[Association between CYP1A1*2A polymorphism and susceptibility to childhood acute lymphoblastic leukemia: a Meta analysis].

OBJECTIVE: To explore the association between CYP1A1*2A polymorphism and susceptibility to childhood acute lymphoblastic leukemia (ALL) through a Meta analysis. METHODS: Inclusion and exclusion criteria were formulated and English and Chinese databases (PubMed, OVID Database, CBM, CNKI, and Wanfang Data) were searched comprehensively. The studies (from January 1999 to April 2015) related to the association between CYP1A1*2A polymorphism and susceptibility to childhood ALL were collected. STATA 12.0 Software was applied to perform the Meta analysis for the articles included. RESULTS: A total of 12 articles were included for analysis (11 English articles and 1 Chinese article), which involved 3 355 cases in total. The results of the Meta analysis showed a significant association between CYP1A1*2A polymorphism and susceptibility to childhood ALL (allele model: OR=1.31, 95% CI: 1.07-1.61; dominant model: OR=1.33, 95% CI: 1.13-1.56; codominant model: OR=1.30, 95% CI: 1.10-1.54). According to the results of a subgroup analysis based on ethnic origin, an increased risk of childhood ALL was observed in both Asian subgroup (dominant model: OR=1.57, 95% CI: 1.19-2.08; codominant model: OR=1.61, 95% CI: 1.20-2.17) and the Caucasian subgroup (allele model: OR=1.31, 95% CI: 1.04-1.63; dominant model: OR=1.22, 95% CI: 1.00-1.49). CONCLUSIONS: CYP1A1*2A polymorphism may be associated with the genetic susceptibility to childhood ALL.

[Correlations between 6-mercaptopurine treatment-related adverse reactions in children with acute lymphoblastic leukemia and polymorphisms of thiopurine methyltransferase gene].

OBJECTIVE: To explore 6-mercaptopurine (6-MP) treatment-related adverse reactions in children with acute lymphoblastic leukemia (ALL), and to assess the association between the polymorphisms of thiopurine methyltransferase (TPMT) gene and these 6-MP related toxicities. METHODS: Total RNA was extracted from bone marrow samples of 46 children with ALL and was then reversed to cDNA. TPMT(*)1S and (*)3C were screened by denaturing gradient gel electrophoresis (DGGE) combining with DNA sequencing. Drug toxicities were classified according to national cancer institute-common toxicity criteria version 3.0 (NCI CTC 3.0). The relationship between TPMT gene polymorphisms and the adverse reactions of 6-MP treatment was analyzed. RESULTS: During the maintenance treatment period, 22% (10/46) of children discontinued 6-MP treatment because of serious adverse reactions. Two children with TPMT(*)3C genotypes presented severe adverse reactions, including 1 child with homozygotic mutation who had 6-MP dose-related myelosuppression and hepatotoxicity. The main side effects of 6-MP were myelosuppression, hepatotoxicity and gastrointestinal reaction. And there were no significant differences between TPMT(*)1S genotypes and severe myelosuppression or hepatotoxicity caused by 6-MP (P>0.05). CONCLUSIONS: TPMT(*)3C may correlate with severe adverse reactions caused by 6-MP.

[Research progress in hypoxanthine-guanine phosphoribosyltrans-ferase].

Hypoxanthine-guanine phosphoribosyltransferase (HPRT) is a cytoplasmic enzyme which is widely distributed in the body. It not only involves in the purine salvage pathway, but also relates to the metabolism of purine analogues drugs. It is a critical transferase regulating the pharmacological effects and toxicity of purine analogues drugs. The mutations of the gene for HPRT, which influence its activity, may cause metabolic diseases with different clinical symptoms, and influence the metabolism of purine analogues. The HPRT gene, also a housekeeping gene, can serve diagnostic markers for many disorders. This paper reviews the recent progresses on HPRT researches in promoting the individual treatment of analogues drugs and the development of new drugs and improving the diagnosis and therapy of inherited metabolic disease caused by HPRT mutations.

[Association of single nucleotide polymorphism of methylenetetrahydrofolate reductase gene with susceptibility to acute leukemia].

OBJECTIVE: To assess whether polymorphisms of methylenetetrahydrofolate reductase (MTHFR) gene is associated with susceptibility to acute lymphoblastic leukemia (ALL) or acute myeloid leukemia (AML) in Chinese Han children. METHODS: The study has included 87 patients with ALL, 22 patients with AML and 120 healthy controls. All subjects were analyzed with reverse transcriptase-polymerase chain reaction-denaturing gradient gel electrophoresis and sequencing. RESULTS: A 677CT genotype of the MTHFR gene was associated with decreased risk of ALL (OR=0.23, 95%CI: 0.07-0.79). However, MTHFR A1298C genotypes were not associated with the risk of either disease. 677TT/1298AA and 677CC/1298AC genotypes were associated with increased risk of ALL(OR=3.78, 95% CI: 1.38-10.40; OR=3.17, 95% CI: 1.18-8.53, respectively), whereas the genotype 677CT/1298AA was associated with susceptibility to AML (OR=0.23, 95% CI: 0.06-0.97). CONCLUSION: Our data suggested that C677T polymorphism of MTHFR gene may increase the risk of childhood AML.

[Relationship between the methylenetetrahydrofolate reductase gene polymorphism and adverse reactions of high-dose methotrexate in children with acute lymphocytic leukemia].

OBJECTIVE: To study the association between methylenetetrahydrofolate reductase (MTHFR) gene polymorphisms and toxicities after high-dose methotrexate (HD-MTX) infusion in children with acute lymphocytic leukemia (ALL). METHODS: MTHFR variants in 52 children with ALL were determined by reverse transcriptase-polymerase chain reaction-denaturing gradient gel electrophoresis and sequencing. Toxicities of children who received HD-MTX chemotherapy were evaluated according to the National Cancer Institute-Common Toxicity Criteria (NCI-CTC). RESULTS: The children carrying MTHFR 1298AC had a higher risk of developing thrombocytopenia compared with the carriers of the 1298 AA genotype (OR=13.7, 95%CI=1.18-159.36, P=0.036). There was no significant difference in HD-MTX chemotherapy-related adverse effects between the patients with different MTHFR C677T or G1793A genotypes. CONCLUSIONS: MTHFR A1298C polymorohism may associate with the toxicity of HD-MTX chemotherapy in children with ALL.

Effect of tumor gangliosides on tyrosine phosphorylation of p125FAK in platelet adhesion to collagen.

The exact mechanisms as to how platelets influence blood-borne metastasis remain poorly understood. Gangliosides, sialic acid-containing glycosphingolipids, are associated with tumor progression and metastasis in humans. Gangliosides isolated from tumor cells promote collagen-stimulated platelet aggregation and ATP secretion and enhance platelet adhesion to immobilized collagen. Gangliosides interact with a number of cell surface receptors including integrin receptors. In this study, we examined the effects of α2ß1 integrin-mediated platelet adhesion to collagen and phosphotyrosine signaling of focal adhesion kinase, p125FAK (FAK). platelets pre-incubated with neuroblastoma tumor gangliosides (NBTGs) or their major component GD2 (disialoganglioside) were more adherent to immobilized collagen (OD570 0.43±0.12, 0.39±0.13) compared to platelets pre-incubated with MTB (0.14±0.06, p<0.001); the effect of NBTGs was blocked by F-17 anti-α2 antibody. Pre-incubation of platelets with NBTGs resulted in a marked increase in the phosphotyrosine content of p125FAK in the adherent platelets compared to the MTB-pre-incubated adherent platelets. F-17 anti-α2 antibody decreased protein tyrosine phosphorylation of NBTG-incubated platelets adherent to collagen. These results indicate that the tumor gangliosides enhance platelet adhesion to extracellular matrix collagen by upregulating integrin α2ß1-mediated tyrosine phosphorylation of p125FAK, thereby providing insight into how this interaction may be involved in neuroblastoma metastasis.

[ALK gene mutations in childhood neuroblastoma].

OBJECTIVE: To investigate mutations of anaplastic lymphoma kinase (ALK) in Chinese children with neuroblastoma (NB). METHODS: Genomic DNA was extracted from 22 cases of paraffin-embedding NB tumor tissues. Gene mutations in the exons 20-26 which were mutational hotspots of ALK were analyzed by PCR-DNA direct sequencing. RESULTS: A novel synonymous mutation C3586T (Leu1196Leu) and a known synonymous mutation C3375A (Gly1125Gly) were found and located at exon 23 and exon 21 of ALK respectively. There were 10 cases (46%) of known synonymous mutation C3375A in 22 cases of NB. The C3375A allelic frequency was 27%. No statistically significant correlation was found between mutation C3375A and clinical parameters of NB such as age, sex, metastasis and tumor differentiation. Mutation was not found in the other 5 exons. CONCLUSIONS: A novel ALK gene synonymous mutation C3586T was identified using PCR-DNA sequencing. A known mutation C3375A in ALK was successfully identified in children, and its incidence is not influenced by the clinical features of childhood NB.

[A novel missense mutation of folypolyglutamate synthetase gene].

OBJECTIVE: To examine allelic frequencies of coding single nucleotide polymorphisms (cSNPs) of folypolyglutamate synthetase (FPGS) gene in Chinese Han children with acute leukemia (AL), in order to provide a basis for detecting the relationship between FPGS genetic polymorphisms and tumor individualized chemotherapy. METHODS: cSNPs of exon 5 were detected with polymerase chain reaction (PCR)-denaturing gradient gel electrophoresis (DGGE) in 91 children with AL and 124 children with upper respiratory infection as controls. Genotypes and allelic frequencies were examined. RESULTS: A novel missense mutation, 502/490 T>C (L151/101P), was found in exon 5 of FPGS from control children. The novel mutation was found in mitochondrial variants in two cases and cytosolic variants in three cases. The cytosolic and mitochondrial variants displayed allelic frequencies of 0.70 % and 0.47 % respectively. The novel mutation was not associated with susceptibility to AL. CONCLUSIONS: A novel missense mutation 502/490 T>C (L151/101P) in exon 5 of FPGS gene is firstly found in Chinese Han children, and the cytosolic and mitochondrial variants display allelic frequencies of 0.70 % and 0.47 % respectively.

[Analysis of a 452C/T single nucleotide polymorphism in γ-glutamyl hydrolase gene in children with acute leukemia].

OBJECTIVE: To investigate the distribution of γ-glutamyl hydrolase gene (GGH) 452C/T genotype and allele frequency in children with acute leukemia (AL) and healthy children. METHODS: Bone marrow samples from 92 children with AL and peripheral blood samples from 124 healthy children were obtained to prepare complementary DNAs (cDNAs). The cDNAs were analyzed for a GGH 452C/T polymorphism by reverse transcriptase-polymerase chain reaction-denaturing gradient gel electrophoresis (RT-PCR-DGGE) and direct sequencing. RESULTS: The frequencies of the AL patients with TT, CT and CC genotypes were 2.2%, 13.0% and 84.8%, and the frequencies of the control children were 1.6%, 16.9% and 81.5%, respectively. There was no significant difference in GGH genotype or T allele frequency between the two groups (P> 0.05). However, the T allele frequency in Han Chinese children was significantly different from those reported in Japanese, Mexican and African-American populations. CONCLUSION: The frequency of 452C/T polymorphism of GGH gene in Han Chinese children has been determined. The results suggested that an ethnic difference may exist.

[Association of single nucleotide polymorphism of reduced folate carrier gene with susceptibility to acute leukemia].

OBJECTIVE: To investigate the allele and genotype frequencies of reduced folate carrier gene (RFC) 80G/A polymorphism in Chinese patients with acute leukemia (AL) and healthy control children, and to provide clue for association between the single nucleotide polymorphism (SNP) of RFC and the occurrence of AL. METHODS: Bone marrow samples from 98 childhood patients with AL and peripheral blood samples from 135 healthy children were obtained to prepare complementary DNAs (cDNAs). The cDNAs were analyzed for the polymorphisms in RFC 80G/A by reverse transcriptase-polymerase chain reaction-denaturing gradient gel electrophoresis (RT-PCR-DGGE) and direct sequencing. RESULTS: The A allele frequencies of the AL patients and control children were 0.515 and 0.415, respectively (P< 0.05). Chi-square test confirmed a statistical significance of the association between RFC80 G/A and AL. CONCLUSION: RFC 80AA or GA genotype may contribute to increasing the susceptibility to AL.

[Studies on the mutation and polymorphism of the TPMT gene in Chinese children with acute leukemia].

OBJECTIVE: To investigate the allelic frequencies and distribution of single-nucleotide polymorphisms within the coding region (cSNPs) of thiopurine S-methyltransferase gene (TPMT) in Chinese children with acute leukemia (AL) and healthy controls, in order to provide genetic references for individual chemotherapy for AL patients by studying the relationship between the cSNP in human TPMT and chemotherapeutic effect of thiopurine drugs. METHODS: The bone marrow samples from 53 children with AL and peripheral blood samples from 115 healthy children were obtained to prepare complementary DNAs (cDNAs). The cDNAs were analyzed for the polymorphisms in the TPMT gene by reverse transcriptase-polymerase chain reaction (RT-PCR)-denaturing gradient gel electrophoresis (DGGE) and DNA sequencing. The distribution of each genotype was evaluated. RESULTS: Two novel heterozygote mutations, 210C>T (C70C, silent) and 622T>C (F208L), were identified in the coding region of the TPMT in a single sample, respectively. The mother of the child with mutation 622T>C was confirmed as the same genotype by DGGE and sequencing (NCBI_ss accession numbers 107796292 and 107795933). Two known polymorphisms, 474T>C (silent) and 719A>G (T240C), were identified. The allelic frequencies were 14.2%, 2.83% and 17.0%, 3.04% in the AL children and control children respectively, with the total allelic frequencies of 16.2% (first reported in the Chinese Han population) and 2.99% respectively. No association with susceptibility to disease was observed. CONCLUSION: Two novel mutations and two known polymorphisms were identified in Chinese children by RT-PCR-DGGE combined with DNA sequencing, which provides the first step to identify genetic markers for predicting variability in response to and toxicity of thiopurine drugs.

[Genetic diagnosis for female carriers of glucose-6-phosphate dehydrogenase deficiency by RT-PCR-DGGE].

OBJECTIVE: To study the feasibility of genetic diagnosis for female carriers of human glucose-6-phosphate dehydrogenase (G6PD) deficiency by reverse transcriptase-PCR-denaturing gradient gel electrophoresis (RT-PCR-DGGE). METHODS: Blood samples were collected from suspected 54 female carriers of G6PD deficiency. Total RNAs of peripheral blood were prepared and reverse-transcripted into cDNA. Design of 6 primer pairs for DGGE was based on 17 mutation sites of G6PD cDNA described in the Chinese population. Mutations in the coding region of G6PD gene were screened and genotyped by combination of PCR-DGGE and DNA sequencing. RESULTS: One case of 1024C/T, 20 cases of 1376G/T and 12 cases of 1388G/A were detected in the 54 samples. The total detection rate was 66.1% (33/54). CONCLUSIONS: Heterozygous mutation rate in female carriers of G6PD deficiency detected by RT-PCR-DGGE is high. RT-PCR-DGGE is value of clinical diagnosis for G6PD-deficiency female carriers.

[Polymorphisms of thymidylate synthase gene detected by RT-PCR-denaturing gradient gel electrophoresis in children with acute leukemia].

OBJECTIVE: Thymidylate synthase (TS) catalyses the conversion of deoxy-uridylate to deoxy-thymidylate and is a key enzyme for DNA synthesis. TS is the target enzyme of 5-fluorouracil (5-FU) and involved in folate metabolism. TS gene polymorphisms play an important role in the efficiency of fluorouracil activity in vivo. This study investigated the allelic frequencies and distribution characters of single-nucleotide polymorphisms within the coding region (cSNPs) of TS gene in Chinese children with acute leukemia (AL) and normal control children in order to explore the possible relationship between the cSNP in human TS gene and chemotherapeutic effects of 5-fluorouracils. METHODS: Bone marrow samples from 53 children with AL and peripheral blood samples from 115 normal children were obtained to prepare complementary DNAs (cDNAs). The cDNAs were analyzed for the polymorphisms in TS gene by reverse transcriptase (RT)-polymerase chain reaction (PCR)-denaturing gradient gel electrophoresis (DGGE) and direct sequencing. The distributive difference of each genotype between AL children and control children was evaluated. RESULTS: A polymorphism 381 A>G (E127E) in the coding region of TS gene was firstly identified in the Chinese population. The 381 A>G allelic frequency in AL children and control children was 12.3% and 13.5% respectively (P>0.05), which were similar to that in the International SNP Bank (12.3%). The allelic frequency of cSNPs was not associated with the susceptibility to AL. CONCLUSIONS: A polymorphism 381 A>G (E127E) in TS gene was successfully identified in children using RT-PCR-DGGE combined with DNA sequencing. There was no significant difference in the allelic frequency of cSNPs in AL children and normal children.

[Study on the relationship between human cytidine deaminase gene polymorphisms and Ara-C sensitivity].

OBJECTIVE: To study the relationship between coding single-nucleotide polymorphisms (cSNPs) in the human cytidine deaminase (CDA) gene and cytosine arabinoside (Ara-C) sensitivity in childhood acute leukemia (AL). METHODS: cDNAs from 87 leukemia and 199 control blood samples were analyzed for the cSNPs in CDA by PCR-denaturing gradient gel electrophoresis (DGGE) and sequencing. Human CDA genes were transformed into E. coli and yeast, respectively. Catalytic activities of the allele CDA and variant CDAs were determined by HPLC assay. The Ara-C sensitivity of the yeast transformants was measured by growth inhibition assays. RESULTS: Three known different polymorphisms, namely, 79A/C (K27Q), 208G/A (A70T) and 435T/C (silent) were identified in the coding region of CDA from an investigated Chinese population and displayed allelic frequencies of 12.1%, 0.5% and 76.2%, respectively. No association with susceptibility to disease was observed. Compared with that of CDA70A, the deamination activities for cytidine and Ara-C substrates of the E. coli transformants carrying human CDA70T were decreased by 53% and 63%, respectively (P<0.01), and the Ara-C IC50 value of the yeast transformants was also significantly decreased by 25% [(973 +/- 61) micromol/L to (735 +/- 31) micromol/L, P<0.05]. CONCLUSIONS: The 3 known cSNPs and their allelic frequencies of CDA are identified in a Chinese childhood AL. The 208A genotype is shown to be more sensitive to Ara-C than 208G genotype.

[Development and clinical application of male human papillomavirus genotyping by membrane DNA chip].

OBJECTIVE: To develop a new method for the detection of male human papillomavirus (HPV) genotypes and to investigate its clinical application value. METHODS: With computer assistance and based on the classical common primers MY09/11, modified PGMY09/11 with 23 HPV subtypes for PCR and Genbank data on HPV, we designed probes for the simultaneous detection of 18 high-risk subtypes (HPV-16, 18, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 73, 83 and MM4) and 5 low-risk subtypes (HPV-6, 11, 42, 43 and 44) and fixed them to the special membrane to make a DNA chip. A total of 112 male urethral samples were collected with swabs and studied for the clinical value. Meanwhile the single subtypes of HPV positive were sequenced and the standard samples detected for their sensitivity. RESULTS: Of the total number, 25 samples were found to be HPV positive, 13 single HPV infection and 12 multiple infection. Nine HPV gene subtypes were noted in the samples: 6, 11, 16, 18, 33, 35, 43, 56 and 73, with sensitivity up to 10 copies of HPV DNA. CONCLUSION: Human papillomavirus genotyping by the membrane DNA chip is applicable to the diagnosis of male HPV infection as well as to the related epidemic and etiological investigation.

[Single-nucleotide polymorphisms of the cytidine deaminase gene in childhood with acute leukemia and normal Chinese children].

OBJECTIVE: Cytidine deaminase (CDA) is a key enzyme for metabolizing chemotherapeutic agent cytosine arabinoside (Ara-C), a deoxycytidine analog used for treatment of acute leukemia and lymphomas. Significant variability in the antitumor efficacy and systemic toxicity of Ara-C has been observed in cancer patients. Two missense mutations changing Ara-C sensitivity and toxicity had been found in the human CDA. Coding single-nucleotide polymorphisms (cSNPs) of CDA had been investigated in Japanese, Europeans Africans and Americans, but not in Chinese. The purpose of this study was to survey the allelic frequencies of CDA cSNPs in Chinese children. METHODS: The bone marrow samples from 87 childhood patients with acute leukemia and peripheral blood samples from 199 non-malignancy-bearing children were obtained to prepare complementary DNAs (cDNAs). The cDNAs were analyzed for the polymorphisms in CDA by polymerase chain reaction (PCR)-denaturing gradient gel electrophoresis (DGGE), PCR-restriction fragment length polymorphism (RFLP) and direct-sequencing. The distributive difference of each genotype was evaluated between children with acute leukemia and control children. RESULTS: Three known different polymorphisms, namely, 79A to C (K27Q), 208G to A (A70T) and 435T to C (silent) were identified in the coding region of CDA from the investigated Chinese population and displayed allelic frequencies of 12.1%, 0.52% and 76.2%, respectively. No association with susceptibility to disease was observed. CONCLUSION: This study demonstrates 3 cSNPs and their allelic frequencies of CDA in Chinese children, and provides the first step to identify genetic markers for predicting variability in Ara-C response and toxicity.