b"Gel & Capillary | RUO AssaysGel and CapillaryIGH Somatic Hypermutation Assays v2.0 Research Use Only (RUO) AssayIGH Somatic Hypermutation Assays v2.0Assay Use Background IGH Somatic Hypermutation Assays are useful for studies involving: Immunoglobulin variable heavy chain gene hypermutation status provides important prognostic information for patients with chronicdentifying clonal rearrangements of the immunoglobulin heavylymphocytic leukemia (CLL) and small lymphocytic lymphoma (SLL). Ichain (IGH) gene The presence of IGH somatic hypermutation (SHM) is defined as Assessing the extent of somatic hypermutation (SHM) in thegreater or equal to 2% difference from the germline variable (V H ) gene sequence, whereas less than 2% difference is considered evidence of variable (V H ) gene sequence (IGHV) in patients with chronicno somatic hypermutation. This has clinical relevance, as there is a lymphocytic leukemia (CLL) and small lymphocytic clear distinction in the median survival of patients with and without lymphoma (SLL) somatic hypermutation. 2Hypermutation of the IGHV gene is strongly Evaluating new research and methods in malignancy studies predictive of a good prognosis while lack of mutation predicts a poor prognosis. This assay aids in identification, sequencing, and analysis Summary and Explanation of the Testof somatic hypermutation status of clonal products. PCR products can be analyzed by gel electrophoresis detection or by differential These assays amplify either genomic DNA or complementary DNAfluorescence detection using capillary electrophoresis followed by gel (cDNA) that lies between the upstream leader (V H L) or framework 1electrophoresis detection. PCR products are sequenced bidirectionally (FR1) regions and the downstream joining (J H ) region of the IGH gene.either directly, after gel extraction, or after cloning into a bacterial The assays employ two different master mixes: Hypermutation Mix 1vector. The resulting sequence is then compared to IGH germline and Hypermutation Mix 2. The Hypermutation Mix 1 targets sequencessequences to determine mutational status.between the leader (V H L) and joining (J H ) regions. Therefore the amplicon product(s) span the entire variable (V H ) region, which contains all framework (FR) and complementarity-determiningSpecimen Requirementsregions (CDR). The Hypermutation Mix 2 targets sequences betweenThese assays test genomic DNA or cDNA from the following sources:the framework 1 (FR1) and joining (J H ) regions. The resulting amplicons include a portion of the FR1 region to the downstream J Hregion. The5 mL of peripheral blood, bone marrow biopsy, or bone marrow primers that target the V H L and FR1 regions have been redesigned to include a universal sequencing tag at the 5'end. This new designaspirate anti-coagulated with heparin or EDTA; or,allows for bi-directional sequencing of clonal PCR products withMinimum 5 mm cube of tissue; or,just one sequencing-tag specific forward primer and one J Hreverse2 g of genomic DNA; or,primer, thus ensuring a more reliable and complete coverage of clonal products. Current ERIC (European Research Initiative on CLL)5 g of total RNA or mRNA; or,guidelines recommend bi-directional sequencing when determining1 g of cDNA; or,the IGH SHM status. Positive and negative DNA, positive RNA, as well as an amplification control are included in the assay. Clonality isFormalin-fixed, paraffin-embedded (FFPE) tissue or slides.indicated if any one of the master mixes generates clonal products.Reference1.P Ghia et al., Leukemia 21: 1-3 (2007).2. P Ghia et al., Blood 105: 1678-1685 (2005).3. F Davi et al., Leukemia 22: 212-214 (2008).Figure Legend: Simple representation of the organization of a rearranged immunoglobulin heavy chain gene on chromosome 14. Black arrows represent the relative positions of primers that target the conserved Leader (L) and Framework 1 (FR1) regions, and the downstream consensus J Hgene segments.128"