Sai-Mei Hou

Molecular characterization of two deletion events involving Alu-sequences, one novel base substitution and two tentative hotspot mutations in the hypoxanthine phosphoribosyltransferase (HPRT) gene in five patients with Lesch-Nyhan syndrome

Mutations identified in the hypoxanthine phosphoribosyltransferase (HPRT) gene of patients with Lesch-Nyhan (LN) syndrome are dominated by simple base substitutions. Few hotspot positions have been identified, and only three large genomic rearrangements have been characterized at the molecular level. We have identified one novel mutation, two tentative hot spot mutations, and two deletions by direct sequencing of HPRT cDNA or genomic DNA from fibroblasts or T-lymphocytes from LN patients in five unrelated families. One is a missense mutation caused by a 610C→T transition of the first base of HPRT exon 9. This mutation has not been described previously in an LN patient. A nonsense mutation caused by a 508C→T transition at a CpG site in HPRT exon 7 in the second patient and his younger brother is the fifth mutation of this kind among LN patients. Another tentative hotspot mutation in the third patient, a frame shift caused by a G nucleotide insertion in a monotonous repeat of six Gs in HPRT exon 3, has been reported previously in three other LN patients. The fourth patient had a tandem deletion: a 57-bp deletion in an internally repeated Alu-sequence of intron 1 was separated by 14 bp from a 627-bp deletion that included HPRT exon 2 and was flanked by a 4-bp repeat. This complex mutation is probably caused by a combination of homologous recombination and replication slippage. Another large genomic deletion of 2969 bp in the fifth patient extended from one Alu-sequence in the promoter region to another Alu-sequence of intron 1, deleting the whole of HPRT exon 1. The breakpoints were located within two 39-bp homologous sequences, one of which overlapped with a well-conserved 26-bp Alu-core sequence previously suggested as promoting recombination. These results contribute to the establishment of a molecular spectrum of LN mutations, support previous data indicating possible mutational hotspots, and provide evidence for the involvement of Alu-mediated recombination in HPRT deletion mutagenesis.

Mutational spectra at the hypoxanthine-guanine phosphoribosyltransferase (HPRT) locus in T-lymphocytes of nonsmoking and smoking lung cancer patients

Molecular analysis of mutations at the hypoxanthine-guanine phosphoribosyltransferase (HPRT) locus in peripheral blood T-lymphocytes can provide information on mechanisms of somatic in vivo mutation in populations exposed to exogenous carcinogens and in individuals with inherent susceptibility to cancer and other diseases. To study possible mutational changes associated with smoking as a risk factor for lung cancer, we analyzed HPRT mutations in T-cells of newly diagnosed, nonsmoking and smoking lung cancer patients before treatment. Reverse transcriptase polymerase chain reaction (RT-PCR) and DNA sequencing methods were used to identify 146 independent mutations, 73 each from 32 nonsmoking and 31 smoking cases. In 35 T-cell mutants, the HPRT cDNA showed loss of an entire exon, indicating a splicing mutation. Among the remaining 111 fully characterized mutations in the coding region, single base pair (bp) substitutions predominated with 79% (48/61) in nonsmokers and 90% (45/50) in smokers. Frameshift and small deletion (1-24 bp) mutations were found in 18 mutants. The distribution of base pair substitutions was nonrandom, with significant clustering at previously identified hotspot positions 143, 197 and 617 in the HPRT coding sequence (P< or =0.008). One additional hotspot, GC-->TA at position 606, was observed only in smokers (P=0.006). The frequency of GC>TA transversions was higher in smokers (13%) than in nonsmokers (6%). Conversely, smokers had a lower frequency of GC>AT transitions (24%) than nonsmokers (35%). This smoking-associated shift of the HPRT mutational spectrum, although not statistically significant, is consistent with the in vitro mutagenicity of benzo(a)pyrene (BaP), a prominent carcinogen of tobacco smoke, and with known differences in the TP53 mutational spectrum in lung tumors of smokers and nonsmokers. Among nonsmokers, the HPRT mutational spectra in healthy population controls and lung cancer patients were similar, but there was a marginally significant difference (P=0.07) in the distribution of base pair substitutions between smoking controls and patients. These results suggest that (i) general mechanisms of somatic mutagenesis in individuals with possible predisposition to cancer (e.g. nonsmoking lung cancer patients) are not different from those in normal healthy individuals, and (ii) the HPRT gene in T-cells is a useful reporter locus for smoking-associated somatic in vivo mutations occurring early in lung cancer development.

Influence of smoking and donor age on the spectrum of in vivo mutation at the HPRT-locus in T lymphocytes of healthy adults

Types and frequencies of in vivo mutation in the hypoxanthine-guanine phosphoribosyl-transferase (HPRT) gene was studied in 142 T cell mutants from 78 healthy nonsmoking and smoking adults with a mean of 65 years. The HPRT mutant frequency in the nonsmokers was 18.7 +/- 12.0 x 10(-6), and in the smokers 26.6 +/- 18.5 x 10(-6) (mean +/- S.D., P < 0.01). Among 107 single base pair substitutions (SBS) in the coding region of the HPRT gene, one new mutable site, one novel nonsense mutation and three not previously reported SBS were identified. Transitions accounted for 59% of the SBS and transversions for 41%. GC > AT transitions were the predominant type of mutation, with 50% of all SBS. The mutations showed a nonrandom distribution along the coding sequence, with three significant hotspots at positions 143, 197 and 617 (13, 14 and 7 mutations, respectively). There was no difference between smokers and nonsmokers with regard to the distribution of mutations at these hotspot positions. However, 85% of the mutations at GC base pairs and 88% of the mutations at AT base pairs in smokers occurred at sites with guanine or thymine, respectively, in the nontranscribed DNA strand. Moreover, smokers had a higher frequency of transversions and lower frequency of transitions than nonsmokers did. Particularly, GC > TA transversions were increased in smokers (11%) compared to nonsmokers (2%), which suggests that tobacco-smoke induced adducts at guanine bases in the nontranscribed DNA strand contributes to the increase of HPRT mutation in smokers. Overall, these results were very similar to the mutational spectra in two younger study populations reported previously [K.J. Burkhart-Schultz, C.L. Thompson, I.M. Jones, Spectrum of somatic mutation at the hypoxanthine phosphoribosyltransferase (HPRT) gene of healthy people, Carcinogenesis 17 (1996) 1871-1883; A. Podlutsky, A.-M. Osterholm, S.-M. Hou, A. Hofmaier, B. Lambert, Spectrum of point mutations in the coding region of the hypoxanthine-guanine phosphoribosyltransferase, Carcinogenesis 19 (1998) 557-566]. With the possible exception of an increase of mutations at hotspot position 143, and a decrease of 5-methylcytosine deamination mediated transitions at CpG-sites in the older individuals, there were no differences between the mutational spectra of old and young adults. In conclusion, both smoking and ageing seem to have minor influences on the spectrum of HPRT mutation in T cells.

Splicing mutations at the HPRT locus in human T-lymphocytes in vivo

We studied 58 splicing mutations originating in vivo at the hypoxanthine guanine phosphoribosyltransferase (HPRT) locus in T‐cells of 30 nonsmoking males. A nonrandom distribution of skipped exons was seen after cDNA sequence analysis, with 71% involving exons 2–3 (15), 4 (11), and 8 (15). The mutations likely to have caused the aberrant splicing were identified in 36 mutants by genomic sequencing. The most frequently observed mutations were simple base substitutions (27) and small deletions (7). Among the base substitutions, 23 occurred in the splice consensus sequences, mainly at the highly conserved dinucleotides (21), and preferentially in the acceptor sites (15). The remaining four base substitutions occurred in the coding sequence where one tandem base substitution, one single bp insertion, and two single bp deletions were also observed. The predicted change in three of the base substitutions would be a stop codon. The tandem mutation (CC → TT) occurred at position 550–551, a possible hotspot for splicing mutations (five of nine previously reported base substitutions at position 551, all C → T, resulted in abnormal splicing). Four of the base substitutions were new HPRT mutations, two in splice sites (IVS7‐3T → G and IVS8 + 3A → C) and two in the coding sequence (307A → T and 594C → G). All the small deletions (>1 bp) affected the acceptor sites. The only three identified mutations related to skipping of exons 2 and 3 were located within exon 3, suggesting a frequent involvement of unknown splicing elements distant from these exons. Environ. Mol. Mutagen. 32:25–32, 1998

Validation of the human T-lymphocyte cloning assay--ring test report from the EU concerted action on HPRT mutation (EUCAHM).

The T-cell cloning assay, which enables the enumeration and molecular analysis of 6-thioguanine resistant (HPRT-negative) mutant T-cells, has been extensively used for studying human somatic gene mutation in vivo. However, large inter-laboratory variations in the HPRT mutant frequency (MF) call for further investigation of inter-laboratory differences in the experimental methodology, and development of an optimal but easy uniform cloning protocol. As part of the EU Concerted Action on HPRT Mutation (EUCAHM), we have carried out two Ring tests for the T-cell cloning assay. For each test, duplicate and coded samples from three buffy coats were distributed to five laboratories for determination of MF using six different protocols. The results indicated a good agreement between split samples within each laboratory. However, both the cloning efficiencies (CEs) and MFs measured for the same blood donors showed substantial inter-laboratory variations. Also, different medium compositions used in one and the same laboratory resulted in a remarkable difference in the level of MF. A uniform operating protocol (UOP) was proposed and compared with the traditional protocols in the second Ring test. The UOP (preincubation) increased the CE in laboratories traditionally using preincubation, but decreased the CE in laboratories traditionally using priming. Adjusted for donor, use of different protocols contributed significantly to the overall variation in lnCE (P = 0.0004) and lnMF (P = 0.03), but there was no significant laboratory effect on the lnCE (P = 0.38) or lnMF (P = 0.14) produced by the UOP alone. Finally, a simplified version of the UOP using the serum-free medium X-Vivo 10 and PMA was tested in one laboratory, and found to produce a considerable increase in CE. This modified UOP needs to be further evaluated in order to be used for future databases on HPRT MFs in various populations.

Analysis of mutation at the hprt locus in human T lymphocytes

Studies of mutation at the hypoxanthine phosphoribosyl transferase (hrpt) locus in human T-cells have the potential to elucidate the molecular basis of in vivo mutagenesis, reveal exposure dependent changes in ther background frequency of mutation, and provide knowledge on individual sensitivity. Styrene exposed lamination workers in Bohemia showed a significantly higher frequency of hprt mutant cells than Swedish control populations studied simultaneously. In a study of 47 healthy, non-smoking male bus maintenance workers exposed to diesel exhausts, soot and oil, and 22 unexposed controls, a significant correlation (P = 0.008) was obtained between the levels of aromatic DNA adducts and frequencies of hprt-mutant T-cells. In the group of workers with the highest exposure, subjects with glutathione S-transferase (GSTM1) deficiency showed significantly higher (P < 0.05) frequency of hprt mutant T-cells than GSTM1-positive subjects. The highest adduct levels were found in subjects with the combined genotype of GSTM1 and NAT2 deficiency (GSTM1-negative slow acetylators). These results indicate that GSTM1 and NAT2 genotypes may play a role in determining the individual levels of hprt mutation and DNA adducts. Using PCR-based screening methods, hprt mutations have been classified in 462 T-cell clones from 43 subjects in this study population. Deletions were found in 3% of the mutants, coding errors in 81% and splice mutations in 17%. Transitions and transversions were equally common, and all types of base substitutions were detected.

Hprt activities and RNA phenotypes in 6-thioguanine resistant human T-lymphocytes

The phenotypic effects of mutation in the hypoxanthine phosphoribosyltransferase (hprt) gene on hprt enzyme activity and hprt mRNA levels were studied in 6-thioguanine (TG) resistant human T-cell clones with various types of hprt mutation. The mean enzyme activity in 16 TG selected clones was less than 1% of the mean in unselected clones. The hprt mRNA levels, measured by a quantitative RNA/RNA solution hybridization assay, were within the normal range in 38% of the mutant clones. Reduced hprt mRNA levels were found in all of three nonsense mutations, four out of five splicing mutations, both of two clones with genomic alterations, three out of five missense mutations and one out of four frameshift mutations caused by 1-4-bp deletions. Intermediate and high enzyme activity and normal hprt mRNA levels were found in two TG selected clones where no hprt mutation was detected. Several clones with very low hprt mRNA levels were found to yield hprt cDNA by PCR amplification. These results show that hprt mutation leads to decreased steady state levels of hprt mRNA in a majority of TG resistant T-cell clones, and that many different types of hprt mutation can have this effect.

Novel types of mutation identified at the hprt locus of human T-lymphocytes

The T-cell cloning assay detecting mutations at the hypoxanthine-guanine phosphoribosyl transferase (hprt) locus provides a well developed system for studying human somatic gene mutation. The hprt mutational spectrum comprises missense, nonsense and splice mutations, as well as large structural alterations including deletions, duplications and insertions. Only few of the hprt deletions, which represent 10-15% of background in vivo mutations in T-cells of adults, have been characterized in detail at the genomic level, and the mechanisms involved in the majority of hprt structural alterations remain unknown. Illegitimate activity of V(D)J recombinase resulting in deletion of hprt exons 2 + 3 has been shown to account for 40% of the hprt mutations in T-lymphocytes of human newborns and a few percent of the mutations in adults. In this report, novel recombinational mechanisms were identified by characterization of two T-cell mutants. One mutant derived from a healthy adult was found to have a 3.2-kb genomic insertion in the first intron of the hprt gene, and a 369-bp T-cell receptort (TCR) alpha gene sequence between exons 1 and 2 of its hprt cDNA. This mutation provides unique and direct evidence for illegitimate recombination between the TCR gene and the hprt gene in human T-lymphocytes in vivo. Moreover, the mutation identifies a novel cDNA sequence for the TCR alpha chain variable region. Another hprt- mutant, obtained from a T-cell culture treated with acetaldehyde, showed that splice mutation can be caused by a large deletion detectable on Southern blot. This 3.4-kb deletion involved both intron 1 and exon 2 sequences and was flanked by 5-bp direct repeats. The utilization of a novel cryptic acceptor site in intron 1, located far upstream from the lost consensus splice site, resulted in a partial inclusion of the intron 1 sequence in the hprt cDNA.

The (CCTTT)n microsatellite polymorphism in the NOS2 gene may influence lung cancer risk and long-term survival, especially in non-smokers

We analyzed the associations of the NOS2 (CCTTT)n promoter polymorphism to lung cancer risk and tumor histology in smokers and non-smokers. We also investigated lung cancer long-term survival in relation to the polymorphism, smoking data, histology, age at diagnosis, and gender. One hundred eighty-five lung-cancer patients and 164 matched controls, where non-smokers were enriched among the lung cancer cases, were genotyped by fragment analysis and sequencing. Genotypes were combined with information on histology, patient smoking status, and cancer-specific death, using a 20-year follow-up. We divided the (CCTTT)n alleles into short (n ≤ 10), intermediate (n = 11–12), and long (n ≥ 13). Patients homozygous for short repeats had significantly increased risk of lung cancer (p = 0.030) compared to carriers of two long alleles (LL). Lack of long allele was associated with a significantly increased lung cancer risk overall (p = 0.011), especially among non-smokers (p = 0.001). A significantly higher lung cancer survival was seen in non-smokers compared to smokers (p = 0.046) and in low-dose smokers compared to high-dose smokers at the time of diagnosis (p = 0.028). Moreover, non-smoking patients with squamous cell carcinoma (p = 0.015) or adenocarcinoma (p = 0.024) showed a significantly lower survival compared to other lung carcinomas. Nitric oxide can induce proliferation as well as apoptosis depending on cellular context. Our results suggest that the (CCTTT)n NOS2 microsatellite may influence the risk of developing lung cancer, especially in non-smokers, possibly by affecting intracellular nitric oxide levels. Our results also give additional information about the yet poorly understood etiological and prognostic differences between lung cancer in non-smokers and smokers.

Influence of XPD variant alleles on p53 mutations in lung tumors of nonsmokers and smokers

Abstract The DNA repair protein XPD is involved in the transcription-coupled nucleotide excision repair of DNA lesions induced by many tobacco and environmental carcinogens. Common polymorphisms in XPD exon 10 (G>A, Asp312Asn) and exon 23 (A>C, Lys751Gln) have been identified, and lung cancer cases homozygous for the variant allele in either exon have been reported to have a reduced repair capacity against benzo[ a ]pyrene-induced DNA damage. We therefore investigated a possible effect of these variant alleles on the p53 mutation frequency and spectrum among 97 Swedish lung cancer patients. Transversions were found to occur more frequently among patients with at least one variant allele than among wild type homozygotes. The XPD variant alleles may thus be associated with reduced DNA repair proficiency. This finding is not in accord with the previous report that associated the exon 23 wild type allele with increased frequency of X-ray-induced chromatid aberrations.