Association of Human Leukocyte Antigen Class 1 genes with Stevens Johnson Syndrome with severe ocular complications in an Indian population

Stevens Johnson syndrome (SJS) is part of a spectrum of adverse drug reactions resulting in the destruction of skin, mucous membranes, and the ocular surface. A similar, more severe form of the disorder included in this spectrum is toxic epidermal necrolysis (TEN). Approximately 35% of patients suffer chronic sequelae such as vascularization, corneal scarring, conjunctival inversion to the cornea, keratinization, symblepharon, scarring of the palpebral conjunctiva, trichiasis, and severe dry eye. We focused on 80 Indian patients with SJS/TEN with severe ocular complications (SOC) and investigated the association of alleles at HLA -A, HLA-B and HLA-C loci; the controls were 50 healthy Indian volunteers. Genotyping at HLA-A, HLA-B, and HLA-C loci showed a significant positive association with HLA-A*33:03, HLA-B*44:03, and HLA-C*07:01 alleles, and a significant negative association with HLA-B*57:01 and HLA-C*06:02. This indicates that HLA-A*33:03, HLA-B*44:03 and HLA-C*07:01 are risk alleles, and HLA-B*57:01 and HLA-C*06:02 are protective alleles in this population. We also found that the haplotypes consisting of HLA-B*44:03 and HLA-C*07:01 were strongly associated with SJS/TEN with SOC in our Indian population (p = 1.1 × 10−7, odds ratio = 11.0). Describing the association of the haplotype could facilitate the understanding of increased risk factors for developing SJS/TEN with SOC.

Common acute ocular manifestations are bilateral severe conjunctivitis, pseudomembrane, and corneal and conjunctival epithelial defects 4 . During the acute phase of SJS/TEN, approximately 50% of patients present with severe ocular involvement 5 .
Common chronic ocular manifestations include vascularization, corneal scarring, conjunctival inversion to the cornea, keratinization, symblepharon, scarring of the palpebral conjunctiva, trichiasis, and severe dry eye 6 . Chronic eye symptoms are red eye, dry eye, pain, itching, grittiness, heavy eyelid, foreign body sensation, decreased vision, a burning sensation, photophobia, and diplopia; they are seen in approximately 35% of patients 3 .
Significant risk factors for ocular involvement in SJS include exposure to drugs such as NSAIDs and cold medications 5,7 . About 80% of Japanese-and 53% of Brazilian SJS/TEN with SOC patients had taken cold medicines [7][8][9] .
Genetic studies involved case-control studies aimed at identifying associations between SJS/TEN with SOC and genetic loci involved in immune response pathways and the detoxication of drugs and xenobiotics. Alleles of various human leukocyte antigen (HLA) loci were the focus of genetic association studies of SJS/TEN in different populations and ethnic differences in genetic associations have been proposed. HLA antigens associated with SJS with ocular complications include HLAB12 or its sub-group HLABw44 in Caucasians 10,11 ; an association has also been reported in a French population with TEN 12 . Elsewhere we reported that in Japanese patients with SJS/TEN with SOC, the disease was associated with specific alleles at various HLA loci including HLA-A*02:06 13 and that HLA-B*44:03 was an independent risk allele for cold-medicine-induced SJS 14 . Our study across different ethnic groups with SJS/TEN with SOC suggested a significant association between HLAB*44:03 and HLA-A*02:06 alleles and SJS/TEN with SOC; in fact, we detected specificity in the associated alleles in different ethnic groups 15 .
The association of HLA-B*5801 with allopurinol-induced SJS/TEN was demonstrated in Han Chinese-16 , European-17 , and Japanese patients 18 . A striking observation in relation to HLA markers in Han-Chinese SJS patients is the association of carbamazepine (CBZ) and HLA-B*15:02 19 ; the predicted specificity and sensitivity for this allele as a marker for CBZ-SJS in this ethnic group was 97% and 100%, respectively. On the other hand, allopurinol-induced SJS/TEN was rarely associated with SOC 20 and only 5% of Japanese SJS/TEN with SOC patients had taken anti-epilepsy drugs such as CBZ 8 .
In the current investigation we focused on Indian patients with SJS/TEN with SOC. We examined genetic factors by investigating the association of alleles at HLA-A, HLA-B, and HLA-C loci.

Methods
Our study was approved by the institutional review board of the L.V. Prasad Eye Institute and Kyoto Prefectural University of Medicine. All experimental procedures were conducted in accordance with the principles set forth in the Helsinki Declaration. The purpose of the experimental protocols was explained to all participants and their prior written informed consent was obtained.
Patients. Between July 2012 and June 2014, 80 patients with SJS/TEN with SOC (43 males, 37 females, age range 5-63 years, mean age 28.1 ± 14.2 years) were recruited at the L.V. Prasad Eye Institute. At the time of this study, their age ranged from 10-50 years. All were in the chronic stage of the disease; the diagnosis of SJS/TEN with SOC was based on a confirmed history of acute-onset high fever, serious mucocutaneous manifestations with skin eruptions, and the involvement of at least 2 mucosal sites, including the oral cavity and ocular surface in the acute stage. In the chronic stage there were the ocular previously reported manifestations such as vascularization, corneal scarring, conjunctival inversion to the cornea, keratinization, symblepharon, scarring of the palpebral conjunctiva, trichiasis, and severe dry eye 14,[21][22][23][24] . The 50 normal Indian controls (27 males, 23 females, median age 36.0 ± 11.4 years) were also recruited at the L.V. Prasad Eye Institute; none had a history of SJS/TEN or related conditions or a history of cutaneous drug reactions.
Samples. Blood samples were collected by venipuncture from all study participants. DNA was extracted from blood leukocytes by the phenol chloroform method and assessed spectrophotometrically.
HLA typing. Genotyping for HLA alleles was performed by subjecting DNA samples to polymerase chain reaction (PCR) assay. This was followed by hybridization to simple-sequence oligonucleotides using a bead-based genotyping kit (Wakunaga, Hiroshima, Japan) 9,13-15,25,26 . Briefly, target DNA was PCR-amplified with biotinylated primers specifically designed for amplified exons 2 and 3 of HLA-A, HLA-B, and HLA-C genes and the PCR amplicon was denatured and hybridized to complementary oligonucleotide probes immobilized on fluorescent-coded microsphere beads. At the same time, the biotinylated PCR product was labeled with phycoerytrin-conjugated streptavidin and immediately examined with Luminex 100 (Luminex, Austin, TX, USA). Genotype determination and data analysis were performed automatically using WAKFLOW typing software (Wakunaga) according to the manufacturer's instructions. Data analysis. We compared the carrier frequency and the gene frequency of individual HLA alleles in the patients and controls based on the dominant model using the Fisher exact test (JMP version.11 software; SAS Institute Japan Ltd., Tokyo, Japan). The odds ratio (OR) with a 95% confidence interval (CI) was calculated with Fisher's exact test (JMP version 11 software). Significance levels were corrected with Bonferroni correction for multiple comparisons.

Results and Discussion
As shown in Supplementary Table 1 Table 1). Our study thus confirms an association of HLA-*B44:03 alleles in Indian patients with SJS/TEN with SOC, and first documents an association of HLA alleles HLA-A*33:03, HLA-B*57:01, HLA-C*06:02, and HLA-C*07:01 in an Indian population.
Although detailed information on drugs used and infections was not available for all patients, we were able to confirm that 23 patients presented with the onset of SJS/TEN after taking cold medicines. HLA analysis of these 23 samples and of 50 control samples showed a significant positive association with HLA-B*44:03 and HLA-C*07:01 alleles and a significant negative association with HLA-C*06:02 (Supplementary Table 2).
In addition to HLA-B*44:03, we identified HLA-C*07:01 as a risk factor for cold medicine-related SJS/TEN with SOC in the current study population and HLA-C*06:02 as a strongly protective HLA allele.
In summary, we first report that in our Indian population, the haplotype comprised of HLA-B*44:03 and HLA-C*07:01 is strongly associated with SJS/TEN with SOC. Our findings also suggest that HLA-B*57:01 and HLA-C*06:02 are protective against this disease.