Discovery of Genetic Markers Associated with Clozapine Induced Agranulocytosis.
Authors: Gerson Stanton , Athanasiou Maria , Reed Carol R. , Dain Bradley , Carr Janet , Whalen Heidi , Lachowicz Michael , Malhotra Anil , Kane John 
Published: April 2020

Abstract
Clozapine is a highly efficacious drug for the treatment of schizophrenia, but its use is limited, in part due to the side effect of agranulocytosis. In order to reduce the incidence of clozapine-induced agranulocytosis (CIA), patients are required to submit to a blood monitoring program. There is evidence that there is a genetic component to CIA, with previously published associations in a number of genes, including HLA-DQB1 (Dettling et. al., Arch Gen Psychiatry2001: 59, 93–94) and HLA-C (Dettling et. al., Pharmacogenetics2001: 11, 135–141). If a genetic test for CIA were developed, it is possible that clozapine could become a safer drug and more patients could benefit from its use. As a first step in developing such a test, we have conducted a case-control study to discover genetic markers associated with CIA. Cases were patients with an ANC&lt;500 during clozapine treatment and who discontinued clozapine. Controls were treated with clozapine at a minimum dose of 250mg/day for with WBC&gt;5000 and ANC&lt;1500 at least one year. For each case, we attempted to enroll 2 age-, gender- and ethnicity-matched controls. We were successful in collecting blood, medical histories and informed consent from 33 CIA cases and 54 controls. Near complete matching was achieved between the cases and controls and ethnicity was balanced by self report and by genomic control. The table below provides clinical and demographic information about the patients.
Characteristics of the Discovery Cases n=33 Controls n=54 Gender Male 17 33 Female 16 21 Ethnicity American Indian 4 4 Black or African American 1 2 White 28 48 Age Median (range) in years 36 (20–58) 35 (18–54) Nadir ANC Median (range) 237 (0–506) N/A Diagnosis Schizophrenia/Schizoaffective Disorder 32 53 Bipolar Disorder 1 1 Time to CIA Median (range) in months 2.2 (0.5–62.6) N/A
Seventy-four candidate genes were sequenced in the exons, intron-exon boundaries, 5′ untranslated region and promoter region for each of the cases and controls. Single nucleotide polymorphisms and small insertion/deletions were scored and a haplotype analysis conducted, comparing the frequency of haplotypes in cases to those in controls using logistic regression with covariates of age, gender and race. Permutation tests were done within each gene and a genetic marker was considered significant with an adjusted p value&lt;0.05. Using these criterian, we have discovered associations between genetic markers in 5 different genes and CIA. Each of the genetic markers is a haplotype with at least 3 polymorphisms. Two of the genes, HLA-C and HLA-DQB1 have been previously reported to be associated with CIA. A combination of genetic markers from 2 genes has 80% sensitivity and specificity for CIA in this discovery population. These findings are sufficient to continue development for a diagnostic test, which will include attempting to replicate the findings in an independently collected cohort. These findings may be applicable to agranulocytosis induced by other drugs.