Long-distance Polymerase Chain Research Articles

A three-way translocation of MLL, MLLT11, and the novel reciprocal partner gene MYO18A in a child with acute myeloid leukemia

Translocations of the MLL gene are common among neonates and infants with acute lymphoblastic and acute myeloid leukemias. We characterized a new three-way translocation involving MLL in an infant with acute myeloid leukemia who subsequently relapsed and underwent a hematopoietic stem cell transplant from an unrelated stem cell donor. The translocation was characterized using karyotyping and fluorescence in situ hybridization. In this patient, a complex rearrangement fused the distal part of 11q23 with 17q11.2, the distal part of 17q11.2 with 1q21, and the distal part of 1q21 with 11q23, resulting in a three-way translocation; t(1;11;17)(q21;q23;q11.2). The two reciprocal MLL fusion sites were cloned by long-distance inverse polymerase chain reaction, which led to the identification of MLL-MLLT11 and the reciprocal MYO18A-MLL fusion alleles. Both fusion genes are in-frame and can be translated into functional fusion proteins. Although the MLL-MLLT11 fusion gene has been described in the literature, the reciprocal MYO18A fusion partner is a novel candidate gene in the growing list of reciprocal MLL fusions.

Identification of chromosomal breakpoints of cancer-specific translocations by rolling circle amplification and long-distance inverse PCR

We describe the use of rolling circle amplification and long-distance inverse polymerase chain reaction (LD-PCR) to identify chromosomal breakpoints and fusion genes in cancer cells carrying acquired translocations. This approach produced enough template for 100 inverse PCR reaction from as little as 20 ng of patient DNA, consequently enabling the use of up to 500 times less patient DNA compared to standard inverse PCR. The method is based on identifying restriction sites in a putative breakpoint area in a cancer-specific translocation, followed by circularization and amplification of the restriction DNA products by using T4 DNA ligase and Phi29 enzyme, respectively. The amplified DNA thus obtained is used as a template in long-distance inverse PCR to amplify and detect the precise breakpoint of the chromosomal rearrangements in question by sequencing of the obtained PCR products. We demonstrate the feasibility of this approach by identifying fusion genes TAF15-ZNF384 (brought about by a (12;17)(p13;q21) translocation) and BCR-ABL1 (produced by a (9:22)(q34;q11.2) translocation) in five leukemia samples. The application of rolling circle amplification before inverse PCR may be particularly useful in the search for chromosomal breakpoints and fusion genes brought about by new translocations when only minute amounts of DNA are available from the sampled malignant lesion.

Prenatal diagnosis strategy of hemophilia A with different clinical backgrounds

Objective To prenatally diagnose HA fetus with different clinical backgrounds. Methods Genetic tests were performed on 15 gravidas subjected for prenatal diagnosis of HA and different methods were employed for diagnosis according to the gestational weeks and clinical data. Amniotic fluid were taken from pregnant women within 23 gestational weeks for direct genotyping and indirect linkage analysis, since these women had probands with clear-cut mutations. Cordocentesis was performed for linkage analysis in pregnant women over 23 gestational weeks with probands whose types of mutation were unknown, while the FⅧ activity tests were carried out simultaneously. For the pregnant women over 23 gestational weeks without proband, cordocentesis was operated for measurement of FⅧ activity and karyotyping, but carriers of hemophilia A could not be detected in these cases. The introns 22 and 1 inversion of F8 gene were identified by long distance-polymerase chain reaction. Nucleotide sequencing was employed if the gene inversion could not be found and linkage analysis of 7 polymorphic markers, including DXS1108, F8Civs13, INTRON22,DXS1073,DXS9901, DXS15, DXS8069 and sex site (Amelo) were applied eventually. Identification of maternal blood contamination must be done before the tests. Results Fifteen samples were identified without maternal blood contamination. Five fetuses were diagnosed with hemophilia A. Meanwhile there were three pregnant women whose cord blood FⅧ activities were less than 1%. One of them was accompanied by trisomy 21; another had inversion mutation in introns 22 of F8 gene; the remaining one was identified with missense mutation in exon 23 (p. Arg2182Cys) of F8 gene. Conclusions Diverse methods should be applied in prenatal diagnosis of hemophilia A with different clinical backgrounds, for the sake of birth defects prevention. Key words: Hemophilia A; Factor Ⅷ; Prenatal diagnosis

Non-immunoglobulin/ BCL6 Gene Fusion in Diffuse Large B-cell Lymphoma: Prognostic Implications

3q27 translocation affecting the BCL6 gene is one of the most common chromosomal abnormalities in diffuse large B-cell lymphoma (DLBCL). BCL6 translocation can involve not only one of the three immunoglobulin gene (Ig ) loci but also another non- Ig chromosomal locus. 5'-rapid amplification of cDNA ends and long-distance inverse polymerase chain reaction (PCR) methods have identified a total of 13 recurrent non- Ig partner genes to date. As the result of non- Ig / BCL6 translocation, many types of regulatory sequences of each partner gene substitute for the 5' untranslated region of the BCL6 and the rearranged BCL6 is presumed to be under the control of the replaced promoter activity. BCL6 translocation occurs more frequently in extranodal DLBCL than in node-based disease. However, the impact of BCL6 translocation on the treatment outcome of DLBCL has been the subject of controversy. We found that survival of DLBCL patients with non- Ig partners was inferior to that of those with Ig / BCL6 translocation, suggesting that non- Ig / BCL6 fusion is a poor prognostic indicator of DLBCL. We next created BCL6 expression plasmids containing a series of non- Ig / BCL6 fusion genes. COS-7 cells transiently transfected with these plasmids expressed high levels of Bcl-6 protein and showed characteristic punctate nuclear staining. These findings suggested that non- Ig / BCL6 translocation plays a pathogenetic role in a proportion of DLBCL.