Identification of seven new BRCA1 germline mutations in Italian breast and breast/ovarian cancer families.

We analyzed 16 Italian breast and breast/ovarian cancer families for BRCA1 germline mutations using a combination of the protein truncation test (PTT) and the single-strand conformation polymorphism techniques. Genomic DNA from the affected proband of each family was analyzed by applying the PTT to exon 11 of the BRCA1 gene. This initial screening led to the identification of truncated protein products in three families that were shown to carry three different frameshift mutations. In the families that scored negative in the PTT, single-strand conformation polymorphism analysis of the entire coding sequence of the gene revealed four additional mutations consisting of one nonsense, one in-frame deletion, one frameshift, and one missense mutation (in a family with a case of male breast cancer). The four frameshift mutations resulted in a decreased expression of the mutant allele, whereas no loss of transcript was associated with the other three mutations. All mutant alleles were shown to cosegregate with the cancer phenotype within the families, and none have previously been reported.

Dominance of a single Epstein-Barr virus strain in SCID-mouse tumors induced by injection of peripheral blood mononuclear cells from healthy human donors.

Severe Combined Immune Deficiency mouse tumors, induced by inoculating peripheral blood mononuclear cells from 11 healthy human donors (hu-PBMC-SCID tumors), were used to analyse Epstein-Barr virus (EBV) type and strain variations. PCR analysis of EBNA 2- and EBNA 3C-specific sequences showed that EBV type A was present in SCID-mouse tumors induced by PBMC from all donors but one, while, using amplimers for a highly polymorphic region within the latent membrane protein (LMP) coding sequence, 5 different strains could be detected among the samples examined. The same LMP fragment was present in different tumors arising in the same animal, as well as in different mice injected with PBMC from any donor. Compared to B95.8 and AG876 prototype viruses, sequence analysis of LMP variants disclosed a higher homology to the latter, with 33 bp additional repetitions and a few point mutations in specific sites. This study confirms and extends previous data on the presence of a single EBV type and strain in the peripheral blood of most normal healthy subjects using the SCID-mouse system.

Three-dimensional clustering of human RAG2 gene mutations in severe combined immune deficiency.

The V(D)J recombination, which leads to the somatic rearrangement of variable, diversity, and joining segments, is the mechanism accountable for the diversity of T cell receptor- and Ig-encoding genes. The products of the RAG1 and RAG2 genes are the lymphoid-specific factors responsible for the initiation of the V(D)J recombination through the generation of a DNA double strand break. RAG1 or RAG2 gene inactivation in the mouse leads to abortion of the V(D)J rearrangement process, early block in both T and B cell maturation, and, ultimately, to severe combined immune deficiency (SCID). A human SCID condition is also characterized by an absence of mature T and B lymphocytes and is associated with mutations in either RAG1- or RAG2-encoding genes. Based on the predicted beta-propeller three-dimensional structure model for RAG2, we found that six out of the seven mutations described to date in T-B-SCID patients are clustered on one side of the propeller, in regions exposed to solvent. This finding reinforces the biological significance of this predicted model and suggests that RAG1 interacts with RAG2 on one of the side of the scaffold formed by the beta-propeller.

Identical mutations in RAG1 or RAG2 genes leading to defective V(D)J recombinase activity can cause either T-B-severe combined immune deficiency or Omenn syndrome.

Omenn syndrome (OS) is an inherited disorder characterized by an absence of circulating B cells and an infiltration of the skin and the intestine by activated oligoclonal T lymphocytes, indicating that a profound defect in the lymphoid developmental program could be accountable for this condition. Inherited mutations in either the recombination activating genes RAG1 or RAG2, resulting in partial V(D)J recombinase activity, were shown to be responsible for OS. This study reports on the characterization of new RAG1/2 gene mutations in a series of 9 patients with OS. Given the occurrence of the same mutations in patients with T-B-severe combined immune deficiency or OS on 3 separate occasions, the proposal is made that an additional factor may be required in certain circumstances for the development of the Omenn phenotype. The nature of this factor is discussed.

Identification of a human endogenous LTR-like sequence using HIV-1 LTR specific primers.

Using 'consensus' primers derived from the LTR region of 15 HIV-1 isolates, a fragment of 583 bp was amplified from human DNA. Even though specificity was confirmed by Southern blot analysis with a conserved LTR oligonucleotide probe, no significant homologies were detected to either retroviral regions or human or non-human published sequences. Nevertheless, when used as a probe, the 583-bp fragment identified a unique DNA sequence in the human genome on chromosome 1, and cross-reactive sequences in monkey, but not mouse, DNA. This novel, unique and conserved sequence of 583 bp was used to isolate a human HS-1 clone in which the structural property of a viral LTR could be identified.

Artemis, a novel DNA double-strand break repair/V(D)J recombination protein, is mutated in human severe combined immune deficiency.

The V(D)J recombination process insures the somatic diversification of immunoglobulin and antigen T cell receptor encoding genes. This reaction is initiated by a DNA double-strand break (dsb), which is resolved by the ubiquitously expressed DNA repair machinery. Human T-B-severe combined immunodeficiency associated with increased cellular radiosensitivity (RS-SCID) is characterized by a defect in the V(D)J recombination leading to an early arrest of both B and T cell maturation. We previously mapped the disease-related locus to the short arm of chromosome 10. We herein describe the cloning of the gene encoding a novel protein involved in V(D)J recombination/DNA repair, Artemis, whose mutations cause human RS-SCID. Protein sequence analysis strongly suggests that Artemis belongs to the metallo-beta-lactamase superfamily.

Analysis of Epstein-Barr virus (EBV) type and variant in spontaneous lymphoblastoid cells and Hu-SCID mouse tumours.

Epstein-Barr virus (EBV) type and strain variations were examined using both lymphoblastoid cell lines (LCLs), spontaneously derived in vitro from peripheral blood mononuclear cells (PBMC) of 15 HIV-1-seropositive individuals; and SCID mouse tumours induced by inoculation of PBMC from 11 healthy human donors (Hu-SCID tumours). Polymerase chain reaction (PCR) analysis disclosed that all but one of the 26 EBV + samples harboured EBV nuclear antigen (EBNA) 2 and 3C type A virus. On the other hand, single strand conformation polymorphism (SSCP) analysis using Epstein-Barr encoded RNA (EBER) specific primers detected an AG876-like (type B) band pattern in 21 of the 26 EBV + samples. Three Hu-SCID tumours scored as B95.8-like (type A), and two showed neither a type A nor a type B SSCP migration pattern. Sequence analysis of the amplified EBER fragments confirmed the PCR-SSCP findings; moreover, additional mutations were present not only in the two EBV + samples with anomalous SSCP pattern, but also in two other samples with a standard SSCP profile. Thus, EBER analysis did not correlate with EBNA typing, and appeared to be unsuitable for EBV type assessment. Latent membrane protein (LMP) analysis disclosed, on the whole, sever size variants: as expected, the differences were due to the variable numbers of a 33-bp repeat in the amplified fragment, as assessed by direct sequencing. The broader variability detected by LMP analysis should prove more useful than typing for assessing the presence of single and/or mixed variants resulting from EBV reactivation and/or reinfection.

Epstein-Barr virus-associated post-transplant lympho-proliferative disease of donor origin in liver transplant recipients.

BACKGROUND/AIMS: Post-transplant lymphoproliferative disease, a potential complication of solid organ transplantation, occurs in about 3% of orthotopic liver transplant recipients. We report the genetic and virological characterization of two cases of post-transplant lymphoproliferative disease that occurred early (4 and 6 months) after orthotopic liver transplant as large-cell non-Hodgkin's lymphomas located at the hepatic hilum. METHODS: Lymphomatous tissues were analyzed for clonality and presence of Epstein-Barr virus (EBV) sequences by Southern blot, polymerase chain reaction, and in situ hybridization techniques. RESULTS: The tumors in both cases were sustained by a clonal proliferation of B lymphocytes containing type A EBV DNA. Moreover, in situ hybridization with a digoxigenin-labeled EBV-specific probe evidenced a strong nuclear signal in most of the neoplastic cells. DNA microsatellite analysis at three different loci detected alleles of donor origin in both tumor samples, suggesting that the neoplastic B cells were of donor origin. CONCLUSIONS: EBV-infected donor B lymphocytes might be responsible for intragraft post-transplant lymphoproliferative disease in orthotopic liver transplant recipients. As 20 to 30% of post-transplant lymphomas involve the graft itself, donor-derived post-transplant lymphoproliferative disease might be more frequent than presently appreciated. Prospective studies are needed to assess its real incidence and identify possible risk factors.