Effect of nonsense mutations on PTEN mRNA stability

Hum Genet (2000) 107 : 24–27 Digital Object Identifier (DOI) 10.1007/s004390000317 O R I G I N A L I N V E S T I G AT I O N Anthony M. Raizis · Martin M. Ferguson · Peter M. George Effect of nonsense mutations on PTEN mRNA stability Received: 14 February 2000 / Accepted: 1 May 2000 / Published online: 4 July 2000 © Springer-Verlag 2000 Abstract Cowden disease is an autosomal dominant disorder associated with an elevated risk of breast, thyroid and skin cancers, in which germline mutations of a tumour suppressor gene (PTEN) have been identified. PTEN has a dual-specificity tyrosine phosphatase domain thought to be essential for tumour suppression. Previous genotype/phenotype correlations have identified several potential associations, for example that truncating mutations result in increased breast cancer risk. Such associations are useful in evaluating the phenotypic functions of PTEN sub-domains. However, genotype/phenotype correlations are likely to be complicated by nonsense mediated mRNA degradation. We report here that three out of four mutations do not significantly affect PTEN transcript stability. Furthermore, we show that manual sequencing methods are better than current dye-based sequencing technologies for detecting heterozygous mutations in PTEN transcripts. Introduction Cowden disease (CD; OMIM 158350), also known as multiple hamartoma syndrome, involves a single locus on 10q23 (Nelen et al. 1996) at which PTEN (GenBank AF000734), a tumour suppressor gene with tyrosine phosphatase and tensin homology, has been identified (Li et al. 1997; Steck et al. 1997). The tyrosine phosphatase domain, residues 82–142, is thought to be critical for tumour suppression. A second gene homologous to PTEN has also been discovered called ΨPTEN, but the function of this gene remains unknown (Dahia et al. 1998). A.M. Raizis · P.M. George (✉) Departments of Clinical Biochemistry, Christchurch Hospital, Christchurch New Zealand e-mail: [email protected], Fax: +64-3-3640525 M.M. Ferguson Department of Stomatology, University of Otago, Dunedin, New Zealand It is important to establish whether there is a genotype/phenotype correlation in families with Cowden disease. An exploratory genotype/phenotype association was found between truncating mutations and the presence of cancer and breast fibroadenoma in different families (Marsh et al. 1998). A second association was found between mutations affecting the phosphatase core and multiorgan disease, i.e. affecting more than four organs. Most reported mutations involve the phosphatase core and disrupt phosphatase activity while maintaining the 5′ substrate-binding domain. If these variants are translated, substrates could bind to the mutant PTEN molecules, but without the ability to dephosphorylate, perhaps resulting in a dominant negative effect. In one previous genotype/phenotype analysis no correlation was observed (Nelen et al. 1999). However, a correlation between the loss of the phosphatase core and multiorgan disease is possible, but more numbers are required to confirm this (Marsh 1998). Genotype/phenotype correlations are likely to be complicated by nonsense-mediated decay of RNA, and a previous report using dye-based sequencing technology showed that PTEN transcripts containing nonsense mutations appeared unstable (Lynch et al. 1997). We show here that of four nonsense mutations examined three do not significantly affect PTEN transcript stability. Materials and methods Patients Four local patients, three male and one female, presented with clinical features of Cowden disease. These features included macrocephaly, thyroid abnormalities and mucocutaneous lesions. The woman developed breast cancer which was later treated by mastectomy. Two of the three men showed gastrointestinal hamartomas, and all three males had trichilemmomas and papillomatous papules. Mild retardation was observed in only one patient. In each case nonsense mutations were identified in the PTEN gene (see below). 25 PCR analysis of the PTEN gene and mRNA Genomic DNA was extracted from peripheral blood of the patient as previously described (Ciulla et al. 1988). Total mRNA was extracted from 5 ml whole blood which was taken from patients and immediately placed in 10 ml Trizol (Life Technologies). Subsequent steps followed manufacturer’s instructions. The mRNA was reverse transcribed (RT) with Superscript (200 U, Life Technologies) using random hexamers (100 µM). In order to detect PTEN mutations exons 1–9 were amplified by the polymerase chain reaction (PCR) using intronic primers as previously described (Liaw et al. 1997), except for exon 1 where the primers used were forward 5′-TCTGCCATCTCTCTCTCCTCCT-3′ and reverse 5′-TCCGTCTACTCCCACGTTCT-3′. Two sets of primers were used for PCR amplification of cDNA, i.e. forward 5′-CATCTCTCTCCTCCTTTTTCTTCA-3′, reverse 5′-ATATCATTACACCAGTTCGTCCCT-3′ which amplifies exons 1–5 and gives a 450-bp PCR product (Dahia et al. 1998). To amplify cDNA exons 5–8 by PCR primers, forward 5′-TGACCAATGGCTAAGTGAAGAT-3′ and reverse 5′-GTATGAAGAATGTATTTACCCAA-3′ were used giving a PCR product of 518 bp. Amplification conditions were 94°C for 5 min followed by 39 cycles of 94°C for 1 min, 53°C for 1 min, and 72°C for 1 min. with a final extension time of 7 min at 72°C. The DNA sequence of each PCR product was determined by direct DNA sequencing of the PCR product using the thermocycling technique (Amersham, UK), thermosequenase and 33P-labelled dideoxy-nucleotide triphosphate chain terminators according to manufacturers instructions. In all sequence reactions the sequencing primers were the same forward or reverse primers used for PCR. ABI sequencing was performed using the ABI 377 automated sequencer, version 3.3 and Big Dye terminators. Restriction analysis of PTEN nonsense mutations Nonsense mutations in each case were confirmed using restriction enzyme based analysis as follows: (a) To confirm the base insertion 40^41InsA a restriction enzyme assay was designed using a forward mutagenic primer 5′-CAAAGAGATCGTTAGCAGCCACAA-3′. To generate a Bs1I restriction site “CCN7GG” specific for the mutation nucleotides 33–34 “AA” were replaced by “CC” (underlined above). Bs1I cleaves the mutant (40^41insA) allele, where the number of nucleotides between “CC” and “GG” is seven, as opposed to six in the wild-type allele. (b) The base substitution K267X was confirmed using a reverse mutagenic primer 5′CAATGAAAGTAAAGTACATACGT-3′. To generate a SnaBI restriction site specific for the mutation, two intronic nucleotides (underlined above) were altered such that SnaBI cleaves the mutant (K267X) 5′-TACGTA-3′ allele but not the wild-type 5′TACGTT-3′ allele. (c) The L139X mutation was confirmed using the mutagenic 3′ reverse primer 5′-AAAAATTTGCCCCGATGTGAT-3′. A MboI restriction cutting site specific for the mutation occurs when a “G” (underlined) is positioned (normally “A” on the antisense strand) such that MboI cleaves the mutant (L139X) allele 5′-GATC-3′ but not the wild-type allele 5′-TATC3′. (d) The R130X mutation was confirmed by cleavage of the mutant allele with FokI. This enzyme cleaves the mutant sequence 5′GGATG-3′ but not the wild-type sequence 5′-GGACG-3′. Results Direct DNA sequence analysis of the PTEN gene, exons 1–9, revealed different nonsense mutations in each patient (Fig. 1a–d, i). In each case the presence of the mutation was confirmed by restriction enzyme analysis as described above. To determine the stability of transcripts containing PTEN mutations cDNA produced from total blood RNA from the four patients was amplified by PCR. DNA sequence analysis of the RT-PCR products obtained from the transcript containing the 40^41InsA mutation shows only faint bands corresponding to the frameshift (Fig. 1a, ii). This is in contrast to the DNA sequence obtained from genomic DNA where the allelic distribution of mutant and wild-type fragments are approximately 1:1. This result indicates that the 40^41InsA mutation reduces stability of the PTEN transcript. We then examined three other mutations for their effect on PTEN transcript stability. PCR amplification and sequencing of genomic DNA shows that these mutations, 130X (Fig. 1b, i and ii), L139X (Fig. 1c, i and ii) and K267X (Fig. 1d, i and ii), give sequences where the wildtype band is approximately equal to the intensity of the mutant band. In the corresponding cDNA sequence the mutated transcript is clearly visible, but the ratio of wildtype PTEN transcript to the mutated transcript is greater than 1:1. This skewed allelic distribution of PTEN transcripts is due at least in part to the cDNA sequence containing the PTEN gene and the PTEN pseudogene ΨPTEN superimposed (Dahia et al. 1998), as indicated by the presence of faint ΨPTEN specific bands (Fig. 1b–d, ii). ΨPTEN, however, does not contain any of the four PTEN mutations and does not interfere with the amplification of PTEN mRNA. The results show that for these three mutations the mutated PTEN transcript is stable and thus can potentially influence phenotype. In one CD case we also determined the cDNA sequence using the dye-based approach, i.e. ABI Prism, Big Dye. Using this technique we examined the same template and primer preparation used to obtain the sequence shown in Fig. 1b, ii. However, no mutation was detectable, nor were any bands corresponding to ΨPTEN detected (Fig. 1b, iii). To ensure that the PTEN cDNA data were not an artefact of our RT-PCR and sequencing technique, retinoblastoma transcripts having a H585Y missense mutation were analysed by RT-PCR using the same reagents and conditions as for PTEN. A sequence comparison of the PCR product produced from genomic DNA (Fig. 1e, i) and cDNA (Fig. 1, ii) showed no difference in the ratio of mutant and wild-type bands. This result indicates that our RT-PCR and sequencing technique does not in itself skew the ratio of mutated to wild-type alleles. Discussion In Cowden disease there is a wide phenotypic variability. The factors that influence this variability are largely unknown although extensive genotype/phenotype analysis may help clarify this question. Previous genotype/phenotype correlations (Marsh et al. 1998) have suggested that disruption of the tyrosine phosphatase core may give rise to all organs being affected, i.e. central nervous system, thyroid, breast, skin and gastrointestinal tract. Most reported mutations cause protein truncation and in one simple model, the CD phenotype is determined by the loss of 26 Fig. 1a–e Sequence analysis of PTEN transcripts containing nonsense mutations. Genomic DNA and RNA was extracted from total blood of four patients diagnosed with Cowden disease. Genomic DNA (i) was amplified by PCR and sequenced directly by incorporation of 33P-labelled dideoxynucleotides as described in Methods. Total RNA was reverse transcribed and the cDNA amplified by PCR and sequenced (ii). The four mutations shown are 40^41InsA (a), R130X (b), R139X (c), K267X (d). Point mutations are arrowed, faint bands specific to ΨPTEN are indicated in lower case (dotted arrows). H585Y is a missense mutation of the retinoblastoma gene RB (e). Sequence analysis of the R130X mutation was performed with an ABI Prism sequencer, using Big Dye (b, iii). Asterisk, the nucleotide position expected to show a mutation; arrows expected positions of ΨPTEN specific signals 27 the deleted domains. In another model a premature stop codon causes instability of the mutant transcript. In this case the truncated protein is not translated (Lynch et al. 1997), and the position of mutations is unlikely to influence phenotype. We have found that the 40^41InsA mutation reduces the stability of the PTEN transcript. From a phenotypic perspective this is of little consequence as the mutation causes most of the PTEN gene molecule to be lost through truncation. In contrast, we find that for three other mutations PTEN transcript stability is relatively unaffected. In two of these examples, R139X and K267X, the phosphatase domain is potentially active in the mutant molecule. Our observations contrast with a previous report in which dye-based methods were used and where it was concluded that nonsense mutations appear to destabilise the PTEN transcript (Lynch et al. 1997). In our hands, dye-labelled terminators did not detect nonsense mutations in PTEN mRNA nor sequence bands specific to the PTEN pseudogene (Fig. 1b, iii), which co-amplifies with PTEN (Fig. 1b–d, ii). Our observations suggest that dyelabelled sequencing methods are inadequate for the detection of heterozygous mutations in PTEN transcripts. However, we cannot exclude the possibility that dye-labelled methods could be optimized using appropriate controls. Conclusions from genotype/phenotype correlations in Cowden disease will only be possible once more examples are examined in detail. Clearly, however, each nonsense mutation will need to be assessed for its potential to cause nonsense mediated decay of mRNA. However, nonsense-mediated decay can only be determined in fresh blood or tissues where the gene is heterozygous. Tumours cannot be used for nonsense mediated decay analysis since homozygous PTEN inactivation can occur for reasons other than loss of the normal allele. For example, PTEN inactivation in tumours sometimes involves epigenetic or translational inactivation mechanisms, which affect both alleles irrespective of the mutation involved (Gimm et al. 2000). Secondly, the transcripts derived from the normal allele act as an internal standard by which to compare normal and mutated transcripts. PTEN transcripts must therefore be examined in the heterozygous state if nonsense mediated decay is to be reliably assessed. Acknowledgements This work was supported by the McClelland Trust Fund, Christchurch, New Zealand. References Ciulla TA, Sklar RM, Hauser SL (1988) A simple method for DNA purification from peripheral blood. 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