Variation of risks of breast and ovarian cancer associated with different germline mutations of the BRCA2 gene.

The breast cancer susceptibility gene BRCA2 on chromosome 13q12-13 has recently been identified. Germline mutations of BRCA2 are predicted to account for approximately 35% of families with multiple case, early onset female breast cancer, and they are also associated with an increased risk of male breast cancer, ovarian cancer, prostate cancer and pancreatic cancer. Germline mutations of a second cancer susceptibility gene BRCA1 (ref. 5), are associated with a strong predisposition to ovarian cancer as well as female breast cancer. Recent studies have suggested that the phenotype in BRCA1 families with respect to the ratio of breast to ovarian cancer varies with the location of the BRCA1 mutation. To determine whether germline mutations in BRCA2 are associated with a similar variation in phenotypic risk, we have analysed the distribution of mutations in 25 families with multiple cases of breast and/or ovarian cancer ascertained in the United Kingdom and Eire. These mutations all lead to premature truncation of BRCA2 as a result of frameshift deletions/insertions or nonsense mutations. Analysis of the mutation distribution along the length of the gene indicates a significant genotype-phenotype correlation. Truncating mutations in families with the highest risk of ovarian cancer relative to breast cancer are clustered in a region of approximately 3.3 kb in exon 11 (P = 0.0004). Published data on mutations in 45 other BRCA2-linked families provide support for this correlation.

BRCA2 mutations in primary breast and ovarian cancers.

The second hereditary breast cancer gene, BRCA2, was recently isolated. Germline mutations of this gene predispose carriers to breast cancer, and, to a lesser extent, ovarian cancer. Loss of heterozygosity (LOH) at the BRCA2 locus has been observed in 30-40% of sporadic breast and ovarian tumours, implying that BRCA2 may act as a tumour suppressor gene in a proportion of sporadic cases. To define the role of BRCA2 in sporadic breast and ovarian cancer, we screened the entire gene for mutations using a combination of techniques in 70 primary breast carcinomas and in 55 primary epithelial ovarian carcinomas. Our analysis revealed alterations in 2/70 breast tumours and none of the ovarian carcinomas. One alteration found in the breast cancers was a 2-basepair (bp) deletion (4710delAG) which was subsequently shown to be a germline mutation, the other was a somatic missense mutation (Asp3095Glu) of unknown significance. Our results suggest that BRCA2 is a very infrequent target for somatic inactivation in breast and ovarian carcinomas, similar to the results obtained for BRCA1.

Identification of the familial cylindromatosis tumour-suppressor gene.

Familial cylindromatosis is an autosomal dominant genetic predisposition to multiple tumours of the skin appendages. The susceptibility gene (CYLD) has previously been localized to chromosome 16q and has the genetic attributes of a tumour-suppressor gene (recessive oncogene). Here we have identified CYLD by detecting germline mutations in 21 cylindromatosis families and somatic mutations in 1 sporadic and 5 familial cylindromas. All mutations predict truncation or absence of the encoded protein. CYLD encodes three cytoskeletal-associated-protein-glycine-conserved (CAP-GLY) domains, which are found in proteins that coordinate the attachment of organelles to microtubules. CYLD also has sequence homology to the catalytic domain of ubiquitin carboxy-terminal hydrolases (UCH).

Localization to Xq27 of a susceptibility gene for testicular germ-cell tumours.

Testicular germ-cell tumours (TGCT) affect 1 in 500 men and are the most common cancer in males aged 15-40 in Western European populations. The incidence of TGCT has risen dramatically over the last century. Known risk factors for TGCT include a history of undescended testis (UDT), testicular dysgenesis, infertility, previously diagnosed TGCT (ref. 7) and a family history of the disease. Brothers of men with TGCT have an 8-10-fold risk of developing TGCT (refs 8,9), whereas the relative risk to fathers and sons is fourfold (ref. 9). This familial relative risk is much higher than that for most other types of cancer. We have collected samples from 134 families with two or more cases of TGCT, 87 of which are affected sibpairs. A genome-wide linkage search yielded a heterogeneity lod (hlod) score of 2.01 on chromosome Xq27 using all families compatible with X inheritance. We obtained a hlod score of 4.7 from families with at least one bilateral case, corresponding to a genome-wide significance level of P=0.034. The proportion of families with UDT linked to this locus was 73% compared with 26% of families without UDT (P=0.03). Our results provide evidence for a TGCT susceptibility gene on chromosome Xq27 that may also predispose to UDT.

Prevalence of BRCA1 and BRCA2 gene mutations in patients with early-onset breast cancer.

BACKGROUND: Mutations in the BRCA1 and BRCA2 genes are found in most families with cases of both breast and ovarian cancer or with many cases of early-onset breast cancer. However, in an outbred population, the prevalence of BRCA1 and BRCA2 mutations in patients with breast cancer who were unselected for a family history of this disease has not been determined. METHODS: Mutations in the BRCA1 and BRCA2 genes were detected in blood samples from two population-based series of young patients with breast cancer from Britain. RESULTS: Mutations were detected in 15 (5.9%) of 254 women diagnosed with breast cancer before age 36 years (nine [3.5%] in BRCA1 and six [2.4%] in BRCA2) and in 15 (4.1%) of 363 women diagnosed from ages 36 through 45 years (seven [1.9%] in BRCA1 and eight [2.2%] in BRCA2). Eleven percent (six of 55) of patients with a first-degree relative who developed ovarian cancer or breast cancer by age 60 years were mutation carriers, compared with 45% (five of 11) of patients with two or more affected first- or second-degree relatives. The standardized incidence ratio for breast cancer in mothers and sisters was 365 (five observed and 1.37 expected) for 30 mutation carriers and 199 (64 observed and 32.13 expected) for 587 noncarriers. If we assume recent penetrance estimates, the respective proportions of BRCA1 and BRCA2 mutation carriers are 3.1% and 3.0%, respectively, of patients with breast cancer who are younger than age 50 years, 0.49% and 0.84% of patients with breast cancer who are age 50 years or older, and 0.11% and 0.12% of women in the general population. CONCLUSIONS: Mutations in the BRCA1 and BRCA2 genes make approximately equal contributions to early-onset breast cancer in Britain and account for a small proportion of the familial risk of breast cancer.

Low frequency of somatic mutations in the LKB1/Peutz-Jeghers syndrome gene in sporadic breast cancer.

Germ-line mutations in the LKB1 gene on chromosome 19p are responsible for most cases of the Peutz-Jeghers syndrome, in which intestinal hamartomas are associated with elevated risks of several cancer types, including breast cancer. We have evaluated the role of somatic mutations in LKB1 in breast cancer. Of 40 informative primary breast cancers, 3 showed loss of heterozygosity on chromosome 19p in the vicinity of LKB1, and no somatic mutations of LKB1 were observed in 62 primary breast cancers and 17 established breast cancer cell lines. The results indicate that mutations in LKB1 do not play an important role in the development of sporadic breast cancer.

The effect of limited courses of cyclosporine on survival and immunocompetence of allogeneic bone marrow chimeras.

Isolator-maintained CBH/Ola (Rtlc) rats were lethally irradiated and reconstituted with spleen and bone marrow cells from fully allogeneic WAG or Wistar (Rtlu) donors. Hematopoietically reconstituted rats were treated with cyclosporine (CsA)4 as prophylaxis for graft-versus-host disease (GVHD) for periods ranging from 6 to 26 weeks. Following the termination of CsA treatment GVH reactivity developed in all recipients of allogeneic cells regardless of the duration of immunosuppression. Approximately a third of the reconstituted rats survived the post-CsA period of GVH activity; these rats carried peripheral lymphocytes and spleen cells of donor strain origin and were specifically unresponsive to donor strain skin grafts. Surviving chimeras remained healthy for long periods (up to 18 months) after transplantation, although morbidity increased slightly for rats moved to normal animal house conditions. However, all chimeras had some degree of lymphopenia and showed diminished immunological responses to extraneous antigens and third-party skin grafts. Experiments to elucidate the mechanisms by which specific tolerance was maintained in chimeras indicated that neither the deletion of host-reactive lymphocytes from the graft nor an absence of host bone-marrow-derived "stimulator" cells was responsible. It was shown that the potential GVH reactivity of normal donor strain cells was specifically suppressed in vivo (in the chimera) and that this suppression could be transferred to secondary irradiated recipients by transferring chimeric spleen cells. Attempts to demonstrate a role for suppressor cells in the maintenance of the chimeric state yielded inconclusive results.

Case of interstitial 12q deletion in association with Wilms tumor.

A 14-month-old boy presenting with Wilms tumor (WT) was found to have a small de novo deletion of the long arm of chromosome 12 (12q11-12q13.11). Microsatellite analysis of this region from constitutional DNA showed that the paternal allele was absent between the markers D12S331 and D12S1713 (inclusive). In the WT there was no evidence of loss of the maternal chromosome. Constitutional chromosome abnormalities can often point to the presence of genes that are important in disease, and the deletion of chromosome 12 in this patient may indicate a gene involved in WT. To determine whether a WT predisposition locus exists at 12q we examined the region in two familial Wilms tumor (FWT) pedigrees unlinked to the known FWT genes on chromosomes 17q (FWT1), 19q (FWT2), and 11p (WT1). In both families WT did not segregate with chromosome 12q markers located within the deletion boundaries.

Allelotype of uterine leiomyomas.

Uterine leiomyomas are the most common benign tumor that arise from smooth muscle cells of the myometrium. Little is known about the etiology and pathogenesis of this tumor. To investigate the molecular pathogenesis of these tumors, we have conducted an allelotype of 102 leiomyomas from 12 patients, using 67 fluorescently-tagged oligonucleotide primers amplifying microsatellite loci covering all autosomes. No areas of the genome showed frequent loss of heterozygosity (LOH); however, the highest rate of LOH (9%) was observed on 7q, consistent with previous cytogenetic observations. Uterine leiomyomas are sometimes multiple. In general, multiplicity of other types of neoplasm is associated with genetic predisposition to the disease. Because multiple tumors were available from each of the 12 patients studied, we looked for evidence of allele-specific LOH, which might indicate the presence of an underlying predisposition gene. However, no evidence for allele-specific LOH was detected, indicating that if cases of multiple uterine leiomyoma are due to an underlying predisposition gene, it is unlikely to be a recessive oncogene.

TGF beta 1 and IL-4 have opposing effects on the proliferation of chronic phase chronic myeloid leukaemic cells stimulated by G-CSF in vitro.

The effects of transforming growth factor beta 1 (TGF beta 1) have been studied in vitro on the clonogenicity of haemopoietic progenitor cells (CFU-CML) from 14 patients with chronic myeloid leukaemia (CML) in chronic phase and 13 normal donors. In 14/14 patients with CML, 5 ng of TGF beta 1/dish decreased CFU-CML-formation in cultures stimulated with 15 ng of granulocyte colony-stimulating factor (G-CSF)/dish (p < 0.0005) and in 13/14 patients TGF beta 1 reduced CFU-CML-formation induced by G-CSF in combination with 5 ng of recombinant human interleukin-4 (rhIL-4)/dish (p < 0.005). In 10/14 samples the number of CFU-CML were reduced to levels lower than in cultures containing G-CSF alone (p < 0.01). In contrast, TGF beta 1 had no significant inhibitory effect on the G-CSF-directed proliferation of normal donor mononuclear cells (MNC) either alone or in combination with rhIL-4. RhIL-4 increased G-CSF-induced colony formation in 13/14 CML samples (p < 0.001), but did not have the same effect in the normal donor samples. The in vitro clonogenicity of CML peripheral blood MNC stimulated with 15 ng of G-CSF could not be correlated with the white cell count or the percentage of CD34+ cells at diagnosis.

A receptor for monomeric IgG2b on rat macrophages.

The binding to rat splenic and peritoneal macrophages of affinity-purified monoclonal rat IgGs, representing all IgG subclasses, was measured by the direct binding of 125I-labelled proteins using an assay that did not require the removal of unbound Ig by washing. Only rat monomeric IgGs of the subclass IgG2b bound specifically and in large amounts to rat macrophages. The binding was temperature dependent and more IgG2b bound to the cells at 4 degrees than at 37 degrees. Spleen macrophages bound approximately 10 times more IgG2b than the same number of peritoneal macrophages, although the association constants (Kas) for the binding were similar for both types of macrophage. The calculated values for the Kas, which varied slightly with each experiment and increased with decrease in temperature, fell within the range 1.3-5.3 X 10(8) M-1; the number of binding sites was estimated as about 10(5)/splenic macrophage and 10(4)/peritoneal macrophage. The binding of 125I-IgG2b to splenic macrophages was inhibited only by unlabelled proteins of the IgG2b isotype and not by IgG1, IgG2a and IgG2c proteins. Soluble IgG2b-antigen complexes also bound to the FcR for monomer but a soluble IgG2a-antigen complex did not inhibit the binding of monomeric IgG2b.

The rat FcR for monomeric IgG is preferentially expressed on red pulp macrophages in the spleen.

Rat monoclonal IgG2b immunoglobulins labelled with 125I or biotin were localized in the red pulp areas but were not found in the marginal zones of the white pulp of the spleen 8 hr after their i.v. injection. In cell suspensions made from the spleen, 90% of red pulp macrophages (M phi) bound 125I-IgG2b monomeric proteins in vitro, whereas similar estimates for marginal zone M phi were 12-30%. Red pulp and marginal zone M phi were distinguished primarily by monoclonal antibodies (mAb) ED2 and ED3, but also by their ability to take up FITC-labelled Ficoll in vivo.

Effect of cyclosporin A on the anti-leukaemia action associated with graft-versus-host disease.

Graft-versus-host disease (GVHD) was induced in Hooded (Rt1c) strain rats by means of high dose total body irradiation (TBI) and subsequent reconstitution with allogeneic bone marrow and spleen cells from WAG (Rt1u) strain donors. Untreated recipients of allogeneic cells died within 20 days of engraftment, whereas those treated daily with Cyclosporin A (CyA), given either from the day receipt of the graft (Day 0) or from Day 4, survived until the end of the experiment (Day 50). If delayed until Day 7, CyA prophylaxis was totally ineffective. Hooded rats bearing a syngeneic leukaemia were irradiated and reconstituted with allogeneic bone marrow. During the course of the ensuing graft-versus-host response (GVHR) leukaemia cells were eradicated from the spleens of the host animals. However, as a consequence of CyA prophylaxis, whether started on Day 0 or delayed until Day 4, the anti-leukaemia potential of the bone marrow allograft was completely abrogated. Anti-tumour activity after engraftment was detectable first at Day 7, i.e. the time at which the GVHR became intractable to the effects of CyA. The results indicate (1) that CyA suppresses the initial events but not the effector phase of the GVHR, and (2) that the anti-host and anti-tumour action of the GVHR may be temporally inseparable.

IgA antibodies in the bile of rats. IV. Synthesis of secretory antibodies in irradiated rats reconstituted with spleen cells: influence of microenvironment on the regulation of IgA production.

Inbred rats were given sub-lethal doses of irradiation so that they were unable to make antibodies unless they were reconstituted with syngeneic lymphoid cells. The ability of the reconstituted rats to make antibodies of the IgA class was measured in terms of the titres of specific antibodies that appeared in their bile. After syngeneic spleen cells, together with antigen, were injected into the spleens of irradiated rats, the titres of specific antibodies that appeared in the bile were low. After the same dose of spleen cells and antigen had been injected into the GALT of irradiated rats, the titres of biliary antibodies were substantial. These results, together with those of appropriate control experiments, suggest that radio-resistant influences in the microenvironment of the GALT may be important in inducing lymphoid cells to produce antibodies of the IgA class in response to local antigen.

Alleletyping of an oligodendrocyte-type-2 astrocyte lineage derive from a human glioblastoma multiforme.

We have conducted alleletyping of two novel cell lines derived from glioblastoma multiforme, which appear to have arisen from different glial lineages, by using 76 fluorescently labeled oligonucleotide primers amplifying microsatellite loci covering the entire human genome. One cell line, Hu-O-2A/Gb1, expresses antigens and metabolic profiles characteristic of the oligodendrocyte-type-2 astrocyte (0-2A) lineage of the rat central nervous system. This cell line generated, in vitro, cells with characteristics of 0-2A progenitor cells, oligodendrocytes and astrocytes. The second cell line, IN1434, is derived from an astrocyte or a precursor cell restricted to astrocytic differentiation. Hu-O-2A/Gbl cells show allelic losses of loci on chromosomes 2, 5, 6, 7, 8, 9, 10, 11, 13, 15, 16, 17, 20 and 21. IN1434 cells are likely to have allelic losses of loci on chromosomes 1, 3, 8 and 10, although no control DNA is available for this cell line. These results, for the first time, provide a detailed information of the molecular genetic defects occurring in Hu-O-2A/Gb1 and IN1434.