b'Next-Generation Sequencing (NGS) | CE-IVD AssaysIGHV Leader Somatic Hypermutation Assay Next-Generation Sequencing (NGS) | CE-IVD AssaysLymphoTrack Dx IGHV Leader SomaticHypermutation AssayAssay Description During B-cell development, genes encoding the IGH protein are The LymphoTrack Dx IGHV Leader Somatic Hypermutation Assayassembled from multiple polymorphic gene segments that undergo for the IlluminaMiSeqis an in vitro diagnostic product intendedrearrangements and selection, generating cell specific V H -D H -J Hfor next-generation sequencing (NGS) based determination of therearrangements that are unique in both length and sequence. frequency distribution of IGH gene rearrangements, as well as theSince leukemias and lymphomas originate from the malignant degree of somatic hypermutation (SHM) of rearranged genes intransformation of individual lymphoid cells, all leukemias and patients suspected of having lymphoproliferative disease. This assaylymphomas generally share one or more cell-specific or "clonal" aids in the identification of lymphoproliferative disorders as well asantigen receptor gene rearrangements. Therefore, tests that detect providing an aid in determining disease prognosis. If you would likeIGH clonal rearrangements can be useful in the study of B- cell to test for IGHV somatic hypermutation using the Thermo Fisher Ionmalignancies. An additional level of diversity is further generated in PGM or Ion S5 platform, please refer to the LymphoTrack Dx IGH FR1the antigen receptors by introducing point mutations in the variable Assay (9-121-0007). regions, also named SHM. In instances where there is a high degree of SHM, there is the risk that primers located within the variable region will not be able to bind and clonal products will not amplify. In these Summary and Explanation of the Test cases, the leader primers located upstream of the variable region The NGS LymphoTrack Dx IGHV Leader Somatic Hypermutationcan be beneficial for the detection of clonal products, due to the Assay for the Illumina MiSeq represents a significant improvementconserved nature of the V H L region. In addition, the SHM rate of the over clonality assays using fragment analysis as it efficiently detectsentire variable gene can be determined using the V H L primers.the majority of IGH gene rearrangements using a single multiplexDetermining the immunoglobulin variable heavy chain gene (IGHV) master mix, identifies the DNA sequence specific for each clonal genehypermutation rate is considered a gold standard for determining the rearrangement, and assesses the somatic hypermutation rate ofprognosis of patients with chronic lymphocytic leukemia (CLL) and clonal samples in the same workflow. small lymphocytic lymphoma (SLL). In addition, NGS methods can The single multiplex master mixes target the Leader (V H L) and theimprove disease stratification.joining (J H ) gene regions of the IGH locus and are designed with Illumina adapters and indices (8 included in Kit A and 24 included in the Panel). This allows for a one-step PCR reaction and pooling ofSpecimen Requirement amplicons from several different samples and targets into a single50 ng of high-quality genomic DNA.Illumina MiSeq run. No post-PCR ligation step is required.The included LymphoTrack Dx Bioinformatics Software enablesReferencessimplified analysis and visualization of individual sample data. 1.B Stamatopoulous et al., Leukemia 4:837-845 (2017).Positive (clonal positive, SHM negative), negative (polyclonal) and2. F Davi et al., Leukemia 22: 212-214 (2008).SHM (clonal positive, SHM positive) DNA controls are included in the kits. 3. JE Miller et al., Molecular Genetic Pathology (2nd ed.). Springer Science & Business Media. 2013: 302.2.7.13 and 30.2.7.18.Background 4. P Ghia et al., Leukemia 21: 2-3 (2007).The human immunoglobulin heavy chain (IGH) gene locus on5. P Ghia et al., Blood 105: 1678-1685 (2005).chromosome 14 (14q32.3) includes 46-52 functional and 306. S Tonegawa. Nature 302: 575-581 (1983).nonfunctional variable (V H ) gene segments, 27 functional diversity (D H ) gene segments, and 6 functional joining (J H ) gene segments spread over 1,250 kilobases.V H L V H 1 V H 2 V H 3 V H 4 V H 5 V H 6 V H 7 J HSimplified representation of the immunoglobulin heavy chain (IGH) gene locus on chromosome 14. Depicted are the variable (V H ) and downstream consensus joining (J H ) region genes involved in rearrangements. Upstream of the variable gene segments, the leader sequence (V H L) is also depicted. Diversity region genes are not depicted.20'