Association of Cth Genetic Variant with Veno-Occlusive Disease in Children Receiving Intravenous Busulfan Before Hematopoietic Stem Cell Transplantation.

Busulfan (Bu) is a key compound given prior to hematopoietic stem cell transplantation (HSCT), in children. Bu has a narrow therapeutic index, where high Bu exposure often leads to increased risk of veno-occlusive disease (VOD), whereas low Bu exposure has been associated with a higher risk of disease recurrence and graft failure. Interindividual differences in drug response are an important cause of resistance to treatment and toxicity. Identifying pharmacogenetic determinants of a given drug may allow for prospective identification of patients with suboptimal drug responses allowing for complementation of traditional treatment protocols by genotype-based drug dose adjustment.

Bu is primarily eliminated via the GSH conjugation in the liver, catalyzed by glutathione s-transferases (GSTs). The Bu glutathione conjugate is then converted to tetrahydrothiophene by cystathionine gamma lyase (CTH). CTH is mainly expressed in the liver and is also essential for glutathione synthesis. Any defective function with CTH could reduce Bu's elimination by preventing its metabolite formation or by decreasing the amount of glutathione needed for conjugation. Thus we investigated the relationship between genetic variants of CTH and HSCT-related toxicity.

A defective function (caused by genetic variants) of CTH will reduce Bu's metabolism resulting in increased toxicity.

The study was performed on sixty six pediatric patients (50% males, mean age 7.8yrs) who underwent allogeneic HSCT with intravenous BU as part of a myeloablative conditioning regimen. Bu dose was adjusted to target Css of 600–900 ng/ml and ursodeoxycholic acid was provided as a prophylaxis for VOD. Genomic DNA collected prior to myeloablation was used for genotyping rs1021737, using a Taqman SNP genotyping Assay. Rs1021737 is a common non-synonymous SNP, with a 1364G>T transition, resulting in a Ser403Ile (S403I) mutation. In Caucasians this SNP has the following frequencies: GG (54%), GT (38%) and TT (7%). We used PANTHER, a computational method designed to predict the biological impact of SNPs and rs1021737 was predicted to be deleterious.

Patients were followed for up to 5yrs. Neutrophil, platelet recoveries, grade 1–4 acute versus host disease, treatment related mortality incidences were 87.9, 84.9, 19.7 and 10.6%, respectively. Overall survival rate was 77%, Events free Survival resulted in 60.6%, while 21.2% of patients relapsed and 10.6% had primary or secondary graft failure. Incidence of VOD (Seattle criteria), hemorrhagic cystitis and lung toxicity were 12.1, 25.8 and 9.1% respectively. Genotype frequencies for rs1021737 within our sample were GG (54.5%), GT (40.9%) and TT (4.5%), similar to the general population. No association was found with clinical outcomes except with VOD. A Chi-Square test demonstrated that the homozygous TT genotype correlated with VOD (p=0.031), with an odds ratio of 19 (95% Confidence Interval; 1.5–241.7). We extended this analysis by performing a multidimensional reduction analysis which included: rs1021737 and GSTA1*B haplotype (a functional variant which has previously been associated with VOD in our sample). The analysis demonstrated that these two variants provided good predictability of VOD risk (accuracy =86%, specificity = 91%, sensitivity = 50%) with a Chi-square p=0.001 and odds ratio of 12.4 (2.36 – 65.13).

Our results suggest an association between CTH 1364G>T variant and VOD in children receiving IV Bu before HSCT. Loss of function of CTH may result in the accumulation of Bu and depletion of glutathione, which is involved in the removal of Bu, and thus homozygous TT individuals are at risk of VOD compared to GG or GT individuals. CTH is also vital in the production of Hydrogen Sulfide which is a protective factor in kidney and liver function. Diminished function of CTH results in cellular damage and inflammation and thus the pathogenesis of VOD may also be related to this biological mechanism. Furthermore, by including the CTH 1364G>T in a polygenic analysis, with the previously associated GSTA1*B, the predictability of VOD improved. This study suggests that genes involved in the metabolism of Bu may help in identifying patients at risk of developing VOD and who will therefore need a better VOD prophylaxis, maybe by administering defibrotide. A prospective multicentre study is ongoing to confirm these results.

No relevant conflicts of interest to declare.