Pharmacogenetics and Infection – Histocompatibility & Immunogenetics

Immunosuppressive Drug Metabolism

Many immunosuppressive drugs are characterised by a narrow therapeutic index. That is, the difference between a therapeutic dose and a toxic dose is small, often requiring patients on immunosuppressive regimes to have their drug dosage adjusted according to actual levels detected in the blood. Too little of an immunosuppressive drug can contribute to rejection whilst too much can be toxic. A classic example is the seen in the nephrotoxicity of the calcineurin inhibitors cyclosporin and tacrolimus.

Getting the right balance is complicated by the fact that individuals differ significantly their bioavailability, i.e. the fraction of a fixed administered dose of drug that reaches systemic circulation. This variability in drug response has been shown to have a heritable genetic component.

Immunosuppressive drugs are transported across the cellular membrane by the transmembrane pump P-glycoprotein in the liver and gut, where they are metabolised by Cytochrome P450 (CYP) enzymes, a large diverse group of enzymes which account to more than 75% of all drug metabolism. Polymorphisms in the genes for Cytochrome P450 enzymes have been associated with differences in immunosuppressive drug metabolism, particularly metabolism of tacrolimus.

The majority of Cytochrome P450 drug metabolic activity is accounted for by the CYP3A sub family. The genes encoding the CYP3A sub family are located on chromosome 7. The main isoforms encoded are CYP3A4 and CYP3A5. Both isoforms carry a number of single nucleotide polymorphisms (SNPs) resulting in a number of alleles. CYP3A4 polymorphisms include CYP3A4*1 and CYP3A4*1B. CYP3A5 polymorphisms include CYP3A5*1 and CYP3A5*3. Most studies have demonstrated that patients with the CYP3A5*3 allelic variant show higher blood concentrations of tacrolimus therefore allowing lower maintenance doses of the immunosuppressant compared with patients with the CYP3A5*1 allele. In one study, the CYP3A4*1/CYP3A5*1 and CYP3A4*1B/CYP3A5*1 genotypes were significantly more frequently associated with the development of biopsy-proven calcineurin related nephrotoxicity compared to the CYP3A4*1/CYP3A5*3 genotype.

Genetics variants also affect drug pharmocodynimics, i.e. the pharmacologic effect resulting from the interaction between the drug and the biologic system. Mutations in the ACE and Angiotensinogen genes for instance lead to differences in blood pressure reduction for the same dose of ACE inhibitors.

Mutations in the N-acetyltransferase (NAT) is genes (NAT1 and NAT2) lead to differences in the rates of acetylation of common drugs such as caffeine, isoniazid, nitrazepam and sulphonamides. Acetylation is a key step in the metabolism of these drugs.

Drug Hypersensitivity

Drug hypersensitivity is a syndrome characterised by a combination of fever, skin rash and internal organ involvement, generally occurring more than 1 week after exposure to a drug. The most common internal organ manifestation is hepatitis. Other less common manifestations include pancreatitis, myocarditis, nephritis, intestinal lung disease and muscle inflammation. Adverse drug reactions can be classified into those, type A, which are predictable based on the pharmacogenetics of the drug and those, type B, which are not.

Many type B drug reactions are now thought to be immunologically mediated. The discovery of associations between drug hypersensitivity and specific HLA alleles has been a recent major advance and has led to the possibility that type B drug hypersensitivity reactions may be predictable and preventable. Common drugs associated with immunologically mediated drug hypersensitivity include the anticonvulsant Carbamazepine and the antiretroviral agents Nevirapine and Abacavir. Carbamazepine has been shown in one study to be associated with the HLA class I allele HLA-B*15:02. Different HLA class I and II associations have been described for the antiretroviral agent Nevirapine in different populations, including HLA-DRB1*01:01, HLA-B*35:05 and HLA-Cw8. The antiretroviral agent Abacavir has been shown to be associated with the HLA class I allele HLA-B*57:01 in all populations.

Abacavir is a guanosine analogue which works by competitive inhibition of the reverse transcriptase enzyme of HIV. It is effective as an antiretroviral agent when used in combination with other anti-retrovirals. Before its association with HLA*57:01 was discovered, use of Abacavir was associated with drug hypersensitivity in about 8% of patients. Abacavir binds in the cleft of the class I molecule and interacts with residues that form the F-pocket of HLA-B*57:01, altering the amino acid that B*57:01 would normally favour from Tryptophan to Isoleucine or Leucine instead. This the repertoire of peptides presented by B*57:01 is altered by Abacavir leading to the hypersensitivity response. Other B57 alleles are not similarly affected by Abacavir.

Symptoms of Abacavir hypersensitivity have a 9-day post drug initiation onset and typically include fever, malaise, gastrointestinal symptoms and internal organ involvement. A mild to moderate rash occurs in approximately 70% of drug hypersensitivity cases. The characteristic feature of Abacavir drug hypersensitivity is that symptoms completely disappear within 72hours of drug discontinuation. However, in susceptible individuals, re-exposure to Abacavir can result in a severe reaction and even death.

Two recent large scale Abacavir drug studies which incorporated skin patch tests have shown that 100% of patients who develop Abacavir drug hypersensitivity carry the HLA-B*57:01 gene. This positive predictive value supports the use of HLA-B*57:01 genetic screening of patients prior to commencement of treatment with Abacavir even though a proportion of HLA-B57:01 patients do not develop hypersensitivity. Current guidelines in the UK recommend that HLA-B*57:01 patients be put on alternative regimes.

The clinical use of HLA-B*57:01 screening for Abacavir drug hypersensitivity prior to treatment may potentially serve as a blueprint for the application of genetic screening in other drug hypersensitivity scenarios.

Drug Reaction with Eosinophilia and Systemic Symptoms (DRESS)

Drug reaction with eosinophilia and systemic symptoms (DRESS syndrome) is a drug induced hypersensitivity syndrome which involves a severe, adverse reaction to a drug. The skin is one of the organs most often affected. Patients present with a widespread rash, fever, lymphadenopathy and haematological abnormalities. DRESS may present as a Severe Cutaneous Adverse Reaction (SCAR). Two of the most well-defined SCARs include Stevens-Johnson Syndrome (SJS) and its more severe counterpart Toxic Epidermal Necrolysis (TEN).

SJS is characterised by severe cutaneous adverse reactions with epidermal detachment from the underlying dermis resulting in blisters and areas of denuded skin, particularly on the torso.

The implicated drugs in SJS and TEN include Carbamazepine and Allopurinol.

Carbamazepine is mainly used to treat neurological disorders, primarily seizures. It has been strongly associated with susceptibility to SJS and TEN in people of Han Chinese descent who have the HLA-B*15:02 allele. Some association has also been reported with HLA-A*31:01 but not HLA-B*15:02 in Japanese and Caucasoid populations.

Allopurinol is widely used to lower blood uric acid levels and prevent or treat its complications. When compared with the normal population, presence of HLA-B*58:01 is associated with a higher risk of susceptibility to SJS and TEN in patients treated with Allopurinol compared to the normal population.

It is recommended that patients are tested for the relevant HLA alleles before commencing treatment with these drugs and that if DRESS is suspected treatment is withdrawn immediately until symptoms resolve.

Stevens Johnson Syndrome

Stevens Johnson Syndrome (SJS) is a milder form of the Toxic Epidermal Necrosis (TEN) on the spectrum from SJS to SJS/TEN overlap to TEN. SJS is characterised by severe cutaneous adverse reactions with epidermal detachment from the underlying dermis resulting in blisters and areas of denuded skin, particularly on the torso.

SJS and TEN are assumed or identified as drug reactions though herpes has also been implicated. The implicated drugs include Carbamazepine and Allopurinol. Carbamazepine is mainly used to treat neurological disorders, primarily seizures. It has been strongly associated with susceptibility to SJS and TEN in people of Han Chinese descent who have the HLA-B*15:02 allele. Some association has also been reported with HLA-A*31:01 but not HLA-B*15:02 in Japanese and Caucasoid populations. The mechanism of action of Carbamazepine is thought to involve binding of Carbamazepine in the cleft of the class I molecule and interacts with residues of HLA-B*15:02, thus altering the repertoire of peptides presented by B*15:02, leading to a broad range of T cells being activated and a hypersensitive response. The range of T cells activated by Carbamazepine is not thought to be as large as the range activated by Abacavir in B*57:01 individuals (15% compared to 25% for Abacavir).

The H&I laboratory contribution would be to help pre-screen patients for HLA-B15:02 prior to commencing Carbamazepine treatment, especially if they have Chinese heritage and screen for B*58:01 prior to commencing Allopurinol treatment. There may also be some benefit to screening for HLA-A31:01 in non-Chinese patients prior to commencing Carbamazepine treatment. However, where a patient has already commenced Carbamazepine or Allopurinol treatment, typing for HLA could help with diagnosis if SJS and TEN are suspected.

HLA and Infection

Human Immunodeficiency Virus (HIV) is a retrovirus, infection with which causes Acquired Immunodeficiency Syndrome (AIDS). HIV infection occurs via transfer with bodily fluids and is characterised by progressive failure of the immune system and life-threatening opportunistic infections. The virus primarily infects cells of the immune system including CD4+ T cells, macrophages and dendritic cells. The mechanism of action involves direct killing of infected cells, increased rates of apoptosis of infected cells and killing of infected CD4+ cells by CD8+ cytotoxic T cells. The opportunistic infections are as a result of CD4+ T cells falling below critical levels for maintaining cellular immunity.

Genetic variations amongst humans have been shown to be associated with either protection against HIV infection, slow progression to AIDS once infected or rapid progression to AIDS. Several studies of highly exposed seronegative individuals, such as sex workers and the healthy new-borns of HIV infected mothers, have shown that the HLA molecules HLA-A2 and A68 are associated with reduced risk of seroconversion. Generally, concordance of HLA class I alleles between a HIV infected patient and their uninfected sexual partner results in increased likelihood of HIV transmission. Where HIV infection has taken place, a number of studies have shown that some patients do not progress on to full blown AIDS. A correlation has been shown to exist in these long term non progressers between the HLAB*57 and B*27 molecules and delayed AIDS onset. In general, patients heterozygous for HLA class I have delayed progression compared to homozygous individuals. The B*35 allele and the HLA-A1, B8, DR17 haplotypes have conversely been shown to be associated with a more rapid disease progression.

Non-HLA genes associated with delayed or rapid progression include the delta 32 variant of the CCR5 chemokine receptor which is associated with resistance to infection and delayed progression once infected and P1 variant of the CCR5 chemokine receptor which is associated with rapid progression.