Characterization of feline immunoglobulin heavy chain variable region genes for the molecular diagnosis of B-cell neoplasia.
To develop a molecular-based assay so that the diagnosis of feline B-cell neoplasia can be facilitated, we have characterized 24 feline immunoglobulin heavy chain variable region (IGH V) complementary DNA (cDNA) transcripts. Structural homology with rearranged human IGH V genes was found, and the sequence information was used to design a feline-specific polymerase chain reaction (PCR)-based assay to amplify the complementarity determining region 3 as a marker for B-cell clonality. Conserved primers derived from the second and third framework regions of V gene segments were used in conjunction with 2 sequence-specific primers and 1 degenerate primer derived from the J gene segments. Each PCR reaction was run in duplicate, and both native and denatured PCR products were evaluated using polyacrylamide gel electrophoresis. Formalin-fixed, paraffin-embedded (FFPE) tissue sections from cats with confirmed B-cell neoplasia (diffuse large B-cell lymphoma, plasmacytoma, and myeloma) were examined, and 15/22 (68.2%) cats produced results indicative of the presence of a monoclonal population of B cells. The evaluation of denatured PCR products (heteroduplex analysis) facilitated a more accurate interpretation in 3/15 (20%) cats. Pseudoclonality was a major reason for the failure to detect monoclonality. Poor DNA quality is a significant concern and was responsible for the removal of 2 cats from the study. Using this assay, FFPE normal feline lymphoid tissues and unfixed peripheral blood mononuclear cells were determined to be composed of polyclonal populations of B cells. This assay represents a useful adjunctive diagnostic tool for the diagnosis and investigation of feline B-cell lymphoproliferative disorders.
Werner
J A
JA
Department of Veterinary, Pathology, Microbiology, and Immunology, School of Veterinary Medicine, University of California, Davis, CA 95616, USA.
Woo
J C
JC
Vernau
W
W
Graham
P S
PS
Grahn
R A
RA
Lyons
L A
LA
Moore
P F
PF
D000595:Amino Acid Sequence; D000818:Animals; D002371:Cat Diseases / Q000175:diagnosis* / Q000235:genetics* / Q000276:immunology; D002415:Cats; D005260:Female; D007143:Immunoglobulin Heavy Chains / Q000737:chemistry / Q000235:genetics*; D007135:Immunoglobulin Variable Region / Q000737:chemistry / Q000235:genetics*; D016393:Lymphoma, B-Cell / Q000175:diagnosis* / Q000235:genetics / Q000276:immunology / Q000662:veterinary*; D008297:Male; D010954:Plasmacytoma / Q000175:diagnosis / Q000235:genetics / Q000276:immunology / Q000662:veterinary; D017386:Sequence Homology, Amino Acid
D007143:Immunoglobulin Heavy Chains; D007135:Immunoglobulin Variable Region