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Endocrine Care |
Program for Population Genetics (P.A.Z., H.T., X.X.), Harvard School of Public Health, Boston, Massachusetts 02115; Genetics Research Laboratory (P.A.Z., H.S., G.H., S.T.A.), Chronic Care Center, Beirut, Lebanon; Department of Endocrinology (G.H.), Hôtel-Dieu de France, Beirut, Lebanon; and Division of Endocrinology (S.T.A.), American University of Beirut, Beirut, Lebanon
Address all correspondence and requests for reprints to: Sami T. Azar, M.D., Department of Endocrinology, American University of Beirut, Medical Center, Bliss Street, Beirut, Lebanon. E-mail: . sazar{at}aub.edu.lb
Abstract
Type-1 diabetes (T1D) is an autoimmune disease leading to insulin deficiency. Its occurrence is influenced by genetic and environmental factors. The human leukocyte antigen (HLA) region on chromosome 6 accounts for 45% of the genetic susceptibility for the disease, mainly the HLA-DQB1*0201 and HLA-DQB1*0302 alleles. Among the environmental factors involved, early exposure to cow’s milk seems to be a trigger. In this study, we investigated the occurrence of T1D in 253 Lebanese Caucasian patients, in relation to HLA-DQB1*0201, HLA-DQB1*0302, HLA-DQB1*0602, gender, and early exposure to cow’s milk, as well as to family history of T1D and type-2 diabetes (T2D). Our genetic analysis results show that in the patients studied, 77% and 40% were positive for BQ1*0201 and BQ1*0302, respectively. As for BQ1*0602, only 0.8% of patients were positive for this T1D protective allele, compared with 24% among the controls. Furthermore, our results did not show any gender preference of the disease or any effects of early intake of cow’s milk on the age at onset of T1D. When family history of T2D or T1D was studied, our results show a novel finding whereby an immediate family history of T2D, but not T1D, delays the age at onset of T1D.
TYPE-1 DIABETES (T1D) is characterized by an autoimmune process that results in the destruction of insulin-producing ß-cells, leading to insulin deficiency [insulin- dependent diabetes mellitus (IDDM)] (1, 2). The age at onset of T1D varies from birth till the age of around 30 yr, with a peak between 2 and 15 yr of age. T1D is one of the most common chronic childhood diseases. Its incidence seems to be increasing in countries around the world and is predicted to be about 40% higher in 2010 than in 1997 (3, 4).
Gender plays a role in both the transmission and the development of diabetes. In a recent review, Gale and Gillespie (5) show evidence for this role. In patients under the age of 15, a male excess was observed in European countries (in which the incidence of T1D is high). The reverse was observed in non-European countries, where the incidence of T1D is lower (6). As for the transmission of T1D from affected patients, it seems that the risk of transmission from a diabetic father is two to three times higher than that of a diabetic mother (7, 8).
The occurrence of T1D is believed to be triggered by the existence of a genetic susceptibility as well as the presence of certain environmental determinants. Genomic studies have confirmed that the main locus defining the genetic susceptibility to T1D is encoded within the major histocompatibility complex-HLA (human leukocyte antigen) region on human chromosome 6 (9, 10).
The HLA class II, which encodes HLA-DR, -DQ, and -DP, accounts for about 45% of the genetic susceptibility for the disease (11). In addition to HLA-DR3 or DR4, found in about 95% of T1D patients (12, 13), T1D susceptibility is associated with DQA1 and DQB1, which are in linkage disequilibrium with DR3 and DR4 (14, 15). DQB1*0201 and DQB1*0302 present a high risk for disease occurrence, whereas DQB1*0602 seems to be protective (16). Other candidate loci associated with this disease have also been proposed on chromosomes 2, 11, and 15 (17, 18, 19, 20). The IDDM12 locus on chromosome 2 (2q33) has been the focus of several studies recently targeting the candidate gene CTLA4 (the cytotoxic T lymphocyte-associated protein 4) that is involved in immunomodulation (19, 21, 22).
Among the environmental risk factors for T1D, three main categories are under investigation: viral infections, such as coxsackie B4 virus and the cytomegalovirus (23); chemicals, such as N-nitroso derivatives (24); and infant diet (containing cow’s milk), in particular the introduction of cow’s milk (25, 26). Coxsackie B4 virus is one of the viruses most associated with T1D. Recent reports show the presence of immune responses to coxsackie virus antigens in T1D children and suggest its implication with T1D autoimmunity, either by direct inflammatory disruption of islets or induction of an immune response (27, 28).
When compared with controls, T1D patients had an earlier introduction to cow’s milk, a shorter duration of exclusive breast-feeding, and higher levels of antibodies to cow’s milk, lactoglobin, and a BSA peptide (29, 30). Moreover, recent studies suggest that exposure to dietary bovine insulin may be the primary event leading to disease onset in genetically susceptible infants and their siblings (31, 32). The majority of the information on T1D is based on studies performed in the United States and Europe. In Lebanese families, we have no available data on the occurrence of T1D in relation to genetic predisposition, gender, family history of both T1D and type-2 diabetes (T2D), and early infant diet on the age at onset of the disease. In the present study, we investigated these factors in T1D Lebanese patients. Our results are in accordance with those generally reported in the literature. However, we report a new finding, where patients with T2D family history have a significantly delayed onset of T1D.
Materials and Methods
Subjects
Two hundred fifty-three Lebanese Caucasian patients at the Chronic Care Center, presenting T1D during the period of January 1993 and May 2001, participated in the current study. The center was created to accommodate T1D patients in Lebanon, and currently 756 T1D patients come for treatment. Those patients enrolled were those who agreed to participate in this study along with other ongoing studies. The ethics committees of the Chronic Care Center and the Harvard School of Public Health approved the study protocol, and an informed written consent was obtained from the participating subjects or their families.
For the diagnosis, the World Health Organization (WHO) definition of T1D was used (33). It was made on the basis of ketoacidosis or ketosis with severe symptoms of acute onset at presentation and continuous dependence on insulin within 6 months of diagnosis. Individuals with maturity-onset diabetes of the young or with Wolfram syndrome were excluded from the study. All patients were under 26 yr of age at presentation. They were 132 females and 121 males. The age at onset of the disease was as follows: 65 patients with age at onset less or equal to 5 yr, 85 patients with age at onset between 5.01 and 10 yr, and 103 patients with age at onset over 10 yr; the overall mean age at onset being 8.9 yr. 90% Of patients with T1D duration of less than 5 yr and 76% of those with T1D duration of more than 5 yr had positive anti-GAD antibodies. A standardized medical history questionnaire was obtained on all the participating patients. Information on the period, in months, during which the patient was breast fed was obtained as well as on the age, in months, at which first exposure to bovine-based dairy products occurred. Concerning family history of diabetes (either T1D or T2D), the patients were requested to provide information about the disease occurrence in their families and were grouped in three categories: those who have no family history, those who have immediate family history, and those with extended family history. It was assumed that the patients or their guardians could correctly identify other family members with T1D or T2D. Further classification was based on questions and documentation concerning the onset age of diabetes in the affected family member and on his or her medication history, to distinguish between the 2 forms of diabetes. T1D was diagnosed if the onset of diabetes was before the age of 30 yr, with ketoacidosis history and dependence on insulin since diagnosis. T2D was diagnosed if the onset of diabetes was after the age of 30 yr, with response to diet and or oral hypoglycemic agents for a period of at least 2 yr from the time of diagnosis. A second reassessment of all family members with diabetes was done at the end of the study, and it was found that all the subjects reported to have T2D were found to remain on diet or oral hypoglycemic agents, and all subjects reported to have T1D were found to remain on insulin therapy.
One hundred healthy individuals were chosen as controls (males and females) and were selected from the Lebanese population. All control individuals had negative anti-GAD antibodies and were above 25 yr of age, as recommended by the DiaMond protocol (34) and had passed through the high-risk period (0–15 yr of age) during which time T1D is most likely to develop.
HLA genotyping
Sample preparation and DNA extraction. Six milliliters of blood was collected in EDTA-containing tubes after a written consent was read and signed by each individual and (in the case of a child) his or her guardian. For this study, 0.5 ml of each sample was used for DNA extraction. DNA was extracted with a QIAamp Blood Kit (QIAGEN, Hilden, Germany) according to the blood and body fluid protocol recommended by the manufacturer. The DNA was eluted in 50 µl Tris-EDTA buffer (20 mM Tris, 2 mM EDTA) and stored at -20 C until use.
PCR amplification and analysis. The DNA samples were amplified with HLA-DQB1*0201, HLA-DQB1*0302, and HLA-DQB1*0602 sequence-specific primers (35, 36). Each sample was also amplified with a primer pair that detects the third intron of the DRB1 gene. These primers target a conserved region of the gene and thus were used as internal positive amplification controls. The amplification was performed in a total vol of 20 µl, and each PCR mixture consisted of 60 ng genomic DNA, 2 mM MgCl2, PCR buffer [50 mM KCl, 10 mM Tris (pH 8.3), 0.001% (wt/vol gelatin)], 200 µM of each deoxynucleotide triphosphate, 0.02 mM of either the allele-specific or control primers and 0.175 U AmpliTaq Gold (PE Applied Biosystems, Foster City, CA). Absence or presence of PCR products was detected by agarose gel electrophoresis. Five microliters of each amplification product was loaded in the presence of a loading dye on 2% agarose gels and subjected to electrophoresis. After 15 min, the gels were stained in a solution of 0.5 µg/ml ethidium bromide and then examined under UV illumination and documented by photography.
Statistical analysis
Differences between the groups were analyzed by the Student’s t test or ANOVA for multiple comparisons. For binomial comparisons, the binomial test was used. A P value less than 0.05 was considered significant.
Results
Effect of gender on the age at onset of T1D
Among the 253 patients, 121 were males with a mean onset age of 9.30 yr, and 132 were females with a mean onset age of 8.59 yr. When compared, no significant differences were observed between the mean age at onset of disease occurrence of male and female patients (Table 1
).
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Patients were divided into 2 groups according to the initial age of dairy intake as being less or equal to 3 months (n = 140) or more than 3 months (n = 108). Dairy intake information for 5 of the patients included in the study was not available. Our results show that no effect of the early introduction (
3 months) of dairy products in infant diet on the age at onset of T1D was observed (9.25 yr vs. 8.44 yr, P = 0.16; Table 2). Of the 140 patients introduced to dairy products at 3 months or earlier, only 29 were not being breast fed, with an onset age of 9.81 yr and similar to that of breast fed patients of the same group (9.11 yr; P = 0.47; Table 2). The duration of breast-feeding had no effect on the onset age of T1D occurrence in the patients.
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The analysis of the 253 T1D patients and the 100 control subjects for the occurrence of HLA-DQB1*0201, *0302, and *0602 is represented in Table 3. Among the patients, 77% were positive for HLA-DQB1*0201, compared with 31% of controls (P < 0.001). In relation to HLA-DQB1*0302, 40% of patients were positive for this allele, whereas only 19% of controls showed a positive result (P < 0.001). Moreover, when tested for the presence of the protective HLA-DQB1 allele, *0602, 24% of controls showed a positive result, compared with only 0.8% of patients. All observed differences were highly significant, with P values less than 0.001. Among the positive samples for HLA-DQB1*0201 and HLA-DQB1*0302, 74 patients (29%) were positive for both alleles vs. 4% of the controls (P < 0.001). The results were the same when male and female subjects (from patient and control groups) were compared to see whether gender differences exist in the genetic profiles (results not shown).
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shows, 74 patients had both alleles, 121 were positive for *0201 only, 28 were positive for *0302 only, and 30 patients were negative for both alleles. No significant difference was observed among the onset ages of the 4 groups.
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Based on the history of occurrence of T1D in the family, patients were divided into 3 groups: 1) T1D in the immediate family (28 patients; mean age at onset, 7.09 yr); 2) T1D in the extended family (41 patients; mean age at onset, 8.99 yr); and 3) no history of T1D in the family (182 patients; mean age at onset, 9.17 yr). No significant differences were found among the mean ages at onset of T1D in the 3 groups (P > 0.05), despite the apparent tendency toward an earlier onset age of the disease in the patients with an immediate T1D family history. However, when the same procedure was done while studying family history for T2D occurrence, significant differences were observed: 15 patients with an immediate family history of T2D had a mean age at onset (13.03 yr) that was higher than that (8.99 yr, n = 144, P < 0.01) of patients with extended family history of T2D and higher than that of patients with no family history of T2D (8.10 yr, n = 92, P < 0.001) (Fig. 1). The latter two groups were not significantly different from one another. Of the 15 patients with immediate family history of T2D, 11 had absolutely no family history of T1D. There was no difference in body mass index, HLA genotype, and HbA1c between patients with immediate family history of T2D and patients without such family history.
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Several studies have reported on the role of gender in the development and transmission of diabetes (5). The general trend seems to favor an increased prevalence among males, especially in populations of European origin (6) despite the fact that the risk for developing the disease is similar for both genders (37, 38). However, in our study, a gender preference of the disease cannot be definitely concluded because the study population was a select group from a single diabetes center.
It is believed that environmental factors are involved in the susceptibility to T1D. Among the factors studied, early exposure (3 months of age) to cow’s milk protein seems to be important in triggering the disease (29, 30). However, our study demonstrates an absence of a significant difference in mean age at onset between patients who were exposed to dairy products at 3 months compared with those exposed after 3 months of age. Despite the fact that our data depends on recollection by parents, this finding was in accordance with another study, which reported no relation with infant nutrition and/or cow’s milk antibodies and T1D (39). It is of interest to note that among the 140 patients in our study exposed to dairy products at up to 3 months of age, 111 were concomitantly being breast fed and 29 were not. When compared, the age at onset of T1D between these two groups was not significantly different.
In addition to environmental determinants of T1D, underlying genetic susceptibility to the disease is well documented. The HLA region on chromosome 6 accounts for 45% of T1D susceptibility (11). In this study, we investigated the presence of three different HLA-DQB1 alleles (*0201, *0302, and *0602) in T1D Lebanese patients. Studies have shown that HLA-DBQ1*0201 and *0302 are positively associated with the disease, whereas HLA-DQB1*0602 seems to be protective (16). Our results seem to be in accordance with the reported literature. T1D patients had a higher occurrence of *0201 and *0302, compared with nondiabetics. On the other hand, the nondiabetics had a higher occurrence of the protective DQB1 allele *0602. In addition, 29% of patients were positive for both *0201 and *0302, compared with 4% among the control group.
Like T1D, T2D has a genetic component, the proof of which comes from the high concordance rate in identical twins, which varies between 60–90% (40, 41), as well as from the documented lifetime risk of T2D in the offspring of T2D patients (42, 43). Recent reports are in favor of the existence of a genetic interaction between T1D and T2D (44). In 1989, Dahlquist et al. (45) reported that T2D was more prevalent in families of T1D patients, compared with control subjects. However, few reports tackled the association of family history of T2D and its effect on T1D patients. A recent finding suggested that T1D and T2D tend to cluster in the same families (44). In the present study, we have investigated the effect of the presence or the absence of family history of T1D or T2D on the age at onset of T1D. Our data strongly suggest that the occurrence of T2D in the immediate family of the T1D patients tends to delay the age at onset of T1D (13.03 vs. 8.10 yr). This finding significantly suggests that a family history of T2D seems to influence the disease onset of T1D. Fagerudd et al. (46) report that T2D in the immediate family history increases the susceptibility toward diabetic nephropathy in T1D patients, with a risk three times higher than that in patients with no parental T2D. Another study observed a low acute insulin secretory response in adult offspring of T2D patients (47). Recently, Erasnus et al. (41) reported that T1D patients with a positive history for T2D show an earlier onset of the disease. However, our study demonstrated an opposite effect of T2D immediate family history on the age at onset of T1D. This difference may not be attributable to confounding factors (such as obesity, hyperinsulinemia, and insulin resistance) associated with T1D because they were found to be similar among patients with a positive family history of T2D and the patients with a negative family history (unpublished observation). Thus, insulin resistance and obesity do not seem to play a role in delaying the onset of T1D in individuals susceptible to developing T1D. The difference may be attributable to geographic variations, which are usually observed from worldwide epidemiological data on T1D (37, 38). In addition, genetic heterogeneity among populations as well as the methods of analysis employed can play a major role in determining factors affecting susceptibility to T1D in a given study. Such variations have led to the emergence of an alternative hypothesis somewhat complementary to the long accepted polygenic nature of T1D. This hypothesis suggests that T1D is an oligogenic disorder, where a few genes determine the risk for diabetes in individual families but different genes are found in different families (48).
In conclusion, we showed that in the Lebanese population, T1D demonstrates no gender preference and that the age at onset of the disease is not affected by early exposure to dairy products. Furthermore, T1D patients share a high occurrence of T1D predisposing HLA-DQB1 risk alleles. Finally, we report a delay in the onset age of T1D in patients with immediate family history of T2D. Our data did not show that the presence of T2D in parents increases the risk for T1D in their children. However, the delayed onset of T1D in a child of a parent with T2D may suggest the presence of a common genetic predisposition for diabetes. Although it is premature to conclude that there may be some clustering of T1D and T2D in the same families, our data and recent studies strongly favor a possible genetic interaction between T1D and T2D (44). Studying families with simultaneous presence of T1D and T2D may prove important in understanding the genetic predisposition to T1D as well as the genetic and environmental factors involved in the onset of the disease.
Footnotes
Abbreviations: HLA, Human leukocyte antigen; IDDM, insulin- dependent diabetes mellitus; T1D, type-1 diabetes; T2D, type-2 diabetes; WHO, World Health Organization.
Received October 19, 2001.
Accepted March 23, 2002.
References
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