Molecular Genetic Basis for Immunoglobulin Gene Rearrangements

Humoral immunity is achieved by the cognitive and effector functions provided by B lymphocytes. Immunoglobulins are produced exclusively by B lymphocytes and constitute the hallmark of the humoral response. Ig molecules are heterodimeric polypeptides composed of two identical 50 to 70 kDa heavy chains, associated with two identical light chains, kappa (κ) or lambda (λ) of approximately 23 kDa. The Ig chains are linked by noncovalent forces and interchain disulfide bridges, and together form a bilaterally symmetric structure. The heavy and light chain proteins encoded by their corresponding genes are composed of a tandem series of homologous segments consisting of approximately 110 amino acid residues. These segments undergo folding into 12 kDa domains with single intrachain disulfide bridges. Heavy chains contain one variable (V) and three constant (C) domains; light chains contain one N-terminal V and one C domain. In both heavy and light chains, the V domain consists of four relatively invariable framework regions of 15 to 30 amino acids separated by three 9 to 12 amino acid–long hypervariable or complementarity determining regions (CDRs). In both heavy and light chains, the CDR3 region is the farthest away from the N-terminus, and it exhibits the greatest sequence variability among the three CDRs. The C-terminal region of secreted Ig molecules binds to complement components and Fc receptors. The genes encoding the Ig proteins are the target of DNA-based studies for clonality assessment.

The organization of Ig genes in the germline is fundamentally similar in all species studied. Genes encoding the two light chains, (κ and λ), and the single locus containing the various heavy chain genes are located on different chromosomes. To generate the large number of antibody molecules needed, B cells have evolved a unique strategy that involves recombination of germline-encoded immunoglobulin gene segments and a somatic hypermutation mechanism that further diversifies the reactivities of the rearranged antigen receptor molecules.¹⁴⁰ The immunoglobulin heavy and light chain gene loci—IgH, Igκ, and Igλ—are located at chromosomes 14q32, 2p12, and 22q11, respectively. In the germline configuration, all antigen receptor genes are composed of discontinuous DNA segments that are referred to as variable (V), diversity (D), joining (J), and constant (C) regions. Although both Ig heavy and light chain genes contain V, J, and C regions, only the IgH genes contain D regions (Figure 44-1). The IgH gene contains approximately 87 VH, 30 DH, and 6 JH segments. The IgH VH segment genes can be categorized into seven families, based on relatedness of the DNA sequences. The ability of Ig genes to undergo recombination in a manner analogous to shuffling of cards affords the capacity to generate an enormous primary repertoire of 10⁹ Ig molecules (Figure 44-2). The human Ig C region contains 11 C region segments, which define nine functional immunoglobulin classes and subclasses (IgM, IgD, IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, and IgE). The C region genes are initially uninvolved in the rearrangement process, but are subsequently juxtaposed to the VDJ complex during RNA splicing. Incorporation of C regions into the rearranged VDJ recombination permits class switching. Thus, the specific V(D)J rearrangement can be expressed as IgG, IgA, or other Ig classes. Following the formation of a productive IgH rearrangement, the light chain loci undergo a similar recombination process involving the joining of V to J segments, because no D segments are present in the light chain loci.²⁸

The Igκ light chain gene is located on chromosome 2p11-13 and contains up to 1000 Vκ genes, five Jκ segments, and one Cκ gene. A κ-deleting sequence (element) is present 3′ to the Cκ region. In Igλ-expressing B cells in which the Igκ genes are not productively rearranged, the κ-deleting element undergoes rearrangement such that the Cκ locus is deleted. The Igλ light chain is situated on chromosome 22q11 and is composed of several Vλ segments with Jλ and Cλ segments arranged in tandem. The Cλ locus is polymorphic and may be present in one to nine copies per allele in different individuals. The hierarchy of the rearrangement events is such that both alleles of the heavy chain rearrange prior to the light chain genes, and the kappa (κ) light chain locus is productively rearranged before (in most cases, but not always) the λ light chain genes. Thus, the immunoglobulin lambda (λ) light chain gene is not favored for clonality analysis because it frequently rearranges after the κ light chain gene. In addition, because most clonal rearrangements of the immunoglobulin heavy chain gene can be detected in up to 95% of clonal processes, it has become customary to perform only Southern blot analysis for the IgH locus in many laboratories.

Molecular Genetic Basis for T-Cell Receptor Gene Rearrangements

A similar process as described for the Ig genes occurs in the T-cell receptor (TCR) genes, with the notable exception of the absence of a somatic hypermutation mechanism in T-cell receptor gene rearrangements. The developmental hierarchy of T-cell receptor genes is such that the TCR-δ gene is the first to rearrange, followed by the TCRγ, TCRβ, and then the TCRα genes.²⁶ The TCR-δ gene is located on chromosome 14q11 and contains six or fewer Vδ segments, only two or three Dδ segments, and three Jδ segments (Figure 44-3). It is located within the TCR-α gene, immediately 3′ to the last TCR-Vα gene and 5′ to the TCR-Jα segments. This location is important in that productive rearrangements of the TCRδ gene lead to expression of the γ/δ TCR. In the event that the TCRδ rearrangement is nonproductive, Vα to Jα rearrangements obligatorily require that the TCRδ be deleted. The TCRα gene is located on band 14q11 and flanks the TCRδ gene on either side; it contains several Vα segments, an extensive J region composed of up to 50 Jα genes, and one Cα locus. By virtue of the architecture of the TCRα and δ loci, rearrangement of a Vα segment to a Jα segment typically leads to deletion of the intervening TCRδ loci. T cells undergoing such rearrangements may go on to express the TCR-α/β protein. The TCRβ gene is located on band 7q34 and contains 75 to 100 Vβ segments, flanked at the 3′ end by two Dβ, Jβ, and Cβ regions. Each region consists of one Dβ segment, with six or seven Jβ segments and one Cβ segment. Rearrangements may occur within or between segments (e.g., Vβ1 to Jβ1, Vβ2 to Jβ1). Extensive sequence homology is noted between the Cβ1 and Cβ2 segments. This phenomenon is exploited in the utilization of a consensus Cβ probe in the Southern blotting–based clonality assessment of TCR genes. The TCRγ gene is located on band 7q15 and contains about 11 Vγ segments and two Jγ genes.²⁴ The simple structure of the architecture of this gene favors its utilization as a target for TCR receptor PCR, where consensus primers are designed to encompass the relatively few Vγ and the Jγ genes.

Southern Blot Hybridization Analysis for Antigen Receptor Gene Rearrangements

In this procedure, high-quality total cellular DNA extracted from a fresh or fresh-frozen specimen is subjected to digestion using different bacterial restriction endonucleases, which produce DNA fragments of different sizes encompassing the Ig or TCR gene region segments to be interrogated (Figures 44-4 and 44-5). Enzyme-restricted DNA from each enzyme is subjected to gel electrophoresis and is transferred by blotting and immobilized on a membrane. A labeled probe complementary to the Ig J region segment is hybridized to the membrane. Clonal rearrangements are recognized by the identification of one or two novel rearranged bands that are distinct from the germline pattern obtained with a benign sample or placental DNA. One or two novel rearranged (nongermline) bands may be identifiable, depending on whether a clonal monoallelic or biallelic rearrangement is present (Figure 44-6).

Material suitable for Southern blot analysis includes fresh tissue or aspiration biopsy material, as long as sufficient cellular material can be obtained from the specimen of interest. Ethanol-fixed tissue can also be successfully utilized. However, formalin-fixed tissue yields DNA of insufficient quality to permit reliable Southern blotting because of cross-linking of proteins to DNA. A Southern blot hybridization assay using three enzymes requires approximately 1.5 × 10⁷ cells (approximately 15 µg of intact total cellular DNA). Hypothetically, all of the antigen receptor genes could be utilized as potential targets for assessment of clonality status. However, only the immunoglobulin heavy and light chains and the T-cell receptor β-chain loci have gained widespread use for clonality testing. With regard to the other T-cell loci, the TCR-α locus is impractical because it is composed of several J segments that are distributed widely apart, and it would pose practical challenges in the optimization of assays for multiple probes. Evaluation of TCRγ and δ loci is not favored because they have only a limited number of V segments, such that even reactive T-cell populations may yield confusing nongermline bands.

The diagnostic hallmark of a clonal population is the presence a nongermline novel rearranged band (see Figure 44-6). However, it is important to recognize that under certain circumstances, nongermline bands do not indicate monoclonality. For example, single-nucleotide polymorphisms (SNPs) may create or abolish restriction enzyme recognition sites, thus yielding fragments with novel restriction banding patterns. In this scenario, utilization of a different enzyme would yield a germline configuration. The potential of these SNPs to confound the interpretation of Southern blot studies justifies the utilization of up to three or more enzymes for the unequivocal assignment of monoclonality. A second situation in which a nongermline band may occur and yet not indicate monoclonality is partial digestion. This is typically evident in the control sample (placental DNA) and invalidates the significance of any such band in the test sample. Clonal rearrangements of the Ig or TCR genes have been used to ascertain the lineage of hematologic neoplasms that do not express B- or T-cell specific markers. Intuitively, clonal rearrangements of Ig genes would indicate B-cell processes, and clonal rearrangements of TCR genes would indicate T-cell processes. However, some malignant lymphomas and leukemias may demonstrate both Ig and TCR rearrangements. In particular, acute leukemias and lymphoblastic lymphomas may demonstrate both Ig and TCR gene rearrangements. Further, up to 20% of acute myeloid leukemias may demonstrate rearrangements of Ig or TCR genes.^20,59,73 Hence, lineage is best assigned using a combination of immunophenotypical and molecular studies.

Polymerase Chain Reaction Analysis of Antigen Receptor Gene Rearrangements

Although Southern blot hybridization analysis for detection of antigen receptor gene rearrangements is recognized as the “gold standard” for assessment of clonality, PCR-based assays have become the mainstay for the detection of rearrangements of Ig and TCR genes in many molecular diagnostic laboratories. This is because SBH is more labor-intensive and takes longer to complete. Additionally, SBH requires a substantial quantity of intact DNA and hence is less amenable to specimens with suboptimal DNA quality (as is the case with fixed paraffin-embedded tissue samples). PCR on the other hand is well suited for the utilization of such specimens, which constitute the majority of samples analyzed in the clinical laboratory.

Application of PCR for the identification of clonality entails the utilization of consensus V region and J region primers and in vitro amplification of DNA across the V(D)J junction, followed by gel electrophoresis or other methods to determine the size distribution of PCR-generated fragments. In the germline configuration, V and J region segments are located several kilobases apart and are not amplifiable. However, the V(D)J recombination brings the VDJ segments into close proximity, thereby permitting amplification of products that, based on primer location, are usually 80 to 350 bases in length. The V region primers typically are designed to be complementary to the conserved framework (I, II, or III), and the J region primer is typically complementary to consensus sequences present across all six J regions in the immunoglobulin heavy chain gene. Similar strategies may be used for light chain genes as well, but the yield is lower; hence, IgH-PCR is favored. Utilization of framework III consensus primers offers the highest yield but can be complemented by increased diagnostic sensitivity afforded by the inclusion of framework II and/or framework I primer-mediated IgH-PCR for clonality. With regard to T-cell assays, the TCRγ-PCR is the most informative. In these assays, monoclonal populations yield one or two dominant bands (Figure 44-7), while polyclonal cells, each of which carries a unique rearrangement with slightly different band sizes, demonstrate a ladder or smear pattern on gel electrophoresis.^81,130 Thus the specificity of each rearrangement identified by PCR is determined by the junctional sequence; hence the marker of clonality in PCR differs from that in SBH analysis, wherein the marker is the configuration of rearranged antigen receptor genes.

Following PCR, the amplification products are analyzed by agarose or polyacrylamide gel electrophoresis and are visualized by ethidium bromide staining.38,138 Agarose gels, although less expensive, do not provide resolution of polyacrylamide, which can be configured to reliably discriminate bands differing in size by as few as three bases. This is often very useful in the assessment of PCR products for antigen receptor gene rearrangement assays. Further resolution may be achieved by evaluating sequence-specific and melting characteristics of double-stranded DNA using denaturation gradient gel electrophoresis or temperature gradient gel electrophoresis.⁵⁵ More recently, capillary gel electrophoresis has become a preferred method for analysis of antigen receptor gene rearrangement. A successful study would exhibit PCR products in the expected size range, appropriate results in positive and negative controls, and absence of bands in the template-free (H₂O) control.

PCR-based clonality assays are less sensitive in detecting all possible clonal rearrangements when compared with SBH analysis, which approaches 100% if sufficient restriction enzymes and a variety of probes are used. Depending on the assay and the lymphoid neoplasm tested, PCR-based clonality studies may show false-negative rates from 10 to 40% when compared with SBH. False negatives occur for several reasons. First, in contrast to SBH, PCR does not detect partial DJ rearrangements, wherein V and J region genes are not approximated to one another and are too distant for conventional PCR to generate an amplification product. Second, the consensus primers are incapable of hybridizing to all of the different V regions, any one of which could be potentially present in any single neoplasm. Of particular importance to the Ig genes, somatic mutations occurring within the Ig V region genes result in decreased ability for the consensus V region primers to anneal optimally to the V region genes in the template DNA. Thus lymphoid neoplasias with a high somatic hypermutation rate, such as follicular lymphoma, yield lower positive rates by PCR-based Ig clonality tests.125,126 Most of the difficulties described previously can be overcome by the utilization of multiple primer sets. In the event of a negative PCR result, SBH analysis may be performed if sufficient high-quality genomic DNA is available. The main drawback for PCR is the propensity for contamination. Various strategies for contamination control such as maintaining separate DNA extraction and amplification rooms, utilization of laminar flow hoods, and incorporation of uracil DNA glycosylase into PCR should be employed as a rule to prevent contamination problems.

The ability to detect rare DNA species carrying a specific molecular aberration (sensitivity) of PCR is dependent on the particular application. For antigen receptor gene rearrangement studies, PCR can detect a monoclonal population in a background of 10² to 10³ polyclonal cells of the same phenotype (B or T cells). By comparison, PCR can detect one cell harboring a chromosomal translocation within a background of 10⁵ to 10⁶cells negative for the translocation.⁹²

Southern Blot Hybridization Analysis for the Detection of Chromosomal Translocations

Southern blot hybridization analysis is an excellent method for the detection of chromosomal translocations. A sequence-specific probe to the gene of interest is utilized for SBH analysis, as described for the antigen receptor genes. Detection of a pattern of bands of different sizes from those seen in the germline configuration of the gene suggests the presence of a translocation. This approach method does not, however, identify the translocation partner of the gene of interest. As with the antigen receptor gene rearrangement studies by SBH, SNPs at enzyme restriction sites may result in nongermline patterns that may be misinterpreted as translocations. In this scenario, utilization of a different restriction enzyme will correctly show the gene locus to be of germline size. The labor-intensive nature of SBH and the requirement for high-quality DNA have diminished its popularity for routine detection of chromosomal translocations in the clinical laboratory. In this regard, PCR continues to gain in utilization for detection of recurrent chromosomal translocations.

Polymerase Chain Reaction Analysis for Detection of Chromosomal Translocations

PCR is well suited for analysis of chromosomal translocations, particularly when the translocation breakpoints are clustered and the sequences flanking the translocation breakpoints are well characterized.

Recurrent Chromosomal Translocations in Acute Myeloid Leukemias

The translocations that occur in the acute myeloid leukemias target transcription factors or transcriptional coactivators. The transcription factors are involved in the differentiation of hematopoietic cells, and disruption of their normal function by the translocation leads to developmental arrest and maturational block at immature stages of differentiation. Targeted transcription factors include the retinoic acid receptor alpha (RARα) in acute promyelocytic leukemia (APL) and several members of the core binding factor (CBF) complex. Core binding factor is a transcription factor composed of heterodimers including a DNA-binding component known as AML1 (RUNX1/CBFA2/PEBP2A) and a subunit CBFβ that activates the transcription of AML1. CBF is targeted in the t(8;21)⁴³ aberration, which results in the AML/ETO fusion characteristic of acute myelogenous leukemia (AML) with differentiation (AML FAB-M2). CBF is similarly targeted in the inv(16), which leads to generation of a fusion gene (CBFβ/SMMHC) comprised of CBFβ and the smooth muscle myosin heavy chain gene (SMMHC) found in a subset of AML4Eo.⁸⁸ The multilineage importance of CBF is manifest in a subset of precursor B-cell acute lymphoblastic leukemias, where it is targeted in the t(12;21), which leads to the TEL/AML1 fusion.⁵⁴ All chimeric proteins that result from chimeric fusions are dominant negative inhibitors of CBF-mediated transcription.^49,62 The transcriptional repression of CBF target genes caused by CBF-chimeric proteins partially involves recruitment of the histone deacetylase complex.

By comparison, APL is characterized by translocations involving the RARα gene (see Table 44-3). These translocations result in a block of myeloid differentiation at the promyelocytic stage. It has been shown that maturational arrest is due in part to recruitment of the nuclear corepressor–histone deacetylase complex. The ability of all-trans retinoic acid (ATRA) to bind to PML/RARα, resulting in release of the nuclear corepressor complex, explains in part the ability of ATRA to relieve the maturational block in promyelocytes central to the development of APL.

t(8;21)(q22;q22)—AML1-ETO

The t(8;21)(q22;q22) was originally described in a case of acute leukemia by Janet Rowley in 1973.¹¹³ The translocation is found in approximately 7% of cases of de novo AML and is more common in young individuals. The translocation is found in 20 to 40% of AMLs of the FAB-M2 subtype.³ It has been reported infrequently in AML FAB-M1, AML FAB-M4, and rare cases of therapy-associated AML. The translocation leads to fusion of the AML1 gene (acute myeloid leukemia 1 gene), also known as CBFA2 (core binding factor subunit A2) or PEBP2α (polyoma enhancer binding protein 2 subunit α), to the ETO gene (8;21), also known as MTG8 (myeloid translocation gene on chromosome 8).

The t(8;21) portends a good prognosis in cases of de novo AML, with a favorable response to treatment with cytosine arabinoside.⁹⁸ The AML1-ETO chimeric fusion is detectable by RT-PCR in the vast majority of t(8;21)-positive AMLs, thus making RT-PCR a good choice for detection of the t(8;21) in clinical samples. The AML1-ETO fusions join exon 5 of AML1 to exon 2 of ETO (Figure 44-11), and the fusion transcript can be detected in complex translocations in a significant proportion of cases that are t(8;21) negative by conventional cytogenetics.^3,94 Specifically, RT-PCR has been reported to detect AML1–ETO fusions in 8 to 12% of AML.^3,79,94 It is interesting to note that the AML1–ETO fusion transcript may be detected using sensitive end point PCR assays several years after chemotherapy or bone marrow transplant (BMT), thus limiting its predictive value for relapse in affected patients.⁷⁰ Quantitative studies using recently developed real-time PCR protocols (Figure 44-12) provide a better indication of increasing transcript copies and hence disease recurrence.

Figure 44-11 Schematic representation of the organization of the AML1 (21q22) and ETO genes (8q22) involved in the t(8;21) anomaly that is characteristic of FAB AML-M2. The centromeric (cen) and telomeric (tel) directions are indicated. In A, the exons of the AML1 gene are depicted in red rectangles; the exons of the ETO gene are depicted in white rectangles. In B, the configuration of the AML1/ETO chimeric fusion is shown. The numbers below the fusion transcript indicate the position of the first nucleotide in the exon involved, or the last nucleotide of the exon immediately 5′ to the fusion.

Only gold members can continue reading. Log In or Register to continue

Share this:

• Click to share on Twitter (Opens in new window)
• Click to share on Facebook (Opens in new window)

Like this:

Like Loading...

Related

Related posts:

1. Toxic Metals
2. Microfabrication and Microfluidics and Their Application to Clinical Diagnostics
3. Establishment and Use of Reference Values
4. Vitamins and Trace Elements

Stay updated, free articles. Join our Telegram channel

Join