Protein Truncation Test Research Articles

Cortisol Producing Adrenal Adenoma—a New Manifestation of Gardner's Syndrome

INTRODUCTION: Familial adenomatous polyposis (FAP) is an autosomal dominant disorder which typically presents with colorectal cancer in early adult life secondary to extensive adenomatous polyps of the colon. Gardner's syndrome is a variant of FAP in which desmoid tumors, osteomas and pigmented retinal lesions occur together with intestinal manifestations. The APC gene (adenomatous polyposis coli) at 5q21 is a tumor suppressor gene which is mutant in FAP. PATIENT: A 36 year old woman presented with a history of polyposis ventriculi, ovarian desmoid cysts, and disseminated desmoid tumors. Her familial history was unremarkable. On admission she complained weight gain, secondary amenorrhea, and episodes of hypertension followed by paroxysmal headache. RESULTS: Elevated urinary free cortisol (878 μg/24h), suppressed basal ACTH (> 5 pg/ml) and insuppressible serum cortisol after low dose dexamethasone (189 ng/ml) revealed adrenal Cushing's syndrome. Abdominal NMR showed an adrenal mass two centimeter in diameter with inhomogeneous contrast enhancement. Unilateral adrenalectomy was performed and an adrenal adenoma was diagnosed by histological criteria. For mutational detection DNA from peripheral blood leucocytes was extracted. A protein truncation test was performed, which revealed a termination mutation between codon 1099 and 1623 of the APC gene. Direct sequencing showed a point mutation in exon 15 of the APC gene at position 1542 (CAG TAG). This region is known to be altered in patients with extraintestinal manifestation of FAP. CONCLUSION: In patients with Gardner's syndrome adrenal tumors leading to hormonal excess should be considered. Whether mutations in the APC gene have implications in sporadic adrenal tumorigenesis needs to be proven.

Increased risk of breast cancer following irradiation for Hodgkin‚s disease is not a result of ATM germline mutations

Purpose : Long-term survivors of Hodgkin‚s disease who received mantle-field irradiation at a young age have a strongly increased risk of developing breast cancer. The purpose of this study was to investigate whether this increased risk was substantially greater among women heterozygous for a germline mutation in the ataxia-telangiectasia gene (ATM). Materials and methods : Thirty-two patients were selected who had developed breast cancer at least 10 years following irradiation for Hodgkin‚s disease before the age of 45 years. In these patients, the complete open reading frame of the ATM gene was analysed for the presence of germline mutations using the protein truncation test and two mutation-specific tests, followed by genomic sequencing. Results : No A-T disease causing germline mutations were found in these selected Hodgkin patients. However, several alternative splicing events were detected which might influence protein expression levels. Conclusions : The data suggest that truncating mutations in the ATM gene are not a major component underlying the increased risk of breast cancer following Hodgkin‚s disease.

Screening a human population sample for DNA repair gene deficiencies utilizing the protein truncation test

Screening a human population sample for DNA repair gene deficiencies utilizing the protein truncation test

The Genoa experience of prenatal diagnosis in NF1.

Type 1 neurofibromatosis (NF1) is an autosomal dominant disorder with an incidence of about 1 in 3500 live births. Symptoms are highly variable from a few cafè-au-lait spots and axillary freckling to plexiform neurofibromas, optic gliomas, pseudarthrosis, and malignancy. Since disease causing mutations are dispersed throughout the gene, prenatal diagnosis is usually performed in familial cases by linkage analysis and rarely by direct characterization of the mutation. We have characterized 48 families and have performed four prenatal diagnoses. In three cases, the linkage analysis was carried out using informative markers. A direct approach using the protein truncation test (PTT) and sequencing was performed in one case in which a R1947X mutation was identified. The extreme variability of the phenotypic expression of the NF1 gene makes reproductive decisions in NF1 families very difficult, as molecular diagnosis cannot predict clinical expression of the disease. The psychological management of the couple is therefore difficult. In two of the three examined families the reproductive choices were not influenced by the specific manifestations of the disease in that family.

Mutational analysis of BRCA1 and BRCA2 genes in Chinese ovarian cancer identifies 6 novel germline mutations.

Germline mutations in the BRCA1 and BRCA2 genes predispose women to breast and ovarian cancer. An incidence of 5% and 3.3% respectively has been reported of BRCA1 and BRCA2 mutations in women with ovarian cancer unselected for family history. The contribution of BRCA1 and BRCA2 mutations to ovarian cancer in Chinese women is unknown. A total of 60 samples of ovarian cancer diagnosed in Chinese unselected for age or family history were analyzed for BRCA mutations using the protein truncation test. The entire coding exon of BRCA1 of 53 cases and that of exon 11 of BRCA2 of 43 cases were successfully screened. Six germline (11.3%) mutations (633C>T, 1080delT, 1129delA, 2371-2372delTG, 3976-3979delGTGA, and IVS 22+7 A>G) were detected in BRCA1. One germline mutation (3337C>T) (2.1%) was detected in BRCA2. None of these seven cases were associated with strong family history of breast and/or ovarian cancer. Five out of our six BRCA1 mutations and the one BRCA2 mutation identified are novel. Our 11.3% incidence of BRCA1 mutations in ovarian cancer found amongst Chinese with insignificant family history is apparently higher than that previously reported in other populations. It suggests that BRCA1 mutation may play a significant role in the development of sporadic ovarian cancer in Chinese women.

Mutational analyses of BRCA1 and BRCA2 in Ashkenazi and non-Ashkenazi Jewish women with familial breast and ovarian cancer.

In Ashkenazi (East European) Jews, three predominant mutations in BRCA1 (185delAG and 5382insC) and BRCA2 (6174delT) account for the majority of germline mutations in high-risk breast and/or ovarian cancer families. Among non-Ashkenazi Jews, the 185delAG, Tyr978Ter, and a handful of "private" mutations have been reported anecdotally within both genes. In this study we attempted to determine the spectrum of BRCA1 and BRCA2 mutations in high-risk Jewish individuals, non-carriers of any of the predominant Jewish mutations. We employed multiplex PCR and denaturing gradient gel electrophoresis (DGGE) analysis for BRCA2, and combined denaturing high performance liquid chromatography (DHPLC) and protein truncation test (PTT) for BRCA1, complemented by DNA sequencing. We screened 47 high-risk Jewish individuals, 26 Ashkenazis, and 21 non-Ashkenazis. Overall, 13 sequence alterations in BRCA1 and eight in BRCA2 were detected: nine neutral polymorphisms and 12 missense mutations, including five novel ones. The novel missense mutations did not co-segregate with disease in BRCA1 and were detected at rates of 6.25% to 52.5% in the general population for BRCA2. Our findings suggest that except for the predominant mutations in BRCA1 and BRCA2 in Jewish individuals, there are only a handful of pathogenic mutations within these genes. It may imply novel genes may underlie inherited susceptibility to breast/ovarian cancer in Jewish individuals.

NF1 gene and neurofibromatosis 1.

Neurofibromatosis 1 (NF1), also known as von Recklinghausen disease, is an autosomal dominant condition caused by mutations of the NF1 gene, which is located at chromosome 17q11.2. NF1 is believed to be completely penetrant, but substantial variability in expression of features occurs. Diagnosis of NF1 is based on established clinical criteria. The presentation of many of the clinical features is age dependent. The average life expectancy of patients with NF1 is probably reduced by 10-15 years, and malignancy is the most common cause of death. The prevalence of clinically diagnosed NF1 ranges from 1/2,000 to 1/5,000 in most population-based studies. A wide variety of NF1 mutations has been found in patients with NF1, but no frequently recurring mutation has been identified. Most studies have not found an obvious relation between particular NF1 mutations and the resulting clinical manifestations. The variability of the NF1 phenotype, even in individuals with the same NF1 gene mutation, suggests that other factors are involved in determining the clinical manifestations, but the nature of these factors has not yet been determined. Laboratory testing for NF1 mutations is difficult. A protein truncation test is commercially available, but its sensitivity, specificity, and predictive value have not been established. No general, population-based molecular studies of NF1 mutations have been performed. At this time, it appears that the benefits of population-based screening for clinical features of NF1 would not outweigh the costs of screening.

Characterization of ATM mutations in 41 Nordic families with ataxia telangiectasia.

The Ataxia Telangiectasia Mutation (ATM) gene is mutated in the rare recessive syndrome Ataxia Telangiectasia (AT), which is characterized by cerebellar degeneration, immunodeficiency, and cancer predisposition. In this study, 41 AT families from Denmark, Finland, Norway, and Sweden were screened for ATM mutations. The protein truncation test (PTT), fragment length and heteroduplex analyses of large (0.8-1.2 kb) cDNA fragments were used. In total, 67 of 82 (82%) of the disease-causing alleles were characterized. Thirty-seven unique mutations were detected of which 25 have not previously been reported. The mutations had five different consequences for the ATM transcript: mutations affecting splicing (43%); frameshift mutations (32%); nonsense mutations (16%); small in-frame deletions (5%); and one double substitution (3%). In 28 of the probands mutations were found in both alleles, in 11 of the probands only one mutated allele was detected, and no mutations were detected in two Finnish probands. One-third of the probands (13) were homozygous, whereas the majority of the probands (26) were compound heterozygote with at least one identified allele. Ten alleles were found more than once; one Norwegian founder mutation constituted 57% of the Norwegian alleles. Several sequence variants were identified, none of them likely to be disease-causing. Some of them even involved partial skipping of exons, leading to subsequent truncation of the ATM protein.

APC mutation and phenotypic spectrum of Singapore familial adenomatous polyposis patients.

Familial adenomatous polyposis (FAP) is a familial form of colon cancer caused by mutation of the adenomatous polyposis coli (APC) gene. Although the APC gene has been extensively studied in the Caucasian population, it has not been previously described in the Chinese population. In the present study, we investigated APC mutation and phenotypic spectrum in the Singapore FAP families who are predominantly Chinese. The protein truncation test (PTT) was used to screen the entire APC gene for germline mutations in 28 unrelated families. Fifteen different mutations were identified in 22 families. Eight mutations were 1-11 basepair deletions or insertions; three involved deletions of whole exons and four were nonsense mutations. Nine of the mutations, including two complex rearrangements, are novel. Eight families including three de novo cases have the same (AAAGA) deletion at codon 1309, indicating that like the Western families, codon 1309 is also the mutation 'hot spot' for Singapore FAP families. In contrast, we did not find any mutation in codon 1061, the second hot spot for the Western population. Congenital hypertrophy of the retinal pigment epithelium (CHRPE) is consistently associated with the prescribed domain (codons 463 to 1387) and is the only phenotype with no intra-family variation. Other than CHRPE, differences in the type and frequency of extracolonic manifestations within the FAP families suggest the influence of modifying genes and environmental factors.

Screening a human population sample for DNA repair gene deficiencies utilizing the protein truncation test

A significant fraction of human cancers are thought to have a genetic component and several lines of evidence suggest that deficiencies in DNA repair may be a contributing factor. Little is known, however, about the frequency and distribution of variants of DNA repair genes in the general human population. The protein truncation test (PTT) was used to screen 136 healthy volunteers for protein-truncating variants of 10 DNA repair genes: APE, CDK7, ERCC1, WAF1, HOGG1, MGMT, POLB, UNG, HAAG, and CCNH. This sample consisted of 41males (30%) and 95 females (70%) with an average age of 25.3 years, ranging from 17 to 60 years of age. No truncating mutations were found in the 10 genes examined in any of the subjects. The 95% confidence interval for a proportion of 0 over the 272 alleles examined per locus is 0-0.01. The calculated frequency of truncating mutations in each of these genes, among the general population, is thus less than 1%. Among the 10 genes tested in 136 people, a single sample had no PCR product for HAAG, even though PCR products were obtained on all other genes. Total RNA dot hybridization confirmed the presence of HAAG mRNA transcripts in this sample. Despite identification of this single DNA repair variant, these results indicate a low frequency of truncating mutations in DNA repair genes in the general population.

Exon skipping in the ATM gene in normal individuals: the effect of blood sample storage on RT-PCR analysis.

Mutations in ATM, the gene defective in the human genetic disorder ataxia-telangiectasia (A-T), have been described in A-T patients and in a variety of tumor samples. Most of these arise due to exon skipping. We developed an RT-PCR based protein truncation test (PTT) to screen for ATM mutations in breast cancer patients showing adverse response to radiotherapy. An additional PTT product was evident in the ATM gene in peripheral blood mononuclear cells (PBMCs) from blood samples that were collected 2 days or more prior to RNA extraction. Lymphoblastoid cell lines established from the same blood samples showed no evidence of the additional band. Cloning and sequencing of the additional RT-PCR product revealed an exact deletion of exon 20 (2639 del 200), pointing to exon skipping. RT-PCR analysis of RNA extracted from freshly prepared PBMCs from 3 normal individuals showed no evidence of the additional RT-PCR product but when the blood was stored for 2-3 days prior to RNA extraction the lower molecular weight band was evident in every sample. DNA sequencing confirmed this to be due to loss of exon 20. These data suggest that mRNA-based mutation analysis on ATM should be carried out on unstored blood samples to avoid artificial loss of exons that give rise to apparent mutations.

Exhaustive mutation analysis of the NF1 gene allows identification of 95% of mutations and reveals a high frequency of unusual splicing defects.

Neurofibromatosis type 1 (NF1) is one of the most common autosomal dominant disorders and is caused by mutations in the NF1 gene. Mutation detection is complex due to the large size of the NF1 gene, the presence of pseudogenes and the great variety of possible lesions. Although there is no evidence for locus heterogeneity in NF1, mutation detection rates rarely exceed 50%. We studied 67 unrelated NF1 patients fulfilling the NIH diagnostic criteria, 29 familial and 38 sporadic cases, using a cascade of complementary techniques. We performed a protein truncation test starting from puromycin-treated EBV cell lines and, if no mutation was found, continued with heteroduplex, FISH, Southern blot and cytogenetic analysis. We identified the germline mutation in 64 of 67 patients and 32 of the mutations are novel. This is the highest mutation detection rate reported in a study of typical NF1 patients. All mutations were studied at the genomic and RNA level. The mutational spectrum consisted of 25 nonsense, 12 frameshift, 19 splice mutations, six missense and/or small in-frame deletions, one deletion of the entire NF1 gene, and a translocation t(14;17)(q32;q11.2). Our data suggest that exons 10a-10c and 37 are mutation-rich regions and that together with some recurrent mutations they may account for almost 30% of the mutations in classical NF1 patients. We found a high frequency of unusual splice mutations outside of the AG/GT 5 cent and 3 cent splice sites. As some of these mutations form stable transcripts, it remains possible that a truncated neurofibromin is formed.

Novel Germline APC Mutations in Swedish Patients with Familial Adenomatous Polyposis and Gardner Syndrome

Background: Familial adenomatous polyposis (FAP) is a familial cancer syndrome in which affected individuals develop multiple adenomatous polyps and are thereby at greatly increased risk of developing colorectal cancer. Gardner syndrome is a variant of FAP, in which the patients also develop extraintestinal tumors, in particular osteomas and desmoid tumors. An attenuated form of the disease (AFAP) is associated with fewer polyps, but still a high risk for colorectal cancer. Germline mutations in the adenomatosis polyposis coli (APC) gene cause FAP and Gardner syndrome and have recently been associated also with the development of AFAP. Methods: We have analysed the entire APC gene for germline mutations in 7 patients with FAP and in 6 patients with suspected AFAP. Mutation screening was performed by direct sequencing of exons 1-14 and using the protein truncation test for analysis of exon 15. Results: Novel disease-causing germline mutations, all of which resulted in truncation of the APC protein, were identified in 6 of the 7 patients with FAP or Gardner syndrome. No APC mutation was detected in any of the 6 patients with suspected AFAP. Conclusions: This study reports novel FAP- and Gardner syndrome-causing mutations in the APC gene. The lack of APC mutations in patients with multiple polyps at young age indicates that other genetic defects may cause this phenotype.

Screening for BRCA2 mutations in 81 Dutch breast-ovarian cancer families.

We have analysed 81 families with a history of breast and/or ovarian cancer for the presence of germline mutations in BRCA2 with a number of different mutation screening techniques. The protein truncation test (PTT) for exons 10 and 11 detected four different frame-shifting mutations in six of these families. Four of the remaining 75 families had given positive linkage evidence for being due to BRCA2. In these families the entire coding region was analysed by single-strand conformational polymorphism, leading to the detection of a non-sense and a splice-site mutation in two of them. While these studies were in progress, Southern analysis of BRCA1 revealed that in our study-population of 81 families, 15 families were segregating either the exon 13 or exon 22 deletion in BRCA1 (Petrij-Bosch et al (1997) Nat Genet17: 341–345). This prompted us to examine BRCA2 in the remaining 58 families by Southern analysis, using two different restriction enzymes. No aberrations were found in the restriction patterns. Thus, contrary to BRCA1, large genomic rearrangements within the BRCA2 gene do not represent a major mutation mechanism among Dutch breast cancer families. © 2000 Cancer Research Campaign

Multiple APC mutations in sporadic flat colorectal adenomas

Adenomas are established pre-malignant lesions in colorectal carcinogenesis. To date the adenoma-carcinoma sequence for the development of colorectal carcinoma (CRC) has been based largely on molecular data of exophytic, polypoid-type adenomas. Subsequently, a different type of adenoma has been identified: the flat adenoma, so called for its flat, non-exophytic appearance, making it less likely to be detected during conventional endoscopy. However, due to technological advances in endoscopic methods, flat-type adenomas can now frequently be detected and are no longer considered rare colorectal lesions. The phenotype of flat colorectal adenomas differs macroscopically and histologically from exophytic adenomas. Flat colorectal adenomas, as a rule, are tubular structures often revealing high-grade dysplasia, irrespective of the size or villous component. Flat adenomas have also been recognised as pre-cancerous lesions in gastric cancer. Unlike the wealth of clinical and molecular information available for polypoid (exophytic) adenomas, molecular profiles of flat-type lesions have not yet been characterised systematically and the full clinical significance hereto realised. Previous molecular investigation of the K-ras gene in flat colorectal adenomas suggests a distinct pathway in their development. In this study, mutation analysis of the adenomatous polyposis coli (APC) gene using the protein truncation test (PTT) in 20 flat colorectal adenomas in a selected group of 16 patients without hereditary predisposition to colorectal cancer, revealed double truncations of the APC gene in four adenomas. In one of these adenomas a third mutation was detected by DNA sequence analysis.

Protein Truncation Test

AbstractThe protein truncation test detects mutations at the protein level that lead to premature translation termination. The method has evolved considerably since is original publication in this manual. This thoroughly revised unit describes what is now the preferred method for performing the protein truncation test. Transcription and translation are performed in separate reactions; during translation, biotin‐labeled or N‐terminally tagged proteins are synthesized. The translation products are detected on immunoblots via chemiluminescence. An using coupled in vitro transcription/translation and radiolabeled proteins is also presented.

Protein truncation test for screening hamartin gene mutations and report of new disease-causing mutations.

Considering the prevalence of truncating mutations in the tuberous sclerosis (TSC) hamartin gene (TSC1), we devised a protein truncation test (PTT) to analyze the full length coding sequence of TSC1. Studying 12 sporadic cases and three familial forms by a combination of PTT and single-strand conformation polymorphism analysis (SSCA), we found 5/15 mutations while PTT alone detected 4/15 truncating mutations, two of which escaped SSCA analysis. SSCA alone picked up one missense mutation and two mutations also detected by PTT. Interestingly, a TSC1 mutation was identified in all three familial forms (3/3) while the rate of mutation detection was lower in sporadic cases (2/12). In conclusion, PTT proves to be a useful technique for the rapid detection of disease-causing mutations in the TSC1 gene.

Mutation screening of the entire coding regions of the TSC1 and the TSC2 gene with the protein truncation test (PTT) identifies frequent splicing defects.

Mutation analyses in tuberous sclerosis (TSC) have reported a wide variety of disease-causing aberrations in the two known predisposing genes, TSC1 and TSC2 on chromosomes 9q34 and 16p13, comprising mainly small mutations distributed over the entire genes. So far, all known TSC1 mutations as well as the majority of TSC2 mutations truncate the proteins hamartin and tuberin, respectively. We describe for the first time an RNA-based screening of the entire coding regions of both TSC genes for truncating mutations applying the protein truncation test (PTT). Simultaneous investigation of both TSC genes in a group of 48 unassigned TSC patients, which were previously tested to exclude large intragenic TSC2 rearrangements, revealed aberrant migrating polypeptides resulting from truncating mutations in nine TSC1 cases and in 16 TSC2 cases while three TSC2 cases showed enlarged proteins. TSC1 mutations include two nonsense mutations, four insertions, and three splice mutations. Nineteen mutations identified in TSC2 were composed of four different nonsense mutations in five patients, one deletion, one insertion, and seven different splicing aberrations due to at least eight different mutations found in 12 patients. Additional predicted truncating mutations according to PTT without possible identification of the causative alteration allowed assignment to TSC1 in one and TSC2 in seven cases. Twelve patients without abnormalities in the PTT are assumed to harbor missense mutations, probably in TSC2. The high proportion of TSC2 splicing aberrations strengthens the importance of intronic disease-causing mutations and the application of RNA-based screening methods to confirm their consequences.

Point mutations in the dystrophin gene: evidence for frequent use of cryptic splice sites as a result of splicing defects.

Ten different mutations have been identified in patients with Becker (n = 1) or Duchenne (n = 9) muscular dystrophy using reverse transcription of total RNA, polymerase chain reaction amplification of the whole coding region of the gene and protein truncation test (PTT) analysis. Seven mutations had not been reported previously, and these consist in three nonsense mutations (Q2522X, E2726X, R3381X), three frameshifting deletions (3686-3687delGT, 5126delA, 5759delC), and four splicing defects of which the effects on the muscle dystrophin mRNA transcripts have been analyzed. In one case, a 3' splice-site mutation (IVS74-2A-->G) resulted in a complex pattern of exon skipping involving exons of the C-terminal domain. In the three other cases, nucleotide substitutions in splice donor (IVS26+2T-->A, IVS65+1G-->A) or acceptor (IVS8-15A-->G) recognition sequences led to the use of cryptic splice sites, with consequent insertions of intronic sequences in the processed mRNA. Up to 34% (70/203) of the point mutations reported to date in the dystrophin database (http://www.dmd.nl) affect splice sites of the dystrophin gene. However, altered mRNA splicing has been confirmed experimentally in only 23% of cases (16/70). Combined with PTT, the transcript analysis protocol defined in this study permits direct determination of the impact of intronic variations on the structure of dystrophin mRNA and of the resulting consequences on the translational reading frame. We present evidence for a frequent use of cryptic splice sites as a result of splicing defects.

HMLH1, hMSH2 and hMSH6 mutations in hereditary non-polyposis colorectal cancer families from southern Sweden.

We have screened 17 Southern Sweden individuals/families with suspected hereditary non-polyposis colorectal cancer (HNPCC) for mutations in the DNA-mismatch repair genes hMLH1, hMSH2 and hMSH6 using denaturing gradient gel electrophoresis, protein truncation test and direct DNA sequencing. The families were selected on the basis of a family history of HNPCC-related tumors or the occurrence of metachronous colorectal cancer/endometrial cancer at young age in an individual with a weak family history of cancer. Furthermore, we required that tumor tissue from at least one individual in the family had to display microsatellite instability. We identified germ-line mutations in 9 individuals from 8 families. Five families had mutations in hMLH1, 4 of which were splice site mutations, 2 had frameshift mutations in hMSH2 and 1 patient with metachronous endometrial and rectal cancer but with a weak family history of cancer had a nonsense mutation in hMSH6. Our results present novel germ-line DNA-repair gene mutations, one of these in hMSH6, and demonstrate the diversified mutation spectrum in Sweden, where no founder mutation has so far been identified.