Identification of clonotypic IgH VDJ sequences in multiple myeloma.
In multiple myeloma (MM) the rearranged immunoglobulin heavy chain (IgH) variable, diversity, and joining (VDJ) DNA sequence of malignant plasma cells (PCs) serves as a marker for cells in the MM clone. This clonotypic sequence can be isolated from MM PCs by reverse transcriptase polymerase chain reaction (RT-PCR) with consensus primers that amplify the rearranged IgH repertoire. This chapter focuses on the key steps in determining patient-specific clonotypic sequences, including bulk RT-PCR using purified bone marrow mononuclear cell (BMMC) RNA, single-cell RT-PCR using RNA from PCs sorted by flow cytometry, IgH sequence alignments using IMGT or V BASE, and patient-specific primer design. In a test panel of several MM patient BMMCs, primers specific for the proposed sequence must amplify IgH from only the original patient. Furthermore, the proposed IgH sequence is not confirmed as clonotypic until these primers generate positive amplifications in the majority of single PCs from the original patient. This two-part test ensures that the proposed IgH sequence satisfies the definition of the clonotypic sequence as the most frequent, unique IgH sequence in an MM patient PC sample. With this patient-specific MM marker, a better understanding of transformed PCs and their B-lineage predecessors can be developed.
Taylor
Brian J
BJ
Cross Cancer Institute, University of Alberta, Edmonton, Canada.
Kriangkum
Jitra
J
Strachan
Erin R
ER
Wizniak
Juanita
J
Pilarski
Linda M
LM
D001483:Base Sequence; D001854:Bone Marrow Cells / Q000276:immunology / Q000473:pathology; D002469:Cell Separation; D017931:DNA Primers; D014644:Genetic Variation; D006801:Humans; D007143:Immunoglobulin Heavy Chains / Q000235:genetics*; D008969:Molecular Sequence Data; D009101:Multiple Myeloma / Q000201:enzymology / Q000235:genetics* / Q000276:immunology* / Q000473:pathology; D010950:Plasma Cells / Q000276:immunology* / Q000473:pathology; D016133:Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D045567:VDJ Recombinases / Q000235:genetics* / Q000378:metabolism
D017931:DNA Primers; D007143:Immunoglobulin Heavy Chains; D045567:VDJ Recombinases