Cutaneous drug hypersensitivity: immunological and genetic perspective.

Drug hypersensitivity is an unpredictable, immunologically mediated adverse reaction, clustered in a genetically predisposed individual. The role of "hapten concept" in immune sensitization has recently been contested by the "pharmacological interaction" hypothesis. After completion of the "human genome project" and with the availability of high-resolution genotyping, genetic susceptibility to hypersensitivity for certain drugs has been proved beyond doubt though the trend is ethnicity and phenotype dependent. Application of this newly acquired knowledge may reduce or abolish the morbidity and mortality associated with cutaneous drug hypersensitivity.

Introduction

The morbidity and mortality associated with adverse reactions to different drugs remain a major threat to present-day therapeutics. Many drug reactions involve the skin either in an isolated manner or as a component of systemic involvement. Some of these cutaneous adverse drug reactions (CADR) like maculopapular rash (MPR) or urticaria are mild, while some others like drug hypersensitivity syndrome (DHS) and Stevens–Johnson syndrome (SJS) are associated with poorer outcomes. Adverse drug reactions (ADRs) can sometimes be explained by the pharmacological property of the drug and are predictable. This is known as “type A” ADR.[1] However, neither certain drug reactions can be explained by the pharmacological nature of the drug nor their occurrence can be predicted. This is categorized as “type B” ADR.[1] The majority of these are caused by immunological response of the host against the parent drug or its active metabolite. The term “drug hypersensitivity reaction” (DHR) is restricted to these immune-mediated “type B” ADRs. Many of these DHRs show familial and ethnic clustering that can only be explained by genetic susceptibility. Recent research has provided some objective proof to this hypothesis. Thus, drug hypersensitivity is an immunologically mediated response restricted to a few genetically predisposed individuals. If the genetic basis can be used to identify the “at-risk population” or the immunogenic component of the drug can be neutralized, many CADR may be predicted and prevented. This constitutes the basis of “personalized medicine”.

Basic Immunology of Drug Hypersensitivity Reaction

Like all hypersensitivity reactions, DHR can be classified according to Gell and Coomb's classification[2] into four different groups, and the fourth group has recently been classified further into four subgroups (IVa–IVd)[34] [Table 1].[3]

Table 1

Drug hypersensitivity—Gell and Coomb's modified classification

Most CADR are caused by type IV or delayed type hypersensitivity reaction (DTHR). The offending drug or its metabolite either diffuse through stratum corneum or enter via systemic circulation and provoke sensitization (vide infra). This leads to stimulation of Langerhans cells (LC) with liberation of inflammatory signals culminating in maturation of LC and their migration to regional lymph node. Consequently, antigen presentation to T-cells takes place through major histocompatibility complex (MHC), which causes expansion of clonal T-cells. Presence of costimulatory signals potentiates this expansion. Recently, these T-cells have been found to have heterogeneous function (cytokine production, inflammatory cellular response and subsequent nature of CADR)[3] [vide [Table 1]].

Drug Sensitization

Hapten theory

Though sensitization and activation of immune system is thought to occur in all drug hypersensitivities, there is much controversy about the process of sensitization. In 1935, Landsteiner introduced the “hapten concept” to explain the mechanism of sensitization.[5] This concept has so far been used to explain the initiation of sensitization in DHR. A hapten, by definition, is a protein with low molecular weight (<1000 Da) which binds covalently in vivo to some protein structure, be it soluble or cell bound. [Prohaptens are proteins which generate hapten after metabolism in vivo]. This hapten-carrier complex is then taken up and processed by antigen presenting cells (APC) and presented to T-cells through MHC. As a result, T-cell activation takes place and primary immunological response is generated.

Pharmacological-interaction theory

In recent years, this “haptenation” theory has been challenged by a novel concept called pharmacological-interaction (P-I) theory.[36] According to this theory, the drug can bind directly to the T-cell receptor (TCR) via a non-covalent bond. Neither is there a need for the drug to bind to any carrier protein nor the drug needs to be metabolically broken down into “haptens”. It has been postulated that for this concept to be significant, the T-cell should express a TCR that can bind the drug and induce stimulatory signal. The T-cells should also have a lower threshold for stimulation so that they are stimulated by minor signal generated by “drug binding to TCR”. Lastly, the TCR should interact with MHC on APC to enhance the immune response.[7]

Hapten theory or pharmacological-interaction concept?

The proponents of the hapten theory have cited the example of sulfamethoxazole (SMX)-nitroso which is a metabolic derivative of SMX and acts as a hapten to initiate SMX hypersensitivity. This SMX-nitroso is deactivated in vivo by non-protein thiol (glutathione) or ascorbate and it has been shown that people with lower level of thiol and ascorbate (like those suffering from HIV/AIDS) generate more SMX-nitroso and develop SMX hypersensitivity more frequently than those in the normal population.[8] However, using the same SMX model, it was shown that addition of glutathione did not reduce, but rather increased the immune stimulation,[9] though SMX-nitroso generation stopped and this could only be explained by the “P-I concept”. In the past few years, several studies had been undertaken to establish or refute these two concepts. The pros and cons of the two theories are presented in Table 2.

Table 2

Pharmacological-interaction concept versus hapten concept

The nickel model: Futile unifying effort or fact?

These two contradictory views could be unified using the nickel model. In this, the nickel ion is shown to act both as a “hapten” as well as a metal ion directly binding to TCR simultaneously.[15] But the concept that priming of T-cells with reactive metabolites (hapten theory) may result in secondary reactivity toward the parent drug (P-I concept) has recently been refuted.[16]

Danger hypothesis: Redefining hapten theory

There was one chink in the armor of “hapten theory”—if certain drug metabolites can act as a hapten, why does not a drug always lead to drug hypersensitivity? The quest for the answer has generated the “danger hypothesis”.[17] According to this hypothesis, the primary signal initiated by the hapten–carrier complex must be strengthened by co-stimulatory signals (danger signals) lest the primary signal should wean off and “tolerance” develops instead of hypersensitivity.

Further substantiation of danger hypothesis

The actual genre of danger signals is presently indefinite, yet the probable candidates are high mobility group box 1 protein (HMGB1), heat shock proteins (HSPs), and S100 proteins.[18] These molecules act through toll-like receptors. Concurrent presence of physical, chemical or viral stress is known to induce cell death and release these danger signals,[19] explaining the increased occurrence of drug hypersensitivity in these conditions. Recent studies have shown that polymorphism of the genes involved in synthesis of these “danger signals” (HSP,[2021] tumor necrosis factor or TNF[2223] ), their “receptors” (toll-like receptor 3[24]) or the “signaling pathway” (proapoptotic Fas-L[25] and IL-4 receptor/IL-13 signaling pathway[26]) can influence the occurrence and severity of drug hypersensitivity.

Genetic Basis of Drug Hypersensitivity Reaction

The idiosyncratic behavior and familial clustering of the drug hypersensitivities have intrigued researchers to find out a genetic basis. In the earlier days, studies of genetic background in drug hypersensitivity meant twin studies (monozygotic and dizygotic) where both twins suffered from the same disease and got the same treatment. In practical sense, it meant an extremely large and economically unfeasible study. The elementary role of MHC in drug hypersensitivity allured initial researchers to concentrate on human leukocyte antigen (HLA), but the outcome of their studies was confusing. The reason may be small number of cases, inadequate case definition or insensitive serological typing.[27]

A shift of idea: Multigenic concept in drug hypersensitivity reaction

Drug hypersensitivity was previously believed to originate from a single gene defect. But now we know that an intricate interaction between many genes as well as environmental factors culminates in drug hypersensitivity. The different genes can be classified function-wise into four groups: a) bio-activation genes, b) bio-inactivation genes, c) genes controlling immune-responsiveness, and 4) genes controlling tissue injury and repair.[19]

Metabolic gene: A suspect in earlier studies

Many investigators concentrated on polymorphisms of genes responsible for the synthesis of drug metabolizing enzymes. It was postulated that carbamazepine 10-11 epoxide, a major metabolite of carbamazepine (CBZ) both in vitro and in vivo, leads to immune sensitization, and defective detoxification of this metabolite causes CBZ induced DHR. In vitro lymphocyte cytotoxic assay also pointed to a detoxification defect underlying the propensity of CBZ hypersensitivity. But no specific polymorphism in microsomal epoxide hydrolase gene (responsible for CBZ 10-11 epoxide detoxification) could be detected in two separate studies.[2829] Earlier studies demonstrated that slow acetylator phenotype arising from acetylator gene polymorphism is associated with SMX hypersensitivity both in normal population[30] and in HIV infected patients,[31] but recently this claim has been refuted.[3233] Similarly, in abacavir hypersensitivity, polymorphism of metabolically important genes has been found to be insignificant.[22]

Re-exploration of major histocompatibility complex and high-resolution genotyping: Old suspect, new method

After failure of the “metabolic gene hypothesis”, most investigators again turned their focus on MHC. In contrast to earlier studies using serology for HLA-typing, nowadays, high-resolution genotyping is being used for this purpose. Knowledge of linkage disequilibrium pattern[3435] has further helped researchers to reach definitive conclusions, particularly in relation to an anti-retroviral drug, abacavir; antiepileptic drugs (AEDs) like carbamazepine (CBZ), oxcarbamazepine (OXC), phenytoin (PHT), lamotrizine (LTG); and the anti-gout drug, allopurinol.

Abacavir and Pharmacogenomics

A curtain raiser in the field

Abacavir is a neucleoside reverse transcriptase inhibitor with good activity against HIV-1 virus. About 5% of patients develop hypersensitivity reaction to abacavir. Majority of the reactions appear in the initial 6 weeks of drug introduction and present as skin rash, fever, gastrointestinal and respiratory manifestations.[36] They start subsiding within 72 hours of drug withdrawal and recur with more severity within hours of drug reintroduction.[37] Familial clustering and lower risk in Afro-Americans[38] suggested genetic susceptibility in abacavir hypersensitivity. High-resolution genetic assessment has found a strong association of abacavir hypersensitivity with HLA-B*5701, HLA-DR7 and HLA-DQ3 haplotypes (odds ratio>100).[39] Further studies using recombinant haplotype mapping has placed the responsible locus in the ancestral haplotype 57.1. Haplotypic polymorphism in the TNF promoter region has also been associated with abacavir hypersensitivity[2240] and this may control severity by influencing TNF synthesis. It was shown recently[21] that concurrent presence of HLA-B*5701 and haplotypic Hsp 70-Hom variant was more specifically associated with abacavir hypersensitivity (odds ratio 3893, P<0.00001). HLA-B*5701 association to abacavir hypersensitivity was later confirmed in White males.[41]

Does ethnicity really matter in abacavir hypersensitivity?

The strong association of HLA-B*5701 allele to abacavir hypersensitivity could not be demonstrated in Afro-Americans and this discrepancy was attributed to linkage disequilibrium.[41] A different genetic basis in non-White population might be another possibility. But this ethnic bias has lately been questioned. Saag et al. have found that in immunologically (by patch test) confirmed (but not in clinically suspected) abacavir hypersensitivity, HLA-B*5701 allele is a 100% sensitive marker in Afro-Americans.[42] Besides, screening for HLA-B*5701 has been shown to reduce the incidence of abacavir hypersensitivity among ethnically mixed population.[43]

Bridging the gap: Clinical application of pharmacogenomics in abacavir hypersensitivity

Depending on a West Australian HIV cohort, Mallal et al. proposed routine HLA-B*5701 genotyping before prescribing abacavir to Whites.[39] Further studies among predominantly White population confirmed that screening is effective in reducing abacavir hypersensitivity[4445] and is cost-effective.[46] Genetic screening has recently been demonstrated to be cost-effective even in multiethnic French population.[43]

Quest for newer genotyping method: Cheap, easy and quick

The complex and time-consuming HLA typing method using sequence-based primer intrigued scientists to invent an alternative path. Recently, an HIV cohort in Switzerland demonstrated a strong association between HCP5 single-nucleotide polymorphism (SNP) and abacavir hypersensitivity. This SNP, known as rs2395029, is in linkage disequilibrium with HLA-B*5701 in European ancestry and was found in all 98 HLA-B*5701 positive individuals, but absent in 999 of 1005 HLA-B*5701 negative persons. This HCP5 genotyping may be useful in place of sequence-based HLA typing for screening abacavir hypersensitivity.[47] A prospective Spanish study[48] found this method easier, cheaper and quicker compared to the conventional sequence based method.

Carbamazepine Hypersensitivity: Second Milestone in Pharmacogenomics

CBZ, SJS/TEN and the Han Chinese

The strongest genetic predisposition for drug hypersensitivity has been documented with CBZ. It is an AED associated with different clinical manifestations of hypersensitivity reaction including maculopapular rash (MPR), drug hypersensitivity syndrome (DHS) and Stevens-Johnson syndrome/toxic epidermal necrolysis (SJS/TEN). The last adverse effect is noted with excess frequency among people from Southeast Asian lineage, particularly Han Chinese, than their European or Japanese counterparts. In a path-finding study among Han Chinese in Taiwan, HLA-B*1502 allele was found among 100% patients who developed SJS/TEN after taking CBZ. The same allele was present only in 3% of CBZ-tolerant patients and the odds ratio was greater than 2504.49 and corrected P-value (Pc) was 3.13 × 10−21.[49]

Ethnicity matters in carbamazepine hypersensitivity

The allelic predisposition to development of SJS/TEN after CBZ intake could not be demonstrated in patients of Japanese[50] or European origin.[51] But multiple studies in non-Chinese Southeast Asian population could replicate the initial result.[52-54] (Among these, the Indian study[54] was methodologically weak as it did not consider the CBZ-tolerant patients). The exact cause of this ethnic difference is not yet clear. Some have attributed this to low prevalence of HLA-B*1502 allele in non-Southeast Asian population.[50] Another explanation comes from the multigenic concept of drug hypersensitivity which claims that other genes may be responsible in non-Southeast Asian population. A recent study demonstrated that HLA-B*5901 may be one of the alleles responsible for CBZ-induced SJS in Japanese.[55] But the most acceptable concept till date is “linkage disequilibrium” which claims that HLA-B*1502 is in strong linkage disequilibrium in Southeast Asian population but not in Japanese or European.

Genetic predisposition is phenotype specific

It should be remembered that the association of HLA-B*1502 allele is only valid for CBZ-induced SJS/TEN, but not for other CBZ hypersensitivities like MPR[5256] or DHS.[56] They may be caused by different allelic association (MPR has been associated with SNP in the HLA-E region and HLA-A*3101 and DHS with SNP in the motilin gene)[56] or by different immune mechanism.

Black-box warning for carbamazepine use

USFDA has issued a black-box warning stating that persons of genetically high risk ancestry should be screened for HLA-B*1502 allele before initiating treatment with CBZ.[57] Similar warning has been issued by Health Canada.[58]

Newer method of detecting the allele

In USA and Canada, many commercial laboratories now perform high-resolution genetic testing with sequence specific primer (SSP) using patient's blood or buccal swab.[59] These methods are costly and time-consuming (take around 1 week). Recently, a new (loop-mediated isothermal amplification or LAMP) method for detecting HLA-B*1502 genotype has been shown to be accurate (100% concordant with SSP), yet inexpensive, rapid and simple.[60] They may be useful in future in the clinical practice.

Does HLA-B*1502 allele confer a drug effect or a class effect?

There was another unanswered question: “Is this genetic susceptibility restricted to the drug (CBZ) or the class (drugs with similar aromatic ring)?” In clinical practice, cross-reactivity is known to exist between CBZ and three structurally similar drugs, phenytoin (PHT), lamotrigine (LTG) and oxcarbazepine (OXC). A large Thai cohort, in 2008, showed PHT-induced SJS to be associated with HLA-B*1502 allele.[52] In a recently published study, Hung et al. showed that OXC, PHT and LTG share the common risk allele HLA-B*1502 while inducing SJS. They recommended that CBZ, OXC and PHT should be avoided in the HLA-B*1502 carrier and LTG should be used with caution.[61]

Does other gene contribute in CBZ hypersensitivity?

Like abacavir, HSP gene polymorphism was also found to be associated with severe CBZ hypersensitivity, but it was not clear whether the association was causal or related to linkage disequilibrium with another gene in MHC.[20]

Allopurinol Hypersensitivity and Genetic Predisposition

Again the Han Chinese

The anti-gout drug, allopurinol, also has shown strong genetic predisposition for developing hypersensitivity reaction. It is the commonest cause of SJS/TEN in Europe[62] and one of the most frequent causes of DHS in the world.[63] In a case–control association study among Han Chinese, Hung et al.[64] found that the HLA-B*5801 allele was present in all 51 (100%) patients with allopurinol-induced severe cutaneous adverse reaction (30 DHS and 21 SJS/TEN) but only in 20 of 135 (15%) allopurinol-tolerant patients [odds ratio 580.3 (95% confidence interval, 34.4–9780.9), corrected P value = 4.7 × 10-24].

Ethnic bias: Probably present

In Japanese population, a strong association of HLA-B*5801 allele to allopurinol-induced SJS/TEN[5065] and DHS[65] was also evident. The association was moderate [odds ratio = 80 (confidence interval = 34–157), P<10−6] in the people of European ancestry.[66] This weaker association is probably due to lower frequency of the allele in the European population. A study among Thai population which has a high frequency of HLA-B*5801 allele has shown a strong association [odds ratio of 348.3 (95% confidence interval = 19.2–6336.9, P = 1.6 × 10)].[67] Further study with large cohort is needed for a definitive conclusion.

Does phenotype matter?

The study among Han Chinese[64] showed, unlike CBZ hypersensitivity, the genetic predisposition, here, is related to both DHS and SJS/TEN. This was confirmed by one Japanese study.[65] But the other studies[506667] did not look into allopurinol-induced DHS.

Is prior genotyping mandatory for allopurinol use?

Tassaneeyakul et al.[67] demonstrated strong association between genotyping for HLA-B*5801 allele and allopurinol-induced SJS/TEN (sensitivity 100%, specificity 87%, positive predictive value 1.52 and negative predictive value 100%) in Thai population. This proves the allele to be a valid genetic marker among these people. But Fernando and Broadfoot[59] recommended screening of all patients irrespective of ethnicity before allopurinol therapy, without much statistical strength. Till date, there is no recommendation for prior genetic screening. Moreover, genotyping for HLA-B*5801 allele is complex and available only in research laboratories.[59] These have impeded the implementation of routine genetic testing before starting allopurinol.

Other Drug: Newer Allele

Apart from the above, weaker association of rare alleles to SJS/TEN has been documented in European study.[66] These include HLA-B*38 for SMX or LTG and HLA-B*73 for oxicam. Recently, HLA-B*5901 allele has been claimed to be strongly associated with methazolamide-induced SJS/TEN in Korean patients.[68] It is too early to comment on the significance of these studies.

The Future

In the future, newer genetic association of drug hypersensitivity will be explored, focusing on their pathogenesis. However, to translate these research findings into clinical application, several points require stringent persuasion. First of all, different immunological hypotheses of drug hypersensitivity need to be verified, unified and rejected (if needed) by practical experiments. Secondly, the phenotypic diagnostic criteria of individual drug hypersensitivity involving the skin (MPR, SJS/TEN and DHS) must be clearly delineated and the genetic association of each of them separately explored. Thirdly, to add statistical strength to these studies, a large number of cases should be included. Considering the rarity of certain drug hypersensitivities, this may necessitate multicenter and even multinational study. Fourthly, genetic susceptibility to individual drugs should be re-evaluated in the light of ethnic backgrounds. The fifth point to be considered is the availability and cost-effectiveness of the screening test. Designing easier, quicker and cheaper tests is essential in this regard. Finally, experimental bias toward immune genes should be avoided. Genetic susceptibility may be conferred by “metabolic” genes and novel “nonimmune” genes[69] as well.

Glossary

Allele: Alternative forms of genes or other DNA sequences that occupy a specific locus on a particular chromosome.

Haplotype: A set of alleles on a single chromosome which are closely linked and inherited as a unit; commonly used in reference to linked genes of the major histocompatibility (HLA) complex.

Linkage disequilibrium: A situation in which some combinations of alleles or genetic markers occur more or less frequently in a population than would be expected from a random combination of alleles based on their frequencies in the loci.

Polymorphism: The occurrence of two or more clearly different phenotypes in the same population of a species, independent of sexual variations.

Single-nucleotide polymorphism: DNA sequence variation occurring when one base at specific base pair position differs between members of a species or paired chromosomes in an individual; for example, from Cytosine (C) → Thymine (T); Adenine (A) → Guanine (G). (AAGCCTA to AAGCTTA).