Detection of hemizygous deletions in genomic DNA from leukaemia specimens for the diagnosis of patients.
Hemizygous deletions in genomic DNA appear to play an important role in tumorigenesis. The loss or inactivation of tumour suppressor genes (TSGs) is of critical importance in most malignancies, and has been shown to affect response to therapy. Here, we report a quantitative real-time polymerase chain reaction (qPCR) designed to detect two TSGs at the CDKN2A locus, p16(INK4A) and p14(ARF) that allows the detection of hemizygous deletions. Testing by qPCR of 18 bone marrow specimens from paediatric acute lymphoblastic leukaemia (ALL) patients at diagnosis revealed nine to be GG, six to be GD and three to be DD for exon 2 of p14(ARF)/p16(INK4A), concordant with Southern blotting analysis. A panel of 13 ALL cell lines was investigated for deletions at the CDKN2A locus and one of the lines, typed as GD for all exons, was further assessed by fluorescence in situ hybridisation, confirming the qPCR findings. The expression levels of p16(INK4A) and p14(ARF) were measured in all cell lines and these quantitative reverse transcriptase PCR results also agreed with the typing by qPCR. The qPCR method described is suitable for detection of hemizygous loss in primary patient material and the accuracy of the method was verified by three independent techniques.
Kees
Ursula R
UR
Telethon Institute for Child Health Research, and Centre for Child Health Research, The University of Western Australia, Perth, Australia. ursula@ichr.uwa.edu.au
Terry
Philippa A
PA
Ford
Jette
J
Everett
Janet
J
Murch
Ashleigh
A
de Klerk
Nick
N
Baker
David L
DL
D000483:Alleles; D001853:Bone Marrow / Q000473:pathology; D045744:Cell Line, Tumor; D019941:Cyclin-Dependent Kinase Inhibitor p16 / Q000235:genetics; D004247:DNA / Q000235:genetics*; D005091:Exons; D017353:Gene Deletion; D006801:Humans; D017404:In Situ Hybridization, Fluorescence / Q000379:methods; D010641:Phenotype; D054198:Precursor Cell Lymphoblastic Leukemia-Lymphoma / Q000175:diagnosis* / Q000235:genetics* / Q000473:pathology; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D025803:Tumor Suppressor Protein p14ARF / Q000235:genetics; D025521:Tumor Suppressor Proteins / Q000235:genetics
D019941:Cyclin-Dependent Kinase Inhibitor p16; D025803:Tumor Suppressor Protein p14ARF; D025521:Tumor Suppressor Proteins; D004247:DNA