Restriction site mutation (RSM) analysis of 2-acetylaminofluorene (2-AAF)-induced mouse liver mutations and comparison with the measurement of in vivo micronucleus induction in the bone marrows of (2-AAF)-treated mice

We report here the successful application of the restriction site mutation (RSM) assay in detecting 2-acetylaminofluorene (2-AAF)-induced mouse liver mutations. A total of seven 2-AAF-induced liver mutations were detected out of a total of 304 analyses performed on 2-AAF-treated liver tissue. No mutations were detected in the 190 RSM analyses performed on untreated liver tissue. The 2-AAF-induced point mutations comprised 60% GC-->TA transversions, 30% GC-->AT transitions, 10% GC-->CG transversions, and 1 insertional event was also detected. All seven mutations were detected in intron 6 of the mouse p53 gene, with no mutations detectable in exons 4 or 5, supporting our previous data on the greater mutability of intron regions. In addition to the RSM analysis, we also report the application of the in vivo bone marrow micronucleus assay in detecting the clastogenicity of 2-AAF. We detected a small, but statistically significant, increase in the number of micronuclei induced by 2-AAF, but only after 2,000 cells were scored. This also confirms previous data showing that 2-AAF is a weak clastogen. Finally, we attempted to compare the sensitivity of the two assays to 2-AAF-induced genotoxicity, as had been previously undertaken with ENU. Both assays detected genotoxicity in their respective tissues; however, different endpoints were analysed. The RSM assay appears to be more adaptable than the micronucleus assay, due to its tissue and organism independence and has the potential to provide more molecular information on genotoxicity. Teratogenesis Carcinog. Mutagen. 20:107-117, 2000.     

The application of the restriction site mutation assay to compare 1-ethyl-1-nitrosourea-induced mutations between the endogenous p53 gene and the transgenic LacZ gene in MutaMouse testes

Transgenic mouse modelling has provided a new approach to study the various steps involved in spontaneous and induced mutagenesis in rodent somatic and germline tissues in vivo. However, the important question arises as to whether mutations occur at the same rate in transgenes as in endogenous genes. Here, the restriction site mutation (RSM) assay was used to study mutations induced in the endogenous p53 gene and LacZ transgene of MutaMouse testes treated with 1-ethyl-1-nitrosourea (ENU). The aim of these experiments was to compare mutation susceptibility between the endogenous p53 gene and the integrated LacZ gene in the transgenic mouse. ENU-treated and control testes were analysed 102 days after treatment; a total of 297 RSM analyses were performed on ENU-treated and untreated testis DNA. Ten mutational events were detected in the p53 gene (exon 5 and intron 8), two of which occurred in untreated animals and probably represent spontaneous events. Only a single mutation was detected in the LacZ gene of an ENU-treated animal by the RSM assay. Thus the RSM assay can readily detect ENU-induced mutations in the p53 gene, but not in the LacZ transgene. Comparison of the LacZ RSM mutation data with results from a previous study of identically dosed MutaMice in the transgenic selection assay [Ashby, J., Gorelick,N.J. and Shelby,M.D. (1997) Mutat. Res., 388, 111-122] showed that LacZ mutations were far more readily recovered with the MutaMouse transgenic selection assay than by RSM analysis. The reason for the relative inability of the RSM assay to detect LacZ mutations may be the smaller target size of the RSM analysis compared with the transgenic selection assay (16 bases compared with 3000 bases). Taking into account the different target sizes by calculating the mutation frequency per base allowed the RSM data regarding p53 and LacZ to be compared with previously published data from transgenic selection assays. These studies demonstrated that the p53 mutations were present at mutation frequencies (per base) 5- to 70-fold higher than the LacZ gene mutations. In addition, the LacZ mutation frequency per base found in the RSM was an order of magnitude higher than that found in the transgenic selection assay. The transgenic selection assay is more sensitive per locus (due to the larger target of the LacZ gene), as evidenced by ability to detect ENU-induced testes mutations readily.     

Mutations spanning P53 exons 5-9 detected by non-isotopic RNAse cleavage assay and protein expression in human colon cancer

The non-isotopic assay (NIRCA), based on the observation that RNAse is able to specifically cleave a single mismatch in RNA/RNA duplexes, has been recently proposed to detect p53 mutations. To verify the use of this method as a valid screening for P53 mutations in a routinely collected cancer series, we used this assay on 3 cases with normal and 5 cases with abnormal P53 expression detected by Western blots. In all cases, P53 exons 5-6, 7 and 8-9 regions were analyzed. There were mutations only in the five overexpressed cases: two cases showed mutations in exon 5, one between intron 6 and exon 6 and two in the region spanning exons 8 and 9. Our experience showed NIRCA to be fast, reliable and providing the ability to study long target regions in a single step, thus making this assay useful for genetic screenings.     

Analyis of factor VIII mRNA reveals defects in everyone of 28 haemophilia A patients

Haemophilia A is a mutationally heterogeneous disease causedby defects in the large and complex factor VIII gene. Recentstudies examining the putative promoter, all exons and mostintron/exon boundaries have failed to detect mutations in halfthe patients with severe disease leading to hypotheses suchas mutations in remote controlling regions or even in genesother than factor VIII. We have amplified the factor VIII gene(putative promotor, coding region and poly-adenylation/cleavagesignal region) in 8 fragments from reverse transcribed mRNAand genomic DNA. Any mutation is then located by chemical mismatchdetection and characterised by direct sequencing. This rapidand efficient method has been fully successful and has revealedan unusual cluster of mutations causing severe disease. Of the28 patients we have reported, 5 had mild or moderate diseaseand all had a missense mutation. Twenty-three patients wereseverely affected and 13 of these had different detrimentalmutations that were fully characterised at the genomic DNA level.The remaining 10 patients all had mRNA with exon 22 not contiguousto exon 23. Since all exons were normal and so were the splicesites of intron 22, the mutation in these patients should bein the regions of intron 22 that were not screened. These resultsprove that all haemophilia A cases are due to mutations of thefactor VIII gene where, unexpectedly, intron 22 seems to bethe target of approximately 40% of the mutations causing severehaemophilia A.     

c-myc GENE ABNORMALITIES IN MUCOSA-ASSOCIATED LYMPHOID TISSUE (MALT) LYMPHOMAS

c-myc gene abnormalities associated with lymphomagenesis, including rearrangements and mutations in the regulatory region between exon I and intron I, have been studied in 54 MALT lymphomas (43 low and 11 high grade) and 36 nodal lymphomas (27 low and 9 high grade). By Southern blot analysis, none of the 54 MALT lymphomas but 2 of 36 nodal lymphomas had c-myc gene rearrangements. Defined tumour cell populations from all MALT lymphoma cases were isolated by microdissection from frozen tissue sections and analysed by polymerase chain reaction–single-strand conformational polymorphism (PCR–SSCP) and direct sequencing for somatic mutations in the exon I/intron I region of the gene. Point mutations in this region were identified in nine cases of MALT lymphoma (7/43=16·2 per cent of low grade; 2/11=18·1 per cent of high grade). These mutations were located at either the exon I/intron I border of myc intron factor (MIF) binding sites, which are critical in the negative regulation of c-myc expression. Of the nodal lymphomas, only the two cases (5·6 per cent) with c-myc gene rearrangement showed scattered or clustered mutations. These results suggest that c-myc mutations in MALT lymphomas are unlikely to be associated with chromosome translocation, which is the main cause of somatic mutations observed in other types of lymphoma. The mutations involving the c-myc regulatory regions may play a pathogenetic role in at least a proportion of MALT lymphomas. © 1997 by John Wiley & Sons, Ltd.     

Identification of rare and novel mutations in the CFTR genes of CF patients in Southern England

Cystic fibrosis patients referred to two genetics centres in southern England and not found to carry common CF-associated mutations in one or both of their CFTR genes have been subjected to an extensive mutation search. The whole of the coding region of the CFTR gene, all intron–exon boundaries and 5′ and 3′ untranslated regions have been examined by a combination of single stranded conformational polymorphism analysis and chemical mismatch detection; 48 chromosomes with rare mutations have been identified, including 7 novel mutations, 182delT in exon 1, G27X in exon 2, Q151X in exon 4, Q220X in exon 6a, Q525X in exon 10, 3041delG in exon 16, and 4271delC in exon 23. © 1994 Wiley-Liss, Inc.     

A study of ENU-induced mutagenesis in the mouse using the restriction site mutation (RSM) assay.

We report here the application of the restriction site mutation (RSM) assay to study the induction of mutations by the alkylating agent ENU. Specifically, mutations were sought in the spleen and bone marrow of mice 3, 10, and 100 days after being treated with ENU; this was compared to data previously published from our laboratory on ENU-induced testes mutations. It was found that the ENU-induced mutations were all at GC bases implicating the O(6)-ethylguanosine adduct. The mutations detected reached a peak at day 10 in the spleen and were detectable to a lesser extent at 100 days, which is similar to the testes data. In the bone marrow, the mutation level rose until day 100, although the level remained below that of the spleen and testes. However, by studying the mutations detected in control animals, it was found that spontaneous mutational events were detectable at the day 100 time point in all three tissues. Hence the spleen, testes, and bone marrow mutations at day 100 in the ENU-treated samples were probably spontaneous mutational events with very few genuine ENU-induced mutations remaining in any of these tissues after 100 days. This paper also demonstrates the applicability of the inverse RSM methodology in the detection of ENU-induced mutations, whereby mutations can be detected by the conversion of one restriction site to another. The iRSM assay appears to be particularly suitable to studying alkylating agents due to their known sequence specific mutation induction. We also show a comparison of the bone marrow micronucleus data with the RSM assay and show that both assays are capable of detecting the genotoxicity of ENU to the mouse bone marrow in vivo.     

The restriction site mutation (RSM) method: clinical applications

The restriction site mutation (RSM) method has been developed over the past 13 years as a sensitive mutation test which can detect mutations in restriction sites in any gene. Due to the fact that 5/8 of the main mutation hotspots in the TP53 gene fall within restriction sites, RSM can analyse them for the presence of rare mutations (1 mutation in 10000 non-mutated copies). After validating the usefulness of RSM in detecting mutagen-induced mutations, we recently turned our attention to looking for TP53 mutations in pre-malignant tissue. We show here that RSM can detect early TP53 mutations in pre-malignant tissue of the oesophagus, stomach, colon and bladder. We can also use these clinical mutation data to speculate as to the causative mutagens involved in these cancer conditions. We here use an example of mutations detected in gastric tissue and those induced in vitro by hydrogen peroxide.     

非缺失/重复型Duchenne肌营养不良症患者的致病点突变分析

目的检测非缺失／重复突变型Duchenne肌营养不良症（Duchenne muscular dystrophy，DMD）的致病点突变。方法对6个家系的6个无关DMD男性患者的DMD基因的79个外显子及5′-3′-非翻译序列进行PER扩增，产物通过变性高效液相色谱（denaturing high performance liquid chromatography，DHPLC）技术进行突变筛查。结果6例非缺失／重复突变型Duchenne肌营养不良症患者，检测出了5例患者的致病点突变，即697-698insGT，C616T，G1255T，C4279T和C2302T。第1个点突变引起移码突变，后4个致病点突变引起翻译的提前终止，最终导致Duchenne肌营养不良症。患者3除致病点突变外，在第39内含子还发现1个T5586＋61A点突变；患者5还检测出了一个位于第8外显子的错义突变；而没有检出致病点突变的患者6，发现了2个外显子突变及2个内含子序列点突变，即C2168＋13T、G5234A、C5280T和5740-13dupG。所有检出的突变有7个点突变未见报道。结论变性高效液相色谱技术结合测序，可用于检测DMD患者的点突变．该方法具有准确，灵敏的特点。     

Hypermutation of the MYC gene in diffuse large cell lymphomas with translocations involving band 8q24

The sequence of the exon 1/intron 1 boundary region of the MYC gene was determined in two diffuse large cell lymphomas (DLCL), one with t(8;14) (q24;q32) and the other with t(8;22) (q24;q11). Both tumors had multiple mutations in this region. Also, both tumors had mutations in the protein binding site in intron I, which is a frequent target for mutational inactivation in endemic Burkitt's lymphoma (eBL). The translocations at 8q24, and multiple mutations in the exon 1/intron 1 boundary region, are reminiscent of similar findings in eBL. The same underlying oncogenic event that occurs in most eBLs is thus found in some DLCLs. © 1993 Wiley-Liss, Inc.     

Evaluation of a plasmid-based transgenic mouse model for detecting in vivo mutations

To study in vivo somatic mutations a C57BL/6 transgenic mousemodel was constructed harboring multiple chromosomally integratedcopies of the plasmid pUR288, which carried the lacZ reportergene as the mutational target. We previously demonstrated thatlacZ-containing plasmids could be rescued from their integratedstate efficient enough to detect mutations in lacZ by positiveselection. The smaller size of the plasmid vector, as comparedwith our earlier transgenic mouse model based on bacteriophagelambda vectors, should offer considerable advantages in termsof rescue efficiency and sensitivity to large size alterationsin the lacZ gene. To evaluate the plasmid-based mouse modelfor its suitability to detect in vivo mutations, we determinedmutant frequencies in different organs of untreated and ethylnitrosourea (ENU)-treated animals using a new, improved protocol.The rescue efficiencies obtained were as high as 200 000/µggenomic DNA; millions of transformants could be obtained inone single experiment. The average spontaneous mutant frequencyin four different organs of 4- to 8-week-old mice ranged from4.41 to 6.82x10^–5;, compared with a mutant frequency ofthe same plasmid grown in Escherichia coli of     

Glycogen storage disease type Ib: structural and mutational analysis of the microsomal glucose-6-phosphate transporter gene.

Glycogen storage disease type Ib is caused by a mutation in the gene encoding microsomal glucose-6-phosphate (G6P) transporter. We determined the exon/intron organization of the G6P transporter gene. Four overlapping genomic fragments containing the entire coding region of the gene were amplified by polymerase chain reaction (PCR) using exonic primers, and their nucleotide sequences were determined. The gene spans 4.5 kb and has eight exons. All exon/intron boundaries adhered to the canonical AG/GT rule. We then designed eight pairs of PCR primers to amplify all coding exons for a mutational analysis and studied five Japanese patients with the disease. Two novel homozygous mutations were identified in two families: a three-base deletion (delV235) in exon 2 in a consanguineous family and a splicing mutation (IVS7+1G-->T) in intron 7 in a nonconsanguineous family. Patient 3 was a compound heterozygote of W118R and IVS1+1G-->A, both of which we previously identified [Kure et al., 1998: Biochem Biophys Res Commun 248:426-431]. Patients 4 and 5 were homozygotes of W118R. Including our previous study, we found a total of ten W118R alleles in nine Japanese patients. The results support our previous suggestion that W118R is prevalent among Japanese patients. The genomic sequence data and mutation spectrum obtained from the Japanese patients will facilitate genetic diagnosis of glycogen storage disease type Ib.     

Genomic rearrangements of hMSH6 contribute to the genetic predisposition in suspected hereditary non-polyposis colorectal cancer syndrome

Methods: Out of 15 HNPCC or HNPCC-like patients who developed tumours with loss of hMSH6 protein expression, we selected three patients who still had no germline mutations after gene sequencing. Genomic DNA of these patients was analysed using PCR based relative quantification of hMSH6 fragments. Indicated exon deletions and amplifications were characterised by long range PCR and sequencing.

Results: Genomic rearrangements were identified in two of the three patients. Breakpoint analyses showed an Alu repeat mediated deletion of 13.0 kb affecting the promoter region, exon 1, and exon 2 in one patient, and a duplication of 4.9 kb containing 1.6 kb of the 3' end of exon 4 and exon 5, integrated into intron 5, in the other patient.

Conclusions: Although genomic rearrangements of hMSH6 only play a small role in the spectrum of all mutations predisposing to HNPCC, our results suggest that up to 10–20% of patients with hMSH6 negative tumours harbour germline rearrangements in this gene.

     

Mutations and DNA diagnoses of classical citrullinemia

Classical citrullinemia is an autosomal recessive disease caused by a genetic deficiency of argininosuccinate synthetase (ASS). We have previously identified 20 mutations in ASS mRNA of human classical citrullinemia and already established the DNA diagnosis of seven mutations as follows. By Southern blot analysis, each of the alleles with exon 5 or 6 deletion in mRNA appears to involve deletion of genomic DNA from this region. Five mutations involving R304W, G324S, IVS-6⁻² (ΔEx7), IVS-13⁺⁵ (ΔEx13), and Δ13bp Ex15&IVS-15 (ins37b/Ex15&16) are diagnosed by a combination of PCR (or modified PCR) and restriction enzyme digestion. It is important to identify the mutation in genomic DNA for prenatal diagnosis and carrier detection. In the present study, we report a novel missense mutation (R279Q) and a new abnormality in the ASS gene (Δ11bp/IVS-15). As three missense mutations (R272C, R279Q, and G280R) were found in exon 12, we isolated and sequenced the intron regions surrounding exon 12 to establish a DNA diagnostic test. Although a mutation with a deletion of the first seven bases in exon 16 of mRNA (Δ7b/Ex16) was found in both Japanese and American patients, the abnormality on the ASS gene was different between the Japanese allele (Δ11bp/IVS-15) and American allele (IVS-15⁻¹). The DNA diagnosis of 47 Japanese alleles with classical citrullinemia showed that the IVS-6⁻² and R304W mutations were found in 49% and 17% of the mutated alleles, respectively. We now have DNA diagnosis systems to detect 14 out of 22 mutations and are performing prenatal diagnosis and carrier detection using genomic DNA on classical citrullinemia. Hum Mutat 9:250–259, 1997. © 1997 Wiley-Liss, Inc.     

General implications for CpG hot spot mutations: methylation patterns of the human iduronate-2-sulfatase gene locus

The methylation pattern at CpG sites of a housekeeping gene correlates with the likelihood of mutation. Mucopolysaccharidosis (MPS) type II, an X-linked disorder, results from the deficiency of iduronate-2-sulfatase (IDS). In these patients, over 35% of independent point mutations at the IDS gene locus were found at CpG sites as transitional events. To gain insight into the relationship between methylation status and CpG hot spot mutations, we investigated patterns of cytosine methylation in the entire IDS gene, except for introns 4-8. Bisulfite genomic sequencing was performed on the normal leukocyte DNA. Our data show that: 1) cytosine methylation at the CpG sites was extensive, except for those present from the promoter region to a portion of intron 3; 2) a sharp boundary of methylated-nonmethylated regions was observed at the 5'-flanking region, whereas a gradual change in methylation was observed in the 2.0-kb segment in the 3'-flanking region; 3) the boundary of the 5'-flanking region contained multiple Sp1 sites and the TATA box; 4) the CpG sites in exons 1 and 2 were hypomethylated and were associated only with rare transitional mutations, while the CpG sites in exon 3 were also hypomethylated, yet were associated with a high rate of transitional mutations; 5) there was no striking sex difference in the methylation patterns in active alleles; and, 6) the methylation in both strands was symmetrical, except at the boundary of methylated-unmethylated regions.