Frequent polymorphism in BCR exon b2 identified in BCR-ABL positive and negative individuals using fluorescent hybridization probes.
Recently, a polymorphic base in exon 13 of the BCR gene (exon b2 of the major breakpoint cluster region) has been identified in the eighth position before the junctional region of BCR-ABL cDNA. Cytosine replaces thymidine; the corresponding triplets are AAT (T allele) and AAC (C allele), respectively, both coding for asparagine. Therefore, this polymorphism has no implication in the primary structure of BCR and BCR-ABL proteins. However, since the alteration is located close to the fusion region it may have a significant influence on the annealing of PCR primers, probes for real time PCR, and antisense oligonucleotides. We have developed a RT-PCR-based screening method to easily identify polymorphic BCR and BCR-ABL alleles in CML patients and normal individuals in order to estimate their frequency. After amplification from cDNA, a melting curve of a specific fluorogenic probe mapping to the 3' end of BCR exon b2 and spanning the polymorphism readily discriminates between normal and polymorphic BCR and BCR-ABL alleles. This reporter probe is 3' labeled with fluorescein and placed next to 5' LC Red640-labeled anchor probes mapping to the 5' ends of BCR exon b3 or ABL exon a2 so that resonance energy transfer occurs when the probes are hybridized (LightCycler technology). T and C alleles were discriminated by a melting temperature difference of the reporter probe of 3.2 K. We have investigated cDNAs derived from leukocytes from seven cell lines and a total of 229 individuals: normal donors, n = 15; BCR-ABL negative chronic myeloproliferative disorders, n=30; BCR-ABL negative acute leukemias, n= 11; b2a2BCR-ABL positive CML, n = 93; and b3a2BCR-ABL positive CML, n= 80. The frequency of the C allele was 33.0% in BCR-ABL negative individuals, 30.6% in b2a2BCR-ABL, and 23.8% in b3a2BCR-ABL positive CML. In CML patients, 27.7% of BCR-ABL and 27.2% of BCR alleles had the C allele (NS). In total, 132 of 458 (28.8%) exons b2 of BCR or BCR-ABL alleles demonstrated this polymorphism. We conclude that a thymidine/cytosine replacement occurs frequently in BCR exon b2. Probes for real time quantitative RT-PCR should be designed not to map to the critical region in order to avoid underestimation of the number of BCR-ABL transcripts.
Saussele
S
S
III. Medizinische Univesitätsklinik, Fakultät für Klinische Medizin der Universität Heidelberg, Germany.
Weisser
A
A
Müller
M C
MC
Emgi
M
M
La Rosée
P
P
Paschka
P
P
Kuhn
C
C
Willer
A
A
Hehlmann
R
R
Hochhaus
A
A
D000208:Acute Disease; D000483:Alleles; D019943:Amino Acid Substitution; D002892:Chromosomes, Human, Pair 22 / Q000235:genetics*; D004252:DNA Mutational Analysis; D018076:DNA, Complementary / Q000235:genetics; D005091:Exons / Q000235:genetics*; D005188:False Negative Reactions; D005456:Fluorescent Dyes; D016044:Fusion Proteins, bcr-abl / Q000032:analysis / Q000235:genetics*; D006801:Humans; D007938:Leukemia / Q000235:genetics; D015464:Leukemia, Myelogenous, Chronic, BCR-ABL Positive / Q000235:genetics; D009196:Myeloproliferative Disorders / Q000235:genetics; D009363:Neoplasm Proteins / Q000235:genetics; D009691:Nucleic Acid Denaturation; D009693:Nucleic Acid Hybridization; D015345:Oligonucleotide Probes; D017354:Point Mutation; D011110:Polymorphism, Genetic; D011505:Protein-Tyrosine Kinases; D011518:Proto-Oncogene Proteins / Q000235:genetics*; D051562:Proto-Oncogene Proteins c-bcr; D012016:Reference Values; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014407:Tumor Cells, Cultured
D018076:DNA, Complementary; D005456:Fluorescent Dyes; D009363:Neoplasm Proteins; D015345:Oligonucleotide Probes; D011518:Proto-Oncogene Proteins; D011505:Protein-Tyrosine Kinases; D016044:Fusion Proteins, bcr-abl; C496613:BCR protein, human; D051562:Proto-Oncogene Proteins c-bcr