Identification of Anaplastic Lymphoma Kinase Variant Translocations Using 5RACE

Anaplastic lymphoma kinase (ALK) is abnormally expressed in anaplastic large cell lymphoma (ALCL) and its expression associated with chromosomal translocations involving the ALK gene at 2p23. These translocations lead to the synthesis of novel chimeric proteins that retain the C-terminal portion of ALK, where the tyrosine kinase domain is located. In most of these tumors, the t(2;5)(p23;q35) translocation causes fusion of the ALK gene to the 5′ region of the nucleophosmin (NPM ) gene, but other different ALK partners have been identified, including nonmuscle tropomyosin (TPM3 ), TRK-fused gene (TFG ), 5′ aminoimidazole-4-carboxamide ribonucleotide formyltranferase/IMP cyclohydrolase (ATIC ), clathrin heavy chain gene (CLTC ), and moesin (MSN ). The characterization of these ALK partners has been performed using different molecular methods, including the 5′ Rapid Amplification of complementary deoxyribonucleic acid (cDNA) Ends (5′RACE) polymerase chain reaction (PCR)-based technique. This approach allows the potential amplification and identification of either 5′ or 3′ mRNA ends from an internal known sequence. In ALK translocations, identification of the 5′ gene involved has been performed using primers designed within the known 3′ catalytic domain of the ALK . Initial reaction consists in a first-strand cDNA synthesis primed using a gene-specific antisense primer (ALK1), performing the cDNA conversion of specific messenger ribonucleic acids, and maximizing the potential for complete extension to the 5′-end of the message. After cDNA synthesis, the first-strand product is purified from unincorporated dNTPs and ALK1. Terminal deoxynucleotidyl transferase is used to add homopolymeric tails to the 3′ ends of the cDNA. Tailed cDNA is then amplified by PCR using a nested gene-specific primer (ALK2), which anneals 3′ to ALK1, and a complementary homopolymer containing an anchor primer (i.e., AAP), which permits amplification from the homopolymeric tail. This allows amplification of unknown sequences between the ALK2 and the 5′ end of the mRNA. Further, nested PCRs usually are required to confer an adequate level of specificity to the process to permit the characterization of RACE products. The reamplification is achieved by using a nested gene-specific primer (ALK3), which anneals 3′ to ALK2, and a universal amplification primer, which anneals to the 5′ sequence previously introduced by the AAP primer. The result of the 5′ amplification yields a product that corresponds to a fragment of the fusion gene, including a partial fragment of the unknown ALK partner. Hybridization with an internal ALK primer is needed to confirm the specificity of the PCR fragments obtained because unspecific bands frequently are generated in these amplifications. The confirmed specific PCR product is subsequently purified and sequenced. Once this gene is identified, terminal primers could be designed to amplify the entire coding region of the fusion gene to be cloned for a complete sequencing analysis, thus allowing further functional studies of the new chimeric ALK gene.