Molecular Cloning of Immunoglobulin Heavy Chain Gene Translocations by Long Distance Inverse PCR

B-cell functions as a key player in the humoral immunity producing immunoglobulin protein in mammalian. To produce wide-ranged and antigen-specific antibody, the B-cell undergoes genetic rearrangement of immunoglobulin genes during its maturation process. The genetic rearrangements in immunoglobulin genes require unstable steps for genome; breakage and re-ligation of the double strand DNA. In the unstable steps, misconduct can be occurred; leading chromosome translocations involving immunoglobulin genes. Chromosome translocations involving the immunoglobulin heavy chain (IGH) locus on chromosome 14q32 are found in various B-lymphoid malignancies, including B-cell precursor lymphoblastic leukemia (BCP-ALL), B-cell non-Hodgkin's lymphoma (B-NHL), and myeloma. Chromosome translocations involving IGH locus are often associated to disease entities among the B-lymphoid malignancies; thus, identification of certain translocation is clinically important information for definitive diagnosis. Moreover, molecular cloning of the IGH translocation breakpoint allows the identification of genes of physiologically or pathologically importance in B-cells (Willis and Dyer, 2000; Küppers and Dalla-Favera, 2001; Siebert et. al., 2001). The consequence of IGH translocations is the deregulated expression of the target gene controlled by potent B-cell-specific transcriptional enhancers within the IGH locus, resulting from physically close apposition with the IGH locus. The vast majority of target genes of IGH translocations play a fundamental role in Bcell biology, such as cell growth, differentiation, apoptosis and signal transduction (Wills and Dyer, 2000). Thus, deregulated expression of those genes alters B-cell fate and may initiate malignant transformation of the affected B-cell. To date, many IGH translocation breakpoints have been molecularly cloned and the target genes have been identified; however, several recurrent breakpoints remain to be cloned (Heim and Miltelman, 1995). Molecular cloning of IGH translocation breakpoints would reveal the involvement of either genes of unknown biological functions or the unsuspected oncogenic potential of known genes. Moreover, some target genes of IGH translocation have been found to be deregulated by gene amplification or unknown genetic mechanisms rather than IGH translocation, thereby contributing to disease development or progression (Dyer et. al, 2010).


Introduction
B-cell functions as a key player in the humoral immunity producing immunoglobulin protein in mammalian.To produce wide-ranged and antigen-specific antibody, the B-cell undergoes genetic rearrangement of immunoglobulin genes during its maturation process.The genetic rearrangements in immunoglobulin genes require unstable steps for genome; breakage and re-ligation of the double strand DNA.In the unstable steps, misconduct can be occurred; leading chromosome translocations involving immunoglobulin genes.Chromosome translocations involving the immunoglobulin heavy chain (IGH) locus on chromosome 14q32 are found in various B-lymphoid malignancies, including B-cell precursor lymphoblastic leukemia (BCP-ALL), B-cell non-Hodgkin's lymphoma (B-NHL), and myeloma.Chromosome translocations involving IGH locus are often associated to disease entities among the B-lymphoid malignancies; thus, identification of certain translocation is clinically important information for definitive diagnosis.Moreover, molecular cloning of the IGH translocation breakpoint allows the identification of genes of physiologically or pathologically importance in B-cells (Willis and Dyer, 2000;Küppers and Dalla-Favera, 2001;Siebert et. al., 2001).The consequence of IGH translocations is the deregulated expression of the target gene controlled by potent B-cell-specific transcriptional enhancers within the IGH locus, resulting from physically close apposition with the IGH locus.The vast majority of target genes of IGH translocations play a fundamental role in Bcell biology, such as cell growth, differentiation, apoptosis and signal transduction (Wills and Dyer, 2000).Thus, deregulated expression of those genes alters B-cell fate and may initiate malignant transformation of the affected B-cell.To date, many IGH translocation breakpoints have been molecularly cloned and the target genes have been identified; however, several recurrent breakpoints remain to be cloned (Heim and Miltelman, 1995).Molecular cloning of IGH translocation breakpoints would reveal the involvement of either genes of unknown biological functions or the unsuspected oncogenic potential of known genes.Moreover, some target genes of IGH translocation have been found to be deregulated by gene amplification or unknown genetic mechanisms rather than IGH translocation, thereby contributing to disease development or progression (Dyer et. al, 2010).

Structure of IGH and its genetic modifications during B-cell differentiation
The human IGH locus consists of 51 functional variable (V) segments, 27 diverse (D) segments, six joining (J) segments and nine constant (C) segments in noncontiguous fashion starting from the telomeric end of 14q32 and spans 1.4 megabases (Mb) (Honjo and Alt, 1995) (Fig. 1).Each C segment, except Cδ, is preceded by repetitive DNA sequences named switch (S) segments, which play a role in class switch recombination, as discussed below.
During the B-cell maturation process, the IGH gene undergoes three major genetic modifications: VDJ recombination, somatic hypermutation, and class switch recombination.Generally, the former occurs in the bone marrow, whereas the latter two occur in the germinal center of the lymph nodes.As a consequence of these genetic modifications, the IGH gene can produce part of the immunoglobulin protein (antibody), which can bind to a specific antigen with distinct effecter functions.Each B-progenitor cell forms a unique VDJ complex by selecting one V, one D, and one J segment randomly, and this process is called VDJ recombination.The VDJ complex therefore encodes part of the variable domain of immunoglobulin heavy chain protein.After encountering an antigen in the germinal center of a lymph node, VDJ complex is further introduced by nucleotide substitution, deletion or duplication, a process called somatic hypermutation, resulting in increased affinity with the respective antigen.In the germinal center, some B-cells exchange constant segments from Cµ to others by the class switch recombination process, which results in the exchange effecter domain of immunoglobulin protein (Fc-domain) retaining the antigen specific binding domain.This process involves the recombination of S regions preceding the Cµ segment and other C segment, and the intervening DNA segment is looped out.When class switching from IgM to IgG1 occurs, for example, Sµ and Sγ1 are recombined with deletion of the intervening DNA, thereafter, Cγ1 segment positions at 3' flanking the VDJ complex thus produce VDJ-Cγ1 transcript (Figure 1).All VDJ recombination, somatic hypermutation and class switch recombination involve DNA double-strand cleavage and re-ligation; errors in each process may result in chromosome translocation targeted to the IGH locus (Küppers and Dalla-Favera, 2001).The IGH translocations seen in B-cell malignancies commonly take place in either the joining (IGHJ) or the switch (IGHS) segments.

Disease entities and IGH translocations
IGH translocation is a common genetic aberration in B-cell malignancies; however, the incidence various according to the disease entity.Precursor B-cell acute lymphoblastic leukemia shows a low frequency of IGH translocation, but recent studies have revealed diverse target genes of IGH translocation in this disease (Dyer et. al., 2010).Examples include cytokine receptor genes (Russell et.. al., 2009), the CCAAT enhancer-binding protein gene family (Chapiro et. al. 2006;Akasaka et. al., 2007), and microRNA 125b-1 (Sonoki et. al 2005;Chapiro et al, 2010, Tassano et al., 2010).Non-Hodgkin lymphoma often shows specific IGH translocations corresponding to the disease entity.t(14;18)(q32;q21) is seen in ~85% of follicular lymphoma and t(8;14)(q24;q32) is seen in ~90% of Burkitt lymphoma.Myeloma, which arises from terminal differentiated B-cells, shows extensive IGH translocation; however, the IGH translocation seen in myeloma defines distinct molecular subtypes of myeloma and is associated with clinical behavior (Bergsagel and Kuehl, 2005).The vast majority of IGH translocation breakpoints are clustered in joining or switch regions.Since most IGH translocation results from physiological genetic modification errors, the breakpoints correspond to the differentiation stage of the tumor cells.IGH translocation breakpoints seen in B-cell precursor acute lymphoblastic leukemia are clustered in the joining region, whereas those seen in myeloma are clustered in the switch region.In B-NHL, IGH translations are seen in joining as well as switch regions.Follicular lymphoma and Mantle cell lymphoma show breakpoints in the joining region of IGH breakpoints suggesting that IGH translocations occur in the early stage of B-cell differentiation in bone marrow.Diffuse large B-cell lymphoma shows switch region breakpoints, suggesting that the translocation occurs in the late stage of B-cell differentiation in lymph nodes.

Molecular cloning of IGH translocation breakpoints using long distance inverse-PCR
Inverse PCR is a useful method to define unknown nucleotide alignments flanking known nucleotide sequences, such as retroviral DNA integration sites into host chromosome DNA (Takemoto et. al. 1994).The principle of inverse PCR uses self-ligated circular DNA as a PCR template and primers setting in a known sequence in the opposite direction.Inverse PCR is easily applied for determination of unknown partner sequence combined with IGH resulting from chromosome translocation.After thermostable DNA polymerase that can amplify long target DNA is available, the combination of inverse PCR and long target thermostable DNA polymerase (long distance inverse PCR; LDI-PCR) is a powerful tool for molecular cloning of an unknown sequence from IGH.Molecular cloning by LDI-PCR requires a small amount of genomic DNA and only couple of days to determine the translocation breakpoints involving IGH translocation.Using this method, many novel partner genes have been identified as listed in Table 1.
Since PCR may yield non-specific products, Southern blot analysis using IGH-specific probes is informative to interpret LDI-PCR results.The LDI-PCR product can be estimated by rearranging the band detected in Southern blot analysis; however, cloning of IGHJ translocations is simple and clear, and can be performed without reference to Southern blot data (Willis et al. 1997;Akasaka et al., 2007).In contrast, IGHS might undergo complex rearrangements, including insertion, deletion, and inversion (Bergsagel et. al. 1999) To distinguish translocation or other rearrangements, Southern blot assays using several probes are required.Thus, it is necessary to have large amounts of high-molecular-weight DNA available to allow Southern blot analysis before performing LDI-PCR for switch regions (Sonoki et al, 2001).
The detailed protocol of LDI-PCR for molecular cloning of the immunoglobulin heavy chain gene has been published elsewhere (Karran et al. 2005).A brief principle is shown in Figure 2.After complete digestion of high molecular genomic DNA with the restriction enzyme, the DNA is reacted with T4-ligase at low DNA concentration to form self-ligated circular DNA.The circular DNA is then purified by a silica-based column and subjected to PCR using primer pairs set at the IGH sequence.When the PCR product of interest is obtained, the nucleotide alignment can be determined directly.Although genomic DNA can be isolated from paraffin-embedded materials, the DNA is fragmented, and thus is not suitable for LDI-PCR.LDI-PCR allows the rapid cloning of vast majority of translocations targeting IGH in B-cell malignancies.The benefits of LDI-PCR are first the speed and ease with which this cloning can now be performed and second that cloning can be performed when only small amounts of material are available (Willis TG, et al. 1997).The principle for LDI-PCR can be applied for other cloning purpose than IGH translocations.

Fig. 1 .
Fig. 1.A, Schematic diagram of human IGH structure.The coding exons lie on 14q32 as noncontiguous fashion.There are three potent enhancers within the IGH locus (Eµ and two 3'E).B, Schematic diagram of functionally rearranged IGH allele.In this schema, VDJ recombination and switch recombination of IgM to IgG1 are shown.Note Sµ and Sγ1 are combined.C, Schematic diagram of IGH translocation involving Joining region.The target gene on the partner chromosome is deregulated by Eµ enhancer.D, Schematic diagram of IGH translocation involving Switch region.The target gene on the partner chromosome is deregulated by 3'E enhancer.

Fig. 2 .
Fig. 2. Schematic diagram of LDI-PCR for cloning IGH translocation breakpoint.Using long distant DNA polymerase, 4~5 kb PCR products are able to be amplified routinely.E; restriction enzyme site, star; translocation breakpoint.

Table 1 .
Table 1 summarizes known partner genes of IGH translocation seen in B-cell malignancies.Disease entity and IGH chromosome translocation.*, Genes identified by LDI-PCR.