Rare variants in complement system genes associate with endothelial damage after pediatric allogeneic hematopoietic stem cell transplantation.

Introduction: Complement system has a postulated role in endothelial problems after hematopoietic stem cell transplantation (HSCT). In this retrospective, singlecenter study we studied genetic complement system variants in patients with documented endotheliopathy. In our previous study among pediatric patients with an allogeneic HSCT (2001-2013) at the Helsinki University Children´s Hospital, Finland, we identified a total of 19/122 (15.6%) patients with vascular complications, fulfilling the criteria of capillary leak syndrome (CLS), venoocclusive disease/sinusoidal obstruction syndrome (VOD/SOS) or thrombotic microangiopathy (TMA). Methods: We performed whole exome sequencing (WES) on 109 patients having an adequate pre-transplantation DNA for the analysis to define possible variations and mutations potentially predisposing to functional abnormalities of the complement system. In our data analysis, we focused on 41 genes coding for complement components. Results: 50 patients (45.9%) had one or several, nonsynonymous, rare germline variants in complement genes. 21/66 (31.8%) of the variants were in the terminal pathway. Patients with endotheliopathy had variants in different complement genes: in the terminal pathway (C6 and C9), lectin pathway (MASP1) and receptor ITGAM (CD11b, part of CR3). Four had the same rare missense variant (rs183125896; Thr279Ala) in the C9 gene. Two of these patients were diagnosed with endotheliopathy and one with capillary leak syndrome-like problems. The C9 variant Thr279Ala has no previously known disease associations and is classified by the ACMG guidelines as a variant of uncertain significance (VUS). We conducted a gene burden test with gnomAD Finnish (fin) as the reference population. Complement gene variants seen in our patient population were investigated and Total Frequency Testing (TFT) was used for execution of burden tests. The gene variants seen in our patients with endotheliopathy were all significantly (FDR < 0.05) enriched compared to gnomAD. Overall, 14/25 genes coding for components of the complement system had an increased burden of missense variants among the patients when compared to the gnomAD Finnish population (N=10 816). Discussion: Injury to the vascular endothelium is relatively common after HSCT with different phenotypic appearances suggesting yet unidentified underlying mechanisms. Variants in complement components may be related to endotheliopathy and poor prognosis in these patients.

Role of glutathione S-transferase P1 polymorphism in early transplant complications in patients undergoing allogeneic stem cell transplantation.

INTRODUCTION: Complications in patients undergoing allogeneic hematopoietic stem cell transplantation (allo-HSCT) cause serious morbidity and mortality. Predicting patients at risk in advance and changing the symptomatic care and/or preparation regimen according to this risk assessment have been emphasized recently. Several single-nucleotide polymorphisms have been studied, and some were found to be responsible for early complications. Glutathione S-transferase P1 (GSTP1) is an enzyme involved in the detoxification process that reduces oxidative stress by reducing the number of free oxygen radicals. AIM: This study aimed to investigate the relationship between GSTP1 polymorphism and early complications of allo-HSCT, iron parameters, overall survival (OS), and transplantation-related mortality (TRM). MATERIALS AND METHODS: A total of 50 patients diagnosed with acute myeloid leukemia (n = 23) or acute lymphoblastic leukemia (n = 27) who underwent allo-HSCT between May 2008 and February 2011 at Gazi University Faculty of Medicine, Stem Cell Transplantation Unit, were included. RESULTS: Of the 50 patients, 24 (48%) were women and 26 (52%) were men. The median age of the patients was 26 (16-74) years. GSTP1 polymorphism was detected in 23 (46%) patients, and 27 (54%) had no polymorphism (wild type). The two groups were compared in terms of early toxicity after transplantation, according to the preparation regimen. The group with GSTP1 polymorphism was found to have a high transferrin saturation index (P < 0.05). Patients with no GSTP1 polymorphism showed a high grade III-IV anemia ratio (P < 0.05). The presence of sinusoidal obstruction syndrome and graft-versus-host disease was similar in both groups (P > 0.05). OS and TRM were higher in the GSTP1 polymorphism group, but no statistical difference was found between the two groups (P > 0.05). CONCLUSIONS: TSI was higher in the GSTP1 polymorphism group. GSTP1 polymorphism had no effect on early transplantation complications. Although the OS and TRM ratios were higher in the GSTP1 polymorphism group, no statistically significant difference was found between the groups. Further studies with larger sample size are needed.

Analysis of glutathione S-transferase and cytochrome P450 gene polymorphism in recipients of dose-adjusted busulfan-cyclophosphamide conditioning.

Sporadic incidence of veno-occlusive disease (VOD) continues to occur, despite achievement of recommended busulfan (BU) concentrations after real-time BU dose adjustment. To explore the potential influence of glutathione S-transferase (GST) and cytochrome P450 (CYP) genotypes on plasma BU concentration, subsequent VOD, and transplant outcome, we assessed the polymorphisms of multiple GST and CYP genes. Fifty-five patients were included (median age 38 years; range 21-67). Of these, 49 received dose-adjusted BU/CY therapy. Twenty-six patients received transplants from human leukocyte antigen-identical siblings, 26 from unrelated donors. The GSTA1*A/*A genotype was significantly associated with lower BU first-dose area under curve (AUC1st). We found that patients with higher AUC1st showed a significantly higher serum total bilirubin during the first month after transplantation, but this was not necessarily associated with subsequent development of VOD. We further analyzed a possible association of GST and CYP polymorphisms and VOD development, and found none of the polymorphisms investigated was associated with VOD incidence. Regarding transplant outcomes, GSTM1-positive patients showed lower relapse rates and better overall survival in multivariate analyses. These results suggest that a GSTM1-positive genotype in patients receiving BU/CY conditioning protects against relapse of hematological malignancies after allogeneic hematopoietic stem cell transplantation.

Genetic Susceptibility to Hepatic Sinusoidal Obstruction Syndrome in Pediatric Patients Undergoing Hematopoietic Stem Cell Transplantation.

Sinusoidal obstruction syndrome (SOS) is a well-recognized and potentially life-threatening complication of hematopoietic stem cell transplantation (HSCT). SOS arises from endothelial cell damage and hepatocellular injury mostly due to the transplantation conditioning regimens but also to other patient, disease, and treatment-related factors. Understanding risk factors associated with the development of SOS is critical for early initiation of treatment or prophylaxis. The knowledge about genetic contribution is limited; few studies investigated so far selected a set of genes. To get more comprehensive insight in the genetic component, we performed an exome-wide association study using genetic variants derived from whole-exome sequencing. The analyses were performed in a discovery cohort composed of 87 pediatric patients undergoing HSCT following a busulfan-containing conditioning regimen. Eight lead single-nucleotide polymorphisms (SNPs) were identified after correction for multiple testing and subsequently analyzed in a validation cohort (n = 182). Three SNPs were successfully replicated, including rs17146905 (P = .001), rs16931326 (P = .04), and rs2289971 (P = .03), located respectively in the UGT2B10, BHLHE22, and KIAA1715 genes. UGT2B10 and KIAA1715 were retained in a multivariable model while controlling for nongenetic covariates and previously identified risk variants in the GSTA1 promoter. The modulation of associations by conditioning regimens was noted; KIAA1715 was dependent on the intensity of the conditioning regimen, whereas the effect of UGT2B10 was equally applicable to all of them. Combined effect of associated loci was also observed (P = .00006) with a genotype-related SOS risk of 9.8. To our knowledge, this is the first study addressing the genetic component of SOS at an exome-wide level and identifying novel genetic variations conferring a higher risk of SOS, which might be useful for personalized prevention and treatment strategies.

Comprehensive analysis of serum microRNAs in hepatic sinusoidal obstruction syndrome (SOS) in rats: implication as early phase biomarkers for SOS.

Sinusoidal obstruction syndrome (SOS) is a liver injury caused by clinical chemotherapy, of which pathogenesis is associated with the damage in liver sinusoidal endothelial cells (LSEC). The unavailability of appropriate specific biomarkers for the early diagnosis of SOS may potentially overlook SOS patients. In this study, we sought to find serum microRNAs (miRNAs) as non-invasive biomarkers for investigating SOS in rats. Male Sprague-Dawley rats were orally administered monocrotaline, and then, their livers and sera were collected after 0.25, 0.5, 1, 2, 4, and 7 days. The rats showed a typical SOS phenotype including LSEC damage as early as day 0.25, followed by severe hepatocyte damage on day 2, and developed hepatic fibrosis from days 4 to 7. The miRNA microarray showed that 65 serum miRNAs were increased in their levels on day 0.25, when LSEC damage was observed, while hepatocyte damage was absent. Among the increased serum miRNAs on days 0.25-1, miR-511-3p was enriched in normal LSECs and miR-21-5p was in both LSECs and hepatocytes, suggesting that they were released into blood from the damaged LSECs. The miR-122-5p, miR-192-5p, and miR-101b-3p, which were enriched in hepatocytes, reached the highest levels in serum on day 2, suggesting their utility as indicators for hepatocyte damage. No miRNA showing an increasing trend from days 4 to 7 was found as a biomarker for fibrosis. In conclusion, we found that LSEC-derived miR-21-5p and especially miR-511-3p in serum would serve as early phase biomarkers for SOS in response to LSEC damage.

Association of CTH variant with sinusoidal obstruction syndrome in children receiving intravenous busulfan and cyclophosphamide before hematopoietic stem cell transplantation.

Sinusoidal obstruction syndrome (SOS) is a severe complication of hematopoietic stem cell transplantation (HSCT) that can be fatal, often attributed to the conditioning regimen prior to HSCT. We evaluated the association of SOS risk with gene variants in cystathionase (CTH), an enzyme involved in glutathione synthesis, in 76 children receiving intravenous busulfan (Bu) before HSCT. Our results indicated an association with CTHc.1364 G>T (ORTT=10.6, 95% confidence interval (CI)=2.16, 51.54) and SOS risk, which was sex dependent (female patients, ORTT=21.82, 95% CI=3.590-132.649). The interaction between CTHc.1364 G>T and another risk variant (GSTA1*B) was explored. A recessive model with the use of GSTA1*B*B and CTH c.1364 TT genotypes proved to be useful at predicting SOS occurrence, indicating the possibility of using these gene variants as markers of SOS occurrence and to further individualize preemptive treatment aimed at reducing SOS incidence.

FDA-Approved Oligonucleotide Therapies in 2017.

Oligonucleotides (oligos) have been under clinical development for approximately the past 30 years, beginning with antisense oligonucleotides (ASOs) and apatmers and followed about 15 years ago by siRNAs. During that lengthy period of time, numerous clinical trials have been performed and thousands of trial participants accrued onto studies. Of all the molecules evaluated as of January 2017, the regulatory authorities assessed that six provided clear clinical benefit in rigorously controlled trials. The story of these six is given in this review.

Effect of FOXP3 polymorphism on the clinical outcomes after allogeneic hematopoietic stem cell transplantation in pediatric acute leukemia patients.

Forkhead BOX P3 (FOXP3) polymorphisms have recently been investigated as candidate risk factors in several tumors and autoimmune diseases. This study aims to evaluate the potential influence of FOXP3 rs3761548 polymorphism in the donor on the outcome of allogeneic hematopoietic stem cell transplantation (allo-HSCT). A total of 171 patients were enrolled for this study and genotyped using direct sequencing. Patients with rs3761548 CC genotype had higher incidence of hepatic veno-occlusive disease (HVOD) and cytomegalovirus (CMV) infection than that of the individuals with AA or AC genotype (P=0.011, P=0.023). Treatment-related mortality (TRM) rate of patients with AA or AC genotype was lower than that of the patients with CC genotype (P=0.044) resulting in a difference in overall survival (OS). However, there was no difference in graft-versus-host disease (GVHD) relapse or blood stream infection (BSI), depending on the genotype at rs3761548 locus. In multivariate analysis, CC genotype showed as a risk factor in the development of HVOD and CMV infection, with low OS. In conclusion, this is the first report on FOXP3 rs3761548 SNP in allo-HSCT and we suggest that this SNP be considered a candidate marker for predicting the development of HVOD and CMV infection after allo-HSCT.

Genetic predisposition resulting in sinusoidal obstruction syndrome in a patient with resected sigmoid cancer on adjuvant oxaliplatin.

A Chinese man who had undergone a curative high anterior resection for sigmoid cancer was administrated XELOX (capecitabine and oxaliplatin) as postoperative adjuvant chemotherapy. He subsequently developed sinusoidal obstruction syndrome (SOS) that resolved on discontinuation of XELOX treatment. Genetic evaluation determined that he had the GSTT1-null and GSTM1-null genotype, known to be an independent risk factor for developing oxaliplatin-induced SOS.

Splenomegaly and Its Associations with Genetic Polymorphisms and Treatment Outcome in Colorectal Cancer Patients Treated with Adjuvant FOLFOX.

PURPOSE: Splenomegaly is a clinical surrogate of oxaliplatin-induced sinusoidal obstruction syndrome (SOS). We investigated development of splenomegaly and its association with treatment outcome and genetic polymorphisms following adjuvant 5-fluorouracil, leucovorin, and oxaliplatin (FOLFOX) in colorectal cancer (CRC) patients. MATERIALS AND METHODS: Splenomegaly was determined by spleen volumetry using computed tomography images obtained before initiation of chemotherapy and after completion of adjuvant FOLFOX in CRC patients. Ten genetic polymorphisms in 4 SOS-related genes (VEGFA, MMP9, NOS3, and GSTP1) were analyzed using DNA from peripheral blood mononuclear cells. RESULTS: Of 124 patients included, increase in spleen size was observed in 109 (87.9%). Median change was 31% (range, -42% to 168%). Patients with splenomegaly had more severe thrombocytopenia compared to patients without splenomegaly during the chemotherapy period (p < 0.0001). The cumulative dose of oxaliplatin and the lowest platelet count during the chemotherapy period were clinical factors associated with splenomegaly. However, no significant associations were found between genetic polymorphisms and development of splenomegaly. Disease-free survival was similar regardless of the development of splenomegaly. CONCLUSION: Splenomegaly was frequently observed in patients receiving adjuvant FOLFOX and resulted in more severe thrombocytopenia but did not influence treatment outcome. Examined genetic polymorphisms did not predict development of splenomegaly.

Role of Pharmacogenetics in Hematopoietic Stem Cell Transplantation Outcome in Children.

Hematopoietic stem cell transplantation (HSCT) is an established therapeutic procedure for several congenital and acquired disorders, both malignant and nonmalignant. Despite the great improvements in HSCT clinical practices over the last few decades, complications, such as graft vs. host disease (GVHD) and sinusoidal obstructive syndrome (SOS), are still largely unpredictable and remain the major causes of morbidity and mortality. Both donor and patient genetic background might influence the success of bone marrow transplantation and could at least partially explain the inter-individual variability in HSCT outcome. This review summarizes some of the recent studies on candidate gene polymorphisms in HSCT, with particular reference to pediatric cohorts. The interest is especially focused on pharmacogenetic variants affecting myeloablative and immunosuppressive drugs, although genetic traits involved in SOS susceptibility and transplant-related mortality are also reviewed.

Heparanase polymorphisms: influence on incidence of hepatic sinusoidal obstruction syndrome in children undergoing allogeneic hematopoietic stem cell transplantation.

PURPOSE: Sinusoidal obstruction syndrome (SOS) is a life-threatening early complication after hematopoietic stem cell transplantation (HSCT), and until now, examinations about the influence of genetic risk factors are extremely rare. The purpose of this study was to identify an association between heparanase (HPSE) single nucleotide polymorphisms (SNPs) and SOS in children undergoing allogeneic HSCT. METHODS: We retrospectively analyzed the distribution of the both HPSE SNPs rs4693608 and rs4364254 and the occurrence of SOS after allogeneic HSCT in 160 children with malignant and non-malignant diseases. RESULTS: Patients with HPSE genotypes GG or AG of rs4693608 (G>A) had a significantly reduced incidence of SOS on day 100 after HSCT compared to patients with genotype AA (4.7 vs. 14.3 %, P = 0.038). In addition, incidence of SOS in patients with genotype CC or CT of rs4364254 (C>T) was significantly decreased in comparison with patients with genotype TT (2.3 vs. 14.7 %, P = 0.004). Interestingly, no patient with genotype CC developed SOS. Because both SNPs co-occur in vivo, we generated subsets: AA-TT, GG-CC, and a group with remaining SNP combinations. We found significant differences between all three patient groups (P = 0.035). Patients with AA-TT showed the highest incidence of SOS (16.7 %), while SOS did not appear in patients with GG-CC (0 %) and residual combinations were numerically in-between (4.9 %). An impact caused by main patient and donor characteristics, established risk factors for SOS, and conditioning regimen could be excluded in multivariate analyses. CONCLUSIONS: HPSE polymorphisms turned out to be significant independent risk factors (P = 0.030) for development of SOS and should be evaluated in further trials.

TPMT and MTHFR genotype is not associated with altered risk of thioguanine-related sinusoidal obstruction syndrome in pediatric acute lymphoblastic leukemia: a report from the Children's Oncology Group.

Sinusoidal obstruction syndrome is a complication of therapy for pediatric ALL and may be modified by thiopurine methyltransferase activity as well as by MTHFR genotype. We assessed TPMT *3A, *3B, *3C, and MTHFR C677T and A1298C germline genetic polymorphisms among 351 patients enrolled in the thioguanine treatment arm of CCG-1952 clinical trial. TPMT and MTHFR C677T genotypes were not associated with SOS risk. The combination of MTHFR and TPMT variant genotypes was not associated with SOS risk. These suggest that germline genetic variation in TPMT and MTHFR do not significantly alter SOS risk in patients exposed to thioguanine.

The potential contribution of tumour-related factors to the development of FOLFOX-induced sinusoidal obstruction syndrome.

BACKGROUND: Chemotherapy-associated liver injury (CALI) has been linked to increased morbidity and poorer disease-specific outcomes in patients undergoing resection of colorectal liver metastases (CRLM). The aim of this study was to assess the contribution of tumour-related factors to the development of FOLFOX-induced liver injury. METHODS: We assessed the effect of FOLFOX treatment on the murine liver either in the presence or absence of CRLM to evaluate the contribution of both chemotherapy and tumour death to the development of CALI. RESULTS: In the presence of liver metastases, there was increased hepatic expression of plasminogen activator inhibitor-1 (146-fold; P<0.01) and vWF (2.4-fold; P<0.01) transcript as compared with sham-operated controls. In addition, we detected large clusters of megakaryocytes in the spleen of FOLFOX-treated tumour-bearing animals. The livers of FOLFOX-treated animals also showed changes in matrix remodelling genes such as TGFß (P<0.01), MMP2 (P<0.001), TIMP1 (P<0.001) and Pro-Collagen I (P<0.05) which was exacerbated in the presence of tumour. These genes have previously been demonstrated to have a key role in FOLFOX-induced liver injury. CONCLUSION: It appears that the toxicity of FOLFOX chemotherapy is enhanced by tumour-related factors.

The role of hematopoietic stem cell transplantation in SP110 associated veno-occlusive disease with immunodeficiency syndrome.

BACKGROUND: Veno-occlusive disease with immunodeficiency (VODI) is an autosomal recessive disorder of combined immunodeficiency (CID) and hepatic injury. Hematopoietic stem cell transplantation (HSCT) - the only definitive treatment for CID - appeared to have a high rate of complications in a previous report. In this study, we describe a new group of patients with VODI highlighting further clinical and immunologic aspects of this disease and re-evaluating the effectiveness of HSCT for the treatment of this disorder. PATIENTS AND METHODS: Review of clinical data, immunologic features, molecular studies, treatment, and final outcome of eight kindred members with VODI. RESULTS: The patients described had clinical and immunologic findings consistent with VODI. The molecular studies revealed a new mutation in the SP110 gene. HSCT was carried out in five patients and was successful in three. CONCLUSIONS: The diagnosis of VODI should be considered in all patients regardless of ethnicity with a severe combined immunodeficiency (SCID)-like presentation, especially with a normal mitogen response, or with signs of hepatic injury. VODI is a primary immune deficiency, which can be successfully corrected by bone marrow transplantation if applied early in the course of disease using appropriate conditioning.

Hepatic veno-occlusive disease with immunodeficiency (VODI): first reported case in the U.S. and identification of a unique mutation in Sp110.

Familial hepatic veno-occlusive disease with immunodeficiency (VODI, OMIM: 235550), a rare form of severe combined immune deficiency, was first described in Australian Lebanese patients as being associated with homozygous mutations in SP110, a gene encoding a PML nuclear body-associated protein. We present the first case of confirmed VODI in the United States, and identify the first novel missense mutation in SP110. The 3-year-old daughter of Hispanic parents without known consanguinity presented at age 5 months with fever, hepatomegaly, and pancytopenia. Her brother died at age 3 months from hepatic failure of undetermined etiology. Initial T- and B-cell counts were low, but eventually normalized. Serum IgG and IgM levels were low for age. Lymphoproliferation to mitogens and allogenic B-cells was normal, but absent to tetanus and candida antigens. Serum antibody levels against pneumococcal, Hib and tetanus antigens were low. Liver biopsies at ages 5 and 9 months were consistent with hepatic veno-occlusive disease or hVOD (also known as sinusoidal obstruction syndrome or SOS) and broncho-alveolar lavage detected Pneumocystis jiroveci. The patient recovered from her acute disease and has been clinically stable on immunoglobulin replacement therapy and trimethoprim-sulfamethoxazole prophylaxis. T-Cell receptor excision circle (TREC) analysis suggests that VODI will not be detected by newborn screening for severe combined immunodeficiency that relies on this assay. DNA was obtained from the patient, 4 siblings, and both parents, and SP110 was sequenced. The first missense mutation, a homozygous deletion/insertion variation in exon 2 (NM_080424.2 (SP110):c.78_79delinsAT) was detected in the patient. This novel mutation segregated in the heterozygous state in other living unaffected family members. The mechanism by which this SP110 mutation associates with VODI is consistent with the normal length mutated SP110 protein being subject to enhanced proteosome degradation resulting in marked reductions in SP110 protein.

Clinical, molecular, and cellular immunologic findings in patients with SP110-associated veno-occlusive disease with immunodeficiency syndrome.

BACKGROUND: Mutations in the SP110 gene result in infantile onset of the autosomal recessive primary immunodeficiency disease veno-occlusive disease with immunodeficiency syndrome (VODI), which is characterized by hypogammaglobulinemia, T-cell dysfunction, and a high frequency of hepatic veno-occlusive disease. OBJECTIVES: We sought to further characterize the clinical features, B-lineage cellular immunologic findings, and molecular pathogenesis of this disorder in 9 patients with new diagnoses, including 4 novel mutations from families of Italian, Hispanic, and Arabic ethnic origin. METHODS: Methods used include clinical review; Sanger DNA sequencing of the SP110 gene; determination of transfected mutant protein function by using immunofluorescent studies in Hep-2 cells; quantitation of B-cell subsets by means of flow cytometry; assessments of B-cell function after stimulation with CD40 ligand, IL-21, or both; and differential gene expression array studies of EBV-transformed B cells. RESULTS: We confirm the major diagnostic criteria and the clinical utility of SP110 mutation testing for the diagnosis of VODI. Analysis of 4 new alleles confirms that VODI is caused by reduced functional SP110 protein levels. Detailed B-cell immunophenotyping demonstrated that Sp110 deficiency compromises the ability of human B cells to respond to T cell-dependent stimuli and differentiate into immunoglobulin-secreting cells in vitro. Expression microarray studies have identified pathways involved in B-lymphocyte differentiation and macrophage function. CONCLUSION: These studies show that a range of mutations in SP110 that cause decreased SP110 protein levels and impaired late B-cell differentiation cause VODI and that the condition is not restricted to the Lebanese population.