The antipsychotic clozapine is reserved for treatment resistant schizophrenia patients despite its proven superior efficacy, because 0.8% of treated patients develop agranulocytosis, an adverse drug reaction [1–3]. Agranulocytosis is a hematological condition in which the number of neutrophils is severely reduced (less than 500 cells/mm³ of blood), which renders the patient susceptible to infections and is fatal in 2–4% of agranulocytosis cases [4]. This severe adverse event is not dose dependent and few risk factors have been identified, although older age and female gender increase risk slightly [2,5]. Around 80% of agranulocytosis cases occur in the first 18 weeks of clozapine treatment and the risk reduces by a factor of ten after the 1st year [1,2]. To improve safety, patients treated with clozapine are required to undergo regular white blood cell counts throughout the course of treatment. This is an invasive procedure, which may be stressful and inconvenient for the patients, and is an important factor in explaining why clozapine is underused despite its efficacy. The market share of clozapine in the USA was only 5% in 2008, whereas around 40% of patients were eligible [3].

A pharmacogenomic test for clozapine-induced agranulocytosis could be used for two different purposes. A test that identifies patients at very low risk would allow some patients to be exempted from hematological monitoring or to follow a more relaxed monitoring schedule. This should remove a barrier for clozapine usage and increase clinical uptake. Alternatively, a test that picks up patients who are at increased risk could be used to improve safety by ensuring these high-risk patients are monitored more closely or not prescribed clozapine. Keeping in mind that there is no alternative treatment for clozapine, the number of false positive test results should be absolutely minimal. Both applications of genetic tests are important, but a test that identifies the low-risk patients has a potential impact on a much larger group of patients and is therefore our primary focus.

Pharmacogenomics of clozapine-induced agranulocytosis

The emphasis of genetic research in agranulocytosis has been on the HLA region, though other immunologic genes and clozapine metabolizing enzymes have also been investigated. From the 1990s, several candidate gene studies reported associations with HLA-alleles and haplotypes, but few results have been replicated [6,7]. This limited success can partly be explained by the small sample sizes of these studies: around 30 agranulocytosis cases gives only limited power to detect an association. Even more important to note is that many of these statistical findings were not adjusted for multiple testing, thereby increasing the probability of false positive results. The most promising candidate gene study found an association with the SNP HLA-DQB1 6672G>C and replicated this finding in a separate sample, using 82 cases in total [8]. The odds ratio (OR) of this association was 17, with 21.5% sensitivity and 98.4% specificity.

More recently, researchers have broadened the search for genetic associations with clozapine-induced agranulocytosis beyond candidate genes. Exome sequencing in a small Finnish sample investigated SNPs, insertions and deletions but found no significant associations [9]. Goldstein et al. performed the most extensive genetic study to date encompassing a genome-wide association study, rare variant exome sequencing, copy number variants analysis and HLA allele imputation in 163 agranulocytosis cases [10]. Two amino acid changes in HLA-DQB1 126Q (OR = 0.19) and HLA-B 158T (OR = 3.11) reached genome-wide significance levels. Combining these two HLA alleles in a pharmacogenomic test that classifies a patient carrying either variant as being at high risk of agranulocytosis would achieve 36% sensitivity and 89% specificity. However, the authors comment that ‘these ORs do not immediately suggest clinical application in screening’ [10].

Application of pharmacogenomic findings to clinical practice

Despite this growing evidence that the HLA region is associated with agranulocytosis in clozapine patients, this knowledge has not been translated to clinical practice yet. In 2007, a test based on the HLA-DQB1 6672G>C variant was commercially marketed but its low sensitivity meant that only one in five agranulocytosis patients were detected and the risk among the patients classified as low risk by the test was only reduced by 20%. Consequently, the test was not widely used and is no longer available [11,12].

We recently developed a framework to assess the clinical value of pharmacogenomic tests for clozapine-induced agranulocytosis and concluded that sensitivity is the most important parameter for evaluating clinical utility [13]. High sensitivity ensures that patients who are at high risk are correctly identified by the test, and that therefore few patients considered low risk will develop agranulocytosis. The necessity of high sensitivity explains why the HLA-DQB1 6672G>C test failed commercially and why the amino acid changes in HLA-DQB1 and HLA-B are not suitable for predictive testing on their own.

Looking at the whole field of psychiatric drug treatment, we must concede that clinical application of genomic findings is still modest. The US FDA lists 24 psychiatric drugs (including clozapine) with pharmacogenomic information on their drug label; in all cases this relates to cytochrome P450 metabolism and possible consequences on drug dosing [14]. However, pharmacogenomic testing is required by the FDA for only one of these 24 drugs, the antipsychotic pimozide [15]. A test for clozapine-induced agranulocytosis could be the first test for an adverse drug reaction caused by a psychiatric drug.

Challenges of identifying pharmacogenomic test for clozapine-induced agranulocytosis

The monitoring procedures introduced for clozapine have been successful in reducing the number of cases of clozapine-induced agranulocytosis, but this clinical success makes it difficult to attain the large sample size of agranulocytosis cases required for well-powered genetic studies. One alternative would be to use patients with neutropenia given a ‘red flag’ in monitoring (neutrophil count less than 1500 cells/mm³) or even include those with an ‘amber flag’ (neutrophil count 1500–2000 cells/mm³). However, relaxing the case threshold for ascertainment is usually counterproductive in genetic studies as patients who would have developed agranulocytosis may be outnumbered by those whose neutrophil levels dropped for other reasons. A further puzzle: assuming our current knowledge of the genetic underpinnings of clozapine-induced agranulocytosis is incomplete, where do the remaining risk variants lie? Methods to impute HLA alleles from SNP genotypes have substantially expanded our ability to assess this type of variation in large genetic studies [16]. In addition to HLA-DQB1 126Q and HLA-B 158T, lower frequency or lower risk HLA variants may exist, which this study was underpowered to detect, and this is suggested by residual evidence for association in the HLA region [10]. Variants outside the HLA region may also increase agranulocytosis risk, and new genome-wide association studies such as the EU-funded CRESTAR study are in progress [17].

Conclusion

Candidate gene studies have targeted the HLA region and the largest genome-wide study so far has indeed confirmed that amino acid changes in HLA alleles are associated with clozapine-induced agranulocytosis. Hypothesis free genome-wide association studies have proven to be more successful than candidate gene studies in disease genetics and larger studies investigating the whole genome should also enable us to uncover more pharmacogenomic associations. In addition, meta-analyses and multidisciplinary collaboration between researchers worldwide will be essential in progressing this field. If genetic variation controls risk of clozapine-induced agranulocytosis, we now have the tools and technology to identify the variants, and to translate them into a highly sensitive pharmacogenomic test that will improve safety, clinical care and patient experience.