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Family-based association study of HLA class II with type 1 diabetes in Moroccans
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PATBIO-3170; No. of Pages 5 Pathologie Biologie xxx (2014) xxx–xxx
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Original article
Family-based association study of HLA class II with type 1 diabetes in Moroccans E´tude d’association familiale entre les ge`nes HLA classe II et le diabe`te type 1 dans la population marocaine A. Drissi Bourhanbour a,b,*, N. Benseffaj a,b, S. Ouadghiri a,b, R. Razine c, A. Touzani d, A. Belafraj d, M. Essakalli a,b a
UPR d’immunologie, faculte´ de me´decine et de pharmacie, Rabat Institut, avenue Mohamed Belarbi El Alaoui, BP 6203, 10000 Rabat, Morocco Unite´ d’immunologie, service de transfusion sanguine et d’he´movigilance, hoˆpital d’enfants-Rabat, Rabat Institut, CHU Ibn-Sina, rue Lamfadel Cherkaoui, BP 6527, Rabat, Morocco c Laboratoire de biostatistique, recherche clinique et epide´miologie, faculte´ de me´decine et de pharmacie, Rabat Institut, avenue Mohamed Belarbi El Alaoui, BP 6203, 10000 Rabat, Morocco d Service de diabe´tologie pe´diatrique, hoˆpital d’enfants-Rabat, Rabat Institut, CHU Ibn-Sina, rue Lamfadel Cherkaoui, BP 6527, Rabat, Morocco b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 2 April 2014 Accepted 1st December 2014 Available online xxx
Background. – The T1D is a multifactorial disease; with a strong genetic control. The human leukocyte antigen (HLA) system plays a crucial role in the autoimmune process leading to childhood diabetes. About 440,000 of the childhood population of the world (1.8 billion children under 14 years of age), have type 1 diabetes, and each year an additional 70,000 develop this disorder. The objective of this study was to investigate the distribution of HLA class II in Moroccan families of diabetic children to identify susceptibility alleles of the Moroccan population. Subjects and methods. – We included in this study, Moroccan families who have at least one child with T1D. The age of onset of diabetes was less than 15 years. HLA class II (DRB1* and DQB1*) was carried out by molecular biology techniques (PCR-SSP and PCR-SSO). The FBAT test (family-based association test) was used to highlight the association between T1D and the HLA-DRB1* and -DQB1* polymorphism. Results. – The association of HLA class II (DRB1*, DQB1*) in type 1 diabetes was analyzed in fifty-one Moroccan families, including 90 diabetics. The results revealed that the most susceptible haplotypes are the DRB1*03:01–DQB1*02:01, DRB1*04:05–DQB1*03:02 (Z = 3.674, P = 0.000239; Z = 2.828, P = 0.004678, respectively). And the most protective haplotype is the DRB1*15–DQB1*06. Conclusion. – This is the first family-based association study searching for an association between HLA class II and T1D in a Moroccan population. Despite the different ethnic groups forming Morocco, Moroccan diabetics share the most susceptible and protective HLA haplotypes with other Caucasians populations, specifically the European and Mediterranean populations. ß 2014 Published by Elsevier Masson SAS.
Keywords: HLA class II Haplotypes Type I diabetes Family-based association study
R E´ S U M E´
Mots cle´s : HLA classe II Haplotypes diabe`te type 1 E´tude d’association familiale
Introduction. – Le diabe`te de type 1 (DT1) est une maladie multifactorielle avec un important controˆle ge´ne´tique. Le syste`me HLA joue un roˆle primordial dans le processus auto-immun conduisant au DT1. Dans le monde, environ 440 000 enfants (1,8 milliards d’enfants aˆge´s de moins de 14 ans) ont le DT1 et chaque anne´e 70 000 enfants supple´mentaires de´veloppent ce de´sordre. L’objectif de ce travail e´tait d’e´tudier la distribution des mole´cules HLA de classe II (DRB1*, DQB1*) chez des enfants diabe´tiques pour identifier les alle`les de susceptibilite´ et de protection vis-a`-vis du DT1 chez la population marocaine.
* Corresponding author. UPR d’immunologie, faculte´ de me´decine et de pharmacie, Rabat Institut, avenue Mohamed Belarbi El Alaoui, BP 6203, 10000 Rabat, Morocco. E-mail address: [email protected] (A. Drissi Bourhanbour). http://dx.doi.org/10.1016/j.patbio.2014.12.001 0369-8114/ß 2014 Published by Elsevier Masson SAS.
Please cite this article in press as: Drissi Bourhanbour A, et al. Family-based association study of HLA class II with type 1 diabetes in Moroccans. Pathol Biol (Paris) (2014), http://dx.doi.org/10.1016/j.patbio.2014.12.001
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Mate´riel et me´thodes. – On a inclus dans cette e´tude des familles marocaines qui ont au moins un enfant atteint DT1. L’aˆge d’appariation du diabe`te e´tait infe´rieur a` 15 ans. Le typage HLA classe II (DRB1* et DQB1*) a e´te´ re´alise´ par des techniques de biologie mole´culaire (PCR-SSP et PCR-SSO). Le test FBAT (Family-Based Association Test) a e´te´ utilise´ pour mettre en e´vidence l’association entre DT1 et le polymorphisme HLA-DRB1* et HLA-DQB1*. Re´sultats. – L’association HLA classe II (DRB1*, DQB1*) et le diabe`te type 1 a e´te´ e´tudie´e chez 51 familles marocaines, incluant 90 diabe´tiques. L’analyse des donne´s par FBAT montre que les deux haplotypes associe´s au DT1 dans la population marocaine sont le DRB1*03:01–DQB1*02:01, DRB1*04:05– DQB1*03:02 (Z = 3,674, P = 0, 000239; Z = 2,828, P = 0, 004678, respectivement). L’haplotype le plus protecteur est DRB1*15–DQB1*06. Conclusion. – Meˆme si la population marocaine est forme´e de plusieurs groupes ethniques, elle partage les meˆmes haplotypes HLA de susceptibilite´ et de protection vis-a`-vis du DT1 avec d’autres populations caucasiennes et plus spe´cifiquement les populations europe´enne et me´diterrane´enne. ß 2014 Publie´ par Elsevier Masson SAS.
1. Introduction
2.2. HLA typing
Type 1 diabetes (T1D) is a polygenic autoimmune disease in which the insulin-secreting pancreatic beta cells are selectively destroyed by T cells [1]. Type 1 diabetes is the third most prevalent chronic disease of childhood. About 440,000 of the childhood population of the world (1.8 billion children under 14 years of age), have type 1 diabetes, and each year an additional 70,000 develop this disorder [2]. According to the estimates by the Moroccan Ministry of Health, 100,000 Moroccans have type 1 diabetes, including 10,000 children [3]. The autoimmunity of T1D is provided by the presence of antibodies against islet cells (ICA), insulin (IAA), glutamic acid decarboxylase (GADA), and tyrosine-phosphatase (IA2-Ab). Genome-wide association studies have identified over 40 polymorphisms that are associated with T1D [4]. However, the major susceptibility for T1D has been mapped to the HLA class II genes HLA-DRB1* and -DQB1* [5,6]. Both susceptible and protective DRB1*–DQB1*haplotypes exist in all populations [7] but differences between populations have been shown. A strong association of HLA-DRB1*03–DQB1*02:01 and of -DRB1*04:01/04:02/04:05– DQB1*03:02 haplotypes with T1D have been established among Caucasians [8–10], and DRB1*09:01–DQB1*03:03 haplotype in Japanese patients [11]. The majority of these studies are casecontrol. In our work, we have used a family-based association design. Compared to the case-control study designs, family-based association designs are attractive, since they test for both linkage and association. The family-based association designs can also avoid the potential confounding effects of population stratification by using the parents as controls for the case (the affected offspring) and are convenient in refining linkage findings in family samples [12]. Up to now, the association of T1D with HLA locus was not investigated in Moroccan families of diabetic children. The objective of this study was to investigate the distribution of HLA class II DRB1* and DQB1* alleles in Moroccans families of diabetics children, to identify susceptibility alleles and to refine genetic knowledge of the Moroccan population. The aim is to be able to provide early and adequate care for the subjects at risk.
HLA typing was performed on samples derived from both parents, affected children, and unaffected children in all families. Genomic DNA was first extracted and purified from peripheral blood samples collected in 5% EDTA using a commercial kit (Qiagen). Low resolution HLA class II (DRB1* and DQB1*) typing was performed by polymerase chain reaction-sequence specific primers (PCR-SSP) using micro generic HLA DNA typing trays (One Lambda) according to the manufacturer’s protocol for all families. In a second step, the DRB1* high-resolution typing was done by polymerase chain reaction sequence-specific oligonucleotide (PCR-SSO) for 24 families. Whereas, the DQB1* was genotyped by using PCR-SSP. All analyzes were carried out in the laboratory of immunohistocompatibility of the Ibn-Sina University Hospital of Rabat, Morocco.
2. Subjects and methods 2.1. Subjects We included in this study fifty-one Moroccan families with at least one child with type 1 diabetes. The families were recruited between 2009 and 2011 from the department of endocrinology of the Children’s Hospital of Rabat. They consisted of 38 multiplex-sibs families (at least two affected children) and 13 simplex families (only one diabetic child). They contain a total of 229 individuals among which 90 are diabetics. The median age was 17 (range: 2–41). The age of onset of T1D was less than 15 years. All subjects gave consent to participate in this study which was approved by the Ethics Committee for Biomedical.
2.3. Statistical analysis The family-based association test (FBAT) software was used to evidence association between HLA-DRB1* and -DQB1* polymorphisms and T1D. This method is an extension of the original transmission disequilibrium test (TDT) [13], a genetic analysis conducted on family trios (affected child and both parents), which assesses whether one allele is inherited in affected children at a rate greater than that predicted. The FBAT allows inclusion of data that cannot be analyzed in the strict TDT, such as families that are missing a parent. Advantages of these methodologies over other population-based approaches include that they test for both association and linkage, and are not susceptible to population stratification, which can introduce confounding factors in case-control studies [14]. The FBAT statistic was calculated under an additive model and for bi-allelic (each allele against all others) mode of testing. Positive Z-statistic of single locus and haplotype FBAT indicated that a specific single locus and haplotype was more frequently transmitted to patients with DT1 in informative families than expected under the null hypothesis of no linkage and no association. The negative sign of Z-statistic indicates that the frequency of transmitted genotype is negative association with susceptibility to T1D. Haplotype FBAT software was also used to estimate haplotype frequencies. The probability (P) value was considered significant at a P-value under 0.05.
3. Results 3.1. HLA-DRB1* allele association in Moroccan patients with DT1 Table 1 shows the HLA-DRB1* alleles frequencies in Moroccan families of diabetic children. A total of eight HLA-DRB1* alleles have been identified. The most frequent HLA-DRB1* alleles were HLA-DRB1*04 (32.5%) and -DRB1*03 (27.7%). Indeed, the FBAT analyses showed that the DRB1*04 and DRB1*03 alleles were significantly associated with DT1 (Z = 3.942, P = 0.000081; Z = 3.069, P = 0.002146, respectively). At the opposite, DRB1*11 and DRB1*15 were negatively associated with susceptibility to T1D (Z = –3.207, P = 0.001341; Z = –2.751, P = 0.005944, respectively). 3.2. HLA-DQB1* allele association in Moroccan patients with DT1 The distribution of alleles frequencies of HLA-DQB1* in Moroccan families of diabetic children are shown in Table 2. We
Please cite this article in press as: Drissi Bourhanbour A, et al. Family-based association study of HLA class II with type 1 diabetes in Moroccans. Pathol Biol (Paris) (2014), http://dx.doi.org/10.1016/j.patbio.2014.12.001
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Table 1 The association between the HLA-DRB1* polymorphisms and T1D by family-based association test. DRB1*alleles
Allele frequencies (%)
Number of families
S-E(S)
Var(S)
Z*
P
01 03 04 07 11 13 15 16
6.6 27.7 32.5 9.2 5.1 6.3 8.1 1.9
6 26 26 12 8 8 11 3
1.5 10.444 13.611 4.5 6 5.222 6.5 0.167
3.25 11.58 11.923 5.25 3.5 3.451 5.583 0.972
0.832 3.069 3.942 1.964 3.207 2.811 2.751 0.169
0.4 0.002146 0.000081 0.049535 0.001341 0.04934 0.005944 0.86577
T1D: type 1 diabetes; S-E(S) and Var(S) are the expected value and variance of the test statistic. Z: is the test statistic; P: significance level. * The negative sign of Z-statistic indicates that the frequency of transmitted genotype is negative association with susceptibility to T1D.
Table 2 The association between the HLA-DQB1* polymorphisms and T1D by family-based association test. DQB1*alleles
Allele frequencies (%)
Number of families
S-E(S)
Var(S)
Z*
P
02 03 05 06
34.8 40.5 10 13.5
15 18 11 13
7.167 7.267 1.433 12.5
8.072 7.896 5.079 7.25
2.522 2.586 0.636 4.642
0.011654 0.009707 0.524771 0.00000344
T1D: type 1 diabetes; S-E(S) and Var(S) are the expected value and variance of the test statistic. Z: is the test statistic; P: significance level. * The negative sign of Z-statistic indicates that the frequency of transmitted genotype is negative association with susceptibility to T1D.
have identified four alleles on HLA-DQB1* locus. The highest frequencies of the HLA-DQB1* alleles were observed for DQB1*03 (40.5%), and DQB1*02 (34.8%). A significant association between DQB1*03 and DQB1*02 and DT1 was shown (Z = 2.586, P = 0.009707; Z = 2.522, P = 0.011654, respectively). The DQB1*06 is negatively associated with T1D (Z = –4.64, P = 0.0000344). 3.3. HLA-DRB1* -DQB1* haplotypes associations with DT1 The HLA-DRB1* -DQB1* haplotype frequencies were calculated in Moroccan families of diabetic children (Table 3). The analysis of the haplotypes shows that DRB1*04–DQB1*03 and DRB1*03– DQB1*02 were significantly associated with susceptibility to T1D (Z = 4.435, P = 0.00000922; Z = 3.136, P = 0.001713, respectively). Among the protective DRB1*–DQB1* haplotype, the DRB1*15– DQB1*06 was negatively associated with T1D (Z = –3.153, P = 0. 001616). 3.4. Analysis of DRB1* and DQB1* in four digit resolution by FBAT The analysis shows that DRB1* 03:01, DRB1*04:05, DQB1*02:01, and DQB1*03:02 alleles were associated with susceptibility to T1D (Z = 3.381, P = 0.000723; Z = 0.0000963, P = 4.899; Z = 4.243, P = 0.000022; Z = 2.860, P = 0.0045231, respectively) (Table 4). T1D risk associations were observed with DRB1*03:01– DQB1*02:01 and DRB1*04:05–DQB1*03:02 specific haplotypes
(Z = 3.674, P = 0.000239; Z = 2.828, P = 0.004678, respectively) (Table 5). 4. Discussion T1D is a multifactorial disease, with a strong genetic control. Several publications have shown the effect of the HLA-DRB1* and DQB1* allele polymorphisms, on predisposition to T1D. Identifying the polymorphic amino acid residues that distinguish such alleles has been extensively studied. The highly polymorphic amino acid positions in the DRb1 and DQb1 chains, which mostly form peptide-binding grooves for antigen presentation to CD4+ T-helper cells, are suggested to predispose an individual to T1D [15]. Indeed, numerous studies have shown that DRb1Lys71+ provides the highest relative risk for T1D [16–18] and that the DQb1Asp57– provides an additive effect to DRb1Lys71+ [19]. The Moroccan population is a mixed group that includes Arabic, Berber, and African with a strong influence of various immigrations, mainly Mediterranean [20]. The gene profile of all these populations probably contributed to the Moroccans genetic pool. T1D clusters in families with an overall genetic risk ratio of 15 [21]. This is the first family-based association study searching for an association between HLA class II and T1D in a Moroccan population. HLA-DRB1*03 and -DRB1*04 alleles showed a significant association with T1D in the Moroccan families of diabetic children. This positive association between HLA class II molecules and T1D has been well established in most population of the world [22,23]
Table 3 Most frequent HLA-DRB1* -DQB1* haplotypes in Moroccan families of diabetic children. DRB1*–DQB1* haplotypes
Haplotype frequencies (%)
Number of families
S-E(S)
Var(S)
Z*
P
04–03 03–02 15–06 13–06 07–03 11–03
32.8 30.1 7.3 4.5 2.8 2.7
22 21 8 5 5 4
14 10.034 6.5 –5 3.5 3.433
9.967 3.136 4.25 2.5 1.75 1.679
4.435 3.136 3.153 3.162 2.646 2.65
0.00000922 0.001713 0.001616 0.001565 0.008151 0.008055
T1D: type 1 diabetes; S-E(S) and Var(S) are the expected value and variance of the test statistic. Z: is the test statistic; P: significance level. * The negative sign of Z-statistic indicates that the frequency of transmitted genotype is negative association with susceptibility to T1D.
Please cite this article in press as: Drissi Bourhanbour A, et al. Family-based association study of HLA class II with type 1 diabetes in Moroccans. Pathol Biol (Paris) (2014), http://dx.doi.org/10.1016/j.patbio.2014.12.001
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Table 4 HLA-DRB1* and -DQB1* four digit allele frequencies in Moroccan families of diabetic children. Alleles
DRB1* 03:01 04:05 04:02 07:01 DQB1* 02:01 03:02
Allele frequencies (%)
Number of families
S-E (S)
Var(S)
Z*
P
15.7 17.6 13.7 6.6
12 10 4 4
8 12 4 4
5.6 6 2 2
3.381 4.899 2.828 2.828
0.000723 0.000000963 0.004678 0.004678
19.2 8
10 10
10 6
4.5 4.4
4.243 2.860
0.000022 0.0045231
T1D: type 1 diabetes; S-E(S) and Var(S) are the expected value and variance of the test statistic. Z: is the test statistic; P: significance level. * The negative sign of Z-statistic indicates that the frequency of transmitted genotype is negative association with susceptibility to T1D.
susceptibility for T1D within Moroccan patients. The study clearly indicates an association between DRB1*03:01–DQB1*02:01, DRB1*04:05–DQB1*03:02 haplotype and T1D in our population. These data may serve as a baseline; however, it has to be controlled in a larger population. Furthermore, the number of informative families is limited and high-resolution HLA typing was done for a small number of families. It should be done in all families in order to precise more specific alleles involved in Moroccan diabetics. However, the advantage of our study is that the family-based association study and haplotype analysis has been used. We can conclude that despite the different ethnic groups forming Moroccan population, Moroccan diabetics share the most susceptible and protective HLA haplotypes with other Caucasians populations, including European and Mediterranean populations. Disclosure of interest
Table 5 Most frequent HLA-DRB1*–DQB1* haplotypes four digit allele in Moroccan families of diabetic children. DRB1*–DQB1* Haplotypes
Haplotype frequencies (%)
Number of families
S-E (S)
Var(S)
Z*
P
03:01–02:01 04:05–03:01
13.1 5.6
6 6
6 4
2.667 2
3.674 2.828
0.000239 0.004678
T1D: type 1 diabetes; S-E(S) and Var(S) are the expected value and variance of the test statistic. Z: is the test statistic; P: significance level. * The negative sign of Z-statistic indicates that the frequency of transmitted genotype is negative association with susceptibility to T1D.
and suggest a role for DRB1*03 and DRB1*04 in the susceptibility to T1D. Indeed, these results were found in European, Latin American, Chinese and South African [24–28] populations, as well as in Maghreb countries [29,30]. Among DRB1*03 and DRB1*04 alleles, DRB1*03:01 and DRB1*04:05 are consistently associated with T1D [22,23,31,32]. In this work, we have identified the same allelic specificities in Moroccan families of diabetic children. HLADRB1*09 was reported in Asian populations [33,34] and even in a Moroccan previous study [35]. In our case, this association was not found. The DRB1*09 allele is very rare in North Africa, and in Morocco it was described only in a Berber population of the north [36]. Our population has only one family coming from this origin and this may explain why this allele was not found. DRB1*15 allele was universally described to be a protective factor against T1D [22,34]. In our study, besides DRB1*15, the DRB1*11 alleles were underrepresented in the Moroccan families of diabetic children. A similar observation has already been reported in Tunisian, Macedonian, Turkish, Lithuanian and French populations [37–41]. We were also interested in the role of HLA-DQB1* alleles in T1D susceptibility since DQB1*02:01 and DQB1*03:02 were the most frequently reported as involved in susceptibility to risk factor for T1D in multiple populations [22,23]. We have identified the same DQB1* allele associated with the disease in the Moroccan population under study. The analysis of HLA-DRB1* -DQB1* haplotypes revealed a significant association of DRB1*03:01–DQB1*02:01, DRB1*04:05– DQB1*03:02 and T1D in the Moroccan population. These results were in accordance with those found with other ethnic origins [22], probably these haplotypes present the self-epitopes to autoreactive T-cell. The study of protection from T1D confirm the protective effect of the DRB1*15–DQB1*06, which represents the dominant combination associated with protection in most populations [22]. Altogether, our results revealed a genetic
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[31] Rewers A, Babu S, Wang TB, Bugawan TL, Barriga K, Eisenbarth GS, et al. Ethnic differences in the associations between the HLA-DRB1*04 subtypes and type 1 diabetes. Ann N Y Acad Scien 2003;1005(1):301–9. [32] Noble JA, Valdes AM. Genetics of the HLA region in the prediction of type 1 diabetes. Curr Diab Rep 2011;11:533–42. [33] Kanga U, Vaidyanathan B, Jaini R, Menon PS, Mehra NK. HLA haplotypes associated with type 1 diabetes mellitus in North Indian children. Hum Immunol 2004;65(1):47–53. [34] Ikegami H, Kawabata Y, Noso S, Fujisawa T, Ogihara T. Genetics of type 1 diabetes in Asian and Caucasian populations. Diabetes Res Clin Pr 2007;77:116–21. [35] Izaabel H, Garchon H-J, Beaurain G, Biga M, Akhayat O, Bach J-F, et al. Distribution of HLA class II alleles and haplotypes in insulin-dependent Moroccan diabetics. Hum Immunol 1996;49:137. [36] Oumhani K, Canossi A, Piancatelli D, Di Rocco M, Del Beato T, Liberatore G, et al. Sequence-based analysis of the HLA-DRB1 polymorphism in Metalsa Berber and Chaouya Arabic-speaking groups from Morocco. Hum Immunol 2002;63:129–38. [37] Abid Kamoun H, Hmida S, Kaabi H, Abid A, Slimane Houissa H, Maamar M. HLA polymorphism in type 1 diabetes Tunisians. Ann Genet 2002;45:45–50. [38] Ilonen J, Kocova M, Lipponen K, Sukarova-Angelovska E, Jovanovska A, Knip M. HLA-DR-DQ haplotypes and type 1 diabetes in Macedonia. Hum Immunol 2009;70(6):461–3. ¨ , Kor Y, Balat A, et al. Trends in the [39] Keskin M, Aygu¨n A, Pehlivan S, Keskin O frequency of HLA DR-DQ haplotypes among children and adolescents with type 1 diabetes mellitus in the southeast region of Turkey. J Clin Res Pediatr Endocrinol 2012;4(4):189. [40] Skrodeniene E, Marciulionyte D, Padaiga Z, Jasinskiene E, Sadauskaite-Kuehne V, Carani B, et al. HLA class II alleles and haplotypes in Lithuanian children with type 1 diabetes and healthy children (HLA and type 1 diabetes). Medicina (Kaunas) 2010;46(8):505–11. [41] Guerin V, Leniaud L, Pedron B, Guilmin-Cre´pon S, Tubiana-Rufi N, Sterkers G. HLA-associated genetic resistance and susceptibility to type I diabetes in French North Africans and French natives. Tissue Antigens 2007;70(3):214–8.
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PATBIO-3170; No. of Pages 5 Pathologie Biologie xxx (2014) xxx–xxx
Available online at
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Original article
Family-based association study of HLA class II with type 1 diabetes in Moroccans E´tude d’association familiale entre les ge`nes HLA classe II et le diabe`te type 1 dans la population marocaine A. Drissi Bourhanbour a,b,*, N. Benseffaj a,b, S. Ouadghiri a,b, R. Razine c, A. Touzani d, A. Belafraj d, M. Essakalli a,b a
UPR d’immunologie, faculte´ de me´decine et de pharmacie, Rabat Institut, avenue Mohamed Belarbi El Alaoui, BP 6203, 10000 Rabat, Morocco Unite´ d’immunologie, service de transfusion sanguine et d’he´movigilance, hoˆpital d’enfants-Rabat, Rabat Institut, CHU Ibn-Sina, rue Lamfadel Cherkaoui, BP 6527, Rabat, Morocco c Laboratoire de biostatistique, recherche clinique et epide´miologie, faculte´ de me´decine et de pharmacie, Rabat Institut, avenue Mohamed Belarbi El Alaoui, BP 6203, 10000 Rabat, Morocco d Service de diabe´tologie pe´diatrique, hoˆpital d’enfants-Rabat, Rabat Institut, CHU Ibn-Sina, rue Lamfadel Cherkaoui, BP 6527, Rabat, Morocco b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 2 April 2014 Accepted 1st December 2014 Available online xxx
Background. – The T1D is a multifactorial disease; with a strong genetic control. The human leukocyte antigen (HLA) system plays a crucial role in the autoimmune process leading to childhood diabetes. About 440,000 of the childhood population of the world (1.8 billion children under 14 years of age), have type 1 diabetes, and each year an additional 70,000 develop this disorder. The objective of this study was to investigate the distribution of HLA class II in Moroccan families of diabetic children to identify susceptibility alleles of the Moroccan population. Subjects and methods. – We included in this study, Moroccan families who have at least one child with T1D. The age of onset of diabetes was less than 15 years. HLA class II (DRB1* and DQB1*) was carried out by molecular biology techniques (PCR-SSP and PCR-SSO). The FBAT test (family-based association test) was used to highlight the association between T1D and the HLA-DRB1* and -DQB1* polymorphism. Results. – The association of HLA class II (DRB1*, DQB1*) in type 1 diabetes was analyzed in fifty-one Moroccan families, including 90 diabetics. The results revealed that the most susceptible haplotypes are the DRB1*03:01–DQB1*02:01, DRB1*04:05–DQB1*03:02 (Z = 3.674, P = 0.000239; Z = 2.828, P = 0.004678, respectively). And the most protective haplotype is the DRB1*15–DQB1*06. Conclusion. – This is the first family-based association study searching for an association between HLA class II and T1D in a Moroccan population. Despite the different ethnic groups forming Morocco, Moroccan diabetics share the most susceptible and protective HLA haplotypes with other Caucasians populations, specifically the European and Mediterranean populations. ß 2014 Published by Elsevier Masson SAS.
Keywords: HLA class II Haplotypes Type I diabetes Family-based association study
R E´ S U M E´
Mots cle´s : HLA classe II Haplotypes diabe`te type 1 E´tude d’association familiale
Introduction. – Le diabe`te de type 1 (DT1) est une maladie multifactorielle avec un important controˆle ge´ne´tique. Le syste`me HLA joue un roˆle primordial dans le processus auto-immun conduisant au DT1. Dans le monde, environ 440 000 enfants (1,8 milliards d’enfants aˆge´s de moins de 14 ans) ont le DT1 et chaque anne´e 70 000 enfants supple´mentaires de´veloppent ce de´sordre. L’objectif de ce travail e´tait d’e´tudier la distribution des mole´cules HLA de classe II (DRB1*, DQB1*) chez des enfants diabe´tiques pour identifier les alle`les de susceptibilite´ et de protection vis-a`-vis du DT1 chez la population marocaine.
* Corresponding author. UPR d’immunologie, faculte´ de me´decine et de pharmacie, Rabat Institut, avenue Mohamed Belarbi El Alaoui, BP 6203, 10000 Rabat, Morocco. E-mail address: [email protected] (A. Drissi Bourhanbour). http://dx.doi.org/10.1016/j.patbio.2014.12.001 0369-8114/ß 2014 Published by Elsevier Masson SAS.
Please cite this article in press as: Drissi Bourhanbour A, et al. Family-based association study of HLA class II with type 1 diabetes in Moroccans. Pathol Biol (Paris) (2014), http://dx.doi.org/10.1016/j.patbio.2014.12.001
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Mate´riel et me´thodes. – On a inclus dans cette e´tude des familles marocaines qui ont au moins un enfant atteint DT1. L’aˆge d’appariation du diabe`te e´tait infe´rieur a` 15 ans. Le typage HLA classe II (DRB1* et DQB1*) a e´te´ re´alise´ par des techniques de biologie mole´culaire (PCR-SSP et PCR-SSO). Le test FBAT (Family-Based Association Test) a e´te´ utilise´ pour mettre en e´vidence l’association entre DT1 et le polymorphisme HLA-DRB1* et HLA-DQB1*. Re´sultats. – L’association HLA classe II (DRB1*, DQB1*) et le diabe`te type 1 a e´te´ e´tudie´e chez 51 familles marocaines, incluant 90 diabe´tiques. L’analyse des donne´s par FBAT montre que les deux haplotypes associe´s au DT1 dans la population marocaine sont le DRB1*03:01–DQB1*02:01, DRB1*04:05– DQB1*03:02 (Z = 3,674, P = 0, 000239; Z = 2,828, P = 0, 004678, respectivement). L’haplotype le plus protecteur est DRB1*15–DQB1*06. Conclusion. – Meˆme si la population marocaine est forme´e de plusieurs groupes ethniques, elle partage les meˆmes haplotypes HLA de susceptibilite´ et de protection vis-a`-vis du DT1 avec d’autres populations caucasiennes et plus spe´cifiquement les populations europe´enne et me´diterrane´enne. ß 2014 Publie´ par Elsevier Masson SAS.
1. Introduction
2.2. HLA typing
Type 1 diabetes (T1D) is a polygenic autoimmune disease in which the insulin-secreting pancreatic beta cells are selectively destroyed by T cells [1]. Type 1 diabetes is the third most prevalent chronic disease of childhood. About 440,000 of the childhood population of the world (1.8 billion children under 14 years of age), have type 1 diabetes, and each year an additional 70,000 develop this disorder [2]. According to the estimates by the Moroccan Ministry of Health, 100,000 Moroccans have type 1 diabetes, including 10,000 children [3]. The autoimmunity of T1D is provided by the presence of antibodies against islet cells (ICA), insulin (IAA), glutamic acid decarboxylase (GADA), and tyrosine-phosphatase (IA2-Ab). Genome-wide association studies have identified over 40 polymorphisms that are associated with T1D [4]. However, the major susceptibility for T1D has been mapped to the HLA class II genes HLA-DRB1* and -DQB1* [5,6]. Both susceptible and protective DRB1*–DQB1*haplotypes exist in all populations [7] but differences between populations have been shown. A strong association of HLA-DRB1*03–DQB1*02:01 and of -DRB1*04:01/04:02/04:05– DQB1*03:02 haplotypes with T1D have been established among Caucasians [8–10], and DRB1*09:01–DQB1*03:03 haplotype in Japanese patients [11]. The majority of these studies are casecontrol. In our work, we have used a family-based association design. Compared to the case-control study designs, family-based association designs are attractive, since they test for both linkage and association. The family-based association designs can also avoid the potential confounding effects of population stratification by using the parents as controls for the case (the affected offspring) and are convenient in refining linkage findings in family samples [12]. Up to now, the association of T1D with HLA locus was not investigated in Moroccan families of diabetic children. The objective of this study was to investigate the distribution of HLA class II DRB1* and DQB1* alleles in Moroccans families of diabetics children, to identify susceptibility alleles and to refine genetic knowledge of the Moroccan population. The aim is to be able to provide early and adequate care for the subjects at risk.
HLA typing was performed on samples derived from both parents, affected children, and unaffected children in all families. Genomic DNA was first extracted and purified from peripheral blood samples collected in 5% EDTA using a commercial kit (Qiagen). Low resolution HLA class II (DRB1* and DQB1*) typing was performed by polymerase chain reaction-sequence specific primers (PCR-SSP) using micro generic HLA DNA typing trays (One Lambda) according to the manufacturer’s protocol for all families. In a second step, the DRB1* high-resolution typing was done by polymerase chain reaction sequence-specific oligonucleotide (PCR-SSO) for 24 families. Whereas, the DQB1* was genotyped by using PCR-SSP. All analyzes were carried out in the laboratory of immunohistocompatibility of the Ibn-Sina University Hospital of Rabat, Morocco.
2. Subjects and methods 2.1. Subjects We included in this study fifty-one Moroccan families with at least one child with type 1 diabetes. The families were recruited between 2009 and 2011 from the department of endocrinology of the Children’s Hospital of Rabat. They consisted of 38 multiplex-sibs families (at least two affected children) and 13 simplex families (only one diabetic child). They contain a total of 229 individuals among which 90 are diabetics. The median age was 17 (range: 2–41). The age of onset of T1D was less than 15 years. All subjects gave consent to participate in this study which was approved by the Ethics Committee for Biomedical.
2.3. Statistical analysis The family-based association test (FBAT) software was used to evidence association between HLA-DRB1* and -DQB1* polymorphisms and T1D. This method is an extension of the original transmission disequilibrium test (TDT) [13], a genetic analysis conducted on family trios (affected child and both parents), which assesses whether one allele is inherited in affected children at a rate greater than that predicted. The FBAT allows inclusion of data that cannot be analyzed in the strict TDT, such as families that are missing a parent. Advantages of these methodologies over other population-based approaches include that they test for both association and linkage, and are not susceptible to population stratification, which can introduce confounding factors in case-control studies [14]. The FBAT statistic was calculated under an additive model and for bi-allelic (each allele against all others) mode of testing. Positive Z-statistic of single locus and haplotype FBAT indicated that a specific single locus and haplotype was more frequently transmitted to patients with DT1 in informative families than expected under the null hypothesis of no linkage and no association. The negative sign of Z-statistic indicates that the frequency of transmitted genotype is negative association with susceptibility to T1D. Haplotype FBAT software was also used to estimate haplotype frequencies. The probability (P) value was considered significant at a P-value under 0.05.
3. Results 3.1. HLA-DRB1* allele association in Moroccan patients with DT1 Table 1 shows the HLA-DRB1* alleles frequencies in Moroccan families of diabetic children. A total of eight HLA-DRB1* alleles have been identified. The most frequent HLA-DRB1* alleles were HLA-DRB1*04 (32.5%) and -DRB1*03 (27.7%). Indeed, the FBAT analyses showed that the DRB1*04 and DRB1*03 alleles were significantly associated with DT1 (Z = 3.942, P = 0.000081; Z = 3.069, P = 0.002146, respectively). At the opposite, DRB1*11 and DRB1*15 were negatively associated with susceptibility to T1D (Z = –3.207, P = 0.001341; Z = –2.751, P = 0.005944, respectively). 3.2. HLA-DQB1* allele association in Moroccan patients with DT1 The distribution of alleles frequencies of HLA-DQB1* in Moroccan families of diabetic children are shown in Table 2. We
Please cite this article in press as: Drissi Bourhanbour A, et al. Family-based association study of HLA class II with type 1 diabetes in Moroccans. Pathol Biol (Paris) (2014), http://dx.doi.org/10.1016/j.patbio.2014.12.001
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Table 1 The association between the HLA-DRB1* polymorphisms and T1D by family-based association test. DRB1*alleles
Allele frequencies (%)
Number of families
S-E(S)
Var(S)
Z*
P
01 03 04 07 11 13 15 16
6.6 27.7 32.5 9.2 5.1 6.3 8.1 1.9
6 26 26 12 8 8 11 3
1.5 10.444 13.611 4.5 6 5.222 6.5 0.167
3.25 11.58 11.923 5.25 3.5 3.451 5.583 0.972
0.832 3.069 3.942 1.964 3.207 2.811 2.751 0.169
0.4 0.002146 0.000081 0.049535 0.001341 0.04934 0.005944 0.86577
T1D: type 1 diabetes; S-E(S) and Var(S) are the expected value and variance of the test statistic. Z: is the test statistic; P: significance level. * The negative sign of Z-statistic indicates that the frequency of transmitted genotype is negative association with susceptibility to T1D.
Table 2 The association between the HLA-DQB1* polymorphisms and T1D by family-based association test. DQB1*alleles
Allele frequencies (%)
Number of families
S-E(S)
Var(S)
Z*
P
02 03 05 06
34.8 40.5 10 13.5
15 18 11 13
7.167 7.267 1.433 12.5
8.072 7.896 5.079 7.25
2.522 2.586 0.636 4.642
0.011654 0.009707 0.524771 0.00000344
T1D: type 1 diabetes; S-E(S) and Var(S) are the expected value and variance of the test statistic. Z: is the test statistic; P: significance level. * The negative sign of Z-statistic indicates that the frequency of transmitted genotype is negative association with susceptibility to T1D.
have identified four alleles on HLA-DQB1* locus. The highest frequencies of the HLA-DQB1* alleles were observed for DQB1*03 (40.5%), and DQB1*02 (34.8%). A significant association between DQB1*03 and DQB1*02 and DT1 was shown (Z = 2.586, P = 0.009707; Z = 2.522, P = 0.011654, respectively). The DQB1*06 is negatively associated with T1D (Z = –4.64, P = 0.0000344). 3.3. HLA-DRB1* -DQB1* haplotypes associations with DT1 The HLA-DRB1* -DQB1* haplotype frequencies were calculated in Moroccan families of diabetic children (Table 3). The analysis of the haplotypes shows that DRB1*04–DQB1*03 and DRB1*03– DQB1*02 were significantly associated with susceptibility to T1D (Z = 4.435, P = 0.00000922; Z = 3.136, P = 0.001713, respectively). Among the protective DRB1*–DQB1* haplotype, the DRB1*15– DQB1*06 was negatively associated with T1D (Z = –3.153, P = 0. 001616). 3.4. Analysis of DRB1* and DQB1* in four digit resolution by FBAT The analysis shows that DRB1* 03:01, DRB1*04:05, DQB1*02:01, and DQB1*03:02 alleles were associated with susceptibility to T1D (Z = 3.381, P = 0.000723; Z = 0.0000963, P = 4.899; Z = 4.243, P = 0.000022; Z = 2.860, P = 0.0045231, respectively) (Table 4). T1D risk associations were observed with DRB1*03:01– DQB1*02:01 and DRB1*04:05–DQB1*03:02 specific haplotypes
(Z = 3.674, P = 0.000239; Z = 2.828, P = 0.004678, respectively) (Table 5). 4. Discussion T1D is a multifactorial disease, with a strong genetic control. Several publications have shown the effect of the HLA-DRB1* and DQB1* allele polymorphisms, on predisposition to T1D. Identifying the polymorphic amino acid residues that distinguish such alleles has been extensively studied. The highly polymorphic amino acid positions in the DRb1 and DQb1 chains, which mostly form peptide-binding grooves for antigen presentation to CD4+ T-helper cells, are suggested to predispose an individual to T1D [15]. Indeed, numerous studies have shown that DRb1Lys71+ provides the highest relative risk for T1D [16–18] and that the DQb1Asp57– provides an additive effect to DRb1Lys71+ [19]. The Moroccan population is a mixed group that includes Arabic, Berber, and African with a strong influence of various immigrations, mainly Mediterranean [20]. The gene profile of all these populations probably contributed to the Moroccans genetic pool. T1D clusters in families with an overall genetic risk ratio of 15 [21]. This is the first family-based association study searching for an association between HLA class II and T1D in a Moroccan population. HLA-DRB1*03 and -DRB1*04 alleles showed a significant association with T1D in the Moroccan families of diabetic children. This positive association between HLA class II molecules and T1D has been well established in most population of the world [22,23]
Table 3 Most frequent HLA-DRB1* -DQB1* haplotypes in Moroccan families of diabetic children. DRB1*–DQB1* haplotypes
Haplotype frequencies (%)
Number of families
S-E(S)
Var(S)
Z*
P
04–03 03–02 15–06 13–06 07–03 11–03
32.8 30.1 7.3 4.5 2.8 2.7
22 21 8 5 5 4
14 10.034 6.5 –5 3.5 3.433
9.967 3.136 4.25 2.5 1.75 1.679
4.435 3.136 3.153 3.162 2.646 2.65
0.00000922 0.001713 0.001616 0.001565 0.008151 0.008055
T1D: type 1 diabetes; S-E(S) and Var(S) are the expected value and variance of the test statistic. Z: is the test statistic; P: significance level. * The negative sign of Z-statistic indicates that the frequency of transmitted genotype is negative association with susceptibility to T1D.
Please cite this article in press as: Drissi Bourhanbour A, et al. Family-based association study of HLA class II with type 1 diabetes in Moroccans. Pathol Biol (Paris) (2014), http://dx.doi.org/10.1016/j.patbio.2014.12.001
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Table 4 HLA-DRB1* and -DQB1* four digit allele frequencies in Moroccan families of diabetic children. Alleles
DRB1* 03:01 04:05 04:02 07:01 DQB1* 02:01 03:02
Allele frequencies (%)
Number of families
S-E (S)
Var(S)
Z*
P
15.7 17.6 13.7 6.6
12 10 4 4
8 12 4 4
5.6 6 2 2
3.381 4.899 2.828 2.828
0.000723 0.000000963 0.004678 0.004678
19.2 8
10 10
10 6
4.5 4.4
4.243 2.860
0.000022 0.0045231
T1D: type 1 diabetes; S-E(S) and Var(S) are the expected value and variance of the test statistic. Z: is the test statistic; P: significance level. * The negative sign of Z-statistic indicates that the frequency of transmitted genotype is negative association with susceptibility to T1D.
susceptibility for T1D within Moroccan patients. The study clearly indicates an association between DRB1*03:01–DQB1*02:01, DRB1*04:05–DQB1*03:02 haplotype and T1D in our population. These data may serve as a baseline; however, it has to be controlled in a larger population. Furthermore, the number of informative families is limited and high-resolution HLA typing was done for a small number of families. It should be done in all families in order to precise more specific alleles involved in Moroccan diabetics. However, the advantage of our study is that the family-based association study and haplotype analysis has been used. We can conclude that despite the different ethnic groups forming Moroccan population, Moroccan diabetics share the most susceptible and protective HLA haplotypes with other Caucasians populations, including European and Mediterranean populations. Disclosure of interest
Table 5 Most frequent HLA-DRB1*–DQB1* haplotypes four digit allele in Moroccan families of diabetic children. DRB1*–DQB1* Haplotypes
Haplotype frequencies (%)
Number of families
S-E (S)
Var(S)
Z*
P
03:01–02:01 04:05–03:01
13.1 5.6
6 6
6 4
2.667 2
3.674 2.828
0.000239 0.004678
T1D: type 1 diabetes; S-E(S) and Var(S) are the expected value and variance of the test statistic. Z: is the test statistic; P: significance level. * The negative sign of Z-statistic indicates that the frequency of transmitted genotype is negative association with susceptibility to T1D.
and suggest a role for DRB1*03 and DRB1*04 in the susceptibility to T1D. Indeed, these results were found in European, Latin American, Chinese and South African [24–28] populations, as well as in Maghreb countries [29,30]. Among DRB1*03 and DRB1*04 alleles, DRB1*03:01 and DRB1*04:05 are consistently associated with T1D [22,23,31,32]. In this work, we have identified the same allelic specificities in Moroccan families of diabetic children. HLADRB1*09 was reported in Asian populations [33,34] and even in a Moroccan previous study [35]. In our case, this association was not found. The DRB1*09 allele is very rare in North Africa, and in Morocco it was described only in a Berber population of the north [36]. Our population has only one family coming from this origin and this may explain why this allele was not found. DRB1*15 allele was universally described to be a protective factor against T1D [22,34]. In our study, besides DRB1*15, the DRB1*11 alleles were underrepresented in the Moroccan families of diabetic children. A similar observation has already been reported in Tunisian, Macedonian, Turkish, Lithuanian and French populations [37–41]. We were also interested in the role of HLA-DQB1* alleles in T1D susceptibility since DQB1*02:01 and DQB1*03:02 were the most frequently reported as involved in susceptibility to risk factor for T1D in multiple populations [22,23]. We have identified the same DQB1* allele associated with the disease in the Moroccan population under study. The analysis of HLA-DRB1* -DQB1* haplotypes revealed a significant association of DRB1*03:01–DQB1*02:01, DRB1*04:05– DQB1*03:02 and T1D in the Moroccan population. These results were in accordance with those found with other ethnic origins [22], probably these haplotypes present the self-epitopes to autoreactive T-cell. The study of protection from T1D confirm the protective effect of the DRB1*15–DQB1*06, which represents the dominant combination associated with protection in most populations [22]. Altogether, our results revealed a genetic
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Please cite this article in press as: Drissi Bourhanbour A, et al. Family-based association study of HLA class II with type 1 diabetes in Moroccans. Pathol Biol (Paris) (2014), http://dx.doi.org/10.1016/j.patbio.2014.12.001