Recent studies have brought to the forefront the importance of somatic mutations during human fetal development and malignant transformation in children, specifically leukemia. Therefore, a better understanding of the frequency and mutational spectrum of spontaneous in utero mutations is essential for understanding the genetic mechanisms associated with pediatric malignancies. Previously we reported that the frequency of somatic mutations during the late stages of fetal development was dependent on both gestational age and gender. Here we present the hypoxanthine-guanine phosphoribosyltransferase (HPRT) reporter gene mutational spectra analysis for 60 T-cell mutant isolates from the umbilical cord blood of preterm newborns to gain insight into background mutational events during the late stages of fetal development. Logistic regression analyses showed a significant increase in HPRT deletions mediated by V(D)J recombinase in preterm newborns compared with full-term newborns (P = 0.009). A comparative analysis of deletion mutations also revealed that V(D)J recombinase-mediated HPRT deletions increased with decreasing gestational age (P = 0.012) and were significantly higher in females than males of the same developmental status (P = 0.031). Developmental and gender-specific differences in HPRT deletions mediated by V(D)J recombinase provide insight into the gender-specific differences seen in infant leukemia.

Significant progress has been made toward understanding the molecular basis and clinical relevance of germinal mutations and inherited human diseases. In contrast, only recently has there been significant evidence demonstrating that somatic mutational events during fetal development also have direct clinical consequences for both pediatric and adult multifactorial diseases, especially cancer.

Recent studies have linked for the first time specific in utero genetic events and the development of cancer in children. Specifically, in utero somatic mutational events involving the MLL/AF4(1, 2) and TEL-AML1(3, 4) gene fusions in T cells and immunoglobulin heavy chain and T-cell receptor rearrangements in B cells (5) have been correlated with the subsequent development of infant and childhood leukemia.

Previously, we reported developmental and gender-specific differences in the in utero frequency of somatic mutations (Mf) at the HPRT3 reporter gene (6). Specifically, the Mf of preterm newborns was higher compared with full-term newborns, with the mean Mf of female preterm newborns being inversely related to gestational age and significantly higher than that found for male preterm newborns (6).

We report here a comparative analysis of the mutational spectra at the HPRT reporter gene mutations in T cells from this cohort. Statistical analyses revealed a significant increase of V(D)J recombinase-mediated HPRT deletions in preterm newborns compared with full-term infants. In addition, there was a significant increase in V(D)J recombinase-mediated deletions in both preterm and full-term female newborns compared with preterm and full-term male newborns. This gender-specific difference in V(D)J recombinase-mediated events may be relevant to understanding the higher incidence of infant leukemia observed among females.

Study Population.

Heparinized umbilical cord blood samples from 20 preterm newborns (gestation, <36 weeks; Ref. 6) and 33 full-term newborns (gestation, ≥36 weeks; Ref. 7) were acquired from the labor and delivery unit of Fletcher Allen Hospital of the University of Vermont College of Medicine. Informed consents were obtained after the procedure was approved by the Committee on Human Research at the University of Vermont.

HPRT T-Cell Cloning Assay.

Determination of HPRT Mf and the isolation of mutant clones were described previously (6, 7). HPRT mutant isolates were expanded and stored at −80°C at either 1 × 104 cells for reverse transcription-PCR or 5 × 104 cells for genomic multiplex PCR before molecular analysis.

Molecular Analysis of HPRT Mutant Isolates.

The HPRT locus, located at Xq26, contains 9 exons, is 43 Kb in size, and has been completely sequenced. The coding sequence is 657-bp long. Molecular analyses of mutant cells both at the genomic and cDNA level have been well described (7, 8, 9). HPRT mutations observed previously include: (a) base substitutions at more that 270 sites in all nine exons; (b) small deletions and insertions; (c) large structural alterations; (d) splice site changes in introns; and (e) specialized genetic events such as V(D)J recombinase-mediated deletions (9, 10, 11).

Because the HPRT gene is located on the X-chromosome, molecular analysis at the DNA/RNA level is performed in different ways for mutant isolates from males and females (7). Mutant isolates from males first were analyzed by multiplex genomic HPRT PCR to determine the presence or absence of the nine HPRT exons (8). Mutant isolates from males showing no genomic alterations were characterized by reverse transcriptase-mediated production of HPRT cDNA, nested PCR amplification, and DNA sequencing of the amplified products (7). The multiplex PCR primer pairs for exons 1–9 also permitted 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 first were analyzed with specific primers to screen for V(D)J recombinase-mediated exon 2–3 deletion mutants (11). Then, those mutant isolates which showed no V(D)J recombinase-mediated deletions were analyzed by reverse transcription-PCR and DNA sequencing.

Statistical Analysis.

Logistic regression was used to assess the effects of gender and development on the proportions of the different types of mutations. Models with interaction terms were fitted to test whether the effects of gender and development were independent. In utero exposure to tobacco smoke was included in some models to determine whether the effects of development and gender were attributable to differences in transplacental exposure to tobacco smoke. Detailed information about maternal smoking and passive smoke exposure was unavailable for many preterm infants, so smoke exposure was represented as dichotomous variables (exposed or not exposed). All models included a random effect to account for the correlation between multiple mutations from the same newborn.

A summary of mutations at the HPRT locus for each mutant isolate, as well as sex and gestational age of the subjects, is shown in Table 1. A total of 66 mutant isolates representing 60 independent mutations from 20 preterm newborns and 85 mutant isolates representing 78 independent mutations from 33 full-term newborns were characterized. Independent mutations were defined as single HPRT mutational events corrected for in vivo clonal expansion of mutant isolates. Clonal expansion was evident in subjects PS21 (deletion exons 1–9); PS30 (C508→T); PS29 (deletion exon 2); PS5 (exclusion exons 2–6); MFS89 (deletion exons 1–9 and deletion exons 2–9); MFS36 (deletion exons 7–9); and MFS65 (G3→T). Mutational spectrum data for full-term infants was reported previously, except for those indicated in Table 1(7).

Distribution Analyses of HPRT Mutations.

The distribution analysis for HPRT mutations during the late stages of fetal development is summarized in Table 2 and shown graphically in Fig. 1.

Mutations were first designated as small alterations, large alterations, and uncharacterized, as defined previously (7). A comparison between all mutations revealed a greater proportion of large alterations compared with small alterations for both preterm and full-term infants. This is consistent with previously reported HPRT mutational spectra in children from birth through 5 years of age (11). Logistic regression analysis demonstrated that the proportions of small and large alterations were not significantly related to gestational age or gender.

Distribution analysis of small alterations revealed a higher proportion of transition mutations compared with transversion mutations in both preterm and full-term newborns. Logistic regression analysis of the proportions of transitions and transversions did not demonstrate a relationship with either development or gender.

There were two predominant large alterations observed: HPRT deletions of exons 2 and 3 mediated by V(D)J recombinase (V(D)J deletions) as defined previously (9, 11), and exon deletions not mediated by V(D)J recombinase (non-V(D)J deletions). Distribution analysis revealed a higher proportion of V(D)J deletions compared with non-V(D)J deletions among all preterm infants and term females, but not among term males. Notably, logistic regression analysis revealed that the proportion of V(D)J deletions to non-V(D)J deletions was significantly related to both development and gender. Specifically, the proportion of V(D)J deletions were significantly higher compared with non-V(D)J deletions in preterm infants than full-term infants of the same gender (OR, 3.5; P = 0.012) as well as higher in females compared with males of the same developmental status (OR, 4.1; P = 0.043). Similar results were obtained when development was represented as gestational age rather than being classified as preterm or full-term, with ORs indicating that the proportion of V(D)J deletions increased ∼13% with each week of decreasing gestational age from birth (P = 0.038).

Previously, we have reported that transplacental exposure to tobacco smoke results in a significant increase in V(D)J-mediated HPRT deletions in healthy full-term infants (7). In this study, an analysis of transplacental tobacco exposure was not related to any type of alteration in those preterm infants for whom exposure status was available.

Analysis of V(D)J Recombinase-mediated Breakpoints.

Characteristic V(D)J recombinase-mediated sequence signature markings for HPRT V(D)J deletions are summarized in Fig. 2. A total of 10 V(D)J recombinase-mediated mutants from female preterm newborns and 21 V(D)J recombinase-mediated mutant isolates from male preterm newborns were characterized. All but two isolates from male subjects were Class I V(D)J deletion mutants, with the remaining mutant isolates being Class III V(D)J deletion mutants (9). There were five mutant isolates from male subjects and two mutant isolates from female subjects that lacked the N nucleotides additions. The percentage of the breakpoint sequences lacking the N nucleotide in the V(D)J mutant isolates from preterm newborns (22.6%, 7 of 31) was higher than the percentage observed previously for full-term newborns (5.6%; 1 of 18; Ref. 7). In addition, two atypical V(D)J recombinase events were observed. The V(D)J deletion breakpoint sequence from PS7M11 contained a 14-base N nucleotide insertion with an unusually long 26-base nibbling at the 3′ side of the breakpoint, whereas another mutation from subject PS7M20 contained a tandem direct repeat of a motif 5′-CACATCCCTTTCATG-3′, which is separated by four bases upstream of the 5′ breakpoint.

In this study we observed a unique mutational spectra for large alterations during the late stages of fetal development that was not seen for small alterations (including single-bp transversions and transitions). The most significant finding is that the proportion of V(D)J recombinase-mediated HPRT deletions are significantly higher during fetal development compared with after birth, and that this genetic event is related to gestational age and gender. Specifically, 63% (54 of 86) of all large alterations found in preterm and full-term newborns are V(D)J-mediated HPRT deletions, with a significantly higher distribution of V(D)J deletions in females (84%) compared with males (57%). To the best of our knowledge, this is the first demonstration of a gender-specific mutational spectra in humans, especially one with potential clinical consequences. We hypothesize that the high frequency of V(D)J recombinase-mediated HPRT deletions observed are the consequence of aberrant V(D)J genomic recombination events occurring at a time of high V(D)J recombinase activity required for in utero T-cell maturation. The reason for the predominance of these V(D)J recombinase rearrangements in females is unclear. Yet our observations may still be relevant, because an increase in these aberrant V(D)J-mediated genomic rearrangements may be responsible for the higher incidence of infant leukemia among females (12).

V(D)J recombinase-mediated rearrangements have been observed with cytogenetic alterations associated with T- and B-cell leukemia (13, 14, 15, 16). In these studies, sequence analysis of translocation and deletion breakpoint sites in malignant clones demonstrated hallmark V(D)J recombinase signature markings that include palindromic bases (P nucleotides) and nucleotide nibbling and/or insertion of nontemplated bases (N nucleotides) at heptamer (CAC/TGTG)/nonamer (GGTTTTTGT) RSSs containing 12- and 23-bp spacers. Recently, chromosomal rearrangements involving the MLL (ALL-1, HRX-AF-9) gene at chromosome band 11q23 and the TEL-AML1 gene fusion in children with infant leukemia (<12 months of age) have been shown to have occurred in utero(3, 17, 18). In addition, molecular analysis of Guthrie blood spots in monozygotic twins demonstrated clonal MLL fusion rearrangements that were subsequently identified in leukemic cells from these children, providing additional support to the hypothesis that in utero somatic mutational events are associated with the development of pediatric leukemia (1, 19, 20). These and other studies in MLL-AF9 knockin mice (21) also demonstrate that other genetic events and environmental influences likely affect the length of the latent period for tumor development. For example, the expression of mutant isoforms of a transcription factor Ikaros has also been correlated with infant leukemia (22). The etiology of a number of MLL rearrangements observed is not clear. Cell lines established from leukemia patients with t(4;11)(q21;q23) MLL translocations have showed the hallmarks of V(D)J recombinase at the chromosomal breakpoints, which include cryptic RSSs and random base insertions at chromosomal breakpoints (23).

MLL rearrangements have also been associated with chemotherapy using topoisomerase II inhibitors (24, 25). These chemotherapeutic agents have been shown to increase the frequency of V(D)J recombinase-mediated HPRT deletions in the CCRF-CEM lymphoid cell line that constitutively expresses RAG1 and RAG2 (26), therefore suggesting a link between DNA DSB repair and V(D)J recombinase-mediated rearrangements. In addition, some components of the DSB repair system are shared by V(D)J recombinase, including the catalytic subunit of DNA-dependent protein kinase (27), Ku70 (28), and Ku80 (29, 30). Breakpoints associated with some t(4;11) translocations involving MLL genes have recently been reported to display short tandem repeats, inversions, and short homologous sequences at the chromosomal breakpoints, suggesting a DNA repair mechanism (31, 32). Of interest, P nucleotide sequences were observed at some of these breakpoints, which may indicate that V(D)J recombinase activity may have also participated in these translocations.

In this report, 22.6% of V(D)J breakpoints in preterm newborns did not contain N nucleotide insertion compared with 5.6% in full-term newborns. A decrease in the insertion of N nucleotides has been associated with the early stages of murine B-cell (33) and γδ T-cell development (34) as well as in human T-cell development attributable to decreased in utero expression of terminal deoxytransferase. The lower frequency of in utero N nucleotides we observed is in agreement with these previous reports. In addition, V(D)J mutant, PS7M20, contained a tandem repeat (5′-CACATCCCTTTCATG-3′). Such tandem repeats were also observed at MLL breakpoint sites (31, 32).

Therefore, during in utero lymphoid development, a synergistic relationship may exist between components of the V(D)J recombinase and DSB repair systems that increases the frequency of aberrant genomic deletions and chromosome translocations that are responsible for the development of leukemia in infants and children.

Fig. 1.

Distribution analysis of HPRT mutations in T cells from preterm and term infants.

Fig. 1.

Distribution analysis of HPRT mutations in T cells from preterm and term infants.

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Fig. 2.

Breakpoint sequence analysis of HPRT V(D)J recombinase-mediated deletions in preterm infants. Intron 1 and 3 breakpoint regions for both Class I and III regions are shown. Bold nucleotides represent conserved genomic V(D)J RSSs associated with 12- or 23-bp spacer sequences. DNA sequences for Class I and III HPRT mutant clones show the hallmark signature markings of V(D)J recombinase-mediated events at breakpoint sites, including nibbling back, the presence of templated P nucleotides (underline), and the insertion of nongermline templated bases, N nucleotides (italics).

Fig. 2.

Breakpoint sequence analysis of HPRT V(D)J recombinase-mediated deletions in preterm infants. Intron 1 and 3 breakpoint regions for both Class I and III regions are shown. Bold nucleotides represent conserved genomic V(D)J RSSs associated with 12- or 23-bp spacer sequences. DNA sequences for Class I and III HPRT mutant clones show the hallmark signature markings of V(D)J recombinase-mediated events at breakpoint sites, including nibbling back, the presence of templated P nucleotides (underline), and the insertion of nongermline templated bases, N nucleotides (italics).

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1

Research was supported by Child Health and Human Development (NICHD) Grants 1K11HD01010 and 1R29HD35309, National Cancer Institute Grant 1KO1CA77737, and National Cancer Institute Grant P30CA22435 to the University of Vermont Cancer Center DNA Analysis Facility.

3

The abbreviations used are: HPRT, hypoxanthine-guanine phosphoribosyltransferase; Mf, mutation frequency; RSS, recombination signal sequences; OR, odds ratio; N nucleotides, nontemplated nucleotides; DSB, double-strand break.

Table 1

Molecular analysis of in utero HPRT mutations

Mutant isolateSexGAaHPRT MutationTransversion/Transition
cDNAGenomic DNA
Preterm infants      
 PS21M1 26  Deletion exons 1–9b  
 PS21M4    Deletion exons 1–9b  
 PS21M3    V(D)J class I  
 PS21M5    V(D)J class I  
 PS21M6   G143→A  Transition (CpG) 
 PS21M2   T595→G  Transversion 
 PS8M2 27  V(D)J class I  
 PS8M3   G606→C  Transversion 
 PS30M1 29 C508→Tb  Transition (CpG) 
 PS30M6   C508→Tb  Transition (CpG) 
 PS30M10   C508→Tb  Transition (CpG) 
 PS30M5   C454→T  Transition 
 PS30M2    V(D)J class I  
 PS30M3    V(D)J class I  
 PS30M7    V(D)J class I  
 PS9M2 29  V(D)J class I  
 PS23M2 31  Deletion exons 2–9  
 PS26BM2 31 Exclusion exons 2–3 V(D)J class I  
 PS26BM3    Deletion exons 1–9  
 PS26BM4   Exclusion exons 2–6  Insertion GCTGCCG @IVS5+1 
 PS29M1 31  Deletion exons 1–9  
 PS29M2    V(D)J class I  
 PS29M5    V(D)J class I  
 PS29M4    Deletion exon 2b   
 PS29M6    Deletion exon 2b  
 PS26aM1 31  V(D)J class I  
 PS26aM3   Exclusion exons 2–3 V(D)J class I  
 PS26aM6   Exclusion exons 2–3 V(D)J class I  
 PS26aM12   Exclusion exons 2–3 V(D)J class I  
 PS26aM9   Exclusion exon 4 IVS4+1 G→A Transition 
 PS26aM4   Exclusion exons 3–7 A307→T Transversion 
 PS26aM5   T81→C  Transition 
 PS4M1 31  V(D)J class I  
 PS4M3    V(D)J class I  
 PS4M4    V(D)J class I  
 PS4M5    V(D)J class I  
 PS4M7    V(D)J class I  
 PS4M2    Deletion exons 7–8  
 PS4M8   Deletion A381   
 PS4M9   Exclusion exon 8 NCc  
 PS18M1 32  V(D)J class I  
 PS18M2    Deletion 537–564  
 PS19M6 32 Exclusion 429–475 NCc  
 PS19M7   C508→T  Transition (CpG) 
 PS6M1 33  V(D)J class I  
 PS16M2 34  V(D)J class 3  
 PS16M9   C508→T  Transition (CpG) 
 PS20M1 34  V(D)J class I  
 PS20M2    C69→A Transversion 
 PS12BM1 34  V(D)J class I  
 PS12BM2   C508→T  Transition (CpG) 
 PS13M1 35 G207→T  Transversion 
 PS15M2 35  V(D)J class I  
 PS15M5    V(D)J class 3  
 PS15M4   C145→T  Transition 
 PS15M6    Deletion exon 1  
 PS7M11 35  V(D)J class I  
 PS7M20    V(D)J class I  
 PS7M13   Exclusion exon 6 IVS5-1 G→A Transition 
 PS14M2 35  V(D)J class I  
 PS14M4    V(D)J class I  
 PS5M1 35 C508→T  Transition (CpG) 
 PS5M8    V(D)J class I  
 PS5M4   Exclusion exons 2–6b NCc  
 PS5M6   Exclusion exons 2–6b NCc  
 PS5M9   Exclusion exons 2–6b NCc  
Term infantsd      
 MFS61aM2 36 G606→T  Transversion 
 MFS89M2 36 C454→T  Transition 
 MFS89M3    Deletion exons 1–9b  
 MFS89M18    Deletion exons 1–9b  
 MFS89M10   C508→T  Transition (CpG) 
 MFS89M12    Deletion exons 2–9b  
 MFS89M6    Deletion exons 2–9b  
 MFS89M4    V(D)J class I  
 MFS89M14    V(D)J class I  
 MFS89M15    V(D)J class I  
 MFS101M1 36 T536→Ab  Transversion 
 MFS101M2   T536→Ab  Transversion 
 MFS101M3    Deletion exons 6–9  
Mutant isolateSexGAaHPRT MutationTransversion/Transition
cDNAGenomic DNA
Preterm infants      
 PS21M1 26  Deletion exons 1–9b  
 PS21M4    Deletion exons 1–9b  
 PS21M3    V(D)J class I  
 PS21M5    V(D)J class I  
 PS21M6   G143→A  Transition (CpG) 
 PS21M2   T595→G  Transversion 
 PS8M2 27  V(D)J class I  
 PS8M3   G606→C  Transversion 
 PS30M1 29 C508→Tb  Transition (CpG) 
 PS30M6   C508→Tb  Transition (CpG) 
 PS30M10   C508→Tb  Transition (CpG) 
 PS30M5   C454→T  Transition 
 PS30M2    V(D)J class I  
 PS30M3    V(D)J class I  
 PS30M7    V(D)J class I  
 PS9M2 29  V(D)J class I  
 PS23M2 31  Deletion exons 2–9  
 PS26BM2 31 Exclusion exons 2–3 V(D)J class I  
 PS26BM3    Deletion exons 1–9  
 PS26BM4   Exclusion exons 2–6  Insertion GCTGCCG @IVS5+1 
 PS29M1 31  Deletion exons 1–9  
 PS29M2    V(D)J class I  
 PS29M5    V(D)J class I  
 PS29M4    Deletion exon 2b   
 PS29M6    Deletion exon 2b  
 PS26aM1 31  V(D)J class I  
 PS26aM3   Exclusion exons 2–3 V(D)J class I  
 PS26aM6   Exclusion exons 2–3 V(D)J class I  
 PS26aM12   Exclusion exons 2–3 V(D)J class I  
 PS26aM9   Exclusion exon 4 IVS4+1 G→A Transition 
 PS26aM4   Exclusion exons 3–7 A307→T Transversion 
 PS26aM5   T81→C  Transition 
 PS4M1 31  V(D)J class I  
 PS4M3    V(D)J class I  
 PS4M4    V(D)J class I  
 PS4M5    V(D)J class I  
 PS4M7    V(D)J class I  
 PS4M2    Deletion exons 7–8  
 PS4M8   Deletion A381   
 PS4M9   Exclusion exon 8 NCc  
 PS18M1 32  V(D)J class I  
 PS18M2    Deletion 537–564  
 PS19M6 32 Exclusion 429–475 NCc  
 PS19M7   C508→T  Transition (CpG) 
 PS6M1 33  V(D)J class I  
 PS16M2 34  V(D)J class 3  
 PS16M9   C508→T  Transition (CpG) 
 PS20M1 34  V(D)J class I  
 PS20M2    C69→A Transversion 
 PS12BM1 34  V(D)J class I  
 PS12BM2   C508→T  Transition (CpG) 
 PS13M1 35 G207→T  Transversion 
 PS15M2 35  V(D)J class I  
 PS15M5    V(D)J class 3  
 PS15M4   C145→T  Transition 
 PS15M6    Deletion exon 1  
 PS7M11 35  V(D)J class I  
 PS7M20    V(D)J class I  
 PS7M13   Exclusion exon 6 IVS5-1 G→A Transition 
 PS14M2 35  V(D)J class I  
 PS14M4    V(D)J class I  
 PS5M1 35 C508→T  Transition (CpG) 
 PS5M8    V(D)J class I  
 PS5M4   Exclusion exons 2–6b NCc  
 PS5M6   Exclusion exons 2–6b NCc  
 PS5M9   Exclusion exons 2–6b NCc  
Term infantsd      
 MFS61aM2 36 G606→T  Transversion 
 MFS89M2 36 C454→T  Transition 
 MFS89M3    Deletion exons 1–9b  
 MFS89M18    Deletion exons 1–9b  
 MFS89M10   C508→T  Transition (CpG) 
 MFS89M12    Deletion exons 2–9b  
 MFS89M6    Deletion exons 2–9b  
 MFS89M4    V(D)J class I  
 MFS89M14    V(D)J class I  
 MFS89M15    V(D)J class I  
 MFS101M1 36 T536→Ab  Transversion 
 MFS101M2   T536→Ab  Transversion 
 MFS101M3    Deletion exons 6–9  
Table 1A

Continued

 MFS7M1 36  V(D)J class I  
 MFS7M3    V(D)J class I  
 MFS7M4    Deletion exon 1  
 MFS36M1 37  Deletion exons 7–9b  
 MFS36M2    Deletion exons 7–9b  
 MFS36M5    Deletion exons 7–9b  
 MFS36M4    Deletion exon 6  
 MFS12M4 38 Exclusion exons 2–3 V(D)J class I  
 MFS12M37    V(D)J class I  
 MFS12M34   C508→T  Transition (CpG) 
 MFS12M40   A401→G  Transition 
 MFS65M3 38 G3→Tb  Transversion 
 MFS65M19   G3→Tb  Transversion 
 MFS65M4   G3→Tb  Transversion 
 MFS65M5    Deletion exons 7–9  
 MFS65M9   Exclusion 8 IVS8+2 T→C Transition 
 MFS65M10    V(D)J class I  
 MFS65M11   G190→C  Transversion 
 MFS65M15   C151→T  Transition (CpG) 
 MFS65M17    V(D)J class I  
 MFS65M18   T82→G  Transversion 
 MFS94M3e 38 Exclusion exons 2–3 NCc  
 MFS94M1   Deletion429–475   
 MFS3M1 39 A484→C  Transversion 
 MFS3M2    V(D)J class I  
 MFS85M3 39 Exclusion exons 2–3 NCc  
 MFS83M3 39  Deletion exons 1–9  
 MFS87M1 39  Deletion exons 1–9  
 MFS87M2    V(D)J class I  
 MFS1M2 39  Deletion exons 1–9  
 MFS1M4    V(D)J class I  
 MFS43M4 39 ins108AT   
 MFS59M2 39  Deletion exons 7–8  
 MFS5M2 39  V(D)J class I  
 MFS58M1 39 G538→A  Transition 
 MFS58M3   G580→C  Transversion 
 MFS58M4    Deletion exons 1–3  
 MFS72M3 39  V(D)J class I  
 MFS72M8    Deletion exons 2–4  
 MFS37M1 39 G134→A  Transition 
 MFS37M2    Deletion exons 2–9  
 MFS37M4   Del230–234 ACCTG   
 MFS13M4 39 G628→A  Transition 
 MFS8M2 40 Del218–221 AATT   
 MFS8M3   Inclusion of first 49 bp of intron 1 IVS1+1G→A Transition 
 MFS25M4 40 Exclusion exon 8 IVS8+5G→A Transition 
 MFS60M1 40 G606→T  Transversion 
 MFS60M2    Deletion exon 4  
 MFS38M4 40 Exclusion exon 7 IVS6-1G→A Transition 
 MFS53M1 40  V(D)J class I  
 MFS53M2    V(D)J class I  
 MFS84M3 40  Deletion exon 1  
 MFS84M4    Deletion exons 2–9  
 MFS2M1 40  V(D)J class I  
 MFS2M2f   Exclusion exon 6 NCc  
 MFS2M4    V(D)J class I  
 MFS2M12   Exclusion exon 6 IVS6+1G→T Transversion 
 MFS2M13    V(D)J class I  
 MFS79M2 40  V(D)J class I  
 MFS6M1 40 Exclusion exons 2–3 V(D)J class I  
 MFS6M2   TCR α insert intron 1   
 MFS57M2 41  Deletion exon 1  
 MFS57M3   C151→T  Transition (CpG) 
 MFS57M5    V(D)J class I  
 MFS68M1 41 G197→A  Transition 
 MFS68M2   C508→T  Transition (CpG) 
 MFS68M3   C151→T  Transition (CpG) 
 MFS68M5   Inclusion of first 49-bp of intron 1 IVS1+5G→A Transition 
 MFS14M1 42 Ins108.AT   
 MFS88M1 42 C508→T  Transition (CpG) 
 MFS88M2    V(D)J class I  
 MFS88M5    Deletion exons 1–9  
 MFS7M1 36  V(D)J class I  
 MFS7M3    V(D)J class I  
 MFS7M4    Deletion exon 1  
 MFS36M1 37  Deletion exons 7–9b  
 MFS36M2    Deletion exons 7–9b  
 MFS36M5    Deletion exons 7–9b  
 MFS36M4    Deletion exon 6  
 MFS12M4 38 Exclusion exons 2–3 V(D)J class I  
 MFS12M37    V(D)J class I  
 MFS12M34   C508→T  Transition (CpG) 
 MFS12M40   A401→G  Transition 
 MFS65M3 38 G3→Tb  Transversion 
 MFS65M19   G3→Tb  Transversion 
 MFS65M4   G3→Tb  Transversion 
 MFS65M5    Deletion exons 7–9  
 MFS65M9   Exclusion 8 IVS8+2 T→C Transition 
 MFS65M10    V(D)J class I  
 MFS65M11   G190→C  Transversion 
 MFS65M15   C151→T  Transition (CpG) 
 MFS65M17    V(D)J class I  
 MFS65M18   T82→G  Transversion 
 MFS94M3e 38 Exclusion exons 2–3 NCc  
 MFS94M1   Deletion429–475   
 MFS3M1 39 A484→C  Transversion 
 MFS3M2    V(D)J class I  
 MFS85M3 39 Exclusion exons 2–3 NCc  
 MFS83M3 39  Deletion exons 1–9  
 MFS87M1 39  Deletion exons 1–9  
 MFS87M2    V(D)J class I  
 MFS1M2 39  Deletion exons 1–9  
 MFS1M4    V(D)J class I  
 MFS43M4 39 ins108AT   
 MFS59M2 39  Deletion exons 7–8  
 MFS5M2 39  V(D)J class I  
 MFS58M1 39 G538→A  Transition 
 MFS58M3   G580→C  Transversion 
 MFS58M4    Deletion exons 1–3  
 MFS72M3 39  V(D)J class I  
 MFS72M8    Deletion exons 2–4  
 MFS37M1 39 G134→A  Transition 
 MFS37M2    Deletion exons 2–9  
 MFS37M4   Del230–234 ACCTG   
 MFS13M4 39 G628→A  Transition 
 MFS8M2 40 Del218–221 AATT   
 MFS8M3   Inclusion of first 49 bp of intron 1 IVS1+1G→A Transition 
 MFS25M4 40 Exclusion exon 8 IVS8+5G→A Transition 
 MFS60M1 40 G606→T  Transversion 
 MFS60M2    Deletion exon 4  
 MFS38M4 40 Exclusion exon 7 IVS6-1G→A Transition 
 MFS53M1 40  V(D)J class I  
 MFS53M2    V(D)J class I  
 MFS84M3 40  Deletion exon 1  
 MFS84M4    Deletion exons 2–9  
 MFS2M1 40  V(D)J class I  
 MFS2M2f   Exclusion exon 6 NCc  
 MFS2M4    V(D)J class I  
 MFS2M12   Exclusion exon 6 IVS6+1G→T Transversion 
 MFS2M13    V(D)J class I  
 MFS79M2 40  V(D)J class I  
 MFS6M1 40 Exclusion exons 2–3 V(D)J class I  
 MFS6M2   TCR α insert intron 1   
 MFS57M2 41  Deletion exon 1  
 MFS57M3   C151→T  Transition (CpG) 
 MFS57M5    V(D)J class I  
 MFS68M1 41 G197→A  Transition 
 MFS68M2   C508→T  Transition (CpG) 
 MFS68M3   C151→T  Transition (CpG) 
 MFS68M5   Inclusion of first 49-bp of intron 1 IVS1+5G→A Transition 
 MFS14M1 42 Ins108.AT   
 MFS88M1 42 C508→T  Transition (CpG) 
 MFS88M2    V(D)J class I  
 MFS88M5    Deletion exons 1–9  
a

GA, gestational age in weeks.

b

These mutants are considered to represent a single independent HPRT mutational event in this subject with subsequent in vivo clonal expansion.

c

NC, not characterized.

d

Mutational spectra data for term infants has been previously reported (7) except for the following subjects: MFS101; MFS85; MFS5; MFS58; MFS72; MFS37; MFS13; MFS38; MFS53; and MFS6.

e

MFS94M3 showed the exclusion of exons 2 and 3 from cDNA, but is not a V(D)J recombinase-mediated deletion. It is probably a splice alteration mutation, but this could not be confirmed by genomic DNA analysis because the mutant arose in a female.

f

MFS2M2 showed the exclusion of exon 6 from cDNA, but no mutations could be found in the genomic region containing exon 6, therefore it is considered an uncharacterized splicing alteration.

Table 2

Distribution analysis of in utero HPRT mutationsa

PretermTermCombined
MaleFemaleMaleFemaleMaleFemale
n%bn%bn%bn%bn%bn%b
No. of subjects 14 27 11 23 43 10 19 37 70 16 30 
No. of mutants 47 31 19 13 68 45 17 11 115 76 36 24 
Independent mutationsc 43 31 17 12.5 61 44 17 12.5 104 75 34 25 
All mutations 43 100 17 100 61 100 17 100 104 100 34 100 
 Small alterations 11 25 35 23 37 35 34 33 12 35 
  Transitions 16 17.5 13 21 29 20 19 23 
  Transversions 17.5 13 11 11 12 
  ≤2-bp insertions 
  ≤2-bp deletions 
 Large alterations 30 70 10 59 37 61 53 67 64 19 56 
  V(D)J deletions 21 49 10 59 17 28 35 38 36 16 47 
  Non-V(D)J deletionsd 19 20 33 12 28 27 
  >2-bp insertions 
 Uncharacterized 12 
Small alterations 11 100 100 23 100 100 34 100 12 100 
  Transitions 64 50 13 56 83 20 59 67 
  Transversions 27 50 35 17 11 32 33 
  ≤2-bp insertions 
  ≤2-bp deletions 
Large alterations 30 100 10 100 37 100 100 67 100 19 100 
  V(D)J deletions 21 70 10 100 17 46 67 38 57 16 84 
  Non-V(D)J deletionsd 27 20 54 22 28 42 11 
  >2-bp insertions 11 
PretermTermCombined
MaleFemaleMaleFemaleMaleFemale
n%bn%bn%bn%bn%bn%b
No. of subjects 14 27 11 23 43 10 19 37 70 16 30 
No. of mutants 47 31 19 13 68 45 17 11 115 76 36 24 
Independent mutationsc 43 31 17 12.5 61 44 17 12.5 104 75 34 25 
All mutations 43 100 17 100 61 100 17 100 104 100 34 100 
 Small alterations 11 25 35 23 37 35 34 33 12 35 
  Transitions 16 17.5 13 21 29 20 19 23 
  Transversions 17.5 13 11 11 12 
  ≤2-bp insertions 
  ≤2-bp deletions 
 Large alterations 30 70 10 59 37 61 53 67 64 19 56 
  V(D)J deletions 21 49 10 59 17 28 35 38 36 16 47 
  Non-V(D)J deletionsd 19 20 33 12 28 27 
  >2-bp insertions 
 Uncharacterized 12 
Small alterations 11 100 100 23 100 100 34 100 12 100 
  Transitions 64 50 13 56 83 20 59 67 
  Transversions 27 50 35 17 11 32 33 
  ≤2-bp insertions 
  ≤2-bp deletions 
Large alterations 30 100 10 100 37 100 100 67 100 19 100 
  V(D)J deletions 21 70 10 100 17 46 67 38 57 16 84 
  Non-V(D)J deletionsd 27 20 54 22 28 42 11 
  >2-bp insertions 11 
a

Distribution analysis was confined to a comparative analysis between independent characterized HPRT mutation events.

b

Percentage was calculated as the ratio of the number of each specific mutation (n) over the number of independent mutations for each group.

c

Represents single HPRT mutational events by correcting for in vivo clonal expansion.

d

Represent deletions >2 bp that are not mediated by V(D)J recombinase.

We thank Holly Pasackow for obtaining preterm cord blood samples.

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