Protein Truncation Test Research Articles

Truncation of the mismatch repair protein PMS2 during the neoplastic transformation of human breast epithelial cells in vitro

Alterations in known mismatch repair (MMR) genes have been found in many cancers, such as in hereditary non-polyposis colorectal cancer syndrome (HNPCC), in addition to specific oncogenes and tumor suppressor gene abnormalities. We have reported mutations in the MMR genes hMSH2, hPMS2 and hMSH6 in human breast epithelial cell lines (HBEC). In hPMS2 we found a non-sense mutation in codon number 471 by a change of glutamine (CAG) by stop codon (TAG). We also observed that the number of alterations in the MMR genes were increased during the process of cell transformation and tumorigenesis. In the present study in order to determine the role of those mutations in the hPMS2 protein activity, we used western blotting, protein truncation test and MMR activity assay employing HBEC cells. In the transformed and tumor cell lines we found a premature hPMS2 protein truncated by western blot and also we confirmed this truncation using the protein truncation test. In the MCF-10F cells only the wild-type hPMS2 protein was detected. The functionality of the hPMS2-truncated protein was confirmed by DNA heteroduplex assay using specific mispaired bases in mutated M13mp2 phages. DNA repair activity was proficient in MCF-10F cells, partially deficient in the transformed cells, and totally deficient in the tumor cell lines. In order to test whether the MMR deficiency was due to an hPMS2 defect, we used the wild-type heterodimer hPMS2-hMLH1 in the in vitro reaction of DNA heteroduplex assay; we have found that addition of the hPMS2 wild-type increased the MMR repair efficiency by more than 40% in tumor cell lines. Our results indicate that a protein-truncating mutation in the hPMS2 gene had occurred during the process of cell transformation and tumorigenesis and that truncation is related to a lack of function of this gene.

Different expression of BRCA1 status and clinical variables in a sample of Italian women with early onset breast cancer (EOBC)

9670 Background: to estimate the spectrum of genetic alteration, family history (FH) and clinical variables in a EOBC population, diagnosed before 41 yrs, consecutively seen at the Genetic Oncology Service of the Parma Hospital from 1999 to 2003, and to investigate the contribution of BRCA1/2 germline mutation to the phenotype of these tumors. Methods: we analyzed clinical and pathological characteristics of 60 EOBC pts. All women underwent full genetic counseling. Of these, 52 (86%) were tested for BRCA1 and BRCA2 mutation analysis while 8 (14%) pts refused it. Exon 11 was screened for BRCA1 mutations using Protein Truncation test (PTT); mutations detected by PTT were confirmed by Direct Sequencing (DS). All the other exons were analyzed by DS. BRCA2 gene was screened using Denaturing High Performance Liquid Chromatography (DHPLC) for all coding exons. Samples showing a heterozygous DHPLC elution profile were subsequently analyzed by DS. Results: 33 (55%) and 10 (16%) out of the 60 pts had breast and ovarian cancer FH, respectively. Median age of BC diagnosis was 34 yrs (range: 23–41); 7 pts (13%) had Bilateral Breast Cancer (BBC). Two pts had similar history of sarcoma at 11–12 years and EOBC at 31–32 years and one patient had EOBC + Ovarian cancer + Malignant Melanoma. Of the 19 pts with completed mutation analysis, 14 (74%) had wild-type BRCA1(WT) (including 3 pts with EOBC+other tumors), 3 (16%) had variants of unclear significance, 2 (10%) had deleterious mutations in BRCA1. Breast FH was similarly distributed in the 5 mutation carriers (MC) and in the 14 WT (60% vs 78%), while ovarian FH was more frequent in MC (60% vs 21%). One of 5 MC had BBC (age 28 and 42) and 1 had no FH. Stage II-III (80% vs 36%), grade 3 (75 % vs 33%), lack of estrogen (50% vs 20%) and progesterone receptor (67% vs 20%), and high proliferation rate (75% vs 43%) were more common in MC than in WT. BRCA2 analysis is still ongoing. Until now, a deleterious mutation in BRCA2 gene was detected in two pts. Conclusion: our data confirm that in EOBC adverse histopathologic features and ovarian FH are more predictive of being MC than good histopathologic features and breast FH. No significant financial relationships to disclose.

Different expression of BRCA1 status and clinical variables in a sample of Italian women with early onset breast cancer (EOBC)

9670 Background: to estimate the spectrum of genetic alteration, family history (FH) and clinical variables in a EOBC population, diagnosed before 41 yrs, consecutively seen at the Genetic Oncology Service of the Parma Hospital from 1999 to 2003, and to investigate the contribution of BRCA1/2 germline mutation to the phenotype of these tumors. Methods: we analyzed clinical and pathological characteristics of 60 EOBC pts. All women underwent full genetic counseling. Of these, 52 (86%) were tested for BRCA1 and BRCA2 mutation analysis while 8 (14%) pts refused it. Exon 11 was screened for BRCA1 mutations using Protein Truncation test (PTT); mutations detected by PTT were confirmed by Direct Sequencing (DS). All the other exons were analyzed by DS. BRCA2 gene was screened using Denaturing High Performance Liquid Chromatography (DHPLC) for all coding exons. Samples showing a heterozygous DHPLC elution profile were subsequently analyzed by DS. Results: 33 (55%) and 10 (16%) out of the 60 pts had breast and ovarian cancer FH, respectively. Median age of BC diagnosis was 34 yrs (range: 23–41); 7 pts (13%) had Bilateral Breast Cancer (BBC). Two pts had similar history of sarcoma at 11–12 years and EOBC at 31–32 years and one patient had EOBC + Ovarian cancer + Malignant Melanoma. Of the 19 pts with completed mutation analysis, 14 (74%) had wild-type BRCA1(WT) (including 3 pts with EOBC+other tumors), 3 (16%) had variants of unclear significance, 2 (10%) had deleterious mutations in BRCA1. Breast FH was similarly distributed in the 5 mutation carriers (MC) and in the 14 WT (60% vs 78%), while ovarian FH was more frequent in MC (60% vs 21%). One of 5 MC had BBC (age 28 and 42) and 1 had no FH. Stage II-III (80% vs 36%), grade 3 (75 % vs 33%), lack of estrogen (50% vs 20%) and progesterone receptor (67% vs 20%), and high proliferation rate (75% vs 43%) were more common in MC than in WT. BRCA2 analysis is still ongoing. Until now, a deleterious mutation in BRCA2 gene was detected in two pts. Conclusion: our data confirm that in EOBC adverse histopathologic features and ovarian FH are more predictive of being MC than good histopathologic features and breast FH. No significant financial relationships to disclose.

BRCA1 status, molecular markers, clinical variables in breast cancer patients with high probability of having an inherited genetic mutation

9648 Background: to evaluate the clinical features and outcomes of Breast Cancer (BC) patients with genetic susceptibility to this disease and to investigate the contribution of BRCA1 germline mutation to the phenotype of these tumors. Methods: we reviewed clinical and pathological records of 144 women with autosomal dominant inheritance of breast (+/- ovarian) cancer risk. All women underwent full genetic counseling. Of these, 101 selected patients with high probability of having a germ-line mutation were tested for BRCA1 mutation analysis. Exon 11 was screened for BRCA1 mutations using Protein Truncation Test; mutations detected were confirmed by Direct Sequencing. All the other exons were analyzed by DS. Results: the two different risk groups had similar clinical outcomes. Of the 57 patients with completed mutation analysis, 44 (77%) patients had wild type BRCA1, 8 (14%) had variants of unclear significance, 5 (8%) had deleterious mutations in BRCA1. With regard to entry criteria for BRCA1 genetic testing, mutations were detected in 5% (1/20), 2,5% (1/41), 16% (2/12) and 16% (1/6) of women with family history, early onset BC (< 40 years), Breast-Ovarian Cancer (BOC) and early onset plus Bilateral Breast Cancer, respectively. BRCA1 Associated Breast Cancers (BABC) were more likely to have histological grade 3 and high proliferation rate than negative cases (40% v 27%; 60% v 45%). These differences were not statistically significant. BABC were significantly more likely to be estrogen receptor-negative (67% v 16%, P = .04). Though not significant, all valuable tumors with BRCA1 mutations were HER-2/neu negative. In the entire cohort, there were no significant differences between BABC and non-BABC in 5 year relapse free survival (60% v 78%, P = not significant [NS]), 5 year event free survival (60% v 66%, P = NS), or 5 year overall survival. Conclusions: BABC seem to present with adverse molecular and histopathologic features when compared with cases not associated with BRCA1 mutations. However, the prognosis of BABC appears to be similar to that of non associated cancer. No significant financial relationships to disclose.

BRCA1 status, molecular markers, clinical variables in breast cancer patients with high probability of having an inherited genetic mutation

9648 Background: to evaluate the clinical features and outcomes of Breast Cancer (BC) patients with genetic susceptibility to this disease and to investigate the contribution of BRCA1 germline mutation to the phenotype of these tumors. Methods: we reviewed clinical and pathological records of 144 women with autosomal dominant inheritance of breast (+/- ovarian) cancer risk. All women underwent full genetic counseling. Of these, 101 selected patients with high probability of having a germ-line mutation were tested for BRCA1 mutation analysis. Exon 11 was screened for BRCA1 mutations using Protein Truncation Test; mutations detected were confirmed by Direct Sequencing. All the other exons were analyzed by DS. Results: the two different risk groups had similar clinical outcomes. Of the 57 patients with completed mutation analysis, 44 (77%) patients had wild type BRCA1, 8 (14%) had variants of unclear significance, 5 (8%) had deleterious mutations in BRCA1. With regard to entry criteria for BRCA1 genetic testing, mutations were detected in 5% (1/20), 2,5% (1/41), 16% (2/12) and 16% (1/6) of women with family history, early onset BC (< 40 years), Breast-Ovarian Cancer (BOC) and early onset plus Bilateral Breast Cancer, respectively. BRCA1 Associated Breast Cancers (BABC) were more likely to have histological grade 3 and high proliferation rate than negative cases (40% v 27%; 60% v 45%). These differences were not statistically significant. BABC were significantly more likely to be estrogen receptor-negative (67% v 16%, P = .04). Though not significant, all valuable tumors with BRCA1 mutations were HER-2/neu negative. In the entire cohort, there were no significant differences between BABC and non-BABC in 5 year relapse free survival (60% v 78%, P = not significant [NS]), 5 year event free survival (60% v 66%, P = NS), or 5 year overall survival. Conclusions: BABC seem to present with adverse molecular and histopathologic features when compared with cases not associated with BRCA1 mutations. However, the prognosis of BABC appears to be similar to that of non associated cancer. No significant financial relationships to disclose.

The role of muscle biopsy in analysis of the dystrophin gene in Duchenne muscular dystrophy: experience of a national referral centre

Although the majority (65%) of boys with Duchenne muscular dystrophy (DMD) carry a deletion in the dystrophin gene, finding mutations in the remaining families is vital for counselling. We have provided a comprehensive mutation service as a national referral centre for France for over 10 years and we report here our experience. Mutation screening is on mRNA from a muscle biopsy. We have detected 79 mutations in 89 samples referred with a diagnosis of DMD, which is the most comprehensive survey to date of the full range of nondeletion mutations. Although some mutations were nonsense mutations, some frameshift mutations and some splicing mutations, all of them led to the generation of premature stop codons or a shortened product which could be detected using the Protein Truncation Test. We recommend a protocol which is robust and sensitive applied to the entire coding region reverse-transcribed from dystrophin transcripts from muscle biopsy.

Protein Truncation Test

The 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 Alternate Protocol using coupled in vitro transcription/translation and radiolabeled proteins is also presented.

BRCA1 mutations in a population-based study of breast cancer in Stockholm County.

The mutation frequency of BRCA1 and BRCA2 in women with breast cancer varies according to family history, age at diagnosis and ethnicity. The contribution of BRCA1 and BRCA2 mutations in breast cancer populations, unselected for age and family history, has been examined in several studies reporting mutation frequencies between 1% and 12% by screening methods, population sizes, and to what extent the gene/s were screened differed in the studies. We wanted to clarify the proportion of breast cancer attributable to mutations in BRCA1 in an unselected breast cancer population from the Stockholm region. All incident breast cancer patients treated surgically in a 19-month period were eligible for the study and 70% (489/696) participated. Exon 11 of BRCA1 was screened for mutations using the protein truncation test, and the mutation frequency was estimated from that. In previous studies on high-risk families from Stockholm, more than 70% of the mutations were detected in exon 11. Two mutations were found, both in patients with a family history or their own medical history of ovarian cancer, giving a mutation frequency in exon 11 of 0.4% and an estimated BRCA1 mutation frequency of <1%. Mutations in BRCA1 in unselected breast cancer cases in our region are rare and likely to be found only in high-risk families. Our BRCA1 prevalence is the lowest of all studies on unselected breast cancer patients, probably reflecting the comparatively low rates detected also in high-risk breast cancer families from the region.

High frequency of MYH gene mutations in a subset of patients with familial adenomatous polyposis

Background & Aims : Inherited colorectal polyposis has been linked to constitutive mutations of the APC tumor suppressor gene. Recently, germline mutations in the base excision repair gene MYH have been associated with a recessively inherited form of the disease. The aim of this study was to evaluate germline mutation frequencies of both MYH and APC susceptibility genes in Italian patients with attenuated familial adenomatous polyposis. Methods : The analysis was performed in 14 unrelated patients by using the protein truncation test for APC and genomic DNA sequencing for MYH. Results : Overall, we identified 7 of 14 (50%) mutation carriers. Two patients were heterozygotes for an APC truncating mutation (2 of 14 [14%]), whereas 5 proved to be homozygotes or compound heterozygotes for MYH gene alterations (5 of 14 [36%]). Two MYH missense mutations, Y165C and G382D, already found to be frequent among patients from northern Europe, were also preponderant in our survey. Individuals with APC-associated syndrome showed a dominant family history of polyposis, whereas patients with MYH-associated disease were either apparently sporadic cases or had a family history consistent with recessive inheritance. MYH biallelic mutation carriers were up to 60% (5 of 8) among patients showing at least 30 adenomas and a family history with no vertical transmission of polyposis. Conclusions : On the basis of our data, patients with attenuated familial adenomatous polyposis with >30 adenomas and no obvious vertical transmission of the disease should be considered for MYH gene testing.

Comprehensive genetic and endoscopic evaluation may be necessary to distinguish sporadic versus familial adenomatous polyposis–associated abdominal desmoid tumors

Background There are limited data regarding how many patients with desmoid tumors actually represent cases with underlying familial adenomatous polyposis. Methods A proband presenting with desmoid tumors and several of the family members underwent a detailed family history, genetic (adenomatous polyposis coli [APC] gene sequencing), and upper and lower endoscopic evaluation. Results The proband's initial diagnosis was of a sporadic desmoid tumor. Colonoscopy was entirely normal. However, on subsequent esophagogastroduodenoscopy, several gastric polyps were found. The proband's mother subsequently underwent colonoscopy and was found to have multiple colon adenomas. On genetic analysis, a deletion of “T” was identified at codon 2645 of the APC gene in the proband. The proband's mother had a normal APC protein truncation test result. However, on full gene sequencing, the mother was found to harbor the same APC gene mutation. Conclusion A detailed family history and endoscopic and genetic evaluations for patients with desmoid tumors are vital because they may be the sentinel presentation of familial adenomatous polyposis. If confirmed in larger studies, APC full gene sequencing and upper and lower gastrointestinal tract evaluation may need to be part of standard evaluation of patients with abdominal desmoid tumors.

First genotype characterization of Argentinean FAP patients: identification of 14 novel APC mutations.

We examined the adenomatous polyposis coli (APC) gene for disease-causing mutations in 51 unrelated Argentinean probands affected by familial adenomatous polyposis (FAP). Using a combination of the protein truncation test, the single strand conformation polymorphism technique, DNA sequencing and quantitative PCR analysis, we identified the specific mutation in 39 (average age: 28.4 years) of the 51 probands (detection rate: 76.47%); 13 are novel germline mutations and one is a novel sequence variant. There were 27 small deletions, four small duplications, five nonsense mutations in exon 15, three nonsense mutations in exons 6, 11, and 12, and one sequence variant in exon 3 identified in a patient bearing a truncating mutation in exon 15. The most common mutation (found in 10 cases) was at codon 1309. All patients negative for APC mutations were also negative for the MutY homolog (MYH) gene mutation, as expected because of fully penetrant FAP cases. This study enlarges the spectrum of APC gene mutations, and reinforces the concept of mutation heterogeneity. It also sheds light on correlations between the site of APC germline mutations and the clinical manifestations of FAP. Our data indicate that the genotype/phenotype correlations in Argentinean patients are similar to those observed in other populations.

Hereditary ovarian cancer in Poland

Background. Uterine papillary serous carcinoma (UPSC) shares common pathologic, genetic, and clinical features with other serous cancers of müllerian origin. The most common histologic type of ovarian tumor associated with BRCA mutations is papillary serous. Because of these histologic similarities, we postulated that, in some cases, UPSC may be a manifestation of a field defect in BRCA1 carriers, which also includes ovarian carcinoma, fallopian tube carcinoma, and primary peritoneal carcinoma.Methods. Fifty-six living patients with UPSC were contacted through their treating physicians and agreed to a family history interview and to provide a blood specimen for BRCA testing. The protein truncation test was used to detect mutations in exons 10 and 11 of BRCA1 and in exon 11 of BRCA2. The presence of four common mutations was assessed by PCR-based specific assays.Results. A high proportion of patients had a past history of breast cancer (11%) or a first-degree relative with breast cancer (29%). Four patients were from families with site-specific hereditary breast cancer. However, there was no clear example of the hereditary breast–ovarian cancer syndrome, and none of the 56 patients was found to carry a BRCA1 or BRCA2 mutation.Conclusions. BRCA mutations do not appear to predispose to UPSC and this type of cancer does not appear to be a manifestation of the classical hereditary breast–ovarian cancer syndrome. The observed association between UPSC and breast cancer may be due to the presence of mutations in other cancer predisposing genes.

Rapid identification of germline mutations in retinoblastoma by protein truncation testing.

To demonstrate the utility of protein truncation testing (PTT) for rapid detection and sequencing of germline mutations in the retinoblastoma tumor suppressor gene (RB1). We performed PTT, a technique based on the in vitro synthesis of protein from amplified RNA, on 27 probands from 27 kindreds with hereditary retinoblastoma. In 4 kindreds, PTT was also performed on 1 additional affected relative. Ten unrelated patients without retinoblastoma were included as negative control subjects. All PTT-detected mutations were further analyzed by focused sequencing of genomic DNA. When no mutation was detected by PTT, we performed exon-by-exon sequencing, as well as cytogenetic analysis by Giemsa-trypsin-Giemsa banding and by fluorescent in situ hybridization for RB1. The results of proband testing were used for direct genetic testing by polymerase chain reaction and sequencing in 11 relatives from 7 of the 27 kindreds. Of the probands tested, 19 (70%) of 27 tested positive for germline mutations by PTT. In 1 kindred, the proband had negative PTT results but an additional affected relative had positive PTT results. Focused DNA sequencing of 1 patient with positive PTT results from each of the 20 kindreds with positive PTT results revealed truncating mutations in 19 kindreds. Four demonstrated frameshift deletions, 6 had splice site mutations, and 9 showed nonsense mutations. Further analysis by genomic exon-by-exon sequencing and karyotype analysis of the 8 probands who tested negative for germline mutations by PTT revealed 1 splice site mutation, 2 missense mutations, and 1 chromosomal deletion. Focused sequencing based on positive PTT results was successfully used to confirm shared truncating mutations in additional affected family members in 2 kindreds. Using a multitiered approach to genetic testing, 23 (85%) of 27 kindreds had mutations identified and those detected by PTT received a positive result in as few as 7 days. In control subjects, PTT produced no false-positive results. Protein truncation testing is an effective, rapid single-modality screen for germline mutations in patients with retinoblastoma. When used as an initial screen, PTT can increase the yield of additional testing modalities, such as sequencing and chromosomal analysis, providing a timely and cost-effective approach for the diagnosis of heritable germline mutations in patients with retinoblastoma.Clinical Relevance The clinical application of PTT in retinoblastoma will improve detection of germline retinoblastoma mutations, which will supply critical information for prognosis, treatment planning, follow-up care, and genetic counseling.

Mutation scanning for the clinical laboratory-protein truncation test.

AbstractThe protein truncation test (PTT) is a powerful mutation detection technique originally described for mutation screening of the dystrophin gene (1). It is based on in vitro transcription/translation technology, and is capable of directly scanning kilobase-sized fragments of DNA for chain-terminating mutations. PTT should be the mutation detection technique of choice to screen genes in which the majority (if not all) of the disease-causing mutations result in the formation of a truncated protein product.KeywordsPolymerase Chain ReactionPolymerase Chain Reaction ProductDystrophin GeneRabbit Reticulocyte LysatePolymerase Chain Reaction TemplateThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Germline Missense Changes in the APC Gene and Their Relationship to Disease

Familial adenomatous polyposis (FAP) is characterized by the presence of hundreds to thousands of adenomas that carpet the entire colon and rectum. Nonsense and frameshift mutations in the adenomatous polyposis coli (APC) gene account for the majority of mutations identified to date and predispose primarily to the typical disease phenotype. Some APC mutations are associated with a milder form of the disease known as attenuated FAP. Virtually all mutations that have been described in the APC gene result in the formation of a premature stop codon and very little is known about missense mutations apart from a common Ashkenazi Jewish mutation (1307 K) and a British E1317Q missense change. The incidence of missense mutations in the APC gene has been underreported since the APC gene lends itself to analysis using an artificial transcription and translation assay known as the Protein Truncation Test (PTT) or the In Vitro Synthetic Protein assay (IVSP).In this report we have used denaturing high performance liquid chromatography to analyse the entire coding sequence of the APC gene to determine if a cohort of patients adhering to the diagnostic criteria of FAP to assess the frequency of missense mutations in the APC gene. Altogether 112 patients were studied and 22 missense mutations were identified. From the total of 22 missense changes, 13 were silent changes and the remaining 9 resulted in amino acid substitutions. One or more of these changes were identified multiple times in 62.5% of the population under study.The results reveal that missense mutations in the APC gene appear not to radically alter protein function but may be associated with more subtle processing of RNA transcripts which in turn could result in the expression of differentially spliced forms of the APC gene which may interfere with the functional activity of the APC protein.

Germline truncating mutations in both MSH2 and BRCA2 in a single kindred.

There has been interest in the literature in the possible existence of a gene that predisposes to both breast cancer (BC) and colorectal cancer (CRC). We describe the detailed characterisation of one kindred, MON1080, with 10 cases of BC or CRC invasive cancer among 26 first-, second- or third-degree relatives. Linkage analysis suggested that a mutation was present in BRCA2. DNA sequencing from III: 22 (diagnosed with lobular BC) identified a BRCA2 exon 3 542G>T (L105X) mutation. Her sister (III: 25) had BC and endometrial cancer and carries the same mutation. Following immunohistochemical and microsatellite instability studies, mutation analysis by protein truncation test, cDNA sequencing and quantitative real-time PCR revealed a deletion of MSH2 exon 8 in III: 25, confirming her as a double heterozygote for truncating mutations in both BRCA2 and MSH2. The exon 8 deletion was identified as a 14.9 kb deletion occurring between two Alu sequences. The breakpoint lies within a sequence of 45 bp that is identical in both Alu sequences. In this large BC/CRC kindred, MON1080, disease-causing truncating mutations are present in both MSH2 and BRCA2. There appeared to be no increased susceptibility to the development of colorectal tumours in BRCA2 mutation carriers or to the development of breast tumours in MSH2 mutation carriers. Additionally, two double heterozygotes did not appear to have a different phenotype than would be expected from the presence of a mutation in each gene alone.

Novel and recurrent mutations in the NF1 gene in Italian patients with neurofibromatosis type 1.

Neurofibromatosis type 1 (NF1) is one of the most common autosomal dominant disorders in humans, affecting 1 in 3500 individuals. NF1 is a fully penetrant exhibiting a mutation rate some 10-fold higher compared to most other disease genes. As a consequence, a high number of cases (up to 50%) are sporadic. Mutation detection is complex due to the large size of NF1 gene, the presence of pseudogenes and the great variety of lesions. In the present study we attempted to delineate the NF1 mutational spectrum in the Italian population reporting four-year experience with the direct analysis of the whole NF1 coding region in 110 unrelated subjects affected by NF1. For each patient, the whole coding sequence and all splice sites were studied for mutations, either by the protein truncation test (PTT), or, most often, by denaturing high performance liquid chromatography (DHPLC). Mutations were identified in 75 (68%) patients. Twenty-two mutations were found to be novel. The detection rate for the different methods was 7/18 (39%) for PTT, and 68/103 (66%) for DHPLC. The mutations were evenly distributed along the NF1 coding sequence. Thirty-two of the 75 unrelated NF1 patients in which germline mutations were identified (32/75, 43%) harbour 23 different recurrent mutations. Fifteen sequence variants likely to represent non-pathogenic polymorphisms were observed at the NF1 locus. Genotype-phenotype analysis was unable to detect any obvious correlation.

Alternatively spliced, truncated human BRCA2 isoforms contain a novel coding exon.

The protein truncation test (PTT) employs in vitro transcription and translation of amplified cDNA and exonic gDNA to reveal truncating germ-line mutations. In a series of PTT analyses, abnormal splicing in the region encompassing exons 20-23 of BRCA2 was discovered in leucocytes from high-risk breast cancer patients. Although sequencing of the genomic DNA in this region failed to reveal a detectable mutation in these patients, cDNA obtained from this region of BRCA2 uncovered numerous alternative splice isoforms. PTT analysis and nested RT-PCR using RNA from leucocytes from healthy individuals, normal tissue and breast and ovarian cancer tumours demonstrated the presence of these alternatively spliced transcripts in all cases. The splice forms appeared to be more prominent in RNA from aged blood, suggesting that isoform expression was conditional. It is therefore important to distinguish naturally occurring alternative splicing from true splice defects due to mutations when interpreting PTT results.

Five haplotypes account for fifty-five percent of ATM mutations in Brazilian patients with ataxia telangiectasia: seven new mutations.

We have studied the molecular genetics of 27 Brazilian families with ataxia telangiectasia (AT). Five founder effect haplotypes accounted for 55.5% of the families. AT is an autosomal recessive disorder of childhood onset characterized by progressive cerebellar ataxia, ocular apraxia, telangiectasia, immunodeficiency, radiation sensitivity, chromosomal instability, and predisposition to cancer. The ATM gene spans more than 150 kb on chromosome region 11q23.1 and encodes a product of 3056 amino acids. The ATM protein is a member of the phosphatidylinositol 3-kinase (PI-3K) family of proteins and is involved in cell cycle control and DNA repair pathways. DNA was isolated from lymphoblastoid cell lines and haplotyped using four STR markers (D11S1818, NS22, D11S2179, D11S1819) within and flanking the ATM gene; all allele sizes were standardized in advance. In addition to the STR haplotypes, SNP haplotypes were determined using 10 critical polymorphisms. The entire gene was screened sequentially by protein truncation testing (PTT), single strand conformation polymorphism (SSCP), and then denaturing high performance liquid chromatography (dHPLC) to identify the disease-causing mutations. Of the expected 54 mutations, 50 were identified. All mutations but one, led to a truncated or null form of the ATM protein (nonsense, splice site, or frameshift). Five families (18.5%) carried a deletion of 3450nt (from IVS28 to Ex31), making this one of the two most common Brazilian mutations. Mutations were located throughout the entire gene, with no clustering or hotspots. Standardized STR haplotype analysis greatly enhanced the efficiency of mutation screening.

Inherited BRCA2 mutations in African Americans with breast and/or ovarian cancer: a study of familial and early onset cases.

In order to identify the spectrum of BRCA2 mutations in African Americans, breast or ovarian cancer patients from 74 independent families at elevated risk of germline mutations were investigated. The entire coding regions and flanking introns of BRCA2 were screened for germline mutations by single-stranded conformation polymorphism, protein truncation test, or denaturing high performance liquid chromatography followed by DNA sequencing. Eight distinct protein-truncating mutations were detected in six female patients (average age of onset of breast cancer: 37.6 years) and two male patients, but not in 163 unrelated disease-free controls. Two (1993delAA, 8643delAT) of the eight pathogenic mutations observed in African Americans have not been previously described. The other six pathogenic mutations (1882delT, 1991delATAA, 2001delTTAT, 2816insA, 4075delGT, 4088delA) have been detected in Caucasians; only the 2816insA mutation has been reported previously in African Americans. There were no significant differences in the frequency of deleterious BRCA2 mutations in African Americans compared with Caucasians. Six rare variations, not previously reported, were identified in five breast cancer patients but not in 163 disease-free control subjects. Of 11 different polymorphisms identified in high-risk African-American breast cancer patients, four may be unique to African Americans. An intron 10 polymorphism observed in patients was not detected in 163 disease-free African-American control subjects; this difference is statistically significant. Since many different pathogenic mutations and variants of unknown significance are observed in African Americans, BRCA2 genetic testing in high-risk African-American families must include the entire coding and flanking non-coding regions of the gene.