Addressing nonlinearity in the exposure-response relationship for a genotoxic carcinogen: cancer potency estimates for ethylene oxide.

Ethylene oxide (EO) has been identified as a carcinogen in laboratory animals. Although the precise mechanism of action is not known, tumors in animals exposed to EO are presumed to result from its genotoxicity. The overall weight of evidence for carcinogenicity from a large body of epidemiological data in the published literature remains limited. There is some evidence for an association between EO exposure and lympho/hematopoietic cancer mortality. Of these cancers, the evidence provided by two large cohorts with the longest follow-up is most consistent for leukemia. Together with what is known about human leukemia and EO at the molecular level, there is a body of evidence that supports a plausible mode of action for EO as a potential leukemogen. Based on a consideration of the mode of action, the events leading from EO exposure to the development of leukemia (and therefore risk) are expected to be proportional to the square of the dose. In support of this hypothesis, a quadratic dose-response model provided the best overall fit to the epidemiology data in the range of observation. Cancer dose-response assessments based on human and animal data are presented using three different assumptions for extrapolating to low doses: (1) risk is linearly proportionate to dose; (2) there is no appreciable risk at low doses (margin-of-exposure or reference dose approach); and (3) risk below the point of departure continues to be proportionate to the square of the dose. The weight of evidence for EO supports the use of a nonlinear assessment. Therefore, exposures to concentrations below 37 microg/m3 are not likely to pose an appreciable risk of leukemia in human populations. However, if quantitative estimates of risk at low doses are desired and the mode of action for EO is considered, these risks are best quantified using the quadratic estimates of cancer potency, which are approximately 3.2- to 32-fold lower, using alternative points of departure, than the linear estimates of cancer potency for EO. An approach is described for linking the selection of an appropriate point of departure to the confidence in the proposed mode of action. Despite high confidence in the proposed mode of action, a small linear component for the dose-response relationship at low concentrations cannot be ruled out conclusively. Accordingly, a unit risk value of 4.5 x 10(-8) (microg/m3)(-1) was derived for EO, with a range of unit risk values of 1.4 x 10(-8) to 1.4 x 10(-7) (microg/m3)(-1) reflecting the uncertainty associated with a theoretical linear term at low concentrations.

Accumulation of somatic mutations in proliferating T cell clones from children treated for leukemia.

There is continued controversy as to the sequential steps and mechanism(s) responsible for the in vivo acquisition of multiple mutations during neoplastic transformation. We investigated the in vivo clonality and mutational spectra of hypoxanthine-guanine phosphoribosyltransferase (HPRT) mutations in T cells from children with acute lymphocytic leukemia (ALL) to gain insight into the mutagenic mechanisms associated with leukemogenesis. We observed several instances of multiple, independent HPRT mutations accumulating in vivo in T cell receptor (TCR) gene defined clones that had undergone extensive pre- and/or post-thymic expansion following chemotherapy. In addition, we also detected the accumulation of multiple unique single mutations within distinct expanding post-thymic T cell clones. This pattern of clonally restricted hypermutability is compatible with extensive cell proliferation and selection alone without postulating genomic instability. These observations provide a paradigm for a continuum of cellular events that eventually results in the clonal accumulation of mutations in selected populations of cells in vivo and may provide insight into the primary genetic events associated with leukemogenesis, as well as the development of second malignancies and drug resistance following chemotherapy.

HPRT mutations in humans: biomarkers for mechanistic studies.

The X-chromosomal gene for hypoxanthine-guanine phosphoribosyltransferase (HPRT), first recognized through its human germinal mutations, quickly became a useful target for studies of somatic mutations in vitro and in vivo in humans and animals. In this role, HPRT serves as a simple reporter gene. The in vivo mutational studies have concentrated on peripheral blood lymphocytes, for obvious reasons. In vivo mutations in T cells are now used to monitor humans exposed to environmental mutagens with analyses of molecular mutational spectra serving as adjuncts for determining causation. Studies of the distributions of HPRT mutants among T cell receptor (TCR) gene-defined T cell clones in vivo have revealed an unexpected clonality, suggesting that HPRT mutations may be probes for fundamental cellular and biological processes. Use of HPRT in this way has allowed the analyses of V(D)J recombinase mediated mutations as markers of a mutational process with carcinogenic potential, the use of somatic mutations as surrogate markers for the in vivo T cell proliferation that underlies immunological processes, and the discovery and study of mutator phenotypes in non-malignant T cells. In this last application, the role of HPRT is related to its function, as well as to its utility as a reporter of mutation. Most recently, HPRT is finding use in studies of in vivo selection for in vivo mutations arising in either somatic or germinal cells.

Developing sustainable studies on environmental health.

Toxicogenetics, toxicogenomics and proteomics are providing new biomarkers for use in human studies. These, coupled with the more traditional biological responses currently in use, provide a vast armamentarium for assessing exposures, effects and susceptibility factors relating to environmental pollutants. Biomarker availability, however, does not automatically translate to usefulness for studies directed at human health improvement. Transitional studies bridging the gap between laboratory and field, with the biomarker as dependent variable, are required for validation for intended applications. Prospective or cross-sectional studies are usually optimal for validating biomarkers of exposure, where the biomarker response follows the event (exposure) and the entire study population is affected. Biomarkers of effect, where the event (outcome) follows the biomarker response and may be infrequent in the study population, present more complex problems. Identification of genotypes as susceptibility factors requires that additional issues be addressed. Despite these difficulties, new data are emerging from transitional investigations that are providing validated biomarkers for sustainable human studies. It is now possible to envision schemes for integrating the results of molecular epidemiological investigations into the general toxicological evaluations of environmental agents. These will allow intermediate endpoints to be used for making realistic human health assessments and for elucidating pathogenic mechanisms that identify targets for intervention, all with the goal of preventing environmentally mediated human disease. Finally, select biomarker responses that predict the likelihood of disease occurrence will find application in the interpretation of individual medical diagnostic tests, with the goal of improving cancer detection and management.

Transplacental mutagenicity of N-ethyl-N-nitrosourea at the hprt locus in T-lymphocytes of exposed B6C3F1 mice.

Previous studies have compared age-related differences in total mutagenic burden in mice of differing age (preweanling, weanling, or young adult) after single intraperitoneal (i.p.) injections of ethylnitrosourea (ENU). The purpose of the present investigation was to determine the effects of time elapsed since treatment on the frequency of hprt mutant T-cells (Mf) from mice treated transplacentally with single acute vs. multiple split doses of ENU. To this end, pregnant C57BL/6 mice (n = 13-16/group), which had been bred to C3H males, were given i.p. injections of 40 mg ENU/kg bw in a single dose on day 18 of gestation, in a split dose of 6 mg ENU/kg bw on days 12 through 18 of gestation, or DMSO vehicle alone. Groups of pups were necropsied on days 10, 13, 15 (single dose only), 17, 20, 40, and 70 postpartum for T-cell isolations and hprt Mf measurements using the T-cell cloning assay. The time required to reach maximum Mfs in T-cells isolated from thymus of transplacentally treated animals was 2 weeks, the same time span as previously observed after ENU treatment of adult, weanling, and preweanling mice. Mfs in T-cells isolated from spleens of control animals averaged 2.1 +/- 0.3 (SE) x 10(-6). In spleens of mice treated transplacentally with ENU in a single dose, Mfs reached a maximum at 15 days postpartum [84.7 +/- 15.8 (SE) x 10(-6)] and decreased to lower but still elevated levels at 40 days postpartum. In spleens of mice treated transplacentally with ENU in a split dose, Mfs reached a maximum at 13 days postpartum [74.0 +/- 16.3 (SE) x 10(-6)] and decreased to background levels at 40 days postpartum. The areas under the curves describing the change in hprt Mfs over time for ENU-treated vs. control mice estimate the mutagenic potency for transplacental single- and split-dose exposures to be 1.9 and 0.8 x 10(3), respectively. Comparison of the mutagenic potency estimates for mice exposed to ENU in utero to 4-week-old mice given a similar dose of the same lot number of ENU indicates that the mouse is more susceptible to ENU-induced mutagenesis during fetal life.

Biomarkers for assessing occupational exposures to 1,3-butadiene.

The overall objective of this study was to evaluate a continuum of biomarkers in blood and urine for their sensitivities as indicators of low level occupational exposures to 1,3 butadiene (BD). The study design was largely cross-sectional, with biological samples collected within a short timeframe. Personal 8-h BD exposure measures were made on several occasions over a 60-day period for each potentially exposed worker in order provide maximum accuracy for this independent variable and to accommodate the different expression intervals of the several biomarkers. Co-exposures to styrene, toluene and benzene were also measured. The study included 24 BD monomer production workers (mean BD exposure=0.642 mg/m(3)), 34 polymerization workers (mean BD exposure=1.794 mg/m(3)) and 25 controls (mean BD exposure=0.023 mg/m(3)). The several biomarkers were measured by a consortium of investigators at different locations in the US and Europe. These biomarkers included: (1) metabolic genotypes (CYP2E1, EH, GST M1, GST T1, ADH2, ADH3), determined in Prague and Burlington, VT; (2) urinary M1 and M2 metabolites (1,2-dihydroxy-4-[N-acetylcysteinyl]-butane and 1-hydroxy-2-[N-acetylcysteinyl]-3-butene, respectively), determined in Albuquerque, NM and Leiden; (3) hemoglobin adducts (N-[2-dihydroxy-3-butenyl]valine=HBVal and N-[2,3,4-trihydroxybutyl]valine=THBVal), determined in Amsterdam and Chapel Hill, NC, respectively; (4) HPRT mutations determined by autoradiographic assay in Galveston, TX, with slides re-read in Burlington, VT; (6) hypoxanthine-guanine phosphoribosyltransferase (HPRT) mutations determined by cloning assay in Leiden with mutational spectra characterized in Burlington, VT; (7) sister chromatid exchanges and chromosome aberrations determined by standard methods and FISH analysis in Prague. Urinary M1 and M2 metabolites and HBVal and THBVal hemoglobin adducts were all significantly correlated with BD exposure levels, with adducts being the most highly associated. No significant relationships were observed between BD exposures and HPRT mutations or any of the cytogenetic endpoints, regardless of method of assay.

Temporal delineation of sequential HPRT mutations arising in vivo in a T-cell clone with a mutator phenotype.

Recurrent mutations in vivo in T-lymphocytes identify clonally restricted genomic instabilities in some individuals. Cell-based assays allow initial recognition of clones with mutator phenotypes, but genotypic selection is required to determine frequencies and temporal sequences of potentially independent mutational events isolated only as complex changes in the same allele. The present work illustrates how two single-base insertions in the HPRT gene recovered only as a double event in a cell-based assay were shown to arise as separate in vivo mutations, being individually present at frequencies of < or =10(-4) and < or =10(-5), respectively, in peripheral blood. Full characterizations of mutator clones will allow elucidation of the earliest events in the emergence of genomic instability in human somatic cells.

Validated biomarker responses influence medical surveillance of individuals exposed to genotoxic agents.

There is currently a vast armamentarium of biomarkers for evaluating human exposures to environmental carcinogens, the effects of such exposures and/or susceptibility to disease outcome. Before application, however, these biomarkers require validation in terms of truly reflecting what is claimed. Transitional epidemiological studies bridge the gap between laboratory and field. In a transitional study, a biomarker response is the dependent variable being evaluated, while the intended measure, i.e. exposure effect or susceptibility, is the independent variable. Once validated, biomarker responses provide valuable data for use in making human health risk assessments and as guides for individual medical surveillance programmes. An analysis of medical decision-making illustrates how biomarker responses that increase the relative risk of subsequent disease occurrence change the 'pre-test likelihood' of having the disease, thereby influencing interpretation of medical diagnostic tests and even the choice of tests to be performed. This argues that an individual's response using salidated biomarkers should be made part of the medical record.

Hypoxanthine-guanine phosphoribosyltransferase reporter gene mutation for analysis of in vivo clonal amplification in patients with HTLV type 1-associated Myelopathy/Tropical spastic paraparesis.

We tested a surrogate selection approach utilizing mutation at a reporter gene [hypoxanthine-guanine phosphoribosyltransferase (hprt)] as a probe for in vivo cell division, for detection of clonal T cell expansion in human T lymphotropic (HTLV-1) carriers. Peripheral blood samples from HTLV-1-infected individuals with HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) were tested to determine the hprt mutant frequency (Mf). Wild-type and hprt mutant T cell clones were isolated, and clonal identity determined by multiplex PCR and DNA sequencing of T cell receptor (TCR) variable region beta-chain (TCR BV) and third complementarity determining regions (CDR3). Seven samples from HAM/TSP patients were tested, and Mfs were within the normal range for adults (mean 11.3 x 10(-6), max 22.4 x 10(-6), min 5.6 x 10(-6)). The frequency of HTLV-1 infection in wild-type and hprt mutant T cells from HAM/TSP patients was determined to identify enrichment in the mutant fraction of cells. This analysis was performed on 196 isolates from 6 individuals with HAM/TSP. In each case, there is enrichment for virally infected cells in the hprt mutant fraction of isolates. Ten mutant and eight wild-type isolates from sample LS42A (Mf 8.4 x 10(-6)) were tested for clonality by TCR BV PCR and sequencing. Of the 10 hprt mutants, there were two in vivo-expanded clones (four isolates with two identical TCRs, or 80% unique TCR sequences). These studies may provide new insights into the precise mechanism of HTLV-1 leukemogenesis, and aid in the study of mutator phenotypes generated by a combination of Tax-mediated in vivo expansion and mutagenesis.

IPCS guidelines for the monitoring of genotoxic effects of carcinogens in humans. International Programme on Chemical Safety.

The purpose of these guidelines is to provide concise guidance on the planning, performing and interpretation of studies to monitor groups or individuals exposed to genotoxic agents. Most human carcinogens are genotoxic but not all genotoxic agents have been shown to be carcinogenic in humans. Although the main interest in these studies is due to the association of genotoxicity with carcinogenicity, there is also an inherent interest in monitoring human genotoxicity independently of cancer as an endpoint. The most often studied genotoxicity endpoints have been selected for inclusion in this document and they are structural and numerical chromosomal aberrations assessed using cytogenetic methods (classical chromosomal aberration analysis (CA), fluorescence in situ hybridisation (FISH), micronuclei (MN)); DNA damage (adducts, strand breaks, crosslinking, alkali-labile sites) assessed using bio-chemical/electrophoretic assays or sister chromatid exchanges (SCE); protein adducts; and hypoxanthine-guanine phosphoribosyltransferase (HPRT) mutations. The document does not consider germ cells or gene mutation assays other than HPRT or markers of oxidative stress, which have been applied on a more limited scale.

Azathioprine associated T-cell mutations in insulin-dependent diabetes mellitus.

Somatic mutations arise regularly in human T lymphocytes. As these events occur at increased frequencies in several autoimmune disorders, presumably because of increased T-cell proliferation, we investigated if this is also true for insulin-dependent diabetes mellitus (IDDM). Mutations of the hypoxanthine guanine phosphoribosyltransferase (hprt) gene measured by 6-thioguanine (TG) selection were studied in 28 patients (60 determinations) enrolled in a prospective double-blinded placebo-controlled study of azathioprine immunosuppression: 17 patients (34 determinations) were receiving azathioprine and 11 (26 determinations) placebo. Mean hprt T-cell mutant frequencies (MFs) were elevated in both patient groups, but only in the azathioprine group were elevations large and statistically correlated with the duration of the therapy. These results suggest that the organ-specific antigenic stimulus of the T-cell proliferation in IDDM does increase mutant cells in the peripheral blood, but this increase is relatively small. However, azathioprine, which is converted to 6-mercaptopurine in vivo, selects and amplifies the hprt mutants that do arise. Clinical azathioprine resistance may be explained by hprt mutations arising in T cells relevant to the underlying autoimmune process. Monitoring for these mutations should allow more effective use of this immunosuppressive agent.

Emergence of genetic instability in children treated for leukemia.

T cells from patients who had received chemotherapy for B-lineage acute lymphocytic leukemia were studied to determine whether genetic instability, a principal characteristic of cancer cells, can also occur in nonmalignant cells. Consistent with expectations for a genetic instability phenotype, multiple mutations were detected in the hypoxanthine-guanine phosphoribosyltransferase (HPRT) reporter gene in independently isolated mutant T cells expressing identical rearranged T cell receptor beta (TCRbeta) gene hypervariable regions. These results indicate that cancer treatment can lead to genetic instability in nonmalignant cells in some individuals. They also suggest a mechanistic paradigm for the induction of second malignancies and drug resistance.

Biomarker responses in human populations: towards a worldwide map.

The discipline of epidemiology studies the determinants of diseases in human populations, identifies causes, determines outcomes and develops prevention strategies. Traditional epidemiology is most useful for studies of acute, relatively common diseases with short incubation periods but less so for studies of chronic low incidence diseases with long incubation periods. Molecular epidemiology, which employs biological responses or biomarkers as surrogates of exposures or effects, can help with the latter. For this reason, there is a great interest in developing and validating biomarkers. DNA damage underlies an important group of chronic diseases with long incubation periods, i.e., cancer. Biomarkers may measure the exposures that induce the DNA damage, the damage itself, or individual susceptibility to damage. Before they can be used for human population research, however, these measures must be validated. Biomarker validation critically depends on field studies. This is accomplished through transitional epidemiological studies that 'bridge the gap' between laboratory and field. Transitional epidemiological studies are of three varieties: (i) Developmental, (ii) Characterization, and (iii) Applied. Biomarkers are the dependent variables in transitional studies. An international network of laboratories for human population monitoring requires yet another dimension for validation, i.e., the comparability of results among laboratories must be determined. This will be achieved by sample sharing projects, with workshops to compare results. Only then can results in one population be compared with results in another. Interlaboratory standardization of assays for biomarkers validated by transitional studies will have far-reaching benefits. It will allow development of worldwide databases of background values for the various biomarkers-or biomarker maps. This, in turn, will facilitate problem identification and eventually constitute the baselines for area-specific population monitoring. Biomarker databases so developed can be compared with worldwide databases for cancer and heritable diseases, validating the former as statistical surrogates of the latter.

Association among somatic HPRT mutant frequency, peripheral blood T-lymphocyte clonality, and serologic parameters of disease activity in children with juvenile onset dermatomyositis.

Somatic mutant frequencies (Mf) were determined using the HPRT T-cell cloning assay of peripheral blood T-lymphocytes from 14 children with juvenile onset dermatomyositis (JDM). Serologic parameters, specifically muscle enzyme determinations in JDM subjects, were correlated with residual lnMf (delta) in these patients to compare T-cell activation with clinical parameters associated with JDM. In addition TCR analysis was performed to determine T-cell proliferation and clonality on 12 HPRT mutant isolates from two individuals with JDM. Statistically significant correlations were found between residual lnMf and the following serologic parameters: aldolase (r = 0.771, P = 0.015); CPK (r = 0.602, P = 0.023); and SGOT (r = 0.656, P = 0.011) in children with JDM. In addition, identical TCR gene rearrangements were identified in 86 and 40% of the HPRT mutant isolates from the two patient samples analyzed, which is a significantly higher level of clonality than the 10-15% expected in normal individuals. These data suggest that determining HPRT Mf can be a useful antigen-independent method of selecting clonally expanding T-lymphocytes in autoimmune disease where relevant antigens are unknown. Future analysis of HPRT mutant isolates from children with active myositis may increase our understand of the activated T-cells involved in this disease.

Selection of hprt mutant T cells as surrogates for dividing cells reveals a restricted T cell receptor BV repertoire in insulin-dependent diabetes mellitus.

T cells with somatically acquired mutations in the hypoxanthine-guanine phosphoribosyltransferase (hprt) gene were isolated from patients with insulin-dependent diabetes mellitus (IDDM) as representatives of populations potentially enriched for in vivo activated T cells. TCRB gene V region usage among mutant isolates from individual IDDM patients, but not from normal controls, showed a pronounced preference for BV14 and, to a lesser extent, BV6. Wild-type (nonmutant) isolates did not show such preferences. Extensive in vivo clonal expansions of the BV14 expressing mutant T cells from IDDM patients were revealed by sequence identity of TCRB chain junctional regions. These data support restricted TCRB gene usage in T cell populations enriched for in vivo activated clones in patients with IDDM.

HPRT mutations in vivo in human CD 34+ hematopoietic stem cells.

The HPRT mutations in T lymphocytes are widely utilized as biomarkers of environmental exposure and effect. The HPRT gene detects a wide variety of mutation types, many of which are similar at the molecular level to those found in oncogenes in cancers. However, it remains to be determined whether the assay for mutations in T lymphocytes is reflective of mutagenic events in tissues or cells which have high frequencies of malignancy in humans. We now demonstrate that the HPRT gene can be utilized to detect mutations in myeloid stem cells, which are frequent progenitor cells of leukemias. This myeloid stem cell assay shows an age related increase in mutation at HPRT and also detects increases in mutant frequency (M-MF) in patients who have undergone chemotherapy. The myeloid mutants are confirmed to have mutations in the HPRT gene by DNA sequence analysis. Increases in M-MF are seen as expected in the clonally unstable myeloid stem cells of patients with myelodysplastic syndromes; however, unexpectedly these patients also have elevated T-lymphocyte mutant frequencies (T-MF). A good correlation is shown between M-MFs and T-MFs in the same patients. Thus, it appears that the T-lymphocyte assay, which is technically much less demanding than the myeloid assay, appears to faithfully represent the frequency of mutagenic events in the myeloid lineage.

Gene mutations with characteristic deletions in cord blood T lymphocytes associated with passive maternal exposure to tobacco smoke.

We have investigated the molecular effects of passive maternal cigarette exposure in a newborn population and consider the possible implications of the observed genetic changes in the development of neoplastic diseases in children. We present a distribution analysis of somatic mutational events in a reporter gene, HPRT, in cord blood T lymphocytes from newborns after transplacental exposure to cigarette smoke. Analysis of 30 HPRT mutant isolates from 12 newborn infants born to mothers with no evidence of environmental exposure to cigarette smoke and 37 HPRT mutant isolates from 12 infants born to mothers exposed to passive cigarette smoke showed a significant difference in the HPRT mutational spectrum in those exposed in utero to cigarette smoke. The most notable change was an increase in 'illegitimate' genomic deletions mediated by V(D)J recombinase, a recombination event associated with hematopoietic malignancies in early childhood. Recent epidemiological studies of maternal and paternal cigarette smoke exposure and childhood cancers may need to be re-interpreted, given these results.

Genetic instability in human T-lymphocytes.

Mutations arising in vivo in the hypoxanthine-guanine phosphoribosyltransferase (HPRT) gene of T-lymphocytes provide a measure of mutation induction in human somatic cells. Studies of measured background HPRT mutant frequency (MF) values show wide inter-individual variation. At the extremes are individuals with 'outlier' MF values, i.e., non-exposed individuals with MF>100x10-6 [Robinson et al., Mutation Res. 313 (1994) 227-247.]. The elevated HPRT MF in one well-studied outlier is due to the in vivo expansion of mutant cells possessing an identical T-cell receptor (TCR) gene rearrangement pattern. We report here that this in vivo expanding TCR clone shows multiple different HPRT mutations and thus possesses a mutator phenotype. Other individuals with T-cell mutator phenotypes have been found, suggesting that this phenomenon may contribute to the extremes of variation in HPRT MFs in the human population.

Molecular bases of hprt mutations in malathion-treated human T-lymphocytes.

Recently, we reported that 6 of 84 (7.1%) hprt mutants arising in in vitro malathion-treated human T-lymphocytes were characterized by specific genomic deletions in a 125-bp region of exon 3 (Pluth et al., Cancer Research 56 (1996) 2393-2399. We have now extended study to determine whether additional differences in molecular spectrum at a basepair level exist between control and malathion-treated mutations, and investigated whether there is evidence to support the hypothesis that malathion is an alkylating agent. We analyzed 101 hprt mutants (24 from control and 77 from treated cultures) isolated form six in vitro malathion exposures of T-lymphocytes from four healthy male donors. Analysis consisted of: Southern blotting, genomic multiplex PCR, genomic DNA sequencing and reverse transcription of PCR amplification (RT/PCR) and sequencing of the cDNA product. Mutations at several basepair sites were frequent after malathion exposure and were isolated from treated cells from at least two different individuals. Using a human hprt mutation database for comparison, the frequency of mutations at one of these sites (basepair 134) was found to be significantly elevated in the malathion-treated cell (p < 0.0005). Hprt mutations in malathion-treated cells arose preferentially at G:C basepairs, which is consistent with earlier reports that malathion alkylates guanine nucleotides. Assessing molecular changes at both genomic and cDNA levels in the same mutants revealed that many small, partial exon deletions (< 20 bp) in genomic DNA were often represented in the cDNA at the loss of one or more exons. In addition, It was noted that identical genomic mutations can result in different cDNA products in different T-cell isolates. These observations affirm the importance of genomic sequence analysis in combination with RT/PCR for a more accurate definition of the mutation spectrum.