Rapid Molecular Cloning of Rearrangements of the IGHJ Locus Using Long-Distance Inverse Polymerase Chain Reaction

Willis, Tony G.; Jadayel, D; Coignet, Lionel; Abdul-Rauf, Munah; Treleaven, J.; Catovsky, Daniel; Dyer, Martin J. S.

doi:10.1182/blood.v90.6.2456

Cited by 124 publications

(49 citation statements)

References 38 publications

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“…1). Using the distance between mcr and mbr probes as an internal length standard of 35.8 kb (Willis et al, 1997), the total area covered by the color barcode was estimated to be Ͼ 600 kb, ranging from 160 kb upstream to 250 kb downstream of the BCL2 gene. The distance between the 5Ј BCL2 probe and the mbr probe, which roughly equals the size of the second BCL2 intron, was estimated at Ϯ 225 kb.…”

Section: Construction Of a Physical Map Of The Bcl2 Regionmentioning

confidence: 99%

“…Follicular lymphoma (FL) is strongly associated with the translocation t(14;18)(q32;q21) and molecular rearrangement of the BCL2 oncogene with juxtaposition to the immunoglobulin heavy-chain (IGH) locus. As shown by pulsed-field gel electrophoresis (PFGE) and long-range PCR, about 75% of BCL2 breakpoints are clustered in the major breakpoint region (mbr) and minor cluster region (mcr), while the rest are scattered between these clusters (Zelenetz et al, 1991;Willis et al, 1997;Akasaka et al, 1998) or at the 5Ј side of the BCL2 gene (Tsujimoto et al, 1987;Rimokh et al, 1993;Seite et al, 1993). Approximately 25% of breakpoints are not detected by standard PCR for mbr and mcr.…”

Section: Introductionmentioning

confidence: 99%

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Interphase FISH detection of BCL2 rearrangement in follicular lymphoma using breakpoint‐flanking probes

Vaandrager

Schuuring

Raap

et al. 2000

Genes Chromosom. Cancer

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Rearrangement of the BCL2 gene is an important parameter for the differential diagnosis of non-Hodgkin lymphomas. Although a relatively large proportion of breakpoints is clustered, many are missed by standard PCR. A FISH assay is therefore desired. Up to now, a lack of probes flanking the BCL2 gene has limited the possibilities for a FISH assay to an approach based on colocalization of probes for BCL2 and the immunoglobulin heavy chain (IGH) locus. Intrinsically high rates of false positive nuclei and high interobserver variability make such assays unsuitable for use on lymphoma tissue samples, where tumor cells often form only a minority of the cell population. Using YAC end cloning techniques and screening of a PAC library, we have isolated PAC clones flanking the BCL2 gene. Using these PACs, and several cosmid clones in the second BCL2 intron, we developed a segregation-based interphase FISH assay with two probe combinations enabling separate detection of 5' and 3' (mbr/mcr) breakpoints. The assay was applied to a series of 40 follicular lymphomas. To evaluate the results, the same lymphomas were analyzed by DNA fiber FISH with a 600-kb set of BCL2 DNA clones labeled in alternating colors in combination with a color barcode covering the IGH locus. This approach allowed precise mapping of BCL2 breakpoints, and simultaneously showed juxtaposition of IGH genes to BCL2. Comparison of the results of interphase and fiber FISH showed complete correlation. Five cases were negative with both FISH techniques as well as with Southern blotting. Interestingly, all of these 5 cases lacked BCL2 overexpression as determined by immunohistochemistry, against 3 of 35 rearrangement-positive follicular lymphomas. Furthermore, absence of t(14;18) seemed to be correlated with a higher histologic grade (grades 2 and 3 according to Berard). These data indicate that the segregation-based interphase FISH assay detects 100% of BCL2 rearrangements. Because interpretation of the results is straightforward and requires no extensive experience, this assay may be the best available diagnostic test for BCL2 rearrangement. Genes Chromosomes Cancer 27:85-94, 2000.

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Section: Construction Of a Physical Map Of The Bcl2 Regionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Interphase FISH detection of BCL2 rearrangement in follicular lymphoma using breakpoint‐flanking probes

Vaandrager

Schuuring

Raap

et al. 2000

Genes Chromosom. Cancer

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“…This failure could be because the restriction sites are such that the circle is too large or too small. Other groups have combined IPCR and long PCR to advantage [Willis et al, 1997;Akasaka et al, 2000], which we will also attempt in future studies.…”

Section: Resultsmentioning

confidence: 99%

Use of inverse PCR to amplify and sequence breakpoints of HPRT deletion and translocation mutations

Williams

Rainville

Nicklas

2002

Environ and Mol Mutagen

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Deletion and translocation mutations have been shown to play a significant role in the genesis of many cancers. The hprt gene located at Xq26 is a frequently used marker gene in human mutational studies. In an attempt to better understand potential mutational mechanisms involved in deletions and translocations, inverse PCR (IPCR) methods to amplify and sequence the breakpoints of hprt mutants classified as translocations and large deletions were developed. IPCR involves the digestion of DNA with a restriction enzyme, circularization of the fragments produced, and PCR amplification around the circle with primers oriented in a direction opposite to that of conventional PCR. The use of this technique allows amplification into an unknown region, in this case through the hprt breakpoint into the unknown joined sequence. Through the use of this procedure, two translocation, one inversion, and two external deletion hprt breakpoint sequences were isolated and sequenced. The isolated IPCR products range in size from 0.4 to 1.8 kb, and were amplified from circles ranging in size from 0.6 to 7.7 kb. We have shown that inverse PCR is useful to sequence translocation and large deletion mutant breakpoints in the hprt gene.

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“…Inverse PCR. The digestion, ligation and purification of the circularized DNA template and sequences of the IgH primers for inverse PCR are described elsewhere (Willis et al, 1997). One microlitre of ligation product (10 ng) was amplified using the long-range PCR technique described above.…”

Section: Patientsmentioning

confidence: 99%

JH probe real‐time quantitative polymerase chain reaction assay for Bcl‐2/IgH rearrangements

Jenner¹,

Summers²,

Norton³

et al. 2002

Br J Haematol

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Summary. Follicular lymphoma (FL) characteristically bears the t(14;18)(q32;q21). However, only 75% of the consequent Bcl-2 breakpoints lie within the major breakpoint region (MBR) or the minor cluster region (mcr). While these can be quantified by cluster region-specific real-time quantitative polymerase chain reaction (RQ-PCR), a significant proportion of cases are left requiring a customized approach. Therefore, an RQ-PCR assay for the quantification of Bcl-2/IgH breakpoints has been developed that uses germline JH TaqMan probes and germline JH primers in combination with customized forward primers. Validation of this approach by comparison with an established MBR RQ-PCR showed both techniques to be concordant across a wide range of copy numbers with a sensitivity of five copies per 10 5 cells. In addition, to generate standard curves equating to diverse Bcl-2/IgH rearrangements, a strategy for using placental DNA as a surrogate standard was devised. The performance of the assay in detecting molecular evidence of disease in sequential biopsies from five patients (three with atypical Bcl-2/IgH breakpoints identified by long-range or inverse PCR, one MBR + and one mcr + ) was tested. This alternative approach represents a sensitive and specific means of quantifying common and atypical Bcl-2/ IgH rearrangements and maximizes the number of patients with FL suitable for molecular monitoring.

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Rapid Molecular Cloning of Rearrangements of the IGHJ Locus Using Long-Distance Inverse Polymerase Chain Reaction

Cited by 124 publications

References 38 publications

Interphase FISH detection of BCL2 rearrangement in follicular lymphoma using breakpoint‐flanking probes

Interphase FISH detection of BCL2 rearrangement in follicular lymphoma using breakpoint‐flanking probes

Use of inverse PCR to amplify and sequence breakpoints of HPRT deletion and translocation mutations

JH probe real‐time quantitative polymerase chain reaction assay for Bcl‐2/IgH rearrangements

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