Yassmine Akkari

Cell fusion is the principal source of bone-marrow-derived hepatocytes.

Evidence suggests that haematopoietic stem cells might have unexpected developmental plasticity, highlighting therapeutic potential. For example, bone-marrow-derived hepatocytes can repopulate the liver of mice with fumarylacetoacetate hydrolase deficiency and correct their liver disease. To determine the underlying mechanism in this murine model, we performed serial transplantation of bone-marrow-derived hepatocytes. Here we show by Southern blot analysis that the repopulating hepatocytes in the liver were heterozygous for alleles unique to the donor marrow, in contrast to the original homozygous donor cells. Furthermore, cytogenetic analysis of hepatocytes transplanted from female donor mice into male recipients demonstrated 80,XXXY (diploid to diploid fusion) and 120,XXXXYY (diploid to tetraploid fusion) karyotypes, indicative of fusion between donor and host cells. We conclude that hepatocytes derived form bone marrow arise from cell fusion and not by differentiation of haematopoietic stem cells.

Myelomonocytic cells are sufficient for therapeutic cell fusion in liver

Liver repopulation with bone marrow–derived hepatocytes (BMHs) can cure the genetic liver disease fumarylacetoacetate hydrolase (Fah) deficiency. BMHs emerge from fusion between donor bone marrow–derived cells and host hepatocytes. To use such in vivo cell fusion efficiently for therapy requires knowing the nature of the hematopoietic cells that fuse with hepatocytes. Here we show that the transplantation into Fah−/− mice of hematopoietic stem cells (HSCs) from lymphocyte-deficient Rag1−/− mice, lineage-committed granulocyte-macrophage progenitors (GMPs) or bone marrow–derived macrophages (BMMs) results in the robust production of BMHs. These results provide direct evidence that committed myelomonocytic cells such as macrophages can produce functional epithelial cells by in vivo fusion. Because stable bone marrow engraftment or HSCs are not required for this process, macrophages or their highly proliferative progenitors provide potential for targeted and well-tolerated cell therapy aimed at organ regeneration.

DNA replication is required To elicit cellular responses to psoralen-induced DNA interstrand cross-links.

ABSTRACT Following introduction of DNA interstrand cross-links (ICLs), mammalian cells display chromosome breakage or cell cycle delay with a 4N DNA content. To further understand the nature of the delay, previously described as a G2/M arrest, we developed a protocol to generate ICLs during specific intervals of the cell cycle. Synchronous populations of G1, S, and G2 cells were treated with photoactivated 4′-hydroxymethyl-4,5′,8-trimethylpsoralen (HMT) and scored for normal passage into mitosis. In contrast to what was found for ionizing radiation, ICLs introduced during G2 did not result in a G2/M arrest, mitotic arrest, or chromosome breakage. Rather, subsequent passage through S phase was required to trigger both chromosome breakage and arrest in the next cell cycle. Similarly, ICLs introduced during G1 did not cause a G1/S arrest. We conclude that DNA replication is required to elicit the cellular responses of cell cycle arrest and genomic instability after psoralen-induced ICLs. In primary human fibroblasts, the 4N DNA content cell cycle arrest triggered by ICLs was long lasting but reversible. Kinetic analysis suggested that these cells could remove up to ∼2,500 ICLs/genome at an average rate of 11 ICLs/genome/h.

Performance of ACMG-AMP Variant-Interpretation Guidelines among Nine Laboratories in the Clinical Sequencing Exploratory Research Consortium

Evaluating the pathogenicity of a variant is challenging given the plethora of types of genetic evidence that laboratories consider. Deciding how to weigh each type of evidence is difficult, and standards have been needed. In 2015, the American College of Medical Genetics and Genomics (ACMG) and the Association for Molecular Pathology (AMP) published guidelines for the assessment of variants in genes associated with Mendelian diseases. Nine molecular diagnostic laboratories involved in the Clinical Sequencing Exploratory Research (CSER) consortium piloted these guidelines on 99 variants spanning all categories (pathogenic, likely pathogenic, uncertain significance, likely benign, and benign). Nine variants were distributed to all laboratories, and the remaining 90 were evaluated by three laboratories. The laboratories classified each variant by using both the laboratorys own method and the ACMG-AMP criteria. The agreement between the two methods used within laboratories was high (K-alpha = 0.91) with 79% concordance. However, there was only 34% concordance for either classification system across laboratories. After consensus discussions and detailed review of the ACMG-AMP criteria, concordance increased to 71%. Causes of initial discordance in ACMG-AMP classifications were identified, and recommendations on clarification and increased specification of the ACMG-AMP criteria were made. In summary, although an initial pilot of the ACMG-AMP guidelines did not lead to increased concordance in variant interpretation, comparing variant interpretations to identify differences and having a common framework to facilitate resolution of those differences were beneficial for improving agreement, allowing iterative movement toward increased reporting consistency for variants in genes associated with monogenic disease.

Fanconi Anemia Proteins Are Required To Prevent Accumulation of Replication-Associated DNA Double-Strand Breaks

ABSTRACT Fanconi anemia (FA) is a multigene cancer susceptibility disorder characterized by cellular hypersensitivity to DNA interstrand cross-linking agents such as mitomycin C (MMC). FA proteins are suspected to function at the interface between cell cycle checkpoints, DNA repair, and DNA replication. Using replicating extracts from Xenopus eggs, we developed cell-free assays for FA proteins (xFA). Recruitment of the xFA core complex and xFANCD2 to chromatin is strictly dependent on replication initiation, even in the presence of MMC indicating specific recruitment to DNA lesions encountered by the replication machinery. The increase in xFA chromatin binding following treatment with MMC is part of a caffeine-sensitive S-phase checkpoint that is controlled by xATR. Recruitment of xFANCD2, but not xFANCA, is dependent on the xATR-xATR-interacting protein (xATRIP) complex. Immunodepletion of either xFANCA or xFANCD2 from egg extracts results in accumulation of chromosomal DNA breaks during replicative synthesis. Our results suggest coordinated chromatin recruitment of xFA proteins in response to replication-associated DNA lesions and indicate that xFA proteins function to prevent the accumulation of DNA breaks that arise during unperturbed replication.

siRNA depletion of BRCA1, but not BRCA2, causes increased genome instability in Fanconi anemia cells.

BRCA1 and BRCA2 proteins act in repair of interstrand crosslinks (ICLs) and maintenance of genome stability and are known to be part of the Fanconi anemia (FA) pathway. We have investigated the role of the BRCA1 and BRCA2 genes in genome stability following ICL damage in normal and FA cells. To circumvent cell lethality of complete disruptions in BRCA1 or BRCA2, small inhibitory RNA (siRNA) was used to transiently deplete the expression of the proteins. Using chromosomal stability after ICL damage as the end point, we find that BRCA1 functions in more than just the FA pathway for genome maintenance, whereas BRCA2 appears to act predominantly in the FA pathway. Depletion of BRCA1 causes a marked decrease, although not a complete absence of, ubiquitination of FANCD2. In contrast to BRCA1, BRCA2 is not needed for normal ubiquitination of FANCD2 after DNA damage, a requirement for the FA pathway to function. Thus, BRCA2 is epistatic to FA genes for ICL repair, but not for damage-induced modification of FANCD2 and may act downstream form FANCD2.

Cytogenetic Instability in Ovarian Epithelial Cells from Women at Risk of Ovarian Cancer

Fanconi anemia is an inherited cancer predisposition disease characterized by cytogenetic and cellular hypersensitivity to cross-linking agents. Seeking evidence of Fanconi anemia protein dysfunction in women at risk of ovarian cancer, we screened ovarian surface epithelial cells from 25 primary cultures established from 22 patients using cross-linker hypersensitivity assays. Samples were obtained from (a) women at high risk for ovarian cancer with histologically normal ovaries, (b) ovarian cancer patients, and (c) a control group with no family history of breast or ovarian cancer. In chromosomal breakage assays, all control cells were mitomycin C (MMC) resistant, but eight samples (five of the six high-risk and three of the eight ovarian cancer) were hypersensitive. Lymphocytes from all eight patients were MMC resistant. Only one of the eight patients had a BRCA1 germ-line mutation and none had BRCA2 mutations, but FANCD2 was reduced in five of the eight. Ectopic expression of normal FANCD2 cDNA increased FANCD2 protein and induced MMC resistance in both hypersensitive lines tested. No FANCD2 coding region or promoter mutations were found, and there was no genomic loss or promoter methylation in any Fanconi anemia genes. Therefore, in high-risk women with no BRCA1 or BRCA2 mutations, tissue-restricted hypersensitivity to cross-linking agents is a frequent finding, and chromosomal breakage responses to MMC may be a sensitive screening strategy because cytogenetic instability identified in this way antedates the onset of carcinoma. Inherited mutations that result in tissue-specific FANCD2 gene suppression may represent a cause of familial ovarian cancer.

Mammalian SNM1 is Required for Genome Stability

The protein encoded by SNM1 in Saccharomyces cerevisiae has been shown to act specifically in DNA interstrand crosslinks (ICL) repair. There are five mammalian homologs of SNM1, including Artemis, which is involved in V(D)J recombination. Cells from mice constructed with a disruption in the Snm1 gene are sensitive to the DNA interstrand crosslinker, mitomycin (MMC), as indicated by increased radial formation following exposure. The mice reproduce normally and have normal life spans. However, a partial perinatal lethality, not seen in either homozygous mutant alone, can be noted when the Snm1 disruption is combined with a Fancd2 disruption. To explore the role of hSNM1 and its homologs in ICL repair in human cells, we used siRNA depletion in human fibroblasts, with cell survival and chromosome radials as the end points for sensitivity following treatment with MMC. Depletion of hSNM1 increases sensitivity to ICLs as detected by both end points, while depletion of Artemis does not. Thus hSNM1 is active in maintenance of genome stability following ICL formation. To evaluate the epistatic relationship between hSNM1 and other ICL repair pathways, we depleted hSNM1 in Fanconi anemia (FA) cells, which are inherently sensitive to ICLs. Depletion of hSNM1 in an FA cell line produces additive sensitivity for MMC. Further, mono-ubiquitination of FANCD2, an endpoint of the FA pathway, is not disturbed by depletion of hSNM1 in normal cells. Thus, hSNM1 appears to represent a second pathway for genome stability, distinct from the FA pathway.

ERCC1 is required for FANCD2 focus formation

The rare genetic disorder Fanconi anemia, caused by a deficiency in any of at least thirteen identified genes, is characterized by cellular sensitivity to DNA interstrand crosslinks and genome instability. The excision repair cross complementing protein, ERCC1, first identified as a participant in nucleotide excision repair, appears to also act in crosslink repair, possibly in incision and at a later stage. We have investigated the relationship of ERCC1 to the Fanconi anemia pathway, using depletion of ERCC1 by siRNA in transformed normal human fibroblasts and fibroblasts from Fanconi anemia patients. We find that depletion of ERCC1 does not hinder formation of double strand breaks in crosslink repair as indexed by gammaH2AX. However, the monoubiquitination of FANCD2 protein in response to MMC treatment is decreased and the localization of FANCD2 to nuclear foci is eliminated. Arrest of DNA replication by hydroxyurea, producing double strand breaks without crosslinks, also requires ERRC1 for FANCD2 localization to nuclear foci. Our results support a role for ERCC1 after creation of a double strand break for full activation of the Fanconi anemia pathway.

Erratum: Performance of ACMG-AMP Variant-Interpretation Guidelines among Nine Laboratories in the Clinical Sequencing Exploratory Research Consortium (American Journal of Human Genetics (2016) 98(6) (1067–1076) (S0002929716300593) (10.1016/j.ajhg.2016.03.024))

Laura M. Amendola,1,16 Gail P. Jarvik,1,16,* Michael C. Leo,2 Heather M. McLaughlin,3 Yassmine Akkari,4 Michelle D. Amaral,5 Jonathan S. Berg,6 Sawona Biswas,7 Kevin M. Bowling,5 Laura K. Conlin,7 Greg M. Cooper,5 Michael O. Dorschner,8 Matthew C. Dulik,9 Arezou A. Ghazani,10 Rajarshi Ghosh,11 Robert C. Green,3,12,15 Ragan Hart,1 Carrie Horton,13 Jennifer J. Johnston,14 Matthew S. Lebo,3,12 Aleksandar Milosavljevic,11 Jeffrey Ou,1 Christine M. Pak,4 Ronak Y. Patel,11 Sumit Punj,4 Carolyn Sue Richards,4 Joseph Salama,1 Natasha T. Strande,6 Yaping Yang,11 Sharon E. Plon,11 Leslie G. Biesecker,14 and Heidi L. Rehm3,12,15,*