Screening for familial ovarian cancer: poor survival of BRCA1/2 related cancers.

AIM: To assess the effectiveness of annual ovarian cancer screening (transvaginal ultrasound and serum CA125 estimation) in reducing mortality from ovarian cancer in women at increased genetic risk. PATIENTS AND METHODS: A cohort of 3532 women at increased risk of ovarian cancer was screened at five European centres between January 1991 and March 2007. Survival from diagnosis of ovarian cancer was calculated using Kaplan-Meier analysis and compared for proven BRCA1/2 carriers with non-carriers and whether the cancer was detected at prevalence or post-prevalent scan. Screening was performed by annual transvaginal ultrasound and serum CA125 measurement. RESULTS: 64 epithelial ovarian malignancies (59 invasive and 5 borderline), developed in the cohort. 26 tumours were detected at prevalent round; there were 27 incident detected cancers and 11 interval. 65% of cancers were stage 3 or 4, however, stage and survival were little different for prevalent versus post-prevalent cancers. Five year and 10 year survival in 49 BRCA1/2 mutation carriers was 58.6% (95% CI 50.9% to 66.3%) and 36% (95% CI 27% to 45%), which was significantly worse than for 15 non-BRCA carriers (91.8%, 95% CI 84% to 99.6%, both 5 and 10 year survival p = 0.015). However, when borderline tumours were excluded, the difference in survival between carriers and non-carriers was no longer significant. CONCLUSION: Annual surveillance, by transvaginal ultrasound scanning and serum CA125 measurement, in women at increased familial risk of ovarian cancer is ineffective in detecting tumours at a sufficiently early stage to influence substantially survival in BRCA1/2 carriers.

Relative frequency, heterogeneity and geographic clustering of PKU mutations in Norway.

We have analysed 236 Norwegian phenylketonuria (PKU) alleles by a combination of mutation scanning methods, restriction enzyme-based assays and DNA sequencing. Thirty-three different mutations constituted 99.6% of all mutant alleles (only 1 allele remains unidentified), 23 of these have been identified also in other European countries. Twenty were predicted missense mutations, 6 splice mutations, 4 nonsense mutations and 2 deletion mutations and 1 mutation disrupted the start codon. The 8 most common mutations represented 83.5% of the PKU alleles, with single allele frequencies ranging from 5.9 to 15.7%. Four of these mutations (R261Q, R408W, Y414C, and 1VS12nt1) are commonly occurring also in PKU patients in other European countries, while the other 4 (G46S, G272X, F299C, and R408Q) have higher frequencies in Norway than in any other country studied. Six mutations (I65T, L249F, P281L, Y356X, R158Q, and R252W) have frequencies between 0.8% and 2.1%, and 19 mutations were encountered only once. The majority of PKU mutations were found on the same RFLP/VNTR haplotype backgrounds in Norway as in other European populations, suggesting that only a few of the mutations may represent recurrent mutations (< 3.4%). Among 10 mutations only reported for our population, we detected 2 de novo mutations (0.8%) arisen in Norway. From the birthplaces of the probands' grandparents, each mutation seemed to have an individual geographic distribution within Norway, with patterns of local mutation clustering. Our observations are compatible with multiple founder effects and genetic drift for the distribution of PKU mutations within Norway.

Diverse PAH transcripts in lymphocytes of PKU patients with putative nonsense (G272X, Y356X) and missense (P281L, R408Q) mutations.

The majority of mutations in the human phenylalanine hydroxylase (PAH) gene that lead to the recessive disease phenylketonuria (PKU) are believed to affect the activity or stability of the PAH enzyme. In this study we have performed in vivo analyses of lymphocyte PAH mRNA from PKU patients homozygous for the PKU missense mutations P281L and R408Q as well as the nonsense mutations G272X and Y356X. The mutations G272X, P281L and R408Q, which are located outside the consensus splice site sequence, result in transcripts with one or more exons skipped in addition to full-length transcripts. The mutation Y356X results in transcripts with one or more exons skipped, but no full-length transcripts. Our findings question the value of functional and structural predictions of mutations at the protein level without analyses of the corresponding transcript.

The Norwegian founder mutations in BRCA1: high penetrance confirmed in an incident cancer series and differences observed in the risk of ovarian cancer.

We aimed to describe the penetrances of the four Norwegian founder mutations in BRCA1 (816delGT, 1135insA, 1675delA and 3347delAG) with regard to breast and ovarian cancers in families ascertained through cancer family clinics or a consecutive series of women with breast or ovarian cancer. We have extended the families as far as possible and tested all family members that asked for genetic testing. Penetrance is based upon counting the mutation carriers. The series contains sufficient numbers of mutation carriers to minimise variation in the estimates due to a limited sample set. The penetrances for all four mutations were high, both with respect to breast and ovarian cancers. This is in accordance with other reports from cancer family clinics, but contrasts with reports from population-based series of mutation carriers. Risks of first cancer (breast or ovarian), breast cancer, and ovarian cancer at age 50 years were 43, 30 and 17%, respectively. Corresponding risks at age 70 years were 84, 58 and 58%. Risks for breast cancer before age 30 years and for ovarian cancer before 35 years were low. Penetrances with regard to ovarian cancer were different for the four mutations. The risk of ovarian cancer was doubled in carriers of the 1675delA mutation when compared with the 816delGT mutation (24 versus 12% at age 50 years, P=0.004). The mutations analysed are high penetrance alleles. No differences in penetrance between the series ascertained through the cancer family clinic or the series of consecutive cancer patients was observed. There are discrepancies between our findings and the low penetrances reported for other mutations in other populations. This may be due to methodological differences, but may reflect differences between mutations and/or modifying factors in different populations.

The BRCA1 syndrome and other inherited breast or breast-ovarian cancers in a Norwegian prospective series.

Inherited breast cancer is a heterogenous group of diseases. We examined this heterogeneity in a prospective series of inherited breast and ovarian cancers, previously demonstrated to include 84% of inherited cancers. Ninety-two tumours (65 breast and 27 ovarian) in 82 patients from 70 kindreds were prospectively diagnosed. Fifteen of the breast cancers were in situ, 50 were infiltrating. 40 (49%) of the 82 women carried a BRCA1 mutation, whereas no mutation in BRCA2 was found. Approximately, two-thirds of the BRCA1 mutation carriers had one of the four most frequent Norwegian founder mutations. Ninety-five per cent of the epithelial ovarian cancers occurred in BRCA1 mutation carrying women versus 38% of infiltrating breast cancers and 7% of carcinoma in situ of the breast. The BRCA1 syndrome was phenotypically distinct with invasive, high grade, oestrogen receptor-negative breast cancers and epithelial ovarian cancers. Non-BRCA1/2 inherited breast cancers included carcinoma in situ and lobular carcinoma and were frequently bilateral. Non-BRCA1/2 inherited breast cancer is not associated with epithelial ovarian cancer and in breast cancers has distinct biological characteristics, indicating that the different subgroups of inherited breast cancer may need different healthcare services.

Genetic epidemiology of BRCA1 mutations in Norway.

Familial breast-ovarian cancer has been demonstrated to be frequent but unevenly distributed in Norway. This was assumed to be caused by the reduced population size created by the medieval Bubonic plagues 25 generations ago, and by the following rapid expansion. We have previously reported that four mutations account for 68% of the BRCA1 mutation carriers. Subsequent analysis has resulted in a total of 100 separate families carrying one of these founder mutations. The four mutations occurred on one specific BRCA1 haplotype each. The 1675delA, 816delGT and 3347delAG families originated from the South-West coast of Norway with a few families in the north, while the traceable ancestors of the 1135insA families clustered along the historical inland road from the South-East to mid-Norway. The carriers of each of the four mutations today are descendants of one or a few individuals surviving the plagues. We may identify the majority of BRCA1 mutation carriers in Norway by screening for local founder mutations.

Prediction of the outcome of genetic testing in HNPCC kindreds using the revised Amsterdam criteria and immunohistochemistry.

BACKGROUND AND AIMS: Hereditary non-polyposis colorectal cancer (HNPCC) may be caused by mutations in the mismatch repair (MMR) genes MLH1, MSH2 or MSH6. Family history (Amsterdam criteria) has traditionally been used to select patients for mutation testing. It has been demonstrated that germline mutations in the MMR genes are associated with lack of the corresponding gene product as assessed with immunohistochemistry (IHC) in tumour specimens. The aim of the study was to assess the value of the Amsterdam criteria II and IHC in predicting germline mutations. METHODS: Fifty-six families that were previously tested for MLH1, MSH2 and MSH6 mutations were selected for this study. All pedigrees were extended and verified and the families were scored according to the original (I) and the revised Amsterdam criteria (II). The probabilities for MLH1 and MSH2 mutations were calculated by logistic regression. In addition, all available tumour material from indexed family members was examined by IHC for the presence of the three gene products. RESULTS: Three out of seven (39%) families where the mutation could be identified complied with the Amsterdam criteria I, while all seven (100%) met the Amsterdam criteria II. All families carrying a MLH1 or MSH2 mutation had > 15% calculated probability of finding a mutation. Tumours from all seven mutation carriers lacked the immunohistochemical expression of the corresponding MMR gene. CONCLUSION: The results indicate that the Amsterdam criteria II in combination with immunohistochemistry of the mismatch repair proteins in tumours may be a cost-effective approach to select families for mutation analysis.

Efficacy of early diagnosis and treatment in women with a family history of breast cancer. European Familial Breast Cancer Collaborative Group.

BACKGROUND: Surveillance programmes for women at increased genetic risk of breast cancer are being established worldwide but little is known of their efficacy in early detection of cancers and hence reduction in mortality. METHODS: Data were contributed from seven centres participating in the EU Demonstration Programme on Clinical Services for Familial Breast Cancer. All breast tumours (n = 161) detected prospectively, from the time of enrolment of women in a screening programme, were recorded. Analysis took account of age at diagnosis, whether tumours were screen-detected or not, their pathological stage and outcome by Kaplan-Meier survival plots. RESULTS: Mean age at diagnosis was 48.6 years. Overall, 75% of tumours were detected in the course of planned examinations. For women under age 50 at diagnosis, this figure was 68%. Eighteen percent were mammographically negative, (23% in patients under age 50). At first ("prevalence") round and at follow-up screening, 16% and 22% of tumours respectively were carcinoma in situ (CIS) while 27% and 22% respectively had evidence of nodal or distant spread (CaN+). Comparison of screen-detected and other tumours showed that the latter were more frequently mammogram-negative and CaN+. Overall five-year survival was 89% and five-year event-free survival 86%. Five-year event-free survival was 100% for CIS, 88% for invasive cancer without nodal or distant spread and 67% for CaN+. CONCLUSIONS: The majority of cancers arising in women at increased genetic risk of breast cancer can be detected by planned screening, even in those under age 50. Surveillance should include regular expert clinical examination and teaching of "breast awareness" as well as mammography. Attention to the logistics of screening programmes may improve still further the proportion of tumours that are screen-detected. The trend towards earlier pathological stage in tumours detected during follow-up rounds and the preliminary findings on survival analysis suggest that this approach will prove to be of long-term benefit for breast cancer families.

The structural requirements of epitopes with IgE binding capacity demonstrated by three major allergens from fish, egg and tree pollen.

Three major allergens from cod fish, egg white and tree pollen, were characterized by studies on their allergenic and antigenic structures. The major allergen of cod fish, Allergen M "parvalbumins pI 4.75", is composed of 113 amino acid residues with a molecular weight of 12,328 daltons. It comprised three domains, AB, CD and EF, consisting of 3 helices interspaced by one loop. Each of the loops of the CD and EF domains each coordinates one Ca2+. The antigenicity and allergenicity of Allergen M was deduced from studying the modified protein and some particular synthetic peptides. Three sites were encompassing IgE binding epitopes namely peptides 33-44, 65-74 and 88-96. A novel peptide (49-64), of the CD-domain, was demonstrated to be allergenically/antigenically active and cross reactive with birch pollen allergen, which incidentally was used as a negative control. This site encompassed two repetitive sequences (D-E-D-K) and (D-E-L-K), suggested to be mutually critical for the specificity of antibody binding. This hypothesis was reconfirmed by SPPS of several analogous peptides of region 39-64. Furthermore, peptide 88-103 of the EF-domain was similarly synthesized; it functioned as a monovalent hapten, blocking and not eliciting allergic reaction. Moreover, peptide 13-32 of domain AB, the non-calcium binding domain, was thoroughly tested. The results of PK inhibition showed clear activity and the peptide was found to function at the level of a divalent determinant. Ovalbumin (OA) is the most dominant of five major allergens of egg white and universally used as model protein. OA allergenic epitopes were shown to be mainly determined by the primary structure and depend on certain peptide chain length. The N-terminal decapeptide (OA 1-10) was shown to react with reaginic IgE. Direct skin test on egg allergic patients, showed no activity and the site was therefore concluded to encompasses one single Ig binding haptenic epitope. Peptide OA 323-339, was demonstrated to be valuable in studies of T-cell recognition of protein antigens. Three analogous peptides of this region were prepared and clearly shown to be immunogenic in rabbits and to bind specific IgE from patients allergic to egg. OA 323-339 was concluded to encompass an allergenic and antigenic epitope which was recognized by human and rabbit B-lymphocytes. Eight peptides in the region 11-122 were similarly synthesized. A test battery was performed to study this region using rabbit polyclonal antibodies and human specific IgE. Some of these sites were involved in binding of particular Ig paratopes. Five immunogenic peptides from the major allergens of tree pollen extracts (segment 23-38), were synthesized. The selection of those peptides was setteled using two algorithms for providing the optimal hydrophobicity.(ABSTRACT TRUNCATED AT 400 WORDS)

Allergenic structure of allergen M from cod. II. Allergenicity of the limited tryptic hydrolysis peptides of fragment TM 2.

The peptides P1B3 (amino acid residues No. 88-113) and LT3N1 (residues No. 88-108), obtained from the limited tryptic hydrolysis of fragment TM 2 from codfish allergen M inhibited the interaction between allergen M and reaginic IgE antibodies both in vitro (87 and 62% RAST inhibition, respectively) and in vivo (total PK test inhibition). The peptide A7B1 (residues No. 97-113) overlapping the former at the COOH-terminus, was inactive in PK test inhibition experiments and produced only a minor inhibition of RAST (15%). The findings suggest that the nonapeptide residue No. 88-96 is partially indispensible for the allergenic activity of fragment TM 2, and is likely to incorporate allergenic determinant(s).

Immunochemical analysis of cod fish allergen M: locations of the immunoglobulin binding sites as demonstrated by the native and synthetic peptides.

The major allergen of codfish (Allergen M) is a muscle protein belonging to the family of calcium binding parvalbumins. The primary structure of the molecule was established and the molecular weight was estimated from the sequence data to be 12,328. Allergen M consists of 113 amino acid residues and one residue of glucose. A molecular arrangement of three domains (AB, CD and EF--the latter two bind one Ca2+ ion each) was described for Allergen M, analogous to carp parvalbumin pI 4.25. The suggested structure was based on the extensive intramolecular amino acid homologies and the immunochemical cross-reactivities of the intact molecule and the two major isolated fragments. The immunological structure of Allergen M was studied by: 1. Modification of certain amino acids residues and study of the reactivity of the modified derivatives. 2. Examination of the immunochemical reactivity of a large number of overlapping peptides obtained by limited and selective tryptic hydrolyses. 3. Solid phase peptide synthesis (SPPS) of segments selected in regard to the reactivity of pre-examined native peptides. The immunological reactivity of the derivatives of Allergen M was assigned by: 1. Rocket line immunoelectrophoresis and quantitative precipitation using rabbit anti-Allergen M in precipitating antibody-mediated reactions and, 2. RAST/RAST-inhibition and PK test/PK-test inhibition using sera from individuals allergic to codfish in IgE-mediated reactions. The modification of Tyr-30 and Arg-75 in isolated and purified peptides indicated that the former was part of a reactive site whereas the latter did not contribute to the activity. Masking of Arg-residue or unchelating the two calcium ions from the native Allergen M, with the resulting perturbation of the tertiary structure, decreased the allergenicity by approximately 25%. Two major fragments of Allergen M were produced and purified: TM1 (residues 1-75) comprising domains AB and CD, and TM2 (residues 76-113) covering domain EF. Both were immunologically reactive; TM1 showing intermediate reactivity between Allergen M and TM2. A high degree of immunological cross reactivity was evident between TM1 and TM2. The finding was in concert with the high intramolecular amino acid homologies of Allergen M, and suggested that the reactive sites were repetitively distributed along the polypeptide chain. The immunological reactivity of several long-sequence overlapping peptides obtained by limited and selective trypsin hydrolysis of Allergen M was studied. The immunologically reactive sites were accordingly assigned to the following regions of the chain: 1. Residues 33-44 on the junction between the AB and CD domains.(ABSTRACT TRUNCATED AT 400 WORDS)

Correlation between increased bronchial responsiveness to histamine and diminished plasma cyclic adenosine monophosphate response after epinephrine in asthmatic children. Diminished plasma cyclic adenosine monophosphate response after epinephrine in moderate childhood asthma.

The respiratory threshold to histamine and the plasma cyclic adenosine monophosphate (AMP) every 5 min for 40 min after subcutaneous epinephrine were determined in 21 children with moderate bronchial asthma who were without symptoms at the time of study. There was a statistically significant correlation between a high respiratory sensitivity to histamine and a low plasma cyclic AMP response to epinephrine. The plasma cyclic AMP response was compared with that in 16 control subjects. The asthmatic patients had significantly diminished responses; the difference was greatest for the values 25 min after stimulation. This study supports the hypotheses that the bronchial hyperresponsiveness in asthma is due partly to a defective beta adrenergic system and that the defect is permanent, existing also during periods without symptoms or medication and in patients with moderate asthma.

[Gene therapy for monogenic inherited diseases]. / Genterapi ved monogent arvelige sykdommer.

BACKGROUND: Monogenic inherited disorders are caused by a mutation in one single gene. Each of these disorders is very rare, but as there are many thousand different monogenic disorders, they represent a significant health problem. Most monogenic disorders are severe and have no cure. Gene therapy will therefore often be the only possible treatment. MATERIAL AND METHODS: Characterisation of the gene is necessary for the development of gene therapy, and at the present time only a limited number of the genes in relation to the total number of the severe monogenic disorders is known. RESULTS: Clinical trials for some of the monogenic disorders, such as cystic fibrosis, have been going on for many years. INTERPRETATION: In spite of the tremendous effort, it is so far not documented that patients have been cured of a monogenic disorder by gene therapy.

The Vater association: malformations of the male external genitalia.

Eight cases with the VATER association of malformations are presented. Maliformations of the external genitalia were found in 3 out of 7 male patients. Two of them presented with a bifid scrotum and a caudally displaced, dysplastic penis. Two patients had been exposed to progestin in early pregnancy and one of them had hyposadias and testicular atrophy. It is suggested that malformations of the male external genitalia might be part of malformations found in the VATER association.

Comparative studies on tree-pollen allergens. I. Isolation and partial characterization of a major allergen from birch pollen (Betula verrucosa).

A major allergen from birch pollen (Betula verrucosa) was isolated by a combination of gel permeation chromatography and preparative isoelectric focusing, and was found adequate for further immunological and chemical characterization. The crude aqueous pollen extract was eluted in 6 UV-absorbance peaks from an Ultrogel AcA-54 column. The main allergenic components were detected in two peaks of molecular weights 29,000 (BV4) and 40,000 daltons, respectively. BV4 contained one quantitatively dominating antigen as assessed by crossed immunoelectrophoresis, and was selected for further purification by isoelectric focusing. Column isoelectric focusing of BV4 gave four protein fractions with pI values corresponding to 5.18, 5.42, 5.76, and 5.94. The pI 5.18 protein was quantitatively dominating. This protein inhibited the radio-allergosorbent test in 8 out of 10 sera from birch pollen-sensitive individuals. Using a serum pool, the inhibition curve of BV4 pI 5.18 was parallel to that of the crude extract and gave a similar maximum inhibition. This allergen was shown to be homogeneous as judged by a single, symmetrical precipitate in crossed immunoelectrophoresis, one band in high-volt electrophoresis and a sharp strip in thin-layer analytical electrofocusing. Amino acid analysis showed a high content of serine. The N-terminal analysis as well as the carbohydrate concentration were preliminarily reported and will be confirmed in future studies.