Barbara C. Godthelp

The DNA helicase BRIP1 is defective in Fanconi anemia complementation group J.

The protein predicted to be defective in individuals with Fanconi anemia complementation group J (FA-J), FANCJ, is a missing component in the Fanconi anemia pathway of genome maintenance. Here we identify pathogenic mutations in eight individuals with FA-J in the gene encoding the DEAH-box DNA helicase BRIP1, also called FANCJ. This finding is compelling evidence that the Fanconi anemia pathway functions through a direct physical interaction with DNA.

Warsaw Breakage Syndrome, a Cohesinopathy Associated with Mutations in the XPD Helicase Family Member DDX11/ChlR1

The iron-sulfur-containing DNA helicases XPD, FANCJ, DDX11, and RTEL represent a small subclass of superfamily 2 helicases. XPD and FANCJ have been connected to the genetic instability syndromes xeroderma pigmentosum and Fanconi anemia. Here, we report a human individual with biallelic mutations in DDX11. Defective DDX11 is associated with a unique cellular phenotype in which features of Fanconi anemia (drug-induced chromosomal breakage) and Roberts syndrome (sister chromatid cohesion defects) coexist. The DDX11-deficient patient represents another cohesinopathy, besides Cornelia de Lange syndrome and Roberts syndrome, and shows that DDX11 functions at the interface between DNA repair and sister chromatid cohesion.

Impaired DNA damage-induced nuclear Rad51 foci formation uniquely characterizes Fanconi anemia group D1

Fanconi anemia is a hereditary cancer susceptibility disorder characterized at the cellular level by spontaneous chromosomal instability and specific hypersensitivity to DNA cross-linking agents such as mitomycin C. This phenotype suggests a possible role for the Fanconi anemia proteins in the repair of DNA lesions induced by these agents, but the molecular mechanism underlying the defect in this disorder has not yet been identified. Here, we show that amongst eight so far identified complementation groups of Fanconi anemia, only fibroblasts derived from group D1 are defective in the formation of nuclear Rad51 foci after X-ray irradiation or mitomycin C treatment. This indicates that the FANCD1 gene product is uniquely involved in the assembly and/or stabilization of the Rad51 complex. Since DNA damage-induced Rad51 nuclear foci are thought to reflect repair of DNA double-strand breaks by homologous recombination, our results suggest that FANCD1 is likely to be involved in homologous recombination-dependent repair.

The Cellular Phenotype of Roberts Syndrome Fibroblasts as Revealed by Ectopic Expression of ESCO2

Cohesion between sister chromatids is essential for faithful chromosome segregation. In budding yeast, the acetyltransferase Eco1/Ctf7 establishes cohesion during DNA replication in S phase and in response to DNA double strand breaks in G2/M phase. In humans two Eco1 orthologs exist: ESCO1 and ESCO2. Both proteins are required for proper sister chromatid cohesion, but their exact function is unclear at present. Since ESCO2 has been identified as the gene defective in the rare autosomal recessive cohesinopathy Roberts syndrome (RBS), cells from RBS patients can be used to elucidate the role of ESCO2. We investigated for the first time RBS cells in comparison to isogenic controls that stably express V5- or GFP-tagged ESCO2. We show that the sister chromatid cohesion defect in the transfected cell lines is rescued and suggest that ESCO2 is regulated by proteasomal degradation in a cell cycle-dependent manner. In comparison to the corrected cells RBS cells were hypersensitive to the DNA-damaging agents mitomycin C, camptothecin and etoposide, while no particular sensitivity to UV, ionizing radiation, hydroxyurea or aphidicolin was found. The cohesion defect of RBS cells and their hypersensitivity to DNA-damaging agents were not corrected by a patient-derived ESCO2 acetyltransferase mutant (W539G), indicating that the acetyltransferase activity of ESCO2 is essential for its function. In contrast to a previous study on cells from patients with Cornelia de Lange syndrome, another cohesinopathy, RBS cells failed to exhibit excessive chromosome aberrations after irradiation in G2 phase of the cell cycle. Our results point at an S phase-specific role for ESCO2 in the maintenance of genome stability.

Definition of a novel complementation group in MHC class II deficiency

In this study we analyzed fibroblasts derived from an MHC class II deficiency patient (type III bare lymphocyte syndrome). Northern blot analysis showed that upon induction with IFN-γ these fibroblasts did not express HLA class II genes and displayed a strongly reduced level of HLA class I gene expression when compared with fibroblasts of a healthy individual. However, when analyzed by RT-polymerase chain reaction (PCR), residual expression could be detected for HLA-DRA, DPB, and DQA, but not for HLA-DRB, DPA, and DQB. The lack of HLA-DRB transcripts in the patient fibroblasts and the high degree of sequence polymorphism of HLA-DRB were exploited in the further analysis of these fibroblasts. Thus far, at least three, and probably four, complementation groups have been defined among patient-derived and experimentally-derived MHC class II-negative cell lines. Transient heterokaryons between the patient fibroblasts and representative B-lymphoblastoid cell lines from each of the complementation groups were analyzed by RT-PCR and Southern blotting, using HLA-DRB-specific primers and biotin-labeled sequence specific oligonucleotides, respectively. These analyses showed that the fibroblasts of this particular patient belonged to a novel complementation group in MHC class II deficiency.

Rad51C is essential for embryonic development and haploinsufficiency causes increased DNA damage sensitivity and genomic instability.

Homologous recombination is essential for repair of DNA interstrand cross-links and double-strand breaks. The Rad51C protein is one of the five Rad51 paralogs in vertebrates implicated in homologous recombination. A previously described hamster cell mutant defective in Rad51C (CL-V4B) showed increased sensitivity to DNA damaging agents and displayed genomic instability. Here, we identified a splice donor mutation at position +5 of intron 5 of the Rad51C gene in this mutant, and generated mice harboring an analogous base pair alteration. Rad51C(splice) heterozygous animals are viable and do not display any phenotypic abnormalities, however homozygous Rad51C(splice) embryos die during early development (E8.5). Detailed analysis of two CL-V4B revertants, V4B-MR1 and V4B-MR2, that have reduced levels of full-length Rad51C transcript when compared to wild type hamster cells, showed increased sensitivity to mitomycin C (MMC) in clonogenic survival, suggesting haploinsufficiency of Rad51C. Similarly, mouse Rad51C(splice/neo) heterozygous ES cells also displayed increased MMC sensitivity. Moreover, in both hamster revertants, Rad51C haploinsufficiency gives rise to increased frequencies of spontaneous and MMC-induced chromosomal aberrations, impaired sister chromatid cohesion and reduced cloning efficiency. These results imply that adequate expression of Rad51C in mammalian cells is essential for maintaining genomic stability and sister chromatid cohesion to prevent malignant transformation.

T cell immune reconstitution after allogeneic bone marrow transplantation in bare lymphocyte syndrome.

To study the impact of an MHC class II-negative environment on T cell immune reconstitution, we have analyzed the phenotypical and functional characteristics of FACS-sorted cultured CD4(+) and CD8(+) T cells in two Bare Lymphocyte Syndrome (BLS) patients before and after allo-BMT. A similar analysis was performed in two MHC class II expressing pediatric leukemia patients after treatment with an allo-BMT who were included in our study as control. It was observed that CD4(+) T cells displayed cytolytic alloreactivity in both BLS patients prior to and within the first year after allo-BMT, whereas such cells were absent at a later time-point, in the donors and pediatric leukemia controls. In addition, reduced MHC class II expression was observed in CD8(+) T cells of both recipients early after allo-BMT, irrespective of the T cell chimerism pattern. Lack of endogenous MHC class II expression in BLS patients, therefore, results in aberrant T cell selection within the first year after allo-BMT, analogous to T cell selection before transplantation. These T cell selection processes seem to be normalized at a later time point after allo-BMT probably due to migration and integration of graft-derived MHC class II-positive antigen presenting cells to sites of T cell selection.

Acquired cross-linker resistance associated with a novel spliced BRCA2 protein variant for molecular phenotyping of BRCA2 disruption

BRCA2 encodes a protein with a fundamental role in homologous recombination that is essential for normal development. Carrier status of mutations in BRCA2 is associated with familial breast and ovarian cancer, while bi-allelic BRCA2 mutations can cause Fanconi anemia (FA), a cancer predisposition syndrome with cellular cross-linker hypersensitivity. Cancers associated with BRCA2 mutations can acquire chemo-resistance on relapse. We modeled acquired cross-linker resistance with an FA-derived BRCA2-mutated acute myeloid leukemia (AML) platform. Associated with acquired cross-linker resistance was the expression of a functional BRCA2 protein variant lacking exon 5 and exon 7 (BRCA2ΔE5+7), implying a role for BRCA2 splicing for acquired chemo-resistance. Integrated network analysis of transcriptomic and proteomic differences for phenotyping of BRCA2 disruption infers impact on transcription and chromatin remodeling in addition to the DNA damage response. The striking overlap with transcriptional profiles of FA patient hematopoiesis and BRCA mutation associated ovarian cancer helps define and explicate the ‘BRCAness’ profile.

The Circulating Human Peripheral T-Cell Repertoire

The mechanisms controlling the generation of T-cell receptor repertoire and T-cell receptor selection are not clearly understood and seem to occur in an apparently random manner. To address the question to what extent the T-cell receptor repertoire is randomly shaped, i.e. antigen driven or is subject to individual specific genetic influences, we have carried out several studies. These studies included the analysis of the degree of combinatorial diversification in both the CD4+CD8− and CD4−CD8+ subsets of T-lymphocytes in monozygotic twins and HLA non-identical, unrelated individuals. Furthermore, we have also analyzed the development of the T-cell receptor repertoire during fetal life and have evaluated the degree of junctional diversification in great detail within several TCRV-regions expressed by circulating peripheral blood T-lymphocytes by DNA sequence analysis of the CDR3 regions. These studies have led to the following conclusions: 1. The predominant influence shaping the T-cell receptor repertoire is genetically predetermined, of which HLA-predicted selection mechanisms exerted during thymic maturation are important contributing factors. 2. In the periphery, the available T-cell receptor repertoires are seemingly smaller than expected on the basis of non-random usage of TCRBV and TCRBJ elements and amino acid composition of the CDR3 regions. 3. Within the CDR3 regions individual-specific amino acid motifs can be discerned.