Detection of chromosomal translocation in hematologic malignancies by a novel DNA-based Looped Ligation Assay (LOLA)

Shuko Harada, Emily Sizzle, Ming-Tseh Lin, Christopher Gocke

Research output: Contribution to journalArticle

Abstract

BACKGROUND: Disease-defining chromosomal translocations are seen in various neoplasms, especially in lymphomas and leukemias. Translocation detection at the DNA level is often complicated by chromosomal breakpoints that are distributed over very large regions. We have developed a ligation-based assay [the looped ligation assay (LOLA)] to detect translocations from diseases with multiple widely spaced breakpoint hot spots. METHODS: Oligonucleotide sets that probe breakpoints of IGH-BCL2 (immunoglobulin heavy-apoptosis regulator) in follicular lymphoma (FL), MYC-IGH (MYC proto-oncogene, bHLH transcription factor- immunoglobulin heavy) in Burkitt lymphoma (BL) and BCR-ABL1 (RhoGEF and GTPase activating protein- ABL proto-oncogene 1, non-receptor tyrosine kinase) in chronic myelogenous leukemia (CML) were designed. DNA from cell lines with these translocations was mixed with oligonucleotides in a single-step ligation reaction followed by PCR amplification. Detection was by capillary electrophoresis.Wealso tested peripheral blood from 16 CML patients and frozen tissue from 17 FL cases, and the results were compared to reverse transcription (RT)- PCR (CML) or fluorescent in situ hybridization (FISH) and δ-PCR (FL). RESULTS: LOLA produced signals of the expected sizes for the cell lines. Normal control DNA yielded no signals. A dilution series yielded translocation-specific peaks at dilutions as low as 1%. Signal intensity was log linear to the DNA concentration (R2 = 0.94). Furthermore, we were able to detect a LOLA peak inDNAfrom 53.3% of FL patients and 87.5% of CML patients. The concordance between LOLA, FISH, and δ-PCR in FL was also excellent. CONCLUSIONS: Our results indicate that LOLA is a simple method that is useful for DNA-based detection of translocations in challenging situations, particularly where the breakpoints are not tightly clustered. The assay also has the added benefit of permitting rapid mapping of the breakpoints.

Original languageEnglish (US)
Pages (from-to)1278-1287
Number of pages10
JournalClinical Chemistry
Volume63
Issue number7
DOIs
StatePublished - Jul 1 2017

Fingerprint

Genetic Translocation
Hematologic Neoplasms
Ligation
Follicular Lymphoma
Assays
Leukemia, Myelogenous, Chronic, BCR-ABL Positive
DNA
Polymerase Chain Reaction
Proto-Oncogenes
Fluorescence In Situ Hybridization
Oligonucleotides
Dilution
Immunoglobulins
Rho Guanine Nucleotide Exchange Factors
Cells
Basic Helix-Loop-Helix Transcription Factors
GTPase-Activating Proteins
Cell Line
Capillary electrophoresis
Burkitt Lymphoma

ASJC Scopus subject areas

  • Clinical Biochemistry
  • Biochemistry, medical

Cite this

Detection of chromosomal translocation in hematologic malignancies by a novel DNA-based Looped Ligation Assay (LOLA). / Harada, Shuko; Sizzle, Emily; Lin, Ming-Tseh; Gocke, Christopher.

In: Clinical Chemistry, Vol. 63, No. 7, 01.07.2017, p. 1278-1287.

Research output: Contribution to journalArticle

@article{40ed5f2ccfb648a887094a25eb5e3a53,
title = "Detection of chromosomal translocation in hematologic malignancies by a novel DNA-based Looped Ligation Assay (LOLA)",
abstract = "BACKGROUND: Disease-defining chromosomal translocations are seen in various neoplasms, especially in lymphomas and leukemias. Translocation detection at the DNA level is often complicated by chromosomal breakpoints that are distributed over very large regions. We have developed a ligation-based assay [the looped ligation assay (LOLA)] to detect translocations from diseases with multiple widely spaced breakpoint hot spots. METHODS: Oligonucleotide sets that probe breakpoints of IGH-BCL2 (immunoglobulin heavy-apoptosis regulator) in follicular lymphoma (FL), MYC-IGH (MYC proto-oncogene, bHLH transcription factor- immunoglobulin heavy) in Burkitt lymphoma (BL) and BCR-ABL1 (RhoGEF and GTPase activating protein- ABL proto-oncogene 1, non-receptor tyrosine kinase) in chronic myelogenous leukemia (CML) were designed. DNA from cell lines with these translocations was mixed with oligonucleotides in a single-step ligation reaction followed by PCR amplification. Detection was by capillary electrophoresis.Wealso tested peripheral blood from 16 CML patients and frozen tissue from 17 FL cases, and the results were compared to reverse transcription (RT)- PCR (CML) or fluorescent in situ hybridization (FISH) and δ-PCR (FL). RESULTS: LOLA produced signals of the expected sizes for the cell lines. Normal control DNA yielded no signals. A dilution series yielded translocation-specific peaks at dilutions as low as 1{\%}. Signal intensity was log linear to the DNA concentration (R2 = 0.94). Furthermore, we were able to detect a LOLA peak inDNAfrom 53.3{\%} of FL patients and 87.5{\%} of CML patients. The concordance between LOLA, FISH, and δ-PCR in FL was also excellent. CONCLUSIONS: Our results indicate that LOLA is a simple method that is useful for DNA-based detection of translocations in challenging situations, particularly where the breakpoints are not tightly clustered. The assay also has the added benefit of permitting rapid mapping of the breakpoints.",
author = "Shuko Harada and Emily Sizzle and Ming-Tseh Lin and Christopher Gocke",
year = "2017",
month = "7",
day = "1",
doi = "10.1373/clinchem.2016.270140",
language = "English (US)",
volume = "63",
pages = "1278--1287",
journal = "Clinical Chemistry",
issn = "0009-9147",
publisher = "American Association for Clinical Chemistry Inc.",
number = "7",

}

TY - JOUR

T1 - Detection of chromosomal translocation in hematologic malignancies by a novel DNA-based Looped Ligation Assay (LOLA)

AU - Harada, Shuko

AU - Sizzle, Emily

AU - Lin, Ming-Tseh

AU - Gocke, Christopher

PY - 2017/7/1

Y1 - 2017/7/1

N2 - BACKGROUND: Disease-defining chromosomal translocations are seen in various neoplasms, especially in lymphomas and leukemias. Translocation detection at the DNA level is often complicated by chromosomal breakpoints that are distributed over very large regions. We have developed a ligation-based assay [the looped ligation assay (LOLA)] to detect translocations from diseases with multiple widely spaced breakpoint hot spots. METHODS: Oligonucleotide sets that probe breakpoints of IGH-BCL2 (immunoglobulin heavy-apoptosis regulator) in follicular lymphoma (FL), MYC-IGH (MYC proto-oncogene, bHLH transcription factor- immunoglobulin heavy) in Burkitt lymphoma (BL) and BCR-ABL1 (RhoGEF and GTPase activating protein- ABL proto-oncogene 1, non-receptor tyrosine kinase) in chronic myelogenous leukemia (CML) were designed. DNA from cell lines with these translocations was mixed with oligonucleotides in a single-step ligation reaction followed by PCR amplification. Detection was by capillary electrophoresis.Wealso tested peripheral blood from 16 CML patients and frozen tissue from 17 FL cases, and the results were compared to reverse transcription (RT)- PCR (CML) or fluorescent in situ hybridization (FISH) and δ-PCR (FL). RESULTS: LOLA produced signals of the expected sizes for the cell lines. Normal control DNA yielded no signals. A dilution series yielded translocation-specific peaks at dilutions as low as 1%. Signal intensity was log linear to the DNA concentration (R2 = 0.94). Furthermore, we were able to detect a LOLA peak inDNAfrom 53.3% of FL patients and 87.5% of CML patients. The concordance between LOLA, FISH, and δ-PCR in FL was also excellent. CONCLUSIONS: Our results indicate that LOLA is a simple method that is useful for DNA-based detection of translocations in challenging situations, particularly where the breakpoints are not tightly clustered. The assay also has the added benefit of permitting rapid mapping of the breakpoints.

AB - BACKGROUND: Disease-defining chromosomal translocations are seen in various neoplasms, especially in lymphomas and leukemias. Translocation detection at the DNA level is often complicated by chromosomal breakpoints that are distributed over very large regions. We have developed a ligation-based assay [the looped ligation assay (LOLA)] to detect translocations from diseases with multiple widely spaced breakpoint hot spots. METHODS: Oligonucleotide sets that probe breakpoints of IGH-BCL2 (immunoglobulin heavy-apoptosis regulator) in follicular lymphoma (FL), MYC-IGH (MYC proto-oncogene, bHLH transcription factor- immunoglobulin heavy) in Burkitt lymphoma (BL) and BCR-ABL1 (RhoGEF and GTPase activating protein- ABL proto-oncogene 1, non-receptor tyrosine kinase) in chronic myelogenous leukemia (CML) were designed. DNA from cell lines with these translocations was mixed with oligonucleotides in a single-step ligation reaction followed by PCR amplification. Detection was by capillary electrophoresis.Wealso tested peripheral blood from 16 CML patients and frozen tissue from 17 FL cases, and the results were compared to reverse transcription (RT)- PCR (CML) or fluorescent in situ hybridization (FISH) and δ-PCR (FL). RESULTS: LOLA produced signals of the expected sizes for the cell lines. Normal control DNA yielded no signals. A dilution series yielded translocation-specific peaks at dilutions as low as 1%. Signal intensity was log linear to the DNA concentration (R2 = 0.94). Furthermore, we were able to detect a LOLA peak inDNAfrom 53.3% of FL patients and 87.5% of CML patients. The concordance between LOLA, FISH, and δ-PCR in FL was also excellent. CONCLUSIONS: Our results indicate that LOLA is a simple method that is useful for DNA-based detection of translocations in challenging situations, particularly where the breakpoints are not tightly clustered. The assay also has the added benefit of permitting rapid mapping of the breakpoints.

UR - http://www.scopus.com/inward/record.url?scp=85021439120&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=85021439120&partnerID=8YFLogxK

U2 - 10.1373/clinchem.2016.270140

DO - 10.1373/clinchem.2016.270140

M3 - Article

C2 - 28515100

AN - SCOPUS:85021439120

VL - 63

SP - 1278

EP - 1287

JO - Clinical Chemistry

JF - Clinical Chemistry

SN - 0009-9147

IS - 7

ER -