Molecular cloning of IGλ rearrangements using long‐distance inverse PCR (LDI‐PCR). (4th December 2012)

• Record Type: Journal Article
• Authors: Shimanuki, Masaya; Sonoki, Takashi; Hosoi, Hiroki; Watanuki, Jyuri; Murata, Shogo; Kawakami, Keiki; Matsuoka, Hiroshi; Hanaoka, Nobuyoshi; Nakakuma, Hideki
• Is Part Of: European journal of haematology. Volume 90:Number 1(2013:Jan.)
• Page Start: 59
• Page End: 67
• Publication Date: 2012-12-04
• Subjects: Hematology -- Periodicals; Blood -- Diseases -- Periodicals; Blood -- Periodicals; 616.15005
• DOI: 10.1111/ejh.12037
• Languages: English
• ISSNs: 0902-4441

Abstract

<abstract abstract-type="main" id="ejh12037-abs-0001"> <title>Abstract</title> <sec id="ejh12037-sec-0001" sec-type="section"> <title>Objectives</title> <p>Malignant cells of mature B‐cell origin show tumor‐specific clonal immunoglobulin gene (<italic>IG</italic>) rearrangements, including <italic>V</italic>(<italic>D</italic>)<italic>J</italic> recombinations, nucleotide mutations, or translocations. Rapid molecular cloning of the breakpoint sequence by long‐distance inverse PCR (LDI‐PCR) has so far been applied to rearrangements targeted to <italic>IGH</italic> joining, <italic>IGH</italic> switch, and <italic>IGκ</italic> regions. We tended to apply LDI‐PCR method for cloning of <italic>IGλ</italic> rearrangements.</p> </sec> <sec id="ejh12037-sec-0002" sec-type="section"> <title>Methods</title> <p>To identify which <italic>IGλ</italic> isotype segment was rearranged, we performed Southern blot analysis using isotype‐specific probes. We set inverse primers on the telomeric side of each joining region and amplified rearranged bands detected by Southern blot analysis as corresponding PCR products.</p> </sec> <sec id="ejh12037-sec-0003" sec-type="section"> <title>Results</title> <p>All germline <italic>IGλ</italic> segments were successfully amplified as expected PCR products. We determined breakpoint sequences of five chromosome translocations involving <italic>IGλ</italic> locus: three novel t(8;22)(q24;q11), one known t(3;22)(q27;q11), and one partially known t(11;22)(q13;q11). Two of the three t(8;22)(q24;q11) were involved in <italic>Jλ</italic> with a recombination signal sequence and one of three in the first exon of <italic>IGLL5</italic>, which lies upstream of <italic>Jλ1</italic>. Three 8q24 breakpoints were widespread at 132, 260 and 366 kb downstream of <italic>MYC</italic> locus. The t(3;22)(q27;q11) showed a juxtaposition of <italic>Jλ2</italic> and the first intron of <italic>BCL6</italic>, as previously reported. In t(11;22)(q13;q11), 3′UTR of <italic>cyclin D1</italic> fused to the constant region of <italic>λ7</italic> with nucleotide mutations. We also amplified four <italic>Vλ/Jλ</italic> recombination sequences.</p> </sec> <sec id="ejh12037-sec-0004" sec-type="section"> <title>Conclusion</title> <p>Our method is a useful tool for molecular analysis of genetic events in <italic>IGλ</italic>.</p> </sec> </abstract>