Type 1 diabetes in Jewish Ethiopian immigrants in Israel: HLA class II immunogenetics and contribution of new environment

Type 1 Diabetes in Jewish Ethiopian Immigrants in Israel: HLA Class II Immunogenetics and Contribution of New Environment A. Zung, M. Elizur, N. Weintrob, T. Bistritzer, A. Hanukoglu, Z. Zadik, M. Phillip, K. Miller, I. Koren, C. Brautbar, and S. Israel ABSTRACT: The interrelationship between human leukocyte antigen immunogenetics and environmental factors and their contribution to the emergence of type 1 diabetes (T1D) were studied in Jewish immigrants from Ethiopia in Israel. This community displays high incidence of T1D, and is unique both by its ethnic segregation and its rapid exposure to a new environment after the immigration. The study population consisted of 152 Ethiopian Jews living in Israel, 33 with T1D and 119 unrelated controls. Human leukocyte antigen class II susceptible and protective alleles in the Jewish Ethiopian patients were similar to those in patients of other ethnic groups in Israel and in non-Jewish Ethiopian patients, with a few exceptions. Three haplotypes were markedly associated with diabetes in Jewish Ethiopian patients: DRB1*0301 DQA1*05 DQB1*02 (OR 4.4, p ⬍ 0.001); DRB1*0404 DQA1 03 DQB1*0302 (OR 19.2, p ⫽ 0.006), and DRB1*0405 DQA1*03 DQB1*0302 (OR ABBREVIATIONS HLA human leukocyte antigen

INTRODUCTION The beta cell destruction characteristic of type 1 diabetes mellitus (T1D) is controlled by several genes [1, 2]. Human leukocyte antigen (HLA) and genetic linkage From the Pediatric Endocrinology Unit, Kaplan Medical Center, Rehovot, Israel (A.Z., M.E., Z.Z.); Institiute for Endocrinology and Diabetes, National Center for Childhood Diabetes, Schneider Children’s Medical Center of Israel, Petach Tikva, Israel (N.W., M.P.); Department of Pediatrics, Assaf Harofeh Medical Center, Zerifin, Israel (T.B.); Department of Pediatrics, Wolfson Medical Center, Holon, Israel (A.H.); Tissue Typing Unit, Hadassah University Hospital, Jerusalem, Israel (K.M., C.B.. S.I.); and Lin Medical Center, Clalit Health Services, Haifa, Israel (I.K.). Address reprint requests to: Amnon Zung, M.D., Pediatric Endocrinology Unit, Kaplan Medical Center, Rehovot 76100, Israel; Tel: 972-89441276/9; Fax: 972-8-9411942; E-mail: [email protected]. Received April 26, 2004; revised June 22, 2004; accepted September 14, 2004. Human Immunology 65, 1463–1468 (2004) © American Society for Histocompatibility and Immunogenetics, 2004 Published by Elsevier Inc.

87.8, p ⬍ 0.001). The highly susceptible allele DRB1*0301 was more common in the general Ethiopian population (25.2%) than in all other ethnic groups in Israel, which may render this community prone to the disease. The age at onset of disease in patients with two susceptible haplotypes was negatively correlated with the duration of living in Israel (r ⫽ ⫺0.621, p ⫽ 0.04). We concluded that ongoing exposure of genetically predisposed immigrants from Ethiopia to diabetogenic environmental factors eventually leads to a high incidence of overt diabetes in this ethnic group. Human Immunology 65, 1463–1468 (2004). © American Society for Histocompatibility and Immunogenetics, 2004. Published by Elsevier Inc. KEYWORDS: Type 1 diabetes; HLA class II; immigrants; ethiopia; environment

T1D

type 1 diabetes

studies have shown that T1D is strongly associated with specific alleles of HLA type II [1, 3]. In addition, various environmental factors may contribute to its appearance in subjects genetically predisposed to the disease. The study of T1D in immigrants provides a unique opportunity to evaluate the interplay between genetic background and environment in the evolution of the disease, especially when the country of origin and the country of destination differ widely. So far, however, results for the relative importance of the environment have been conflicting. Some studies found that immigrants from countries with a low incidence of T1D attained the incidence common to their country of destination [4], whereas others showed the opposite [5]. 0198-8859/04/$–see front matter doi:10.1016/j.humimm.2004.09.006

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In Israel, the overall annual incidence of T1D in the young population (ages 0 –17 years) ranges between 7.7/ 105 and 7.9/105, with remarkable variations by ethnic group [6, 7]. Shamis et al.[8] documented a marked increase in the incidence of T1D in Israel between 1965 and 1993, in line with changes reported worldwide [9, 10]. Their survey did not reflect, however, the incidence of T1D among Ethiopian Jews, most of whom immigrated to Israel during the last two decades. According to the Israeli Insulin-Dependent Diabetes Mellitus Registry [6, 7], the Jewish Ethiopian community has one of the highest annual incidences of T1D of all ethnic groups in Israel. The aim of the present study was to examine HLA class II immunogenetics and environmental factors in Jewish patients with T1D whose families emigrated from rural areas in Ethiopia to Israel. We also compared the genetic profile of these patients with that of other ethnic groups in Israel, reported by our team [11], and nonJewish patients in Ethiopia, reported by others [12, 13].

PATIENTS AND METHODS Population The study group included 33 patients (15 male, 18 female) with T1D attending seven diabetes clinics in Israel. One hundred nineteen unrelated healthy individuals served as controls. None of the control subjects had a first-degree relative with T1D. Both parents of the patients and the controls were born in Ethiopia. Average age at disease onset was 8.6 ⫾ 4.9 years (range 0.4 –20 years). Five of the study patients were born in Ethiopia and emigrated to Israel at an average age of 9.1 ⫾ 3.3 years. In all five, the diabetes was diagnosed in Israel. A diagnosis of T1D was made based on the presence of diabetes-associated antibodies or ketoacidosis either at presentation of the disease or later during follow-up. Levels of diabetes-associated autoantibodies (islet cell antibodies, insulin autoantibodies, and antiglutamic acid decarboxylase autoantibodies) at disease onset were obtained from the medical records. These data were available in 19 patients only, because autoantibodies are not routinely checked in all diabetes clinics in Israel. Blood samples were collected from patients for HLA typing during planned visits in diabetes clinics and in addition to routine blood tests. Human leukocyte antigen class II alleles and genotypes in the patients and control subjects were compared with those obtained in other ethnic groups in Israel [11] and to non-Jewish patients with T1D in Ethiopia [12, 13]. The study protocol was approved by the ethics committees of the hospitals that participated in this study and the Israel

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Ministry of Health Committee on Human Experimentation. HLA Class II Typing Genomic DNA was isolated by the salting-out method [14]. DRB1 and DQB1 loci were typed using the Lipa HLA kit (Murex Innogenetics, Ghent Belgium). DQA1 was typed by the polymerase chain reaction–sequencespecific oligonucleotide probe method using the Olerup kit (Saltsjoboden, Sweden). The Lipa HLA tests are based on the reverse hybridization principle [15]. In brief, amplified biotinylated DNA material by polymerase chain reaction was chemically denatured, and the single strands were hybridized with specific oligonucleotide probes immobilized as parallel lines on membrane-based strips. After hybridization, streptavidin labeled with alkaline phosphatase was added and bound to any biotinylated hybrid previously formed. Incubation with BCIP/ NBT substrate solution resulted in a purple-brown precipitate. The reaction was stopped by wash step and the reactivity pattern of the probes was recorded. Statistical Analysis ␹2 test or Fisher’s exact test (as applicable) was used to examine the significance of the relative distribution of T1D cases for each allele or genotype. The p values were adjusted by Bonferroni’s method within each group of alleles or genotypes: only adjusted values of 0.05 or less are presented. The odds ratio [OR] was calculated by the logit method for case-control studies. In cases of zero incidence, 0.5 was added to each of the frequency table values before calculating the OR. The statistical analysis was performed by SAS software. RESULTS HLA Class II Susceptible and Protective Alleles Six alleles were found to be associated with T1D in Ethiopian Jews: three in the DRB1 group, two in DQB1 group, and one in the DQA1 group (Table 1). All six susceptible alleles were also associated with T1D in the other ethnic groups in Israel, namely Ashkenazi and non-Ashkenazi Jews and Israeli Arabs (Table 1). Differences in susceptible alleles between the Ethiopian Jews and the T1D patients of the other ethnic groups were restricted to the DRB*04 group. Neither patients nor controls of Ethiopian origin harbored DRB1*0402, a susceptible allele common to many ethnic groups in Israel; in the Ethiopian patients, the alleles DRB1*0404 and DRB1*0405 were associated with T1D. Two alleles showed a negative association with T1D in Ethiopian Jews: DQA1*01 and DQB1*0301. This was true for the other ethnic groups in Israel as well, but

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TABLE 1 HLA class II alleles positively associated with type 1 diabetes (comparison between Ethiopian Jews and other ethnic groups in Israel) Ethiopian Jews

Ashkenazi Jews

Non-Ashkenazi Jews

Arabs

(Pt: 33/C: 119)*

(Pt: 70/C: 132)

(Pt: 95/C: 293)

(Pt: 37/C: 109)

Allele

OR

p value

OR

p value

OR

p value

OR

p value

DRB1*03 DRB1*0404 DRB1*0405 DQA1*03 DQB1*02 DQB1*0302 DRB1*0402

4.5 (2.4–8.3) 3.8 (1.2–12.4) 11.4 (4.2–30.7) 4.2 (2.2–8.0) 3.0 (1.7–5.3) 6.0 (2.7–13.2) (n ⫽ 0)a

⬍0.001 0.40 ⬍0.001 ⬍0.001 0.009 ⬍0.001 NS

4.5 0.9 12.2 3.5 2.6 5.1 7.5

⬍0.001 NS NS ⬍0.01 ⬍0.01 ⬍0.001 ⬍0.001

11.2 1.8 6.1 9.7 4.2 9.8 4.5

⬍0.001 NS ⬍0.001 ⬍0.001 ⬍0.001 ⬍0.001 ⬍0.001

5.8 3.0 13 6.2 3.8 4.9 8.2

⬍0.001 NS ⬍0.01 ⬍0.001 ⬍0.05 ⬍0.01 ⬍0.01

Abbreviations: *Pt ⫽ patients, C ⫽ controls; OR ⫽ odds ratio; NS ⫽ not significant. Parentheses include 95% confidence intervals. a DRB1*0402 allele was not detected in any of the Ethiopian Jews.

it did not reach a statistical significance in all of them (Table 2). An additional five alleles that were negatively associated with T1D in all ethnic groups in Israel, including Ethiopian Jews, reached statistical significance in non-Ashkenazi patients only (Table 2). Among the 119 Ethiopian control subjects, 30 (25%) carried the highly susceptible allele DRB1*0301. This prevalence was similar to that reported by us in healthy Yemenite Jews [16], and significantly higher than the reported prevalence in healthy subjects of other ethnic groups in Israel [13] (Table 3). Distribution of Three Putative Locus Haplotypes Three haplotypes were markedly associated with T1D in Jewish Ethiopian patients: DRB1*0301 DQA1*05 DQB1*02 (OR ⫽ 4.4, p ⬍ 0.001, patients vs controls); DRB1*0404 DQA1 03 DQB1*0302 (OR ⫽ 19.2, p ⫽

0.006), and DRB1*0405 DQA1*03 DQB1*0302 (OR ⫽ 87.8, p ⬍ 0.001). Five patients (15%) carried no susceptible haplotypes, 15 patients (45%) carried one, and 13 patients (40%) carried two of those haplotypes. Several other haplotypes that carried the DRB1*04 allele combined with various DQA1 and DQB1 alleles (i.e., DRB1*0401 DQA1*03 DQB1*0302 [OR ⫽ 2.4] and DRB1*0405 DQA1*03 DQB1*02 [OR ⫽ 3.1]) did not reach a statistical significance, though they are known as T1D susceptible haplotypes. This may derive from the relatively small sample size of T1D in our study. A similar study in Yemenite Jews [16], who have the highest incidence of T1D of all ethnic groups in Israel [6], revealed two susceptible haplotypes in patients with T1D: DRB1*0301 DQA1*05 DQB1*02, which is common also in Ethiopian patients, and DRB1*0402 DQA1*03

TABLE 2 HLA class II alleles negatively associated with type 1 diabetes (comparison between Ethiopian Jews and other ethnic groups in Israel)a Ethiopian Jews

Ashkenazi Jews

Non-Ashkenazi Jews

Arabs

(Pt: n ⫽ 33/C: n ⫽ 119)

(Pt: n ⫽ 70/C: n ⫽ 132)

(Pt: n ⫽ 95/C: n ⫽ 293)

(Pt: n ⫽ 37/C: n ⫽ 109)

Allele

OR

p value

OR

p value

OR

p value

OR

p value

DQA1*01 DQB1*0301 DRB1*0701 DRB1*11 DQA1*0201 DQB1*0503 DQB1*0601

0.3 (0.2–0.6) 0.2 (0.1–0.7) 0.1 0.1 0.1 (n ⫽ 0)a 0.7

0.0013 0.025 NS NS NS NS NS

0.6 0.1 0.6 0.04 0.6 0.1 0.6

NS ⬍0.001 NS ⬍0.001 NS NS NS

0.2 0.01 0.3 0.02 0.3 0.06 0.2

⬍0.01 ⬍0.001 ⬍0.01 ⬍0.001 ⬍0.01 ⬍0.05 ⬍0.05

0.4 0.05 0.3 0.05 0.3 0.1 0.2

NS ⬍0.001 NS ⬍0.05 NS NS NS

Abbreviations as in Table 1. Parentheses include 95% confidence intervals. a DRB1*0503 allele was not detected in any of the Ethiopian Jews.

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TABLE 3 Prevalence of the type 1 diabetes–susceptible allele DRB1*0301 among healthy subjects of various ethnic groups in Israel Ethnic groups

Percentage of DRB1*0301 allele carriers

Non-Ashkenazi Jews (n ⫽ 95) Ashkenazi Jews (n ⫽ 132) Yemenite Jews (n ⫽ 76) Ethiopian Jews (n ⫽ 119)

7.4% (n ⫽ 7)a 12.8% (n ⫽ 17)a 23.7% (n ⫽ 18)b 25.2% (n ⫽ 30)b

Evaluation of the possible effect of the period since immigration on the age at onset of the disease revealed (by linear regression analysis) that in the patients with two susceptible haplotypes, the age at diagnosis was negatively correlated with the time the family had lived in Israel before the birth of the diabetic offspring (r ⫽ ⫺0.621, p ⫽ 0.04). This association was not found in the other haplotype groups or in the whole study population.

DISCUSSION The immigration of a Jewish Ethiopian community from a developing African country to Israel, a Westernized country, afforded us a unique opportunity to learn about the genetic predisposition and environmental influences that contribute to the pathogenesis of T1D. Previous studies that addressed this issue dealt mainly with the incidence of T1D in the country of destination compared with the country of origin. In most cases, the two countries were similar in terms of culture, industrialization, urbanization, and average annual income. In a report on the incidence of T1D in Montreal, the risk of diabetes among French Canadians and Jewish Canadians was about double that in France and Israel, respectively [4]. By contrast, the annual incidence rate of T1D in young immigrants to Germany from former Yugoslavia, Italy, and Greece was closer to that in their country of origin than in German children [5]. Although the latter study emphasized the importance of genetics in T1D, its assessment of the impact of environment was limited, because the environmental shift imposed by immigration was much less dramatic than the one experienced by our subjects, who immigrated from Ethiopia to Israel. Indeed, according to the Human Development Report of 2003, commissioned by the United Nations Development Programme, there are major differences in economic, educational, and health aspects between these

p ⬍ 0.05 for a versus b (␹2 and Fisher’s exact test).

a ,b

DQB1*0302, which is similar to two susceptible haplotypes in Ethiopian patients (in which DRB1*0402 is replaced by DRB1*0404 and DRB1*0405). No remarkable differences were found either in susceptible alleles or in negatively associated alleles between Jewish and non-Jewish Ethiopian patients with T1D (Table 4). Some statistically significant differences between these populations may derive from the relatively small size of the groups studied. HLA DRB1 Genotype Analysis In the study group, the well-known susceptible genotypes DR3/DR3, DR3/DR4, and DR4/DR4 were associated with T1D at prevalence rates of 12.1%, 39.4%, and 12.1%, respectively. However, only the first two reached statistical significance. In an analysis that included all genotypes with DR3 and DR4 or either one of them in both haplotypes, 63.6% of T1D patients carried high risk genotypes compared with 9.1% of controls. Factors Affecting the Age at Onset of Diabetes When stratifying by number of susceptible haplotypes, the study population showed a tendency for a lower average age at onset of diabetes with an increase in the number of susceptible haplotypes.

TABLE 4 HLA class II–susceptible and negatively correlated haplotypes (comparison between Jewish and non-Jewish Ethiopian patients with type 1 diabetes)

DRB1 0301 0404 0405 0405 07

Jewish Ethiopians

Non-Jewish Ethiopiansa

[Pt ⫽ 33; controls ⫽ 119]

[Pt ⫽ 39; controls ⫽ 39]

DQA1

DQB1

OR

p value

OR

p value

05 03 03 03 0201

02 0302 0302 0201 0201

4.4 (2.4–8.2) 19.2 (2.2–168) 87.9 (5.0–285) 3.1 0.2

⬍0.001 0.006 ⬍0.001 NS NS

4.8 3 14 4.9 0.2

⬍0.0005 NS ⬍0.05 ⬍0.025 ⬍0.001

Abbreviations as in Table 1. Parentheses include 95% confidence intervals. a Data on non-Jewish Ethiopians from Abdulkadir et al. [13].

Diabetes in Ethiopian Jews: Genetics and Environment

countries. By comparing the two countries, it was reported that life expectancy was 46 years versus 79 years, gross domestic product per capita was US$810 versus US$19,790, infant mortality rate (per 1000 live births) was 116 versus 6, 1 year olds fully immunized against measles were 52% versus 94%, and health expenditure per capita was US$14 versus US$2,338 in Ethiopia and Israel, respectively. For centuries, the Jewish community in Ethiopia, located in the rural area of Gondar in the northern part of the country, was ethnically, geographically, and genetically isolated from the rest of the Jewish diaspora. Immigration to Israel began in 1980, at which time there were fewer than 500 Ethiopian Jews in Israel. By the end of 2001, this number reached 62,000 [17]. Most Ethiopian Jews currently live in inner city congregations and intermarry little with other ethnic groups. Their move to Israel exposed them not only to a new culture, but also to new health practices, such as vaccination programs and dietary habits, and to infectious agents typical to Westernized countries. The effect of diet in the evolution of type 2 diabetes is well known, although a few studies in animal models of BB rats and nonobese diabetic mice have also demonstrated its effect in the prevention or induction of T1D [18 –20]. However, the precise diabetogenic and protective components in these diets were not elucidated. Two consecutive reports by the Israeli group of the T1D Registry highlighted the relatively high incidence of T1D in Ethiopian immigrants [6, 7]. Unfortunately, information on the incidence of T1D in non-Jewish Ethiopians residing in Ethiopia is lacking. Our search for unique genetic factors that may render Jewish Ethiopians susceptible to T1D yielded no major differences in HLA class II susceptible and negatively associated alleles between diabetic Ethiopian Jews and other ethnic groups in Israel [13] or non-Jewish Ethiopians [14, 15]. The sole exception was DRB1*0402, which was positively associated with T1D in all other ethnic groups in Israel, but was not detected in either patients or controls of Ethiopian origin. Interestingly, the prevalence of the highly susceptible allele DRB1*0301 was remarkably higher in the general Jewish Ethiopian population in Israel than among healthy Ashkenazi and non-Ashkenazi Jews. Furthermore, a similarly high prevalence of the allele was reported in healthy Yemenite Jews, another ethnic group with a high annual incidence of T1D [6]. We therefore postulate that the genetic makeup that characterizes both Ethiopian and Yemenite Jews may contribute to their high incidence of T1D. In the subgroup of patients with the heaviest genetic load (i.e.,with two susceptible haplotypes), we found a negative correlation between age at onset of diabetes and

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interval between time of arrival in Israel and birth of the T1D child. Apparently, the adoption of a Western lifestyle, with increasing exposure to diabetogenic factors over time, contributed to the increased incidence of T1D in genetically predisposed individuals. By contrast, the newer immigrants, who still adhered to the religious and cultural customs that marked their way of life in Ethiopia, preserved a traditional microenvironment that provided temporary protection from the disease. Similarly, in a study of Yemenite Jews, Weintrob et al. [21] found that part of the remarkable increase in the incidence of T1D in the Israeli immigrants compared with their Yemenite-resident counterparts could be partly attributed to environmental factors. In conclusion, there is a similarity in susceptibility alleles between Jewish diabetic patients of Ethiopian origin and patients from other ethnic groups in Israel. The high frequency of the susceptibility allele DRB1*0301 in the healthy Ethiopian and Yemenite Jews may make it one of the genetic factors which render these populations prone to T1D. We assume that the ongoing process of urbanization/Westernization that accompanied the immigrants’ resettlement in Israel exposed this already genetically predisposed population to diabetogenic environmental factors, leading eventually to overt diabetes. ACKNOWLEDGMENT Special thanks are due Dr. Zeev Josefsberg, Ms. Lea Malach, and Ms. Regina Ofan for their contribution to this study. The study was supported by a grant to Dr. Zung from the Intramural Division of Research Funds of the Hebrew University of Jerusalem.

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