NT5C2 Gene Research Articles

Clinical Utility of a Novel Triplex Digital PCR Assay for Clone Monitoring in Sequential and Relapsed Pediatric B-Cell Acute Lymphoblastic Leukemia Patients.

Digital polymerase chain reaction (PCR) studies for clonal disease monitoring in B-acute lymphoblastic leukemia patients are currently limited due to the heterogeneous nature of mutations, which limit cost-effective assay designs. In this study, 70 samples (14 relapse and 56 sequential therapy samples) were tested for 13 recurrent mutations identified on deep sequencing in our published cohort (KRAS, NRAS, NT5C2, PMS2, UHRF1, KMT2D, and TP53 genes) via a novel triplex digital PCR assay. A total of seven major clones of NRAS [five] and NT5C2 [two] were noted in six out of 14 (43%) relapse patients, accounting for 44% of early relapses. In addition, 10 minor clones (PMS2 [two], NRAS [four], NT5C2 [three], and TP53 [one]) were noted in five out of 14 (36%) patients. In the 56 sequential therapy samples, six major clones were noted (NRAS [five], KRAS [one]) in four out of 14 (28.5%) patients, with two increasing in size in maintenance samples, leading to subsequent relapse in both cases. In addition, therapy-acquired minor clones in NT5C2 [four] and PMS2 [one] were seen to emerge in maintenance samples in four out of 14 (28.5%) patients, with concordant detection of such major and minor clones in unpaired relapse samples, indicating the need for their active surveillance during therapy. Overall, digital PCR validated NRAS and NT5C2 major clones in one-third (10 out of 27; 37%) of cases, driving nearly half of early relapses.

Schizophrenia risk proteins ZNF804A and NT5C2 interact in cortical neurons.

The zinc finger protein 804A (ZNF804A) and the 5'-nucleotidase cytosolic II (NT5C2) genes are amongst the first schizophrenia susceptibility genes to have been identified in large-scale genome-wide association studies. ZNF804A has been implicated in the regulation of neuronal morphology and is required for activity-dependent changes to dendritic spines. Conversely, NT5C2 has been shown to regulate 5' adenosine monophosphate-activated protein kinase activity and has been implicated in protein synthesis in human neural progenitor cells. Schizophrenia risk genotype is associated with reduced levels of both NT5C2 and ZNF804A in the developing brain, and a yeast two-hybrid screening suggests that their encoded proteins physically interact. However, it remains unknown whether this interaction also occurs in cortical neurons and whether they could jointly regulate neuronal function. Here, we show that ZNF804A and NT5C2 colocalise and interact in HEK293T cells and that their rodent homologues, ZFP804A and NT5C2, colocalise and form a protein complex in cortical neurons. Knockdown of the Zfp804a or Nt5c2 genes resulted in a redistribution of both proteins, suggesting that both proteins influence the subcellular targeting of each other. The identified interaction between ZNF804A/ZFP804A and NT5C2 suggests a shared biological pathway pertinent to schizophrenia susceptibility within a neuronal cell type thought to be central to the neurobiology of the disorder, providing a better understanding of its genetic landscape.

Abstract 2058: Identifications of novel variants using system biology-based approaches in NT5C2, its association to relapse acute lymphoblastic leukaemia

Abstract Acute Lymphoblastic Leukaemia (ALL) patients experience relapse despite having a good prognosis; approximately 90% of children and 75% to 85% of adolescents and young adults survive for five years. Chemotherapy resistance and a poor prognosis are linked to relapsed acute lymphoblastic Leukaemia (R-ALL). Evidence suggests that the cytosolic 5 nucleotidase II (NT5C2) gene mutations play a crucial role in the emergence of recurrent ALL; The early R-ALL is usually accompanied by 6-MP resistance, which is typically brought on by gain-of-function mutations impacting NT5C2-driven mutation in relapse mutations by different mechanisms is still unknown. The study aims to identify novel top missense variants of NT5C2 that play a crucial role in the resistance of chemotherapy drugs, altered biological pathways and their impact on the treatment of relapse leukaemia. NT5C2 (5'-Nucleotidase, Cytosolic II) is a Protein Coding gene. This gene induces resistance to chemotherapeutic drug 6 mercaptopurine Which causes leukaemia cell growth and leukaemia-initiating cell activity. The NT5C2 gene variants are extracted from Genome Aggregation Database (gnomAD) and ExAC. The extracted data contained 2280 variants with chromosome number, position reference allele, and altered allele and these were functionally annotated using the ANNOVAR tool. The variants are screened through various computational tools to narrow down based on non-synonymous and stopgain mutations in the exonic region. The predicted 519 variants narrow down by scoring GERP (>2.0) and CADD PHRED (cut-off score >10), ClinVar, SIFT score, Polyphen, PhyloP, PhastCons, LRT score predictions and which indicates the top 10% deleterious variants including reported and unknown variants. The SNPs, insertions, deletions, CNVs or structural variants of top missense variants on genes, transcripts, and protein sequence determined on Variants Effect Predictor on the ensemble. These variants were verified on dbSNP and Clinvar through their variant IDs. The functional prediction of these variants was made to detect their significant role in R-ALL. These findings examine the future role of NT5C2 mutations in the chemoresistance of relapse ALL. This may help to find a new therapeutic combination of drugs to treat relapse acute lymphoblastic leukaemia to improve overall survival. Citation Format: Sugunakar V, Obul Reddy Bandapalli, Ramita Sharma. Identifications of novel variants using system biology-based approaches in NT5C2, its association to relapse acute lymphoblastic leukaemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2058.

Therapy-Acquired Clonal Mutations in Thiopurine Drug-Response Genes Drive Majority of Early Relapses in Pediatric B-Cell Precursor Acute Lymphoblastic Leukemia

Forty pediatric (0-12 years) B-ALL DNA samples (20 paired Diagnosis-Relapse) and an additional six B-ALL DNA samples (without relapse at 3 years post treatment), as the non-relapse arm, were retrieved from the biobank for advanced genomic analysis. Deep sequencing (1050-5000X; mean 1600X) was performed using a custom NGS panel of 74 genes incorporating unique molecular barcodes. A total 47 major clones (>25% VAF) and 188 minor clones were noted in 40 cases after bioinformatic data filtering. Of the forty-seven major clones, eight (17%) were diagnosis-specific, seventeen (36%) were relapse-specific and 11 (23%) were shared. In the control arm, no pathogenic major clone was noted in any of the six samples. The most common clonal evolution pattern observed was therapy-acquired (TA), with 9/20 (45%), followed by M-M, with 5/20 (25%), m-M, with 4/20 (20%) and unclassified (UNC) 2/20 (10%). The TA clonal pattern was predominant in early relapses 7/12 (58%), with 71% (5/7) having major clonal mutations in the NT5C2 or PMS2 gene related to thiopurine-dose response. In addition, 60% (3/5) of these cases were preceded by an initial hit in the epigenetic regulator, KMT2D. Mutations in common relapse-enriched genes comprised 33% of the very early relapses, 50% of the early and 40% of the late relapses. Overall, 14/46 (30%) of the samples showed the hypermutation phenotype, of which the majority (50%) had a TA pattern of relapse. Our study highlights the high frequency of early relapses driven by TA clones, demonstrating the need to identify their early rise during chemotherapy by digital PCR.

Direct-to-Consumer Genetic Test and Lifestyle Questionnaire Analysis of Body Mass Index and Body Fat Percentage in a Large Korean Population

Background: Direct-to-Consumer (DTC) genetic testing provides genetic risk to consumers and motivates consumers to take care of their own customized health care. In 2018, we developed and provided a DTC genetic testing service (GENESTART™) in collaboration with Herbalife Korea Co. Ltd. Methods: The analyzed dataset consisted of the body fat percentage (BFP), body mass index (BMI), 31 genetic polymorphism genotypes, and responses to 19 questionnaire items of 24,447 individuals. The genetic main effects for BFP and BMI were examined by linear regression analysis, and the interaction effects were examined using a generalized linear model that controlled age and sex as covariates. Results: In the case of BFP, the sample average was 31.47% overall, 24.76% for men, and 32.79% for women, showing that men had an average BFP that was 8 percentage points lower than that of women. The average BMI was 25.38 overall, 26.45 for men, and 25.17 for women, showing that men had an average BMI of 1.2 kg/m2 higher than that of women. The FTO and MC4R genes, well-known obesity markers, showed a significant correlation with both phenotypes, and the BDNF gene, which is related to stress obesity, showed a highly significant association with BMI but only a weak association with BFP. Among the remaining genes, TRIB1, ABCA1, MYL2, G6PC, GCKR, GLIS3, CYP17A1, HECTD4, and NT5C2 genes showed significant associations with the obesity-related phenotypes. In this study, we found four interaction results for BFP (ABO and fruits, CYP1A2 and sugary foods, FTO and muscle exercise, MC4R and vitamins) and five interactions for BMI (MC4R and proteins, CSK and fruits, MC4R and calcium, DGKB and calcium, CSK and water). Conclusions: This study is expected to enable the provision of personalized and accurate solutions for BFP and BMI management to customers who have undergone genetic testing.

CRISPR/Cas9-Mediated Induction of Relapse-Specific NT5C2 and PRPS1 Mutations Confers Thiopurine Resistance as a Relapsed Lymphoid Leukemia Model

6-Mercaptopurine (6-MP) is a key component in maintenance therapy for childhood acute lymphoblastic leukemia (ALL). Recent next-generation sequencing analysis of childhood ALL clarified the emergence of the relapse-specific mutations of the NT5C2 and PRPS1 genes, which are involved in thiopurine metabolism. In this scenario, minor clones of leukemia cells could acquire the 6-MP-resistant phenotype as a result of the NT5C2 or PRPS1 mutation during chemotherapy (including 6-MP treatment) and confer disease relapse after selective expansion. Thus, to establish new therapeutic modalities overcoming 6-MP resistance in relapsed ALL, human leukemia models with NT5C2 and PRPS1 mutations in the intrinsic genes are urgently required. Here, mimicking the initiation process of the above clinical course, we sought to induce two relapse-specific hotspot mutations (R39Q mutation of the NT5C2 gene and S103N mutation of the PRPS1 gene) into a human lymphoid leukemia cell line by homologous recombination (HR) using the CRISPR/Cas9 system. After 6-MP selection of the cells transfected with Cas9 combined with single-guide RNA and donor DNA templates specific for either of those two mutations, we obtained the sublines with the intended NT5C2-R39Q and PRPS1-S103N mutation as a result of HR. Moreover, diverse in-frame small insertion/deletions were also confirmed in the 6-MP-resistant sublines at the target sites of the NT5C2 and PRPS1 genes as a result of nonhomologous end joining. These sublines are useful for molecular pharmacological evaluation of the NT5C2 and PRPS1 gene mutations in the 6-MP sensitivity and development of therapy overcoming the thiopurine resistance of leukemia cells. SIGNIFICANCE STATEMENT: Mimicking the initiation process of relapse-specific mutations of the NT5C2 and PRPS1 genes in childhood acute lymphoblastic leukemia treated with 6-mercaptopurine (6-MP), this study sought to introduce NT5C2-R39Q and PRPS1-S103N mutations into a human lymphoid leukemia cell line by homologous recombination using the CRISPR/Cas9 system. In the resultant 6-MP-resistant sublines, the intended mutations and diverse in-frame small insertions/deletions were confirmed, indicating that the obtained sublines are useful for molecular pharmacological evaluation of the NT5C2 and PRPS1 gene mutations.

A comprehensive in silico analysis of pathogenic nsSNPs in the NT5C2 gene involved in relapsed ALL

Background: About 10-20% of children suffering from acute lymphoblastic leukemia (ALL), experience a relapse, which is a major cause of their death. Purine nucleotide analogs are frequently prescribed to maintain the treatment of ALL. Cytosolic 5´-nucleotidase (NT5C2) catalyzes the 5´ dephosphorylation of purine analogs. Gain-of-function mutations in the NT5C2 gene result in resistance to the treatment with purine analogs and subsequently in the relapse of the disease.
 Materials and Methods: In this descriptive study, bioinformatics tools were used to assess the effect of single nucleotide polymorphisms (SNPs) in the NT5C2 gene on the function and structure of the protein. So, 352 missense variants were retrieved from the NCBI database and analyzed by SIFT, PROVEAN, PMut, PANTHER, PolyPhen2, SNPs & Go, and PhD-SNP servers. Then, structural evaluations were performed using HOPE, NetSurp-2.0, and PyMOL. Moreover, stability and evolutionary preservation were assessed by I-Mutant2.0 and ConSurf, respectively.
 Results: As many as 31 nsSNPs were predicted to be affecting the protein function and stability. Also, the native residues were found to be evolutionarily preserved. The structural evaluation demonstrated that a change of hydrophobicity, flexibility, size, charge, or surface accessibility due to 24 nsSNPs would lead to the change of noncovalent interactions and then the conformation of the protein.
 Conclusion: Identification of biomarkers is significant in the prediction of relapses in ALL children. In this study, bioinformatics tools served to identify 24 high-risk deleterious nsSNPs in the NT5C2 gene. These mutations can be used to predict resistance to chemotherapy and relapse in ALL patients.

Putative regulatory functions of SNPs associated with bronchial asthma, arterial hypertension and their comorbid phenotype.

Linkage disequilibrium (LD) of single nucleotide polymorphisms (SNPs) of TLR4/AL160272.2 (rs1927914, rs1928298, rs7038716, rs7026297, rs7025144) was estimated in the Slavs of West Siberia. We further investigated an association of SNPs in TLR4/AL160272.2 (rs1927914, rs7038716, rs7025144), SERPINA1 (rs1980616), ATXN2/BRAP (rs11065987), IL2RB (rs2284033), NT5C2 (rs11191582), CARD8 (rs11669386), ANG/RNASE4 (rs1010461), and ABTB2/ САТ (rs2022318) genes with bronchial asthma (BA), arterial hypertension (AH) and their comorbidity. Then, the disease-associated SNPs were annotated in silico in relation to their potential regulatory functions. Strong LD was detected between rs1928298 and rs1927914, as well as rs7026297 and rs7038716 in the Slavs of West Siberia. It was found that the rs1927914 G allele of the TLR4 gene and the rs1980616 C allele of the SERPINA1 gene are associated with the predisposition to BA. These SNPs can affect binding affinity of transcription factors of the Pou and Klf4 families, as well as the expression levels of the TLR4 and SERPINA1 genes. The rs11065987 allele A of the ATXN2/BRAP genes, the rs11669386 A allele of the CARD8 gene, the rs2284033 allele G of the IL2RB gene, and the rs11191582 allele G of the NT5C2 gene were associated with the risk of AH. These variants can alter binding affinity of the Hoxa9, Irf, RORalpha1 and HMG-IY transcription factors, as well as the expression levels of the ALDH2, CARD8, NT5C2, ARL3, and SFXN2 genes in blood cells/vessels/heart, respectively. The risk of developing a comorbid phenotype of AD and AH is associated with the A allele of rs7038716 and the T allele of rs7025144 of the TLR4/AL160272.2 genes, the A allele of rs1010461 of the ANG gene and the C allele of rs2022318 of the ABTB2/CAT genes. Variants rs7038716 and rs7025144 can change the expression levels of the TLR4 gene in blood cells, while rs1010461 and rs2022318 influence the expression levels of the ANG and RNASE4 genes as well as the CAT and ABTB2 genes in blood cells, lungs/vessels/heart.

NUDT15 polymorphism and NT5C2 and PRPS1 mutations influence thiopurine sensitivity in acute lymphoblastic leukaemia cells.

In chemotherapy for childhood acute lymphoblastic leukaemia (ALL), maintenance therapy consisting of oral daily mercaptopurine and weekly methotrexate is important. NUDT15 variant genotype is reportedly highly associated with severe myelosuppression during maintenance therapy, particularly in Asian and Hispanic populations. It has also been demonstrated that acquired somatic mutations of the NT5C2 and PRPS1 genes, which are involved in thiopurine metabolism, are detectable in a portion of relapsed childhood ALL. To directly confirm the significance of the NUDT15 variant genotype and NT5C2 and PRPS1 mutations in thiopurine sensitivity of leukaemia cells in the intrinsic genes, we investigated 84 B‐cell precursor‐ALL (BCP‐ALL) cell lines. Three and 14 cell lines had homozygous and heterozygous variant diplotypes of the NUDT15 gene, respectively, while 4 and 2 cell lines that were exclusively established from the samples at relapse had the NT5C2 and PRPS1 mutations, respectively. Both NUDT15 variant genotype and NT5C2 and PRPS1 mutations were significantly associated with DNA‐incorporated thioguanine levels after exposure to thioguanine at therapeutic concentration. Considering the continuous exposure during the maintenance therapy, we evaluated in vitro mercaptopurine sensitivity after 7‐day exposure. Mercaptopurine concentrations lethal to 50% of the leukaemia cells were comparable to therapeutic serum concentration of mercaptopurine. Both NUDT15 variant genotype and NT5C2 and PRPS1 mutations were significantly associated with mercaptopurine sensitivity in 83 BCP‐ALL and 23 T‐ALL cell lines. The present study provides direct evidence to support the general principle showing that both inherited genotype and somatically acquired mutation are crucially implicated in the drug sensitivity of leukaemia cells.

Limited Therapeutical Impact of Extensive Genomic Profiling in Relapsed Pediatric B-Cell Precursor Acute Lymphoblastic Leukemia: Population-Based Four-Year Consecutive Cohort

High throughput genomic technologies, such as high-density SNP arrays and whole exome and transcriptome sequencing (WES, RNAseq), significantly deepened our insight into the genomic background and biology of acute lymphoblastic leukemia (ALL) during the past decade. Novel genetic aberrations and genomic and gene expression patterns have been described which led to the definition of several novel ALL subtypes and risk-predictive factors and to the identification of novel druggable and/or resistance-associated lesions potentially impacting therapy tailoring. Anecdotal evidence of therapeutical interventions based on the up-to-date genetic data has resulted into a great demand for these investigations. However, it is unclear what proportion of pediatric ALL patients, especially the cases of therapy-resistant leukemia, would really benefit from extensive genomic profiling.Between V/2013 and VIII/2017, relapse of Ph-negative B-cell precursor (BCP) ALL was diagnosed in 20 children (1-18 years) within the Czech Republic. In addition to the routine genetics, 18/20 cases were profiled by high density SNP array (18/18), WES (18/18) and RNAseq (17/18). The examined cohort comprised 10 B-other, 4 hyperdiploid, 3 ETV6-RUNX1 -positive and 1 KMT2A -rearranged ALL. At primary leukemia manifestation, all children were treated according to the ALL-BFM protocols [BFM 95 (n=1), BFM 2000 (n=1) and BFM 2009 (n=16)] and suffered very early (n=1), early (n=4) or late relapses (n=13). Upon disease relapse, 5, 11 and 2 cases were treated according to ALL-REZ BFM 2002 and IntReALL 2010 SR and HR protocols, respectively.Bulk bone marrow (BM) samples or sorted leukemic blasts were analyzed in isolated BM (n=15) and combined BM+CNS (n=2) relapses, while tissue from uterus infiltrated with blasts was analyzed in a single case of isolated extramedullary relapse. In addition to leukemic blasts, remission samples were analyzed by WES in all and by SNP array in selected cases to distinguish germline genetic variants. Somatic copy number aberrations and regions of uniparental disomy were called from SNP array data using commercial softwares. WES and RNAseq data were processed and somatic single nucleotide variants, indels and fusion transcripts were called using in-house pipeline based on publicly available software tools. Targeted analysis was performed to identify DUX4 gene rearrangements (r.). Hierarchical clustering based on gene expression profiles (unsupervised clustering and clustering with gene sets defining DUX4 -r., Ph-like and ETV6-RUNX1 -like ALL) was performed using RNAseq data. To facilitate this analysis, gene expression data from relapse cohort were combined with primary diagnosis data from >50 B-other ALL cases. Clinically important findings were reported to national study centre.Two B-other cases were classified as DUX4 -r. and 2 other cases as Ph-like ALL. Of note, 2 cases (both treated as standard risk in primary diagnosis) had iAMP21. Apart from 3 CRLF2 -rearrangements (1 present in Ph-like case), 6 KRAS and 2 NRAS genes mutations, no potentially druggable lesions were found. While the frequency of CRLF2 -rearrangements and RAS mutations found in relapse was comparable to that seen in primary disease, relapse cohort was enriched for lesions associated with unfavorable prognosis and therapy resistance. Three and four cases harbored deletion of TP53 gene and IKZF1 plus deletion pattern; however, in 1/2 cases with available data from primary diagnosis the IKZF1 plus pattern was identified only in relapse. Deletion of CREBBP and TBL1XR1 genes, potentially associated with resistance to glucocorticoids, were found in 2 cases and 1 case, respectively. Mutations of NT5C2 gene resulting in resistance to mercaptopurine were found in 2 cases. Three out of 18 analyzed cases did not harbor any of the above mentioned lesions. None of the genetic findings directly impacted patients' treatment.To conclude, we successfully implemented advanced genomics into centralized diagnostics of resistant ALL at the national level. Although it improved our understanding of disease relapse (yet, not in 100% of cases) the results were not translated to therapy tailoring in patients treated according to the large international protocols, thus, questioning the cost/benefit ratio of such extensive genetic diagnostics.Support: AZV 15-30626A and 16-30186A, GACR 15-06049Y, Primus/MED/28 DisclosuresNo relevant conflicts of interest to declare.

Effects of NT5C2 Germline Variants on 6‐Mecaptopurine Metabolism in Children With Acute Lymphoblastic Leukemia

6-mercaptopurine (6-MP) is widely used in the treatment of acute lymphoblastic leukemia (ALL), and its cytotoxicity is primarily mediated by thioguanine nucleotide (TGN) metabolites. A recent genomewide association study has identified germline polymorphisms (e.g., rs72846714) in the NT5C2 gene associated with 6-MP metabolism in patients with ALL. However, the full spectrum of genetic variation in NT5C2 is unclear and its impact on 6-MP drug activation has not been comprehensively examined. To this end, we performed targeted sequencing of NT5C2 in 588 children with ALL and identified 121 single nucleotide polymorphisms nominally associated with erythrocyte TGN during 6-MP treatment (P<0.05). Of these, 61 variants were validated in a replication cohort of 372 children with ALL. After considering linkage disequilibrium and multivariate analysis, we confirmed two clusters of variants, represented by rs72846714 and rs58700372, that independently affected 6-MP metabolism. Functional studies showed that rs58700372 directly altered the activity of an intronic enhancer, with the variant allele linked to higher transcription activity and reduced 6-MP metabolism (lower TGN). By contrast, rs72846714 was not located in a regulatory element and instead its association signal was explained by linkage disequilibrium with a proximal functional variant rs12256506 that activated NT5C2 transcription in-cis. Our results indicated that NT5C2 germline variation significantly contributes to interpatient variability in thiopurine drug disposition.

NT5C2 methylation regulatory interplay between DNMT1 and insulin receptor in type 2 diabetes

Epigenetics alternation of non-genetic variation and genome-wide association study proven allelic variants may associate with insulin secretion in type 2 diabetes (T2D) development. We analyzed promoter DNA methylation array to evaluate the associated with increased susceptibility to T2D (30 cases, 10 controls) and found 1,091 gene hypermethylated in promoter regions. We performed the association study of T2D and found 698 single nucleotide polymorphisms in exon and promoter sites by using 2,270 subjects (560 cases, 1,710 controls). A comparison of DNA hypermethylation and gene silencing of mouse T2D results in our T2D patients’ results showed that the 5′-nucleotidase, cytosolic II (NT5C2) and fucosyltransferase 8 (FUT8) genes were strongly associated with increased susceptibility to T2D. DNA hypermethylation in promoter regions reduced NT5C2 gene expression, but not FUT8 in T2D patients. NT5C2 protein expression was decreased in pancreatic β-cells from T2D mice. Transient transfection NT5C2 into RIN-m5F cells down-regulated DNA methyltransferase I (DNMT1) expression and up-regulation of the insulin receptor. Moreover, NT5C2 knockdown induced in DNMT1 overexpression and insulin receptor inhibition. Taken together, these results showed that NT5C2 epigenetically regulated insulin receptor in patients and mice with T2D, and maybe provide for T2D therapy strategy.

Ras pathway mutation feature in the same individuals at diagnosis and relapse of childhood acute lymphoblastic leukemia.

Acute lymphoblastic leukemia (ALL) is the most common malignancy in children, while relapse and refractory ALL remains a leading cause of death in children. However, paired ALL samples of initial diagnosis and relapse subjected to next-generation sequencing (NGS) could construct clonal lineage changes, and help to explore the key issues in the evolutionary process of tumor clones. Therefore, we aim to analyze gene alterations during the initial diagnosis and relapse of ALL patients and to explore the underlying mechanism. Targeted exome sequencing technology was used to detect molecular characteristic of initial diagnosis and relapse of ALL in 12 pediatric patients. Clinical features, treatment response, prognostic factors and genetic features were analyzed. In our 12 paired samples, 75% of pre-B-cell acute lymphoblastic leukemia (B-ALL) patients had alterations in the Ras pathway (NRAS, KRAS, NF1, and EPOR), and Ras mutation are very common in patients with ALL relapse. TP53 mutations mainly existed in the primary clones and occurred at the initial diagnosis and relapse of ALL. Relapse-associated genes such as NT5C2 and CREBBP were observed in patients with ALL relapse; however, all patients included in this study had gene abnormalities in the Ras pathway, and NT5C2 and CREBBP genes may collaboratively promote ALL relapse. Among the 12 ALL patients, Ras pathway mutations are common in ALL relapse and may be associated with other recurrence-related genes alterations. The study with paired samples could improve the understanding of ALL relapse.

NT5C2 Gene Polymorphisms and the Risk of Coronary Heart Disease

Background: Increasing studies have reported that 5′-nucleotidase cytosolic II (NT5C2) has a strong relationship with coronary heart disease (CHD) development. This study was designed to examine the relationship between NT5C2 polymorphisms and CHD in the Chinese Han population. Methods: We studied 501 CHD patients and 496 healthy controls from the Second Affiliated Hospital of Hainan Medical University in Hainan Province, China. Four single nucleotide polymorphisms (SNPs) in NT5C2 were selected and genotyped using Agena MassARRAY technology. Odds ratios and 95% confidence intervals were calculated using logistic regression after adjusting for age and gender. Stratification analysis was performed by age and gender in all individuals; we especially investigated the effects of NT5C2 SNPs on hypertension and diabetes among CHD patients. Results: rs2148198 of NT5C2 was strongly associated with an increased risk of CHD (allele: p = 0.045; codominant: p = 0.007; additive: p = 0.016). Stratified analysis revealed that rs2148198 was associated with increased CHD risk in individuals aged ≤61 years and males. For CHD patients, rs2148198 significantly affected the risk of hypertension and diabetes (p < 0.05). Further, rs79237883 of NT5C2 was associated with decreased susceptibility to hypertension in multiple genetic models for individuals with CHD (allele: p = 0.007; codominant: p = 0.001; dominant: p = 0.001; additive: p = 0.008). Conclusion: This study reports the association of NT5C2 gene variants and CHD susceptibility in the Chinese Han population. Especially, NT5C2 rs2148198 was significantly associated with CHD risk in the subgroups of males, hypertension, and diabetes.

S130 TRANSIENT SUBCLONES CARRYING NT5C2 MUTATIONS DEFINE A HIGH‐RISK PATIENT GROUP WITH POOR OUTCOME IN PEDIATRIC RELAPSED B‐CELL PRECURSOR ACUTE LYMPHOBLASTIC LEUKEMIA

Background:Acute lymphoblastic leukemia (ALL) is the most common cancer in children. Despite an overall favorable prognosis, 15–20% of patients suffer a relapse. In the treatment of relapsed ALL, resistance to chemotherapy is a major challenge. The purine nucleotide metabolizing cytosolic 5́‐nucleotidase II (NT5C2) harbors activating mutations in 20% of pediatric relapsed T‐ALL and in 3–10% of relapsed B‐cell precursor ALL cases, which are considered to drive relapse formation by causing resistance to purine analogue‐based maintenance treatment of ALL. However, the impact of NT5C2 mutations for second‐line treatment and long‐term outcome of relapsed patients is unknown.Aims:To elucidate the clinical significance of NT5C2 mutations in relapsed ALL, we studied the NT5C2 gene in a large cohort of 455 patients with relapsed B‐cell precursor ALL and assessed correlations with clinical presentation, genetics, response to treatment and outcome.Methods:We detected NT5C2 mutations by Sanger sequencing and amplicon‐based next generation sequencing. To detect subclonal NT5C2 mutations, we established allele‐specific quantitative real‐time PCR assays following minimal residual disease measurement guidelines. All relapsed patients were treated according to the German ALL‐REZ BFM 2002 protocol.Results:By sequencing, we found n = 36 NT5C2 mutations in n = 34 samples. About 30% of NT5C2 mutations appeared subclonal with an estimated allele frequency of less than 20%. The variants p.R39Q (8/36, 22%) and p.R367Q (14/36, 39%) were the most prevalent mutations in our cohort. Using allele‐specific quantitative real‐time PCR targeted to p.R39Q and p.R367Q, we identified additional 69 subclonal NT5C2 mutations. In total, NT5C2 mutations were present in 16% of patients with relapsed B‐cell precursor ALL (74/455), but the majority of cases (66%) showed subclonal mutations only. NT5C2 mutations most frequently occurred in relapses of so‐called B‐other‐ALL that lacks established cytogenetic changes (P &lt; 0.001) and were correlated with CDKN2A/B loss. In addition, predominant, but not subclonal NT5C2 mutations were associated with NRAS mutations indicating that RAS signaling may be involved in NT5C2‐driven relapse formation. Both, subclonal and predominant NT5C2 mutations, were associated with inferior event‐free survival rates of relapsed patients compared to wild‐type NT5C2 (0.189±0.058 and 0.333 ± 0.096, respectively, versus 0.575 ± 0.026; P &lt; 0.001). Response to relapse treatment was particularly poor in relapsed patients with subclonal NT5C2 mutations only. This group showed an increased rate of nonresponse of more than 30% (P &lt; 0.001). Moreover, patients of this group who responded to relapse treatment, showed high minimal residual disease levels at the end of relapse induction treatment in 80% of cases. To investigate whether outgrowth of NT5C2 mutant subclones during relapse treatment contributes to the poor response and outcome, we analyzed follow‐up samples and found that subclonal NT5C2 mutations never grew out to predominant mutations at the time of nonresponse or second relapse. In fact, analysis of samples taken after relapse induction treatment showed that NT5C2 mutant clones were frequently eradicated early during relapse treatment (14/20, 70%), whereas NT5C2 wild‐type leukemic cells persisted at minimal residual disease levels higher than 10E‐03 (16/20, 80%).Summary/Conclusion:Subclonal NT5C2 mutations define a patient group with poor response to treatment and outcome in relapsed B‐cell precursor ALL, but due to their transient character they may not play a driving role in this clinical phenotype.

Whole genome sequencing identifies a novel homozygous exon deletion in the NT5C2 gene in a family with intellectual disability and spastic paraplegia

Hereditary spastic paraplegias are a rare group of clinically and genetically heterogeneous neurodegenerative diseases, with upper motor neuron degeneration and progressive lower limb spasticity as their main phenotypic features. Despite that 76 distinct loci have been reported and some casual genes identified, most of the underlying causes still remain unidentified. Moreover, a wide range of clinical manifestations is present in most hereditary spastic paraplegias subtypes, adding further complexity to their differential clinical diagnoses. Here, we describe the first exon rearrangement reported in the SPG45/SPG65 (NT5C2) loci in a family featuring a complex hereditary spastic paraplegias phenotype. This study expands both the phenotypic and mutational spectra of the NT5C2-associated disease.

Pharmacogenetic analysis of hepatitis C virus related mixed cryoglobulinemia.

Mixed cryoglobulinemia (MC) is an extra hepatic hepatitis C virus related problem and different studies suggested genetics' role in predicting this complication. We evaluated the influence of SNPs in IL-28B, SLC29A1, SLC28A2, NT5C2, HNF4 and ABCB1 genes in MC prediction. SNPs were evaluated through real-time PCR. ABCB1 (gene encoding P-glycoprotein) 3435C>T SNP was associated with MC presence (p = 0.034): related to T allele carriers (CC vs CT/TT), we reached a p-value of 0.013. In the logistic regression analysis baseline viral load >600.000 IU/ml (p < 001), IL28B rs8099917/rs12979860 TT/CC (p < 0.001), NT5 (gene encoding for 5' nucleotidase) 153 TC (p = 0.012) and ABCB1 3435 CT/TT (p = 0.034) genotypes predicted MC presence. These data could help clinicians to identify patients with higher probability to show MC extra hepatic complication.

Whole Exome Analysis of Relapsing Patients with Acute Promyelocytic Leukemia

Background : APL is, in the vast majority of cases, driven by t(15 ;17) translocation, which leads to PML/RARA rearrangement. Remarkably, APL is an uncommon genetically simple disease and only few additional alterations, cooperating with PML/RAR, have been described at diagnostic (Welch et al, Cell 2012). Most APL can be cured with targeted therapy combining all-trans retinoic acid (ATRA) and chemotherapy (CT). However, genetic mechanisms underlying the 10-15% relapses observed with this regimen remain unclear. The goal of the present study was to identify mutations that cooperate with PML/RAR and those responsible for acquired resistance to ATRA-CT treatment in APL patients by whole-exome sequencing of diagnostic/ remission/relapse trios.Methods: Newly diagnosed APL patients included in clinical trials of the French Swiss Belgian APL group between 1994 and 2008, treated with ATRA-CT, before the introduction of first-line ATO, who experienced at least one relapse and had adequate material, were studied. We collected retrospectively 64 samples from 23 patients, including 23 diagnostic samples, 18 at first complete remission (CR) and 23 at relapse (22 first relapse and 1 second relapse). Whole exome-sequencing was performed on all samples. DNA libraries were prepared with the SureSelect human v5 kit (Agilent) and sequenced on Hiseq1000 (Illumina). The bioinformatic analysis was performed by GECO/integragen using CASAVA variant calling (Illumina) and dedicated pipeline. 18 trios and 5 duos passed the stringent quality control and were analyzed for somatic variants and copy number variations (CNV).Results :After elimination of polymorphisms, the median number of somatic variants corresponding to de novo mutation at diagnosis was 14, while only 3 new somatic variants appeared at relapse (figure 1). Notably, we failed to detect oncogene alterations other than PML/RARA in 7/23 (30%) patients. At diagnostic, 39% of patients (9/23) presented the common FLT3 alterations and at relapse 22% (5/23) of patients presented the known RARA mutations. Moreover, recurrent alterations were observed in activators of the MAPK signaling (22%): NRAS (2 patients), BRAF (1 patient), KRAS (1 patient), SPRY1 (1 patient). Mutations in the NT5C2 gene (3 patients), coding a 5'nucleotidase implicated in resistance to nucleoside-analog therapy, were solely observed at relapse, as in acute lymphoblastic leukemia (ALL). Abnormalities of epigenetic regulators were also detected at diagnostic and/or relapse: WT1 (7 patients, 30%), NSD1 (2 patients), TET2 (1 patient), ASXL1 (1 patient) and MED12 (2 patients). Homozygote WT1 inactivation by mutation plus neutral copy LOH occurred in 3 patients at relapse.The genetic markers identified allowed us to construct several evolution models. In 8 patients (35%), the diagnostic and relapse clones were clearly distinct, supporting the fact that they independently derived from pre-leukemic cells that survived ATRA/chemotherapy. In contrast, other relapses appeared to derive from the diagnostic clone.Conclusion: Our data highlight the genetic simplicity of APL with very few alterations detected and 30% patients without identified mutations in addition to PML/RARa. Our results support the existence of two prototypic mechanisms of relapse: re-emergence of a new APL from persisting pre-leukemic cells and relapse from APLs often expressing strong oncogenes at diagnosis, impeding therapy response and favoring the acquisition of resistance mutations at relapse, including PML/RARA or NT5C2. It will be interesting to assess the prevalence of those two mechanisms in the exceptional cases of relapse in patients treated with more recent frontline regimens that combine ATRA and arsenic in APL. [Display omitted] DisclosuresAdes:Celgene, Takeda, Novartis, Astex: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Fenaux:Celgene, Janssen,Novartis, Astex, Teva: Honoraria, Research Funding.

The rs11191580 variant of the NT5C2 gene is associated with schizophrenia and symptom severity in a South Chinese Han population: evidence from GWAS.

Objective:Recent genome-wide association studies have identified a significant relationship between the NT5C2 variant rs11191580 and schizophrenia (SCZ) in European populations. This study aimed to validate the association of rs11191580 polymorphism with SCZ risk in a South Chinese Han population. The relationship of this polymorphism with the severity of SCZ clinical symptoms was also explored.Methods:A case-control study was performed in 462 patients with SCZ and 598 healthy controls. rs11191580 was genotyped by the Sequenom MassARRAY iPLEX platform. A total of 459 SCZ patients completed the Positive and Negative Syndrome Scale (PANSS) evaluation. Data were analyzed by PLINK software.Results:We confirmed an association of the rs11191580 polymorphism with SCZ risk in South Chinese Han under a dominant genetic model (ORadj = 0.769; 95%CIadj = 0.600-0.984; padj = 0.037). PANSS scores showed a significant association between variant rs11191580 and total score (padj = 0.032), lack of response scale score (padj = 0.022), and negative scale score (additive: padj = 0.004; dominant: padj = 0.016; recessive: padj = 0.021) after data were adjusted for age and sex.Conclusion: NT5C2 variant rs11191580 conferred susceptibility to SCZ and affected the clinical symptoms of SCZ in a South Chinese Han population.

Abnormal NT5C2 gene expression in acute lymphoblastic leukemia

Objective To investigate the expression and significance of mRNAand exon mutationof NT5C2 gene in acute lymphoblastic leukemia (ALL)bone marrow. Methods Case control study design was used in this study. Bone marrow samples were collected from ALL patients in Anhui Provincial Cancer Hospital in recent 4 years. The patientswere divided into the initial diagnosis group, the complete remission group and the recurrence group.And they could specifically be divided into 36 patients initially diagnosed, 36 patients who achievedcomplete remission and 16 patients who relapsed with children B-ALL, 15 patients initially diagnosed, 15 patients who achievedcomplete remission and 9 patients who relapsed with children T-ALL, 18 patients initially diagnosed, 18 patients who achievedcomplete remission and 12 patients who relapsed with adult B-ALL, and 11 patients initially diagnosed, 11 patients who achievedcomplete remission and 6 patients who relapsed with adult B-ALL. The initial diagnosis, complete remission and recurrence samples were matched. 8 children and 8 adults without hematologic malignanciewere used as controls.Real-time PCR was performed to detect the level of NT5C2 mRNAin ALL patients. The exons of NT5C2 gene were cloned and sequenced for the common mutations in all cases. The results of NT5C2 mRNA levels in different groups were performed using non-parametric test by SPSS16.0 analytics software, and then non-parametric test together with correlation analysis was analyzed between NT5C2 mRNA levels of different initial diagnosis groups and gender, age, leukocyte level and risk classification . Results (1)The expression of NT5C2 mRNA levels of recurrence group were higher than that of initial diagnosis group, complete remission group and controls in children and adult B-ALL respectively(P 0.05). (3)NT5C2 mRNA expression of initial diagnosis group in children and adult B-ALL and T-ALL was not correlated with risk classification(P>0.05). (4)A newheterozygousmutation p. P414A of NT5C2 was discovered in a recurrencesample. Conclusions (1)High expression ofNT5C2 mRNA is associated with recurrence inchildren and adult B-ALL, and it may be an indicator of monitoring recurrence.(2)The incidence of exons mutation of NT5C2 gene in ALL is low in China.(Chin J Lab Med, 2016, 39: 501-505) Key words: Precursor cell lymphoblastic leakemia-ymphoma; 5'-Nucleotidase; Mutation; Recurrence