The role of Hemochromatosis C282Y and H63D mutations in the development of type 2 diabetes mellitus in Greece

INTRODUCTION

Hereditary Hemochromatosis (HH) is an autosomal recessive disorder characterized by excessive intestinal iron absorption that leads to iron overload of parenchymal cells in many organs, including the liver and the pancreas and several mutations of the HFE gene (C282Y and H63D) have been linked to HH^1-6. It seems that homozygosity for the C282Y mutation of the HFE and compound heterozygosity C282Y/H63D can cause HH, but the prevalence of the above mutations varies significantly in different ethnic groups^1-6. Several studies have tried to test the hypothesis that heterozygosity for hereditary hemochromatosis-causing mutations could be a risk factor for the development of diabetes Mellitus type 2 (DM2). There are studies in favor of such an association and others that disprove it^7-13. Although the penetrance of the C282Y mutation has recently been reported to be low¹⁴, heterozygosity might affect the iron load of the body^15-16. As expected, Iron overload can negatively affect insulin secretion as well as insulin sensitivity^17-19. There are indications that patients with DM2 are iron overloaded^20-22 and blood letting in high ferritin type 2 diabetic patients improves insulin sensitivity and beta cell function^23-24. The prevalence of hereditary hemochromatosis in patients with type 2 diabetes varies significantly in various populations^25-26. The aim of our study was to determine the prevalence of the C282Y and H63D mutations in the general Greek population and in patients with type 2 diabetes and to examine if the aforementioned mutations contribute to the iron load of the body in patients with type 2 diabetes in the Hellenic population.

METHODS

Study design

Part 1: 100 unselected patients with type 2 diabetes and 100 non-diabetics matched for age, sex and BMI were genotyped for C282Y and H63D mutations of the HFE gene. Participants were recruited from the diabetes center and the endocrine clinic of the university hospital of Patras.

Part 2: 587 outpatients with type 2 diabetes attending the diabetes center were screened for hemochromatosis by measuring iron and total iron binding capacity (TIBC) in the fasting state. 502 non dialetic outpatients attending the endocrine clinic for goiter served as the control group. Subjects with transferrin saturation more than 38% were genotyped for C282Y and H63D mutations. All participants were tested for hemoglobin, hematocrit, mcv, ferritin, aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, urea , and creatinine. Inclusion criteria for the diagnosis of diabetes were either fasting plasma glucose concentration greater than 126 mg/dl or plasma glucose concentration greater than 200 mg/dl at 2 hours in an oral glucose tolerance test.

Exclusion criteria for participation in the study were anemia, renal failure or any known inflammatory disease, which might have affected the iron metab
olism, and alcohol consumption of more than 2-3 glasses of wine daily. Using these criteria, 87 diabetics and 79 controls were excluded from the study. The remaining 500 diabetics and 423 controls were enrolled.

Laboratory methods

Serum iron, total iron binding capacity (TIBC), ferritin, aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, urea and creatinine were measured with routine hospital laboratory assays from fasting samples. Transferrin saturation index was calculated according to the formula Ironx100/TIBC.

DNA was extracted from whole blood using Nucleospin Blood QuickPure kit (Machery-Nagel). Oligonucleotides were constructed according to published DNA sequence (GenBank U630319): forward primer 5'-GGAGTTCGAACCTAAAGACGT-3' and reverse primer 5'-AGGGCTCCCAGATCACAATG-3' for the C282Y mutation, and forward primer 5'-tcagagcaggaccttggtctt-3' and reverse primer 5'-actctgactcagctgcagcca-3' for the h63d mutation. 100 ng of genomic DNA was used as template in a PTC-200 thermocycler. PCR was performed in a total volume of 50 ml, using 10 pmol of each appropriate primer, 200 mM of each dNTP, 1.5 mM MgCl, 2.5 Units of Taq DNA polymerase and 10 x reaction buffer. After an initial denaturing at 95°C for 5 min, reactions were subjected to 30 cycles of 45 sec denaturing at 95°C, 45 sec annealing at 60°C and 45 sec extension at 72°C, followed by a final extension at 72°C for 5 minutes.

The two fragments (one for each mutation) were amplified by polymerase chain reaction (PCR), then purified from the gel and subsequently digested with RsaI (C282Y) and DpnII. (H63D), respectively. Their products were analyzed on 2% agarose gel.

Statistical analysis

SPPSS 10 was used for the statistical analyses. Student's t test and Pearson's x 2 test were used and p values less than 0.05 were considered significant.

RESULTS

1. Prevalence of C282Y and H63D among patients with type 2 diabetes and controls

In part 1 of the study, the prevalence of the above mutations in the general population and in patients with DM2 were determined. The two groups were matched for age, sex, and BMI, and were unselected for transferrin saturation. The characteristics of these two groups as well as the frequencies of the two mutations are shown in table 1 .The frequencies of the two mutations in diabetes type 2 group did not differ significantly from those in the general population (controls). Pooled data from the two groups were used to calculate the allele frequency for the two mutations. For the C282Y allele, 3 mutations were found out of 400 chromosomes tested and the allele frequency was estimated to be 3/400=0,0075. For the H63D allele, 46 mutations were found out of 400 chromosomes tested and the allele frequency was estimated to be 46/400=0,115.

2. Iron indices among type 2 diabetics

In part two of the study, 587 diabetes type 2 patients and 502 controls were screened for transferrin saturation, hemoglobin, hematocrit, MCV, aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, blood urea and creatinine. From this pool, 87 diabetics and 79 controls were excluded from the study because they did not meet the inclusion criteria. The remaining 500 diabetics and 423 controls were enrolled. All of them had a repeat measurement of fasting transferrin saturation as well as serum ferritin determination.

The age, sex and BMI as well as the transferring saturation and the serum ferritin values in patients and controls are shown in table 2 . Patients and controls were comparable in terms of age and BMI. Among men, the diabetics had higher transferrin saturation (31,8±11 vs 29,5±8, p<0,05) compared to controls and their ferritin values were also significantly higher compared to controls (159±80 vs 87±51, p<0,001). Among women, the differences in terms of transferrin saturation were more striking. Women with type 2 diabetes had mean transferrin saturation 28,5±10 compared to 25,5±9,6 of the control group, (p value <0,001) and their ferritin values were also significantly higher compared to controls (87±60 vs 57±52, p<0,01). 

The allocation of people according to transferrin saturation is presented in tables 4 and 5 . At every level of transferrin saturation, the percentage of diabetics was higher compared to controls. For both males and females, the percentage of patients with diabetes type 2 and transferrin saturation more than 38% was significantly greater than that in the controls. 

HFE mutations, transferrin saturation and Ferritin values (Table 6 )

Subjects with even one mutation(C282Y or H63D) had higher transferrin saturation compared to the those with no such mutations. This seems to apply to both diabetics (49± 8,6 vs 44,5± 5,4, p<0,01) and controls (49,3± 7,3 vs 42,6± 3,3 p<0,01). Ferritin was higher in control subjects carrying a mutation compared to those with no such mutation (99± 43 vs 68± 29, p<0,01). However, no such difference was detected among diabetics.

Hereditary hemochromatosis among patients with type 2 diabetes

The frequency of the C282Y allele was very low in the Greek population tested (0.0075) and homozygotes (C282Y/C282Y) were not detected in either group. We found 4 compound heterozygotes C282Y/H63D in the diabetics and none in the control group. This difference did not not reach statistical significance (p=0.06).

DISCUSSION

The allele frequency for the C282Y mutation in the Hellenic population was much lower (0.0075) than the one reported in people of Celtic origin³. Analogous findings to our study were reported from southern Europe⁴. The allele frequency for the H63D mutation (0.115) was comparable to the one in other populations^3,4. In our subjects, Homozygotes for hemochromatosis mutations were not detected, possibly because of the low frequency of C282Y mutation in our group. Based on these data, we cannot draw conclusions as to whether hereditary hemochromatosis is more prevalent among patients with diabetes type 2 in our population. Mutations of the transferrin receptor and the ferroportin 1 have recently been reported^5-6 in Italian people. These mutations may account for at least a fraction of HH patients in whom the C282Y/C282Y or C282Y/H63D genotypes are not detected. We assume that the Greek population is genetically closer to the southern Italian population in terms of hereditary hemochromatosis causing mutations, and most likely a greater percentage of HH patients carry mutations other than the C282Y and H63D.

We did not detect any differences in the frequencies of C282Y or H63D alleles between type 2 diabetics and controls. This finding does not support the view that these mutations are in linkage disequilibrium for diabetes type 2. The discordance between our findings and those of other studies might be attributed to the different genetic background of the tested subjects.

It seems that the presence of these mutations even in the heterozygous state increases the iron load of the organism.^27-28 The iron load could render peripheral tissues, and especially the liver, less sensitive to the action of insulin and consequently speed up the progression of the natural course of diabetes.

The mean serum ferritin level was found to be higher in patients with diabetes type 2 in accordance with other studies. This finding could either represent an elevated iron body stores in diabetes type 2 or simply reflect inflammation without true iron load, or else reflect a decreased clearance of glycosylated ferritin. Patients with diabetes type 2 seem to have higher transferrin saturation compared to the general population. This difference is even greater in women. The increased ferritin level is seen in the group of type 2 diabetes as a whole, as well as in the subgroup of people with transferrin saturation more than 38%. The increased serum ferritin combined with the increased transferrin saturation could represent increased iron load with a potential effect on the hepatocyte and the beta cell of the pancreas. Increased iron load of the liver could lead to resistance to the action of insulin and hyperinsulinemia. There are very few studies focused on this question so that a conclusive answer as to whether diabetics type 2 have increased iron deposition at the hepatocyte²⁹ cannot be offered. In a recent study, we found that the iron load was significantly increased in offspring of type 2 diabetic subjects and was proportional to the insulin resistance index (unpublished observations). In conclusion, the allele frequency for the C282Y mutation was found lower (0.0075) in the Greek population than that reported in people of Celtic origin. The allele frequency for the H63D mutation (0.115) is comparable to other populations. Patients with diabetes type 2 have higher serum ferritin values as well as transferring saturation compared to the general population. The presence of either C282Y or H63D mutations contribute to the iron load of the organism. No difference in the frequency of C282Y and H63D alleles between patients with diabetes type 2 and the general population was detected in Greece.

REFERENCES
1. Feder JN, Thomas W, Tsuchihashi Z, et al, 1996 novel MHC class I-like gene is mutated in patients with hereditary haemochromatosis. Nat Genet 13: 399-408.
2. Beutler E, Gelbart T, West C, et al, 1996 Chorney M. Mutation analysis in hereditary hemochromatosis. Blood Cells Mol Dis 22: 187-194.
3. The UK Haemochromatosis Consortium, 1997 A simple genetic test identifies 90% of UK patients with haemochromatosis. Gut 41: 841-844.
4. Carella M, D'Abrosio L, Totaro A, et al, 1997 Mutation analysis of the HLA-H gene in Italian hemochromatosis patients. Am J Hum Genet 60: 828-832.
5. Camaschella C, Gasparini P, 2000 The gene TFR2 is mutated in a new type of haemochromatosis mapping to 7q22. Nature genetics 25: 14-15.
6. Pietrangelo A, Montosi G, Torato A, et al, 1999 Hereditary hemochromatosiss in adults without pathogenic mutations in the hemochromatosis gene. N Engl J Med 341: 725-732.
7. Kwan T, Leber B, Ahuja S, Carter R, Gerstein HC, 1998 Patients with type 2 diabetes have a high frequency of the C282Y mutation of the hemochromatosis gene. Clin Invest Med 21: 251-257.
8. Moczulski DK, Grzeszczak W, Gawlik B, 2001 Role of hemochromatosis C282Y and H63D mutations in HFE gene in development of type 2 diabetes and diabetic nephropathy. Diabetes Care 24: 1187-1191.
9. Frayling T, Ellard S, Grove J, Walker M, Hattersley AT, 1998 C282Y mutation in HFE gene and type II diabetes. Lancet 351: 1933-34.
10. Behn PS, Glaser B, Permutt A, 1998 Contribution of the C282Y hereditary hemochromatosis mutation to type II diabetes mellitus in ashkenzai Jews. Diabetes 47: 1523.
11. Braun J, Donner H, Plock K, Rau H, Usadel KH, Baden-hoop K, 1998 Hereditary haemochromatosis mutations (HFE) in patients with type II diabetes mellitus. Diabetelogia 41: 983-84.
12. Sampson MJ, Williams T, Heyburn PJ, et al, 2000 Prevalence of HFE mutations in unselected male patients with type 2 diabetes. J Lab Clin Med 135: 170-173.
13. Dubois-Laforgue D, Caillat-Zucman S, Djilali-Saiah I, et al, 1998 Mutations in HFE, the hemochromatosis candidate gene, in patients with NIDDM. Diabetes Care 21: 1371-1372.
14. Beutler E, Felitti VJ, Koziol JA, Ho NJ, Gelbart T, 2002 Penetrance of 845G®A (C282Y) HFE hereditary haemochromatosis mutation in the USA. Lancet 359: 211-218.
15. Dubois-Laforgue D, Caillat-Zucman S, Boitard C, Timsit J, 2000 Clinical characteristics of type 2 diabetes in patients with mutations of HFE. Diabetes Metab 26: 65-68.
16. Bulaj ZJ, Griffen LM, Jorde LB, Edwards CQ, Kushner JP, 1996 Clinical and biochemical abnormalities in people heterozygous for hemochromatosis. N Engl J Med 335: 1799-805.
17. Niederau C, Berger M, Stremmel W, et al, 1984 Hyperinsulinaemia in non-cirrhotic haemochromatosis: impaired hepatic insulin degradation? Diabetologia 26: 441-444.
18. Merkel PA, Simonson DC, Amiel SA, et al, 1988 Insulin resistance and hyperinsulinemia in patients with thalassemia major treated by hypertransfusion. N Engl J Med 318: 809-814.
19. Hramiak IM, Finegood DT, Adams PC, 1997 Factors affecting glucose tolerance in hereditary hemochromatosis. Clin Invest Med 20:110-118.
20. Fernandez-Real JM, Ricart-Engel W, Arroyo E, et al, 1998 Serum ferritin as a component of the insulin resistance syndrome. Diabetes Care 21: 62-68.
21. Ford ES, Cogswell ME, 1999 Diabetes and serum ferritin concentration among U.S. adults. Diabetes Care 22: 1978-1983.
22. Kaye TB, Guay AT, 1994 Increased serum ferritin levels in patients with diabetes mellitus. Mayo Clin Proc 69: 498-499.
23. Facchini FS, Hua NW, Stoohs RA, 2002 Effect of iron depletion in carbohydrate-intolerant patients with clinical evidence of nonalcoholic fatty liver disease. Gastroenterology 122: 931-939.
24. Fernandez-Real JM, Penarroja G, Castro A, Garcia-Bragado F, Hernandez-Aguado I, Ricart W, 2002 Blood letting in high-ferritin type 2 diabetes: effects on insulin sensitivity and beta-cell function. Diabetes 51: 1000-1004.
25. Conte D, Manachino D, Colli A, 1998 Prevalence of genetic haemochromatosis in a cohort of Italian patients with diabetes mellitus. Ann Intern Med 128: 370-373.
26. Turnbull AJ, Mitchison HC, Peaston RT, et al, 1997 The prevalence of haemochromatosis in a diabetic population 7. Q J Med 90: 271-275.
27. Jackson HA, Carter K, Darke C, et al, 2001 HFE mutations, iron deficiency and overload in 10,500 blood donors. Br J Haematol 114: 474-484.
28. Van Lerberghe S, Hermans MP, Dahan K, Buysschaert M, 2002 Clinical expression and insulin sensitivity in type 2 diabetic patients with heterozygous mutations for haemochromatosis. Diabetes Metab 28: 33-38.
29. Turlin B, Mendler MH, Moirand R, Guyader D, Guillygomarc'h A, Deugnier Y, 2001 Histologic features of the liver in insulin resistance-associated iron overload. A study of 139 patients. Am J Clin Pathol 116: 263-270.

Address correspondence and requests for reprints to:
Ioannis G Habeos, Department of Internal Medicine, Division of Endocrinology,
University of Patras, Medical School, Patras, Greece 26500,
Tel/Fax: 2610999690, e-mail: ihabeos@med.upatras.gr

Received 06-12-02, Revised 27-12-02, Accepted 03-01-03