A new method to detect loss of heterozygosity using cohort heterozygosity comparisons.
BACKGROUND
Loss of heterozygosity (LOH) is an important marker for one of the 'two-hits' required for tumor suppressor gene inactivation. Traditional methods for mapping LOH regions require the comparison of both tumor and patient-matched normal DNA samples. However, for many archival samples, patient-matched normal DNA is not available leading to the under-utilization of this important resource in LOH studies. Here we describe a new method for LOH analysis that relies on the genome-wide comparison of heterozygosity of single nucleotide polymorphisms (SNPs) between cohorts of cases and un-matched healthy control samples. Regions of LOH are defined by consistent decreases in heterozygosity across a genetic region in the case cohort compared to the control cohort.


METHODS
DNA was collected from 20 Follicular Lymphoma (FL) tumor samples, 20 Diffuse Large B-cell Lymphoma (DLBCL) tumor samples, neoplastic B-cells of 10 B-cell Chronic Lymphocytic Leukemia (B-CLL) patients and Buccal cell samples matched to 4 of these B-CLL patients. The cohort heterozygosity comparison method was developed and validated using LOH derived in a small cohort of B-CLL by traditional comparisons of tumor and normal DNA samples, and compared to the only alternative method for LOH analysis without patient matched controls. LOH candidate regions were then generated for enlarged cohorts of B-CLL, FL and DLBCL samples using our cohort heterozygosity comparison method in order to evaluate potential LOH candidate regions in these non-Hodgkin's lymphoma tumor subtypes.


RESULTS
Using a small cohort of B-CLL samples with patient-matched normal DNA we have validated the utility of this method and shown that it displays more accuracy and sensitivity in detecting LOH candidate regions compared to the only alternative method, the Hidden Markov Model (HMM) method. Subsequently, using B-CLL, FL and DLBCL tumor samples we have utilised cohort heterozygosity comparisons to localise LOH candidate regions in these subtypes of non-Hodgkin's lymphoma. Detected LOH regions included both previously described regions of LOH as well as novel genomic candidate regions.


CONCLUSIONS
We have proven the efficacy of the use of cohort heterozygosity comparisons for genome-wide mapping of LOH and shown it to be in many ways superior to the HMM method. Additionally, the use of this method to analyse SNP microarray data from 3 common forms of non-Hodgkin's lymphoma yielded interesting tumor suppressor gene candidates, including the ETV3 gene that was highlighted in both B-CLL and FL.
Green
Michael R
MR
Genomics Research Centre, Griffith Institute for Health & Medical Research, Griffith University, Parklands Drive, Southport, Queensland, Australia.
Jardine
Paul
P
Wood
Peter
P
Wellwood
Jeremy
J
Lea
Rod A
RA
Marlton
Paula
P
Griffiths
Lyn R
LR
D016022:Case-Control Studies; D002874:Chromosome Mapping; D002878:Chromosomes, Human, Pair 1; D015331:Cohort Studies; D004273:DNA, Neoplasm / Q000032:analysis; D020869:Gene Expression Profiling / Q000379:methods; D015973:Gene Expression Regulation, Leukemic; D015972:Gene Expression Regulation, Neoplastic; D005787:Gene Frequency; D016147:Genes, Tumor Suppressor; D006801:Humans; D015451:Leukemia, Lymphocytic, Chronic, B-Cell / Q000235:genetics*; D019656:Loss of Heterozygosity; D008224:Lymphoma, Follicular / Q000235:genetics*; D016403:Lymphoma, Large B-Cell, Diffuse / Q000235:genetics*; D008390:Markov Chains; D020411:Oligonucleotide Array Sequence Analysis; D020641:Polymorphism, Single Nucleotide; D015203:Reproducibility of Results
D004273:DNA, Neoplasm
J Immunol. 2007 Dec 1;179(11):7215-9
18025162
Bioinformatics. 2004 May 22;20(8):1233-40
14871870
Blood Rev. 2008 Sep;22(5):261-81
18456377
Nat Rev Cancer. 2001 Nov;1(2):157-62
11905807
Hum Genet. 2006 Jul;119(6):659-68
16738949
Genome Res. 2007 Nov;17(11):1665-74
17921354
Proc Natl Acad Sci U S A. 1973 Dec;70(12):3324-8
4202843
Oncogene. 2001 Apr 19;20(17):2153-60
11360199
EMBO J. 2008 Feb 20;27(4):589-605
18285820
Nat Genet. 1999 Jan;21(1 Suppl):20-4
9915496
J Biol Chem. 2004 Dec 3;279(49):50986-93
15377654
Haematologica. 1999 Jul;84(7):594-9
10406899
Cancer Sci. 2007 May;98(5):698-706
17374122
Annu Rev Immunol. 1994;12:141-79
8011280
Nucleic Acids Res. 2007;35(6):2013-25
17341461
PLoS Comput Biol. 2006 May;2(5):e41
16699594
Blood. 2002 Oct 1;100(7):2414-20
12239151
Cancer Epidemiol Biomarkers Prev. 2006 Feb;15(2):258-65
16492913
Curr Opin Oncol. 2002 Jan;14(1):65-72
11790983
Blood. 2006 Nov 1;108(9):3161-7
16857995
Blood. 2007 Feb 1;109(3):1202-10
17053054