Mutation analysis of RAD51D in non-BRCA1/2 ovarian and breast cancer families.

BACKGROUND: Recent data show that mutations in RAD51D have an aetiological role in ovarian carcinoma, yet mutations do not appear to be associated with an increased risk for breast cancer. We studied ovarian and breast cancer families having at least one woman affected by ovarian carcinoma, to assess the importance of RAD51D mutations in such families. METHODS: The coding region of the RAD51D gene was analysed in 175 BRCA1/2-negative families with family histories of both ovarian and breast cancer ascertained from two Canadian and two Belgian institutions. RESULTS: We identified one previously reported deleterious mutation, p.Arg186(*) (c.556C>T), and two novel variants; missense substitution p.Cys119Arg and an intronic variant c.83-26A>G. p.Arg186(*) segregated with the disease in the family and two ovarian carcinomas available for analysis showed loss of the wild-type allele, but the novel variants are likely neutral. CONCLUSION: RAD51D should be included in genetic screening of ovarian cancer families that do not have BRCA1/BRCA2 mutations. We show that mutations are more likely to be found in families with two or more ovarian cancers, or in probands with first-degree relatives with ovarian cancer, and we feel testing should be preferentially offered to affected women from such families.

A founder BRCA2 mutation in non-Afrikaner breast cancer patients of the Western Cape of South Africa.

Founder mutations in BRCA1 and BRCA2 have been reported in many different populations. We studied 105 Coloured and 16 Black Xhosa women residing in the Western Cape of South Africa diagnosed with breast cancer. We screened these patients using our standard panel of six previously reported SA Afrikaner and Ashkenazi Jewish BRCA1/2 mutations and identified only two Afrikaner mutations. Further screening by the protein truncation test (BRCA1 exon 11, and BRCA2 exons 10 and 11) revealed an additional four deleterious mutations (BRCA1 c.1504_ 1508del,p.Leu502AlafsX2, BRCA2 c.2826_2829del,p.Ile943LysfsX16, c.6447_6448dup,p.Lys2150IlefsX19 and c.5771_5774del,p.Ile1924Argfs X38). The latter, also known in Breast Cancer Information Core nomenclature as 5999del4, was identified in 4 of 105 (3.8%) Coloureds and 4 of 16 (25%) Xhosa women, which makes it a frequent founder mutation in the Western Cape Province. Although this mutation was previously reported to occur in the Netherlands, haplotype analysis indicated two distinct origins for the Dutch and South African mutations, excluding the possibility of a common Dutch ancestor and suggesting gene flow from the indigenous tribes such as the Xhosa to the Coloured population instead. Further studies to determine the carrier rate of this variant in the Xhosa and other SA populations are warranted.

Genetic architecture of prostate cancer in the Ashkenazi Jewish population.

BACKGROUND: Recently, numerous prostate cancer risk loci have been identified, some of which show association in specific populations. No study has yet investigated whether these single nucleotide polymorphisms (SNPs) are associated with prostate cancer in the Ashkenazi Jewish (AJ) population. METHODS: A total of 29 known prostate cancer risk SNPs were genotyped in 963 prostate cancer cases and 613 controls of AJ ancestry. These data were combined with data from 1241 additional Ashkenazi controls and tested for association with prostate cancer. Correction for multiple testing was performed using the false discovery rate procedure. RESULTS: Ten of twenty-three SNPs that passed quality control procedures were associated with prostate cancer risk at a false discovery rate of 5%. Of these, nine were originally discovered in studies of individuals of European ancestry. Based on power calculations, the number of significant associations observed is not surprising. CONCLUSION: We see no convincing evidence that the genetic architecture of prostate cancer in the AJ population is substantively different from that observed in other populations of European ancestry.

A link between diabetes and atherosclerosis: Glucose regulates expression of CD36 at the level of translation.

Both the risk and the rate of development of atherosclerosis are increased in diabetics, but the mechanisms involved are unknown. Here we report a glucose-mediated increase in CD36 mRNA translation efficiency that results in increased expression of the macrophage scavenger receptor CD36. Expression of CD36 was increased in endarterectomy lesions from patients with a history of hyperglycemia. Macrophages that were differentiated from human peripheral blood monocytes in the presence of high glucose concentrations showed increased expression of cell-surface CD36 secondary to an increase in translational efficiency of CD36 mRNA. We obtained similar data from primary cells isolated from human vascular lesions, and we found that glucose sensitivity is a function of ribosomal reinitiation following translation of an upstream open reading frame (uORF). Increased translation of macrophage CD36 transcript under high glucose conditions provides a mechanism for accelerated atherosclerosis in diabetics.

Binding of tissue plasminogen activator to cultured human endothelial cells.

Tissue plasminogen activator (t-PA) and urokinase (u-PA), the major activators of plasminogen, are synthesized and released from endothelial cells. We previously demonstrated specific and functional binding of plasminogen to cultured human umbilical vein endothelial cells (HUVEC). In the present study we found that t-PA could bind to HUVEC. Binding of t-PA to HUVEC was specific, saturable, plasminogen-independent, and did not require lysine binding sites. The t-PA bound in a rapid and reversible manner, involving binding sites of both high (Kd, 28.7 +/- 10.8 pM; Bmax, 3,700 +/- 300) and low (Kd, 18.1 +/- 3.8 nM; Bmax 815,000 +/- 146,000) affinity. t-PA binding was 70% inhibited by a 100-fold molar excess of u-PA. When t-PA was bound to HUVEC, its apparent catalytic efficiency increased by three- or fourfold as measured by plasminogen activation. HUVEC-bound t-PA was active site-protected from its rapidly acting inhibitor: plasminogen activator inhibitor. These results demonstrate that t-PA specifically binds to HUVEC and that such binding preserves catalytic efficiency with respect to plasminogen activation. Therefore, endothelial cells can modulate hemostatic and thrombotic events at the cell surface by providing specific binding sites for activation of plasminogen.

The HNPCC associated MSH2*1906G-->C founder mutation probably originated between 1440 CE and 1715 CE in the Ashkenazi Jewish population.

The MSH2*1906G-->C mutation was recently shown to be a rare yet highly penetrant mutation leading to colorectal cancer. The mutation was only found among Ashkenazi Jewish individuals and lies on an extended haplotype that is common in that population. This study determined that the mutation probably arose between 11 and 22 generations ago, during the time when the Ashkenazim were living in eastern Europe.

Prevalence and penetrance of BRCA1 and BRCA2 gene mutations in unselected Ashkenazi Jewish women with breast cancer.

BACKGROUND: Approximately 2.0%-2.5% of Ashkenazi Jewish women carry one of three founding mutations in the BRCA1 and BRCA2 genes, and each mutation is associated with a high lifetime risk of invasive breast cancer. We investigated the extent to which these three mutations contribute to breast cancer incidence in the Ashkenazi Jewish population. METHODS: We ascertained 457 Jewish women with prevalent cases of breast cancer who were unselected for age or family history of the disease; 412 of these women were tested for the three founder mutations (case patients). Control subjects consisted of 360 non-Jewish women with breast cancer (control patients) and 380 healthy Jewish women with no history of cancer (control subjects). RESULTS: Mutations were found in 48 (11.7%) of 412 Jewish case patients. Forty-six of 48 mutations occurred in women with early-onset breast cancer (<50 years) or a history of ovarian or early-onset breast cancer in a first-, second-, or third-degree relative. The estimated penetrance to age 70 years for breast cancer was 59.9% for the BRCA1 gene mutations and 28.3% for the BRCA2 gene mutation. Compared with Jewish control subjects, the relative risk (RR) of breast cancer for first-degree relatives of mutation carriers was 5.16 (95% confidence interval [CI] = 3.14-8. 48), but risk was also increased for relatives of noncarriers (RR = 1.66; 95% CI = 1.18-2.33). The RR of prostate cancer for first-degree relatives of Jewish case patients was 3.36 (95% CI = 1. 49-7.56). CONCLUSIONS: Approximately 12% of breast cancers in the Ashkenazi Jewish population are attributable to mutations in the BRCA1 or BRCA2 gene. Genetic testing may be useful when Jewish women with breast cancer are diagnosed before age 50 years or have a close relative with ovarian or early-onset breast cancer. An association between breast and prostate cancers was observed in our study population.

TP53 mutations in breast cancer associated with BRCA1 or BRCA2 germ-line mutations: distinctive spectrum and structural distribution.

Several groups have studied the molecular pathology of inherited breast cancer. By combining several such studies, we show in this study that somatic TP53 abnormalities are more common in breast cancer associated with BRCA1 or BRCA2 germ-line mutations than in sporadic breast cancers (odds ratio, 2.8; P = 0.0003). Then, we compared the spectrum of TP53 mutations for breast cancers in the IARC TP53 mutation database with the 82 mutations reported in BRCA1/2-associated breast cancers. The spectrum differed significantly both in distribution (P < 1 x 10(-6)) and in base changes (P = 0.025). Mutations at A:T bp were more common in BRCA1/2-associated tumors and strand bias suggesting DNA repair abnormalities was found. Changes were common at TP53 codons that are not mutation hotspots. Structural modeling showed that most of these p53 non-hotspot amino acids characterized in breast tumors isolated from patients with deficient BRCA1/2 function are distributed in a region of the protein on the opposite side of the p53 DNA-binding surface. Our results suggest that BRCA1/2 mutations influence the type and distribution of TP53 mutations seen in breast cancer.

Magnetoelectric relaxor and reentrant behaviours in multiferroic Pb(Fe2/3W1/3)O3 crystal.

Significant quenched disorder in crystal structure can break ferroic (magnetic or electric) long-range order, resulting in the development of ferroic glassy states at low temperatures such as magnetic spin glasses, electric dipolar glasses, relaxor ferroelectrics, etc. These states have been widely studied due to novel physical phenomena they reveal. Much less known are the effects of quenched disorder in multiferroics, i.e. the materials where magnetic and electric correlations coexist. Here we report an unusual behaviour in complex perovskite Pb(Fe2/3W1/3)O3 (PFW) crystals: the coexistence of electric relaxor, magnetic relaxor and antiferromagnetic (AFM) states. The most striking finding is the transformation of the AFM phase into a new reentrant-type magnetic glassy phase below Tg ≅ 10 K. We show that the behaviour at this transformation contrasts the typical behaviour of canonical spin glasses and is similar to the behaviour of relaxor ferroelectrics. Magnetoelectric effect is also observed in the AFM phase in the temperature range of the transition into electric relaxor phase at Tf ≅ 200. The mechanism of magnetic relaxor behaviour is supposed to arise from the frustrated interactions among the spins located at the AFM domain walls. Our results should inspire further studies of multirelaxor behaviour in other multiferroic systems.

Nuclear genes associated with a single Brassica CMS restorer locus influence transcripts of three different mitochondrial gene regions.

Previous studies have shown that the mitochondrial orf224/atp6 gene region is correlated with the Polima (pol) cytoplasmic male sterility (CMS) of Brassica napus. We now extend this correlation by showing that the effects of nuclear fertility restoration on orf224/atp6 transcripts cosegregate with the pol restorer gene Rfp1 in genetic crosses. We also show, however, that the recessive rfp1 allele, or a very tightly linked gene, acts as a dominant gene, designated Mmt (modifier of mitochondrial transcripts), in controlling the presence of additional smaller transcripts of the nad4 gene and a gene possibly involved in cytochrome c biogenesis. A common sequence, TTGTGG, maps immediately downstream of the 5' termini of both of the transcripts specific to plants with the Mmt gene and may serve as a recognition motif in generation of these transcripts. A similar sequence, TTGTTG, that may be recognized by the product of the alternate allele (or haplotype), Rfp1, is found within orf224 just downstream of the major 5' transcript terminus specific to fertility restored plants. Our results suggest that Rfp1/ Mmt is a novel nuclear genetic locus that affects the expression of multiple mitochondrial gene regions, with different alleles or haplotypes exerting specific effects on different mitochondrial genes.

An MLH1 haplotype is over-represented on chromosomes carrying an HNPCC predisposing mutation in MLH1.

BACKGROUND: The mismatch repair gene, MLH1, appears to occur as two main haplotypes at least in white populations. These are referred to as A and G types with reference to the A/G polymorphism at IVS14-19. On the basis of preliminary experimental data, we hypothesised that deviations from the expected frequency of these two haplotypes could exist in carriers of disease associated MLH1 germline mutations. METHODS: We assembled a series (n=119) of germline MLH1 mutation carriers in whom phase between the haplotype and the mutation had been conclusively established. Controls, without cancer, were obtained from each contributing centre. Cases and controls were genotyped for the polymorphism in IVS14. RESULTS: Overall, 66 of 119 MLH1 mutations occurred on a G haplotype (55.5%), compared with 315 G haplotypes on 804 control chromosomes (39.2%, p=0.001). The odds ratio (OR) of a mutation occurring on a G rather than an A haplotype was 1.93 (95% CI 1.29 to 2.91). When we compared the haplotype frequencies in mutation bearing chromosomes carried by people of different nationalities with those seen in pooled controls, all groups showed a ratio of A/G haplotypes that was skewed towards G, except the Dutch group. On further analysis of the type of each mutation, it was notable that, compared with control frequencies, deletion and substitution mutations were preferentially represented on the G haplotype (p=0.003 and 0.005, respectively). CONCLUSION: We have found that disease associated mutations in MLH1 appear to occur more often on one of only two known ancient haplotypes. The underlying reason for this observation is obscure, but it is tempting to suggest a possible role of either distant regulatory sequences or of chromatin structure influencing access to DNA sequence. Alternatively, differential behaviour of otherwise similar haplotypes should be considered as prime areas for further study.

Double primary cancers of the breast and thyroid in women: molecular analysis and genetic implications.

Multiple primary cancers are characteristic of hereditary cancer syndromes. A familial association between breast and thyroid cancer has been suggested, but a genetic basis for this association has not yet been established. To determine the extent to which double primary cancers of the breast and thyroid are due to common hereditary factors, we conducted a registry- and hospital-based study in Ontario and Quebec. We obtained family histories of 74 women diagnosed with both cancer of the breast and thyroid before 70 years of age. Cancer histories were obtained for the 533 first- degree relatives of these women. The observed cancer rate in the relatives was compared with the expected number, based on age- standardized Canadian cancer incidence rates, and relative risks were estimated. A total of 87 cancers were observed in the relatives, compared to 93.7 expected cancers, giving a relative risk of 0.9 (95% confidence interval (CI): 0.7-1.1). The risk for breast cancer was 1.1 (95% CI: 0.6-1.7) and the risk for thyroid cancer was 0.7 (95% CI: 0-3.8). Blood samples were collected on 53 patients for mutational analysis of the BRCA1, BRCA2, and PTEN genes. One woman was found to be a carrier of a BRCA1 mutation (exon 11 3227delT). Our findings do not support the hypothesis that a significant proportion of double primary cancers of the breast and thyroid are due to hereditary factors.

Tracer disequilibrium in CO2 compartments during NaH14CO3 infusion.

The failure of labeled CO2 to equilibrate between extracellular and intracellular CO2 compartments may influence the accuracy of substrate oxidation measurements during infusion of carbon-labeled tracers because it may generate errors in estimate of fixation of labeled CO2 derived from control experiments in which labeled bicarbonate is infused. In this study, normal volunteers received a 14-hour overnight primed continuous infusion of NaH14CO3. Over the last 4 hours of the study, steady-state conditions were achieved in the specific activities (SAs) of expired 14CO2 and plasma urea, which was used as a probe for hepatic intracellular CO2 SA. Plasma urea SA was approximately 17% lower than expired CO2 SA (46.4 +/- 5.6 v 56.8 +/- 3.9 disintegrations per minute.mumol-1, P < .02). Fractional 14CO2 recovery was 94.8% +/- 0.8%; when corrected for failure to equilibrate with intracellular CO2, fractional recovery was 89.5% +/- 1.9%. These data indicate that compartmentalization of CO2 may occur in humans. The duration of our experiments, required because of the long half-life of plasma urea, may have minimized the apparent magnitude of compartmentalization. Furthermore, it is possible that compartmentalization in extrahepatic tissues could be of either lesser or greater magnitude than that which we observed in liver. Whether this phenomenon contributes to incomplete recovery of 14CO2 during NaH14CO3 infusion cannot be determined from our results. Additional studies using different experimental approaches will be required to better measure CO2 compartmentalization.

Dielectric characterization of (1-x)PMN-xPT (x = 0.07 and 0.10) ceramics synthesized by an ethylene glycol-based soft chemical route.

Materials based on relaxor ferroelectrics have become one of the most important families of functional materials being explored for such applications as sensors/actuators, micro-electromechanical systems (MEMS), non-volatile random access memories, and high-energy-density capacitors. Fabrication of high-quality relaxor-based ceramics remains, however, a challenging task. In this work, a new soft chemical synthetic method for the preparation of the complex perovskite-based relaxor ferroelectric solid solutions, (1-x)Pb(Mg(1/3)Nb(2/3))O(3)-xPbTiO(3) was developed using ethylene glycol as the solvent. Ceramics with compositions of x = 0.07 and 0.10 were prepared and it was found that a 10% stoichiometric excess of Pb(2+) was required to compensate for lead oxide volatility at the high temperatures used for sintering. The ceramics produced by this method show excellent dielectric properties at room temperature, such as a high dielectric constant (~20 000) and low loss over a large temperatures range (tan δ < 0.01 between 20 and 200°C). The temperature dependence of the dielectric constant exhibits typical relaxor ferroelectric behavior, fitting a quadratic law which describes the high-temperature slope of ε'(T) peak. The frequency dispersion of the temperature of maximum permittivity satisfies the Vogel-Fulcher law.

Identification and characterization of human endothelial cell membrane binding sites for tissue plasminogen activator and urokinase.

Cultured human endothelial cells synthesize and secrete two types of plasminogen activator, tissue plasminogen activator (t-PA) and urokinase (u-PA). Previous work from this laboratory (Hajjar, K.A., Hamel, N. M., Harpel, P. C., and Nachman, R. L. (1987) J. Clin. Invest. 80, 1712-1719) has demonstrated dose-dependent, saturable, and high affinity binding of t-PA to two sites associated with cultural endothelial cell monolayers. We now report that an isolated plasma membrane-enriched endothelial cell fraction specifically binds 125I-t-PA at a single saturable site (Kd 9.1 nM; Bmax 3.1 pmol/mg membrane protein). Ligand blotting experiments demonstrated that both single and double-chain t-PA specifically bound to a Mr 40,000 membrane protein present in detergent extracts of isolated membranes, while high molecular weight, low molecular weight, and single-chain u-PA associated with a Mr 48,000 protein. Both binding interactions were reversible and cell-specific and were inhibitable by pretreatment of intact cells with nanomolar concentrations of trypsin. The relevant binding proteins were not found in subendothelial cell matrix, failed to react with antibodies to plasminogen activator inhibitor type 1 and interacted with their respective ligands in an active site-independent manner. The isolated t-PA binding site was resistant to reduction and preserved the capacity for plasmin generation. In contrast, the isolated u-PA binding protein was sensitive to reduction, and did not maintain the catalytic activity of the ligand on the blot. The results suggest that in addition to sharing a matrix-associated binding site (plasminogen activator inhibitor type 1), both t-PA and u-PA have unique membrane binding sites which may regulate their function. The results also provide further support for the hypothesis that plasminogen and t-PA can assemble on the endothelial cell surface in a manner which enhances cell surface generation of plasmin.

Increased dietary branched-chain amino acids do not improve growth in developing rats with chronic biliary obstruction.

We studied dietary branched-chain amino acid enrichment in cholestatic weanling rats. Growth was assessed with body weight, muscle weight and nitrogen balance. Systemic metabolic measurements that reflect liver function were evaluated, including plasma ammonia, albumin, amino acids, glucose, triglyceride and branched-chain ketoacids, as well as urinary carnitine excretion. Twenty-two rats underwent bile-duct ligation at 14 d of age. At weaning, 11 rats were fed a control diet and 11 an isoenergetic, isonitrogenous branched-chain amino acid-enriched diet for 3 wk, each with a sham-operated, pair-fed control. Body weights were similar in all four groups. Changes due to bile-duct ligation and not affected by the diet manipulation included lower plasma glucose, nitrogen balance and muscle weight, and higher triglyceride concentration, carnitine excretion and liver weight. Changes due to ligation that were normalized by dietary manipulation included plasma albumin, ammonia and total amino acid concentrations. The ratio of branched-chain to aromatic amino acids was decreased in ligated animals fed both diets; however, branched-chain amino acids were lower in the two groups fed more branched-chain amino acids.