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• W2017233521 abstract "Deletions of chromosome 13q14 are common in chronic lymphocytic leukemia and other cancers, demonstrating the importance of this region in tumorigenesis. We report the use of two single-nucleotide polymorphism (SNP)-based techniques to determine 13q loss of heterozygosity (LOH) status in 15 patients with CLL: (i) digital SNP (dSNP), where analysis of heterozygous SNPs detects allelic imbalances, and (ii) DNA sequencing, where LOH is identified by comparison of allelic peak heights in normal and neoplastic cells. The SNP-based techniques were compared with established molecular techniques, fluorescence in situ hybridization and multiplex ligation-dependent probe amplification, to determine their utility and relative sensitivity. dSNP proved to be the most sensitive technique, identifying 13q14 LOH in 11 of 13 (85%) patients (95% CI: 55%, 98%) without the need for neoplastic cell enrichment. Three cases showed evidence of LOH by dSNP that was not apparent by other techniques. In 8 of 13 (62%) cases, partial or interstitial patterns of LOH were observed by dSNP. Our findings demonstrate that dSNP represents a useful, sensitive technique for the analysis of chromosomal aberrations that result in LOH. It may have applications for the analysis of other malignancies that are difficult to assess by conventional molecular techniques. Deletions of chromosome 13q14 are common in chronic lymphocytic leukemia and other cancers, demonstrating the importance of this region in tumorigenesis. We report the use of two single-nucleotide polymorphism (SNP)-based techniques to determine 13q loss of heterozygosity (LOH) status in 15 patients with CLL: (i) digital SNP (dSNP), where analysis of heterozygous SNPs detects allelic imbalances, and (ii) DNA sequencing, where LOH is identified by comparison of allelic peak heights in normal and neoplastic cells. The SNP-based techniques were compared with established molecular techniques, fluorescence in situ hybridization and multiplex ligation-dependent probe amplification, to determine their utility and relative sensitivity. dSNP proved to be the most sensitive technique, identifying 13q14 LOH in 11 of 13 (85%) patients (95% CI: 55%, 98%) without the need for neoplastic cell enrichment. Three cases showed evidence of LOH by dSNP that was not apparent by other techniques. In 8 of 13 (62%) cases, partial or interstitial patterns of LOH were observed by dSNP. Our findings demonstrate that dSNP represents a useful, sensitive technique for the analysis of chromosomal aberrations that result in LOH. It may have applications for the analysis of other malignancies that are difficult to assess by conventional molecular techniques. Chronic lymphocytic leukemia (CLL) is the most common leukemia among adults in the Western world, occurring predominantly in individuals over the age of 65 years.1O'Brien SM Kantarjian H Thomas DA Giles FJ Freireich EJ Cortes J Lerner S Keating MJ Rituximab dose-escalation trial in chronic lymphocytic leukemia.J Clin Oncol. 2001; 19: 2165-2170Crossref PubMed Scopus (560) Google Scholar It is a heterogeneous disease associated with a highly variable clinical course.2Chiorazzi N Rai KR Ferrarini M Chronic lymphocytic leukemia.N Engl J Med. 2005; 352: 804-815Crossref PubMed Scopus (1337) Google Scholar At present, the molecular etiology of CLL remains largely undetermined. However, it is widely acknowledged that the disease, like most cancers, is associated with marked chromosomal instability.3Dohner H Stilgenbauer S Benner A Leupolt E Krober A Bullinger L Dohner K Bentz M Lichter P Genomic aberrations and survival in chronic lymphocytic leukemia.N Engl J Med. 2000; 343: 1910-1916Crossref PubMed Scopus (2743) Google Scholar Fluorescence in situ hybridization (FISH) has identified recurrent chromosomal abnormalities in approximately 80% of CLL patients, and specific genomic aberrations now serve as prognostic indicators for disease progression.3Dohner H Stilgenbauer S Benner A Leupolt E Krober A Bullinger L Dohner K Bentz M Lichter P Genomic aberrations and survival in chronic lymphocytic leukemia.N Engl J Med. 2000; 343: 1910-1916Crossref PubMed Scopus (2743) Google Scholar The most frequent chromosomal aberration in CLL is the loss of genetic material from the long arm of chromosome 13. Chromosome 13q14 deletions are reported in over 50% of CLL cases and are associated with a favorable prognosis when present as the sole genomic abnormality3Dohner H Stilgenbauer S Benner A Leupolt E Krober A Bullinger L Dohner K Bentz M Lichter P Genomic aberrations and survival in chronic lymphocytic leukemia.N Engl J Med. 2000; 343: 1910-1916Crossref PubMed Scopus (2743) Google Scholar. The deletion of chromosome band 13q14 has also been reported in a variety of other malignancies,4Facon T Avet-Loiseau H Guillerm G Moreau P Genevieve F Zandecki M Lai JL Leleu X Jouet JP Bauters F Harousseau JL Bataille R Mary JY Chromosome 13 abnormalities identified by FISH analysis and serum beta2-microglobulin produce a powerful myeloma staging system for patients receiving high-dose therapy.Blood. 2001; 97: 1566-1571Crossref PubMed Scopus (309) Google Scholar5La Starza R Wlodarska I Aventin A Falzetti D Crescenzi B Martelli MF Van den Berghe H Mecucci C Molecular delineation of 13q deletion boundaries in 20 patients with myeloid malignancies.Blood. 1998; 91: 231-237PubMed Google Scholar6Lu W Takahashi H Furusato M Maekawa S Nakano M Meng C Kikuchi Y Sudo A Hano H Allelotyping analysis at chromosome 13q of high-grade prostatic intraepithelial neoplasia and clinically insignificant and significant prostate cancers.Prostate. 2006; 66: 405-412Crossref PubMed Scopus (14) Google Scholar7Schlade-Bartusiak K Stembalska A Ramsey D Significant involvement of chromosome 13q deletions in progression of larynx cancer, detected by comparative genomic hybridization.J Appl Genet. 2005; 46: 407-413PubMed Google Scholar leading researchers to postulate that this chromosomal region harbors tumor suppressor gene(s) involved in the etiology of these diseases. Delineation of the minimal deleted region at 13q14 may facilitate the identification of such genes. Several well-established techniques are available for the detection and characterization of chromosome 13q14 deletions. These include metaphase cytogenetics,8Juliusson G Oscier DG Fitchett M Ross FM Stockdill G Mackie MJ Parker AC Castoldi GL Guneo A Knuutila S Elonen E Gahrton G Prognostic subgroups in B-cell chronic lymphocytic leukemia defined by specific chromosomal abnormalities.N Engl J Med. 1990; 323: 720-724Crossref PubMed Scopus (560) Google Scholar interphase-FISH,3Dohner H Stilgenbauer S Benner A Leupolt E Krober A Bullinger L Dohner K Bentz M Lichter P Genomic aberrations and survival in chronic lymphocytic leukemia.N Engl J Med. 2000; 343: 1910-1916Crossref PubMed Scopus (2743) Google Scholar array-based comparative genome hybridization,9Schwaenen C Nessling M Wessendorf S Salvi T Wrobel G Radlwimmer B Kestler HA Haslinger C Stilgenbauer S Dohner H Bentz M Lichter P Automated array-based genomic profiling in chronic lymphocytic leukemia: development of a clinical tool and discovery of recurrent genomic alterations.Proc Natl Acad Sci USA. 2004; 101: 1039-1044Crossref PubMed Scopus (203) Google Scholar single nucleotide polymorphism (SNP) arrays10Pfeifer D Pantic M Skatulla I Rawluk J Kreutz C Martens UM Fisch P Timmer J Veelken H Genome-wide analysis of DNA copy number changes and LOH in CLL using high-density SNP arrays.Blood. 2007; 109: 1202-1210Crossref PubMed Scopus (188) Google Scholar and multiplex ligation-dependent probe amplification (MLPA).11Coll-Mulet L Santidrian AF Cosialls AM Iglesias-Serret D de Frias M Grau J Menoyo A Gonzalez-Barca E Pons G Domingo A Gil J Multiplex ligation-dependent probe amplification for detection of genomic alterations in chronic lymphocytic leukaemia.Br J Haematol. 2008; 142: 793-801Crossref PubMed Scopus (39) Google Scholar While extremely useful, each technique has limitations and no technique is universally applicable. Conventional cytogenetic analysis has been traditionally hampered by the low mitotic activity of CLL cells. FISH analysis is limited to the genomic regions covered by the probes; therefore, deletions not covering these specific regions may remain undetected. Low density SNP arrays may fail to identify cases where the deleted region is very small,12Li X Self SG Galipeau PC Paulson TG Reid BJ Direct inference of SNP heterozygosity rates and resolution of LOH detection.PLoS Comput Biol. 2007; 3: e244Crossref PubMed Scopus (18) Google Scholar whereas both array-based comparative genome hybridization and MLPA are acknowledged to have limited sensitivity for the detection of aberrations present in a low percentage of cells.9Schwaenen C Nessling M Wessendorf S Salvi T Wrobel G Radlwimmer B Kestler HA Haslinger C Stilgenbauer S Dohner H Bentz M Lichter P Automated array-based genomic profiling in chronic lymphocytic leukemia: development of a clinical tool and discovery of recurrent genomic alterations.Proc Natl Acad Sci USA. 2004; 101: 1039-1044Crossref PubMed Scopus (203) Google Scholar,11Coll-Mulet L Santidrian AF Cosialls AM Iglesias-Serret D de Frias M Grau J Menoyo A Gonzalez-Barca E Pons G Domingo A Gil J Multiplex ligation-dependent probe amplification for detection of genomic alterations in chronic lymphocytic leukaemia.Br J Haematol. 2008; 142: 793-801Crossref PubMed Scopus (39) Google Scholar Consequently, there is a need for a sensitive technique that allows high-resolution screening of chromosome band 13q14. We report here the novel application of two molecular techniques, digital single nucleotide polymorphism (dSNP) and loss of heterozygosity (LOH) DNA sequencing, for the characterization of LOH at chromosome 13q14 in patients with CLL. Both techniques rely on the genotypic analysis of heterozygous SNPs to determine LOH status. Digital SNP is a sophisticated innovative technique that allows identification of LOH through the direct counting of alleles.13Zhou W Goodman SN Galizia G Lieto E Ferraraccio F Pignatelli C Purdie CA Piris J Morris R Harrison DJ Paty PB Culliford A Romans KE Montgomery EA Choti MA Kinzler KW Vogelstein B Counting alleles to predict recurrence of early-stage colorectal cancers.Lancet. 2002; 359: 219-225Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar Briefly, the technique relies on the identification of heterozygous SNPs in patient samples and subsequent separation of the heterozygous alleles through serial dilution of the patient DNA. PCR amplification of the heterozygous alleles is performed and the allelic frequencies counted. Deviation from the expected 50:50 ratio for heterozygous alleles represents LOH at that specific locus and is highly suggestive of a deletion. Sequential probability ratio testing (SPRT)14Piantadosi S Clinical trials. Wiley-Interscience, New York1997: 236-237Google Scholar confirms the significance of such deviations. DNA sequencing may be used to determine LOH status through the comparison of heterozygous SNPs in neoplastic and normal cells. Briefly, paired patient DNA samples from isolated mononuclear cells and non-neoplastic buccal mucosa cells are obtained for each patient. Heterozygous SNPs present in the buccal DNA samples are compared with mononuclear cell DNA samples by PCR/sequence analysis. The quantification of heterozygous alleles is determined using Mutation Surveyor software (version 3.2, SoftGenetics, State College, PA). Tumor cells harboring LOH show reduced quantities of a heterozygous allele. The reduced allele quantity is not noted in the normal buccal mucosa cells. The aim of this study was to assess the utility and relative sensitivity of SNP-based technology to identify and characterize 13q14 LOH status in a cohort of 15 patients with CLL. High-resolution screening of chromosome band 13q14 may allow for the delineation of the minimal deleted region and identification of candidate tumor suppressor genes for further investigation. Fifteen patients with CLL under the care of the Hematology Unit at the Royal Devon and Exeter Hospital were enrolled in this study. Each patient had an established diagnosis of CLL based on current World Health Organization classification guidelines.15Müller-Hermelink H Montserrat E Catovsky D Harris N Chronic lymphocytic leukaemia/small lymphocytic lymphoma.in: Jaffe ES Harris NL Stein H Vardiman JW World Health Organization classification of tumours: pathology and genetics of tumours of haematopoietic and lymphoid tissues. IARC, 2001: 127-130Google Scholar Patients had a history of persistent lymphocytosis >5 × 109/L and an immunophenotypic profile typical of CLL, confirmed by a flow cytometry score of ≥4 in all cases.16Matutes E Owusu-Ankomah K Morilla R Garcia Marco J Houlihan A Que TH Catovsky D The immunological profile of B-cell disorders and proposal of a scoring system for the diagnosis of CLL.Leukemia. 1994; 8: 1640-1645PubMed Google Scholar The majority of patients (13/15) had received treatment for their disease before involvement in this study. The study protocol had received prior approval by the local research ethics committee and all patients provided written informed consent in accordance with the Declaration of Helsinki. DNA was extracted from whole blood using the Wizard Genomic DNA purification kit (Promega, Southampton, UK) according to the manufacturer's instructions. Mononuclear cells were isolated through Lymphoprep gradient centrifugation (Axis Shield, Olso, Norway) and processed for DNA extraction using the Wizard Genomic DNA purification kit (Promega, Southampton, UK). DNA concentrations were measured using a ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington). Normal genomic DNA was extracted from non-neoplastic buccal mucosa cells using standard procedures. Cells were collected by rubbing a cytology brush against the inside of the patient's cheek. The cells were lysed in 50 mmol/L NaOH and DNA was precipitated in 1M/L Tris/HCL (pH 7.5). A panel of 10 SNP markers covering the 13q14.2 to 13q34 region of chromosome 13 (Figure 1A) was chosen on the basis of high population heterozygosity (0.3 to 0.5). Probes and primers to these polymorphisms were designed by the Applied Biosystems Assays-on-Demand service (ABI, Foster City, CA) (probe and primer sequences are available in Table 1). In each case, allele-specific probes were labeled 5′ with either 6-FAM or VIC and 3′ with a minor groove binding (MGB) protein serving as a non-fluorescent quencher. Assays were validated using standard curves generated from seven serial (1:2) dilutions of heterozygous DNA to ensure equivalent amplification and probing efficiency, and by amplification of homozygous individuals of both genotypes to ensure that background fluorescence was within acceptable limits.Table 1Sequences of Probes and Primers Used for Digital SNP AnalysisNameSequenceLabelsRB-1 F5′-GTTGCTGGACAGCCTATGGAT-3′NoneRB-1 R5′-AAGGAATTATACCAAAGCAGCTAACTGAA-3′NoneRB-1 P(1)5′-TCAGGTGACTATCTTTTGT-3′5′ VIC, 3′ MGBRB-1 P(2)5′-CAGGTGACTATGTTTTGT-3′5′ 6-FAM, 3′ MGBEBPL F5′-CCCACCCTTCTACTTTTCTGGATTT-3′NoneEBPL R5′-CACCAGCCGGCTTAAAGC-3′NoneEBPL P(1)5′-AAATGGAAAGATTGTCAGAGTA-3′5′ VIC, 3′ MGBEBPL P(2)5′-ATGGAAAGATTGTCGGAGTA-3′5′ 6-FAM, 3′ MGBKPNA3 F5′-CAAGTCTGTTTGCTTAATGTCCCATTG-3′NoneKPNA3 R5′-CCAGTCCCGCACTGACT-3′NoneKPNA3 P(1)5′-CCAAAGCAAGTCACAC-3′5′ VIC, 3′ MGBKPNA3 P(2)5′-CAAAGCGAGTCACAC-3′5′ 6-FAM, 3′ MGBC13Orf1 F5′-CTCATTAGATCCAATCCCACTGCTT-3′NoneC13Orf1 R5′-CCACTGTAAATGACTAAATTTGAACTGCTT-3′NoneC13Orf1 P(1)5′-AAACTCCCAAACAAAG-3′5′ VIC, 3′ MGBC13Orf1 P(2)5′-AACTCCCAGACAAAG-3′5′ 6-FAM, 3′ MGBKCNRG F5′-GATCTCTCAAAAATTGATGAGTGTTGGTT-3′NoneKCNRG R5′-TCGAGACCTTGCTGTGATTCTG-3′NoneKCNRG P(1)5′-AAAGTCATTTGAATTCC-3′5′ VIC, 3′ MGBKCNRG P(2)5′-AAGTCATTTCAATTCC-3′5′ 6-FAM, 3′ MGBDLEU2 F5′-ACTTCCCTACCTATTCTCCTGGAAAA-3′NoneDLEU2 R5′-GGAGCTAGAGGCATTTTGTAAGCAA-3′NoneDLEU2 P(1)5′-AATCAAGAAATGACACTTT-3′5′ VIC, 3′ MGBDLEU2 P(2)5′-CAATCAAGAAATTACACTTT-3′5′ 6-FAM, 3′ MGBDLEU1 F5′-GCCAGTGTCTAAACTCCAAACAAC-3′NoneDLEU1 R5′-GGCGGTTTTAAATGCACGTGTATC-3′NoneDLEU1 P(1)5′-CAATGAGACCTAGTATATG-3′5′ VIC, 3′ MGBDLEU1 P(2)5′-CAATGAGACCTTGTATATG-3′5′ 6-FAM, 3′ MGBDLEU7 F5′-GGGTCTTGAAAGAAAAGTACAGAGCTA-3′NoneDLEU7 R5′-CCACTCAGTTTTCCCACACCTAA-3′NoneDLEU7 P(1)5′-AAAGAAAGGCATCCCCCCAG-3′5′ VIC, 3′ MGBDLEU7 P(2)5′-AAAGGCATCGCCCCAG-3′5′ 6-FAM, 3′ MGBSLC15A1 F5′-GCTGCTGATTTCAGTGGAGACA-3′NoneSLC15A1 R5′-TTTTGGATTAATCACCTCCCAGCTT-3′NoneSLC15A1 P(1)5′-TCTAGATGCAAGTATCTG-3′5′ VIC, 3′ MGBSLC15A1 P(2)5′-TAGATGCAAATATCTG-3′5′ 6-FAM, 3′ MGBUBAC2 F5′-GAATCCTAAGATGCTTAATTTTGTAAGTTTGCA-3′NoneUBAC2 R5′-GGTGCATGATGAGCAGTGAAAA-3′NoneUBAC2 P(1)5′-CTCGGATCATATTTAG-3′5′ VIC, 3′ MGBUBAC2 P(2)5′-CTCGGATCGTATTTAG-3′5′ 6-FAM, 3′ MGBProbes are labeled 5′ with 6-FAM or VIC and 3′ with a MGB to enhance probe specificity. Nucleotides representing the SNPs under investigation are highlighted in bold. Open table in a new tab Probes are labeled 5′ with 6-FAM or VIC and 3′ with a MGB to enhance probe specificity. Nucleotides representing the SNPs under investigation are highlighted in bold. Real-time PCR amplification using the ABI 7900HT platform (ABI) was initially performed for all 15 patient samples to identify individuals who were heterozygous for each SNP. PCR was performed in 10-μl reactions including 5 μl universal master mix, (no AMPerase) (ABI), assay mix (0.9 μmol/L probe, 1.8 μmol/L each primer), and 40 ng of DNA. PCR reaction conditions were 95°C for 20 seconds, followed by 60 cycles of 95°C for 1 second and 60°C for 20 seconds. Following amplification, appropriate baseline and threshold levels were set and the genotype of each sample was determined for each SNP. The optimal DNA concentration to yield good separation of alleles was calculated from the average molecular weight of a mole of base (342 g), the number of molecules in a mole of substance (the Avogadro constant, 6.023 × 1023) and the number of bases in the human genome (3 × 109). However, since this figure takes no account of the presence of PCR inhibitors or DNA degradation in the sample, optimal dilution factors were further determined empirically. Optimally diluted DNA samples were distributed to the wells of 96-well optical plates. Real-time amplification of the DNA was performed. PCR reactions were performed in a total volume of 8 μl including 4 μl universal master mix (no AMPerase) (ABI), assay mix (0.9 μmol/L probe, 1.8 μmol/L each primer), and 2 μl appropriately diluted DNA. PCR cycles were 95°C for 20 seconds, followed by 60 cycles of 95°C for 1 second and 60°C for 20 seconds. Following amplification, each well was genotyped as allele 1, allele 2, allele 1 + 2, or no product detected (nd). Likelihood analysis14Piantadosi S Clinical trials. Wiley-Interscience, New York1997: 236-237Google Scholar was applied to assess the strength of evidence for deviation from a 50:50 distribution of alleles. The specific method used was the SPRT, as described in previous studies.13Zhou W Goodman SN Galizia G Lieto E Ferraraccio F Pignatelli C Purdie CA Piris J Morris R Harrison DJ Paty PB Culliford A Romans KE Montgomery EA Choti MA Kinzler KW Vogelstein B Counting alleles to predict recurrence of early-stage colorectal cancers.Lancet. 2002; 359: 219-225Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar,17Zhou W Galizia G Lieto E Goodman SN Romans KE Kinzler KW Vogelstein B Choti MA Montgomery EA Counting alleles reveals a connection between chromosome 18q loss and vascular invasion.Nature Biotechnol. 2001; 19: 78-81Crossref Scopus (676) Google Scholar This technique allows two probabilistic hypotheses to be compared as data accumulates. A heterozygous sample is expected to have allelic balance, with the two heterozygous alleles appearing at approximately the same frequency (null hypothesis H0) (Figure 2A). A sample with LOH will have allelic imbalance (alternative hypothesis H1) with over-representation of one allele (Figure 2B). The degree of imbalance will be directly proportional to the number of clonal B-lymphocytes that are present in the sample and that harbor the aberration. Threshold curves representing the two hypotheses are generated for a specific tumor load based on a predetermined likelihood ratio. Analysis is concluded by plotting the number of informative alleles tested against the major allele ratio (Figure 3A). If the point is plotted above the upper threshold curve, it is considered as evidence of allelic imbalance and hence LOH at that locus. Points plotted below the lower threshold curve are categorized as having allelic balance and therefore no LOH.Figure 3Digital SNP analysis of 13q in patients with CLL A: Sequential probability ratio test (SPRT) to detect LOH at 13q. The x axis represents the number of alleles tested and the y axis represents the major allele frequency. Threshold curves are calculated for a 60% tumor load based on the likelihood ratio of 8. Analysis is conducted by plotting the number of alleles tested against the major allele frequency. Points plotted above the upper curve represent LOH, while those below the lower curve represent no LOH. B: Schematic representation of chromosome 13 from band 13q14.2 to band 13q32. The sample number is given at the left side of the figure and the markers tested are shown at the top. Markers colored in black represent loci with LOH determined by dSNP analysis, while those in white represent loci with no LOH.View Large Image Figure ViewerDownload Hi-res image Download (PPT) A likelihood ratio of >8 was accepted as evidence of LOH since it provides a post-test probability of LOH of 89%, if the pre-test probability of the presence of LOH at 13q is assumed to be 50%. Since SPRT allows for repeat testing, the post-test probability of LOH can be increased further by testing each sample with several allelic probes. The proportions (P) of the major allele for a likelihood ratio of 8 for the presence of LOH in test samples with varying proportions of B-lymphocytes were calculated according to the formula P = log (16)/(n*log[(100−l)/100]) + log([100−l)/(100−l/2)]/log([100−l]/100), where n = total number of informative alleles counted and l = the percentage of lymphocytes in the peripheral blood sample. The results of the equation were tabulated with Microsoft Excel 2000 software. Primers were designed to amplify the SNPs rs3809325 and rs9535499 within EBPL and DLEU7 respectively (Table 2). Paired patient DNA samples from isolated mononuclear cells and non-neoplastic buccal mucosa cells were amplified by PCR and sequenced in one direction by Big-Dye terminator chemistry using the ABI 3730 genetic analyzer (ABI). The LOH status of each sample was determined through the quantification of each allele by peak height analysis using the DNA quantification tool of Mutation Surveyor software (version 3.2, SoftGenetics, State College, PA) (Figure 1B). Coefficient of variation analysis was applied to establish normal variation observed for peak height quantification.Table 2Sequences of Primers Used for DLEU7 and EBPL LOH SequencingNameSequenceDLEU7 F5′-TGTAAAACGACGGCCAGTGTGAGGTGGG CCAGAAATAA-3′DLEU7 R5′-CAGGAAACAGCTATGACCAATTACAAT AGGGTAGGCAGCA-3′EBPL F5′-TGTAAAACGACGGCCAGTTATCGCCAAG CCAATAGGAC-3′EBPL R5′-CAGGAAACAGCTATGACCAGTAGACCGG GGCAGAATTT-3′ Open table in a new tab FISH analysis for the detection of chromosome 13q deletions was performed by Bristol Genetics Laboratory as a routine clinical test using LSI S13S319 (Spectrum Orange) and LSI 13q34 (Spectrum Aqua) (Vysis Inc., Abbott Molecular, Maidenhead, UK) probes mapping at 13q14 and 13q34 respectively. FISH was conducted according to the manufacturer's protocol using co-denaturation and rapid wash procedures. MLPA was performed according to the manufacturer's protocol (MRC-Holland, Amsterdam, Holland). Two kits, Salsa MLPA kit PO37 CLL1 and PO38 CLL2, provided a total of 12 probes that specifically target 13q14. The locations of the probes are shown in Figure 1A. Ligation products were amplified using a common 6-FAM labeled primer set. Products were analyzed on an ABI 3100 genetic analyzer (ABI) and dosage analysis was performed using Genemarker software (Version 1.70, SoftGenetics, State College, PA). The software calculates dosage quotients by comparing peak height values for each sample with those produced by normal controls (Figure 1C). Real-time PCR assays were designed and validated for 10 SNPs spanning the 13q14.2 to 13q34 region. The median distance between the SNP probes at 13q14 was 92 kb (range, 10 kb to 1348 kb). All assays were deemed suitable for further analysis on the basis of yielding reliable and specific amplification with an efficiency difference of less than 0.5 between alleles, and minimal cross allele reactivity. All assays demonstrated a linear relationship between crossing point and DNA concentration over seven serial 1:2 dilutions (data not shown). Of the 15 patients studied, 13 were heterozygous for at least one of the 10 SNPs within the panel and were therefore suitable for dSNP analysis. The median number of heterozygous SNPs per patient was 3.0 (range, 2.0 to 5.0). In patients with more than one informative SNP at 13q14, the median distance between SNPs per patient was 169 kb (range, 42 kb to 2478 kb). The average mass of one molecule of DNA was calculated as 2.6 × 10−12g. Therefore, a DNA concentration of 2.6 × 10−12pg/μl should yield one template molecule per 1 μl. In practice, the presence of PCR inhibitors and the occurrence of variable amounts of DNA degradation in the samples meant that the optimal DNA concentrations needed to be determined empirically. Optimal DNA concentrations ranged from 2 to 40 pg/μl. The CLL load was calculated for each patient as the percentage lymphocytes of the total leukocytes at the time of sample collection. In the majority of cases, the CLL load ranged from 70% to 100% (Table 3). In several cases, the CLL load was notably lower at 26% (CLL 08), 37% (CLL 09), and 41% (CLL 11).Table 3Summary of 13q14 Status as Determined by dSNP, LOH Sequencing, FISH, and MLPA13q14 statusIdentifierCLL LoaddSNPLOH SequencingFISHMLPACLL 0173%LOHLOHDeletedDeletedCLL 0295%LOH−DeletedDeletedCLL 0370%No LOHNo LOHNot deletedNot deletedCLL 0478%−−Not deletedNot deletedCLL 0597%LOHNo LOHDeletedDeletedCLL 0662%LOHLOHDeletedDeletedCLL 0798%LOHNo LOHNot deletedNot deletedCLL 0826%LOHNo LOHNot deletedNot deletedCLL 0937%LOHLOHDeletedDeletedCLL 1089%LOHLOHDeletedDeletedCLL 1141%No LOHNo LOHNot deletedNot deletedCLL 12100%−−DeletedDeletedCLL 1388%LOH−Not deletedNot deletedCLL 1484%LOH−DeletedDeletedCLL 1591%LOHLOHDeletedDeletedDiscrepant results are highlighted in bold. Open table in a new tab Discrepant results are highlighted in bold. The LOH status of selective informative markers spanning the region 13q14.2 to 13q34 was determined using dSNP analysis for each of the 13 CLL patients with heterozygous SNPs. Allelic imbalances at 13q14 were identified in 11/13 (85%) (95% CI: 55%, 98%) CLL patients using this technique (Figure 3B). When analysis was extended to cover the 13q34 region, allelic imbalances were observed in all 13 patients. Deviations from the expected balanced allele frequency were tested for statistical significance by SPRT analysis, thus confirming the LOH status (Figure 3A). The extent of LOH varied considerably between patients and no common region of LOH was evident. Six cases (CLL 03, CLL 05, CLL 07, CLL 10, CLL 11, and CLL 13) showed evidence of LOH extending along just part of the chromosome arm. Two cases (CLL 02 and CLL 15) showed evidence complex chromosomal rearrangements, with regions of LOH disrupted by small, interstitial regions of heterozygosity. Digital SNP analysis allowed the identification of LOH in patients with low CLL loads (CLL 08, CLL 09, and CLL11) without the need for mononuclear cell enrichment techniques. FISH and MLPA allowed direct assessment of the 13q14 deletion status at specific loci in all 15 patients. FISH and MLPA showed complete concordance, both identifying chromosome 13q14 deletions in 9/15 (60%) patients. Of the nine patients with a confirmed deletion at 13q14 by FISH and MLPA, eight showed evidence of LOH by dSNP analysis. The LOH status of the remaining patient with a confirmed 13q14 deletion could not be determined by dSNP analysis due to the lack of informative heterozygous SNPs within our panel. Three discrepancies (CLL 07, CLL 08, and CLL 13) were observed between the dSNP, FISH, and MLPA techniques (Discrepancies highlighted in Table 3). Each discrepancy showed evidence of LOH by dSNP analysis that was not apparent by FISH or MLPA. Of the 14 CLL patients for whom both buccal and mononuclear cell samples were available, 10 were heterozygous for either rs3809325 (EBPL) or rs9535499 (DLEU7) and were tested by DNA sequencing to identify LOH. The mean coefficient of variation for repeat measurements of allele peak height was 6.4 (95% CI: 3.7, 9.1). We therefore considered a reduction of ≥6.5% in the allele quantification in mononuclear cell DNA, when compared with that observed for the corresponding allele in buccal mucosa DNA, to be indicative of LOH at that locus. DNA sequencing identified 13q14 LOH in 5/10 (50%) patients with CLL. There was good concordance between the DNA sequencing, FISH and MLPA techniques, with just one discordant result reported (CLL 05) (discrepancy highlighted in Table 3). This case had a 13q14 deletion confirmed by FISH and MLPA but did not show LOH by DNA sequencing analysis. Digital SNP and DNA sequencing were both used to determi" @default.
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• W2017233521 title "Evaluation of 13q14 Status in Patients with Chronic Lymphocytic Leukemia Using Single Nucleotide Polymorphism-Based Techniques" @default.
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