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Editor—Familial adenomatous polyposis (FAP) is an autosomal dominantly inherited disorder characterised by the development of hundreds to thousands of adenomatous polyps in the colon and rectum. If left untreated, there is a very high risk of colorectal cancer. Adenomatous polyps may also develop proximally in the stomach and the distal part of the duodenum. FAP is also associated with a variety of extracolonic benign and malignant manifestations, including congenital hypertrophy of the retinal pigment epithelium (CHRPE), dental abnormalities, desmoid tumours, osteomas, epidermoid cysts, hepatoblastoma, and thyroid neoplasia.1 Germline mutations of the APC gene localised on chromosome 5q21.22 are responsible for FAP.2 3 APC is a tumour suppressor gene encoding a 2843 amino acid protein, which contains multiple functional domains and which mediates growth regulatory signals by its association with a variety of cytoplasmic proteins. More than 300 differentAPC mutations have so far been identified distributed throughout the whole gene, with a higher concentration in the 5′ part of exon 15 (codons 713-1597).4 5 The majority of mutations are predicted to introduce premature termination signals resulting from single nucleotide alterations, small insertions or deletions, or splice site mutations that lead to truncation of the normal protein product.4 Missense mutations have rarely been reported and their functional implications are often unclear.6 7 Larger deletions and insertions have been described, as well as genomic rearrangements resulting from recombinations mediated by Alu elements which cause inappropriate exon splicing.8-10 Isoforms of APCtranscripts lacking exon 9, exon 10A, and exon 14 encoded sequences have been reported.2 11-13 Isoforms lacking exon 9 or exon 14 owing to splice site mutations have also been associated with a FAP phenotype.14-17
In this study, we describe a G→T transversion at nucleotide position 1869 in exon 14 which gives rise to a silent mutation, since both normal and mutated alleles encode an arginine residue at codon 623. This exonic mutation induces complete skipping of exon 14, leading to truncated APC protein and resulting in a FAP phenotype.
Materials and methods
The index case of family GE08 (IV.1, fig 1, table 1) was referred to our institution for genetic counselling. The patient underwent a total colectomy for diffuse polyposis. A diagnosis of FAP was made on the basis of family history and histopathological results. After informed consent had been obtained from numerous family members, medical records were reviewed to confirm the diagnosis of polyposis, polyposis and cancer, and the age of occurrence. The clinical information obtained is shown in table 1. The pedigree was constructed as shown in fig 1. Peripheral blood samples were obtained from the proband and family members III.5, IV.1, IV.2, IV.3, IV.4, IV.5, IV.6, IV.15, IV.16, IV.18, IV.19, IV.20, IV.21, IV.22, IV.26, V.1, V.2, V.3, V.4, V.11, and V.12.
Mutation analysis was carried out on genomic DNA prepared from isolated leucocytes using SNAP Whole Blood DNA Isolation Kit (Invitrogen, Carlsbad, CA). DNA was also extracted from paraffin embedded normal tissue from subject III.5.18 The protein truncation test (PTT) was performed to detect truncating mutations in exon 15.19 Mutation screening of the entireAPC coding sequence was performed on genomic DNA using single strand conformation polymorphism (SSCP). Thirty-seven different segments of the APC gene were amplified by PCR using the primer pairs reported elsewhere.2 SSCP was carried out as described previously.18 The variant conformer was confirmed in at least three different samples from the same person and by direct DNA sequence analysis using an ABI PRISM TM 377 DNA Sequencer (Perkin Elmer, Foster City, CA).
mRNA was isolated from IL-2 transformed lymphoblastoid cell lines by using the Micro-Fast Track mRNA Isolation Kit (Invitrogen, Carlsbad, CA). First strand cDNA was synthesised with 2 μg of polyA+ RNA in a reaction mixture containing 1.5 mmol/l MgCl2, 10 mmol/l Tris HCl, 50 mmol/l KCl (pH 8.3), 200 μmol/l each dNTP, 200 U of M-MLV Reverse Transcriptase (Ambion, Austin, TX), 25 U RNase OUT (Life Technologies, Gaithersburg, MD), 50 μmol/l of primer RV7-A20 and primer 15A-RP,2 and 0.25 U Taqpolymerase in a volume of 50 μl. Primers RV7-A and 15A-RP enabled theAPC region corresponding to codon 493 to 759 to be amplified. The PCR products were visualised by gel electrophoresis on a 1.5% agarose gel stained with ethidium bromide. The band intensity was measured directly on the agarose gel by Gene Snap 4.00.00 and Gene Tools Version 3.00.13 Syn Gene software (Synoptics Ltd, Cambridge, UK). Amounts of each mRNA were expressed both as absolute values and as the ratio between the 793 band, which corresponds to cDNA encompassing the APCexon 11-15A, and the 578 band which corresponds to the alternatively spliced transcript lacking exon 14. GAPDHgene coamplification was carried out as an internal control. Moreover, the PCR products were separated by 6% silver stained polyacrylamide gel electrophoresis. The bands of 793 and 578 nucleotides were cut off and eluted in water overnight at 37°C. One μl of each mixture was then used to amplify the two bands separately with primers RV7-A and 15A-RP. The identity of the PCR products was ascertained by direct sequencing. A stretch of intronic 40 nucleotides upstream of exon 14 was also sequenced for analysing the acceptor site branch point.
Protein extraction and western blot analysis were performed according to Gismondi et al 21 using 10% SDS PAGE gel. EBV transformed cells of subjects IV.1, IV.16, a normal control, and a control carrying a nonsense mutation at codon 563 were analysed.
The index patient had a clinical diagnosis of FAP and underwent colectomy at the age of 46. The pedigree of family GE08 (fig 1) shows some peculiarities regarding the age of occurrence and the multiplicity of polyps. The age at which polyps and cancer appeared is later than in classical polyposis, except for IV.14 and IV.16 (table 1). These patients belonged to a branch of the family in which a consanguineous marriage had taken place between first cousins, both possible carriers of the disease. The number of adenomatous polyps ranged from six to more than 100 in affected members. No upper gastrointestinal tract lesions, CHRPE, or other extracolonic manifestations are present in the family.
During mutation screening of the APC coding region, a G→T transversion at nucleotide position 1869 was detected by SSCP and direct sequencing of exon 14 in the index patient IV.1. This mutation changes codon 623 from CGG to CGT, both encoding arginine (R623R).22 Since the entire open reading frame of theAPC gene was analysed by SSCP and PTT several times without detecting any additional sequence variation, this is the only detectable DNA alteration unique to this patient.
The sequence at the branch point of the exon 14 acceptor site also did not show any alteration. Subsequent screening for the mutation in 21 additional relatives showed the presence of the mutation in three successive generations, as shown in fig 1, and its segregation with the disease. In patient III.5, the mutation was detected on paraffin embedded tissue. Direct sequencing of DNA from patients IV.14 and IV.16 did not show homozygosity for the R623R mutation. This allelic variant was not observed in any of 100 DNA samples from normal controls.
Messenger RNA purified from transformed lymphoblastoid cell lines from three affected subjects (IV.1, IV.4, IV.16) and from three normal controls was examined. The analysis of cDNA with primer sets RV7-A and 15A-RP showed the two isoforms representing the mRNA transcript containing exon 14 and the alternatively spliced transcript lacking exon 14 leading to a stop codon in exon 15A.
The densitometric ratio between the two isoforms of 793 and 578 bp amplicons was comparable in all normal controls, being 4.2. By contrast, the band intensity of 578 bp was greater in the three patients examined, with a densitometric ratio of 0.6 (p<0.03) showing a drastic increase in the alternatively spliced isoform of exon 14 (fig2A).
Direct DNA sequencing of the two bands showed only the wild type allele in the 793 bp mRNA isoform containing the normal spliced RNA while the G→T transversion was not present. In the retrotranscripted, short 578 bp isoform, the mRNA showed a complete lack of exon 14.
Western blot analysis was performed on protein extracts from a normal control and from subjects IV.1 and IV.16. In addition, a FAP patient with a nonsense mutation at codon 563 was analysed as an APC protein truncated control (fig 2B). Using 10% SDS-PAGE analysis, the APC wild type protein was not detectable; the only truncated protein was observed in subjects IV.1 and IV.16 and in the APC mutated control indicating that the mutant mRNA is translated into a stable truncated protein. No truncated APC protein was detected in the normal control.
The exonic silent mutation R623R reported here creates a complete skipping of exon 14 of the APC gene and gives rise to a stable truncated APC protein. This mutation has not been described previously and does not represent a polymorphic gene variant as it has been tested on more than 100 subjects. This mutation occurs as the only DNA change in all affected members of a large three generation FAP family. Clinically, the family is characterised by an unaggressive form of the disease in terms of age of onset and number of polyps (table 1, fig 1), except for the branch derived from a consanguineous marriage, both parents being possible carriers of FAP. This difference might occur because of the intrafamilial variability of FAP or because of some unknown recessive modifier gene inherited from both parents. Although alternative skipping of exon 14 is a physiological event observed in normal subjects, a splice acceptor site mutation leading to increased expression of anAPC mRNA isoform without exon 14 has been previously described in a FAP family.16 In our family, splice sites do not show any modification and, furthermore, to rule out the possibility of an intron alteration in the tract near the acceptor splice site, which might eliminate lariat formation at the branch point, a stretch of 40 nucleotides upstream of the exon 14 acceptor site was also sequenced.
Our data indicate that the mutation harbouring allele is expressed exclusively in the alternative isoform and that the DNA exonic silent mutation might to be able to produce a complete skipping of exon 14, suggesting the existence of a mechanism of splicing regulation distinct from splice junctions. Silent mutations not leading to amino acid change are generally considered to be normal variants and are thought to have no role in disease. However, a few papers have described exonic silent mutations able to induce exon skipping in the fibrillin-1 gene, the MLH1 gene, and the human phenylalanine hydroxylase gene, all associated with the corresponding diseases.23-25
Recent studies have indicated that sequence elements that are distinct from the splice sites are also needed for normal splicing. These elements are required for efficient splicing and may affect splice site recognition during constitutive and alternative splicing.26 27 They are found within coding exons and are called exonic splicing enhancers (ESE). Since no consensus sequence that describes ESE is recognised, these elements are difficult to identify, not least because they are not as purine rich as was originally thought.28 Silent point mutations that affect ESE and lead to inappropriate exon skipping have been described in human genetic diseases. Frontotemporal dementia linked to chromosome 17 missense mutation affects splicing of tau gene exon 10 by acting on an ESE.29 Carbohydrate deficient glycoprotein syndrome is associated with a missense mutation that disrupts a splicing enhancer sequence, resulting in exon 5 skipping.30 Splicing difference between the two forms of spinal muscular atrophy, SMN1 and SMN2, is attributed to a silent mutation in an ESE located in the centre of SMN exon 7.31 Very recently, Liu et al 32 reported aberrant exon skipping in the BRCA1 gene resulting from nonsense, missense, or translationally silent mutations, which have disrupted a critical ESE. The G→T silent genomic DNA mutation is the only change found in family GE08 and it is located in a purine rich DNA sequence (ACCGGAGCCAG). This region in the middle of exon 14 might represent an ESE sequence necessary for the control of the alternative splicing and the one base substitution might disrupt this, thus provoking total exon 14 skipping. Experiments are being carried out to test enhancer activity of the exon 14APC sequences by inserting them into another transcript that could be assayed by in vitro splicing and/or by transfection,33 followed by mutagenesis of the sequence, to identify the elements that compose the putative enhancer.
We describe a silent third base codon mutation in exon 14 of the APC gene, a G→T transversion at nucleotide 1869 (R623R), associated with complete skipping of exon 14 and production of a stable truncated APC protein. The R623R mutation segregates with the disease in a large FAP family. This mutation is the sole variation found in the entire APC coding sequence, is not a polymorphism, and has not been described before.
To evaluate whether the silent genomic DNA variation could affect RNA transcription, RT-PCR analysis was carried out in affected subjects. Although exon 14 is subject to alternative splicing, an unbalanced expression of the two exon 14 isoforms was found. Sequence analysis of the two isoforms showed that the mutant APC allele expressed exclusively mRNA lacking exon 14. Western blot analysis showed that the mutant mRNA was translated into a stable truncated protein.
Our findings show a possible new model of APC mutation causing disease and suggest that exonic elements might modulate the splicing of APC gene exon 14. They therefore underline the importance of investigating the significance of silent mutations.
This work was supported by MURST, Ministero dell'Università e della Ricerca Scientifica e Tecnologica, Cofinanziamento 1998, Ricerca Finalizzata-Ministero Sanità 1999, ICS030.1RF99.34. We also acknowledge Galliera Genetic Bank, Progetto Telethon C42.