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A NOVEL TECHNIQUE TO ANALYZE THE RELATIVE CONCENTRATION OF THE EXPRESSED IMMUNOGLOBULIN (IG) VH GENES   L RASSENTI AND TJ KIPPS   [Adapted from an original report by Rassenti et al. (1)]   INTRODUCTION  
    B-cells are lymphocytes that have rearranged certain genes in order to produce the light and heavy chain peptides that comprise antibody molecules, or immunoglobulins (Ig) (see Bauer, Chapter 4). Although there are about 100 V H genes in the human haploid genome, they may be grouped into 7 subgroups based on similarities in their DNA nucleotide sequences (and the
resulting amino acid sequences of the V H peptides they produce) (2,3,4,5,6).  Previous studies have shown that these V H subgroups are not used randomly in the human adult B-cell repertoire (6,7,8). A number of genetic and somatic mechanisms account for this non-stochastic use (see 1).
 
    Conventional methods for analyzing the Ig V H gene repertoire have relied on methods that are cumbersome and not readily suited for serial or large-scale population studies. A rapid and reproducible method for analysis of the Ig repertoire would allow application in such studies.
Therefore, we have developed a rapid, nonradioactive method to analyze the relative concentration of the messenger RNA (mRNA) from each of these subgroups and applied this method to peripheral blood lymphocytes from normal and B-CLL samples. This chapter is a brief overview of our methods and findings.
  METHODS  
    Total RNA was isolated from peripheral blood cell preparations. First-strand cDNA was synthesized and amplified via an “anchored PCR” technique (10) and then subjected to a nested PCR reaction. The single-stranded anti-sense DNA from each sample was tethered to ELISA microtiter plate wells.  These wells were each probed with a digoxigenin-labeled oligonucleotide corresponding to the leader sequence sense strand of an Ig V H subgroup 1 through 6. Because the leader sequences of V H 7 are so similar to those of V H 1, additional oligonucleotide probes corresponding to the sense strand sequence of V H 1 or V H 7 were used to distinguish these two subgroups.
  RESULTS AND DISCUSSION  
    When this technique was applied to peripheral blood B-cells from normal adults, a distinctive and consistent pattern of relative expression was observed (Figure 1): V H 3 was the dominant subgroup, followed by V H 4, V H 1, and V H 5.  The remaining subgroups represented only small contributions. This distribution, which is based on mRNA analysis, is quite similar to the V H subgroup distribution previously determined by analysis of DNA from B-cells (7, 8, 9) (Figure 1).
 
    Peripheral blood samples from 11 randomly-selected patients with B-cell chronic lymphocytic leukemia (B-CLL) showed a very different pattern of V H expression: the V H distribution was limited entirely to the one V H subgroup represented by the leukemic clone. Moreover, the distribution of V H subgroups among these B-CLL patients was quite different than the distribution of V H subgroups among B-cells of normal individuals (Figure 2). In particular, the V H 1 subgroup was detected in 45% of the CLL cases we analyzed. We have extended that observation and shown that one gene among those in the V H 1 subgroup is particularly common in B-CLL (11). These results show that B-CLL does not arise with equal probability among all B-cells, but rather that B-cells expressing immunoglobulins from certain subgroups have a larger propensity for leukemic transformation.
 
    Reconstitution experiments in which B-CLL cells were mixed with normal blood showed that our technique is highly sensitive to changes in the Ig repertoire cause by a small subpopulation of monoclonal B-cells. The technique therefore could be used to test for emerging monoclonal populations among normal B-cells in samples such as the ones presented at this workshop.
With the availability of simple, rapid methods to prepare cDNA, this technique is highly suitable for population surveys seeking risk factors for B-cell lymphoproliferative disorders. Departure from the normal expression of the Ig V H repertoire may be associated with increased risk for autoimmune disease.  Shifts in V H expression associated with aging, with other chronic diseases, and with HIV-related lymphomas may also be of considerable interest.
      REFERENCES  
  1. Rassenti L, Kipps TJ. A novel technique to analyze the relative concentration of the
      expressed immunoglobulin (Ig) V H genes. Ann New York Acad Sci 1995; 764:463-473.
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      serments dispersed throughout the V H locus. Eur J Immunol 1993; 23:832-839.
  7. Huang C, Stewart AK, Schwartz RS, Stollar BD. Immunoglobulin heavy chain gene
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      the human immunoglobulin V H locus completed by analysis of the telomeric region of
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10. Loh EY, Elliott JF, Cwirla S, Lanier LL, Davis MM. Polymerase chain reaction with
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11. Sasso EH, Johnson T, Kipps TJ. Expression of the immunoglobulin V H gene 51p1 is
      proportional to its germline gene copy number. J Clin Invest 1996; 97:2074-2080.
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