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Association of HLA-B*51:01, HLA-B*55:01, CYP2C9*3, and Phenytoin-Induced Cutaneous Adverse Drug Reactions in the South Indian Tamil Population

Phenytoin (PHT) is one of the most commonly reported aromatic anti-epileptic drugs (AEDs) to cause cutaneous adverse reactions (CADRs), particularly severe cutaneous adverse reactions (SCARs). Although human leukocyte antigen (HLA)-B*15:02 is associated with PHT-induced Steven Johnson syndrome/toxic epidermal necrosis (SJS/TEN) in East Asians, the association is much weaker than it is reported for carbamazepine (CBZ). In this study, we investigated the association of pharmacogenetic variants of the HLA B gene and CYP2C9*3 with PHT-CADRs in South Indian epileptic patients. This prospective case-controlled study included 25 PHT-induced CADRs, 30 phenytoin-tolerant patients, and 463 (HLA-B) and 82 (CYP2C9*3) normal-controls from previous studies included for the case and normal-control comparison. Six SCARs cases and 19 mild-moderate reactions were observed among the 25 cases. Pooled data analysis was performed for the HLA B*51:01 and PHT-CADRs associations. The Fisher exact test and multivariate binary logistic regression analysis were used to identify the susceptible alleles associated with PHT-CADRs. Multivariate analysis showed that CYP2C9*3 was significantly associated with overall PHT-CADRs (OR = 12.00, 95% CI 2.759–84.87, p = 003). In subgroup analysis, CYP2C9*3 and HLA B*55:01 were found to be associated with PHT-SCARs (OR = 12.45, 95% CI 1.138–136.2, p = 0.003) and PHT-maculopapular exanthema (MPE) (OR = 4.041, 95% CI 1.125–15.67, p = 0.035), respectively. Pooled data analysis has confirmed the association between HLA B*51:01/PHT-SCARs (OR = 6.273, 95% CI 2.24–16.69, p = <0.001) and HLA B*51:01/PHT-overall CADRs (OR = 2.323, 95% CI 1.22–5.899, p = 0.037). In this study, neither the case nor the control groups had any patients with HLA B*15:02. The risk variables for PHT-SCARs, PHT-overall CADRs, and PHT-MPE were found to be HLA B*51:01, CYP2C9*3, and HLA B*55:01, respectively. These alleles were identified as the risk factors for the first time in the South Indian Tamil population for PHT-CADRs. Further investigation is warranted to establish the clinical relevance of these alleles in this population with larger sample size.


Introduction
Phenytoin (PHT) is still the most effective treatment for generalized tonic-clonic seizures (GTCS) despite newer anti-epileptic drugs' (AED) availability [1,2]. However, cutaneous adverse drug reactions (CADRs) may limit its use; the estimated relative risk of PHT-severe cutaneous adverse reactions (SCARs) was reported to be 13% [3]. The prevalence of CADRs ranges from 2-5% in India [4]. Further, PHT and carbamazepine (CBZ) are the worst offenders of CADRs, with incidence rates of 13 and 18%, respectively [5]. The contributing factors to CADRs can be both genetic and non-genetic. In 2004, the association between HLA B*15:02 and CBZ-Steven Johnson syndrome/toxic epidermal necrosis (SJS/TEN) was reported among the Han Chinese populations [6]. Later this association has been confirmed with PHT-SJS/TEN in the Thai and Chinese Asian population [7,8]. As a result, the FDA issued a warning for HLA B*15:02/PHT-SJS/TEN cross-reactivity. However, the strength of this association is weaker than CBZ-SJS/TEN and not demonstrated well enough in many populations [9,10].
Polymorphisms in genes that encode drug-metabolizing enzymes, in addition to HLA-B alleles, play a key role in the initiation of CADRs by slowing drug metabolism. PHT is metabolized primarily by CYP 450 in phase I reactions and mostly by UDP-glucuronosyl transferase (UGT) in phase II reactions. The enzymes CYP2C9 and CYP2C19 are responsible for 90% and 10% of the metabolism of PHT, respectively [11].In CYP2C9, 2* and 3* variants are responsible for reduced PHT clearance [12,13].
Due to distinct waves of immigration, dissimilar genetic patterns in the Indian population have been extensively documented: North Indians are genetically closer to Caucasians, whilst Central Indians are closer to Asians than the European group [14,15]. The South Indians, or Dravidians, who are distributed on the southern side of India (peninsular India), are the original inhabitants of the Indian sub-continent; thus, the distribution of polymorphic alleles is not homogenous [16,17]. For example, the frequency of HLA B*15:02 is lower (0%) among West Coast Parsi and higher (6%) among Pawra in the Khandesh region [18,19]. Similarly, the frequency of CYP2C9*3 varies by location in India: a higher frequency (7-9%) is seen in the Dravidian population, whereas the prevalence is relatively low (0-2%) in the North Indian population [20].
A few studies in India have reported the association of HLA alleles and AEDs induced CADRs [21][22][23][24], and one study from South India reported a high serum PHT concentration in healthy volunteers carrying CYP2C9*3 [19]. There has been little research into the genetic risk factors for PHT-CADRs in the South Indian Tamils, who inhabit primarily in Tamil Nadu, India, and also scantily in Malaysia, Sri Lanka, Singapore, and Mauritius. We aimed to look at the relationship between HLA-B alleles, CYP2C9*3, and PHT-CADRs in the South Indian Tamil population for the first time in this study.

Study Design and Settings
This study was conducted as a prospective case-controlled study over a period of 13months in the Neurology-Outpatient Department (OPD), Neurology ward, and Neurology-Intensive Care Unit (ICU) of Meenakshi Mission Hospital and Research Center (MMHRC) in Madurai, Tamil Nadu, India. This study was approved by the Institutional Ethics Committee Board of MMHRC (MMHRC/IEC/07/2018). DNA analysis of both cases and tolerant controls was performed at the Immunology Department, School of Biological Sciences, Madurai Kamaraj University, Madurai, Tamil Nadu, India ( Figure 1).

Participants
Patients who had reported CADRs within 12 weeks of using PHT were included in the study. Patients on PHT for more than 3 months and had no signs or symptoms of CADRs were considered tolerant controls. The patients who signed the consent form and agreed to give 3 mL of whole blood were included. Patients experiencing other PHT-ADRs and with established skin problems, such as psoriasis and contact dermatitis, were eliminated. Normal-control data was retrieved only from South Indian populations, but no such restrictions were kept for pooled data analysis.
The dermatologist at MMHRC diagnosed all of the PHT-CADRs as well as additional diagnostic criteria, such as the temporal relationship with phenytoin, clinical morphology of the skin, and mucosal and systemic involvement. SJS/TEN, acute generalized exanthematous pustulosis (AGEP), exfoliative dermatitis (ED), drug rash with eosinophilia and systemic symptoms (DRESS) reactions were considered as SCARs. The remaining reactions with no/less mucosal involvement were considered to be mild-moderate reactions in this study [25]. Maculopapular exanthema was defined as a rash characterized by fine pink macules/papules/lesions on the skin with no mucosal or systemic involvement (MPE) [26]. Acneiform drug eruption was defined as a monomorphic eruption without comedones (AFDE) [27].
Fixed drug eruption (FDE) was defined as a single round and oral, sharply demarcated, red-lined lesion with a diameter of 1-10 cm [28]. PHT-lichenoid drug eruption was defined as skin lesions characterized by scaling and hypertrophic pigmentation, generally in combination with oral eruption (LDE) [29]. Patients with rapidly developing dark purpuric macules, atypical target lesions, blisters accompanied by mucosal and skin detachment were diagnosed with SJS, according to Roujeau's criteria [30]. The RegiSCAR criteria were used to diagnose DRESS and DHS, which included an acute skin rash with at least one internal organ lymphadenopathy; hematologic abnormalities, such as eosinophilia and atypical lymphocytosis; and fever [25]. Patients with erythematic inflammatory skin disease, scaling on the cutaneous surface of the skin, thickened skin, itching, swollen lymph nodes, fever, and fluid loss were considered as PHT-ED [31].
Once the patient was identified as a case or control, the consent form was handed to them along with a patient information document created in the local language of Tamil. The normal-control data for the HLA-B association study was obtained from Leenam Dedhia et al. (2015), who investigated HLA diversity and its significance in South Indians [32]. The normal control data for CYP2C9*3 and PHT-CADRs association testing is from the published literature (Nahar R et al. 2013) [33].

Causality Assessment
The causality of PHT-CADRs was assessed using Naranjo's scale, with patients scoring 9 (definite) and 5-8 (probable) included in this study [34]. Patients reported with DRESS were included if the RegiSCAR score was definite >5 and probable (4-5) [35]. The ALDEN score was used to assess the drug's causality with SJS: patients scoring very probable >6 or probable 4-5 were included [36]. DNA was extracted from 3mL of peripheral blood using the salting-out method [37,38]. A UV spectrophotometer was used to measure the concentration and purity of DNA by measuring its optical density (OD) at 260 nm. The polymerase chain reactions-sequencespecific primer technique was used to genotype HLA-B. (PCR-SSP) (Applied Biosystems Verti-Thermal cycler, Thermo Fisher Scientific, Waltham, MA, USA) [39].

CYP2C9*3 Sequencing
The reference DNA sequence of the target variant CYP2C9*3 was retrieved (rs1057910), and the genomic DNA was amplified using the selected forward primer from chromosome passion at 94981018 to 94981037 bp (GTGCATCTGTAACCATCCTC) and the reverse complementary primer from chromosome passion at 94981455 to 94981476 bp (GAGTTAT-GCACTTCTCTCACCC). The PCR DNA was purified according to the manufacturer's protocol by MinElute PCR Purification Kit (Cat. No. 28006, Qiagen, Valencia, CA, USA) and sequenced using a 3500 automatic DNA segmentation analyser (3730 DNA analyzer, Applied Biosystems, Thermo Fisher Scientific, Waltham, MA, USA). Sequence scanner software was used to obtain sequential sequencing.

Statistical Analysis
To compare the demographics and clinical features of the case and control groups, a Student's t-test was performed. The Fisher exact or Pearson chi-square tests were used if the demographics are categorical variables. The OR was calculated to see if there was an association between specific pharmacogenetic risk factors and PHT-CADRs. The Fisher exact test was used to get the p-value. Woolf's logit method was used for any cells in a contingency table that had zero. The bivariate analysis was carried out on alleles with a prevalence of more than 5% in the case group. To examine the association of various risk alleles with PHT-induced CADRs, PGx variants that exhibit significant association in bivariate analysis were included in a multivariate binary logistic regression analysis.
We used pooled data analysis for the HLA B*51:01 to boost the study's power. The HLA B*51:01 case and tolerant control data were obtained from one of the North Indian studies that reported the relationship between HLA alleles and AED-induced CADRs [20].
All the statistical analysis was performed using GraphPad Prism 8. After Bonferroni correction, a p-value 0.008 (<0.05/6-two-tailed) was considered significant.

Patient Demographics
This study included 30 PHT-tolerant and 25 PHT-induced CADRs epileptic cases in this study. Six (23.07%) of the 25 cases were SCARs, including two cases of SJS, three cases of DRESS, and one case of ED. The remaining 19 cases (76.92%) were mild-moderate reactions, comprised of 15 MPE, 2 AFDE, 1 LDE, and 1 FDE. The case group included 14 males and 11 females with a mean age of 40.60 ± 18.15 years, while the PHT-tolerant group included 18 males and 12 females with a mean age of 36.21 ± 14.71 years. Epilepsy, seizure, cerebrovascular accidents, and CNS infections were the common indications for PHT in both the case and tolerant groups (Table 1) (Supplementary Table S1). The CYP2C9 normal-control data from the previous study included 82 healthy people (40 males and 42 females) from Dravidian or South Indian populations, such as Tamils (25), Andhra Pradesh (32), and Kerala [35]. This study included the data of 463 HLA-B normalcontrols (Tamils) that was retrieved from past literature [34]. A total of 52 PHT-CADRs patients (31 MPE, 7 SJS/TEN, 8 DRESS, and 5 FDE) and 100 PHT-tolerant control patients with ages ranging from 6 to 72 were included in the pooled analysis. There were 22/30 and 41/59 females and males in their case and tolerant groups, respectively [20].

Clinical Features of PHT-CADRs
The itching was more common among mild-moderate reactions (13/20) than SCARs (1/6). Maculopapular rash/exanthematous rash or lesions/skinredness/burning sensation or warmth while touching were the most common cutaneous clinical manifestations in mildmoderate reactions (6/6 in SCARs and 13/20). Papules, pustules, blisters, and erythema were other serious features of skin reactions that were more common with SCARs (4/6) than mild to moderate reactions (5/20). The most commonly impacted mucosal sites were the mouth, eyes, genitals, and anogenital mucosa. SCARs were the only ones that showed systemic involvement. The most commonly affected systems were the liver and hematological systems. All three DRESS patients showed lymphadenopathy, abnormal lymphocytes, and eosinophilia. In one ED patient, neutrophilic leukocytosis was observed ( Table 1).
The onset latency period ranged from 7 to 42 days, with a mean of 21.7 days. In Naranjo's causation assessment, all cases received a likely score of ≥5. According to the RegiSCAR and ALDEN criteria, all DRESS and SJS patients had a definite (>5) and very probable (>6) connection.    Table 2).
A study from South India reported that HLA B*07 was the most common allele (6-13%) in the HLA-B gene, and its association with cervical cancer along with HLA DQ8 was found to be significant. The next common alleles reported in South Indians were the split antigens of the HLA B5 serotype, HLA B* 51 (8-12.5%) and HLA B* 52 (5-10%). Their association with different vasculitides was reported (HLA B*51 and Behchet'sdiseases, HLA B* 52, and Takayasu's arteritis) [45].
In this study, the association between HLA B*40:01 and PHT-mild moderate reactions in particular, MPE, was found to be stronger when compared to normal healthy controls, confirming previous findings (Sukesm et al., 2020) [46], which confirmed HLA B*40:01 as a risk factor for PHT-induced MPE (OR 3.647; 95% CI, 1.193-11.147; p = 0.023).This allele could be a drug-specific HLA genetic marker for PHT-MPE. However, a study with a larger cohort is needed to confirm this finding.
HLA B*55:01 was not shown to be susceptible to PHT-induced CADRs in any previous association studies that looked into the relationship between HLA and AEDs. The current study is the first to confirm this association in the South Indian-Tamil population. However, a Han-Chinese study also showed a correlation between HLA B*55:01 and LTG-induced MPE (OR = 24.78, 95% CI 1.50-408.76, p = 0.08) [47]. This genotype has been linked to penicillin hypersensitivity and nevirapine-induced SCARs in addition to AEDs [48,49]. In this study, HLA B*15:01 and HLA B*35:01 were found in a higher percentage of PHT-tolerant people than in cases and normal controls. This finding is consistent with other Indian studies that indicated a higher prevalence of the HLA B*15:01 allele in the control group than in the case group [23,24], whereas HLA B*35:01 was associated with LTG-induced MPE [50]. In the Tamil population, HLA B*15:01 and HLA B*35:01 may be protective alleles for PHT-CADRs.
The mutant allele (AC) was present in 48 and 7.14 percent of cases and tolerant-control groups, respectively, with no homozygosity (CC), which is similar to a study that found no frequency of CC genotype in the South Indian Dravidian community [19]. The current investigation found a substantial link between the CYP2C9*3 heterozygous condition and PHT-induced CADRs. In this investigation, patients with CYP2C9*3 alleles were almost 13 times more vulnerable to PHT-CADRs than the tolerant group. This finding is comparable to that of a Thai study, which found that patients with CYP2C9*3 have a 14.5 times higher incidence of PHT-SCARs. A subgroup analysis of this study within the SCARs group reveals a higher correlation between CYP2C9*3 and PHT-induced DRESS, which was also corroborated in another Thai investigation [46,51,52].
There may be some limitations to this research. We did not rule out patients who were using CYP2C9 inhibitors, which could have contributed to the rise in PHT levels. Despite this, only three patients were prescribed VPA (CYP2C9 inhibitor), and no other known CYP2C9 inhibitors were prescribed in this group. Although a few studies have found a relationship between CYP2C9*2 and PHT-CADRs, we did not investigate this allele in this study because it is extremely rare (1-2%) in the South Indian population. Another limitation of our research is the small sample size. The rare outcome of interest is the reason for it. In addition to genetic defects, clinical and non-clinical factors may play a role in the initiation of PHT-CADRs, and these should be examined alongside genetic variants.

Conclusions
CYP2C9*3 and HLA B*51:01 were found to be associated with PHT-SCARs and PHT-DRESS. On the other hand, PHT-mild/moderate cutaneous reactions are linked with HLA B*55:01 and HLA B*40:01 in this study. This is the first study in South India, specifically among Tamils, to show a correlation between HLA B*51:01, HLA B*55:01, and CYP2C9*3 alleles and PHT-CADRs. These alleles can be employed as genetic markers to identify individuals who are susceptible to PHT-CADRs and to ensure that PHT is as safe as possible for Tamil epileptic patients. Furthermore, our findings highlight the necessity of including the HLA B*5101 and CYP2C9*3 alleles into a pre-emptive genetic testing panel for Asians with PHT-CADRs.