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ANALYSIS OF IMMUNOGLOBULIN VARIABLE HEAVY AND LIGHT GENE EXPRESSION IN CHILEAN B-CHRONIC LYMPHOCYTIC LEUKEMIA PATIENTS PA BERTIN, E SANTIBANEZ, C RISUENO, C MELLER, F BARRIGA, G GREBE, AND P LIRA This work was supported by Fondecyt 1920758 and Fondecyt 1940606 INTRODUCTION
    B-Chronic lymphocytic leukemia (B-CLL) is the most prevalent form of leukemia of the western world and accounts for 30% of all the leukemias. B-CLL represents a malignancy of a subset of B lymphocytes that coexpress CD5 and CD19 and/or CD 20 antigens (1-4). Malignant B lymphocytes, like normal B lymphocytes, express surface immunoglobulin. Immunoglobulin
molecules have two identical light chains and two identical heavy chains each with a variable and a constant region. The heavy chain is encoded by four germline gene segments, three for the variable region and one for the constant region. The heavy chain locus is located in chromosome 14q23 and is 2500 kb in length. It contains genes that encode for variable (V H ), diversity (D), joining (J H ) and constant (C H ) regions. The variable region of the heavy chain is encoded by one V H , one D and one J H gene that undergo rearrangement. The human germline V H repertoire consists of approximately 50 functional V H segments. Functional and nonfunctional members of each of the seven V H families are interspersed throughout the locus (5,6). There are two type of light chains: Kappa and Lambda. The locus of the kappa light chain is located in chromosome 2p11-12 comprises a single constant gene (C-kappa), five joining (J-kappa) and approximately 76 variable (V-kappa) genes that belong to four subgroups. The lambda locus is located in chromosome 22q11 and comprises 7 constant genes (C-lambda) each with a joining gene (J-lambda) and over 70 variable genes (V-lambda) that belong to 10 subgroups. Several investigators have reported biased usage of V H families in B-CLL (7-11). Analysis of larger databases demonstrates that V H 3 is the most frequent used fam-ily, probably because of family size (27). There is no database of V L usage in B-CLL. We have studied the V H and V L gene expression of 30 Chilean B-CLL patients on the basis of a PCR method.
Patients
    We studied 30 patients, 16 males and 14 females, with an age range of 48 to 82 years (mean 68). Blood was drawn and a ficoll-hypaque gradient was done to obtain lymphocytes. DNA and RNA were obtained by the guanidintiocyanate method. PCR reactions were carried out with specific primers for each of the V H families and a J H consensus primer. Also PCR reactions were carried out with primers for the different V L subgroups.

    The primers used are as listed.

VH1      5'      C C T C A G T G A A G G T C T C C T G C A A G G       3'
VH2      5'      T C C T G C G C T G G T G A A A G C C A C A C A       3'
VH3      5'      G G T C C C T G A G A C T C T C C T G T G C A G        3'
VH4A   5'      T C G G A G A C C C T G T C C C T C A C C T G C        3'
VH4B   5'      C G C T G T C T C T G G T T A C T C C A T C A G          3'
VH5      5'      G A A A A A G C C C G G G G A G T C T C T G A A       3'
VH6      5'      C C T G T G C C A T C T C C G G G G A C A G T G        3'
JHA       5'      A C C T G A G G A G A C G G T G A C C A G G G T       3'
VK1      5'       T G T C T G C A T C T G T A G G A G A C A G               3'
VK2      5'       T C T C C C T G C C C G T C A C C C T T G G A G       3'
VK3      5'      C C C T G T C T T T G T C T C C A G G G G A                3'
VK4      5'      C C C T G G C T G T G T C T C T G G G C G A G            3'
VK5      5'      G A A A C G A C A C T C A C G C A G T C T C C          3'
VK6      5'      A G T C T G T G A C T C C A A A G G A G                      3'
JKA       5'      T A G T A C C T T G G T C C C T T G G C C G A            3'
VL1       5'      G T G T C T G C G G C C C C A G G A C A G                  3'
VL2       5'      G C C T C C C T G G G A G C C T C G G T C                  3'
VL3       5'      T C C T A T G T G C T G A C T C A G C C                       3'
JLA        5'     G G T C A G C T T G G T C C C T C C G C C G A            3'
PCR conditions
    A reaction volume of 50 ml, with 50mM KCl; 10mM Tris-HCl, pH 8.4; 2.5 mM MgCl2; and 200 mM dNTPs was used. Each primer was 0.5 mM and 0.5 mg DNA and 0.5 U of Taq pol were used. The temperatures of the reaction were 94º C for 4 min for the first cycle, 68ºC for 1.5 min, 94º for 1 min (30 cycles), and 72º for 10 min at the end of the last cycle. Some primers required some variations in temperatures and MgCl 2 concentration. The PCR products were analyzed by agarose gel electrophoresis and visualized using ethidium bromide.
RESULTS
    The PCR products from 30 patients with documented B-CLL are summarized in Table 1.

    The PCR products for the heavy chain were V H 3 in 27 cases, V H 1 in 9 cases and V H 4 in one patient. No PCR products were obtained with primers for the V H 2, V H 5 and V H 6 families. We did not have primers for family V H 7 at the time these patients were studied. In 23 patients just one PCR product was obtained and in 7 patients 2 PCR products were observed. The PCR products for the light chains belong to Kappa in 14 patients and Lambda in 16 patients. The Kappa products were subgroup 1 in 6 cases, subgroup 2 in 3 cases, subgroup 3 in 4 cases and subgroup 4 in two cases. The Lambda products are 5 each for subgroups 1, 2, and 3. In one Lambda patient, we were unable to assign a known subgroup.
DISCUSSION
    We have studied the variable heavy and light immunoglobulin gene expression in 30 Chilean patients with documented B-CLL using a PCR based method. V H gene expression in B-CLL has been analyzed by different investigators by several molecular techniques: Northern blot with specific probes, PCR amplification, cloning and sequencing of PCR products, and monoclonal antibodies against specific gene products. Some investigators agree in the biased expression of V H genes, with preferential use of V H families and certain V H genes (7-13, 21, 22; see also Chapter 9). The normal repertoire of V H genes in normal B lymphocytes has been described (23). As we communicated previously, the distribution of V H gene expression in B-CLL is proportional to family size (24).

    Reports of V L gene expression are few in the literature. Klein and Zachau (14) described 4 V K repertoire subgroups (sg): sg1= 33%, sg2=21%, sg3=38% and sg4=8%. The V K expression in our 30 B-CLL patients has almost a normal distribution. The V L repertoire reported by Blomberg et al., is composed of 10 subgroups; sg1, sg2 and sg3 are the most relevant with 70% of all the V L genes (15). Most of the B cell neoplasia studies to date express one of these 3 subgroups. Our data support that the V L expression in B-CLL is not biased. We could not establish the V L subgroup in one patient because of insufficient sequence data.

    One patient had a Richter’s transformation. From a gastric large cell lymphoma we were able to obtain paraffin embedded tissue and the V H family differed from the original one of the B-CLL cells. With respect to the literature and this observation, there are two hypothesis, a new clone or a clonal evolution (25, 26). Our data suggest that V H and V L expression in B-CLL is proportional to gene expression in normal B cells.
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