Retrovirus-Like Long-Terminal Repeat DQ-LTR13 and Genetic Susceptibility to Type 1 Diabetes and Autoimmune Addison’s Disease
http://www.100md.com
糖尿病学杂志 2005年第3期
1 Department of Internal Medicine, University of Perugia, Perugia, Italy
2 Department of Molecular Medicine, Karolinska Institute, Stockholm, Sweden
3 Department of Gynecologic, Obstetrics and Paediatric Sciences, University of Perugia, Perugia, Italy
4 Department of Medical and Surgical Sciences, University of Padova, Padova, Italy
5 Division of Endocrinology, Department of Internal Medicine, University of Turin, Turin, Italy
6 Institute of Endocrine Sciences, University of Milan, Ospedale Maggiore IRCCS, Milan, Italy
ABSTRACT
Controversial data are available on the association between the retrovirus-like long-terminal repeat (LTR) DQ-LTR13 and genetic susceptibility to type 1 diabetes and other autoimmune diseases. We analyzed DNA samples from 315 type 1 diabetic patients, 166 autoimmune Addison’s disease (AAD) patients, 1,054 healthy subjects, and 144 families of type 1 diabetic offspring. DQ-LTR13 was more frequent among patients than healthy subjects (Pc < 0.0006), and a preferential transmission of DQB10302-LTR13+ from parents to type 1 diabetic offspring was observed. DQ-LTR13 was in linkage disequilibrium (LD) with DQB10302 but not DQB10201. The presence of DQ-LTR13 increased the odds ratio of DQB10302 2.9- to 3.2-fold for type 1 diabetes and AAD. DRB10403 was absent in all of the 169 DRB104-positive patients but present in 27% (34 of 127) DRB104-positive healthy subjects (Pc < 0.001). DQ-LTR13 was detected in 1 of 34 (3%) DRB10403-positive healthy subjects and 36 of 93 (39%) individuals carrying another DRB104 allele (Pc = 0.002). Multivariate logistic regression analysis revealed that DQ-LTR13 is not independently associated with type 1 diabetes and AAD after correction for DQB10302 and DRB10403. Conversely, DQB10201, DQB10302, DRB10401, and DRB10403 were all significantly associated with disease risk also after correction for DQ-LTR13. We provide conclusive evidence that the genetic association of DQ-LTR13 with type 1 diabetes and AAD is primarily due to a LD with DQB10302 and DRB10403.
Human endocrine autoimmune diseases, which include type 1 diabetes, autoimmune Addison’s disease (AAD), thyroid diseases, autoimmune hypophysitis, hypoparathyroidism, and premature ovarian failure, are complex genetic traits with major contribution of HLA gene polymorphism (1,2). HLA-DRB103-DQA10501-DQB10201 is positively associated with most endocrine autoimmune diseases (2,3), while DRB104-DQA10301-DQB10302 is preferentially associated with type 1 diabetes (3), although it has been also found to be associated with AAD (4,5).
The highest genetic risk for childhood type 1 diabetes identified to date is that of the DRB103-DQA10501-DQB10201/DRB104-DQA10301-DQB10302 genotype, which is associated with clinical signs of the disease in 1:25eC1:20 subjects (6). Genetic risk conferred by HLA class II haplotypes is modulated by the DRB104 subtype (7). DRB10403 confers dominant protection from type 1 diabetes even when part of the high-risk DRB103-DQA10501-DQB10201/DRB104-DQA10301-DQB10302 genotype(7eC9).
At least 8% of the human genome consists of retrovirus-like elements (10), corresponding to 50eC1,000 proviral copies of different full-length human endogenous retrovirus elements per haploid human genome, with up to 25,000 copies of solitary long-terminal repeats (LTRs) distributed on most chromosomes. One LTR (DQ-LTR3), integrated 15 kb upstream of the DQB1 gene, was initially found to be associated with genetic susceptibility for type 1 diabetes (11). However, some DQB1 alleles are characterized by a massive deletion of >5 kb, with absence of LTR3, and DQ-LTR3 is associated with human diseases only because of its presence with some DQB1 alleles (12). Another retrovirus-like element (LTR13) is located much closer to the DQB1 gene (1.3 kb upstream) (13), outside the deletion in the 5' flanking region. LTR13s are primate specific and do not display sequence similarity to any known retroviral LTRs but have a structure that resembles that of other retroviral LTRs with a potential promoter, polyadenylation signal, and a tandemly repeated 53-bp enhancer-like sequence (14). An association between presence of DQ-LTR13 and risk for both type 1 diabetes and AAD has preliminarily been reported (15,16), and it has been hypothesized that DQ-LTR13 might play a role in the pathogenesis of human autoimmunity (17). However, it is still unclear to what extent this association results from linkage disequilibrium (LD) with DRB1 and DQB1 alleles. Pascual et al. (12,18) reported that DQ-LTR13 was invariably associated with DQB10302, 0303, and 0402 in the Spanish population, while Krach et al. (19) reported the presence of DQ-LTR13 in German and Belgian subjects negative for these three alleles.
With the aim of testing whether DQ-LTR13 is independently associated with endocrine autoimmune diseases and/or modulates genetic risk conferred by DQB1 alleles, we analyzed a large set of genomic DNA samples from type 1 diabetic patients, AAD patients, and healthy control subjects from continental Italy, as well as complete families of type 1 diabetic children. Both type 1 diabetes and AAD were studied because of common HLA associations, similarities in pathogenesis, and previous reports on the role of DQ-LTR13 in genetic susceptibility for these diseases (15,16).
To address the problem of the association of DQ-LTR13 with DQB1 alleles in the Italian population, we first studied 200 healthy control subjects completely genotyped for HLA-DR and DQ and found 30 positive for DQ-LTR13 (15%). In subjects carrying DQ-LTR13, 0301 was the most frequent HLA-DQB1 allele (detected in 15 subjects [50%]), in line with its frequent occurrence in the Italian population (46% of our healthy control subjects). DQB10302 was found in five (17%) and DQB10303 in three (10%) DQ-LTR13-positive individuals. No DQ-LTR13-positive sub-jects were positive for DQB10402. A total of eight subjects (27%) were negative for DQB10301, 0302, and 0303, and, in this subgroup, 0201 was the most frequent DQB1 allele (detected in five subjects). In three DQ-LTR13-positive subjects, the DQB10501-0602, DQB10502-0503, and DQB10501-0604 genotypes were detected. Thus, in continental Italy, DQ-LTR13 is not exclusively associated with DQB10302, 0303, or 0402 but can be found to be associated with DQB10201,0301, and other DQB1 alleles.
We then expanded our analysis to determine the frequencies of high-risk alleles and haplotypes in 1,056 healthy control subjects, 315 type 1 diabetic patients, and 166 AAD patients. DQ-LTR13 was detected in 32% of type 1 diabetic patients and 28% of AAD patients compared with 17% of healthy control subjects (P < 0.0001 and P = 0.0005, respectively) (Table 1).
A permutation test using the EM algorithm showed that DQ-LTR13 is in LD with DQA10301-DQB10302 (exact P and 2 P < 0.0001) but not with DRB103-DQA10501-DQB10201. None of the tested variables showed signifi-cant deviations from Hardy-Weinberg equilibrium. Coexistence of DRB104-DQA10301-DQB10302 and DQ-LTR13 was demonstrated in 22% of type 1 diabetic patients and 20% of AAD patients compared with only 2% of healthy control subjects (Table 1). Frequency of DQ-LTR13 among DQA10301-DQB10302-positive individuals increased significantly from 26% (22 of 86) in healthy control subjects to 77% (69 of 90) in type 1 diabetic and 82% (33 of 40) in AAD patients (P < 0.0001). Accordingly, presence of DQ-LTR13 increased the odds ratio (OR) of DQA10301-DQB10302 2.9- to 3.2-fold. Frequencies of simultaneous presence of DRB103-DQA10501-DQB10201 and DQ-LTR13 were similar to those predicted by random association (Table 1).
A total of 192 parents of type 1 diabetic offspring were found to be heterozygous for HLA-DR-DQ-LTR haplotypes. DRB104-DQA10301-DQB10302 was more often transmitted to affected offspring than expected, only in the presence of DQ-LTR13 (PTDT = 0.0003; TDT, transmission distortion test) (Table 2). No significant transmission was observed for DQA10301-DQB10302-LTR13eC, DRB103-DQA10501-DQB10201-LTR13eC, and X-LTR13+, in agreement with the results of the German-Belgian study (15).
A major genetic factor influencing susceptibility for type 1 diabetes is DRB10403, which has not been taken into consideration in previous works on DQ-LTR13 (15,16,18,19). We analyzed the frequency of different DRB104 sub-types and the distribution of DQ-LTR13 among these subtypes (Table 3). DRB10403 was absent in type 1 diabetic and AAD patients but was the most frequent DRB104 allele in healthy control subjects (27% of DRB104-positive individuals) (P < 0.0001). This high frequency of 0403 in healthy control subjects was somewhat unexpected, as much lower frequencies have been observed in other European countries (7eC9). The high prevalence of 0403 among DRB104-positive healthy control subjects, along with the low prevalence of high-risk class II haplotypes, can in part explain the lower incidence of type 1 diabetes in continental Italy compared with North European countries (20).
DRB10404 was not significantly increased in Italian AAD patients (Table 3), which is at variance with previous reports from the U.S. (4) and Norway (5). DRB10404 was not skewed toward non-DQB10302 alleles in our population, as it was found in 5 of 40 (12%) DQB10302-positive and 2 of 16 (12%) non-DQB10302 AAD patients. Similarly, DRB10404 was detected in 15 of 86 (17%) DQB10302-positive and 8 of 41 (19%) non-DQB10302 healthy control subjects. Differences in DRB10403 frequency in the general population may be responsible for the discrepancy between our study and other studies from different geographical areas.
DQ-LTR13 was detected in 1 of 34 (2.9%) DRB10403-positive healthy control subjects, a frequency that was lower than expected, as 36 of 93 (39%) subjects carrying another DRB104 allele were positive for this element (Pc = 0.002). This could not be explained by a low frequency of DQB10302, as 25 of 34 (74%) DRB10403-positive healthy control subjects were carrying DQA10301-DQB10302. The reasons why DQ-LTR13 is preferentially associated with some HLA haplotypes are not completely elucidated. We speculate that DRB10403 is in LD with a DQB10302 allele not carrying this element.
The low frequency of DQ-LTR13 in DRB10403-positive individuals is an important novel finding and raises the possibility that the association of DQ-LTR13 with type 1 diabetes and AAD is secondary to the negative association of DRB10403 with human diseases. To address this question, we performed multivariate logistic regression analysis. When DRB10403 was not included in the model, DQA10501-DQB10201 (P < 0.001, OR 5.91 [95% CI 4.40eC7.94]), DQA10301-DQB10302 (P < 0.001, 3.91 [2.69eC5.69]), and DQ-LTR13 (P = 0.014, 1.52 [1.09eC2.13]) were all independently associated with type 1 diabetes after correction for age and sex. However, inclusion of DRB10403 substantially modified the outcome of logistic regression, as DQ-LTR13 no longer entered the model (Table 4). Inclusion of DRB10401 confirmed the secondary association of DQ-LTR13 with type 1 diabetes and showed the independent association of DRB10401 with type 1 diabetes. Similar results were observed with AAD patients (Table 4). Analysis of OR associated with DQ-LTR13 revealed a reduction of genetic risk by 51eC55% (in AAD and type 1 diabetes, respectively) when DQB10302 was included and by 78eC80% (in type 1 diabetes and AAD, respectively) when DRB10403 was also included. Thus, at least 80% of genetic association between DQ-LTR13 and endocrine autoimmune diseases is explained by LD with DRB1 and DQB1 alleles.
In conclusion, although the risk conferred by DQB10302 is increased by the presence of DQ-LTR13, presumably because of the absence of DRB10403 in subjects positive for DQ-LTR13, this element did not provide any independent genetic risk in our study, and association of DQ-LTR13 with both type 1 diabetes and AAD appears to be secondary to LD with the DQB1 and DRB1 alleles. Although a minimal genetic contribution of DQ-LTR13 cannot be completely excluded, it is unlikely that this retrovirus-like element plays a relevant independent role in genetic susceptibility for type 1 diabetes or other endocrine autoimmune diseases.
RESEARCH DESIGN AND METHODS
Genomic DNA was obtained from 315 type 1 diabetic patients, 166 AAD patients, and 1,056 healthy control subjects. Type 1 diabetic patients (median age at diagnosis 13 years [range 1eC49], M:F ratio 1.37) were consecutively recruited between 1 January 1993 and 31 March 2004 and were all residents of Umbria (central Italy). Diagnosis was made according to National Diabetes Data Group criteria (21). Islet cell autoantibodies were detected in >95% of type 1 diabetic patients. Our type 1 diabetic group included 85% of incident cases in Umbria during the period 1993eC2003 who were <30 years of age. Type 1 diabetic case subjects who were >30 years of age at disease onset were consecutively recruited at the Department of Internal Medicine, Perugia, Italy. No type 1 diabetic patients were positive for adrenal autoantibodies.
For transmission analysis, 144 families of type 1 diabetic children were also studied. In each family, genomic DNA samples from the diabetic proband (median age at diagnosis 9 years [range 1eC19], M:F ratio 1.29) and both parents were analyzed.
Between January 1998 and April 2004, the Italian Addison Network (22) enrolled 316 patients with primary adrenal insufficiency. According to a recent update of diagnostic criteria, based on autoantibody levels, imaging data, and biochemical parameters (22), 217 cases (69%) were classified as AAD. Of these 217 cases, DNA was available for 166. All 166 patients included in this study (median age at diagnosis 34 years [range 8eC73], M:F ratio 0.63) were resident in central-northern Italy. Of these 166 patients, 78 (47%) had other autoimmune diseases. More specifically, 24 AAD patients (14%) also had type 1 diabetes. No APS I patient was included in our study.
Blood samples collected between March 1994 and February 2004 were available for 1,056 unrelated healthy control subjects (median age 32 years [range 4eC63], M:F ratio 1.16) with no family history of endocrine autoimmune diseases. All subjects gave their informed consent for the study.
HLA-DR-DQ genotyping.
HLA-DR-DQ genotyping was performed by SSO-dot blot analysis with modifications of a previously described method (23) using sequence-specific oligonucleotides that were 3' end labeled with digoxigenin (Roche Diagnostics, Monza, Italy). A chemiluminescent signal, generated by using alkaline phosphatase-labeled anti-digoxigenin (Roche) and CSPD (Roche), was measured in a microplate scintillation/luminescence counter (TopCount NXT; Packard Instrument, Meriden, CT). Each membrane contained 10 control samples with known HLA genotype. HLA-DRB104 subtyping was performed by PCR-SSP (sequence-specific primer) according to Zetterquist and Olerup (24).
Detection of DQ-LTR13.
Presence/absence of DQ-LTR13 was tested by a nested PCR approach as described by Pani et al. (16). External primers (5'-GGTCAGAAGTAATGTTTGCC-3' and 5'-TAATGGTTATAAAGCAATTAGAAC-3') were used to generate a 1,057-bp fragment in the presence or a 51-bp fragment in the absence of DQ-LTR13. The results were confirmed by using a pair of internal primers (5'-AGTAATGTTTGCCAGTCTGTAG-3' and 5'-AATTAGAACAATGCCTGGTGTG-3') that generates a 1,035-bp fragment. A third PCR with primers 5'-CCAGTCTCAGGTGCTCTAGAA-3' and 5'-AGAAGCATTTCCTAGGTCCTGA-3' generated a 1,530-bp fragment in the presence and a 532-bp fragment in the absence of DQ-LTR13. PCR fragments were separated in a 1.5% agarose gel and stained with ethidium bromide.
Statistical analysis.
Differences in allele/haplotype/genotype frequencies between patients and healthy control subjects were tested by the 2 method. Yates’ correction or Fisher’s exact test was used when necessary. Probability values were corrected (Pc) for the number of comparisons according to the number of HLA-DRB1, -DQA1, and -DQB1 alleles or haplotypes observed or for the degree of freedom for DQ-LTR13 allele- and genotype-wise comparisons. Association of the dichotomous variables presence/absence of DQA10501-DQB10201, DRB10401, DRB10403, DQA10301-DQB10302, and DQ-LTR13 with endocrine autoimmune diseases, and dependence on other variables such as sex and age at diagnosis, was tested by multivariate logistic regression analysis using SPSS for Windows (SPSS, Chicago, IL). The transmission distortion test (25) was used to detect preferential transmission of DQA1-DQB1-LTR13-extended haplotypes to type 1 diabetic offspring. The 2 test was used to compare different subgroups and observed transmissions to random expected transmissions of 50%. Pairwise LD was tested by permutation test using the EM algorithm. Deviations from Hardy-Weinberg equilibrium were tested by comparison of observed and expected genotype frequencies. A P (or Pc) value <0.05 was considered significant in all tests.
ACKNOWLEDGMENTS
This study was supported in part by a grant from the Ministry of University and Scientific Research project "Morbo di Addison: forme cliniche, eziopatogenesi e storia naturale," PRIN 20001063439 (to C.B. and A.F.), and funds from the Svenska Institutet, Stockholm, Sweden (to G.G.).
The following members of the Italian Addison Network contributed to the collection of data and blood samples from patients with primary adrenal insufficiency: B. Ambrosi (Milan), A. Angeli (Turin), G. Arnaldi (Ancona), E. Arvat (Turin), A. Baccarelli (Milan), L. Barbetta (Milan), P. Beck-Peccoz (Milan), A. Bellastella (Naples), A. Bizzarro (Naples), M. Boscaro (Ancona), F. Cavagnini (Milan), C. Dal Pre?(Padova), A. De Bellis (Naples), F. Dotta (Rome), E. Ghigo (Turin), S. Laureti (Perugia), F. Loree (Siena), M. Mannelli (Florence), F. Mantero (Padova), G. Mantovani (Milan), P. Paccotti (Turin), F. Pecori-Gilardi (Milan), R. Perniola (Lecce), F. Santeusanio (Perugia), M. Terzolo (Turin), C. Tiberti (Rome), P. Toja (Milan), M. Torlontano (S. Giovanni Rotondo), V. Toscano (Rome), V. Trischitta (S. Giovanni Rotondo), R. Zanchetta (Padova).
The following members of the Umbria Type 1 Diabetes Registry contributed to the collection of data and blood samples from patients with type 1 diabetes: A. Angeli (Gubbio), E. Baiocchi (Assisi), D. Belladonna (Todi), R. Bellanti (Citte?di Castello), G. Berioli (Spoleto), M. Bracaccia (Orvieto), G. Campanelli (Citte?di Castello), G. Campolo (Todi), C. Cicioni (Terni), S. Coaccioli (Terni), A. Coletti (Gualdo Tadino), M. Cozzari (Cascia), G. Di Matteo (Perugia), G. Divizia (Spoleto), Ad. Falorni (Perugia), G. Fracassi (Marsciano), A. Fragasso (Foligno), A. Frascarelli (Assisi), S. Gagliardo (Narni), G. Giannico (Marsciano), A. Lilli (Gubbio), E. Madeo (Citte?della Pieve), G. Mancini (Orvieto), R. Marcacci (Castiglione del Lago), C. Marino (Gubbio), M. Napolitano (Foligno), G. Pennoni (Gualdo Tadino), E. Picchio (Perugia), M.L. Picchio (Gualdo Tadino), S. Pocciati (Foligno), F. Santeusanio (Perugia), M. Scattoni (Narni), E. Vignai (Citte?della Pieve).
AAD, autoimmune Addison’s disease; LD, linkage disequilibrium; LTR, long-terminal repeat
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2 Department of Molecular Medicine, Karolinska Institute, Stockholm, Sweden
3 Department of Gynecologic, Obstetrics and Paediatric Sciences, University of Perugia, Perugia, Italy
4 Department of Medical and Surgical Sciences, University of Padova, Padova, Italy
5 Division of Endocrinology, Department of Internal Medicine, University of Turin, Turin, Italy
6 Institute of Endocrine Sciences, University of Milan, Ospedale Maggiore IRCCS, Milan, Italy
ABSTRACT
Controversial data are available on the association between the retrovirus-like long-terminal repeat (LTR) DQ-LTR13 and genetic susceptibility to type 1 diabetes and other autoimmune diseases. We analyzed DNA samples from 315 type 1 diabetic patients, 166 autoimmune Addison’s disease (AAD) patients, 1,054 healthy subjects, and 144 families of type 1 diabetic offspring. DQ-LTR13 was more frequent among patients than healthy subjects (Pc < 0.0006), and a preferential transmission of DQB10302-LTR13+ from parents to type 1 diabetic offspring was observed. DQ-LTR13 was in linkage disequilibrium (LD) with DQB10302 but not DQB10201. The presence of DQ-LTR13 increased the odds ratio of DQB10302 2.9- to 3.2-fold for type 1 diabetes and AAD. DRB10403 was absent in all of the 169 DRB104-positive patients but present in 27% (34 of 127) DRB104-positive healthy subjects (Pc < 0.001). DQ-LTR13 was detected in 1 of 34 (3%) DRB10403-positive healthy subjects and 36 of 93 (39%) individuals carrying another DRB104 allele (Pc = 0.002). Multivariate logistic regression analysis revealed that DQ-LTR13 is not independently associated with type 1 diabetes and AAD after correction for DQB10302 and DRB10403. Conversely, DQB10201, DQB10302, DRB10401, and DRB10403 were all significantly associated with disease risk also after correction for DQ-LTR13. We provide conclusive evidence that the genetic association of DQ-LTR13 with type 1 diabetes and AAD is primarily due to a LD with DQB10302 and DRB10403.
Human endocrine autoimmune diseases, which include type 1 diabetes, autoimmune Addison’s disease (AAD), thyroid diseases, autoimmune hypophysitis, hypoparathyroidism, and premature ovarian failure, are complex genetic traits with major contribution of HLA gene polymorphism (1,2). HLA-DRB103-DQA10501-DQB10201 is positively associated with most endocrine autoimmune diseases (2,3), while DRB104-DQA10301-DQB10302 is preferentially associated with type 1 diabetes (3), although it has been also found to be associated with AAD (4,5).
The highest genetic risk for childhood type 1 diabetes identified to date is that of the DRB103-DQA10501-DQB10201/DRB104-DQA10301-DQB10302 genotype, which is associated with clinical signs of the disease in 1:25eC1:20 subjects (6). Genetic risk conferred by HLA class II haplotypes is modulated by the DRB104 subtype (7). DRB10403 confers dominant protection from type 1 diabetes even when part of the high-risk DRB103-DQA10501-DQB10201/DRB104-DQA10301-DQB10302 genotype(7eC9).
At least 8% of the human genome consists of retrovirus-like elements (10), corresponding to 50eC1,000 proviral copies of different full-length human endogenous retrovirus elements per haploid human genome, with up to 25,000 copies of solitary long-terminal repeats (LTRs) distributed on most chromosomes. One LTR (DQ-LTR3), integrated 15 kb upstream of the DQB1 gene, was initially found to be associated with genetic susceptibility for type 1 diabetes (11). However, some DQB1 alleles are characterized by a massive deletion of >5 kb, with absence of LTR3, and DQ-LTR3 is associated with human diseases only because of its presence with some DQB1 alleles (12). Another retrovirus-like element (LTR13) is located much closer to the DQB1 gene (1.3 kb upstream) (13), outside the deletion in the 5' flanking region. LTR13s are primate specific and do not display sequence similarity to any known retroviral LTRs but have a structure that resembles that of other retroviral LTRs with a potential promoter, polyadenylation signal, and a tandemly repeated 53-bp enhancer-like sequence (14). An association between presence of DQ-LTR13 and risk for both type 1 diabetes and AAD has preliminarily been reported (15,16), and it has been hypothesized that DQ-LTR13 might play a role in the pathogenesis of human autoimmunity (17). However, it is still unclear to what extent this association results from linkage disequilibrium (LD) with DRB1 and DQB1 alleles. Pascual et al. (12,18) reported that DQ-LTR13 was invariably associated with DQB10302, 0303, and 0402 in the Spanish population, while Krach et al. (19) reported the presence of DQ-LTR13 in German and Belgian subjects negative for these three alleles.
With the aim of testing whether DQ-LTR13 is independently associated with endocrine autoimmune diseases and/or modulates genetic risk conferred by DQB1 alleles, we analyzed a large set of genomic DNA samples from type 1 diabetic patients, AAD patients, and healthy control subjects from continental Italy, as well as complete families of type 1 diabetic children. Both type 1 diabetes and AAD were studied because of common HLA associations, similarities in pathogenesis, and previous reports on the role of DQ-LTR13 in genetic susceptibility for these diseases (15,16).
To address the problem of the association of DQ-LTR13 with DQB1 alleles in the Italian population, we first studied 200 healthy control subjects completely genotyped for HLA-DR and DQ and found 30 positive for DQ-LTR13 (15%). In subjects carrying DQ-LTR13, 0301 was the most frequent HLA-DQB1 allele (detected in 15 subjects [50%]), in line with its frequent occurrence in the Italian population (46% of our healthy control subjects). DQB10302 was found in five (17%) and DQB10303 in three (10%) DQ-LTR13-positive individuals. No DQ-LTR13-positive sub-jects were positive for DQB10402. A total of eight subjects (27%) were negative for DQB10301, 0302, and 0303, and, in this subgroup, 0201 was the most frequent DQB1 allele (detected in five subjects). In three DQ-LTR13-positive subjects, the DQB10501-0602, DQB10502-0503, and DQB10501-0604 genotypes were detected. Thus, in continental Italy, DQ-LTR13 is not exclusively associated with DQB10302, 0303, or 0402 but can be found to be associated with DQB10201,0301, and other DQB1 alleles.
We then expanded our analysis to determine the frequencies of high-risk alleles and haplotypes in 1,056 healthy control subjects, 315 type 1 diabetic patients, and 166 AAD patients. DQ-LTR13 was detected in 32% of type 1 diabetic patients and 28% of AAD patients compared with 17% of healthy control subjects (P < 0.0001 and P = 0.0005, respectively) (Table 1).
A permutation test using the EM algorithm showed that DQ-LTR13 is in LD with DQA10301-DQB10302 (exact P and 2 P < 0.0001) but not with DRB103-DQA10501-DQB10201. None of the tested variables showed signifi-cant deviations from Hardy-Weinberg equilibrium. Coexistence of DRB104-DQA10301-DQB10302 and DQ-LTR13 was demonstrated in 22% of type 1 diabetic patients and 20% of AAD patients compared with only 2% of healthy control subjects (Table 1). Frequency of DQ-LTR13 among DQA10301-DQB10302-positive individuals increased significantly from 26% (22 of 86) in healthy control subjects to 77% (69 of 90) in type 1 diabetic and 82% (33 of 40) in AAD patients (P < 0.0001). Accordingly, presence of DQ-LTR13 increased the odds ratio (OR) of DQA10301-DQB10302 2.9- to 3.2-fold. Frequencies of simultaneous presence of DRB103-DQA10501-DQB10201 and DQ-LTR13 were similar to those predicted by random association (Table 1).
A total of 192 parents of type 1 diabetic offspring were found to be heterozygous for HLA-DR-DQ-LTR haplotypes. DRB104-DQA10301-DQB10302 was more often transmitted to affected offspring than expected, only in the presence of DQ-LTR13 (PTDT = 0.0003; TDT, transmission distortion test) (Table 2). No significant transmission was observed for DQA10301-DQB10302-LTR13eC, DRB103-DQA10501-DQB10201-LTR13eC, and X-LTR13+, in agreement with the results of the German-Belgian study (15).
A major genetic factor influencing susceptibility for type 1 diabetes is DRB10403, which has not been taken into consideration in previous works on DQ-LTR13 (15,16,18,19). We analyzed the frequency of different DRB104 sub-types and the distribution of DQ-LTR13 among these subtypes (Table 3). DRB10403 was absent in type 1 diabetic and AAD patients but was the most frequent DRB104 allele in healthy control subjects (27% of DRB104-positive individuals) (P < 0.0001). This high frequency of 0403 in healthy control subjects was somewhat unexpected, as much lower frequencies have been observed in other European countries (7eC9). The high prevalence of 0403 among DRB104-positive healthy control subjects, along with the low prevalence of high-risk class II haplotypes, can in part explain the lower incidence of type 1 diabetes in continental Italy compared with North European countries (20).
DRB10404 was not significantly increased in Italian AAD patients (Table 3), which is at variance with previous reports from the U.S. (4) and Norway (5). DRB10404 was not skewed toward non-DQB10302 alleles in our population, as it was found in 5 of 40 (12%) DQB10302-positive and 2 of 16 (12%) non-DQB10302 AAD patients. Similarly, DRB10404 was detected in 15 of 86 (17%) DQB10302-positive and 8 of 41 (19%) non-DQB10302 healthy control subjects. Differences in DRB10403 frequency in the general population may be responsible for the discrepancy between our study and other studies from different geographical areas.
DQ-LTR13 was detected in 1 of 34 (2.9%) DRB10403-positive healthy control subjects, a frequency that was lower than expected, as 36 of 93 (39%) subjects carrying another DRB104 allele were positive for this element (Pc = 0.002). This could not be explained by a low frequency of DQB10302, as 25 of 34 (74%) DRB10403-positive healthy control subjects were carrying DQA10301-DQB10302. The reasons why DQ-LTR13 is preferentially associated with some HLA haplotypes are not completely elucidated. We speculate that DRB10403 is in LD with a DQB10302 allele not carrying this element.
The low frequency of DQ-LTR13 in DRB10403-positive individuals is an important novel finding and raises the possibility that the association of DQ-LTR13 with type 1 diabetes and AAD is secondary to the negative association of DRB10403 with human diseases. To address this question, we performed multivariate logistic regression analysis. When DRB10403 was not included in the model, DQA10501-DQB10201 (P < 0.001, OR 5.91 [95% CI 4.40eC7.94]), DQA10301-DQB10302 (P < 0.001, 3.91 [2.69eC5.69]), and DQ-LTR13 (P = 0.014, 1.52 [1.09eC2.13]) were all independently associated with type 1 diabetes after correction for age and sex. However, inclusion of DRB10403 substantially modified the outcome of logistic regression, as DQ-LTR13 no longer entered the model (Table 4). Inclusion of DRB10401 confirmed the secondary association of DQ-LTR13 with type 1 diabetes and showed the independent association of DRB10401 with type 1 diabetes. Similar results were observed with AAD patients (Table 4). Analysis of OR associated with DQ-LTR13 revealed a reduction of genetic risk by 51eC55% (in AAD and type 1 diabetes, respectively) when DQB10302 was included and by 78eC80% (in type 1 diabetes and AAD, respectively) when DRB10403 was also included. Thus, at least 80% of genetic association between DQ-LTR13 and endocrine autoimmune diseases is explained by LD with DRB1 and DQB1 alleles.
In conclusion, although the risk conferred by DQB10302 is increased by the presence of DQ-LTR13, presumably because of the absence of DRB10403 in subjects positive for DQ-LTR13, this element did not provide any independent genetic risk in our study, and association of DQ-LTR13 with both type 1 diabetes and AAD appears to be secondary to LD with the DQB1 and DRB1 alleles. Although a minimal genetic contribution of DQ-LTR13 cannot be completely excluded, it is unlikely that this retrovirus-like element plays a relevant independent role in genetic susceptibility for type 1 diabetes or other endocrine autoimmune diseases.
RESEARCH DESIGN AND METHODS
Genomic DNA was obtained from 315 type 1 diabetic patients, 166 AAD patients, and 1,056 healthy control subjects. Type 1 diabetic patients (median age at diagnosis 13 years [range 1eC49], M:F ratio 1.37) were consecutively recruited between 1 January 1993 and 31 March 2004 and were all residents of Umbria (central Italy). Diagnosis was made according to National Diabetes Data Group criteria (21). Islet cell autoantibodies were detected in >95% of type 1 diabetic patients. Our type 1 diabetic group included 85% of incident cases in Umbria during the period 1993eC2003 who were <30 years of age. Type 1 diabetic case subjects who were >30 years of age at disease onset were consecutively recruited at the Department of Internal Medicine, Perugia, Italy. No type 1 diabetic patients were positive for adrenal autoantibodies.
For transmission analysis, 144 families of type 1 diabetic children were also studied. In each family, genomic DNA samples from the diabetic proband (median age at diagnosis 9 years [range 1eC19], M:F ratio 1.29) and both parents were analyzed.
Between January 1998 and April 2004, the Italian Addison Network (22) enrolled 316 patients with primary adrenal insufficiency. According to a recent update of diagnostic criteria, based on autoantibody levels, imaging data, and biochemical parameters (22), 217 cases (69%) were classified as AAD. Of these 217 cases, DNA was available for 166. All 166 patients included in this study (median age at diagnosis 34 years [range 8eC73], M:F ratio 0.63) were resident in central-northern Italy. Of these 166 patients, 78 (47%) had other autoimmune diseases. More specifically, 24 AAD patients (14%) also had type 1 diabetes. No APS I patient was included in our study.
Blood samples collected between March 1994 and February 2004 were available for 1,056 unrelated healthy control subjects (median age 32 years [range 4eC63], M:F ratio 1.16) with no family history of endocrine autoimmune diseases. All subjects gave their informed consent for the study.
HLA-DR-DQ genotyping.
HLA-DR-DQ genotyping was performed by SSO-dot blot analysis with modifications of a previously described method (23) using sequence-specific oligonucleotides that were 3' end labeled with digoxigenin (Roche Diagnostics, Monza, Italy). A chemiluminescent signal, generated by using alkaline phosphatase-labeled anti-digoxigenin (Roche) and CSPD (Roche), was measured in a microplate scintillation/luminescence counter (TopCount NXT; Packard Instrument, Meriden, CT). Each membrane contained 10 control samples with known HLA genotype. HLA-DRB104 subtyping was performed by PCR-SSP (sequence-specific primer) according to Zetterquist and Olerup (24).
Detection of DQ-LTR13.
Presence/absence of DQ-LTR13 was tested by a nested PCR approach as described by Pani et al. (16). External primers (5'-GGTCAGAAGTAATGTTTGCC-3' and 5'-TAATGGTTATAAAGCAATTAGAAC-3') were used to generate a 1,057-bp fragment in the presence or a 51-bp fragment in the absence of DQ-LTR13. The results were confirmed by using a pair of internal primers (5'-AGTAATGTTTGCCAGTCTGTAG-3' and 5'-AATTAGAACAATGCCTGGTGTG-3') that generates a 1,035-bp fragment. A third PCR with primers 5'-CCAGTCTCAGGTGCTCTAGAA-3' and 5'-AGAAGCATTTCCTAGGTCCTGA-3' generated a 1,530-bp fragment in the presence and a 532-bp fragment in the absence of DQ-LTR13. PCR fragments were separated in a 1.5% agarose gel and stained with ethidium bromide.
Statistical analysis.
Differences in allele/haplotype/genotype frequencies between patients and healthy control subjects were tested by the 2 method. Yates’ correction or Fisher’s exact test was used when necessary. Probability values were corrected (Pc) for the number of comparisons according to the number of HLA-DRB1, -DQA1, and -DQB1 alleles or haplotypes observed or for the degree of freedom for DQ-LTR13 allele- and genotype-wise comparisons. Association of the dichotomous variables presence/absence of DQA10501-DQB10201, DRB10401, DRB10403, DQA10301-DQB10302, and DQ-LTR13 with endocrine autoimmune diseases, and dependence on other variables such as sex and age at diagnosis, was tested by multivariate logistic regression analysis using SPSS for Windows (SPSS, Chicago, IL). The transmission distortion test (25) was used to detect preferential transmission of DQA1-DQB1-LTR13-extended haplotypes to type 1 diabetic offspring. The 2 test was used to compare different subgroups and observed transmissions to random expected transmissions of 50%. Pairwise LD was tested by permutation test using the EM algorithm. Deviations from Hardy-Weinberg equilibrium were tested by comparison of observed and expected genotype frequencies. A P (or Pc) value <0.05 was considered significant in all tests.
ACKNOWLEDGMENTS
This study was supported in part by a grant from the Ministry of University and Scientific Research project "Morbo di Addison: forme cliniche, eziopatogenesi e storia naturale," PRIN 20001063439 (to C.B. and A.F.), and funds from the Svenska Institutet, Stockholm, Sweden (to G.G.).
The following members of the Italian Addison Network contributed to the collection of data and blood samples from patients with primary adrenal insufficiency: B. Ambrosi (Milan), A. Angeli (Turin), G. Arnaldi (Ancona), E. Arvat (Turin), A. Baccarelli (Milan), L. Barbetta (Milan), P. Beck-Peccoz (Milan), A. Bellastella (Naples), A. Bizzarro (Naples), M. Boscaro (Ancona), F. Cavagnini (Milan), C. Dal Pre?(Padova), A. De Bellis (Naples), F. Dotta (Rome), E. Ghigo (Turin), S. Laureti (Perugia), F. Loree (Siena), M. Mannelli (Florence), F. Mantero (Padova), G. Mantovani (Milan), P. Paccotti (Turin), F. Pecori-Gilardi (Milan), R. Perniola (Lecce), F. Santeusanio (Perugia), M. Terzolo (Turin), C. Tiberti (Rome), P. Toja (Milan), M. Torlontano (S. Giovanni Rotondo), V. Toscano (Rome), V. Trischitta (S. Giovanni Rotondo), R. Zanchetta (Padova).
The following members of the Umbria Type 1 Diabetes Registry contributed to the collection of data and blood samples from patients with type 1 diabetes: A. Angeli (Gubbio), E. Baiocchi (Assisi), D. Belladonna (Todi), R. Bellanti (Citte?di Castello), G. Berioli (Spoleto), M. Bracaccia (Orvieto), G. Campanelli (Citte?di Castello), G. Campolo (Todi), C. Cicioni (Terni), S. Coaccioli (Terni), A. Coletti (Gualdo Tadino), M. Cozzari (Cascia), G. Di Matteo (Perugia), G. Divizia (Spoleto), Ad. Falorni (Perugia), G. Fracassi (Marsciano), A. Fragasso (Foligno), A. Frascarelli (Assisi), S. Gagliardo (Narni), G. Giannico (Marsciano), A. Lilli (Gubbio), E. Madeo (Citte?della Pieve), G. Mancini (Orvieto), R. Marcacci (Castiglione del Lago), C. Marino (Gubbio), M. Napolitano (Foligno), G. Pennoni (Gualdo Tadino), E. Picchio (Perugia), M.L. Picchio (Gualdo Tadino), S. Pocciati (Foligno), F. Santeusanio (Perugia), M. Scattoni (Narni), E. Vignai (Citte?della Pieve).
AAD, autoimmune Addison’s disease; LD, linkage disequilibrium; LTR, long-terminal repeat
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