- Email: [email protected]
The Genetic Basis of Alopecia Areata: HLA Associations with Patchy Alopecia Areata Versus Alopecia Totalis and Alopecia Universalis
The Genetic Basis of Alopecia Areata: HLA Associations with Patchy Alopecia Areata Versus Alopecia Totalis and Alopecia Universalis Beth W. Colombe, Calvin D. Lou,* and Vera H. Price²
Thomas Jefferson University Department of Medicine and Hospital, Philadelphia, Pennsylvania, U.S.A.; *University of California San Francisco Department of Surgery; ²University of California San Francisco Department of Dermatology, San Francisco, California, U.S.A.
Many diseases, notably those having a strong autoimmune component, have been shown to have an association with speci®c human leukocyte antigens (HLA). The molecular basis for this genetic association with disease is the fact that HLA bind and present peptides derived from self and foreign protein antigens to the immune system for recognition and activation of the immune response. Previous studies with heterogeneous groups of alopecia areata (AA) patients have suggested associations with some HLA class I and class II antigens. For this study we selected only patients with long-standing disease and strati®ed them into two groups by strict de®nitions of duration and extent of disease: those with patchy AA and those with either alopecia totalis (AT) or alopecia universalis (AU). The patients were tissue
typed for HLA class II antigens by biomolecular methods that provided antigen discrimination at an allele level. More than 80% of all of the AA patients typed were positive for the antigen DQB1*03 (DQ3), suggesting that this antigen is a marker for general susceptibility to AA. In addition, two other antigens were found signi®cantly increased in frequency only in the group of AT/AU patients, DRB1*0401 (DR4) and DQB1*0301(DQ7). This strongly suggests that the two clinical types of AA, namely patchy AA versus AT/AU, can be distinguished by a genetically based predisposition to extent of disease. Key words: genetic susceptibility to AA/histocompatibility. Journal of Investigative Dermatology Symposium Proceedings 4:216± 219, 1999
T
THE ASSOCIATION OF DISEASES WITH SPECIFIC HLA
here is consensus that alopecia areata (AA) is a disease with a strong autoimmune component. A number of genes that in¯uence the susceptibility and severity of AA have been described in animal models (Zhang and Oliver, 1994; Sundberg et al, 1995). In humans, it is also evident that an immune response modulates the condition of the hair follicles in affected individuals; e.g., activated T cells in®ltrate affected skin sites (Todes-Taylor et al, 1984; Khoury et al, 1992), cytokines in¯uence follicular cycling (McDonagh et al, 1995), and autoantibodies to cellular components have been demonstrated (Tobin and Orentreich, 1994). Compelling evidence has accumulated that the inheritance of speci®c human leukocyte antigens (HLA) can predispose some individuals to a given disease, frequently a disease of autoimmune character such as diabetes mellitus, psoriasis, thyroiditis, multiple sclerosis, or rheumatoid arthritis (Tiwari, 1985). It is not surprising therefore that there is evidence that HLA are an important genetic in¯uence in AA.
The relationship between HLA and disease has only become clear during the past decade as biomolecular methods have revealed the molecular structure of the HLA molecule and its role in the immune response (Bjorkman et al, 1987; Brodsky and Gugliardi, 1991). HLA are the products of approximately a dozen closely linked genes located on chromosome 6 in a region known as the major histocompatibility complex (MHC). These antigens, abundant on the surface of most cells, are a functional component of the immune response. HLA molecules can bind both exogenous and endogenous antigenic peptide fragments in a surface groove and present them to T cells for recognition and subsequent activation of immune system effector mechanisms. In autoimmunity, it is presumed that a self-derived antigen peptide or a peptide that mimics self (molecular mimicry; Oldstone, 1987) is being presented to self T cells, resulting in self destructive responses (Lechler, 1994). The MHC-HLA genes comprise the most polymorphic genetic system of the human. The polymorphism resides in the variation in the sequence of DNA bases in and around the peptide antigen binding groove of the HLA molecule (Brown et al, 1993). Functionally, the structure of the groove varies with the individual HLA antigen and thus affects the speci®city of peptide binding (Stern et al, 1994). Therefore, for example, certain HLA antigens may have a high binding af®nity for a given autoantigenic peptide and may confer increased susceptibility to a process of autoimmune destruction in certain diseases, or to a successful immune resistance to a pathogen
Manuscript received January 11, 1999; revised April 8, 1999; accepted for publication April 9, 1999. Reprint requests to: Dr. Beth W. Colombe, Immunogenetics and Tissue Typing Laboratory, Thomas Jefferson University Hospital, 111 S 11th St. Room 8245 Gibbon Bldg., Philadelphia, PA 19107. Email: Beth. [email protected] Abbreviations: AA, alopecia areata; AT, alopecia totalis; AU, alopecia universalis; HLA, human leukocyte antigen(s). 1087-0024/99/$14.00
´ Copyright # 1999 by The Society for Investigative Dermatology, Inc. 216
VOL. 4, NO. 3, DECEMBER 1999
GENETIC BASIS OF ALOPECIA AREATA
(Nepom et al, 1992). Our working hypothesis in looking for an HLA association for AA is that a limited number of HLA have the capacity to preferentially bind a restricted number of immunodominant AA peptides. The molecular complex of AA peptide plus HLA can then be recognized by T cell clones leading to disease pathogenesis. Any individual having inherited the speci®c HLA will have increased susceptibility for AA. Previous serologic-based studies of possible HLA class I (HLA-A, -B, and -C loci) associations with AA (Kianto et al, 1977; HachamZadeh et al, 1981; Averbakh and Pospelo, 1986; Orecchia et al, 1987) have failed to show consistency or signi®cance for any particular HLA; however, several population studies exploring HLA class II antigen (HLA-DR and -DQ loci) associations (Mikesell et al, 1986; Frentz et al, 1986; Orecchia, 1987; Odum et al, 1990; Morling et al, 1991; Zhang et al, 1991; Duvic et al, 1991; Welsh et al, 1994; Colombe et al, 1995; Price et al, 1996) using both serologic and DNA techniques have implicated DR4, DR5, and DQ3 as likely susceptibility antigens. It is notable that many of these studies saw trends that failed to achieve statistical signi®cance. Possible reasons for the lack of signi®cance include the small numbers of patients studied, the use of serologic typing to de®ne the HLA class II antigens and the heterogeneity of extent and duration of disease in the patient groups. DEFINITION OF TWO GROUPS OF AA PATIENTS BASED UPON EXTENT AND DURATION OF DISEASE Building upon this previous work on HLA associations with AA, our group undertook a study of AA patients strati®ed by a strict de®nition of extent and duration of disease. We selected patients with long-standing disease because we hypothesized that they had an established pattern for their disease presentation. Our intent was to minimize heterogeneity among the patients. All patients were over 18 y of age and of Caucasian ethnicity. We de®ned three groups of patients: Group I: Patients newly diagnosed with AA for 6 mo or less, with hair loss of any extent. The future duration and extent of their disease is unknown. Group I is a random group of patients and serves as a control group for patients in Groups II and III who have disease of speci®ed extent and duration. Group II: Patients with greater than 25% scalp hair loss for 18 mo or longer but never with AT/AU. Group III: Patients with 100% scalp hair loss with or without body hair loss, and duration of AT/AU greater than 2 y at any time during their lives.
Table I. HLA associated with long-standing AA HLA: p values (corrected)
Patient group
DQB1*3
I (mixed) II (patchy AA) III (AT/AU)
NS 1.5 3 10±2 6.3 3 10±6
DQB1*0301 DRB1*1104 DRB1*0401 NS NS 1.2 3 10±2
4.1 3 10±9 1.8 3 10±3 3.3 3 10±8
NS NS 1.0 3 10±3
A normal control group was composed of North American Whites from data taken from the 11th International Histocompatibility Workshop (Tsuji et al, 1992). Patients were typed for HLA-DR and -DQ antigens by molecular methods, mainly by the method of sequence speci®c priming. This technique allows for de®nition of HLA at the allele level. The frequencies of HLA class II antigens were compiled separately for each group. Statistical treatment of the HLA frequency data: HLA frequencies and p-values were determined by chi square analysis of twoby-two contingency tables. The p values were corrected by multiplying each by 126, a factor equal to the number of HLA class II alleles typed for (63) multiplied by the number of comparisons made (2). These data were corrected by this stringent method because we wanted to establish the HLA associations de novo for each of our clinically de®ned AA patient groups. Normally, once an HLA association is published for a disease, such corrections are no longer necessary. RESULTS AND DISCUSSION Frequencies of HLA in patients with long-standing AA A total of 283 patients have been typed for HLA-DR and -DQ antigens; 96 patients had patchy AA (referred to as Group II); 152 patients had either AT or AU (referred to as Group III); 35 patients had AA of recent diagnosis and random extent of disease (referred to as Group I). Four speci®c HLA alleles were found to occur with a statistically signi®cant increase in frequency, namely DQB1*03 (DQ3); DQB1*0301 (DQ7), a subtype of DQB1*03; DRB1*1104 (DR11); DRB1*0401 (DR4). Table I shows the p values attained after correcting the data for the number of HLA class II alleles typed for. The broad antigen DQB1*03, known as antigen DQ3 by serology, is signi®cant for both the patchy AA and AT/AU groups, whereas DQB1*0301 (DQ7 by serology), a subtype (allele) of DQB1*03, is signi®cantly elevated only in the AT/AU patients. In addition to DQB1*0301, the HLA allele, DRB1*0401, is found with signi®cantly increased frequency uniquely in the AT/AU group of patients, suggesting that these two alleles may serve as special genetic markers for susceptibility for the more severe manifestations of AA. Most striking is the highly signi®cant increase in the occurrence of DRB1*1104, a subtype of DR11, in all groups of AA patients. An increase in this relatively rare allele has also been noted in AA patients by Duvic and colleagues (Welsh et al, 1994). Table II summarizes the frequencies of these four HLA in the three patient groups and in a control group of normal North American Caucasians. There is a clear trend for all AA patients to have the DQB1*03 antigen with an overall frequency of more than 80%, although the size of Group I is too small (N = 35) to achieve statistical signi®cance by our strict criteria. The DR11 subtype DRB1*1104 is found in all patient groups with an average frequency of 16%. Although not characteristic of the majority of AA patients, DRB1*1104 is more than 10 times more frequent in patients than in the control population. Although DRB1*0401 and DQB1*0301 appear to have comparable frequencies in Groups II and III, these antigens are signi®cantly increased only in the AT/ AU group (Group III) once the proper statistical tests are applied (Table I).
Table II. Frequencies of HLA associated with long-standing patchy AA and long-standing AT/AU Patient group Controls I (mixed) II (patchy AA) III (AT/AU)
217
HLA frequencies (%) (N)
DQB1*03
DQB1*0301
DRB1*1104
DRB1*0401
(DQ 191; DR 286) (35) (96) (152)
48.2
32.0
1.0
13.2
82.2 79.2 87.6
54.2 52.0 56.6
22.8 10.4 17.2
17.2 25.0 32.2
218
COLOMBE ET AL
JID SYMPOSIUM PROCEEDINGS
These HLA frequency data strongly suggest that there is both a general and a group-speci®c HLA association with AA: the broad antigen DQB1*03 and the allele DRB1*1104 serve as markers of general susceptibility to AA, whereas other speci®c HLA alleles further differentiate the two groups of long-standing AA patients that are distinguished by extent of hair loss (patchy AA versus AT/ AU). The ®nding of the general association of DQB1*03 with AA con®rms that of Welsh et al (1994) who reported the same 80% incidence of DQB1*03 in a group of 85 white European AA patients. We ®nd that the more severe AT/AU form of the disease is associated with the presence of the inherited HLA alleles DRB1*0401 and DQB1*0301, strongly suggesting that a genetic predisposition for disease is related to the presence of certain HLA antigens. Previous studies have found increased frequency of these same antigens. DR4 has been found associated with long-standing and severe disease in a study of 22 Danish AA patients (Frentz, 1986), and DQB1*0301 (DQ7) has been previously reported to be signi®cantly increased in a cohort of 20 Danish AA patients (Morling et al, 1991) and in a group of 85 North American white AA patients with both patchy and extensive disease of extended duration (Welsh et al, 1994). DR5 (DR11) was found to be associated with susceptibility to long standing disease and with early onset of disease in a group of 127 Italian AA patients (Orecchia et al, 1987). Possible mechanisms for susceptibility are discussed in the Conclusions. Follow up on patients from Group I Group I patients were a group of AA patients diagnosed for only 6 mo or less and therefore of uncertain future duration; they could have had any extent of disease at the time of study. As such, they represented a random collection of AA patients and served as controls for our criteria for selecting our Group II and Group III patients with long-standing disease. They also served as a cohort of patients to be used to validate the ®ndings of our study. We have obtained current information on the clinical course of 17 of the original 35 patients. The antigen frequencies (number of patients in parentheses) in these 17 patients were: DQB1*03, 82% (14); DRB1*1104, 35% (six); DRB1*0401, 24% (four), and DQB1*0301, 59% (10). These frequencies are comparable with those of the original group of 35 patients (Table II). Eight of the patients have gone on to have long-standing AT or AU over the past 2±4 y after their initial visit. Of these eight patients, seven had either DRB1*0401 or DQB1*0301, the susceptibility alleles for AT/AU. There were ®ve patients who were DQB1*0301-positive who have not developed either AT or AU during the 4 y since they were ®rst diagnosed (two of these were also DRB1*0401-positive). There were six patients who were both DQB1*0301-positive and DRB1*1104-positive, and four of these six patients have gone on to long-standing AT or AU. Both the presence of DQB1*0301 and the highly signi®cant p value for the association of DRB1*1104 and AT/AU (Table I) would predict a strong likelihood of progression to AT/AU for patients of this phenotype. Two groups of long-standing AA patients are further distinguished by age of onset of disease and increased familial incidence In our initial study of 100 patients (Colombe
et al, 1995) with long-standing disease, we noted that early onset of disease was characteristic of those patients with AT/AU. We looked at the age of onset of the ®rst patch of AA in the AT/AU patient group compared with the patchy AA group (Table III). By age 10, 45% of the AT/AU patients had their ®rst patch as compared with only 10% of the patients with patchy AA; by age 30, 82% of AT/AU patients had their ®rst manifestation of disease compared with 51% of patchy AA patients. Thus, in addition to a particular HLA association, the long-standing AT/AU patients have early onset as a second distinguishing feature. Other autoimmune diseases such as psoriasis (Henseler and Christophers, 1985; Elder et al, 1994), diabetes mellitus type 1 (Nepom and Erlich, 1991), and juvenile rheumatoid arthritis (Nepom, 1992), have shown a similar grouping of characteristics, namely early onset, severity and persistence, HLA association, and, in addition, familial incidence. In our initial group of 100 patients, we found that patients having their ®rst AA patch by age 30 had a 37% family incidence, whereas there was only a 7% incidence of familial disease in the group of patients with onset after age 30. Thus, AA as manifested in the group of long-standing AT/AU patients resembles some of the classic autoimmune diseases. CONCLUSIONS Our results show that speci®c HLA are associated with susceptibility to AA. Our data indicate a strong association of DQB1*03 and DRB1*1104 with a general susceptibility to AA. In addition, we ®nd two other HLA, DRB1*0401 and DQB1*0301, to be associated with long-standing AT/AU. It is indeed striking to ®nd the identical HLA as have been previously reported in both serologic and DNA-based studies of HLA and AA. Many of these HLA had been found to be associated with a similar extent and duration of AA. In our study of Caucasian AA patients of mixed European ethnicity, these antigens now all attain statistically signi®cant associations with speci®c clinical parameters of the disease. By identifying these HLA, we are a step closer to understanding the structure of the putative AA antigen(s) that is being recognized by T lymphocytes, cells that are crucial to the initiation and perpetuation of an in¯ammatory immune response localized to the hair follicle. The topography of the peptide antigen binding groove of the HLA molecule implies the structure and sequence of the AA antigen and provides knowledge useful for further study to identify the antigen. We can speculate that the structure of the binding grooves of the HLA identi®ed here have special af®nity for some self (auto) antigen, or for an exogenous antigen that may mimic a self antigen found in the hair follicle or its environment. Identi®cation of the AA antigen(s) will be a major step in the understanding of the mechanisms of AA and in the design of therapies for prevention and treatment (Nepom, 1992). By presentation of this antigenic peptide, HLA antigens become a partner in the events of an autoimmune response; however, from HLA and disease studies in the literature, it is clear that for the individual, the phenotypic presence of predisposing HLA is but one component of a constellation of factors leading to autoimmune disease. Other genetic in¯uences as well as environmental factors certainly contribute to the pathogenesis of AA. REFERENCES
Table III. Early age of onset for patients with long-standing AT/AU Patient group Age of onset of ®rst patch (y) By age 10 By age 20 By age 30
Long-standing patchy (Group II, N = 42)
Long-standing AT/AU (Group III, N = 58)
10% 27% 51%
45% 66% 82%
Averbakh EV, Pospelo LE: HLA antigens in patients with alopecia areata. Vestn Dermatol Venerol 1:24, 1986 Bjorkman PJ, Saper MA, Samraoui B, et al: Structure of the human class I histocompatibility antigen, HLA-A2. Nature 329:506, 1987 Brodsky FM, Gugliardi L: The cell biology of antigen processing and presentation. Ann Rev Immunol 9:707, 1991 Brown JH, Jardetzky TS, Gorga JC, et al: Three-dimensional structure of the human class II histocompatibility antigen HLA-DR1. Nature 364:33, 1993 Colombe BW, Price VH, Khoury EL, et al: HLA class II antigen associations help to de®ne two types of alopecia areata. J Am Acad Dermatol 33:757, 1995 Duvic M, Hordinsky MK, Fiedler VC, et al: HLA±D locus associations in alopecia areata. Arch Dermatol 127:64, 1991 Elder JT, Nair RP, Guo S, et al: The genet psoriasis. Arch Dermatol 130:216, 1994 Frentz G, Thomsen K, Jakobsen BK, et al: HLA-DR4 in alopecia areata. J Am Acad Dermatol 14:129, 1986
VOL. 4, NO. 3, DECEMBER 1999
Hacham-Zadeh S, Brautbar C, Cohen C, et al: HLA and alopecia areata in Jerusalem. Tissue Antigens 18:71, 1981 Henseler T, Christophers E: Psoriasis of early and late onset: Characterization of two types of psoriasis vulgaris. J Am Acad Dermatol 13:450, 1985 Khoury EL, Price VH, Abdel-Salam MM, et al: Topical minoxidil in alopecia areata: no effect on the perifollicular lymphoid in¯itration. J Invest Dermatol 99:40, 1992 Kianto U, Reunala T, Karvonen J, et al: HLA-B12 in alopecia areata. Arch Dermatol 113:1716, 1977 Lechler R (ed.): HLA and Disease. San Diego: Academic Press, 1994 McDonagh A, Elliott KR, Messenger AG: Cytokines and dermal papilla function in alopecia areata. J Invest Dermatol 104:9S, 1995 Mikesell JF, Bergfeld WF, Braun WE: HLA-DR antigens in alopecia areata. Cleve Clin Q 53:189, 1986 Morling N, Frentz G, Fugger L, et al: DNA polymorphism of HLA class II Genes in alopecia areata. Dis Markers 9:35, 1991 Nepom G: Disease markers and new therapeutics. dual objectives of genetics in rheumatology. Rheum Dis Clin No Amer 18:719, 1992 Nepom GT, Erlich H: MHC class-II molecules and autoimmunity. In: Ann Rev Immunol 1991, Vol. 9, eds WE Paul, CG Fathman, H Metzger. Palo Alto, CA: Annal Reviews, 1991, p. 493 Odum N, Morling J, Georgsen BK, et al: HLA-DP antigens in patients with alopecia areata. Tissue Antigens 35:114, 1990 Oldstone M: Molecular mimicry and autoimmune disease. Cell 50:819, 1987
GENETIC BASIS OF ALOPECIA AREATA
219
Orecchia G, Cuccia Belvedere M, Martinetti M, et al: Human leukocyte antigen region involvement in the genetic predisposition to alopecia areata. Dermatologica 175:10, 1987 Price VH, Colombe BW: Heritable factors distinguish two types of alopecia areata. Dermatologic Clinics 14:679, 1996 Stern LJ, Brown JH, Jardetzky TS, et al: Crystal structure of the human class II MHC protein HLA-DR1 complexed with an in¯uenza virus peptide. Nature 368:215, 1994 Sundberg JP, Boggess D, Montagutelli X, Hogan M, King LL Jr: C3H/HeJ Mouse Model for Alopecia Areata. J Invest Dermatol 104:16s, 1995 Tiwari J, Terasaki P: HLA disease associations. New York: Springer-Verlag, 1985 Tobin DJ, Orentreich N, Fenton DA, Bystryn J-C: Antibodies to hair follicles in alopecia areata. J Invest Dermatol 102:591, 1994 Todes-Taylor N, Turner R, Wood GS, et al: T cell subpopulations in alopecia areata. J Am Acad Derm 11:216, 1984 Tsuji K, Aizawa M, Sasazuki T (eds): HLA 1991. New York: Oxford University Press, 1992, pp. 1073 and 1166 Welsh EA, Clark HA, Epstein SZ, et al: Human leukocyte antigen ± DQB1*03 alleles are associated with alopecia areata. J Invest Dermatol 103:758, 1994 Zhang JG, Oliver RF: Immunohistological study of the development of the cellular in®ltrate in the pelage follicles of the DEBR model for alopecia areata. Br J Derm 130:405±414, 1994 Zhang L, Weetman AP, Friedmann PS, et al: HLA associations with alopecia areata. Tissue Antigens 38:89, 1991
Thomas Jefferson University Department of Medicine and Hospital, Philadelphia, Pennsylvania, U.S.A.; *University of California San Francisco Department of Surgery; ²University of California San Francisco Department of Dermatology, San Francisco, California, U.S.A.
Many diseases, notably those having a strong autoimmune component, have been shown to have an association with speci®c human leukocyte antigens (HLA). The molecular basis for this genetic association with disease is the fact that HLA bind and present peptides derived from self and foreign protein antigens to the immune system for recognition and activation of the immune response. Previous studies with heterogeneous groups of alopecia areata (AA) patients have suggested associations with some HLA class I and class II antigens. For this study we selected only patients with long-standing disease and strati®ed them into two groups by strict de®nitions of duration and extent of disease: those with patchy AA and those with either alopecia totalis (AT) or alopecia universalis (AU). The patients were tissue
typed for HLA class II antigens by biomolecular methods that provided antigen discrimination at an allele level. More than 80% of all of the AA patients typed were positive for the antigen DQB1*03 (DQ3), suggesting that this antigen is a marker for general susceptibility to AA. In addition, two other antigens were found signi®cantly increased in frequency only in the group of AT/AU patients, DRB1*0401 (DR4) and DQB1*0301(DQ7). This strongly suggests that the two clinical types of AA, namely patchy AA versus AT/AU, can be distinguished by a genetically based predisposition to extent of disease. Key words: genetic susceptibility to AA/histocompatibility. Journal of Investigative Dermatology Symposium Proceedings 4:216± 219, 1999
T
THE ASSOCIATION OF DISEASES WITH SPECIFIC HLA
here is consensus that alopecia areata (AA) is a disease with a strong autoimmune component. A number of genes that in¯uence the susceptibility and severity of AA have been described in animal models (Zhang and Oliver, 1994; Sundberg et al, 1995). In humans, it is also evident that an immune response modulates the condition of the hair follicles in affected individuals; e.g., activated T cells in®ltrate affected skin sites (Todes-Taylor et al, 1984; Khoury et al, 1992), cytokines in¯uence follicular cycling (McDonagh et al, 1995), and autoantibodies to cellular components have been demonstrated (Tobin and Orentreich, 1994). Compelling evidence has accumulated that the inheritance of speci®c human leukocyte antigens (HLA) can predispose some individuals to a given disease, frequently a disease of autoimmune character such as diabetes mellitus, psoriasis, thyroiditis, multiple sclerosis, or rheumatoid arthritis (Tiwari, 1985). It is not surprising therefore that there is evidence that HLA are an important genetic in¯uence in AA.
The relationship between HLA and disease has only become clear during the past decade as biomolecular methods have revealed the molecular structure of the HLA molecule and its role in the immune response (Bjorkman et al, 1987; Brodsky and Gugliardi, 1991). HLA are the products of approximately a dozen closely linked genes located on chromosome 6 in a region known as the major histocompatibility complex (MHC). These antigens, abundant on the surface of most cells, are a functional component of the immune response. HLA molecules can bind both exogenous and endogenous antigenic peptide fragments in a surface groove and present them to T cells for recognition and subsequent activation of immune system effector mechanisms. In autoimmunity, it is presumed that a self-derived antigen peptide or a peptide that mimics self (molecular mimicry; Oldstone, 1987) is being presented to self T cells, resulting in self destructive responses (Lechler, 1994). The MHC-HLA genes comprise the most polymorphic genetic system of the human. The polymorphism resides in the variation in the sequence of DNA bases in and around the peptide antigen binding groove of the HLA molecule (Brown et al, 1993). Functionally, the structure of the groove varies with the individual HLA antigen and thus affects the speci®city of peptide binding (Stern et al, 1994). Therefore, for example, certain HLA antigens may have a high binding af®nity for a given autoantigenic peptide and may confer increased susceptibility to a process of autoimmune destruction in certain diseases, or to a successful immune resistance to a pathogen
Manuscript received January 11, 1999; revised April 8, 1999; accepted for publication April 9, 1999. Reprint requests to: Dr. Beth W. Colombe, Immunogenetics and Tissue Typing Laboratory, Thomas Jefferson University Hospital, 111 S 11th St. Room 8245 Gibbon Bldg., Philadelphia, PA 19107. Email: Beth. [email protected] Abbreviations: AA, alopecia areata; AT, alopecia totalis; AU, alopecia universalis; HLA, human leukocyte antigen(s). 1087-0024/99/$14.00
´ Copyright # 1999 by The Society for Investigative Dermatology, Inc. 216
VOL. 4, NO. 3, DECEMBER 1999
GENETIC BASIS OF ALOPECIA AREATA
(Nepom et al, 1992). Our working hypothesis in looking for an HLA association for AA is that a limited number of HLA have the capacity to preferentially bind a restricted number of immunodominant AA peptides. The molecular complex of AA peptide plus HLA can then be recognized by T cell clones leading to disease pathogenesis. Any individual having inherited the speci®c HLA will have increased susceptibility for AA. Previous serologic-based studies of possible HLA class I (HLA-A, -B, and -C loci) associations with AA (Kianto et al, 1977; HachamZadeh et al, 1981; Averbakh and Pospelo, 1986; Orecchia et al, 1987) have failed to show consistency or signi®cance for any particular HLA; however, several population studies exploring HLA class II antigen (HLA-DR and -DQ loci) associations (Mikesell et al, 1986; Frentz et al, 1986; Orecchia, 1987; Odum et al, 1990; Morling et al, 1991; Zhang et al, 1991; Duvic et al, 1991; Welsh et al, 1994; Colombe et al, 1995; Price et al, 1996) using both serologic and DNA techniques have implicated DR4, DR5, and DQ3 as likely susceptibility antigens. It is notable that many of these studies saw trends that failed to achieve statistical signi®cance. Possible reasons for the lack of signi®cance include the small numbers of patients studied, the use of serologic typing to de®ne the HLA class II antigens and the heterogeneity of extent and duration of disease in the patient groups. DEFINITION OF TWO GROUPS OF AA PATIENTS BASED UPON EXTENT AND DURATION OF DISEASE Building upon this previous work on HLA associations with AA, our group undertook a study of AA patients strati®ed by a strict de®nition of extent and duration of disease. We selected patients with long-standing disease because we hypothesized that they had an established pattern for their disease presentation. Our intent was to minimize heterogeneity among the patients. All patients were over 18 y of age and of Caucasian ethnicity. We de®ned three groups of patients: Group I: Patients newly diagnosed with AA for 6 mo or less, with hair loss of any extent. The future duration and extent of their disease is unknown. Group I is a random group of patients and serves as a control group for patients in Groups II and III who have disease of speci®ed extent and duration. Group II: Patients with greater than 25% scalp hair loss for 18 mo or longer but never with AT/AU. Group III: Patients with 100% scalp hair loss with or without body hair loss, and duration of AT/AU greater than 2 y at any time during their lives.
Table I. HLA associated with long-standing AA HLA: p values (corrected)
Patient group
DQB1*3
I (mixed) II (patchy AA) III (AT/AU)
NS 1.5 3 10±2 6.3 3 10±6
DQB1*0301 DRB1*1104 DRB1*0401 NS NS 1.2 3 10±2
4.1 3 10±9 1.8 3 10±3 3.3 3 10±8
NS NS 1.0 3 10±3
A normal control group was composed of North American Whites from data taken from the 11th International Histocompatibility Workshop (Tsuji et al, 1992). Patients were typed for HLA-DR and -DQ antigens by molecular methods, mainly by the method of sequence speci®c priming. This technique allows for de®nition of HLA at the allele level. The frequencies of HLA class II antigens were compiled separately for each group. Statistical treatment of the HLA frequency data: HLA frequencies and p-values were determined by chi square analysis of twoby-two contingency tables. The p values were corrected by multiplying each by 126, a factor equal to the number of HLA class II alleles typed for (63) multiplied by the number of comparisons made (2). These data were corrected by this stringent method because we wanted to establish the HLA associations de novo for each of our clinically de®ned AA patient groups. Normally, once an HLA association is published for a disease, such corrections are no longer necessary. RESULTS AND DISCUSSION Frequencies of HLA in patients with long-standing AA A total of 283 patients have been typed for HLA-DR and -DQ antigens; 96 patients had patchy AA (referred to as Group II); 152 patients had either AT or AU (referred to as Group III); 35 patients had AA of recent diagnosis and random extent of disease (referred to as Group I). Four speci®c HLA alleles were found to occur with a statistically signi®cant increase in frequency, namely DQB1*03 (DQ3); DQB1*0301 (DQ7), a subtype of DQB1*03; DRB1*1104 (DR11); DRB1*0401 (DR4). Table I shows the p values attained after correcting the data for the number of HLA class II alleles typed for. The broad antigen DQB1*03, known as antigen DQ3 by serology, is signi®cant for both the patchy AA and AT/AU groups, whereas DQB1*0301 (DQ7 by serology), a subtype (allele) of DQB1*03, is signi®cantly elevated only in the AT/AU patients. In addition to DQB1*0301, the HLA allele, DRB1*0401, is found with signi®cantly increased frequency uniquely in the AT/AU group of patients, suggesting that these two alleles may serve as special genetic markers for susceptibility for the more severe manifestations of AA. Most striking is the highly signi®cant increase in the occurrence of DRB1*1104, a subtype of DR11, in all groups of AA patients. An increase in this relatively rare allele has also been noted in AA patients by Duvic and colleagues (Welsh et al, 1994). Table II summarizes the frequencies of these four HLA in the three patient groups and in a control group of normal North American Caucasians. There is a clear trend for all AA patients to have the DQB1*03 antigen with an overall frequency of more than 80%, although the size of Group I is too small (N = 35) to achieve statistical signi®cance by our strict criteria. The DR11 subtype DRB1*1104 is found in all patient groups with an average frequency of 16%. Although not characteristic of the majority of AA patients, DRB1*1104 is more than 10 times more frequent in patients than in the control population. Although DRB1*0401 and DQB1*0301 appear to have comparable frequencies in Groups II and III, these antigens are signi®cantly increased only in the AT/ AU group (Group III) once the proper statistical tests are applied (Table I).
Table II. Frequencies of HLA associated with long-standing patchy AA and long-standing AT/AU Patient group Controls I (mixed) II (patchy AA) III (AT/AU)
217
HLA frequencies (%) (N)
DQB1*03
DQB1*0301
DRB1*1104
DRB1*0401
(DQ 191; DR 286) (35) (96) (152)
48.2
32.0
1.0
13.2
82.2 79.2 87.6
54.2 52.0 56.6
22.8 10.4 17.2
17.2 25.0 32.2
218
COLOMBE ET AL
JID SYMPOSIUM PROCEEDINGS
These HLA frequency data strongly suggest that there is both a general and a group-speci®c HLA association with AA: the broad antigen DQB1*03 and the allele DRB1*1104 serve as markers of general susceptibility to AA, whereas other speci®c HLA alleles further differentiate the two groups of long-standing AA patients that are distinguished by extent of hair loss (patchy AA versus AT/ AU). The ®nding of the general association of DQB1*03 with AA con®rms that of Welsh et al (1994) who reported the same 80% incidence of DQB1*03 in a group of 85 white European AA patients. We ®nd that the more severe AT/AU form of the disease is associated with the presence of the inherited HLA alleles DRB1*0401 and DQB1*0301, strongly suggesting that a genetic predisposition for disease is related to the presence of certain HLA antigens. Previous studies have found increased frequency of these same antigens. DR4 has been found associated with long-standing and severe disease in a study of 22 Danish AA patients (Frentz, 1986), and DQB1*0301 (DQ7) has been previously reported to be signi®cantly increased in a cohort of 20 Danish AA patients (Morling et al, 1991) and in a group of 85 North American white AA patients with both patchy and extensive disease of extended duration (Welsh et al, 1994). DR5 (DR11) was found to be associated with susceptibility to long standing disease and with early onset of disease in a group of 127 Italian AA patients (Orecchia et al, 1987). Possible mechanisms for susceptibility are discussed in the Conclusions. Follow up on patients from Group I Group I patients were a group of AA patients diagnosed for only 6 mo or less and therefore of uncertain future duration; they could have had any extent of disease at the time of study. As such, they represented a random collection of AA patients and served as controls for our criteria for selecting our Group II and Group III patients with long-standing disease. They also served as a cohort of patients to be used to validate the ®ndings of our study. We have obtained current information on the clinical course of 17 of the original 35 patients. The antigen frequencies (number of patients in parentheses) in these 17 patients were: DQB1*03, 82% (14); DRB1*1104, 35% (six); DRB1*0401, 24% (four), and DQB1*0301, 59% (10). These frequencies are comparable with those of the original group of 35 patients (Table II). Eight of the patients have gone on to have long-standing AT or AU over the past 2±4 y after their initial visit. Of these eight patients, seven had either DRB1*0401 or DQB1*0301, the susceptibility alleles for AT/AU. There were ®ve patients who were DQB1*0301-positive who have not developed either AT or AU during the 4 y since they were ®rst diagnosed (two of these were also DRB1*0401-positive). There were six patients who were both DQB1*0301-positive and DRB1*1104-positive, and four of these six patients have gone on to long-standing AT or AU. Both the presence of DQB1*0301 and the highly signi®cant p value for the association of DRB1*1104 and AT/AU (Table I) would predict a strong likelihood of progression to AT/AU for patients of this phenotype. Two groups of long-standing AA patients are further distinguished by age of onset of disease and increased familial incidence In our initial study of 100 patients (Colombe
et al, 1995) with long-standing disease, we noted that early onset of disease was characteristic of those patients with AT/AU. We looked at the age of onset of the ®rst patch of AA in the AT/AU patient group compared with the patchy AA group (Table III). By age 10, 45% of the AT/AU patients had their ®rst patch as compared with only 10% of the patients with patchy AA; by age 30, 82% of AT/AU patients had their ®rst manifestation of disease compared with 51% of patchy AA patients. Thus, in addition to a particular HLA association, the long-standing AT/AU patients have early onset as a second distinguishing feature. Other autoimmune diseases such as psoriasis (Henseler and Christophers, 1985; Elder et al, 1994), diabetes mellitus type 1 (Nepom and Erlich, 1991), and juvenile rheumatoid arthritis (Nepom, 1992), have shown a similar grouping of characteristics, namely early onset, severity and persistence, HLA association, and, in addition, familial incidence. In our initial group of 100 patients, we found that patients having their ®rst AA patch by age 30 had a 37% family incidence, whereas there was only a 7% incidence of familial disease in the group of patients with onset after age 30. Thus, AA as manifested in the group of long-standing AT/AU patients resembles some of the classic autoimmune diseases. CONCLUSIONS Our results show that speci®c HLA are associated with susceptibility to AA. Our data indicate a strong association of DQB1*03 and DRB1*1104 with a general susceptibility to AA. In addition, we ®nd two other HLA, DRB1*0401 and DQB1*0301, to be associated with long-standing AT/AU. It is indeed striking to ®nd the identical HLA as have been previously reported in both serologic and DNA-based studies of HLA and AA. Many of these HLA had been found to be associated with a similar extent and duration of AA. In our study of Caucasian AA patients of mixed European ethnicity, these antigens now all attain statistically signi®cant associations with speci®c clinical parameters of the disease. By identifying these HLA, we are a step closer to understanding the structure of the putative AA antigen(s) that is being recognized by T lymphocytes, cells that are crucial to the initiation and perpetuation of an in¯ammatory immune response localized to the hair follicle. The topography of the peptide antigen binding groove of the HLA molecule implies the structure and sequence of the AA antigen and provides knowledge useful for further study to identify the antigen. We can speculate that the structure of the binding grooves of the HLA identi®ed here have special af®nity for some self (auto) antigen, or for an exogenous antigen that may mimic a self antigen found in the hair follicle or its environment. Identi®cation of the AA antigen(s) will be a major step in the understanding of the mechanisms of AA and in the design of therapies for prevention and treatment (Nepom, 1992). By presentation of this antigenic peptide, HLA antigens become a partner in the events of an autoimmune response; however, from HLA and disease studies in the literature, it is clear that for the individual, the phenotypic presence of predisposing HLA is but one component of a constellation of factors leading to autoimmune disease. Other genetic in¯uences as well as environmental factors certainly contribute to the pathogenesis of AA. REFERENCES
Table III. Early age of onset for patients with long-standing AT/AU Patient group Age of onset of ®rst patch (y) By age 10 By age 20 By age 30
Long-standing patchy (Group II, N = 42)
Long-standing AT/AU (Group III, N = 58)
10% 27% 51%
45% 66% 82%
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