation in IPF1. We identified two mutations resulting in the synthesis of a mutant protein including a new missense mutation, E224K, two new silent mutations, and a new mutation in the 5'-untranslated region. We report the clinical features of patients with the E224K mutations and the effect of this mutation on IPF-1 function.


   Subjects and Methods
Top
 Abstract
 Introduction
 Subjects and Methods
Results
 Discussion
 References
 
Subjects

Diabetic patients were recruited from the diabetic outpatient clinics of the two major hospitals in Trinidad: the Port of Spain and San Fernando General Hospitals. Approval for the study was obtained from the Ethics Committees of both hospitals, and sampling took place over a 3-month period. Additionally, 12 diabetic individuals visiting a private clinic were recruited. The patient group consisted of 169 unrelated subjects of East Indian descent (Indo-Trinidadian), 66 of African descent (Afro-Trinidadian), and 29 of mixed ancestry. All individuals were diagnosed with type 2 diabetes before 40 yr of age and had a history of family type 2 diabetes in at least two generations. Race was assigned on the basis of the subject possessing at least three grandparents belonging to the same racial group (9). Subjects of mixed ancestry were predominantly of African and East Indian descent but also included some with European, Chinese, or Amerindian backgrounds. Nondiabetic subjects (60 Indo-Trinidadian, 60 Afro-Trinidadian, and 11 of mixed ancestry) were recruited from the Chest Clinic at Mt. Hope Hospital (Champs Fleurs, Trinidad and Tobago) and from San Fernando General Hospital (San Fernando, CA). Additional control subjects were obtained from various health centers throughout the island. After giving written consent, subjects were interviewed further to complete a medical questionnaire, and anthropometric measurements were taken. Assisting physicians examined the patients to determine their health status and the presence of any diabetes-related complications and to obtain a blood sample for DNA isolation. The nondiabetic subjects were nondiabetic by history and had normal urine or fasting blood glucose levels. In the absence of oral glucose tolerance testing (OGTT), we cannot exclude the presence of some individuals with have unrecognized diabetes.

Mutation screening

The minimal promoter, coding region, and adjacent introns of IPF1 were screened for mutations in diabetic and nondiabetic subjects by direct sequencing of the PCR product as described previously (10). Mutations discovered were confirmed by sequencing the opposite strand and by cloning the PCR product into pGEM-3Z (Promega, Madison, WI) and sequencing individual clones.

OGTT

After an overnight 12-h fast, blood samples were obtained at 0 min. Dextrose (75 g) was then administered orally, and blood samples were obtained at 30, 60, and 120 min for measurement of glucose, insulin, and C peptide concentrations. Plasma glucose was measured using a glucose analyzer (Model 2300 STAT, YSI Inc., Yellow Springs, OH). The coefficient of variation of this method is less than 2%. Serum insulin was assayed by a double-antibody technique with a lower limit of sensitivity of 20 pmol/liter and an average intraassay coefficient of variation of 6%. The cross-reactivity of proinsulin in the RIA for insulin is approximately 40%. Plasma C peptide was measured by RIA. The lower limit of sensitivity of the assay is 0.02 pmol/ml, and the intraassay coefficient of variation is 6%.

Functional studies of wild-type (WT) and mutant IPF-1

The E224K mutation was generated using the pALTER-1 system (Promega, Southampton, UK). Mutagenesis was confirmed by direct sequencing of both strands of the plasmid. The WT and mutant (E224K) forms of human IPF-1 were expressed in vitro using the vector pcDNA3 (Invitrogen, Carlsbad, CA). They were also ligated into the simian virus 40 (SV40) promoter-enhancer-driven GAL4 expression plasmid pSG24 (11) to create in-frame GAL4 fusion proteins. The reporter gene construct pGAL4TKCAT contains five copies of the GAL4 DNA binding site cloned upstream of the thymidine kinase (TK) chloramphenicol acetyl transferase (CAT) reporter gene. The FFCAT plasmid contains 5 copies of the sequence -247 to -197 of the rat insulin I gene spanning the A2 element (-212 to-208) that binds IPF-1 and the E2 element (-239 to-228) that binds E47 (12). E47 was expressed using the SV40 promoter enhancer of plasmid pJ3{Omega} (13). We used pRSV-luciferase as an internal control. Plasmid DNA was prepared using the QIAGEN (Valencia, CA) endotoxin-free method and quantified spectrophotometrically.

{alpha}TC-1 cells, a pancreatic {alpha}-cell line (14), were cultured in DMEM containing 25 mM glucose and supplemented with 10% heat-inactivated fetal calf serum (Invitrogen, Paisley, UK). HeLa cells were cultured in DMEM containing 10% calf serum. Cells were subcultured into six-well plates (5 x 105 cells/well) and grown for 24 h until they reached 80% confluence. {alpha}TC-1 cells were first transfected with 1 µg/well of the WT or E224K IPF-1 construct using Lipofectamine-plus reagent (Invitrogen, Paisley, UK). Cells were harvested 48 h after transfection in 1 ml PBS and then centrifuged for 1 min at 12,000 x g. The cell pellets were resuspended in 100 µl buffer containing 10 mM HEPES (pH 7.9), 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM dithiothreitol, and 1x Protease Inhibitor Cocktail Solution (Roche, Mannheim, Germany) and allowed to swell for 25 min on ice before adding 6 µl of 10% (vol/vol) Nonidet P-40. Cells were then vortexed for 15 sec and centrifuged for 1 min at 12,000 x g at 4 C. The supernatant was removed and stored as cytoplasmic extract. The nuclear pellets were resuspended in 50 µl ice-cold buffer containing 20 mM HEPES (pH 7.9), 0.4 M NaCl, 1 mM EDTA, 1 mM dithiothreitol, 1x Protease Inhibitor Cocktail Solution, and 5% (vol/vol) glycerol and shaken for 1 h. The nuclear extract was then centrifuged for 1 min at 12,000 x g at 4 C and the supernatant removed and stored at-70 C. Protein concentrations were determined using the Bio-Rad Protein Assay II System (Bio-Rad, Hercules, CA).

Western blot analysis was carried out using a specific IPF-1 antibody (a kind gift from Dr. C. V. Wright, Vanderbilt University). Three micrograms of nuclear or cytoplasmic protein extract from transfected cells were fractionated by SDS-PAGE on a 10% acrylamide gel and blotted onto ECL-nitrocellulose membrane (Amersham, Amersham, UK). The membrane was incubated overnight in a buffer containing 20 mM Tris-HCl (pH 7.4) containing 0.2 M NaCl, 0.1% Tween 20, 5% nonfat milk powder, and an 1:5,000 dilution of anti-IPF-1 antibody. The antigen-antibody complex was detected by incubating the membrane for 60 min in buffer containing a 1:10,000 dilution of horseradish-peroxidase-conjugated antirabbit IgG secondary antibody.

Analysis of IPF-1 binding activity was done by EMSA using the A3 site of the human insulin gene promoter (15). Three micrograms of transfected nuclear and cytoplasmic extract were incubated with 32P-labeled probe for 20 min at room temperature in buffer containing 10 mM Tris (pH 7.5), 50 mM KCl, 5 mM dithiothreitol, 1 mM EDTA, 0.5 µg dI.dC, and 5% (vol/vol) glycerol. The samples were then run on a 6% acrylamide gel at 150 V for 2 h. The labeled IPF-1 binding complex was detected by autoradiography.

The subcellular localization of WT and E224K IPF-1 in {alpha}TC-1 cells was determined by immunohistochemical analysis. Cells were grown on two-chamber slides for 24 h and then transfected as described above. After transfection, the cells were washed twice with PBS and then fixed in 4% formaldehyde, 0.1% Tween 20, and PBS for 1 h at room temperature. The fixed cells were then incubated for 1 h at room temperature in blocking buffer containing 10 mg/ml BSA and 0.1% Tween 20 in PBS. Primary anti-IPF-1 antibody (1:800 dilution) was added to each chamber in 1 mg/ml BSA and 0.1% Tween 20 in PBS and incubated for 1 h at room temperature. After three washes in 0.1% Tween 20 in PBS, fluorescein isothiocyanate-conjugated second antibody (1:400 dilution) was added to each chamber and incubated at room temperature for 1 h in the dark. Cells were finally washed three times with 0.1% Tween 20 in PBS and mounted with Vectashield (Vector Laboratories, Burlingame, CA) mounting medium containing 4',6-diamidino-2-phenylindole for nuclear staining. Fluorescence microscopy was carried out using a Zeiss Axioplan II microscope (Carl Zeiss, Thornwood, NY). Control samples containing only primary antibody or second antibody stained negative.

The transactivation properties of WT and E224K IPF-1 were analyzed in transfected {alpha}TC-1 and HeLa cells using the Gal4 DNA binding domain system (16). The GAL4WT and GAL4E224K fusion constructs described above (0.5 µg/well) were cotransfected into {alpha}TC-1 cells together with the reporter plasmid pGAL4TKCAT (0.5 µg/well). CAT activity was measured by using the QUAN-T-CAT Assay System (Amersham). The effect of IPF-1 and E224K on FFCAT activity was examined in HeLa cells transfected in triplicate in six-well plates using Lipofectamine reagent and 500 ng FFCAT, 250 ng pRSV-Luc, 250 ng of pcDNA3-IPF1, pcDNA3-E224K, or the pCMV4 vector (Invitrogen). The Rous Sarcoma Virus enhancer-driven luciferase construct (pRSV-LUC) served as an internal transfection control in these assays. Forty-eight hours later, the cells were harvested and cell extracts assayed for CAT and/or luciferase activity (13).

Data analysis

In transfection studies, significant differences were assessed using the unpaired t test. The insulinogenic index at 30 min (Insulinogenic index30) was determined as described by Seltzer et al. (17): Insulinogenic index30 = Insulin30 (pmol/liter) - Insulin0 (pmol/liter)/Glucose30 (mg/dl) - Glucose0 (mg/dl). Homeostatic model assessment insulin resistance (HOMA-R) was estimated as described by Matthews et al. (18): Insulin0 min (µU/ml) x Glucose0 min (mg/dl)/405. Group differences for Insulinogenic index30 and HOMA-R were assessed using an unpaired t test. A P-value < 0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
Discussion
 References
 
Clinical characteristics

Clinical characteristics of the study population are described in Table 1Go. Diabetic subjects, the Indo-Trinidadians had a lower body mass index than Afro-Trinidadians although the difference was not significant.


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  		TABLE 1. Clinical characteristics of study population

 
Mutations in the IPF-1 gene

We screened 395 unrelated diabetic (n = 264) and nondiabetic subjects (n = 131) for mutations in IPF1 using PCR and direct sequencing. We found five mutations (Table 2Go). They included the previously described type 2 diabetes-predisposing mutation IPF1-c.724 726dupCCG (P242 P243dupP) which was present in 3 of 66 Afro-Trinidadians with early-onset diabetes, 1 of 60 nondiabetic Afro-Trinidadian controls, and 1 of 29 individuals of mixed ancestry with early-onset diabetes (7, 8). The other four mutations have not been previously reported and include a substitution in the 5'-untranslated region (c.-18C>T), two silent mutations (c.162G>A and c.768C>A), and a missense mutation E224K (c.670G>A). All of the mutations were found in the heterozygous state, and we observed no compound heterozygotes.


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  		TABLE 2. Sequence variation in the IPF-1 gene in early-onset diabetes in Trinidad

 
The E224K mutation was found in two of 169 unrelated Indo-Trinidadians (TT001 and TR006) and none of 60 controls. Genotyping with other markers in the region of IPF1, including the microsatellite DNA polymorphism D13S787 and reference single nucleotide polymorphisms rs2293942 and rs2293943, suggest that the mutant allele (E224K) is on the same haplotype [D13S787-rs2293942-rs2293943: 255 bp (allele 2)-C-T] in these two individuals, consistent with them sharing a common ancestor at some point in the past.

The E224K mutation cosegregated with early-onset diabetes/IGT in the TT001 pedigree (Fig. 1Go). In generation III, four carriers were diagnosed with diabetes or IGT between 13–21 yr. The only carrier in the TT001 pedigree who did not have diabetes or IGT was an 11-yr-old girl (subject III-8). She presently has normal glucose tolerance, by OGTT, but may develop diabetes in the future. The early-onset and cosegregation of the E224K mutation with diabetes in the TT001 pedigree is consistent with a diagnosis of MODY4 (6). There are also noncarriers in the TT001 pedigree who have diabetes or IGT, indicating that the E224K in IPF1 is not the only cause of diabetes in this family. Such genetic heterogeneity has been noted in other MODY families (19, 20).



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  		FIG. 1. Pedigree of the TT001 family. Individuals with diabetes are indicated by filled symbols (squares are male, circles are female). Gray symbols indicate individuals with IGT. Genotype: N, normal IPF-1 allele, M, mutant E224K allele. FBG, Fasting blood glucose; N/A, not applicable; 2hBG, 2-h blood glucose.

 
In the TR006 pedigree, the proband’s father (deceased) was diagnosed with diabetes at 17 yr of age. The proband (deceased) was diagnosed with diabetes at 25 yr of age. Both the proband and his father were treated with insulin. The only other family member with glucose intolerance is an older brother who has IGT but did not inherit the E224K mutation. The proband has three children, ages 23, 21, and 9 yr, and none have diabetes. The two older children are not diabetic and did not inherit the E224K mutation (the 9-yr-old was not available for testing).

OGTT in subjects with the E224K mutation

We carried out OGTT in diabetic (subjects II-4, II-8, III-1, and III-3) and nondiabetic (III-8) E224K carriers in the TT001 pedigree (Fig. 1Go) and five unrelated nondiabetic/nonmutation subjects. We used the Insulinogenic index30 as a measure of ß-cell function and HOMA-R to assess insulin resistance (17, 18). The diabetic carriers exhibited a significant reduction (P < 0.05) in ß-cell function compared with nondiabetic/nonmutation subjects (Fig. 2Go). There was no significant difference in HOMA-R between these two groups. The results are consistent with a ß-cell defect that is present even in the nondiabetic carrier. However, we will need to identify additional nondiabetic carriers to confirm this result. If there is a founder effect for this mutation in the Indo-Trinidadian population, as our genetic data suggest, we may be able to identify other families with the E224K mutation that will allow us to carry out additional clinical studies.



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  		FIG. 2. Results of OGTT in subjects with (NM) and without the E224K mutation (NN). A, Insulinogenic index (30 min) as a function of genotype. The genotypes of the three groups of subjects are indicated. B, HOMA-R as a function of genotype.

 
Functional studies of E224K IPF-1

Residue 224 is in a highly conserved region of IPF1. It is glutamic acid in human, hamster, rat, mouse, frog, and zebra fish IPF-1, implying an important functional role. To study the effect of the E224K mutation on the different properties of IPF-1, we first expressed constructs encoding the WT and mutant (E224K) forms of human IPF-1 in {alpha}TC-1 cells. These cells were chosen for this study because they are IPF-1 negative and originate from the endocrine pancreas (14). Western blotting analysis showed that both WT and E224K IPF-1 generated a 46-kDa protein that was localized predominantly (90%) in the nuclear fraction (Fig. 3AGo). The same subcellular distribution was observed by immunocytochemistry (Fig. 3BGo), and no detectable staining was found with the vector alone (pcDNA3).



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  		FIG. 3. Expression of WT and E224K IPF-1 in {alpha}TC-1 cells. A, Western blot analysis using an anti-IPF-1 antibody of nuclear (N) and cytoplasmic (C) extracts prepared from WT or E224K IPF-1-transfected {alpha}TC-1 cells. This result is representative of data from three separate experiments. B, Immunocytochemical localization of WT and E224K IPF-1 in {alpha}TC-1 cells. IPF-1 antibody immunohistochemical analysis was performed with WT IPF-1-, E224K IPF-1-, and vector control (pcDNA3)-transfected cells. IPF-1 antibody staining in green was visualized using a fluorescein-conjugated second antibody that appears green in the image. Panel C, DNA-binding activity of WT and E224K IPF-1. EMSA analysis was performed with nuclear (N) and cytoplasmic (C) extracts from {alpha}TC-1 cells transfected with WT and E224K IPF-1. The samples were normalized for protein content. Results are representative of data from three separate experiments. Mr, Relative molecular mass.

 
We also studied the effect of the E224K mutation on binding to a double-stranded oligodeoxynucleotide corresponding to the A3 site of the human insulin gene (14). A single major band of similar intensity was observed using nuclear and cytoplasmic extracts of {alpha}TC-1 cells transfected with the WT and E224K plasmids (Fig. 3CGo). This band was shown to contain IPF-1 using the anti-IPF-1 antibody and mutant A3 oligonucleotides in competition studies (data not shown).

To specifically study effects of the E224K mutation on the transactivation properties of IPF-1, the WT and E224K IPF-1 cDNAs were cloned in frame with the GAL4 DNA binding domain of pSG424 and cotransfected into {alpha}TC-1 cells along with the GAL4 DNA binding site linked to the CAT reporter gene. Activation of the CAT reporter gene was 20% less efficient in cells transfected with GAL4:E224K than with GAL4:WT (P < 0.05) (Fig. 4Go). The transactivation properties of IPF-1 were also studied in HeLa cells using FFCAT, a rat insulin I enhancer-driven reporter whose activity is dependent on addition of both IPF-1 and E47 (12, 21). As observed with the GAL4:IPF chimera, E224K was significantly less active than the WT (P < 0.001) (Fig. 5AGo). In both transfection systems, the reduced activation properties were not due to a change in the expression or stability of the E224K mutant (Fig. 5BGo; data not shown).



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  		FIG. 4. The E224K mutation reduces the transactivation potential of IPF-1. CAT activity measured in {alpha}TC-1 cells cotransfected with GAL4:WT IPF-1 or GAL4:E224K IPF-1 and the pGAL4TKCAT reporter. The pSG24 vector containing only the GAL4 DNA binding domain was used as a negative control. CAT activity was measured 48 h after transfection and normalized against protein concentration. The data are expressed relative to the CAT activity of the pSG24 plasmid and represent the mean ± SE of three separate experiments.

 


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  		FIG. 5. E224K IPF-1 is less able to cooperatively stimulate insulin expression with the E47 activator. A, HeLa cells were transfected with the indicated constructs. Results are expressed relative to CAT activity of the FFCAT construct cotransfected with WT IPF-1 and E47. Data are from five separate experiments showing mean ± SE. Lane 1, FFCAT alone; lane 2, + E47; lane 3, + WT IPF-1; lane 4, + E224K IPF-1; lane 5, + E47 + WT IPF-1; lane 6, + E47 + E224K IPF-1. B, Representative Western blot analysis of nuclear extracts from HeLa cells transfected with the indicated DNA constructs. Lane assignments are as for A except: lane 4, + E47 + WT IPF-1; lane 5, + E224K IPF-1.

 

   Discussion
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
References
 
Mutations in the IPF-1 gene have been associated with three different forms of diabetes. The form that results depends on whether there is complete (permanent neonatal diabetes) or partial deficiency (MODY4 and type 2 diabetes) (3, 5, 6, 7, 8). In the case of partial deficiency, the specific effect of a given mutations on IPF-1 function may determine whether MODY or type 2 diabetes develops. We have examined the extent of sequence variation in the IPF-1 gene in Indo-, Afro-, and racially mixed Trinidadians with familial early-onset type 2 diabetes. Our results indicate that mutations in this gene are not a common cause of diabetes in any of these groups. However, they may be a contributing factor in 1.2% of cases in the Indo-Trinidadians and perhaps as much as 4.6% in the Afro-Trinidadians studied here (Table 2Go). One limitation of our study is that we recruited patients from the diabetic outpatient clinics at the major hospitals. Although this protocol ensures a nationwide sampling, it only captures subjects whose glucose control is very poor or who develop serious complications. Mild mutations in IPF-1 might thus be underestimated.

We identified a new mutation in IPF1, E224K, which may result in MODY4. Preliminary clinical studies of diabetic and nondiabetic carriers suggest that this mutation affects ß-cell function. We identified this mutation in two ostensibly unrelated individuals. However, it occurred on the same extended haplotype, suggesting a common origin for the mutant allele (founder effect). Thus, additional individuals and families with this mutation may be found among the Indo-Trinidadian population.

We characterized the effect of the E224K mutation on human IPF-1 function. It does not appear to affect the expression, nuclear localization, or DNA-binding properties of IPF-1. This is not surprising because sequences involved in DNA-binding and nuclear localization have been mapped within the homeodomain, amino acids 146–206 (22, 23), and deletion of COOH-terminal sequences (amino acids 206–283) have no effect on these parameters (24). The E224K mutation resulted in a modest but significant reduction in IPF-1 transactivation as a GAL4 fusion protein and in synergistic action with E47 (Figs. 4Go and 5Go). The major transactivation sequences within IPF-1 have been mapped to a proline-rich region at the NH2 terminus (16, 24). The activity of this transactivation domain, which contains three subdomains, can be stimulated by glucose (25, 26). Sequences toward the COOH terminus have no intrinsic transactivation activity. However, successive deletion of these sequences results in both enhancement and diminution of transactivation. This suggests that the COOH-terminal region of IPF-1 may be involved in protein-protein interactions that act synergistically to enhance or modulate the activity of the N-terminal domain (24). A possible candidate for this COOH-terminal interacting protein is a recently described 41-kDa nuclear protein, named PCIF1 (27).

Although our results show that the E224K mutation affects the transactivation properties of IPF-1, they do not explain why this defect leads to the development of diabetes. It is possible that the modest effect on IPF-1 activity reduces the levels of the IPF-1 target genes that are important in regulating normal ß-cell function and mass (28, 29, 30).

In summary, mutations in the IPF-1 gene may be associated with MODY4 (E224K) in the Indo-Trinidadian population and a contributing factor in the development of type 2 diabetes in Afro-Trinidadians (P242 P243dupP). A registry of patients with these mutations could lead to a better understanding of the role of this gene in the development of diabetes in this community and improve diagnosis and treatment.


   Acknowledgments
 
We thank Sunita Ramnarine for recruitment of family members and Paul Rue for measuring insulin and C peptide levels. We also thank Chris Wright for providing the anti-IPF-1 and Michael German for the FFCAT construct.


   Footnotes
 
This work was supported by grants from the University of the West Indies Research and Publications Fund, the U.S. Public Health Service (DK-20595, DK-50203, and DK-44840), and the Wellcome Trust. G.I.B. is an Investigator of the Howard Hughes Medical Institute.

Abbreviations: CAT, Chloramphenicol acetyl transferase; HOMA-R, homeostatic model assessment insulin resistance; IGT, impaired glucose tolerance; IPF-1, insulin promoter factor-1; MODY, maturity-onset diabetes of the young; OGTT, oral glucose tolerance testing; PDX-1, pancreatic duodenal homeobox-1; SV, simian virus; TK, thymidine kinase; WT, wild-type.

Received July 23, 2003.

Accepted October 20, 2003.


   References
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 

  1. King H, Aubert RE, Herman WH 1998 Global burden of diabetes, 1995–2025: prevalence, numerical estimates, and projections. Diabetes Care 21:1414–1431[Abstract]
  2. Brereton B 1981 A history of modern Trinidad: 1783–1962. Oxford, UK: Heinemann International; 103–106
  3. Fajans SS, Bell GI, Polonsky KS 2001 Molecular mechanisms and clinical pathophysiology of maturity-onset diabetes of the young. N Engl J Med 345:971–980[Free Full Text]
  4. Gloyn AL, McCarthy MI 2001 The genetics of type 2 diabetes. Best Pract Res Clin Endocrinol Metab 15:293–308[CrossRef][Medline]
  5. Stoffers DA, Zinkin NT, Stanojevic V, Clarke WL, Habener JF 1997 Pancreatic agenesis attributable to a single nucleotide deletion in the human IPF1 gene coding sequence. Nat Genet 15:106–110[CrossRef][Medline]
  6. Stoffers DA, Ferrer J, Clarke WL, Habener JF 1997 Early-onset type-II diabetes mellitus (MODY4) linked to IPF1. Nat Genet 17:138–139[CrossRef][Medline]
  7. Macfarlane WM, Frayling TM, Ellard S, Evans JC, Allen LI, Bulman MP, Ayres S, Shepherd M, Clark P, Millward A, Demaine A, Wilkin T, Docherty K, Hattersley AT 1999 Missense mutations in the insulin promoter factor-1 gene predispose to type 2 diabetes. J Clin Invest 104:R33–R39
  8. Hani EH, Stoffers DA, Chevre JC, Durand E, Stanojevic V, Dina C, Habener JF, Froguel P 1999 Defective mutations in the insulin promoter factor-1 (IPF-1) gene in late-onset type 2 diabetes mellitus. J Clin Invest 104:R41–R48
  9. Miller GJ, Beckles GL, Byam NT, Price SG, Carson DC, Kirkwood BR, Baker IA, Bainton D 1984 Serum lipoprotein concentrations in relation to ethnic composition and urbanization in men and women of Trinidad, West Indies. Int J Epidemiol 13:413–421[Abstract/Free Full Text]
  10. Hara M, Lindner TH, Paz VP, Wang X, Iwasaki N, Ogata M, Iwamoto Y, Bell GI 1998 Mutations in the coding region of the insulin promoter factor 1 gene are not a common cause of maturity-onset diabetes of the young in Japanese subjects. Diabetes 47:845–846[Medline]
  11. Lillie JW, Green MR 1989 Transcription activation by the adenovirus E1a protein. Nature 338:39–44[CrossRef][Medline]
  12. German MS, Moss LG, Wang J, Rutter WJ 1992 The insulin and islet amyloid polypeptide genes contain similar cell-specific promoter elements that bind identical ß-cell nuclear complexes. Mol Cell Biol 12:1777–1788[Abstract/Free Full Text]
  13. Peshavaria M, Henderson E, Sharma A, Wright CV, Stein R 1997 Functional characterization of the transactivation properties of the PDX-1 homeodomain protein. Mol Cell Biol 17:3987–3996[Abstract/Free Full Text]
  14. Powers AC, Efrat S, Mojsov S, Spector D, Habener JF, Hanahan D 1990 Proglucagon processing similar to normal islets in pancreatic {alpha}-like cell line derived from transgenic mouse tumor. Diabetes 39:406–414[Abstract]
  15. Boam DS, Docherty K 1989 A tissue-specific nuclear factor binds to multiple sites in the human insulin-gene enhancer. Biochem J 264:233–239[Medline]
  16. Sadowski I, Ptashne M 1989 A vector for expressing GAL4(1–147) fusions in mammalian cells. Nucleic Acids Res 17:7539[Free Full Text]
  17. Seltzer HS, Allen EW, Herron Jr AL, Brennan MT 1967 Insulin secretion in response to glycemic stimulus: relation of delayed initial release to carbohydrate intolerance in mild diabetes mellitus. J Clin Invest 46:323–335
  18. Matthews DR, Hosker JP, Rudenski AS, Naylor BA, Treacher DF, Turner RC 1985 Homeostasis model assessment: insulin resistance and ß-cell function from fasting plasma glucose and insulin concentrations in man. Diabetologia 28:412–419[CrossRef][Medline]
  19. Bell GI, Xiang KS, Newman MV, Wu SH, Wright LG, Fajans SS, Spielman RS, Cox NJ 1991 Gene for non-insulin-dependent diabetes mellitus (maturity-onset diabetes of the young subtype) is linked to DNA polymorphism on human chromosome 20q. Proc Natl Acad Sci USA 88:1484–1488[Abstract/Free Full Text]
  20. Vionnet N, Stoffel M, Takeda J, Yasuda K, Bell GI, Zouali H, Lesage S, Velho G, Iris F, Passa P, Froguel P, Cohen D 1992 Nonsense mutation in the glucokinase gene causes early-onset non-insulin-dependent diabetes mellitus. Nature 356:721–722[CrossRef][Medline]
  21. Peers B, Leonard J, Sharma S, Teitelman G, Montminy MR 1994 Insulin expression in pancreatic islet cells relies on cooperative interactions between the helix loop helix factor E47 and the homeobox factor STF-1. Mol Endocrinol 8:1798–1806[Abstract/Free Full Text]
  22. Moede T, Leibiger B, Pour HG, Berggren P, Leibiger IB 1999 Identification of a nuclear localization signal, RRMKWKK, in the homeodomain transcription factor PDX-1. FEBS Lett 461:229–234[CrossRef][Medline]
  23. Hessabi B, Ziegler P, Schmidt I, Hessabi C, Walther R 1999 The nuclear localization signal (NLS) of PDX-1 is part of the homeodomain and represents a novel type of NLS. Eur J Biochem 263:170–177[Medline]
  24. Lu M, Miller C, Habener JF 1996 Functional regions of the homeodomain protein IDX-1 required for transactivation of the rat somatostatin gene. Endocrinology 137:2959–2967[Abstract]
  25. Petersen HV, Peshavaria M, Pedersen AA, Philippe J, Stein R, Madsen OD, Serup P 1998 Glucose stimulates the activation domain potential of the PDX-1 homeodomain transcription factor. FEBS Lett 431:362–366[CrossRef][Medline]
  26. Shushan EB, Cerasi E, Melloul D 1999 Regulation of the insulin gene by glucose: stimulation of trans-activation potency of human PDX-1 N-terminal domain. DNA Cell Biol 18:471–479[CrossRef][Medline]
  27. Liu A, Stoffers D 2002 Down-regulation of PDX-1 by a novel C-terminus interacting factor, PCIF1. Diabetes 51(Suppl 2):A55 (Abstract)
  28. Wilson ME, Scheel D, German MS 2003 Gene expression cascades in pancreatic development. Mech Dev 120:65–80[CrossRef][Medline]
  29. Thomas MK, Devon ON, Lee JH, Peter A, Schlosser DA, Tenser MS, Habener JF 2001 Development of diabetes mellitus in aging transgenic mice following suppression of pancreatic homeoprotein IDX-1. J Clin Invest 108:319–329[CrossRef][Medline]
  30. Johnson JD, Ahmed NT, Luciani DS, Han Z, Tran H, Fujita J, Misler S, Edlund H, Polonsky KS 2003 Increased islet apoptosis in Pdx1+/- mice. J Clin Invest 111:1147–1160[CrossRef][Medline]



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