Label Name: IGH SOM MUTN
Lab Discipline: Molecular Diagnostics
Institution:  Duke University Health System 
EAP ID:  LAB6578 
Last Review:  3/17/2017 10:12:01 AM
Container & Volume
  Age Group   Container   Volume  
  0  - 18 Years LAVENDER TOP TUBE 3  ML
Label Reminders
  Be sure to include the patient's name, history #, date and time of collection, and collector's initials.
Collection Notes
  • Peripheral blood: One lavender-top EDTA tube (minimum of 3 ml) is required. A recent peripheral blood count (with differential) is recommended to ensure involvement by CLL. Send unprocessed peripheral blood to the laboratory promptly at ambient temperature.

    Bone Marrow: One lavender-top EDTA tube (minimum of 1 ml) is required. Send unprocessed bone marrow to the laboratory promptly at ambient temperature.

  Deliver to laboratory at ambient temperature. If there is a delay of more than 24 hours in delivery, please refrigerate the sample. Samples must arrive within 48 hours of collection. DO NOT FREEZE.
Turn Around Time -  Routine: 14 days   Stat: N/A
Reference Values
  This assay uses multiplexed PCR followed by Sanger DNA sequencing to detect somatic mutations in the Ig heavy chain variable genes (IgVH). Genomic DNA is first extracted from the patient sample. This DNA is then used in two multiplex PCR reactions. One reaction targets and amplifies DNA sequences between the leader and joining regions. Amplification products from this reaction contain FR1, CDR1, FR2, CDR2 and FR3 regions. The second PCR reaction targets and amplifies DNA sequences between FR1 and joining regions. Amplification products from this reaction contain CDR1, FR2, CDR2 and FR3 regions. A specimen quality control multiplex PCR is also performed for sample quality assurance. PCR products are first visualized using capillary electrophoresis (QIAxcel, Qiagen). Samples containing one or two distinct clonal amplification products are then resolved by agarose gel electrophoresis and visualized using ethidium bromide staining. Clonal amplification products are excised from the gel and recovered using a QIAquick gel extraction kit (Qiagen). PCR products are treated with an exonuclease/phosphatase mixture (ExoSAP-IT) and sequenced bi-directionally with the Big Dye Terminator v3.1 Cycle Sequencing Kit. Sequencing products are purified with the Big Dye XTerminator Purification Kit and resolved using the ABI 3130xl Genetic Analyzer. Sequences are first analyzed using the ABI Data Collection software v3.0. Sequence data files are exported using the ABI Sequence Scanner (v1.0) software, manually edited, and aligned by the ImMunoGeneTics Information System V-QUEry and Standardization Alignment Tool (IMGT/V-Quest, http://imgt.cines.fr). IMGT determines variable gene usage and percent homology to germ line sequences. Samples with a percent homology of less than 98.0% are designated as mutated (M), and those with a percent homology greater than 98.0% are designated as unmutated (U).

This test was developed and its performance characteristics determined by the DUHS Clinical Molecular Diagnostics Laboratory. It has not been cleared or approved by the U.S. Food and Drug Administration. This test is used for clinical purposes. It should not be regarded as investigational or for research. This laboratory is certified under the Clinical Laboratory Improvement Amendments of 1988 ("CLIA") as qualified to perform high complexity clinical testing.
Clinical Significance and Interpretive Data

The human immunoglobulin heavy chain locus (IGH), mapped to 14q32.3, spans 1250 kb and encodes the immunoglobulin heavy chain. This locus contains an extended repertoire of germline variable segments (V), with approximately 52 functional V segments, 27 functional diversity (D) segments, and 6 functional joining (J) segments. The heavy chain V segments contain three framework regions (FR1, FR2 and FR3), and two complementarity-determining regions (CDR1 and CDR2). Immature B-cells undergo somatic recombination that joins individual D and H segments, followed by V segment rearrangement, creating a functional V-D-J IgH gene. This can happen in either or both of the IgH loci. Thus, circulating B lymphocytes contain either one or two copies of a recombinant V-D-J heavy chain, unique in both sequence and length. Nave B-cells enter germinal centers where further diversification occurs through affinity maturation. This includes somatic hypermutation, which incorporates single nucleotide changes (also rare deletions and duplications) into the V segment, targeting mainly the CDR regions and less frequently the FR regions. Somatically mutated memory B-cells represent around 40% of all B-cells in normal adult peripheral blood, while pre-germinal nave B-cells account for the other 60%.

In lymphoproliferative disorders, Ig clonality assessment can be useful for diagnosis based on the principle that all cells of a malignancy have a common clonal origin. Through PCR-based methods, the presence of a clonally rearranged expansion provides a detectable molecular marker distinguishable from the diverse Ig repertoire of normal cells. A clonal IGH population can be further evaluated to determine the somatic hypermutation (SH) status of the IgVH gene in malignant cells.

B-cell chronic lymphocytic leukemia (B-CLL) is a neoplastic disease of CD5+ lymphocytes. Two distinct subsets of B-CLL have emerged based on the mutation status of the expressed IgVH in clonal cells. Approximately 50% of B-CLL patients carry germline VH genes, while the other half demonstrate somatically mutated VH genes, where somatic mutation is defined by >2% difference in sequence homology from the germline V gene. Presumably, the two disease subsets arise from either nave B-cells or post-germinal center memory cells. Mutational status analysis of the IgVH gene thus provides an indicator for the cellular origin of disease, which can add prognostic value and aid in management.


The IgVH mutational status in B-CLL patients has been shown to distinguish two subsets of patients with differing prognoses. Mutated CLL cases have a more favorable prognosis with a longer survival requiring less treatment as compared to unmutated cases. Mutational status serves as an independent, highly significant prognostic factor for clinical outcome in B-CLL, correlating well with disease severity and overall survival; unlike many of the other markers, the mutation status remains relatively constant through disease progression. In addition to mutation status, sequencing of the immunoglobulin heavy chain provides information on variable segment usage. Recent studies have shown that estimates of prognosis can be further refined based on this data. Specifically, patients with B-CLL in which the variable segment IgHV3-21 is used have a worse prognosis independent of mutation status or other prognostic indicators. This information along with IGH mutational status can be used in conjunction with other B-CLL prognostic indicators including CD38 expression, ZAP-70 expression, cytogenetic abnormalities, clinical staging, leukocyte doubling time, and LDH levels to provide the best possible, patient specific, approximation of prognosis.


Damle RN, et. al. IG V gene mutation status and CD38 expression as novel prognostic indicators in chronic lymphocytic leukemia. Blood 1999; 94:1840-7.

Giudicelli V. et. al. IMGT/V-QUEST, an integrated software program for immunoglobulin and T cell receptor V-J and V-D-J rearrangement analysis. Nucleic Acids Res 2004; 32:W435-40.

Hamblin TJ et .al. CD38 expression and immunoglobulin variable region mutations are independent prognostic variables in chronic lymphocytic leukemia, but CD38 expression may vary during the course of the disease. Blood 2002; 99:1023-9.

Hamblin TJ, et. al. Unmutated Ig VH genes are associated with a more aggressive form of chronic lymphocytic leukemia. Blood 1999:94; 1848-54.

Kuppers R, et. al. Cellular origins of human B-cell lymphomas. New Eng J Med 1999; 341:1520-9.

Marasca R, et. al. Immunoglobulin mutational status detected through single-round amplification of partial V region represents a good prognostic marker for clinical outcome in chronic lymphocytic leukemia. J Mol Diagn 2005; 7:566-74.

Matthews C, et. al. Routine analysis of IgVH mutational status in CLL patients using BIOMED-2 standardized primers and protocols. Leukemia Lymphoma 2004; 45:1899-1904.

Van Dongen JJ, et. al. Design and standardization of PCR primers and protocols for detection of clonal immunoglobulin and T-cell receptor gene recombination in suspect lymphoproliferations: Report of the BIOMED-2 Concerted Action BMH4-CT98-3936. Leukemia 2003; 17:2257-2317.

Weinberg JB et al. Clinical and molecular predictors of disease severity and survival in chronic lymphocytic leukemia. American Journal of Hematology. 2007; 82(12): 1063-1070.
    This test can be used for:

Prognostic testing in patients with chronic lymphocytic leukemia (CLL).
    This assay determines only B-cell heavy chain mutation status.

A small number of IGH clonal rearrangements are not detectable with this assay, and cannot be assessed for mutation status.

Mutation status determination is based on variable segment sequencing of the FR1 to FR3 region in PCR Mix 1 and the CDR1 to FR3 region in PCR Mix 2. A sample containing a clonal band in Mix 2 only will have a mutation status determination based on a partial sequence of the variable segment. Percent homology derived from entire versus partial variable segment sequence is usually concordant.

Multiple factors can contribute to prognosis in patients with B-CLL including, clinical stage, chromosomal deletions (del 17p), and expression of prognostic markers including ZAP-70 and CD38. Thus, this assay is intended for use as an aid in determining patient prognosis, but is not a substitute for a complete pathologic and clinical evaluation, or physician's judgment and clinical experience.
Test Synonyms
  Synonym(s): CLL
Molecular Diagnostics Laboratory

Medical Director:
 Michael Datto, M.D., Ph.D.
 Phone: 919-684-6965
 Email: michael.datto@duke.edu
Lab Director:
 Catherine Rehder Ph.D, FACMG
 Phone: 919-613-8434
 Email: catherine.rehder@duke.edu
Lab Director:
 Siby Sebastian Ph.D., DABMG
 Phone: 919-613-8432
 Email: siby.s@duke.edu

 Wadsworth Bldg, Cytogenetics, Rm 0220
 2351 Erwin Rd
 Durham,  NC  27705
 Phone: 919-684-2698
 FAX: 919-668-5424

Performing Times: