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Subgrouping of patients with oral lichen planus according to cytochrome P450 enzyme phenotype and genotype
Vol. 118 No. 4 October 2014
Subgrouping of patients with oral lichen planus according to cytochrome P450 enzyme phenotype and genotype C. Kragelund, DDS, PhD,a S.B. Jensen, DDS, PhD,a C. Hansen Cand Scient,b K. Broesen, DMS, Dr Med,c L.A. Torpet, DDS, PhD, Dr Odont,a and J. Reibel, DDS, PhD, Dr Odonta University of Copenhagen, Copenhagen, Denmark; University of Southern Denmark, Odense, Denmark
Objective. This study aimed to determine if the activity of the environmentally influenced cytochrome P450 enzyme CYP1A2, alone or in combination with CYP2D6*4 genotype, discriminates subgroups of oral lichen planus (OLP) according to lifestyle factors and clinical manifestations. Study Design. A total of 111 patients with OLP were categorized according to normal, low, or high CYP1A2 activity and CYP2D6*4 genotype. Lifestyle parameters influencing the CYP1A2 activity and symptoms and manifestations of OLP were recorded. Results. Of the 111 patients, 21% had low, 65% normal, and 14% high CYP1A2 activity. The high-CYP1A2-activity group was more exposed to CYP1A2 inducers than the low-CYP1A2-activity group. In the normal-CYP1A2-activity group, more patients had a CYP2D6*4 genotype (58%) (P ¼ .02), and they presented more symptoms (P ¼ .003) and gingival lesions (P ¼ .03). More patients in the low-CYP1A2-activity group and without CYP2D6*4 genotype presented red lesions (P ¼ .04). Conclusions. We suggest CYP2D6*4 genotype as a disease-susceptible genotype and low or high CYP1A2 activity levels as indicators of environmental influence in OLP subgroups. (Oral Surg Oral Med Oral Pathol Oral Radiol 2014;118:469-474)
Oral lichen planus (OLP) is an inflammatory disease with unknown etiology. The prevalence of OLP is 2% in Scandinavia, and middle-aged women are most frequently affected.1 Drugs and environmental factors have been associated with OLP, in which case the manifestations are referred to as oral lichenoid lesions (OLL).2 Apart from contact lesions in close contact with dental restorations, it is difficult, if not impossible, to differentiate between OLP and OLL, and drugs from all therapeutic classes have been implicated in causing OLL, in which case the condition is termed lichenoid drug eruptions (LDE).1,3 To date, the diagnosis of OLP is made by a combined clinical and histopathologic examination, the most important finding of the latter being a subepithelial band-like lymphocytic infiltration and liquefaction degeneration in the basal part of the
This study was presented as an oral and poster presentation at the 10th Biennial Congress of the European Association of Oral Medicine, Athens, Greece, 2012. The authors thank Novo Nordisk Foundation (Apotekerfonden af 1991), the Danish Medical Research Council, the Danish Dental Association (F.U.T./Calcin Fonden), and the Forskningsfond for financial support. a Section of Oral Medicine, Clinical Oral Physiology, Oral Pathology & Anatomy, Department of Odontology, Faculty of Health and Medical Sciences, University of Copenhagen. b Wilhelm Johannsen Center for Functional Genome Research, Department of Medical Genetics, Faculty of Health and Medical Sciences, University of Copenhagen. c Centre of Clinical Pharmacology, University of Southern Denmark. Received for publication Feb 6, 2014; returned for revision Jun 16, 2014; accepted for publication Jun 22, 2014. Ó 2014 Elsevier Inc. All rights reserved. 2212-4403/$ - see front matter http://dx.doi.org/10.1016/j.oooo.2014.06.008
epithelium.4,5 The World Health Organization categorizes OLP as a disorder with a malignant potential.6 Subgrouping of patients into OLP and OLL groups according to clinical and histopathologic criteria has been suggested by many, most recently by van der Meij and van der Waal,7 but still a relation to etiologic or prognostic differences has not been clarified. However, asymmetrical and erythematous manifestations have been suggested as being features of LDE,3,8 in contrast to OLP, characterized by white reticular and papular lesions symmetrically distributed in the posterior part of the buccal mucosa.9 A subgroup of female patients with gingival lichenoid lesions and simultaneous genital lichenoid lesions are suggested to have vulvovaginal gingival syndrome.10 Thus, lichen planus (LP) can vary in clinical presentation and have widespread manifestations, and apart from the skin and the oral and genital mucous membranes, the esophageal, anal, and conjunctival tissues can be affected.9 Some patients presenting erythematous and ulcerative oral lesions experience severe morbidity including symptoms such as stinging and burning sensations,
Statement of Clinical Relevance Oral lichen planus (OLP) has unknown etiology, but genetic and environmental factors may be involved. Based on minor differences in lifestyle and OLP manifestations, we suggest CYP2D6*4 genotype as a disease-susceptible genotype and low or high CYP1A2 activity levels as indicators of environmental influence in OLP subgroups. 469
ORAL AND MAXILLOFACIAL PATHOLOGY 470 Kragelund et al.
commonly resulting in impairment of normal oral function.9 As the etiology and pathogenesis of OLP are unknown, only symptomatic treatment can be initiated, with treatment response being unpredictable.11,12 Women have a lower activity of the drug metabolizing cytochrome P450 enzyme (CYP) CYP1A2,13 but environmental and genetic factors also influence CYP1A2 activity, which may have clinical significance for drug metabolism.14 CYP1A2 metabolizes caffeine, 8% to 10% of marketed drugs, and endogenous compounds (e.g., estradiol). No polymorphisms or genetic haplotype have been identified in the CYP1A2 gene that can predict the metabolic phenotype.14 Environmental factors inducing CYP1A2 activity include coffee, smoking, charcoal-grilled meat, and cruciferous vegetables.15,16 Oral contraceptives/estrogens and some other drugs are CYP1A2 inhibitors.13 In cancer research, CYP1A2 has been a topic of interest, because this enzyme, like CYP1A1, activates procarcinogens (such as polycyclic aromatic hydrocarbons).14 Thus, low CYP1A2 activity increases the risk of reduced clearance of xenobiotics, and high activity increases the bioactivation of procarcinogens.14 Earlier, in a study on polymorphic CYP enzymes, our group found that patients with OLP had a higher prevalence of the CYP2D6*4 genotype (50%) than did the background population (30%) (P ¼ .0001). We suggested that CYP2D6*4 was a disease-susceptible genotype in OLP, and molecular mimicry was hypothesized as a possible mechanism.17 Molecular mimicry may happen when the immune system reacts to self-proteins having sequence similarities with pathogenic proteins. Intolerance toward self-proteins, such as CYP enzymes, may cause organ-specific or multiorgan manifestations, depending on protein expression in the organs or if a subsequent activator, local or systemic, is present.18 Thus, it is interesting to further examine the influence of CYP2D6*4, given that it has sequence similarity with herpes simplex virus type 1 and Candida albicans, which both commonly can be detected in the oral cavity.17 To assess whether CYP1A2 activity level can be used as an indicator of environment-associated OLP and whether CYP2D6*4 contributes as a susceptibility gene in OLP, we investigated whether it is possible to subgroup patients with OLP with respect to clinical manifestations and symptoms according to the CYP1A2 activity alone or in combination with the CYP2D6*4 genotype, because it may reflect differences in etiology and be of prognostic importance.
MATERIAL AND METHODS A total of 217 patients with OLP with unknown etiology (contact lesions were excluded) were invited to participate in the study, but 97 refrained for different
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reasons,17 and 9 did not complete the phenotype tests. Thus, 111 patients were included in the study. No pregnant or lactating women or patients with other autoimmune mucocutaneous diseases were included. All patients were regularly monitored at the Clinic for Oral Medicine at the School of Dentistry in Copenhagen for an average period of 4.5 years (range, 6 months to 26 years). No investigation of hepatitis C virus (HCV) was performed. The study was approved by the Ethical Committee of Copenhagen and Frederiksberg Counties (KF 03-005/02), and written and verbal information was given before written consent was obtained from all patients. Health and drug data All drugs taken by the patients were classified according to the Anatomic, Therapeutic, and Chemical classification index (ATC code)19 and were analyzed according to CYP1A2 metabolism and CYP1A2 influence (i.e., induction and inhibition).14,20 Lifestyle data Smoking habits were recorded as the average number per day of cigarettes, cheroots, and cigars plus daily grams of pipe tobacco (if any). One cigarette was set as equal to 1 g, one cheroot as 3 g, and one cigar as 5 g of tobacco. Smoking habits were categorized as follows: never smokers, former smokers, light smokers (1-14 g/d), moderate smokers (15-24 g/d), and heavy smokers (25 g/d).21 The patients’ intake of coffee and tea was registered as number of cups daily. CYP1A2 phenotyping Directly after emptying their bladder, the patients were instructed to ingest 200 mg of caffeine (probe for CYP1A2) together with 100 mg of sparteine sulfate (probe for CYP2D6) and 100 mg of mephenytoin (probe for CYP2C19). Results for CYP2D6 and CYP2C19 have been published previously.22 The patients abstained from consuming food, drugs, and beverages containing caffeine for 48 hours before the phenotyping was performed and during the 4 hours of the test. For the caffeine analysis, a 15-mL urine sample was collected in a tube containing 100 mL N HCl approximately 4 hours after the probe drugs were ingested. The concentrations of caffeine and of the caffeine metabolites 5-acetylamino-6-formylamino-3-methyluracil (AFMU), 1-methyluric acid (1-MU), 1-methylxanthine (1-MX), and 1,7-dimethyluric acid (17-DMU) were determined by high-performance liquid chromatography. The CYP1A2 activity was calculated as the ratio of (AFMU þ 1-MU þ 1-MX)/17-DMU. The
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CYP1A2 activity was defined differently for the 2 genders, and for both genders “normal” was defined as the mean metabolic ratio 1 SD in the persons in whom no CYP1A2 inducers or inhibitors were identified, in accordance with findings in the Danish twin study on CYP1A2 activity by Rasmussen et al. (2002).13 Among the female participants, the CYP1A2 activity was defined as low when the ratio was <2.4, as high when it was >7.4, and as normal when it was in between. Among the male participants, the CYP1A2 activity was defined as low when the ratio was <2.9, as high when it was > 9.1, and as normal when it was in between.13 CYP2D6*4 genotype Finger blood was obtained from all patients, and genomic DNA was extracted for CYP2D6, CYP2C19, and CYP2C9 genotype analyses, as described earlier.17 In brief, all patients were tested for the most common alleles of CYP2D6, CYP2C9, and CYP2C19 resulting in intermediate (IM) or poor (PM) drug metabolism: CYP2D6 (*3,*4, *6, *9, and *10), CYP2C9 (*1 and *2), and CYP2C19 (*1 and *2). Furthermore, based on our previous hypothesis, we suggest the CYP2D6*4 genotype as a possible disease-susceptible genotype. OLP and related registrations The day before the phenotype test, an oral clinical examination was performed as described earlier.22 OLPrelated symptoms were recorded as present or absent. The patient’s self-reported history of extraoral LP (cutaneous, esophageal, genital, nail, and other areas) was recorded. OLP lesions were documented at the consultation according to the number of affected regions, clinical types present, and degree of symmetrical distribution. The clinical types were graded according to appearance: (1) ulcerative, (2) erythematous, (3) plaque, (4) reticular, and (5) papular type. No patients had the bullous type. Ulcerative and erythematous lesions were categorized as red lesions and the remainder types as white lesions. The degree of symmetrical distribution was evaluated as symmetrical or asymmetrical (bilateral with a side difference or unilateral lesions), and the number of anatomic regions with OLP manifestations was recorded as described earlier.22 To investigate for secondary candidal infection, a cytosmear was taken from areas that were erythematous or symptomatic (or both). Statistics The SAS software package (version 9.2; SAS Institute Inc) was used for statistical analyses. For nonnormally distributed data, the Kruskal-Wallis test or the Wilcoxon rank sum test was used. Difference in
ORIGINAL ARTICLE Kragelund et al. 471
proportion was evaluated by the c2 test or the Fisher exact test when expected numbers were <10. An unpaired t test was used to analyze the age distributions of the groups. The level of statistical significance was set at P < .05. There were no adjustments for multiple comparisons.
RESULTS CYP1A2 phenotype The majority of patients with OLP (65%; n ¼ 73) had normal CYP1A2 activity, compared to low activity (21%; n ¼ 23) or high activity (14%; n ¼ 15). Various data are given in Table I, including the caffeine ratio; gender; age; CYP2D6*4 genotype; exposure to CYP1A2 substrates, inducers, and inhibitors; and number of CYP2D6, CYP2C9, and CYP2C19 IMs and PMs in the phenotype groups. CYP2D6*4 genotype More patients (58%; 42 of 73) had a CYP2D6*4 genotype in the normal-CYP1A2-activity group compared with the remainder (34%; 13 of 38) (c2; P ¼ .02), but not compared with the low-CYP1A2-activity group (35%; 8 of 23) (Fisher exact test; P ¼ .093) or the highCYP1A2-activity group (33%; 5 of 15) (Fisher exact test; P ¼ .098). Thus, 58% of the patients with OLP had the disease-susceptible CYP2D6*4 genotype and no apparent CYP1A2 environmental stimulus. CYP1A2 substrates, inducers, and inhibitors Significantly more CYP1A2-inducing drugs were taken in the high-CYP1A2-activity group (Fisher exact test; P ¼ .032). Likewise, there were significantly more smokers in the high-CYP1A2-activity group compared with the low-CYP1A2-activity group (Fisher exact test; P ¼ .032) (see Table I), and the smoking habits were reflected in the CYP1A2 activity in a dose-dependent manner. Also, the patients in the high-CYP1A2-activity group reported a higher daily intake of coffee than the remainder (Wilcoxon rank sum test; P ¼ .024) and in particular compared with the low-CYP1A2-activity group (Kruskal-Wallis test; P ¼ .003). Clinical parameters, CYP1A2 phenotype, and CYP2D6*4 genotype Both the low- and the high-CYP1A2-activity groups are believed to be at risk of an environment-related OLP because of drug accumulation and reactive metabolites, respectively. Interestingly, less CYP1A2 substrates were taken by the normal-CYP1A2-activity group with CYP2D6*4 genotype (Fisher exact test; P ¼ .03). There were no significant differences in the distribution of symptoms and clinical characteristics of
ORAL AND MAXILLOFACIAL PATHOLOGY 472 Kragelund et al.
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Table I. Subgrouping of CYP1A2 phenotype in patients with OLP according to activity and demography, potential CYP1A2 modifiers (inducers and inhibitors), and CYP2D6*4 genotype Low CYP1A2 activity
Normal CYP1A2 activity
High CYP1A2 activity
n ¼ 23
n ¼ 73
n ¼ 15
4.47 1.53 (2.37-9.03) 45/28 59.9 (31-86) 42 (58) 30%
13.31 10.37 (8.12-48.61) 11/4 57.2 (31-77) 5 (33) 40%
Variable
Caffeine ratio SD (range) 1.47 0.74 (0.10-2.73) Women/men 15/8 Age (y), mean (range) 58.6 (30-74) CYP2D6*4 genotype, n (%) 8 (35) CYP1A2 substrates (%) 52% CYP1A2 induction CYP1A2-inducing drugs (%) 0 Smokers (%) 13% Moderate smokers (%) 0 Heavy smokers (%) 0 Coffee drinkers (%) 91% Heavy coffee drinkers (3 cups per day) 65% Coffee, cups, median (range) 3 (1-7) Tea drinkers (%) 57% Tea, cups, median (range) 6 (1-10) CYP1A2 inhibitors Drugs (%) 22% CYP2D6 IM or PM 12 (52) CYP2C9 IM or PM 6 (26) CYP2C19 IM or PM 4 (17)
P
1% 22% 7% 3% 92% 74% 4 (1-20) 45% 2 (0-11)
20% 47% 20% 0 87% 80% 6 (2-10) 27% 2
16% 48 (66) 22 (30) 17 (23)
20% 6 (40) 5 (33) 3 (20)
.02* .03y <.03y
.003y; .024z
CYP, cytochrome P450 enzyme; IM, intermediate metabolizers; OLP, oral lichen planus; PM, poor metabolizers. *NormalCYP1A2activity>< the remainders.
(Fisher exact test; P ¼ .01). More patients with low CYP1A2 activity and no CYP2D6*4 genotype (possible environmental OLP) presented red lesions (Fisher exact test; P ¼ .04), and none had oral candidiasis.
OLP between the CYP1A2-activity groups. However, analyzing according to the CYP2D6*4 genotype, a few interesting results were found (Table II). More patients with normal CYP1A2 activity and CYP2D6*4 genotype (possible genetic OLP) reported symptoms (Fisher exact test; P ¼ .003) and more had gingival lesions (Fisher exact test; P ¼ .03) compared with the remainder with a CYP2D6*4 genotype. Secondary candidal infections were diagnosed in 4 patients, which could influence the presence of symptoms and erythema. When these patients were excluded, more symptoms were recorded in the normal-CYP1A2activity group members with CYP2D6*4 genotype
DISCUSSION Our findings suggest that a subgroup of patients with OLP may have a susceptible genotype for OLP and another subgroup may have an environmentally provoked disease (i.e., OLL or LDE). When subgrouping patients with OLP according to CYP2D6*4 genotype and CYP1A2 phenotype, we found that a large proportion of
Table II. CYP1A2 activity and CYP2D6*4 genotype status in relation to symptoms and clinical parameters in patients with OLP Phenotype Genotype No. of patients, n Symptoms, n (%) Buccal LP, n (%) Gingival LP, n (%) Red lesions, n (%) Asymmetrical lesions, n (%) No. of anatomic regions affected, median (range) Extraoral LP, n (%) Cutaneous LP, n (%)
Low CYP1A2 activity CYP2D6*4 6 7 5 2 3 5.5
8 (75) (88) (63) (25) (38) (2-17)
1 (13) 1 (13)
No-CYP2D6*4 14 14 10 9 4 6
15 (93) (93) (67) (60) (27) (1-22)
2 (13) 1 (7)
Normal CYP1A2 activity CYP2D6*4 35 37 31 15 19 7
42 (83) (88) (74) (36) (45) (1-26)
12 (29) 11 (26)
CYP, cytochrome P450 enzyme; OLP, oral lichen planus; LP, lichen planus.
No-CYP2D6*4 21 27 23 9 9 8
31 (68) (87) (74) (29) (29) (1-21)
6 (19) 5 (16)
High CYP1A2 activity CYP2D6*4 2 4 2 2 5 7
5 (40) (80) (40) (40) (100) (1-13)
1 (20) 1 (20)
No-CYP2D6*4 8 10 7 4 4 10
10 (80) (100) (70) (40) (40) (3-33)
1 (10) 2 (10)
P .003 .03 .04
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the patients could be subdivided into either having a CYP2D6*4 genotype (possible genetic OLP) or having low or high CYP1A2 activity (possible environmental OLP), with some overlap. However, 28% of all the patients with OLP had neither CYP2D6*4 genotype nor low or high CYP1A2 activity. More patients with possible genetic OLP (normal CYP1A2 activity and CYP2D6*4 genotype) reported symptoms, and more had gingival lesions. The low-CYP1A2-activity and CYP2D6*4-negative patients presented more red lesions, which has been associated with LDE in a previous study.8 There were no obvious clinical differences between the different groups. Still, it may be naive to anticipate finding a clinical difference between OLP and OLL, given that the clinical course of OLP fluctuates.23 It is possible that there are differences in the course of the disease and the prognosis between the possible genetic OLP and the environmentally provoked OLP, but prospective studies are needed. CYP2D6*4, being a disease-susceptible genotype, has also been suggested by other groups. CYP2D6*4 genotype was associated with an accelerated rate of liver fibrosis in patients infected with HCV.24,25 Liver fibrosis develops after chronic inflammation, and chronic inflammation is the key element in OLP.4,26 OLP, as an oral manifestation of a systemic disease27,28, could possibly be related to the CYP2D6*4 genotype. However, we did not find significantly more patients with extraoral LP in the possible genetic OLP group (CYP2D6*4 genotype and normal-CYP1A2activity group); but the presence of local and systemic activators may vary in the group. As 30% of the background population has a CYP2D6*4 genotype, additional factors must be in play in the development of OLP. In some geographic areas, there seems to be an association between OLP and HCV infection. In these areas, OLP is suggested to be an extrahepatic HCV manifestation in HCV-positive patients.29 In 10% of patients with chronic HCV infection, liver-kidney microsome-1 (LKM-1) autoantibodies are detected.30 Molecular mimicry is believed to be involved in the pathogenesis of autoimmune hepatitis type 2 (AIH-2) because of epitope homology between CYP2D6, HCV, herpes simplex virus type 1, and cytomegalovirus.24,31 In AIH-2, CYP2D6 is targeted by LKM-1 autoantibodies, CD4þ T cells, and CD8þ T cells.26 In this respect, it is interesting that CD4þ T cells and CD8þ T cells account for the majority of the cells in the bandlike lymphocytic infiltrate immediately subjacent to the epithelium in OLP,5 and autoantibodies have been found in subsets of OLP.3,32,33 One case report has been published reporting LKM-1 autoantibodies in a patient with OLP and hypothyroidism,32 but 2 case series did not find any LKM-1 autoantibodies in patients with LP.34,35 However, it is not clear if these studies
ORIGINAL ARTICLE Kragelund et al. 473
investigated OLP or cutaneous LP. Cutaneous LP has a more self-limiting nature compared with OLP, and they may be pathogenetically different.9,32 Autoantibodies have been sporadically detected in OLP,3,32,33 but it seems appropriate also to search for autoreactive T cells in OLP. However, subgrouping of patients is fundamental, as multiple etiologies cause lichenoid lesions.2
CONCLUSION Subgrouping of patients with OLP/OLL is crucial for better understanding of the pathogenesis, progression, treatment, and prognosis. Surprisingly, the present study found that CYP2D6*4 genotype was more prevalent in patients with normal CYP1A2 activity, suggesting that genetic susceptibility might have an influence in this subgroup and lifestyle factors in the low- and highCYP1A2-activity groups. For treatment purposes it would be logical to pay attention to environmental factors and drugs influencing CYP1A2 activity, as these may influence the clinical presentation and prognosis. Thus, OLP subgrouping may allow for more personalized treatment with less unpredictability and variation in treatment response.12 Future projects should involve searching for particular autoreactive T cells against CYP2D6*4 in CYP2D6*4-positive patients with OLP. The authors thank the Research Unit of Clinical Pharmacology, University of Southern Denmark, where the CYP1A2 phenotyping was performed.
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OOOO October 2014 25. Fishman S, Lurie Y, Peretz H, et al. Role of CYP2D6 polymorphism in predicting liver fibrosis progression rate in Caucasian patients with chronic hepatitis C. Liver Int. 2006;26:279-284. 26. Vergani D, Mieli-Vergani G. Aetiopathogenesis of autoimmune hepatitis. World J Gastroenterol. 2008;14:3306-3312. 27. Ebrahimi M, Lundqvist L, Wahlin YB, Nylander E. Mucosal lichen planus, a systemic disease requiring multidisciplinary care: a cross-sectional clinical review from a multidisciplinary perspective. J Low Genit Tract Dis. 2012;16:377-380. 28. Nakai K, Tanaka H, Hanada K, et al. Decreased expression of cytochromes P450 1A2, 2E1, and 3A4 and drug transporters Naþ-taurocholate-cotransporting polypeptide, organic cation transporter 1, and organic anion-transporting peptide-C correlates with the progression of liver fibrosis in chronic hepatitis C patients. Drug Metab Dispos. 2008;36:1786-1793. 29. Carrozzo M. Oral diseases associated with hepatitis C virus infection, part 2: lichen planus and other diseases. Oral Dis. 2008;14:217-228. 30. Sugimura T, Obermayer-Straub P, Kayser A, et al. A major CYP2D6 autoepitope in autoimmune hepatitis type 2 and chronic hepatitis C is a three-dimensional structure homologous to other cytochrome P450 autoantigens. Autoimmunity. 2002;35:501-513. 31. Manns MP, Griffin KJ, Sullivan KF, Johnson EF. LKM-1 autoantibodies recognize a short linear sequence in P450 IID6, a cytochrome P-450 monooxygenase. J Clin Invest. 1991;88: 1370-1378. 32. Cottoni F, Tedde G, Solinas A, Deplano A. Lichen planus associated with anti-liver-kidney microsome-positive chronic active hepatitis and hyperthyroidism. Arch Dermatol. 1991;127:17301731. 33. McCartan BE, Lamey P. Lichen planusdspecific antigen in oral lichen planus and oral lichenoid drug eruptions. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2000;89:585-587. 34. Divano MC, Parodi A, Rebora A. Anti-liver-kidney microsome type 1 antibodies in lichen planus. Arch Dermatol. 1992;128:991. 35. Rebora A, Robert E, Rongioletti F. Clinical and laboratory presentation of lichen planus patients with chronic liver disease. J Dermatol Sci. 1992;4:38-41. Reprint requests: Camilla Kragelund Section of Oral Medicine Clinical Oral Physiology Oral Pathology & Anatomy Faculty of Health and Medical Sciences University of Copenhagen 20, Norre Allé, DK-2200 Copenhagen N Denmark [email protected]
Subgrouping of patients with oral lichen planus according to cytochrome P450 enzyme phenotype and genotype C. Kragelund, DDS, PhD,a S.B. Jensen, DDS, PhD,a C. Hansen Cand Scient,b K. Broesen, DMS, Dr Med,c L.A. Torpet, DDS, PhD, Dr Odont,a and J. Reibel, DDS, PhD, Dr Odonta University of Copenhagen, Copenhagen, Denmark; University of Southern Denmark, Odense, Denmark
Objective. This study aimed to determine if the activity of the environmentally influenced cytochrome P450 enzyme CYP1A2, alone or in combination with CYP2D6*4 genotype, discriminates subgroups of oral lichen planus (OLP) according to lifestyle factors and clinical manifestations. Study Design. A total of 111 patients with OLP were categorized according to normal, low, or high CYP1A2 activity and CYP2D6*4 genotype. Lifestyle parameters influencing the CYP1A2 activity and symptoms and manifestations of OLP were recorded. Results. Of the 111 patients, 21% had low, 65% normal, and 14% high CYP1A2 activity. The high-CYP1A2-activity group was more exposed to CYP1A2 inducers than the low-CYP1A2-activity group. In the normal-CYP1A2-activity group, more patients had a CYP2D6*4 genotype (58%) (P ¼ .02), and they presented more symptoms (P ¼ .003) and gingival lesions (P ¼ .03). More patients in the low-CYP1A2-activity group and without CYP2D6*4 genotype presented red lesions (P ¼ .04). Conclusions. We suggest CYP2D6*4 genotype as a disease-susceptible genotype and low or high CYP1A2 activity levels as indicators of environmental influence in OLP subgroups. (Oral Surg Oral Med Oral Pathol Oral Radiol 2014;118:469-474)
Oral lichen planus (OLP) is an inflammatory disease with unknown etiology. The prevalence of OLP is 2% in Scandinavia, and middle-aged women are most frequently affected.1 Drugs and environmental factors have been associated with OLP, in which case the manifestations are referred to as oral lichenoid lesions (OLL).2 Apart from contact lesions in close contact with dental restorations, it is difficult, if not impossible, to differentiate between OLP and OLL, and drugs from all therapeutic classes have been implicated in causing OLL, in which case the condition is termed lichenoid drug eruptions (LDE).1,3 To date, the diagnosis of OLP is made by a combined clinical and histopathologic examination, the most important finding of the latter being a subepithelial band-like lymphocytic infiltration and liquefaction degeneration in the basal part of the
This study was presented as an oral and poster presentation at the 10th Biennial Congress of the European Association of Oral Medicine, Athens, Greece, 2012. The authors thank Novo Nordisk Foundation (Apotekerfonden af 1991), the Danish Medical Research Council, the Danish Dental Association (F.U.T./Calcin Fonden), and the Forskningsfond for financial support. a Section of Oral Medicine, Clinical Oral Physiology, Oral Pathology & Anatomy, Department of Odontology, Faculty of Health and Medical Sciences, University of Copenhagen. b Wilhelm Johannsen Center for Functional Genome Research, Department of Medical Genetics, Faculty of Health and Medical Sciences, University of Copenhagen. c Centre of Clinical Pharmacology, University of Southern Denmark. Received for publication Feb 6, 2014; returned for revision Jun 16, 2014; accepted for publication Jun 22, 2014. Ó 2014 Elsevier Inc. All rights reserved. 2212-4403/$ - see front matter http://dx.doi.org/10.1016/j.oooo.2014.06.008
epithelium.4,5 The World Health Organization categorizes OLP as a disorder with a malignant potential.6 Subgrouping of patients into OLP and OLL groups according to clinical and histopathologic criteria has been suggested by many, most recently by van der Meij and van der Waal,7 but still a relation to etiologic or prognostic differences has not been clarified. However, asymmetrical and erythematous manifestations have been suggested as being features of LDE,3,8 in contrast to OLP, characterized by white reticular and papular lesions symmetrically distributed in the posterior part of the buccal mucosa.9 A subgroup of female patients with gingival lichenoid lesions and simultaneous genital lichenoid lesions are suggested to have vulvovaginal gingival syndrome.10 Thus, lichen planus (LP) can vary in clinical presentation and have widespread manifestations, and apart from the skin and the oral and genital mucous membranes, the esophageal, anal, and conjunctival tissues can be affected.9 Some patients presenting erythematous and ulcerative oral lesions experience severe morbidity including symptoms such as stinging and burning sensations,
Statement of Clinical Relevance Oral lichen planus (OLP) has unknown etiology, but genetic and environmental factors may be involved. Based on minor differences in lifestyle and OLP manifestations, we suggest CYP2D6*4 genotype as a disease-susceptible genotype and low or high CYP1A2 activity levels as indicators of environmental influence in OLP subgroups. 469
ORAL AND MAXILLOFACIAL PATHOLOGY 470 Kragelund et al.
commonly resulting in impairment of normal oral function.9 As the etiology and pathogenesis of OLP are unknown, only symptomatic treatment can be initiated, with treatment response being unpredictable.11,12 Women have a lower activity of the drug metabolizing cytochrome P450 enzyme (CYP) CYP1A2,13 but environmental and genetic factors also influence CYP1A2 activity, which may have clinical significance for drug metabolism.14 CYP1A2 metabolizes caffeine, 8% to 10% of marketed drugs, and endogenous compounds (e.g., estradiol). No polymorphisms or genetic haplotype have been identified in the CYP1A2 gene that can predict the metabolic phenotype.14 Environmental factors inducing CYP1A2 activity include coffee, smoking, charcoal-grilled meat, and cruciferous vegetables.15,16 Oral contraceptives/estrogens and some other drugs are CYP1A2 inhibitors.13 In cancer research, CYP1A2 has been a topic of interest, because this enzyme, like CYP1A1, activates procarcinogens (such as polycyclic aromatic hydrocarbons).14 Thus, low CYP1A2 activity increases the risk of reduced clearance of xenobiotics, and high activity increases the bioactivation of procarcinogens.14 Earlier, in a study on polymorphic CYP enzymes, our group found that patients with OLP had a higher prevalence of the CYP2D6*4 genotype (50%) than did the background population (30%) (P ¼ .0001). We suggested that CYP2D6*4 was a disease-susceptible genotype in OLP, and molecular mimicry was hypothesized as a possible mechanism.17 Molecular mimicry may happen when the immune system reacts to self-proteins having sequence similarities with pathogenic proteins. Intolerance toward self-proteins, such as CYP enzymes, may cause organ-specific or multiorgan manifestations, depending on protein expression in the organs or if a subsequent activator, local or systemic, is present.18 Thus, it is interesting to further examine the influence of CYP2D6*4, given that it has sequence similarity with herpes simplex virus type 1 and Candida albicans, which both commonly can be detected in the oral cavity.17 To assess whether CYP1A2 activity level can be used as an indicator of environment-associated OLP and whether CYP2D6*4 contributes as a susceptibility gene in OLP, we investigated whether it is possible to subgroup patients with OLP with respect to clinical manifestations and symptoms according to the CYP1A2 activity alone or in combination with the CYP2D6*4 genotype, because it may reflect differences in etiology and be of prognostic importance.
MATERIAL AND METHODS A total of 217 patients with OLP with unknown etiology (contact lesions were excluded) were invited to participate in the study, but 97 refrained for different
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reasons,17 and 9 did not complete the phenotype tests. Thus, 111 patients were included in the study. No pregnant or lactating women or patients with other autoimmune mucocutaneous diseases were included. All patients were regularly monitored at the Clinic for Oral Medicine at the School of Dentistry in Copenhagen for an average period of 4.5 years (range, 6 months to 26 years). No investigation of hepatitis C virus (HCV) was performed. The study was approved by the Ethical Committee of Copenhagen and Frederiksberg Counties (KF 03-005/02), and written and verbal information was given before written consent was obtained from all patients. Health and drug data All drugs taken by the patients were classified according to the Anatomic, Therapeutic, and Chemical classification index (ATC code)19 and were analyzed according to CYP1A2 metabolism and CYP1A2 influence (i.e., induction and inhibition).14,20 Lifestyle data Smoking habits were recorded as the average number per day of cigarettes, cheroots, and cigars plus daily grams of pipe tobacco (if any). One cigarette was set as equal to 1 g, one cheroot as 3 g, and one cigar as 5 g of tobacco. Smoking habits were categorized as follows: never smokers, former smokers, light smokers (1-14 g/d), moderate smokers (15-24 g/d), and heavy smokers (25 g/d).21 The patients’ intake of coffee and tea was registered as number of cups daily. CYP1A2 phenotyping Directly after emptying their bladder, the patients were instructed to ingest 200 mg of caffeine (probe for CYP1A2) together with 100 mg of sparteine sulfate (probe for CYP2D6) and 100 mg of mephenytoin (probe for CYP2C19). Results for CYP2D6 and CYP2C19 have been published previously.22 The patients abstained from consuming food, drugs, and beverages containing caffeine for 48 hours before the phenotyping was performed and during the 4 hours of the test. For the caffeine analysis, a 15-mL urine sample was collected in a tube containing 100 mL N HCl approximately 4 hours after the probe drugs were ingested. The concentrations of caffeine and of the caffeine metabolites 5-acetylamino-6-formylamino-3-methyluracil (AFMU), 1-methyluric acid (1-MU), 1-methylxanthine (1-MX), and 1,7-dimethyluric acid (17-DMU) were determined by high-performance liquid chromatography. The CYP1A2 activity was calculated as the ratio of (AFMU þ 1-MU þ 1-MX)/17-DMU. The
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CYP1A2 activity was defined differently for the 2 genders, and for both genders “normal” was defined as the mean metabolic ratio 1 SD in the persons in whom no CYP1A2 inducers or inhibitors were identified, in accordance with findings in the Danish twin study on CYP1A2 activity by Rasmussen et al. (2002).13 Among the female participants, the CYP1A2 activity was defined as low when the ratio was <2.4, as high when it was >7.4, and as normal when it was in between. Among the male participants, the CYP1A2 activity was defined as low when the ratio was <2.9, as high when it was > 9.1, and as normal when it was in between.13 CYP2D6*4 genotype Finger blood was obtained from all patients, and genomic DNA was extracted for CYP2D6, CYP2C19, and CYP2C9 genotype analyses, as described earlier.17 In brief, all patients were tested for the most common alleles of CYP2D6, CYP2C9, and CYP2C19 resulting in intermediate (IM) or poor (PM) drug metabolism: CYP2D6 (*3,*4, *6, *9, and *10), CYP2C9 (*1 and *2), and CYP2C19 (*1 and *2). Furthermore, based on our previous hypothesis, we suggest the CYP2D6*4 genotype as a possible disease-susceptible genotype. OLP and related registrations The day before the phenotype test, an oral clinical examination was performed as described earlier.22 OLPrelated symptoms were recorded as present or absent. The patient’s self-reported history of extraoral LP (cutaneous, esophageal, genital, nail, and other areas) was recorded. OLP lesions were documented at the consultation according to the number of affected regions, clinical types present, and degree of symmetrical distribution. The clinical types were graded according to appearance: (1) ulcerative, (2) erythematous, (3) plaque, (4) reticular, and (5) papular type. No patients had the bullous type. Ulcerative and erythematous lesions were categorized as red lesions and the remainder types as white lesions. The degree of symmetrical distribution was evaluated as symmetrical or asymmetrical (bilateral with a side difference or unilateral lesions), and the number of anatomic regions with OLP manifestations was recorded as described earlier.22 To investigate for secondary candidal infection, a cytosmear was taken from areas that were erythematous or symptomatic (or both). Statistics The SAS software package (version 9.2; SAS Institute Inc) was used for statistical analyses. For nonnormally distributed data, the Kruskal-Wallis test or the Wilcoxon rank sum test was used. Difference in
ORIGINAL ARTICLE Kragelund et al. 471
proportion was evaluated by the c2 test or the Fisher exact test when expected numbers were <10. An unpaired t test was used to analyze the age distributions of the groups. The level of statistical significance was set at P < .05. There were no adjustments for multiple comparisons.
RESULTS CYP1A2 phenotype The majority of patients with OLP (65%; n ¼ 73) had normal CYP1A2 activity, compared to low activity (21%; n ¼ 23) or high activity (14%; n ¼ 15). Various data are given in Table I, including the caffeine ratio; gender; age; CYP2D6*4 genotype; exposure to CYP1A2 substrates, inducers, and inhibitors; and number of CYP2D6, CYP2C9, and CYP2C19 IMs and PMs in the phenotype groups. CYP2D6*4 genotype More patients (58%; 42 of 73) had a CYP2D6*4 genotype in the normal-CYP1A2-activity group compared with the remainder (34%; 13 of 38) (c2; P ¼ .02), but not compared with the low-CYP1A2-activity group (35%; 8 of 23) (Fisher exact test; P ¼ .093) or the highCYP1A2-activity group (33%; 5 of 15) (Fisher exact test; P ¼ .098). Thus, 58% of the patients with OLP had the disease-susceptible CYP2D6*4 genotype and no apparent CYP1A2 environmental stimulus. CYP1A2 substrates, inducers, and inhibitors Significantly more CYP1A2-inducing drugs were taken in the high-CYP1A2-activity group (Fisher exact test; P ¼ .032). Likewise, there were significantly more smokers in the high-CYP1A2-activity group compared with the low-CYP1A2-activity group (Fisher exact test; P ¼ .032) (see Table I), and the smoking habits were reflected in the CYP1A2 activity in a dose-dependent manner. Also, the patients in the high-CYP1A2-activity group reported a higher daily intake of coffee than the remainder (Wilcoxon rank sum test; P ¼ .024) and in particular compared with the low-CYP1A2-activity group (Kruskal-Wallis test; P ¼ .003). Clinical parameters, CYP1A2 phenotype, and CYP2D6*4 genotype Both the low- and the high-CYP1A2-activity groups are believed to be at risk of an environment-related OLP because of drug accumulation and reactive metabolites, respectively. Interestingly, less CYP1A2 substrates were taken by the normal-CYP1A2-activity group with CYP2D6*4 genotype (Fisher exact test; P ¼ .03). There were no significant differences in the distribution of symptoms and clinical characteristics of
ORAL AND MAXILLOFACIAL PATHOLOGY 472 Kragelund et al.
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Table I. Subgrouping of CYP1A2 phenotype in patients with OLP according to activity and demography, potential CYP1A2 modifiers (inducers and inhibitors), and CYP2D6*4 genotype Low CYP1A2 activity
Normal CYP1A2 activity
High CYP1A2 activity
n ¼ 23
n ¼ 73
n ¼ 15
4.47 1.53 (2.37-9.03) 45/28 59.9 (31-86) 42 (58) 30%
13.31 10.37 (8.12-48.61) 11/4 57.2 (31-77) 5 (33) 40%
Variable
Caffeine ratio SD (range) 1.47 0.74 (0.10-2.73) Women/men 15/8 Age (y), mean (range) 58.6 (30-74) CYP2D6*4 genotype, n (%) 8 (35) CYP1A2 substrates (%) 52% CYP1A2 induction CYP1A2-inducing drugs (%) 0 Smokers (%) 13% Moderate smokers (%) 0 Heavy smokers (%) 0 Coffee drinkers (%) 91% Heavy coffee drinkers (3 cups per day) 65% Coffee, cups, median (range) 3 (1-7) Tea drinkers (%) 57% Tea, cups, median (range) 6 (1-10) CYP1A2 inhibitors Drugs (%) 22% CYP2D6 IM or PM 12 (52) CYP2C9 IM or PM 6 (26) CYP2C19 IM or PM 4 (17)
P
1% 22% 7% 3% 92% 74% 4 (1-20) 45% 2 (0-11)
20% 47% 20% 0 87% 80% 6 (2-10) 27% 2
16% 48 (66) 22 (30) 17 (23)
20% 6 (40) 5 (33) 3 (20)
.02* .03y <.03y
.003y; .024z
CYP, cytochrome P450 enzyme; IM, intermediate metabolizers; OLP, oral lichen planus; PM, poor metabolizers. *NormalCYP1A2activity>
(Fisher exact test; P ¼ .01). More patients with low CYP1A2 activity and no CYP2D6*4 genotype (possible environmental OLP) presented red lesions (Fisher exact test; P ¼ .04), and none had oral candidiasis.
OLP between the CYP1A2-activity groups. However, analyzing according to the CYP2D6*4 genotype, a few interesting results were found (Table II). More patients with normal CYP1A2 activity and CYP2D6*4 genotype (possible genetic OLP) reported symptoms (Fisher exact test; P ¼ .003) and more had gingival lesions (Fisher exact test; P ¼ .03) compared with the remainder with a CYP2D6*4 genotype. Secondary candidal infections were diagnosed in 4 patients, which could influence the presence of symptoms and erythema. When these patients were excluded, more symptoms were recorded in the normal-CYP1A2activity group members with CYP2D6*4 genotype
DISCUSSION Our findings suggest that a subgroup of patients with OLP may have a susceptible genotype for OLP and another subgroup may have an environmentally provoked disease (i.e., OLL or LDE). When subgrouping patients with OLP according to CYP2D6*4 genotype and CYP1A2 phenotype, we found that a large proportion of
Table II. CYP1A2 activity and CYP2D6*4 genotype status in relation to symptoms and clinical parameters in patients with OLP Phenotype Genotype No. of patients, n Symptoms, n (%) Buccal LP, n (%) Gingival LP, n (%) Red lesions, n (%) Asymmetrical lesions, n (%) No. of anatomic regions affected, median (range) Extraoral LP, n (%) Cutaneous LP, n (%)
Low CYP1A2 activity CYP2D6*4 6 7 5 2 3 5.5
8 (75) (88) (63) (25) (38) (2-17)
1 (13) 1 (13)
No-CYP2D6*4 14 14 10 9 4 6
15 (93) (93) (67) (60) (27) (1-22)
2 (13) 1 (7)
Normal CYP1A2 activity CYP2D6*4 35 37 31 15 19 7
42 (83) (88) (74) (36) (45) (1-26)
12 (29) 11 (26)
CYP, cytochrome P450 enzyme; OLP, oral lichen planus; LP, lichen planus.
No-CYP2D6*4 21 27 23 9 9 8
31 (68) (87) (74) (29) (29) (1-21)
6 (19) 5 (16)
High CYP1A2 activity CYP2D6*4 2 4 2 2 5 7
5 (40) (80) (40) (40) (100) (1-13)
1 (20) 1 (20)
No-CYP2D6*4 8 10 7 4 4 10
10 (80) (100) (70) (40) (40) (3-33)
1 (10) 2 (10)
P .003 .03 .04
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the patients could be subdivided into either having a CYP2D6*4 genotype (possible genetic OLP) or having low or high CYP1A2 activity (possible environmental OLP), with some overlap. However, 28% of all the patients with OLP had neither CYP2D6*4 genotype nor low or high CYP1A2 activity. More patients with possible genetic OLP (normal CYP1A2 activity and CYP2D6*4 genotype) reported symptoms, and more had gingival lesions. The low-CYP1A2-activity and CYP2D6*4-negative patients presented more red lesions, which has been associated with LDE in a previous study.8 There were no obvious clinical differences between the different groups. Still, it may be naive to anticipate finding a clinical difference between OLP and OLL, given that the clinical course of OLP fluctuates.23 It is possible that there are differences in the course of the disease and the prognosis between the possible genetic OLP and the environmentally provoked OLP, but prospective studies are needed. CYP2D6*4, being a disease-susceptible genotype, has also been suggested by other groups. CYP2D6*4 genotype was associated with an accelerated rate of liver fibrosis in patients infected with HCV.24,25 Liver fibrosis develops after chronic inflammation, and chronic inflammation is the key element in OLP.4,26 OLP, as an oral manifestation of a systemic disease27,28, could possibly be related to the CYP2D6*4 genotype. However, we did not find significantly more patients with extraoral LP in the possible genetic OLP group (CYP2D6*4 genotype and normal-CYP1A2activity group); but the presence of local and systemic activators may vary in the group. As 30% of the background population has a CYP2D6*4 genotype, additional factors must be in play in the development of OLP. In some geographic areas, there seems to be an association between OLP and HCV infection. In these areas, OLP is suggested to be an extrahepatic HCV manifestation in HCV-positive patients.29 In 10% of patients with chronic HCV infection, liver-kidney microsome-1 (LKM-1) autoantibodies are detected.30 Molecular mimicry is believed to be involved in the pathogenesis of autoimmune hepatitis type 2 (AIH-2) because of epitope homology between CYP2D6, HCV, herpes simplex virus type 1, and cytomegalovirus.24,31 In AIH-2, CYP2D6 is targeted by LKM-1 autoantibodies, CD4þ T cells, and CD8þ T cells.26 In this respect, it is interesting that CD4þ T cells and CD8þ T cells account for the majority of the cells in the bandlike lymphocytic infiltrate immediately subjacent to the epithelium in OLP,5 and autoantibodies have been found in subsets of OLP.3,32,33 One case report has been published reporting LKM-1 autoantibodies in a patient with OLP and hypothyroidism,32 but 2 case series did not find any LKM-1 autoantibodies in patients with LP.34,35 However, it is not clear if these studies
ORIGINAL ARTICLE Kragelund et al. 473
investigated OLP or cutaneous LP. Cutaneous LP has a more self-limiting nature compared with OLP, and they may be pathogenetically different.9,32 Autoantibodies have been sporadically detected in OLP,3,32,33 but it seems appropriate also to search for autoreactive T cells in OLP. However, subgrouping of patients is fundamental, as multiple etiologies cause lichenoid lesions.2
CONCLUSION Subgrouping of patients with OLP/OLL is crucial for better understanding of the pathogenesis, progression, treatment, and prognosis. Surprisingly, the present study found that CYP2D6*4 genotype was more prevalent in patients with normal CYP1A2 activity, suggesting that genetic susceptibility might have an influence in this subgroup and lifestyle factors in the low- and highCYP1A2-activity groups. For treatment purposes it would be logical to pay attention to environmental factors and drugs influencing CYP1A2 activity, as these may influence the clinical presentation and prognosis. Thus, OLP subgrouping may allow for more personalized treatment with less unpredictability and variation in treatment response.12 Future projects should involve searching for particular autoreactive T cells against CYP2D6*4 in CYP2D6*4-positive patients with OLP. The authors thank the Research Unit of Clinical Pharmacology, University of Southern Denmark, where the CYP1A2 phenotyping was performed.
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