Journal of the Pancreas Open Access

Keywords

Genetics; Mexico; Polymorphism, Genetic

Abbreviations

GCK: glucokinase; HRP: horseradish peroxidase; MODY: maturityonset diabetes of the young; NP-40: Nonidet P-40; T2DM: type 2 diabetes mellitus

INTRODUCTION

Type 2 diabetes mellitus (T2DM), a chronic disease affecting glucose metabolism, is characterized by both insulin resistance and pancreatic beta-cell dysfunction. It is estimated that 150 million people are affected worldwide [1]. In Mexico, diabetes has become the first cause of death [2] and prevalence estimations are as high as 12% [3]. Characteristically, the age of onset in the Mexican population is earlier than expected, as almost 15% of the cases are diagnosed before age 40 (early-onset T2DM) [4]. The premature morbidity and mortality associated with early-onset T2DM has a much greater socio-economic impact than the late-onset form.

Maturity-onset diabetes of the young (MODY) is a genetically and clinically heterogeneous subtype of diabetes mellitus characterized by early-onset, autosomal dominant inheritance and beta cell dysfunction. Six genes have been identified as responsible for MODY: HNF-4alpha, glucokinase (GCK), HNF-1alpha, IPF-1, HNF-1beta and NEUROD1 [5, 6, 7, 8, 9, 10]. Additionally, in several populations other yet unknown genes are responsible for the disease in 16-45% of MODY families [11]. This group is designated as MODY X. MODY gene sequence variations have been associated with the more common polygenic forms of T2DM in several populations [12, 13, 14, 15]. The aim of this study was to establish whether mutations in genes encoding the transcription factors HNF- 1alpha, HNF-4alpha, IPF-1, HNF-1beta or NEUROD1 are involved in the etiology of early-onset diabetes in a group of Mexican families previously screened for the presence of glucokinase mutations, including five families fulfilling the MODY criteria [16]. No mutations were identified in HNF-4alpha, IPF-1, HNF-1beta and NEUROD1 genes in any of the analyzed families. However, a new HNF-1alpha gene mutation (P379H) was found in a MODY family.

MATERIALS AND METHODS

Subjects

A total of 23 extended families were included in the analysis, recruited from the diabetes clinics of three different major referral centers in Mexico City. The families selected were those which included a proband with early onset diabetes initially not requiring insulin treatment, and at least one affected first degree relative diagnosed before the age of 40. Glucokinase gene mutations had been screened for and ruled out in this cohort in a previous study [16]. Five families showed a typical MODY inheritance pattern. The clinical characteristics of the probands from the 23 families studied are shown in Table 1; the family structures and phenotypic profile of the probands were described elsewhere [16]. Islet cell antibodies were positive in 3 cases who fulfilled the inclusion criteria and had pedigrees compatible with autosomal dominant inheritance. Family history was positive for diabetes on both the paternal and maternal lines in 18 cases, while an autosomal dominant transmission pattern was evident through one of the parental lines in only 5 cases. Moreover, 60.9% of the probands were overweight (n=10; body mass index (BMI) ranging 25.6-28.5 kg/m2) or obese (n=4; BMI greater than 30 kg/m2), while the BMI was less than 25 kg/m2 in the remaining 9 cases.

DNA Extraction and PCR Amplification

Genomic DNA was obtained from peripheral blood or lymphocyte cell lines using the proteinase-K, phenol-chloroform extraction method as described [16]. PCR primers used for amplification have been previously reported [5, 6, 7, 8, 9, 10]. The analysis included the exons and intron-exon boundaries of the HNF-4alpha, HNF-1alpha, IPF-1, HNF-1beta and NEUROD1 genes. Minimal promoter regions (500 bp) of the HNF-4alpha and HNF-1alpha genes [17] as well as the alternative promoter P2 of HNF- 4alpha were also screened [18]. PCR amplifications were carried out using 50-100 ng of DNA, 10 pmoles of each primer, 0.1 μCi of alpha32PdCTP (Amersham Biosciences, Buckinghamshire, England); specific activity: 3,000 Ci/mmol), 1 mM dNTPs, 1.5 mM MgCl2.

PCR-SSCP and Automated Sequencing

SSCP analysis was performed as described [16]. Samples were run in 6% polyacrylamide gels in the presence or absence of 10% glycerol at room temperature at 8 watts for 12 h. Control samples were analyzed simultaneously. PCR fragments were sequenced using the 3100 ABI PRISM Genetic Sequence Analyzer following the manufacturer’s recommendations (Applied Biosystems, Foster City, CA, USA).

Transactivation Assay

RINm5f and HeLa cell lines were grown in RPMI containing 10% fetal bovine serum, 200 mM L-glutamine and 1X non-essential aminoacids to approximately 80% confluency. 1.5 x 105 cells were transfected using Lipofectamine 2000 Reagent (Invitrogen, Carlsbad, CA, USA) with 50 ng of HNF1alpha-cDNA3.1 wild type, mutant or the empty vector, together with 0.5 μg human insulin promoter/pGL3-basic luciferase vector and 10 ng of pRL-TK internal control vector (Promega, Madison, WI, USA). The transcriptional activity was measured 48 hours later using the Dual Luciferase Assay System (Promega, Madison, WI, USA) in a LumicountTM luminometer (Packard, Meriden, Connecticut, USA). Transactivation results were derived from two independent experiments, each carried out in sixtuplicate. The results were normalized for transfection efficiency and by subtracting the value of the empty vector.

Preparation of Nuclear Extracts and Western Blot Analysis

RINm5f and HeLa cells were transfected with 300 ng of the wild type or mutant HNF1alpha-pcDNA 3.1 construct. After 48 h, the cells were lysed in a buffer containing 50 mM Tris-HCL (pH 8), 150 mmol/L NaCl, 1% Nonidet P-40 (NP-40), 0.5% deoxycolate, 1 mmol/L sodium orthovanadate, 15 μg/mL aprotinin and 1 mmol/L phenylmethylsulphonyl fluoride. Cell lysate protein concentrations were measured using a protein assay kit (BioRad, Hercules, CA, USA). Samples (30 μg protein) were run in 10% SDS-PAGE and transferred to a Hybond-PTM (Amersham Biosciences, Buckinghamshire, England) membrane. The membrane was blocked with a solution containing 5% milk powder, 0.05% Tween-20TM (BioRad, Hercules, CA, USA) in PBS and incubated in a 1:500 dilution of anti-HNF-1alpha (Geneka Biotechnology, Montreal, Quebec, Canada) for 1.5 h, followed by incubation in a 1:10,000 dilution of horseradish peroxidase (HRP) goat anti-rabbit IgG as a secondary antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA). The membrane was developed using the ECL quimioluminesence kit (Amersham Biosciences, Buckinghamshire, England) following the manufacturer’s recommendations.

ETHICS

Informed consent was obtained from all participants through their attending physicians. The protocol was approved by the Ethics Committee in each of the referral Institutions.

STATISTICS

Data are expressed as mean±standard deviation and frequencies, and they are compared by means of the Student’s t and the Fisher's exact tests. Two-tailed P values less than 0.05 are considered statistically significant.

RESULTS

No HNF-4alpha, IPF-1, NEUROD1 or HNF- 1beta gene mutations were identified in any of the families analyzed. Sequence polymorphisms found in the HNF-4alpha, HNF-1alpha and IPF-1 genes are shown in Table 2.

A previously unreported mutation (P379H) in exon 6 of the HNF-1alpha gene was identified in one MODY family (Figure 1). The mutation was found in a heterozygous form in the proband, the affected father and a paternal great-aunt, but not in the unaffected siblings or mother. The proband was diagnosed with diabetes at age 13, and is currently on oral antihyperglycemic therapy (glibenclamide/ metformin) and a diet/exercise regime. On diagnosis, her BMI was 27.3 kg/m2, but is currently normal (22.9 kg/m2). She and the other affected relatives have not developed ketoacidosis. Her father was diagnosed at age 38; his weight is normal (BMI is 23.0 kg/m2) and he is on oral antihyperglycemic therapy. Both the proband and her father are free of chronic complications 5 and 12 years after diagnosis, respectively, although the paternal great-aunt has developed hypertension. The proband was negative for anti-beta cell autoantibodies.

Table 1 displays the clinical characteristics of the probands of MODY X, early-onset diabetes and the P379H mutation families. A higher proportion of early-onset diabetes patients required insulin treatment (P=0.046) and had suffered ketoacidosis (although not significant: P=0.554) as compared to the MODY families. Age at diagnosis, BMI and glycemia at diagnosis and after treatment were not significantly different between MODY X and early-onset diabetes patients. RINm5f and HeLa cell transfection studies revealed that the transactivation activity of the HNF-1alpha P379H mutant protein on the human insulin promoter was lower than that of the wild-type protein (38 and 42% respectively) (Figure 2 A and B). This decrease was not related to the reduced expression of the mutant protein, as ascertained by Western blot analysis of nuclear extracts (Figure 2C). Among the 11 polymorphisms identified (4 intronic, 5 synonymous and 2 nonsynonymous), four are new: A58A in HNF- 4alpha, L54L in IPF-1, and P394P and IVS6nt+27C>T in HNF-1alpha. Polymorphisms L254M, G288G, L459L, S487N , IVS6nt-2A>G and IVS7nt+7G>A in HNF-1alpha, and IVS2nt-4C>T in HNF- 4alpha were reported previously [19]. Interestingly, three unrelated patients showed the same 3 polymorphisms in exon and intron 7 of the HNF-1alpha gene (L459L, S487N and IVS7nt+7G>A). Genotype analysis of the available relatives showed that the haplotype was carried as a block in a single allele.

DISCUSSION

We evaluated the contribution of the MODY genes to the etiology of early-onset diabetes in twenty-three unrelated Mexican families (including at least 5 with autosomal dominant inheritance) recruited from three different major referral centers. The Mexican population can thus be included among the population groups assessed for the contribution of MODY genes in early-onset type 2 diabetes.

Mutations in known MODY genes were not found in 4 of the five MODY families studied. Only one with apparent autosomal dominant inheritance carried a new P379H HNF-1alpha gene mutation. This finding is consistent with the observed frequency of HNF-1alpha mutations in autosomal dominant early-onset T2DM families or single affected carriers from other ethnic groups [17, 19, 20, 21, 22, 23, 24, 25]. The mutation lies within exon 6, encoding the transactivation domain of the protein. Codon 379 is probably a mutational hotspot of the gene, since other mutations have been reported at this site in several families [19]. However, all previously reported mutations at this position were either deletions and/or insertions which resulted in truncated HNF- 1alpha forms (i.e., P379fsdelCT).

In vitro expression studies demonstrated that the transactivation potential of the recombinant P379H mutant protein on the human insulin promoter is reduced both in HeLa and in the pancreatic RINm5f cell lines. Cosegregation of the mutant allele and diabetes in this family is consistent with the results of the in vitro expression studies. Moreover, the clinical phenotype is similar to that reported for other HNF-1alpha mutation patients [20, 26].

The four putative MODY families in which no mutations were identified, are likely a valuable source in identifying additional MODY genes. In this regard, a recent whole genome scan for MODY X reported linkage to chromosomes 3, 5, 6 and 10 in European families [27] and to 8p23 and 2q37 in U. S. families [28], providing further evidence for the high genetic heterogeneity underlying MODY etiology.

An alternative but less likely explanation for the low mutation rate found is the low sensitivity of PCR-SSCP to detect sequence variations [29, 30, 31]. In this regard, even though intronic and 5' regulatory sequence changes may have been missed with the designed method, the number of both new and previously reported polymorphisms identified, particularly in the HNF-1alpha gene, is noteworthy.

Among the 11 MODY gene polymorphisms identified during the course of the study, of particular interest were L459L, S487N and IVS7nt+7G>A in exon and intron 7 of the HNF-1alpha gene since they segregated as a haplotype in three unrelated probands. Whether this haplotype is in fact associated with diabetes risk in our population remains to be determined.

In summary, this is the first report in which all known MODY genes were screened for sequence variations in an early-onset diabetes Latin American cohort. We identified a new HNF-1alpha mutation (P379H) in a MODY family, and proved that it alters the transactivation potential of the mutant protein on the human insulin promoter. Our results are consistent with findings in Brazilian MODY families who show a high proportion of relatives without HNF-4alpha, HNF-1alpha or glucokinase gene mutations [32], and with a recent study failing to find HNF-4alpha, HNF-1alpha or glucokinase gene mutations in Mexican-American early-onset diabetes families [33]. This suggests that there are still unidentified genes in the majority of MODY families in Latin American populations. Moreover, the thorough analysis of all known MODY genes led to the identification of a putative HNF-1alpha risk haplotype in this cohort, while HNF-4alpha, glucokinase (GCK), IPF-1, HNF-1beta and NEUROD1 apparently fail to contribute to the etiology of early-onset diabetes in Mexican families, providing further support for the high heterogeneity of this disease.

Acknowledgements

We thank Dr. Doris Stoffers for providing the insulin promoter/pGL3 luciferase vector. This study was supported by grant IN-207493 of the Dirección General de Asuntos del Personal Académico, Universidad Nacional Autónoma de México and by the Consorcio Promotor del INMEGEN (Instituto Nacional de Medicina Genómica), México. ADL, VFO and SCQ were supported through a fellowship from CONACyT (Consejo Nacional de Ciencia y Tecnología, México).

References

1. Zimmet P, Alberti KG, Shaw J. Global and social implications of the diabetic epidemic. Nature 2001; 414:782-7. [PMID 11742409]
2. Aguilar-Salinas CA, Rojas RF, Gómez-Pérez J, García E, Valles V, Ríos-Torres JM, et al. Prevalence and characteristic of early-onset type 2 diabetes in Mexico. Am J Med 2002; 113:569-74. [PMID 12459403]
3. Olaiz G, Rojas R, Barquera S, Shamah T, Aguilar C, Cravioto P, López P, Hernández M, Tapia R, Sepúlveda J. Encuesta Nacional de Salud 2000. Tomo 2. La Salud de los adultos.Cuernavaca Morelos, México Instituto Nacional de Salud Pública.
4. Aguilar-Salinas CA, Velazquez Monroy O, Gomez-Perez FJ, Gonzalez Chavez A, Esqueda AL, Molina Cuevas V, et al. Characteristics of the patients with type 2 diabetes in Mexico: results from a large population-based, nation wide survey. Diabetes Care 2003; 26:2021-6. [PMID 12832306]
5. Froguel P, Zouali H, Vionnet N, Velho G, Vaxillaire M, Sun F, et al. Familial hyperglycemia due to mutations in glucokinase: definition of a subtype of diabetes mellitus. N Engl J Med1993; 328:697-702. [PMID 8433729]
6. Yamagata K, Furuta H, Oda N, Kaisaki PJ, Menzel S, Cox NJ, et al. Mutations in the hepatocyte nuclear factor-4 alpha gene in maturity-onset diabetes of the young (MODY1). Nature 1996; 384:458-60. [PMID 8945471]
7. Yamagata K, Oda N, Kaisaki PJ, Menzel S, Furuta H, Vaxillaire H, et al. Mutations in the hepatocyte nuclear factor-1 alpha gene in maturity-onset diabetes of the young (MODY3). Nature 1996; 384:455-8. [PMID 8945470]
8. Stoffers DA, Ferrer J, Clarke WL, Habener HL. Early-onset type- II diabetes (MODY4) linked to IPF- 1. Nat Genet 1997; 17:138-9. [PMID 9326926]
9. Horikawa Y, Iwasaki N, Hara M, Furuta H, Hinokio Y, Cockburn BN, et al. Mutations in the hepatocyte nuclear factor-1 beta gene (TCF2) associated with MODY. Nat Genet 1997; 17:384-5. [PMID 9398836]
10. Malecki MT, Jhala US, Antonellis A, Fields L, Doria A, Orban T, et al. Mutations in NEUROD1 are associated with the development of type 2 diabetes mellitus. Nat Genet 1999; 23:323-8. [PMID 10545951]
11. Velho G, Robert JJ. Maturity-Onset Diabetes of the Young (MODY): Genetic and Clinical Characteristics. Horm Res 2002; 57:29-33. [PMID 11979019]
12. Triggs-Raine BL, Kirkpatrick RD, Kelly SL, Norquay LD, Cattini PA, Yamagata K, et al. HNF- 1alpha G319S, a transactivation-deficient mutant, is associated with altered dynamics of diabetes onset in an Oji-Cree community. Proc Natl Acad Sci USA 2002; 99:4614-9 [PMID 11904371]
13. Hani EH, Suaud L, Boutin P, Chevre JC, Durand E, Philippi A, et al. A missense mutation in hepatocyte nuclear factor-4 alpha, resulting in a reduced transactivation activity, in human late-onset noninsulin- dependent diabetes mellitus. J Clin Invest 1998; 101:521-6. [PMID 9449683]
14. Sakurai K, Seki N, Fujii R, Yagui K, Tokuyama Y, Shimada F, et al. Mutations in the hepatocyte nuclear factor-4alpha gene in Japanese with non-insulindependent diabetes: a nucleotide substitution in the polypyrimidine tract of intron 1b. Horm Metab Res 2000; 32:316-20. [PMID 10983627]
15. Macfarlane WM, Frayling TM, Ellard S, Evans JC, Allen LI, Bulman MP, et al. Missense mutations in the insulin promoter factor-1 gene predispose to type 2 diabetes. J Clin Invest 1999; 104:R33-9. [PMID 10545530]
16. Del Bosque-Plata L, García-García E, Ramírez- Jiménez S, Cabello-Villegas J, Riba L, Gomez-León A. et al. Analysis of the glucokinase gene in Mexican families displaying early-onset non-insulin dependent diabetes mellitus including MODY families. Am J Med Genet 1997; 72:387-93. [PMID 9375718]
17. Lehto M, Wipemo C, Ivarsson SA, Linfgren C, Lipsanen-Nyman M, Weng J. et al. High frequency of mutations in MODY and mitochondrial genes in Scandinavian patients with familial early-onset diabetes. Diabetologia 1999; 42:1131-7. [PMID 10447526]
18. Thomas H, Jaschkowitz K, Bulman M, Frayling TM, Mitchell SMS, Roosen S, et al A distant upstream promoter of the HNF-4alpha gene connects the transcription factors involved in maturity-onset diabetes of the young. Hum Mol Genet 2001; 10:2089- 97. [PMID 11590126]
19. Ellard S. Hepatocyte nuclear factor 1 alpha (HNF- 1 alpha) mutations in maturity-onset diabetes of the young. Hum Mut 2000; 16:377-85. [PMID 11058894]
20. Shih DQ, Stoffel M. Molecular etiologies of MODY and other early-onset forms of diabetes. Curr Diab Rep 2002; 2:125-34. [PMID 12643132]
21. Frayling TM, Evans JC, Bulman MP, Pearson E, Allen L, Owen K, et al. Beta-cell genes and diabetes: molecular and clinical characterization of mutations in transcription factors. Diabetes 2001; 50:S94. [PMID 11272211]
22. Barrio R, Bellanne-Chantelot C, Moreno JC, Morel V, Calle H, Alonso M, Mustieles C. Nine novel mutations in Maturity-Onset Diabetes of the Young (MODY) Candidate genes in 22 Spanish families. J Clin Endo Metab 2002; 87:2532-9. [PMID 12050210]
23. Pruhova S, Ek J, Lebl J, Sumnik Z, Saudek F, Andel M, et al. Genetic epidemiology of MODY in the Czech republic: new mutations in the MODY genes HNF-4 alpha, GCK and HNF-1alpha. Diabetologia 2003; 46:291-5. [PMID 12627330]
24. Tonooka N, Tomura H, Takahashi Y, Onigata K, Kikuchi N, Horikawa Y, et al. High frequency of mutations in the HNF-1alpha gene in non-obese patients with diabetes of youth in Japanese and identification of a case of digenic inheritance. Diabetologia 2002; 45:1709-12. [PMID 12488961]
25. Bjorkhaug L, Sagen JV, Thorsby P, Sovik O, Molven A, Njolstad PR Hepatocyte nuclear factor-1 alpha gene mutations and diabetes in Norway. J Clin Endocrinol Metab 2003; 88:920-31. [PMID 12574234]
26. Velho G, Froguel P. Genetic, metabolic and clinical characteristics of maturity onset diabetes of the young. EurJ Endocrinol 1998; 138:233-9. [PMID 9539292]
27. Frayling TM, Lingren CM, Chevre JC, Menzel S, Wishart M, Benmerzroua Y, et al. A genome-wide scan in families with maturity-onset diabetes of the young. Diabetes 2003; 52:872-81. [PMID 12606533]
28. Kim SH, Ma X, Weremowicz S, Ercolino T, Powers C, Mlynarski W, et al. Identification of a locus for maturity-onset diabetes of the young on chromosome 8p23. Diabetes 2004; 53:1375-84. [PMID 15111509]
29. Hayashi K, Yandell DW. How sensitive is PCRSSCP? Hum Mutat 1993; 2:338-46. [PMID 8257985]
30. Bunn CF, Lintott CJ, Scott RS, George PM. Comparison of SSCP and DHPLC for the detection of LDLR mutations in a New Zealand cohort. Hum Mutat 2002, 19:311-3. [PMID 11857755]
31. Fan E, Levin DB, Glickman BW, Logan DM. Limitations in the use of SSCP analysis Mutat Res 1993; 283:85-92 . [PMID 7686269]
32. Moises SR, Reis AF, Morel V. Prevalence of Maturity-onset diabetes of the young mutations in Brazilian families with autosomal dominant early-onset type 2 diabetes. Diabetes Care 2001; 24:786-7. [PMID 11315851]
33. Allayee H, Parker D, Stammreich A, Canales J, Scheel G, Lusis A, Ipp E. Mutations in the genes for MODY are not a common cause of early-onset type 2 diabetes in minority populations. American Society of Human Genetics, Annual Meeting 2004 Abstract 2177.