Jordi Surrallés

Disease-corrected haematopoietic progenitors from Fanconi anaemia induced pluripotent stem cells

The generation of induced pluripotent stem (iPS) cells has enabled the derivation of patient-specific pluripotent cells and provided valuable experimental platforms to model human disease. Patient-specific iPS cells are also thought to hold great therapeutic potential, although direct evidence for this is still lacking. Here we show that, on correction of the genetic defect, somatic cells from Fanconi anaemia patients can be reprogrammed to pluripotency to generate patient-specific iPS cells. These cell lines appear indistinguishable from human embryonic stem cells and iPS cells from healthy individuals. Most importantly, we show that corrected Fanconi-anaemia-specific iPS cells can give rise to haematopoietic progenitors of the myeloid and erythroid lineages that are phenotypically normal, that is, disease-free. These data offer proof-of-concept that iPS cell technology can be used for the generation of disease-corrected, patient-specific cells with potential value for cell therapy applications.

Induction of micronuclei by five pyrethroid insecticides in whole-blood and isolated human lymphocyte cultures

Five pyrethroid insecticides: cypermethrin, deltamethrin, fenpropathrin, fenvalerate and permethrin, were tested for their ability to induce micronuclei in both whole-blood (WB; three donors) and isolated human lymphocyte (IL, 2 donors) cultures, by using the cytokinesis-block method with 6 micrograms/ml cytochalasin B (Cyt-B). Fenvalerate and permethrin were tested with two different concentrations of Cyt-B (3 and 6 micrograms/ml). At the concentration ranges tested, all the five pyrethroids induced clear dose dependent cytotoxic effects, fenpropathrin being the most toxic. Nuclear division index (NDI) and the newly introduced index of cytotoxicity, the cytokinesis block proliferation index (CBPI), reflected the dose dependency more accurately than the percentage of binucleated cells did. CBPI is similar to NDI except that it estimates the average number of cell divisions that the cell population has gone through, and, therefore, classifies both trinucleate and tetranucleate cells into the same category. Cypermethrin and fenpropathrin slightly increased the number of MN and micronucleated cells in WB lymphocyte cultures from two out of the three donors. Deltamethrin produced a positive response only in WB cultures of one donor and in IL cultures of another donor. Permethrin gave mostly negative results, although it increased the MN frequency in WB cultures of one donor when 6 micrograms/ml Cyt-B was used. Fenvalerate did not significantly induce MN. With certain reservations to the purity and isomer composition of each pesticide, the existing information appears to support the idea that pyrethroid insecticides have a weak (cypermethrin, deltamethrin and fenpropathrin) or nule (fenvalerate and permethrin) genotoxic activity in vitro.

Mutations in ERCC4, Encoding the DNA-Repair Endonuclease XPF, Cause Fanconi Anemia

Fanconi anemia (FA) is a rare genomic instability disorder characterized by progressive bone marrow failure and predisposition to cancer. FA-associated gene products are involved in the repair of DNA interstrand crosslinks (ICLs). Fifteen FA-associated genes have been identified, but the genetic basis in some individuals still remains unresolved. Here, we used whole-exome and Sanger sequencing on DNA of unclassified FA individuals and discovered biallelic germline mutations in ERCC4 (XPF), a structure-specific nuclease-encoding gene previously connected to xeroderma pigmentosum and segmental XFE progeroid syndrome. Genetic reversion and wild-type ERCC4 cDNA complemented the phenotype of the FA cell lines, providing genetic evidence that mutations in ERCC4 cause this FA subtype. Further biochemical and functional analysis demonstrated that the identified FA-causing ERCC4 mutations strongly disrupt the function of XPF in DNA ICL repair without severely compromising nucleotide excision repair. Our data show that depending on the type of ERCC4 mutation and the resulting balance between both DNA repair activities, individuals present with one of the three clinically distinct disorders, highlighting the multifunctional nature of the XPF endonuclease in genome stability and human disease.

Histone H2AX and Fanconi anemia FANCD2 function in the same pathway to maintain chromosome stability

Fanconi anemia (FA) is a chromosome fragility syndrome characterized by bone marrow failure and cancer susceptibility. The central FA protein FANCD2 is known to relocate to chromatin upon DNA damage in a poorly understood process. Here, we have induced subnuclear accumulation of DNA damage to prove that histone H2AX is a novel component of the FA/BRCA pathway in response to stalled replication forks. Analyses of cells from H2AX knockout mice or expressing a nonphosphorylable H2AX (H2AXS136A/S139A) indicate that phosphorylated H2AX (γH2AX) is required for recruiting FANCD2 to chromatin at stalled replication forks. FANCD2 binding to γH2AX is BRCA1‐dependent and cells deficient or depleted of H2AX show an FA‐like phenotype, including an excess of chromatid‐type chromosomal aberrations and hypersensitivity to MMC. This MMC hypersensitivity of H2AX‐deficient cells is not further increased by depleting FANCD2, indicating that H2AX and FANCD2 function in the same pathway in response to DNA damage‐induced replication blockage. Consequently, histone H2AX is functionally connected to the FA/BRCA pathway to resolve stalled replication forks and prevent chromosome instability.

The suitability of the micronucleus assay in human lymphocytes as a new biomarker of excision repair.

The cytokinesis blocked micronucleus assay is relatively insensitive to detect agents that predominantly induce excision repairable DNA lesions. However, it has been recently proposed that excision-repairable DNA lesions induced in G0/G1 phase can be converted to micronuclei by using inhibitors of the gap filling step of excision repair so that unfilled gaps are converted to double stranded breaks after S phase and micronuclei (MN) at completion of mitosis. As it has been recently demonstrated this process could be improved by combining cytosine arabinoside (ARA-C) and hydroxyurea (HU). In the present work, we have investigated the suitability of this new approach by studying its ability to detect excision repairable DNA lesions induced by 10 pesticides (alachlor, atrazine, cypermethrin, deltamethrin, fenpropathrin, fenvalerate, maleic hydrazide, paraquat, permethrin and trifluralin) and 3 well-known mutagenic agents (ethyl methane sulphonate, EMS; methylnitrosourea, MNU; and mytomicin C, MMC). Our results showed that the combination of ARA-C and HU substantially increased the level of MN in whole blood lymphocyte cultures, but it provided an excess of toxicity when further treatments, such as MNU, were performed. When ARA-C alone was used, the ARA/CBMN assay appeared to be highly sensitive and specific in detecting agents known to induce excision repairable DNA lesions. Thus, EMS and MNU but not MMC greatly induced DNA excision repair. On the other hand, alachlor, permethrin and, to a lesser extent, trifluralin and fenpropathrin also increased the ratio of excision repairable DNA lesions converted to MN. On the contrary, atrazine, cypermethrin, deltamethrin, fenvalerate, maleic hydrazide and paraquat did not induce excision repair.

Accelerated Telomere Shortening in the Human Inactive X Chromosome

Telomeres are nucleoprotein complexes at the end of eukaryotic chromosomes, with important roles in the maintenance of genomic stability and in chromosome segregation. Normal somatic cells lose telomeric repeats with each cell division both in vivo and in vitro. To address a potential role of nuclear architecture and epigenetic factors in telomere-length dynamics, the length of the telomeres of the X chromosomes and the autosomes was measured in metaphases from blood lymphocytes of human females of various ages, by quantitative FISH with a peptide nucleic-acid telomeric probe in combination with an X-chromosome centromere-specific probe. The activation status of the X chromosomes was simultaneously visualized with antibodies against acetylated histone H4. We observed an accelerated shortening of telomeric repeats in the inactive X chromosome, which suggests that epigenetic factors modulate not only the length but also the rate of age-associated telomere shortening in human cells in vivo. This is the first evidence to show a differential rate of telomere shortening between and within homologous chromosomes in any species. Our results are also consistent with a causative role of telomere shortening in the well-documented X-chromosome aneuploidy in aging humans.

Fanconi anemia: a model disease for studies on human genetics and advanced therapeutics.

Fanconi anemia (FA) is characterized by bone marrow failure, malformations, and chromosome fragility. We review the recent discovery of FA genes and efforts to develop genetic therapies for FA in the last five years. Because current data exclude FANCM as an FA gene, 15 genes remain bona fide FA genes and three (FANCO, FANCR and FANCS) cause an FA like syndrome. Monoallelic mutations in 6 FA associated genes (FANCD1, FANCJ, FANCM, FANCN, FANCO and FANCS) predispose to breast and ovarian cancer. The products of all these genes are involved in the repair of stalled DNA replication forks by unhooking DNA interstrand cross-links and promoting homologous recombination. The genetic characterization of patients with FA is essential for developing therapies, including hematopoietic stem cell transplantation from a savior sibling donor after embryo selection, gene therapy, or genome editing using genetic recombination or engineered nucleases. Newly acquired knowledge about FA promises to provide therapeutic strategies in the near future.

Clinical and Molecular Characteristics of Squamous Cell Carcinomas From Fanconi Anemia Patients

Fanconi anemia is a recessively inherited disease that is characterized by congenital abnormalities, bone marrow failure, and a predisposition to develop cancer, particularly squamous cell carcinomas (SCCs) in the head and neck and anogenital regions. Previous studies of Fanconi anemia SCCs, mainly from US patients, revealed the presence of high-risk human papillomavirus (HPV) DNA in 21 (84%) of 25 tumors analyzed. We examined a panel of 21 SCCs mainly from European Fanconi anemia patients (n = 19 FA patients; 16 head and neck squamous cell carcinomas [HNSCCs], 2 esophageal SCCs, and 3 anogenital SCCs) for their clinical and molecular characteristics, including patterns of allelic loss, TP53 mutations, and the presence of HPV DNA by GP5+/6+ polymerase chain reaction. HPV DNA was detected in only two (10%) of 21 tumors (both anogenital SCCs) but in none of the 16 HNSCCs. Of the 18 tumors analyzed, 10 contained a TP53 mutation. The patterns of allelic loss were comparable to those generally found in sporadic SCCs. Our data show that HPV does not play a major role in squamous cell carcinogenesis in this cohort of Fanconi anemia patients and that the Fanconi anemia SCCs are genetically similar to sporadic SCCs despite having a different etiology.

Modelling Fanconi anemia pathogenesis and therapeutics using integration-free patient-derived iPSCs

Fanconi anaemia (FA) is a recessive disorder characterized by genomic instability, congenital abnormalities, cancer predisposition and bone marrow (BM) failure. However, the pathogenesis of FA is not fully understood partly due to the limitations of current disease models. Here, we derive integration free-induced pluripotent stem cells (iPSCs) from an FA patient without genetic complementation and report in situ gene correction in FA-iPSCs as well as the generation of isogenic FANCA-deficient human embryonic stem cell (ESC) lines. FA cellular phenotypes are recapitulated in iPSCs/ESCs and their adult stem/progenitor cell derivatives. By using isogenic pathogenic mutation-free controls as well as cellular and genomic tools, our model serves to facilitate the discovery of novel disease features. We validate our model as a drug-screening platform by identifying several compounds that improve hematopoietic differentiation of FA-iPSCs. These compounds are also able to rescue the hematopoietic phenotype of FA patient BM cells.

Germline Mutations in FAN1 Cause Hereditary Colorectal Cancer by Impairing DNA Repair

Identification of genes associated with hereditary cancers facilitates management of patients with family histories of cancer. We performed exome sequencing of DNA from 3 individuals from a family with colorectal cancer who met the Amsterdam criteria for risk of hereditary nonpolyposis colorectal cancer. These individuals had mismatch repair-proficient tumors and each carried nonsense variant in the FANCD2/FANCI-associated nuclease 1 gene (FAN1), which encodes a nuclease involved in DNA inter-strand cross-link repair. We sequenced FAN1 in 176 additional families with histories of colorectal cancer and performed in vitro functional analyses of the mutant forms of FAN1 identified. We detected FAN1 mutations in approximately 3% of families who met the Amsterdam criteria and had mismatch repair-proficient cancers with no previously associated mutations. These findings link colorectal cancer predisposition to the Fanconi anemia DNA repair pathway, supporting the connection between genome integrity and cancer risk.