Prospective multicenter cohort study of estrogen and insulin-like growth factor system in BRCA mutation carriers.

PURPOSE: Major clinical characteristics of BRCA1/2-related cancers include association with estrogen and metabolic consequences. We aimed to evaluate serum estradiol (E2) and insulin-like growth factor 1 (IGF-1) levels as a marker of insulin resistance in BRCA1/2 mutation carriers and high-risk, BRCA-negative controls. METHODS: Eligible cancer-free women (age 18-42 with regular menstrual cycles) who had been screened for BRCA1/2 mutations between 2005 and 2013 completed a questionnaire and underwent a single blood draw. E2 was measured with radioimmunoassay, and IGF-1 was measured with enzyme-linked immunosorbent assay. RESULTS: Eighty-six women participated (44 carriers and 42 non-carriers) in this study. BRCA mutation carriers were significantly younger than non-carriers (p = 0.0002). Age-adjusted basal (menstrual cycle days 2-5) serum E2 level was not significantly different between BRCA mutation carriers and non-carriers (30.4 vs. 24.7 pg/mL, p = 0.07). BRCA mutation carriers have significantly lower age-adjusted serum IGF-1 levels compared to non-carriers (89.7 vs. 112.6 ng/mL, p < 0.001). In women with BRCA mutations, the risk of having low serum IGF-1 level (IGF-1: ≤85 ng/mL) was 10.7 times as great as that of women without BRCA mutations (95 % CI 2.5, 46.2). There was a significant inverse association between basal E2 and IGF-1 levels in BRCA mutation carriers after adjusting age and BMI (p = 0.03). CONCLUSIONS: IGF-1 level is significantly lower in cancer-free BRCA mutation carriers versus BRCA-negative controls, and there is a potential association between E2 and IGF-1 in cancer-free BRCA mutation carriers. Our findings may instigate future studies evaluating the role of both E2 and IGF-1 in BRCA mutation carriers.

A pilot study of BRCA mutation carriers' knowledge about the clinical impact of prophylactic-oophorectomy and views on fertility consultation: a single-center pilot study.

BRCA mutation carriers will experience early surgically induced menopause following prophylactic bilateral salpingo-oophorectomy (PBSO). This pilot study aimed to investigate their (1) knowledge about the clinical impact of PBSO; (2) views on fertility consultation (FC)/fertility preservation (FP) treatment; and (3) difficulties in conceiving compared to non-carriers. A cross-sectional, single institution web-survey was performed at a university-based IVF center. Women aged 18-50 years who were screened for BRCA gene mutations from 2005 to 2013 were recruited via mail. Forty-one BRCA-positive and 110 BRCA-negative women completed the survey (response rate: 50 %). The knowledge about the reproductive impact of PBSO was limited, with the majority of women in this highly educated sample only identifying the correct response 64 % of the time. Among BRCA mutation carriers, 24 (59 %) had positive views about FC/FP treatments. A larger proportion of women with no children at the time of BRCA testing, and those who were non-white tended to have positive views toward FP. Women with, versus without, BRCA mutations were more likely to have difficulty in conceiving (p = 0.08). This well-educated group had limited knowledge about the reproductive clinical impact of PBSO, or the benefit of a FP before PBSO. Most women with BRCA mutations were interested in FC/FP treatment if they had not completed childbearing at the time of screening. Targeted referrals for FC at the time of BRCA screening may help women improve knowledge and allow improved decision-making about reproductive options.

Genetic testing for colorectal carcinoma susceptibility: focus group responses of individuals with colorectal carcinoma and first-degree relatives.

BACKGROUND: Colorectal carcinoma (CRC) may be the most frequent form of hereditary cancer. Genetic counseling and testing for heritable CRC is a promising approach for reducing the high incidence and mortality rates associated with the disease. Patients with CRC or those with at least one family member with the disease are the most likely persons to request or be offered genetic testing in the clinical or research setting. Currently, however, little is known about the behavioral, psychosocial, ethical, legal, and economic outcomes of CRC genetic counseling and testing. METHODS: Eight focus group interviews, four for CRC patients (n = 28) and four for first-degree relatives (n = 33), were conducted to obtain insights into attitudes, beliefs, and informational needs about genetic testing for hereditary CRC. RESULTS: Focus group interviews revealed a general lack of knowledge about cancer genetics and genetic testing; worry about confidentiality issues; strong concern for family members, particularly children; and a need for primary care providers to be informed about these issues. Major perceived advantages of genetic testing included improving health-related decisions, guiding physicians in making recommendations for surveillance, and informing relatives about risk potential. Disadvantages included potential discrimination, adverse psychologic effects, and financial costs associated with testing. CONCLUSIONS: As knowledge and media coverage of genetics continue to expand, it becomes increasingly important to continue efforts on behalf of, and in partnership with, those individuals most affected by genetic testing for hereditary cancer syndromes. These findings provide data needed to develop and implement informational, educational, counseling, and research-oriented programs that are sensitive to individuals' concerns and preferences.

In-frame deletions of BRCA1 may define critical functional domains.

The identification of genomic rearrangements involving more than 0.5 kb of the BRCA1 gene has confirmed a more complex mutation spectrum than was initially appreciated. Genomic rearrangements in BRCA1 represent 15% of all mutations in a group of French and American breast and ovarian cancer families and 36% of all mutations in a group of Dutch families. The rearrangements described to date range in size from 510 bp to 23.8 kb, are found throughout the gene, and are most frequently attributable to homologous recombination. We describe the identification of rearrangements in two breast and ovarian cancer families that involve 3.4 and 11.5 kb of the BRCA1 gene and span multiple exons but maintain the reading frame. Both gene rearrangements appear to result from Alu-mediated homologous recombination and have been detected by using a combination of protein truncation analysis and Southern blot analysis. These rearrangements result in the loss of amino acids that lie at the carboxy-terminus of the protein and that have previously been shown to have functional significance. Because these rearrangements result in the deletion of exons but maintain the reading frame, they may provide insights into specific regions and amino acids that have functional significance for the BRCA1 protein.

An Alu-mediated 7.1 kb deletion of BRCA1 exons 8 and 9 in breast and ovarian cancer families that results in alternative splicing of exon 10.

Constitutive large deletions and duplications of BRCA1 resulting from Alu-mediated recombination account for a significant proportion of disease-causing mutations in breast and/or ovarian cancer families. Using Southern blot analysis and a protein truncation test (PTT), we have identified a 7.1 kb germline deletion in two families with breast and ovarian cancer. This deletion, which includes exons 8 and 9 and leads to a frameshift at the mRNA level, appears to result from homologous recombination between closely related Alu repeats, one in intron 7 and one in intron 9. In addition to the transcript without exons 8 and 9, analysis of RNA by protein truncation test from individuals with the deletion also identified the presence of alternative splicing of exon 10 from the mutant allele, which results in a transcript that lacks exons 8, 9, and 10. Of interest is that the two American families who carry this deletion are of northern European ancestry and share a common haplotype, suggesting that this deletion may represent a founder mutation. Genes Chromosomes Cancer 28:300-307, 2000.

A temperature-sensitive mutation of Coprinus cinereus, hyt1-1, that causes swelling of hyphal tips.

The TU25 mutant strain of the basidiomycete Coprinus cinereus grows well at 28 degrees C but not at 37 degrees C. Microscopic examination revealed that TU25 exhibited swelling at hyphal apices after a shift-up from 28 degrees C to 37 degrees C. The temperature sensitivity and hyphal swelling co-segregated through meiosis as expected for a single Mendelian factor, designated hyt1 (hyphal tip). Both defects could be suppressed by the presence of osmotic stabilizers in the medium, suggesting that the hyt1 gene product is required for proper cell-wall function. Chemical analysis of the cell walls, however, failed to detect any clear difference in the composition of the cell-wall polysaccharides between the wild-type and TU25. A DNA fragment which complements the hyt1-1 mutation was cloned and sequenced. The predicted protein in the ORF essential for complementation of hyt1-1 is a novel protein.

Frequent changes in the number of reiterated ribosomal RNA genes throughout the life cycle of the basidiomycete Coprinus cinereus.

We have examined the stability of the tandemly repeated genes that encode the ribosomal RNA in Coprinus cinereus. These genes are contained within two linked HindIII fragments in a 3.0-Mb chromosome. We monitored the size of these fragments in both mitotic and meiotic segregants using the contour-clamped homogeneous electric field (CHEF) method. No length changes were observed in the smaller HindIII fragment (100 kb; 10 repeats) among the DNAs prepared from 46 asexual spore derivatives (oidia) or 128 meiotic segregants (basidiospores from 32 tetrads). However, the larger HindIII fragment (1100 kb; 120 repeats) did exhibit variability. Substantial changes, involving up to 40% of the larger HindIII fragment were recorded in 7 of 46 oidial isolates (including 4 of 22 transformed derivatives). To learn if the changes were confined to the vegetative portion of the life cycle, we examined transmission of HindIII variants through three crosses. In the first two crosses (16 tetrads total), no changes were observed in the large HindIII fragment. However, in the third cross (16 tetrads), each tetrad showed at least one alteration. In half of the tetrads from the third cross, the altered patterns segregated 2:2, suggesting that the changes occurred after mating but prior to premeiotic DNA replication. We conclude that breakage and rejoining reactions within the rDNA are frequent and are not confined to any particular stage of the life cycle. It also appears that certain repeats are sheltered from these events. Finally, marked differences in rDNA stability were observed in the cross analyzed.

Targeted transformation in Coprinus cinereus.

We examined the influence of DNA form and size on the arrangement and genomic location of transforming DNA sequences in the basidiomycete Coprinus cinereus. Protoplasts with either single or double mutations in the tryptophan synthetase (TRP1) gene were transformed with cloned copies of this gene which contained only a single DNA strand, contained a specific single nick within the C. cinereus sequences (4.8 kb), contained a specific double-strand break, or contained an additional 35 kb of flanking genomic sequences. Gene replacement events were recovered when each DNA type was used. However, none of these substrates offers a substantial improvement in transformation or targeting frequency when compared to supercoiled circular DNA, which has allowed recovery of both gene replacements as well as homologous insertions in 5% of the transformants analyzed. The frequency of transformants carrying tandem insertions with multiple copies of the transforming DNA was reduced when single-stranded DNA was used, and increased when DNA containing double-strand breaks was used. These results have important implications for the efficient design of targeted transformation and co-transformation experiments.

Molecular characterization of TRP1, a gene coding for tryptophan synthetase in the basidiomycete Coprinus cinereus.

We utilized a cloned gene (TRP5) encoding tryptophan synthetase (TSase) from Saccharomyces cerevisiae to identify and clone the corresponding gene (TRP1) from the basidiomycete Coprinus cinereus. The primary nucleotide (nt) sequence of this gene was determined and compared to sequences from other filamentous fungi, as well as to other genes coding for TSase. A transformation assay was used to demonstrate that 321 nt, which do not include CAAT or TATAAA elements and precede the translation initiation codon, are sufficient for expression in a variety of chromosomal locations. The coding region (2584 nt) is interrupted at nine positions, and putative splicing signals (5'-GTRNGT...YAG-3') are present in each case. The predicted translation product contains 702 amino acids (aa) and is very similar to other TSases, except in the region of aa 257-296 that connects the alpha and beta functional domains. Both the number and the identity of the aa differ in this region between C. cinereus. S. cerevisiae, and Neurospora crassa. Comparison of exon boundaries in the C. cinereus sequence to the three-dimensional structure of Salmonella typhimurium TSase indicates that there is no simple correlation between exons and major functional domains in this protein.

Characterization of cDNA clones for human glycophorin A. Use for gene localization and for analysis of normal of glycophorin-A-deficient (Finnish type) genomic DNA.

Glycophorin A is the major membrane sialoglycoprotein of human erythrocytes and represents a typical example of a transmembrane glycoprotein. The functional role of this cell-surface component is not known but it represents a receptor for viruses, bacteria and parasites like Plasmodium falciparum. 1. Two cDNA clones encoding glycophorin A have been characterized from human fetal cDNA libraries. The longer cDNA extended from the coding region of glycophorin A (residues 4-131) to the 3' untranslated region which included two polyadenylation signals and a poly(A) tail. 2. The structural gene for glycophorin A is located on chromosome 4, q28-q31 as shown by in situ hybridization, thus confirming the previous localization by genetic linkage analysis. 3. Three distinct mRNA species (1.0 kb, 1.7 kb and 2.2 kb) have been identified in erythroid spleen. Northern blot analyses with a probe directed against the 3' untranslated region of the mRNAs indicated that all these species share a homologous 3' non-coding region and that the first polyadenylation signal downstream the stop codon is not used. 4. Preliminary studies by Southern blot analysis of the genomic DNA from normal En(a+) and rare En(a-) donors suggest that the glycophorin A gene has a complex organization and is largely deleted in donors of the En(a-) phenotype (Finnish type) who lack glycophorin A on their red cells.

DNA-mediated transformation of the basidiomycete Coprinus cinereus.

We have developed a simple and efficient transformation system for the agaric fungus, Coprinus cinereus. Protoplasts were prepared from asexual spores that harbor one or two mutations in the structural gene for tryptophan synthetase. The protoplasts can be stably transformed using the cloned Coprinus gene at a frequency of 1 in 10(4) viable protoplasts. A variety of molecular events accompanies the formation of stable transformants, including insertion of the transforming DNA at the homologous locus. The transforming DNA is stable through cell division, mating, fruiting body formation, and meiosis.

Characterization of the catalytic subunit of factor XIII by radioimmunoassay.

Plasma factor XIII is composed of two subunits, a and b, whereas platelet and other intracellular zymogens have only a-subunits. The catalytic subunit, a, is the same in all forms. In order to characterize the interactions of 1- with b-chains in the plasma zymogen and a-chains with other molecules and to correlate factor XIII activity with a-protein, a specific, sensitive radioimmunoassay was developed. With the polyclonal antisera used, the assay recognizes all molecular forms of a (zymogens, activation intermediates, enzyme) equally well. The assay can be used to determine a-chain concentration in plasma and serum and in purified test systems. Fibrinogen in high concentrations affects the assay, probably by interfering with the interactions of 125I-a with antibody. However, at the plasma dilutions used in the assay, there is no significant fibrinogen effect. With this assay, the a-chain concentration in normal plasma is approximately 15 micrograms/ml. This compares with 14 micrograms/ml b-chain in plasma and indicates that all of the a- and b-chains in plasma probably circulate in the form of an equimolar zymogen complex. The serum concentration of a-protein is about 6% of the plasma concentration. There is a high correlation between a-protein and factor XIII activity.

Immunochemical studies of human factor XIII.

Plasma factor XIII circulates as a noncovalently associated, tetrameric zymogen (a2b2). The b subunit may act as a carrier protein for the a subunit, which possesses the potential catalytic function. In order to define interactions that occur between the two subunits, a sensitive and specific RIA for the b subunit has been developed. Purified plasma factor XIII was incubated with thrombin and chromatographed on organomercurial agarose to separate the subunits. Pure b chain eluted in buffer containing CaCl2. This material was used as the standard b preparation, both for preparing a monospecific antiserum and for establishing the assay. The linear range of the assay is 7 to 700 ng/ml (Ca. 0.1 to 10 nM b subunit), with a minimum detectable dose of l. Data were analyzed by use of the logit-log transformation of antigen-binding curves. The dose-response slope for standard b is -2.76 + or - 0.20, with a potency (ED50) of 74.9 + or - 6.5 ng/ml. This RIA is also valid for determining b subunit concentration of plasma and serum. The dose-response slope for the plasma system is -2.69 + or - 0.20 with an ED50 identical to that of the purified system. By this method the mean b subunit concentration in normal human plasma (corrected for anticoagulant) is 13.8 micro g/ml (ca. 0.15 micro M). The concentration in serum is 13.6 micro g/ml, which shows that b subunit is quantitatively recovered after coagulation has occurred.

The levels of corticosterone-binding proteins in rat milk and coincidental serum, and the dissociation rates of the corticosterone.protein complexes.

The corticosterone-binding protein present in rat whey was further characterized by determining, with the aid of a dextran-coated charcoal procedure, the apparent rate of dissociation of the corticosterone.protein complex. The half-time values for the dissociation of the corticosterone.protein complexes in rat whey and serum were compared and found to be identical, i.e. 23 min at 0 C, when the measurements were made over a period of 40 min. The possible presence in small amounts of a corticosterone.protein complex in whey with the much slower dissociation rate characteristic of mammary glucocorticoid receptor could not be detected even when the dissociation was followed over a much longer period. The charcoal adsorption method also provided independent estimates of the molar concentrations of the corticosterone-binding proteins in rat serum and whey. The mean concentration of corticosterone-binding protein in whey was found to be 15% of that in coincidental serum during early lactation. The serum levels of corticosterone-binding protein decline markedly at parturition and then rise from day 2 to day 6 of lactation in rats with small litters. The results of this and a previous study suggest that the corticosterone-binding protein in whey is probably derived from that in serum. The mode of transport of the corticosterone-binding protein from the bloodstream across the mammary epithelium into milk as well as the concentrations of the corticosterone-binding proteins in serum and whey may be factors influencing the uptake of the glucocorticoid by its target cells.

The renaturation of reduced polyalanyl-chymotrypsinogen and chymotrypsinogen.

Chymotrypsinogen has been successfully renatured in solution, after reduction of its 5 disulfide bonds in 6 M guanidine-HCl. This has been made possible by the study of the renaturation of a model derivative, polyalanyl-chymotrypsinogen. The reduced derivative is shown to refold and reoxodize spontaneously, with a 30-40% yield, into molecules which are monomeric and fully susceptible to activation by trypsin. Chymotrypsinogen can also be renatured but only in the presence of reagents allowing disulfide interchange and of moderate concentrations of guanidine-HCl or urea. These results illustrate how the kinetic trapping of incorrectly folded molecules by wrong S-S bonds and aggregation can be overcome, thus allowing the correct refolding of the protein.