Mutations In Perforin Gene In Chinese Patients With Acute Lymphoblastic Leukemia And Lymphoma

Backgroundperforin(PRF1) was secreted by T cells, NK cells and other active killer cells. Mutations in PRF1 result in the decrease or absence of the protein and its activity, so that immunological surveillance to virus-infected and transformed tumor cells is compromised. Mutations in PRF1 are found in about 60% cases of familial hemaphagocytic lymphohistocytisis (FHLH) due to severely impaired CTL and NK functions.Knock-out mice for perforin have a high incidence of lymphocytic tumors. In recent years, mutations and polymorphisms in PRF1 have been detected in aplastic anemia, lymphomas and autoimmune lymphoproliferative syndromes. For example, a total of 6 different perforin mutations were identified in 12 of 44 cases with childhood anaplastic large cell lymphoma. In a group of 29 lymphoma patients, biallelic mutations were found in 4 patients and monoallelic mutations presented in 4 other patients. In another report, one case with mutations in PRF1 and FAS genes developed T-cell lymphoblastic lymphoma, and his brother carried the same mutations but manifested hemophagocytic lymphohistiocytosis, suggesting that additional genetic or environmental factors may have impact on clinical symptoms of the patients with PRF1 mutations. Acute lymphoblastic leukemia (ALL) is a malignant monoclonal lymphoproliferative disease, often associated with chromosomal abnormalities or mutations in several genes. A91V mutation in PRF1 has been investigated in childhood ALL with paradoxical results. This mutation was a frequent predisposing factor for childhood ALL from screening A91V mutation in 100 patients. However, in another study on 2272 ALL children, A91V polymorphism was not found to be a risk factor for childhood ALL. Here we want to investigated mutations and SNPs in PRF1 in 111 children and adults with ALL and 77 with lymphoma.ObjectiveTo identify whether mutations and single nucleotide polymorphism (SNP) in perforin gene (PRF1) are correlated with acute lymphoblastic leukemia (ALL) and lymphoma.Patients and methods1. ALL/lymphoma patients and healthy candidate recruitment111 ALL patients, who treated in Beijing Daopei Hospital or Peking University First Hospital during the period from February 2006 to December 2008, were recruited; The patients were diagnosed as ALL and supposed for hematopoietic stem cell transplantation.77 lymphoma patients, who treated in Peking University First Hospital and the General Hospital of Armed Police during the period from October 2005 to February 2010, were recruited; In addition,63 healthy medical students were recruited as controls. The research project was approved by the Medical Ethics Committee of Peking University First Hospital and the General Hospital of Armed Police and the Institutional Review Board of Beijing Daopei Hospital.2.Methods(1)DNA preparationGenomic DNA was extracted from mononuclear cells in peripheral blood or nail and hair (2)Sequencing of PCR products amplified from coding exons and their flanking introns in PRF1(3)Karyotype analysis and BCR-ABL fusion gene examination(4)Immunophenotype(5)EBER-1 in situ hybridizationResults 11. Karyotype and BCR-ABL fusion gene analysis Among111 ALL cases,93 cases are B-ALL,18 cases are T-ALL.We performed karyotype analysis in 93 cases of the 111 ALL patients. t(9;22)(q34;q11) trnaslocation, i.e., positive Ph chromosome, was found in 32 (34.4%) cases, of whom all were B-ALL patients. We also performed fusion gene examinations in 111 ALL cases. BCR-ABL fusion transcript was found in 36 cases, of whom 32 cases were Ph chromosome positive, and 4 cases were not performed karyotype analysis.2.Four novel monoallelic missense point mutations in PRF1 were found in 4 B-ALL cases with abnormal karyotype.The resultant amino acid changes are G198R,R225Q,D486G and R509K.3. Two novel monoallelic synonymous point mutations S388S and Q540Q in PRF1 were detected in 5 B-ALL cases with abnormal karyotype or fusion gene.4. No significant differences in the heterozygosity ratio of reported SNPs in PRF1 between ALL patients and normal controls. Results 21. Eight novel missense point mutations in PRF1 were found in 77 lymphomas In the 77 lymphoma cases,8 point mutations were found in PRF1. L11P,Q164L,I125R are biallelic missense mutations, P188L,R385W,L6P,A109G,F169S are monoallelic missense mutations.2. Three novel synonymous point mutations in PRF1 were found in 77 lymphomas In the 77 lymphoma cases,3 point mutations were found in PRF1. c.9 C>T (A3A)and c.216 C> A (T72T) are biallelic mutations, c.180 A> G (P60P) is monoallelic mutation.3. No significant differences in the heterozygosity ratio of reported SNPs in PRF1 between lymphoma patients and normal controls. Genotype and allele frequencies of rs885821, rs885822, rs10999427, rs10999426 and rs12161733 conform to Hardy-Weinberg equilibrium, and have no significant differences between lymphoma patients and normal individuals (P> 0.05)4. The same mutations could also be detected in nail or hair follicles in 4 lymphoma cases.5. Only one is EBER positive among 8 lymphoma cases with PRF1 mutations.Conclusions1.Mutations in PRF1 may occur in B-ALL patients, frequently seen in those with Ph chromosome or other karyotype abnormalities.2.Mutations in PRF1 can also occur in lymphoma patients and has no correlationship with EB virus.3. Mutations in PRF1 in ALL/lymphoma are germline mutations.