pii: sp-00758-14
http://dx.doi.org/10.5665/sleep.4344
SHORT NOTE
HLA-DQ Allele Competition in Narcolepsy: Where is the Evidence? Mehdi Tafti, PhD Center for Integrative Genomics (CIG) University of Lausanne, Lausanne, Switzerland; Center for Investigation and Research in Sleep (CIRS), Centre Hospitalier Universitaire Vaudois (CHUV), Lausanne, Switzerland
Functional DQ molecules are indeed formed by heterodimerization of a DQα with a DQβ chain. The heterodimerization is not random and depends on the relative affinity between the two proteins, as well as their respective level of expression. The affinity is primarily dictated by structural constraints. Not only DQB1 gene is structurally polymorphic, but also the expression of its various alleles varies according to their promoter polymorphism so that not all alleles are expressed at the same level. For instance, DQB1*03:01 is expressed at substantially higher levels than DQB1*05:01 and DQB1*06:02.6 Unfortunately, the relative expression level of all DQB1 alleles (in homozygous and heterozygous states) has not been determined (neither at mRNA nor at protein level), so that the relative proportion of differently expressed heterodimers remains unknown. Moreover, Dr. Mignot’s group reported that in homozygous DQB1*06:02 subjects, the expression of this allele is increased by approximately 50%,7 and not 100% as postulated by their model. Additionally, the level of DQB1 expression varies according to the cell type (not all antigen presenting cells express the same level of DQB1 molecules) and condition (e.g., increased expression by cytokines).6 Obviously, the expression level of different DQ molecules is regulated in a complex manner rather than the simple gene dosage as proposed by the allele competition model. As recognized by Ollila et al., the allele competition model does not explain why DQB1*03:01 increases the risk of narcolepsy. The model fails to explain either our finding (which escaped the attention of Ollila et al.) of a protective role for DQB1*02, which has been also recently reported by van der Heide and coworkers.8 Nor does it take into account our suggestion that DQB1*06:09 that heterodimerizes with the susceptibility DQA1*01:02 might also be protective. Although we agree that our finding that DQB1*06:09 is protective has diminished statistical robustness because this allele is rare, we reject the suggestion that this occurred because of population stratification. We included only patients and controls of European origin and analyzed the allele frequencies of each country separately. Finally, protective HLA alleles act in a dominant and not competitive manner.9 DQB1*06:02/06:03 subjects are not at a lower relative risk for narcolepsy, they are actually strongly protected against narcolepsy (by at least 5-fold and not 2 as anticipated by the allele competition model). The same holds true for the protective role of DQB1*06:02 in type 1 diabetes and of other HLA alleles in many other HLA-associated disorders without any allele competition. In conclusion, the allele competition model is not based on experimental data and suffers several exceptions concerning DQB1 alleles (i.e., DQB1*03:01, DQB1*02, and DQB1*06:09). Even in the case of heterozygous DQB1*06:02/DQB1*05:01, 06:01, and 06:03 subjects, a direct quantification of different
Published in this issue is a comment by Ollila et al.1 on our study2 in which they disagree with our interpretation and instead propose a so-called allele competition model as a better interpretation of our results. Their comment raises several points that I would like to clarify. First, Ollila et al.1 disagree with the title of our study “DQB1 locus alone explains most of risk and protection in narcolepsy” because we did not look at other HLA loci, particularly the DQA1 locus. We concede that the title appears somewhat ambiguous. However, the term “alone” in our title should not imply that DQB1 is the sole locus but that “by looking at this locus only, one can explain an overwhelming portion of risk and protection.” Obviously, due to the tight linkage disequilibrium within the HLA Class II region, the respective contribution of a single locus is difficult to single out. For instance, in a given population of western European origin as in our study, over 99% of subjects carry a single cis haplotype: DRB5*01:01, DRB1*15:01, DQA1*01:02, DQB1*06:02. Nevertheless, DQA1*01:02, DQB1*06:02 can be found in rare DRB1*15:01 negative cases (4 out of 682 patients [0.6%] in our study). Together with more frequent African American patients with such haplotypes, it was concluded that DQ more than DR molecules might be critically involved in narcolepsy.3–5 The same argument holds true for the DQA1 and DQB1 loci. While DQA0102 heterodimerizes with several DQB1 molecules, DQB10602 almost exclusively (in European populations) heterodimerizes with DQA10102, strongly suggesting that DQB1 more than DQA1 molecules might be critically involved. The allele competition model posits that the relative risk is proportional to the amount of the DQA10102/DQB10602 (DQ0602) heterodimer expressed by antigen presenting cells. Accordingly, the model explains why doubling the amount of this heterodimer in homozygous subjects doubles the risk while heterozygotes carrying DQB1*05:01, 06:01, and 06:03 in trans are protected because they form additional DQ heterodimers and reduce the availability of DQ0602 molecules. In other terms, and as reported in their comment by reshuffling our original data, relative risks can be basically 1 (neutral), 2 (homozygous), or 0.5 (heterozygous with DQB1*05:01, 06:01, and 06:03). These theoretical assumptions lack sound experimental evidence on transdimerization and allele-dependent surface expression of DQ molecules, and they ignore several exceptions. Submitted for publication December, 2014 Accepted for publication December, 2014 Address correspondence to: Mehdi Tafti, PhD, University of Lausanne, Center for Integrative Geromics, Le Genopode, Lausanne, 1015 Switzerland; Tel: (41) 21-692-3971; Fax: (41) 21-692-3965; Email: mehdi.tafti@ unil.ch SLEEP, Vol. 38, No. 1, 2015
153
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DISCLOSURE STATEMENT Dr. Tafti has indicated no financial conflicts of interest.
DQ heterodimers in relevant antigen presenting cells is needed to support the model. Rather, a simple HLA-DQB1 typing assay is sufficient to account for almost all of the susceptibility to and protection from the disease, although it is evident that the antigen-presenting specificity for putative pathogenic peptides of the DQ antigens is dictated by α/β heterodimers. Finally I also would like to comment on the caution raised about our “overinterpretation of the relative risk of 251, in the absence of verified hypocretin deficiency in all of our patients.” First, if the authors claim that the narcolepsy-cataplexy diagnosis is valid only if the CSF hypocretin 1 level is measured, then over 80% of reported cases in the literature are not valid (including their own previous reports). Secondly, we have previously published hypocretin 1 levels in the largest-ever reported homogeneous sample of narcolepsy with cataplexy patients (294 European patients) and showed that over 96% had either low or undetectable levels,10 and believe this figure is representative of our overall sample. Obviously, measuring the CSF hypocretin 1 level in 1,218 patients in the presence of well-characterized narcolepsy with cataplexy is unrealistic, and this has never been done in any previous HLA study. Also the allegation that our “clinicians used HLA typing to confirm diagnosis” is pure speculation. Indeed over 55% of our reported patients had HLA typing by my laboratory or the laboratory of Dr. Tiercy for our published paper,2 and the typing was performed one to several years after the diagnosis. No diagnosis has been changed based on HLA results. Therefore, we consider that our risk estimate is the most accurate one available and would suggest that the previously reported lower risks might be due to the inclusion of less well-characterized patients without cataplexy. Finally, I had difficulty finding the reference concerning evidence that HCRT56–68 and HCRT87–99 are the “culprit antigens” in narcolepsy.
SLEEP, Vol. 38, No. 1, 2015
CITATION Tafti M. HLA-DQ allele competition in narcolepsy: where is the evidence? SLEEP 2015;38(1):153–154. REFERENCES
1. Ollila HM, Fernandez-Vina M, Mignot E. HLA-DQ allele competition in narcolepsy: a comment on Tafti et al. DQB1 locus alone explains most of the risk and protection in narcolepsy with cataplexy in Europe. Sleep 2015;38:147–51. 2. Tafti M, Hor H, Dauvilliers Y, et al. DQB1 locus alone explains most of the risk and protection in narcolepsy with cataplexy in Europe. Sleep 2014;37:19–25. 3. Matsuki K, Grumet FC, Lin X, et al. DQ (rather than DR) gene marks susceptibility to narcolepsy. Lancet 1992;339:1052. 4. Mignot E, Kimura A, Lattermann A, et al. Extensive HLA class II studies in 58 non-DRB1*15 (DR2) narcoleptic patients with cataplexy. Tissue Antigens 1997;49:329–41. 5. Mignot E, Lin L, Rogers W, et al. Complex HLA-DR and -DQ interactions confer risk of narcolepsy-cataplexy in three ethnic groups. Am J Hum Genet 2001;68:686–99. 6. Ferstl B, Zacher T, Lauer B, Blagitko-Dorfs N, Carl A, Wassmuth R. Allele-specific quantification of HLA-DQB1 gene expression by realtime reverse transcriptase-polymerase chain reaction. Genes Immun 2004;5:405–16. 7. Weiner Lachmi K, Lin L, Kornum BR, et al. DQB1*06:02 allele-specific expression varies by allelic dosage, not narcolepsy status. Hum Immunol 2012;73:405–10. 8. van der Heide A, Verduijn W, Haasnoot GW, Drabbels JJ, Lammers GJ, Claas FH. HLA dosage effect in narcolepsy with cataplexy. Immunogenetics 2014 Oct 3. [Epub ahead of print]. 9. Tsai S, Santamaria P. MHC Class II polymorphisms, autoreactive T-cells, and autoimmunity. Front Immunol 2013;4:321. 10. Luca G, Haba-Rubio J, Dauvilliers Y, et al. Clinical, polysomnographic and genome-wide association analyses of narcolepsy with cataplexy: a European Narcolepsy Network study. J Sleep Res 2013;22:482–95.
154
HLA-DQ Allele Competition—Tafti
http://dx.doi.org/10.5665/sleep.4344
SHORT NOTE
HLA-DQ Allele Competition in Narcolepsy: Where is the Evidence? Mehdi Tafti, PhD Center for Integrative Genomics (CIG) University of Lausanne, Lausanne, Switzerland; Center for Investigation and Research in Sleep (CIRS), Centre Hospitalier Universitaire Vaudois (CHUV), Lausanne, Switzerland
Functional DQ molecules are indeed formed by heterodimerization of a DQα with a DQβ chain. The heterodimerization is not random and depends on the relative affinity between the two proteins, as well as their respective level of expression. The affinity is primarily dictated by structural constraints. Not only DQB1 gene is structurally polymorphic, but also the expression of its various alleles varies according to their promoter polymorphism so that not all alleles are expressed at the same level. For instance, DQB1*03:01 is expressed at substantially higher levels than DQB1*05:01 and DQB1*06:02.6 Unfortunately, the relative expression level of all DQB1 alleles (in homozygous and heterozygous states) has not been determined (neither at mRNA nor at protein level), so that the relative proportion of differently expressed heterodimers remains unknown. Moreover, Dr. Mignot’s group reported that in homozygous DQB1*06:02 subjects, the expression of this allele is increased by approximately 50%,7 and not 100% as postulated by their model. Additionally, the level of DQB1 expression varies according to the cell type (not all antigen presenting cells express the same level of DQB1 molecules) and condition (e.g., increased expression by cytokines).6 Obviously, the expression level of different DQ molecules is regulated in a complex manner rather than the simple gene dosage as proposed by the allele competition model. As recognized by Ollila et al., the allele competition model does not explain why DQB1*03:01 increases the risk of narcolepsy. The model fails to explain either our finding (which escaped the attention of Ollila et al.) of a protective role for DQB1*02, which has been also recently reported by van der Heide and coworkers.8 Nor does it take into account our suggestion that DQB1*06:09 that heterodimerizes with the susceptibility DQA1*01:02 might also be protective. Although we agree that our finding that DQB1*06:09 is protective has diminished statistical robustness because this allele is rare, we reject the suggestion that this occurred because of population stratification. We included only patients and controls of European origin and analyzed the allele frequencies of each country separately. Finally, protective HLA alleles act in a dominant and not competitive manner.9 DQB1*06:02/06:03 subjects are not at a lower relative risk for narcolepsy, they are actually strongly protected against narcolepsy (by at least 5-fold and not 2 as anticipated by the allele competition model). The same holds true for the protective role of DQB1*06:02 in type 1 diabetes and of other HLA alleles in many other HLA-associated disorders without any allele competition. In conclusion, the allele competition model is not based on experimental data and suffers several exceptions concerning DQB1 alleles (i.e., DQB1*03:01, DQB1*02, and DQB1*06:09). Even in the case of heterozygous DQB1*06:02/DQB1*05:01, 06:01, and 06:03 subjects, a direct quantification of different
Published in this issue is a comment by Ollila et al.1 on our study2 in which they disagree with our interpretation and instead propose a so-called allele competition model as a better interpretation of our results. Their comment raises several points that I would like to clarify. First, Ollila et al.1 disagree with the title of our study “DQB1 locus alone explains most of risk and protection in narcolepsy” because we did not look at other HLA loci, particularly the DQA1 locus. We concede that the title appears somewhat ambiguous. However, the term “alone” in our title should not imply that DQB1 is the sole locus but that “by looking at this locus only, one can explain an overwhelming portion of risk and protection.” Obviously, due to the tight linkage disequilibrium within the HLA Class II region, the respective contribution of a single locus is difficult to single out. For instance, in a given population of western European origin as in our study, over 99% of subjects carry a single cis haplotype: DRB5*01:01, DRB1*15:01, DQA1*01:02, DQB1*06:02. Nevertheless, DQA1*01:02, DQB1*06:02 can be found in rare DRB1*15:01 negative cases (4 out of 682 patients [0.6%] in our study). Together with more frequent African American patients with such haplotypes, it was concluded that DQ more than DR molecules might be critically involved in narcolepsy.3–5 The same argument holds true for the DQA1 and DQB1 loci. While DQA0102 heterodimerizes with several DQB1 molecules, DQB10602 almost exclusively (in European populations) heterodimerizes with DQA10102, strongly suggesting that DQB1 more than DQA1 molecules might be critically involved. The allele competition model posits that the relative risk is proportional to the amount of the DQA10102/DQB10602 (DQ0602) heterodimer expressed by antigen presenting cells. Accordingly, the model explains why doubling the amount of this heterodimer in homozygous subjects doubles the risk while heterozygotes carrying DQB1*05:01, 06:01, and 06:03 in trans are protected because they form additional DQ heterodimers and reduce the availability of DQ0602 molecules. In other terms, and as reported in their comment by reshuffling our original data, relative risks can be basically 1 (neutral), 2 (homozygous), or 0.5 (heterozygous with DQB1*05:01, 06:01, and 06:03). These theoretical assumptions lack sound experimental evidence on transdimerization and allele-dependent surface expression of DQ molecules, and they ignore several exceptions. Submitted for publication December, 2014 Accepted for publication December, 2014 Address correspondence to: Mehdi Tafti, PhD, University of Lausanne, Center for Integrative Geromics, Le Genopode, Lausanne, 1015 Switzerland; Tel: (41) 21-692-3971; Fax: (41) 21-692-3965; Email: mehdi.tafti@ unil.ch SLEEP, Vol. 38, No. 1, 2015
153
HLA-DQ Allele Competition—Tafti
DISCLOSURE STATEMENT Dr. Tafti has indicated no financial conflicts of interest.
DQ heterodimers in relevant antigen presenting cells is needed to support the model. Rather, a simple HLA-DQB1 typing assay is sufficient to account for almost all of the susceptibility to and protection from the disease, although it is evident that the antigen-presenting specificity for putative pathogenic peptides of the DQ antigens is dictated by α/β heterodimers. Finally I also would like to comment on the caution raised about our “overinterpretation of the relative risk of 251, in the absence of verified hypocretin deficiency in all of our patients.” First, if the authors claim that the narcolepsy-cataplexy diagnosis is valid only if the CSF hypocretin 1 level is measured, then over 80% of reported cases in the literature are not valid (including their own previous reports). Secondly, we have previously published hypocretin 1 levels in the largest-ever reported homogeneous sample of narcolepsy with cataplexy patients (294 European patients) and showed that over 96% had either low or undetectable levels,10 and believe this figure is representative of our overall sample. Obviously, measuring the CSF hypocretin 1 level in 1,218 patients in the presence of well-characterized narcolepsy with cataplexy is unrealistic, and this has never been done in any previous HLA study. Also the allegation that our “clinicians used HLA typing to confirm diagnosis” is pure speculation. Indeed over 55% of our reported patients had HLA typing by my laboratory or the laboratory of Dr. Tiercy for our published paper,2 and the typing was performed one to several years after the diagnosis. No diagnosis has been changed based on HLA results. Therefore, we consider that our risk estimate is the most accurate one available and would suggest that the previously reported lower risks might be due to the inclusion of less well-characterized patients without cataplexy. Finally, I had difficulty finding the reference concerning evidence that HCRT56–68 and HCRT87–99 are the “culprit antigens” in narcolepsy.
SLEEP, Vol. 38, No. 1, 2015
CITATION Tafti M. HLA-DQ allele competition in narcolepsy: where is the evidence? SLEEP 2015;38(1):153–154. REFERENCES
1. Ollila HM, Fernandez-Vina M, Mignot E. HLA-DQ allele competition in narcolepsy: a comment on Tafti et al. DQB1 locus alone explains most of the risk and protection in narcolepsy with cataplexy in Europe. Sleep 2015;38:147–51. 2. Tafti M, Hor H, Dauvilliers Y, et al. DQB1 locus alone explains most of the risk and protection in narcolepsy with cataplexy in Europe. Sleep 2014;37:19–25. 3. Matsuki K, Grumet FC, Lin X, et al. DQ (rather than DR) gene marks susceptibility to narcolepsy. Lancet 1992;339:1052. 4. Mignot E, Kimura A, Lattermann A, et al. Extensive HLA class II studies in 58 non-DRB1*15 (DR2) narcoleptic patients with cataplexy. Tissue Antigens 1997;49:329–41. 5. Mignot E, Lin L, Rogers W, et al. Complex HLA-DR and -DQ interactions confer risk of narcolepsy-cataplexy in three ethnic groups. Am J Hum Genet 2001;68:686–99. 6. Ferstl B, Zacher T, Lauer B, Blagitko-Dorfs N, Carl A, Wassmuth R. Allele-specific quantification of HLA-DQB1 gene expression by realtime reverse transcriptase-polymerase chain reaction. Genes Immun 2004;5:405–16. 7. Weiner Lachmi K, Lin L, Kornum BR, et al. DQB1*06:02 allele-specific expression varies by allelic dosage, not narcolepsy status. Hum Immunol 2012;73:405–10. 8. van der Heide A, Verduijn W, Haasnoot GW, Drabbels JJ, Lammers GJ, Claas FH. HLA dosage effect in narcolepsy with cataplexy. Immunogenetics 2014 Oct 3. [Epub ahead of print]. 9. Tsai S, Santamaria P. MHC Class II polymorphisms, autoreactive T-cells, and autoimmunity. Front Immunol 2013;4:321. 10. Luca G, Haba-Rubio J, Dauvilliers Y, et al. Clinical, polysomnographic and genome-wide association analyses of narcolepsy with cataplexy: a European Narcolepsy Network study. J Sleep Res 2013;22:482–95.
154
HLA-DQ Allele Competition—Tafti
