IGH Somatic Hypermutation Assays are Research Use Only tests. Historically, IGH Somatic Hypermutation Assays have been used to:

1. Identify clonal rearrangements of the immunoglobulin heavy (IGH) chain gene
2. Determine the extent of somatic hypermutation in the variable region of the immunoglobulin heavy chain gene in patients with chronic lymphocytic leukemia (CLL) and small lymphocytic lymphoma (SLL)

InVivoScribe Technologies' assay are standardized PCR-based tests. Each test comes with a Standard Operating Procedure (SOP), an interpretation guide, master mixes, and controls. Master mixes are composed of a buffered magnesium solution, deoxynucleotides, and multiple primers that target the gene segments of interest. Multiple primers are used to ensure a more comprehensive testing approach necessary to reliably identify clonal rearrangements. These tests are complete with the exception of Taq DNA Polymerase, which is not provided. A single thermocycler program and similar detection methods are used within each series of test to improve consistency, reduce human error, and facilitate cross training.

Standard Protocol.
50ul Total PCR Reaction Volume:

1. Using gloved hands, remove the master mixes from the freezer. Allow the tubes to thaw; then gently vortex to mix.
2. In a containment hood or dead air box remove an appropriate aliquot to clean, sterile microfuge tube (one tube for each of the master mixes). Aliquot volumes should be 45ul for each sample + 135ul for the positive, negative and no template controls. We recommend adding an additional 20ul to correct for pipetting errors.
3. Add the appropriate amount of either AmpliTaq Gold or AmpliTaq DNA polymerase (0.25ul of either AmpliTaq Gold or AmpliTaq @ 5U/ul per 50ul total PCR reaction volume) to each of the master mixes and gently mix by inverting several times or gently vortexing.
4. Aliquot 45ul of master mix to individual wells of a PCR plate.
5. Add 5ul of DNA from the unknown and control samples to individual tubes or wells containing the respective master mix reactions, and pipette up and down several times to mix. Amplify target DNA using the universal thermocycler program.

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Rearrangements of the antigen receptor genes occur during ontogeny in B and T lymphocytes. These gene rearrangements are unique in length and sequence for each cell. Therefore, polymerase chain reaction (PCR) assays can be used to identify lymphocyte populations derived from a single cell by detecting the unique V-J gene rearrangements present within these antigen receptor loci. This PCR-based assay employs multiple consensus DNA primers that target conserved genetic regions within the immunoglobulin heavy chain (IGH) gene. This test is used to detect and sequence the majority of clonal IGH rearrangements from either genomic DNA or complementary DNA (cDNA). Clonal products can be detected using a variety of methods, including gel and capillary electrophoresis.

Immunoglobulin variable heavy chain gene hypermutation status provides important prognostic information for patients with chronic lymphocytic leukemia (CLL) and small lymphocytic lymphoma (SLL). The presence of IGH somatic hypermutation (SHM) is defined as greater or equal to 2% difference from the germline variable (V) gene sequence, whereas less than 2% difference is considered evidence of no somatic hypermutation. This has clinical relevance, as there is a clear distinction in the median survival of patients with and without somatic hypermutation. Hypermutation of the IGHV gene is strongly predictive of a good prognosis while lack of mutation predicts a poor prognosis. It should be emphasized that the results of any molecular test should always be interpreted in the context of clinical, histological and immunophenotypic data.

Polymerase Chain Reaction (PCR)
PCR assays are routinely used for the identification of clonal lymphocyte populations. Each B-cell has a single productive IGH gene rearrangement (consisting of the combination of a variable (V) region, a diversity (D) region, and a joining (J) region) that is unique in both length and sequence. Therefore, when genomic DNA or cDNA from a normal or polyclonal population is amplified using primers that flank the V-J region, a bell-shaped curve (Gaussian distribution) of amplicon products within an expected size range is produced. On a gel, this distribution of products is seen as a smear. This Gaussian distribution reflects the heterogeneous population of V-D-J rearrangements. In cases where lymphocytes are not present, no product is seen. For genomic DNA or cDNA from samples containing a clonal population, the yield is one or two prominent amplified products (amplicons) within a diminished polyclonal background. Two products are produced in cases where the initial rearrangement was non-productive and was followed by rearrangement of the other homologous chromosome.

Since the antigen receptor genes are polymorphic (consisting of a heterogeneous population of related DNA sequences), it is difficult to employ a single set of primer sequences to target all of the conserved flanking regions around the V-D-J rearrangement. N-region diversity and somatic mutation further diversify the genetic sequences in these regions. Therefore multiplex master mixes, which target several regions such as the leader (L) or framework (FR) regions, are required to identify the majority of clonal rearrangements. As indicated, clonal rearrangements are identified as prominent, single-sized products within the background of different-sized amplicon products that form a Gaussian distribution around a statistically favored, average-sized rearrangement. As expected, primers that amplify from the L or FR regions, produce a correspondingly different size-range of V-D-J products.

This test amplifies either genomic DNA or cDNA that lies between the upstream leader (L) or framework 1 (FR1) regions and the downstream joining (J) region of the IGH gene. The test employs two different master mixes: Hypermutation Mix 1 and Hypermutation Mix 2. The Hypermutation Mix 1 targets sequences between the leader and joining regions. Therefore the amplicon product(s) span the entire variable (V) region, which contains the FR1, CDR1 (complementarity-determining region 1), FR2, CDR2, and FR3 regions. The Hypermutation Mix 2 targets sequences between the framework 1 (FR1) and joining (J) regions. The resulting amplicons include a portion of the FR1 region to the downstream J region. Accordingly products do not include the complete FR1 sequence.

Gel Electrophoresis Detection
Gel electrophoresis, such as agarose gel electrophoresis or non-denaturing polyacrylamide gel electrophoresisis (PAGE), is commonly used to resolve the different amplicon products based on their size, charge, and conformation. Since DNA is negatively charged, when an electrical potential (voltage) is applied across the gel containing PCR products, the electrical field causes the amplicons to migrate through the gel. Smaller DNA fragments are able to easily migrate through the gel matrix, whereas larger DNA fragments migrate more slowly. This causes a separation of the amplicon products based on size. Ethidium bromide or other DNA intercalating dye can then be used to stain and detect these products in the gel.

A heteroduplex analysis can also be performed and run on a polyacrylamide gel to separate clonal and non-clonal PCR products. A heteroduplex analysis involves denaturing the PCR products at a high temperature, then quickly re-annealing the DNA strands by suddenly reducing the temperature. This causes a large portion of DNA strands to incorrectly bind to other non-homologous strands creating loops in the DNA. These loops cause a significant reduction in the ability of the DNA to migrate through a polyacrylamide gel. However, if the majority of the PCR products are clonal, when a heteroduplex analysis is performed, most of these PCR products will correctly re-anneal with a homologous strand. These PCR products will run normally through the polyacrylamide gel. Therefore in a clonal sample with a polyclonal background, a heteroduplex analysis will cause most of the polyclonal product to run much slower through the polyacrylamide gel, thereby increasing their separation and the ability to identify the clonal band(s).

Differential Fluorescence Detection
Differential fluorescence detection is commonly used to resolve the different-sized amplicon products using a capillary electrophoresis instrument. Primers can be conjugated with several different fluorescent dyes (fluorophors) so that they can produce different emission spectra upon excitation by a laser in the capillary electrophoresis instrument. In this manner, different fluorescent dyes can correspond to different targeted regions. This detection system results in unsurpassed sensitivity, single nucleotide resolution, differential product detection, and relative quantification. In addition, the use of agarose and polyacrylamide gels, as well as the use of carcinogens such as ethidium bromide, can virtually be eliminated. Further, differential detection allows accurate, reproducible and objective interpretation of primer-specific products and automatic archiving of data. Inter-assay and intra-assay reproducibility in size determination using capillary electrophoresis is approximately 1 to 2 nucleotides. This reproducibility and sensitivity coupled with the automatic archiving of specimen data allows for the monitoring, tracking, and comparison of data from individual patients over time.

IGH Somatic Hypermutation (SHM) Analysis
The degree of somatic mutation in the immunoglobulin heavy (IGH) chain variable (V) genes is one of the best prognostic tools in the treatment of patients with Chronic Lymphocytic Leukemia (CLL) and Small Lymphocytic Lymphoma (SLL). Clonal PCR products are identified and they are gel extracted and sequenced. For somatic hypermutation (SHM) analysis the full variable region (FR1-FR3) or a partial variable region (CDR1-FR3) are sequenced to determine mutational status. Mutational status is determined by comparing the sequence of the IGH V region of the patient sample to the most homologous germline V sequence. Sequences that differ by more than 2% from their corresponding germline sequences are considered highly mutated whereas sequences that differ by less than 2% are considered unmutated. Listed below are several websites available to aid in IGH Somatic Hypermutation Analyses:

IMGT (The International ImMunoGeneTics information system)
http://imgt.cines.fr
Analysis tools: IMGT/V-QUEST and IMGT/Junction Analysis

VBASE at The MRC Centre for Protein Engineering’s Database of human antibody genes
http://vbase.mrc-cpe.cam.ac.uk and http://www.vbase2.org
Analysis tools: DNAPLOT

NCBI (National Center for Biotechnology Information)
http://www.ncbi.nlm.nih.gov/igblast
Analysis tools: IgBLAST (Basic Local Alignment Search Tool)
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This assay tests genomic DNA or complementary DNA (cDNA) from the following sources:

1. 5cc of peripheral blood, bone marrow biopsy, or bone marrow aspirate anti-coagulated with heparin or EDTA
2. Minimum 5mm cube of tissue
3. 2ug of genomic DNA
4. 5ug of total RNA or mRNA
5. 1ug of cDNA
6. Formalin-fixed paraffin embedded tissue or slides

This product and the methods employed are covered by United States Letters Patent Numbered 5,296,351 and 5,418,134; Australian Patent Number 626,601 and Japanese Patent Number 2,781,438, all of which are licensed exclusively to InVivoScribe Technologies (“IVS”).

Purchase of this product includes a limited sublicense for non-commercial practice of this technology for use within (or with respect to data or product that are transmitted to) the United States, Japan or Australia only when the purchaser is registered with IVS as an exclusively non-commercial user of IVS products. No sublicense is granted simply by purchase of these products. Non-commercial practice of the technology means sample testing done for teaching and basic research. Non-Commercial practice excludes testing if any of the following apply: (a) test results, products or information derived from the tests are used for or in support of patient care, or are transferred to a healthcare professional involved in patient care; (b) test results are clinically utilized to determine cause of death; (c) compensation, in any form or manner, is received for performing the tests.

To request a form for registration as an exclusively non-commercial product user, to discuss terms for a potential sublicense for broader practice of these methods, or for any questions concerning the scope or content of the non-commercial sublicense please contact our legal department by email at legal@invivoscribe.com, or by telephone at (858) 623-8105.

These methods also require nucleic acid amplification methods such as Polymerase Chain Reaction (PCR), which is covered by patents owned by Hoffmann-LaRoche, Inc. and F. Hoffmann-LaRoche Ltd. No license under these patents to use the PCR process is conveyed expressly or by implication to the purchaser by the purchase of these products. The assays described herein are not approved by any regulatory agency for clinical use. These assays are not for diagnostic or therapeutic use. This product is sold FOR RESEARCH USE ONLY; not for use in diagnostic procedures.