Mutational spectral analysis at the HPRT locus in healthy children

Mutation Research 505 (2002) 27–41

Mutational spectral analysis at the HPRT locus in healthy children Barry A. Finette a,b,c,∗ , Heather Kendall a,b , Pamela M. Vacek d a

b

Department of Pediatrics, University of Vermont, Burlington, VT 05405, USA Microbiology and Molecular Genetics, University of Vermont, Burlington, VT 05405, USA c Vermont Cancer Center, University of Vermont, Burlington, VT 05405, USA d Department of Medical Biostatistics, University of Vermont, Burlington, VT 05405, USA

Received 26 December 2001; received in revised form 3 April 2002; accepted 16 May 2002

Abstract There is growing evidence linking somatic mutational events during fetal development and childhood to an increasing number of multifactorial human diseases. Despite this, little is known about the relationship between endogenous and environmentally induced exogenous mutations during human development. Here we describe a comparative spectral analysis of somatic mutations at the hypoxanthine-guanine phosphoribosyltransferase (HPRT) reporter gene locus in healthy children. We observed an age-specific decrease in the proportion of large alterations and a corresponding increase in the proportion of small alterations with increasing age following birth (P < 0.001). The age specific decrease in the proportion of large alterations (67–30%) was mainly due to a decrease in the proportion of aberrant variable (V), diversity (D) and joining (J) (V(D)J) recombinase mediated HPRT deletions (P < 0.001). The increase in the proportion of small alterations with age (28–64%) was associated with an increase in transversions from 8% in children at the late stages of fetal development to 31% in children 12–16 years old (P = 0.003). Transitions decreased with age, especially at CpG dinucleotides (P = 0.010), as transversions increased (P = 0.009). These patterns of mutations provide insight into important spontaneous, genotoxic, and site-specific recombinational somatic mutational events associated with the age-specific development of human disease in children as well as adults. © 2002 Elsevier Science B.V. All rights reserved. Keywords: HPRT; Mutational spectrum; Children

1. Introduction Somatic mutational events play a central role in the pathogenesis of human diseases and aging. Despite this, there is limited information regarding the relationship between endogenous background and environmental genotoxic induced mutations, human development, and the age specific incidence of human diseases. The relevance of these genetic events has become increasingly important with the growing ∗ Corresponding author. Tel.: +1-802-656-20; fax: +1-802-656-2077. E-mail address: [email protected] (B.A. Finette).

body of evidence linking transplacental exposures and in utero somatic mutational events with the development of a variety of human diseases. With respect to children, the association between age/development and the incidence of diseases is best observed with pediatric malignancies. Acute lymphocytic leukemia (ALL) in children is strictly age dependent with a bell shaped distribution that peaks between 3 and 5 years of age [1]. In contrast, the incidence of embryonic tumors such as retinoblastoma and neuroblastoma are highest at the time of birth and decrease with age. Tumors such as sarcomas and Hodgkin’s disease are exceedingly rare in young children but increase dramatically with age [1]. Still other malignancies such

0027-5107/02/$ – see front matter © 2002 Elsevier Science B.V. All rights reserved. PII: S 0 0 2 7 - 5 1 0 7 ( 0 2 ) 0 0 1 1 9 - 7

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B.A. Finette et al. / Mutation Research 505 (2002) 27–41

as colon, lung and breast cancer, are virtually absent in children. Similar age specific patterns also exist for a variety of other diseases and most likely reflect developmentally specific genetic and cellular events associated with the pathogenesis of these diseases. For this reason, a fundamental understanding of the spectrum of endogenous background and exogenous induced somatic mutational events in children is critical in order to gain insight into the relationship between the age/developmental patterns of pediatric and adult diseases. The hypoxanthine-guanine phosphoribosyltransferase (HPRT) T-cell cloning assay has been extensively used as a reporter gene assay for determining the frequency and molecular characteristics of somatic mutational events in humans [2]. The HPRT gene product is a phosphoribosylation enzyme in the purine salvage pathway, which will also phosphoribosylate cytotoxic purine analogues such as 6-thioguanine (6-TG). Cells containing an inactivating HPRT gene mutation, however, are able to proliferate in the presence of 6-TG, providing an effective system for mutant selection. The frequency of background HPRT mutant frequency (Mf) in adult humans is approximately 5 × 10−6 at 20 years of age with a subsequent age related linear 2–5% increase per year [3,4]. In contrast, for healthy term newborns, the mean somatic Mf is approximately 10-fold lower than adults [5,6] whereas the Mf of children ranging from birth through adolescents increases more rapidly with age than is found with adults [7]. In addition, developmental and gender specific differences in Mf have also been observed during the late stages of fetal development [8]. The main focus of this study is to determine if there is also age/developmentally specific pattern for somatic mutational events in children. HPRT mutant isolates can be expanded in vitro for extensive molecular analysis. The HPRT gene is 43 Kb in size, composed of nine exons and is located on the X-chromosome at position Xq26. Molecular analysis of HPRT mutations can be performed with a variety of cDNA and genomic methods allowing for detailed molecular characterization of mutational events arising in vivo in humans, and thus providing insight into specific genetic mechanisms and mutational spectra. Mutational spectral analysis of HPRT mutant clones has captured a wide variety of somatic mutation events, which are associated with human

diseases. These events include single-base substitutions and small deletions, large structural alterations, multiple base losses, translocations and double strand breakpoint mediated deletions including variable (V), diversity (D) and (J) joining (V(D)J) recombinase mediated rearrangements [9–12]. It is important to note that somatic mutations at the HPRT reporter gene have no direct clinical consequences and are primarily used as a biomarker of effect, reflective of genome wide mutational events. We report here the first comparative HPRT mutational spectral analysis of somatic mutations in healthy children. We observe that somatic genetic alterations in children follow divergent developmentally specific mutational spectral patterns reflective of spontaneous, genotoxic, and site-specific recombinational genetic events. These findings provide insight into the development of human disease in children as well as in adults.

2. Materials and methods 2.1. Subjects The HPRT mutant frequency was determined with T-cell isolated from heparinized peripheral blood samples from 44 pediatric subjects recruited from the general and subspecialty pediatric clinics of the Department of Pediatrics at the University of Vermont [7]. Subjects were under 17 years of age between the 10th and 90th percentile for height, weight, and head circumference, and had no history or clinical evidence of an autoimmune or systemic disorder, tobacco use, acute/chronic use of immunosuppressive medications, or significant radiation exposure. These subjects were recruited after it was determined that they would require blood analysis for clinical screening or diagnostic evaluation. Informed consents were obtained for all subjects, following the procedure approved by the Committee on Human Research at the University of Vermont. 2.2. HPRT T-cell cloning assay Determination of HPRT Mf and the isolation of mutant isolates were previously described [7,8,12,13]. HPRT mutant isolates were expanded and stored at

B.A. Finette et al. / Mutation Research 505 (2002) 27–41

−80 ◦ C at either 1 × 104 cells for reverse transcriptase (RT)-PCR or 5×104 cells for genomic multiplex-PCR prior to molecular analysis. In addition, HPRT mutant T-cell isolates also included in our analysis were from umbilical cord blood samples from 20 preterm newborns (gestation <36 weeks) [8] and 34 full-term newborns (gestation ≥36 weeks) [13] acquired from the labor and delivery unit of Fletcher Allen Hospital of the University of Vermont College of Medicine. 2.3. Molecular analysis of HPRT mutant isolates Molecular analyses of HPRT mutant isolates both at the genomic and cDNA level have been well described [10,13,14]. Since the HPRT gene is located on the X-chromosome, molecular analysis at the DNA/RNA level is performed in a different way for mutant isolates from males and females [13]. Mutant isolates from males were first analyzed by multiplex genomic HPRT-PCR to determine the presence or absence of the nine HPRT exons [14]. Of importance is that a vast majority of HPRT exon 2–3 deletion mutant isolates from children and newborns have been shown to display intronic breakpoint regions containing the molecular signatures of V(D)J recombinase mediated events (classes I–III) [10]. As a result, mutant isolates that were missing exons 2 and 3 as determined by multiplex genomic-PCR were analyzed with V(D)J specific primers that amplified V(D)J recombinase mediated deletion segments followed by DNA sequence analysis of the specific deletion breakpoint. Mutant isolates from males showing no genomic alterations were characterized by reverse transcriptase (RT) mediated production of HPRT cDNA, nested-PCR amplification and DNA sequencing of the amplified products [13]. The multiplex-PCR primer pairs for exons 1–9, also permitted genomic sequence analyses of both intron and exon segments involved in most splice sequence mutations, reflected as exon exclusions or intron inclusions in cDNA. For mutant isolates from females, multiplex genomic-PCR analysis was not performed because the inactive X-chromosome precludes deletion determination. Therefore, HPRT mutant isolates from females were first analyzed with specific primers to screen for V(D)J recombinase mediated exon 2–3 deletion mutants [12]. Those mutant isolates that showed no V(D)J recombinase mediated deletions were then analyzed by RT-PCR and DNA sequencing.

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2.4. Statistical analysis The independent effects of age and gender on the proportions of differing types of mutations were assessed by logistic regression. In some analyses, age was included as an ordinal variable, with the values 1–5 corresponding to the five age groups: preterm newborns, term newborns, ages 0–5, 6–11, and 12– 16 years. In other analyses, each child’s actual age in years was used. Both representations of age gave similar results, but only the results based on age group are presented below because of the differences in mutational spectra observed between preterm and term newborns, which are indistinguishable in the analyses based on years of age. Models with interaction terms were fitted to test whether age-related changes in mutational spectra differ for males and females. All models included a random effect for subject to account for a correlation between multiple mutations from the same child.

3. Results 3.1. Analysis of HPRT mutant isolates from children A summary of mutations at the HPRT locus for mutant isolates, as well as HPRT Mf, gender and age of subjects from birth to 16 years of age is shown in Table 1. A total of 119 mutant isolates representing 108 independent mutations from 44 subjects were characterized from children less than 1–16 years of age. Independent mutations were defined as single HPRT mutational events corrected for in vivo clonal proliferation by T-cell receptor cDNA sequence analysis, Table 1. 3.2. Distribution analyses of HPRT mutations The distribution analysis for HPRT mutations, as a percent of total, in children from birth to 16 years of age for males and females is summarized in Table 2 and graphically presented in Fig. 1. Mutational spectral data from preterm infants [15] and term newborns [13] is also included to provide a baseline for comparative analysis after birth. Mutations were designated as small alterations, large alterations and uncharacterized as previously defined [13].

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B.A. Finette et al. / Mutation Research 505 (2002) 27–41

Table 1 Molecular analysis of HPRT mutant isolates from children Mutant isolate

Mf × 10−6

Sex

Agea

Mutation cDNA

0–5 years BF20 M1 BF20 M2 BF 20 M4 BF20 M5 BF8 M2 BF25 M1 BF25 M2 BF25 M3 BF67 M2 BF67 M3 BF9 M1 BF9 M3 BF59 M1 BF59 M3 BF59 M5 BF66 M4 BF66 M5 BF22 M2 BF22 M4 BF39 M1 BF40 M3 BF117 M2 BF49 M2 BF10 M3 BF10 M4 BF56 M1 BF131 M1 BF69 M2 BF130 M3 BF130 M2 BF130 M1 BF65 M1 BF65 M2 BF55 M1 BF119 M1 BF119 M2 6–11 years BF53 M1 BF53 M4 BF105 M2 BF105 M4 BF23 M1 BF23 M2 BF23 M3 BF15 M1 BF15 M3 BF15 M4 BF64 M1 BF64 M1 BF64 M4

1.3

M

0.08

Genomic DNA V(D)J; class I V(D)J; class I V(D)J; class I

C508 → T 0.3 0.75

M M

0.2 0.75

1.1

M

0.8

0.4

F

1.0

4.4

M

1.0

1.3

M

1.0

0.77

F

1.20

0.85 1.7 1.2 0.7 1.8

M M M F F

1.5 1.7 2.2 3.1 3.2

2.5 1.7 2.6 2.1

M F M M

4.2 4.2 4.4 4.5

0.4

F

5.0

2.2 2.1

M M

5.2 5.8

Exclusion exon 6 G47 → A del610–626 5 exon 9 C222 → A Exclusion exons 2–3 C377 → T

CpG transition V(D)J; class I V(D)J; class I IVS6 + 1G → A

Transversion NC Transition V(D)J; class III V(D)J; class I

Exclusion exons 2–3 Exclusion403–414 Exclusion exon 6 Exclusion116–125 Exclusion533–553 Exclusion exons 2–3 Exclusion of exon 8 C430 → T Exclusion exon 4 Exclusion exons 2–3 Exclusion exons 2–3 Exclusion exon 6 Exclusion610–626 A404 → T

Transversion V(D)J; class I IVS2–1G → C 12 bp del. @ 403–414 IVS6+1G → A V(D)J; class I Del116–125 Del. exon 5 NC V(D)J; class I T592 → A IVS3-1G → A NC NC Del. exon 6 V(D)J; class I 17 bp del. IVS8-13-+4

6.4

2.0

M

6.8

1.4

F

7.5

8.7

F

7.7

4.1

M

8.1

Transversion Transition

Transversion Transition Transition

Transversion Del. exons 1–9 Del. exon 5

T614 → G M

Transition Transition

NCb

G481 → C

5.4

Transition/ transversion

Transversion

V(D)J; class I G3 → A A466 → T A484 → C Exclusion exons 2–3 InsC305 C434 → A T389 → G Exclusion bp 1–14 Exclusion bp 1–6 C151 → T

Transition Transversion Transversion V(D)J; class I Transversion Transversion 18 bpIVS-1del-14 to +4 or-12 to +6 17 bp del. IVS0-11-+6 CpG transition Del. exon 1

C575 → A

Transversion

B.A. Finette et al. / Mutation Research 505 (2002) 27–41

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Table 1 (Continued ) Mutant isolate

Mf × 10−6

Sex

Agea

Mutation cDNA

BF64 M5 BF64 M8 BF64 M10 BF64 M12 BF64 M15 BF64 M16 BF64 M17 BF64 M18 BF64 M19 BF64 M20 BF18 M1 BF48 M4 BF12 M2 BF12 M13 BF12 M9 BF12 M3 BF12 M1 BF12 M4 BF12 M6 BF12 M7 BF12 M8 BF12 M10 BF101 M3 BF124 M1 BF124 M2 BF109 M1 BF109 M9 BF109 M16 BF109 M18 BF109 M21 BF109 M3 BF1 M1 BF1 M2 BF1 M3 BF1 M4 BF58 M1 12–16 years BF7 M1 BF7 M2 BF7 M5 BF7 M6 BF7 M7 BF7 M8 BF7 M9 BF7 M10 BF6 M3 BF6 M5 BF6 M6 BF6 M7 BF19 M5 BF19 M10 BF4 M2

Genomic DNA

Transition/ transversion

Del. exon 6 V(D)J; class I G568 → A C151 → T T284 → G InsA496–499

Transition CpG Transition Transversion Del. exon 3 Del. exon 2

G569 → C 0.5 2.2 9.0

M M F

8.7 8.9 9.0

3.3 8.0

M F

9.13 10

21.0

M

10.5

2.0

M

10.7

Exclusion exon 8 G209 → A C113 → T G212 → A GG568–569 → AAc GG568–569 → AAc GG568–569 → AAc GG568–569 → AAc GG568–569 → AAc GG568–569 → AAc GG568–569 → AAc GG568–569 → AAc Deletion 2–9, +1.83 Mb frag. InsG503 Exclusion of exon 8

F

10.9

Exclusion exons 2–3

4.0

M

12.5

C648 → G Exclusion exon 7 G135 → T G212 → A Exclusion exon 8

F

12.5

4.0

F

13.2

7.0

M

13.2

DelGGAGA165–169 DelA401/402 Exclusion of exon 8 Exclusion of exon 2–3 Exclusion of exon 8 49 bp inclu. 3 exon 1 Exclusion610–626

Transition Transition Transition Transition Transition Transition Transition Transition Transition Transition Transition Transition

NC V(D)J; class I V(D)J; class Ic V(D)J; class Ic V(D)J; class Ic V(D)J; class Ic Del. exon 1–9

DelT549 InsT590 C486 → A

0.8

1.9

Transversion Del. exon 5 IVS8+5G → A

Transversion Del. exon 5 IVS3-1G → A

IVS6-3C → G Del. exon 4

Transition Transversion Transversion Transversion Transition

NC Del. 1–9 Del. exon 7–9

NC V(D)J; class I T592 → A IVS1 + 1 G → A IVS8-2A → T

Transversion Transition Transversion

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B.A. Finette et al. / Mutation Research 505 (2002) 27–41

Table 1 (Continued ) Mutant isolate

Mf × 10−6

Sex

Agea

Mutation cDNA

BF4 M3 BF113 M1 BF113 M2 BF113 M5 BF113 M4 BF102 M2 BF102 M4 BF5 M2 BF5 M3 BF21 M3 BF21 M5 BF21 M10 BF104 M1 BF104 M5 BF103 M2 BF103 M6 BF103 M7 BF112 M1 BF112 M3

Genomic DNA

Transition/ transversion

Deletion exon 6 6.5

F

13.5

3.4

F

13.5

1.2

F

13.9

4.3

F

15.2

4.2

M

15.7

3.1

F

16.0

2.7

M

16.2

G568 → A G580 → A G190 → C Del A483/484 Exclusion exons 2–4 G617 → T A136 → C G617 → A Exclusion exon 7 Exclusion exon 7 C508 → T Exclusion exon 7 InsA216 Exclusion exon 4 Exclusion bp1–7 exon 1 Del. AA217–219

Transition Transition Transversion del369–384 + IVS2-1–3

IVS7 + 1G → Tc IVS7 + 1G → Tc IVS6-3C → G Del. exons 4–9 IVS3-2A → G 12bp del. IVS0-4-+8

Transversion Transversion Transition Transversion Transversion CpG Transition Transversion

Transition

Deletion exons 1–9

a

Age in years. NC: not characterized. c These mutants are considered to represent a singe independent HPRT mutational event in this subject with subsequent in vivo clonal proliferation. b

Specifically, small alterations included all transversions and transitions as well as insertions and deletions ≤2 base pairs. Large alterations included all deletion mutations >2 base pairs, as well as exon deletions as determined by HPRT multiplex-PCR. Deletion mutations were further sub-classified as those deletions mediated by V(D)J recombinase (V(D)J deletions) and those not mediated by V(D)J recombinase (non-V(D)J deletions). In addition, >2 base pair insertions were also classified as large alterations. A comparative distribution analysis of all mutations revealed a significant age-related decrease in the proportion of large alterations and a corresponding increase in the proportion of small alterations with increasing age (P < 0.001), Table 2, Fig. 1. The percentage of large alterations steadily declined with age from 67% in preterm infants to 30% for children 12–16 years old (P < 0.001). This trend did not differ significantly between males and females but females had lower percentages of large alterations overall during this time interval (P = 0.024).

The increase in the proportion of small alterations with age was due to a significant increase in transversions from 8% in preterm newborns to 31% in children 12–16 years old (P = 0.003), Table 2 and Fig. 1. As a proportion of all mutations, transitions (CpG and non-CpG) did not show a significant age-related trend but as a proportion of the small alterations, transitions decreased significantly with age as transversions increased (P = 0.009). These results are in agreement to those observed in adults (>19 years of age) in which the frequency of transversions, specifically A:T to C:G, increase with age [16]. In contrast, 60% of small alterations in fullterm newborns were transitions, compared to 33% in children 12–16 years old. The decrease in transitions with age was significant only for CpG transitions, 38–6% (P = 0.010), not for non-CpG transitions Table 2 and Fig. 1. The proportion of CpG transitions in children 12–16 years of age (3%) is not significantly different to that observed for adults (5.1%) [17]. These age specific patterns for transitions do not differ significantly for males and females except that non-CpG transitions comprise a

B.A. Finette et al. / Mutation Research 505 (2002) 27–41

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Fig. 1. Distribution of somatic mutations at the HPRT locus in children.

B.A. Finette et al. / Mutation Research 505 (2002) 27–41

significantly larger proportion of the small alterations in females than males (P = 0.050), as well as a larger proportion of all alterations (P = 0.021). The decline in large alterations with age was mainly due to a significant decrease in the proportion of V(D)J deletions (P < 0.001). In addition, a gender specific difference was observed in large alterations due to the lower proportion of non-V(D)J deletions in females (P = 0.004), Table 2 and Fig. 1. There were no gender specific differences in V(D)J deletions in children from birth to 16 years of age. This is in contrast to what was recently reported for preterm and fullterm newborn infants in which a significant increase in V(D)J recombinase mediated HPRT deletions was observed in females during the late stages of fetal development [15]. The proportion of all mutations that were non-V(D)J deletions did not show a significant age specific trend over the five age groups. Of interest is that with the age-related decrease in V(D)J deletions, non-V(D)J deletions became an increasing proportion of the large alterations (P < 0.001), ranging from 20% in preterm newborns to 90% in children aged 12–16, Table 2 and Fig. 1.

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3.3. Frequency of HPRT mutations We have previously reported that there is an age specific increase in the overall frequency of HPRT mutations in children [4,7]. Therefore, the frequency of specific mutational events would be affected not only by its percentage of total but by the mean Mf for a specific group as well. The frequency of a particular type of mutation was estimated by multiplying its proportion by the mean HPRT Mf for each specific age group of subjects, Table 3 and Fig. 2. Although overall mutant frequencies were obtained for each subject, there were not enough mutant isolates from each individual to determine within-subject distributions of mutant types. Hence, the frequencies of the differing mutant types could not be calculated for each subject and inter-subject variability could not be estimated. This precludes statistical comparison of differing groups of subjects. The frequency of specific HPRT mutational events in children followed similar age specific trends that were observed for the distribution analysis of HPRT mutants described above. There is an increase in the frequency of small alterations with age that included an increase in

Table 3 Frequency of somatic mutational events at the HPRT locus in childrena Groups (years)

Mean Mf × 10−6

Single base substitution Transitions

Transversions

CpG transitions

All

V(D)J deletions

Non-V(D)J deletions

All

Pretermb

0.8 0.7 1.1 0.8 1.0 0.5 1.5 1.7 1.0 3.5 2.8 4.7 3.8 4.5 3.3

0.06 0.03 0.19 0.11 0.11 0.11 0.25 0.20 0.30 0.74 0.42 1.60 0.68 0.31 0.86

0.06 0.05 0.13 0.09 0.13 0.03 0.25 0.25 0.20 0.70 0.62 0.80 1.19 1.62 0.86

0.08 0.08 0.07 0.07 0.10 0.03 0.03 0.07 0.00 0.17 0.20 0.00 0.11 0.00 0.16

0.20 0.16 0.39 0.27 0.34 0.17 0.53 0.52 0.50 1.61 1.24 2.40 1.87 1.93 1.88

0.42 0.34 0.65 0.24 0.28 0.17 0.46 0.66 0.10 0.45 0.39 0.38 0.11 0.00 0.16

0.11 0.13 0.00 0.22 0.33 0.06 0.28 0.32 0.20 0.87 0.84 0.80 1.03 1.93 0.53

0.52 0.48 0.65 0.46 0.61 0.23 0.74 0.98 0.30 1.32 1.23 1.18 1.14 1.93 0.69

Male Female Fulltermb Male Female 0–5 years Male Female 6–11 years Male Female 12–16 years Male Female

Large alterations

a Frequency of HPRT mutations (×10−6 ) are the product of the mean HPRT mutant frequency for subjects in which mutant clones were analyzed and the % distribution of the specific mutation. HPRT Mf values for mutants BF21, BF12, and BF109 were not included for the calculation of mean Mf because they are considered outliers as a result of in vivo clonal proliferation. b Mean HPRT Mf for preterm and fullterm infants were based on data previously reported (Finette, 1994. Mut. Res; Yoshioka, 1999 PNAS).

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Fig. 2. Frequency of somatic mutations at the HPRT locus in children.

B.A. Finette et al. / Mutation Research 505 (2002) 27–41

transversions and non-CpG transitions. The frequency of CpG transitions did not have an age specific pattern. These observations are consistent with a very high percentage of CpG transitions occurring in newborns compared with adults. The frequency of large alterations increased with age, which is in contrast to the distribution analysis but the frequency of V(D)J deletions decreased with age while the frequency of non-V(D)J deletions increased with age which is in agreement with the distribution patterns described above.

4. Discussion A significant body of knowledge exists on the genetic and cellular basis of inherited human diseases. Recently, somatic mutations during the early stages of human development and childhood have been shown to be associated with the pathogenesis of neurologic [18,19], endocrine [20,21], cardiovascular [22], and autoimmune multifactorial diseases [23], as well as cancer [24–26]. Molecular epidemiologic studies have demonstrated clinically important somatic mutational events in specific disease genes as a consequence of both endogenous as well as genotoxic environmental genetic mechanisms. Because children have large body surface areas, high dietary intake and basal metabolic rates, as well as developmentally immature detoxification and excretory systems, they are especially susceptible to the genetic and cellular consequences of environmental genotoxic exposures. Children, especially infants, consume about seven times the fluids and up to four times the amount of food per pound per day than adults [27,28]. The high surface to volume ratio of children and increased baseline respiratory rate, also results in an O2 consumption two fold higher than adults [27,28]. In addition, the behavior and developmental characteristics of children place them in direct contact with high levels of environmental pollutants, which they metabolize and excrete at reduced rates compared to adults. During fetal development and early childhood, there is also a decreased capacity to metabolize genotoxic compounds as a result of changing metabolic profiles of phase I and II enzyme systems [29,30]. As a result, children are more sensitive and vulnerable to expo-

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sure to exogenous as well as endogenous mutagenic and carcinogenic chemicals [31]. In this study, we demonstrate that there is an age-specific background spectrum of somatic mutations at the HPRT reporter gene locus in children from birth through adolescence. Comparable studies in the lacZ transgenic murine models have also demonstrated that somatic mutational events not only increase with age but that the spectrum of genetic alterations is organ specific [32]. We observed a significant increase in the proportion of large mutational events early during human development, especially aberrant V(D)J recombinase mediated deletions that decreased with age. The proportion of large alterations is quite striking for children less than 5 years of age (59–67% of all mutations screened), Table 2. Of significance is that between 50 and 78% of all the deletions observed in children 0–5 years of age were the result of aberrant site-specific recombination events mediated by the enzyme V(D)J recombinase. V(D)J recombinase normally mediates the genomic rearrangement of V, D and J regions of T-cell receptor (TCR), and immunoglobulin (Ig) genes to generate diversity for antigen specific recognition [33–36]. These site-specific rearrangements represent developmental genetic events that occur during normal immunological maturation. Aberrant V(D)J recombinase mediated rearrangements such as those demonstrated at the HPRT locus have been associated with cytogenetic alterations at disease specific genes associated with T and B-cell leukemia [37–43]. We have previously postulated that during normal immunologic development, V(D)J recombinase can cause genetic changes, with and without clinical consequences analogous to the clinically relevant mutations associated with hematopoietic malignancies [12]. With respect to small alterations the most significant findings was the increase in transversions with increasing age (Table 2 and Fig. 1). The age specific increase in transversions with age may be the result of the accumulation of background mutations as a consequence of exposure to ubiquitous environmental mutagenic/carcinogenic compounds. Multiple animal and human reporter gene model systems have demonstrated a significant increase in transversions as a consequence of bulky DNA adducts following exposure to a variety of environmental genotoxic chemicals that include heterocyclic amines [44,45], butadiene

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[46], polycyclic aromatic hydrocarbons and aromatic amines [47], as well as ultraviolet light, aflatoxin B1 and tobacco smoke [48]. In these studies, spectral analysis demonstrated the largest increase to be GC → TA transversions, although other transversions as well as some transitions were also increased following genotoxic exposure. Recently, transplacental exposure to heterocyclic amines and polycyclic hydrocarbons has demonstrated an increase in transversions as well as a high susceptibility to lung tumor formation in murine models [49]. In this study, the total number of transversions observed did not allow us to perform a comparative analysis to determine if statistically significant differences existed between different transversion events with age. Spectral analysis did demonstrate that GC → TA transversions were the most common with a relatively equal distribution between the remaining transversion events (Table 1) supporting our premise that the increase of transversions with age probably reflects somatic mutations as a result of environmental exposures. With respect to transitions, there was a significant increase in the proportion of CpG transitions during in utero and early human development that decreased with age. This same trend was not true for transitions in general. These observations are consistent with previously published results [17] and supports the view that CpG transitions occur at a higher frequency during early human development because of an increase in lymphocyte proliferation. This has clinical significance since CpG transitions have been shown to be the most common transition event in germline HPRT mutations in children with Lesch-Nyhan syndrome [50] and p53 mutations associated with Li-Fraumeni syndrome [51]. CpG transitions are also the most common single base substitution seen at the p53 gene associated with a variety of adult cancers [51]. It has been estimated that transitions at CpG dinucleotides occur at a frequency 42-fold higher than that predicted from random mutations in coding regions of genes that cause human diseases [52]. Based on our observations, many of the CpG transition events seen with diseases in adults may be occurring early during human development. One of our most intriguing observations is the age/gender-specific spectrum pattern seen for some mutational events. Females had a lower proportion of large alterations overall as well as non-V(D)J

deletions. The proportion of V(D)J recombinase mediated deletions decreases in females and increases in males between 0 and 5 years of age, Table 2 and Fig. 1. Of interest is that the incidence of ALL in males also increases during this age interval. The increase in the proportion of V(D)J deletions in children 0–5 years of age is the opposite to that recently reported for preterm infants where there was a significant increase in V(D)J deletions in females [15]. The reason why gender specific differences exist for V(D)J recombinase mediated events is unclear. In this study we also observed an increased proportion of non-CpG transitions for males. In humans, gender/developmental specific differences have been described during fetal development for neural differentiation [53], growth [54], lung maturation [55], cardiac development [56], and glutathione-reductase expression [57]. With respect to molecular differences, adult females have been found to have gender specific differences in the spectrum of p53 mutations and levels of DNA adducts in lung cancer tumors [58,59] as well as a more active immune response [60]. A link between these gender specific mutational events with gender specific autoimmune diseases such as juvenile rheumatic arthritis, juvenile psoriatic arthritis, systemic lupus erythematosus and fybromyalgia [61] is intriguing but to date unclear. These investigations suggest that somatic mutational events in children are a dynamic process that is similar to the developmental maturation of other critical cellular and organ specific systems that can be associated with the incidence of age-specific diseases. These observations support the need for future studies integrating gene-environmental interactions at various stages of human development to gain insight into the expression, susceptibility, and mechanisms of human diseases.

Acknowledgements We thank Holly Pasackow R.N., and the clinical providers in the Department of Pediatrics at the General Pediatrics Clinic of the University of Vermont for obtaining blood samples. Research was supported by the National Institute of Child Health and Human Development (NICHD) grants 1K11HD01010 and 1R29HD35309, and National Cancer Institute

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(NCI) 1KO1CA77737 grant. In addition, NCI grant P30CA22435 to the University of Vermont Cancer Center DNA Analysis Facility.

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