VDJ recombinase-mediated TCR ß locus gene usage and coding joint processing in peripheral T cells during perinatal and pediatric development.

The generation of TCR proteins is the result of V(D)J recombinase-mediated genomic rearrangements at recombination signal sequences (RSS) in human lymphocytes. V(D)J recombinase can also mediate rearrangements at nonimmune or "cryptic" RSS in normal and leukemic human peripheral T cells. We previously demonstrated age- and gender-specific developmental differences in V(D)J coding joint processing at cryptic RSS within the HPRT locus in peripheral T cells from healthy children (Murray et al. 2006. J. Immunol. 177: 5393-5404). In this study, we investigated developmentally specific V(D)J recombinase TCRß immune gene rearrangements and coding joint processing at RSS in peripheral T cells in the same pediatric population. This approach provided a unique opportunity to investigate site-specific V(D)J recombinase rearrangements and coding joint processing at immune and nonimmune genes from the same individual T cell population. We determined the genomic sequence of 244 TCRß coding junctions from 112 (63 male, 49 female) subjects from the late stages of fetal development through 9 y of age. We observed both age- and gender-specific V(D)J recombinase-mediated TCRß gene usage and coding joint processing at immune RSS. To the best of our knowledge, these data represent the first description of age- and gender-specific developmental differences in TCR gene usage and coding joint processing that could directly influence TCR diversity and immune specificity. It will be important for future studies to ascertain the mechanistic etiology of these developmental and gender differences in TCR diversity and specificity, as well as their importance with respect to the age and gender risks for infectious and autoimmune diseases in humans.

V(D)J recombinase-mediated processing of coding junctions at cryptic recombination signal sequences in peripheral T cells during human development.

V(D)J recombinase mediates rearrangements at immune loci and cryptic recombination signal sequences (cRSS), resulting in a variety of genomic rearrangements in normal lymphocytes and leukemic cells from children and adults. The frequency at which these rearrangements occur and their potential pathologic consequences are developmentally dependent. To gain insight into V(D)J recombinase-mediated events during human development, we investigated 265 coding junctions associated with cRSS sites at the hypoxanthine-guanine phosphoribosyltransferase (HPRT) locus in peripheral T cells from 111 children during the late stages of fetal development through early adolescence. We observed a number of specific V(D)J recombinase processing features that were both age and gender dependent. In particular, TdT-mediated nucleotide insertions varied depending on age and gender, including percentage of coding junctions containing N-nucleotide inserts, predominance of GC nucleotides, and presence of inverted repeats (Pr-nucleotides) at processed coding ends. In addition, the extent of exonucleolytic processing of coding ends was inversely related to age. We also observed a coding-partner-dependent difference in exonucleolytic processing and an age-specific difference in the subtypes of V(D)J-mediated events. We investigated these age- and gender-specific differences with recombination signal information content analysis of the cRSS sites in the human HPRT locus to gain insight into the mechanisms mediating these developmentally specific V(D)J recombinase-mediated rearrangements in humans.

Analysis of genetic alterations and clonal proliferation in children treated for acute lymphocytic leukemia.

The development of risk-directed treatment protocols over the last 25 years has resulted in an increase in the survival rates of children treated for cancer. As a consequence, there is a growing population of pediatric cancer survivors in which the long-term genotoxic effects of chemotherapy is unknown. We previously reported that children treated for acute lymphocytic leukemia have significantly elevated somatic mutant frequencies at the hypoxanthine-guanine phosphoribosyltransferase (HPRT) gene in their peripheral T cells. To understand the molecular etiology of the increase in mutant frequencies following chemotherapy, we investigated the HPRT mutation spectra and the extent of clonal proliferation in 562 HPRT T cell mutant isolates of 87 blood samples from 47 subjects at diagnosis, during chemotherapy, and postchemotherapy. We observed a significant increase in the proportion of CpG transitions following treatment (13.6-23.3%) compared with healthy controls (4.0%) and a significant decrease in V(D)J-mediated deletions following treatment (0-6.8%) compared with healthy controls (17.0%). There was also a significant change in the class type percentage of V(D)J-mediated HPRT deletions following treatment. In addition, there was a >5-fold increase in T cell receptor gene usage-defined mean clonal proliferation from diagnosis compared with the completion of chemotherapeutic intervention. These data indicate that unique genetic alterations and extensive clonal proliferation are occurring in children following treatment for acute lymphocytic leukemia that may influence long-term risks for multifactorial diseases, including secondary cancers.

Somatic mutant frequency at the HPRT locus in children associated with a pediatric cancer cluster linked to exposure to two superfund sites.

The somatic mutant frequency (Mf) of the hypoxanthine phosphoribosyl transferase (HPRT) gene has been widely used as a biomarker for the genotoxic effects of exposure but few studies have found an association with environmental exposures. We measured background Mfs in 49 current and former residents of Dover Township, New Jersey, who were exposed during childhood to industrially contaminated drinking water. The exposed subjects were the siblings of children who developed cancer after residing in Dover Township, where the incidence of childhood cancer has been elevated since 1979. Mfs from this exposed group were compared to Mfs in 43 age-matched, presumably unexposed residents of neighboring communities with no known water contamination and no increased cancer incidence. Statistical comparisons were based on the natural logarithm of Mf (lnMF). The mean Mf for the exposed group did not differ significantly from the unexposed group (3.90 x 10(-6) vs. 5.06 x 10(-6); P = 0.135), but unselected cloning efficiencies were higher in the exposed group (0.55 vs. 0.45; P = 0.005). After adjustment for cloning efficiency, lnMf values were very similar in both groups and age-related increases were comparable to those previously observed in healthy children. The results suggest that HPRT Mf may not be a sensitive biomarker for the genotoxic effects of environmental exposures in children, particularly when substantial time has elapsed since exposure.

Genotoxicity of therapeutic intervention in children with acute lymphocytic leukemia.

The survival rates of children treated for cancer have dramatically increased after the development of standardized multiple-modality treatment protocols. As a result, there is a rapidly growing population of pediatric cancer survivors in which the long-term genotoxic effects of chemotherapeutic intervention is unknown. To study the genotoxic effects of antineoplastic treatment in children, we performed a comparative analysis of the changes in the frequency of somatic mutations (Mfs) at the hypoxanthine-guanine phosphoribosyltransferase (HPRT)-reporter gene in children treated for acute lymphocytic leukemia (ALL). We measured HPRT Mfs from 130 peripheral blood samples from 45 children with ALL (13, low risk; 22, standard risk; and 10, high risk) from the time of diagnosis, as well as during and after the completion of therapy. We observed a significant increase in mean HPRT Mfs during each phase of therapy (diagnosis, 1.4 x 10(-6); consolidation, 52.1 x 10(-6); maintenance, 93.2 x 10(-6); and off-therapy, 271.7 x 10(-6)) that were independent of the risk group treatment protocol used. This 200-fold increase in mean somatic Mf remained elevated years after the completion of therapy. We did not observe a significant difference in the genotoxicity of each risk group treatment modality despite differences in the compositional and clinical toxicity associated with these treatment protocols. These findings suggest that combination chemotherapy used to treat children with ALL is quite genotoxic, resulting in an increased somatic mutational load that may result in an elevated risk for the development of multi-factorial diseases, in particular second malignancies.

Comparative analysis of HPRT mutant frequency in children with cancer.

The link between exposure to environmental mutagens and the development of cancer is well established. Yet there is a paucity of data on the relationship between gene-environment interactions and the mechanisms associated with the somatic mutational events involved with malignant transformation, especially in children. To gain insight into somatic mutational mechanisms in children who develop cancer, we determined the background mutant frequency (Mf) in the hypoxanthine phosphoribosyl transferase (HPRT) reporter gene of peripheral blood lymphocytes from pediatric cancer patients at the time of diagnosis and prior to therapeutic intervention. We studied 23 children with hematologic malignancies and 31 children with solid tumors prior to initial therapeutic intervention. Children with solid tumors, specifically sarcomas, and Hodgkin's disease were significantly older and had elevated HPRT Mfs (6.1 x 10(-6) and 3.7 x 10(-6), respectively) at the time of diagnosis, compared to normal controls (2.3 x 10(-6)) and other pediatric tumor groups including children with acute lymphocytic leukemia and non-Hodgkin's lymphoma (ALL/NHL, 1.7 x 10(-6)), central nervous system tumors (CNS, 3.6 x 10(-6)), and neuroblastoma (1.9 x 10(-6)). Of importance is that the significant differences observed in HPRT Mfs between these groups no longer existed after correcting for the effects of age. These data demonstrate that in children who develop cancer there appears to be no significant increase in background HPRT Mf that would indicate significant exposure to genotoxic chemicals or an underlying DNA repair defect resulting in genomic instability. In addition, these data demonstrate the importance of correcting for the effect of age when comparing the frequency of somatic mutations in children and should provide baseline data for future longitudinal biomonitoring studies on the genetic effects of chemotherapy in children treated for cancer.