A Molecular Characterization of the Allelic Expression of the BRCA1 Founder Δ9-12 Pathogenic Variant and Its Potential Clinical Relevance in Hereditary Cancer.

Hereditary breast and ovarian cancer (HBOC) syndrome is a genetic condition that increases the risk of breast cancer by 80% and that of ovarian cancer by 40%. The most common pathogenic variants (PVs) causing HBOC occur in the BRCA1 gene, with more than 3850 reported mutations in the gene sequence. The prevalence of specific PVs in BRCA1 has increased across populations due to the effect of founder mutations. Therefore, when a founder mutation is identified, it becomes key to improving cancer risk characterization and effective screening protocols. The only founder mutation described in the Mexican population is the deletion of exons 9 to 12 of BRCA1 (BRCA1Δ9-12), and its description focuses on the gene sequence, but no transcription profiles have been generated for individuals who carry this gene. In this study, we describe the transcription profiles of cancer patients and healthy individuals who were heterozygous for PV BRCA1Δ9-12 by analyzing the differential expression of both alleles compared with the homozygous BRCA1 control group using RT-qPCR, and we describe the isoforms produced by the BRCA1 wild-type and BRCA1Δ9-12 alleles using nanopore long-sequencing. Using the Kruskal-Wallis test, our results showed a similar transcript expression of the wild-type allele between the healthy heterozygous group and the homozygous BRCA1 control group. An association between the recurrence and increased expression of both alleles in HBOC patients was also observed. An analysis of the sequences indicated four wild-type isoforms with diagnostic potential for discerning individuals who carry the PV BRCA1Δ9-12 and identifying which of them has developed cancer.

Profiling Chromosome Topological Features by Super-Resolution 3D Structured Illumination Microscopy.

Three-dimensional structured illumination microscopy (3D-SIM) and fluorescence in situ hybridization on three-dimensional preserved cells (3D-FISH) have proven to be robust and efficient methodologies for analyzing nuclear architecture and profiling the genome's topological features. These methods have allowed the simultaneous visualization and evaluation of several target structures at super-resolution. In this chapter, we focus on the application of 3D-SIM for the visualization of 3D-FISH preparations of chromosomes in interphase, known as Chromosome Territories (CTs). We provide a workflow and detailed guidelines for sample preparation, image acquisition, and image analysis to obtain quantitative measurements for profiling chromosome topological features. In parallel, we address a practical example of these protocols in the profiling of CTs 9 and 22 involved in the translocation t(9;22) in Chronic Myeloid Leukemia (CML). The profiling of chromosome topological features described in this chapter allowed us to characterize a large-scale topological disruption of CTs 9 and 22 that correlates directly with patients' response to treatment and as a possible potential change in the inheritance systems. These findings open new insights into how the genome structure is associated with the response to cancer treatments, highlighting the importance of microscopy in analyzing the topological features of the genome.

Characterizing Chemotherapy/Radiotherapy-Induced Genome Chaos in Hodgkin's Lymphoma Patients Using M-FISH.

Hodgkin lymphoma (HL) is one of the most common lymphomas, with an incidence of 3 per 100,000 persons. Current treatment uses a cocktail of genotoxic agents, including adriamycin, bleomycin, vinblastine, and dacarbazine (ABVD), along with or without radiotherapy. This treatment regimen has proved to be efficient in killing cancer cells, resulting in HL patients having a survival rate of >90% cancer-free survival at five years. However, this therapy does not have a specific cell target, and it can induce damage in the genome of non-cancerous cells. Previous studies have shown that HL survivors often exhibit karyotypes characterized by complex chromosomal abnormalities that are difficult to analyze by conventional banding. Multicolor fluorescence in situ hybridization (M-FISH) is a powerful tool to analyze complex karyotypes; we used M-FISH to investigate the presence of chromosomal damage in peripheral blood lymphocytes from five healthy individuals and five HL patients before, during, and one year after anti-cancer treatment. Our results show that this anti-cancer treatment-induced genomic chaos that persists in the hematopoietic stem cells from HL patients one year after finishing therapy. This chromosomal instability may play a role in the occurrence of second primary cancers that are observed in 10% of HL survivors. This chapter will describe a protocol for utilizing M-FISH to study treatment-induced genome chaos in Hodgkin's lymphoma (HL) patients, following a brief discussion.

Reversion from basal histone H4 hypoacetylation at the replication fork increases DNA damage in FANCA deficient cells.

The FA/BRCA pathway safeguards DNA replication by repairing interstrand crosslinks (ICL) and maintaining replication fork stability. Chromatin structure, which is in part regulated by histones posttranslational modifications (PTMs), has a role in maintaining genomic integrity through stabilization of the DNA replication fork and promotion of DNA repair. An appropriate balance of PTMs, especially acetylation of histones H4 in nascent chromatin, is required to preserve a stable DNA replication fork. To evaluate the acetylation status of histone H4 at the replication fork of FANCA deficient cells, we compared histone acetylation status at the DNA replication fork of isogenic FANCA deficient and FANCA proficient cell lines by using accelerated native immunoprecipitation of nascent DNA (aniPOND) and in situ protein interactions in the replication fork (SIRF) assays. We found basal hypoacetylation of multiple residues of histone H4 in FA replication forks, together with increased levels of Histone Deacetylase 1 (HDAC1). Interestingly, high-dose short-term treatment with mitomycin C (MMC) had no effect over H4 acetylation abundance at the replication fork. However, chemical inhibition of histone deacetylases (HDAC) with Suberoylanilide hydroxamic acid (SAHA) induced acetylation of the FANCA deficient DNA replication forks to levels comparable to their isogenic control counterparts. This forced permanence of acetylation impacted FA cells homeostasis by inducing DNA damage and promoting G2 cell cycle arrest. Altogether, this caused reduced RAD51 foci formation and increased markers of replication stress, including phospho-RPA-S33. Hypoacetylation of the FANCA deficient replication fork, is part of the cellular phenotype, the perturbation of this feature by agents that prevent deacetylation, such as SAHA, have a deleterious effect over the delicate equilibrium they have reached to perdure despite a defective FA/BRCA pathway.

Chromosome analysis of 303 pregnancy losses in Mexico.

BACKGROUND: Chromosomal abnormalities are present in 50 to 60% of miscarriages and in 6 to 19% of stillbirths. Although microarrays are preferred for studying chromosomal abnormalities, many hospitals cannot offer this methodology. OBJECTIVE: To present the results of the cytogenetic analysis of 303 products of conception (POC), which included 184 miscarriages, 49 stillbirths and 17 cases of undefined age. MATERIAL AND METHODS: Karyotyping, fluorescence in situ hybridization, short tandem repeats and microarrays were used, depending on the type of loss and available sample. RESULTS: In 29 POCs we found maternal tissue and were eliminated from the analyses. Informative results were obtained in 250 (91.2 %)/274 cases; the karyotyping success rate was 80.7%; that of single nucleotide polymorphism microarrays, 94.5%; and that of fluorescence in situ hybridization and short tandem repeat, 100%. Cytogenetic abnormalities were observed in 57.6% of miscarriages and in 24.5% of stillbirths; 94% of total anomalies were numerical and 6% were submicroscopic. CONCLUSIONS: Karyotyping with simultaneous short tandem repeat study to rule out contamination of maternal cells is effective for studying miscarriages; in stillbirths, microarrays are recommended.

ANTECEDENTES: Las alteraciones cromosómicas están presentes en 50 a 60 % de los abortos espontáneos y en 6 a 19 % de los mortinatos. Aunque se prefieren los microarreglos para estudiarlos, numerosos hospitales no pueden ofrecerlos. OBJETIVO: Presentar los resultados del estudio citogenético de 303 productos de la concepción (POC), 184 se obtuvieron de abortos espontáneos, 49 fueron mortinatos y en 17 no se identificó la de edad gestacional. MATERIAL Y MÉTODOS: Se empleó cariotipo, hibridación in situ con fluorescencia, secuencias cortas repetidas en tándem y microarreglos, según el tipo de pérdida y la muestra disponible. RESULTADOS: En 29 POC se encontró tejido materno, por lo que fueron eliminados de los análisis. En 250 (91.2 %)/274 casos se obtuvieron resultados informativos; la tasa de éxito del cariotipo fue de 80.7 %; la de los microarreglos de SNP, de 94.5 %; y la de la hibridación fluorescente in situ y la repetición corta en tándem, de 100 %. Se observaron anomalías citogenéticas en 57.6 % de los abortos espontáneos y en 24.5 % de los mortinatos; 94 % de las anomalías fueron numéricas y 6 %, submicroscópicas. CONCLUSIONES: El cariotipo en conjunto con el estudio de secuencias cortas repetidas en tándem para descartar contaminación de células maternas es efectivo para estudiar abortos espontáneos; los microarreglos se recomiendan en los mortinatos.

Estudio cromosómico de 303 pérdidas gestacionales en México / Chromosome analysis of 303 pregnancy losses in Mexico

Resumen Antecedentes: Las alteraciones cromosómicas están presentes en 50 a 60 % de los abortos espontáneos y en 6 a 19 % de los mortinatos. Aunque se prefieren los microarreglos para estudiarlos, numerosos hospitales no pueden ofrecerlos. Objetivo: Presentar los resultados del estudio citogenético de 303 productos de la concepción (POC), 184 se obtuvieron de abortos espontáneos, 49 fueron mortinatos y en 17 no se identificó la de edad gestacional. Material y métodos: Se empleó cariotipo, hibridación in situ con fluorescencia, secuencias cortas repetidas en tándem y microarreglos, según el tipo de pérdida y la muestra disponible. Resultados: En 29 POC se encontró tejido materno, por lo que fueron eliminados de los análisis. En 250 (91.2 %)/274 casos se obtuvieron resultados informativos; la tasa de éxito del cariotipo fue de 80.7 %; la de los microarreglos de SNP, de 94.5 %; y la de la hibridación fluorescente in situ y la repetición corta en tándem, de 100 %. Se observaron anomalías citogenéticas en 57.6 % de los abortos espontáneos y en 24.5 % de los mortinatos; 94 % de las anomalías fueron numéricas y 6 %, submicroscópicas. Conclusiones: El cariotipo en conjunto con el estudio de secuencias cortas repetidas en tándem para descartar contaminación de células maternas es efectivo para estudiar abortos espontáneos; los microarreglos se recomiendan en los mortinatos.

Abstract Background: Chromosomal abnormalities are present in 50 to 60 % of miscarriages and in 6 to 19 % of stillbirths. Although microarrays are preferred for studying chromosomal abnormalities, many hospitals cannot offer this methodology. Objective: To present the results of the cytogenetic analysis of 303 products of conception (POC), which included 184 miscarriages, 49 stillbirths and 17 cases of undefined age. Material and methods: Karyotyping, fluorescence in situ hybridization, short tandem repeats and microarrays were used, depending on the type of loss and available sample. Results: In 29 POCs we found maternal tissue and were eliminated from the analyses. Informative results were obtained in 250 (91.2 %)/274 cases; the karyotyping success rate was 80.7 %; that of single nucleotide polymorphism microarrays, 94.5 %; and that of fluorescence in situ hybridization and short tandem repeat, 100 %. Cytogenetic abnormalities were observed in 57.6 % of miscarriages and in 24.5 % of stillbirths; 94 % of total anomalies were numerical and 6 % were submicroscopic. Conclusions: Karyotyping with simultaneous short tandem repeat study to rule out contamination of maternal cells is effective for studying miscarriages; in stillbirths, microarrays are recommended.

A Boolean network model of the double-strand break repair pathway choice.

Double strand break (DSB) repair is critical to maintaining the integrity of the genome. DSB repair deficiency underlies multiple pathologies, including cancer, chromosome instability syndromes, and, potentially, neurodevelopmental defects. DSB repair is mainly handled by two pathways: highly accurate homologous recombination (HR), which requires a sister chromatid for template-based repair, limited to S/G2 phases of the cell cycle, and canonical non-homologous end joining (c-NHEJ), available throughout the cell cycle in which minimum homology is sufficient for highly efficient yet error-prone repair. Some circumstances, such as cancer, require alternative highly mutagenic DSB repair pathways like microhomology-mediated end-joining (MMEJ) and single-strand annealing (SSA), which are triggered to attend to DNA damage. These non-canonical repair alternatives are emerging as prominent drivers of resistance in drug-based tumor therapies. Multiple DSB repair options require tight inter-pathway regulation to prevent unscheduled activities. In addition to this complexity, epigenetic modifications of the histones surrounding the DSB region are emerging as critical regulators of the DSB repair pathway choice. Modeling approaches to understanding DSBs repair pathway choice are advantageous to perform simulations and generate predictions on previously uncharacterized aspects of DSBs response. In this work, we present a Boolean network model of the DSB repair pathway choice that incorporates the knowledge, into a dynamic system, of the inter-pathways regulation involved in DSB repair, i.e., HR, c-NHEJ, SSA, and MMEJ. Our model recapitulates the well-characterized HR activity observed in wild-type cells in response to DSBs. It also recovers clinically relevant behaviors of BRCA1/FANCS mutants, and their corresponding drug resistance mechanisms ascribed to DNA repair gain-of-function pathogenic variants. Since epigenetic modifiers are dynamic and possible druggable targets, we incorporated them into our model to better characterize their involvement in DSB repair. Our model predicted that loss of the TIP60 complex and its corresponding histone acetylation activity leads to activation of SSA in response to DSBs. Our experimental validation showed that TIP60 effectively prevents activation of RAD52, a key SSA executor, and confirms the suitable use of Boolean network modeling for understanding DNA DSB repair.

Frequent copy number variants in a cohort of Mexican-Mestizo individuals.

BACKGROUND: The human genome presents variation at distinct levels, copy number variants (CNVs) are DNA segments of variable lengths that range from several base pairs to megabases and are present at a variable number of copies in human genomes. Common CNVs have no apparent influence on the phenotype; however, some rare CNVs have been associated with phenotypic traits, depending on their size and gene content. CNVs are detected by microarrays of different densities and are generally visualized, and their frequencies analysed using the HapMap as default reference population. Nevertheless, this default reference is inadequate when the samples analysed are from people from Mexico, since population with a Hispanic genetic background are minimally represented. In this work, we describe the variation in the frequencies of four common CNVs in Mexican-Mestizo individuals. RESULTS: In a cohort of 147 unrelated Mexican-Mestizo individuals, we found that the common CNVs 2p11.2 (99.6%), 8p11.22 (54.5%), 14q32.33 (100%), and 15q11.2 (71.1%) appeared with unexpectedly high frequencies when contrasted with the HapMap reference (ChAS). Yet, while when comparing to an ethnically related reference population, these differences were significantly reduced or even disappeared. CONCLUSION: The findings in this work contribute to (1) a better description of the CNVs characteristics of the Mexican Mestizo population and enhance the knowledge of genome variation in different ethnic groups. (2) emphasize the importance of contrasting CNVs identified in studied individuals against a reference group that-as best as possible-share the same ethnicity while keeping this relevant information in mind when conducting CNV studies at the population or clinical level.

Inherited bone marrow failure syndromes: phenotype as a tool for early diagnostic suspicion at a major reference center in Mexico.

Introduction: The inherited bone marrow failure syndromes (IBMFSs) are a group of rare disorders characterized by bone marrow failure (BMF), physical abnormalities, and an increased risk of neoplasia. The National Institute of Pediatrics (INP) is a major medical institution in Mexico, where patients with BMF receive a complete approach that includes paraclinical tests. Readily recognizable features, such as the hematological and distinctive physical phenotypes, identified by clinical dysmorphologists, remain crucial for the diagnosis and management of these patients, particularly in circumstances where next-generation sequencing (NGS) is not easily available. Here, we describe a group of Mexican patients with a high clinical suspicion of an IBMFS. Methods: We performed a systematic retrospective analysis of the medical records of patients who had a high IBMFS suspicion at our institution from January 2018 to July 2021. An initial assessment included first ruling out acquired causes of BMF by the Hematology Department and referral of the patient to the Department of Human Genetics for physical examination to search for specific phenotypes suggesting an IBMFS. Patients with high suspicion of having an IBMFS were classified into two main groups: 1) specific IBMFS, including dyskeratosis congenita (DC), Diamond-Blackfan anemia (DBA), Shwachman-Diamond syndrome (SDS), thrombocytopenia with absent radii (TAR), and severe congenital neutropenia (SCN); 2) undefined IBMFS (UI). Results: We established a high suspicion of having an IBMFS in 48 patients. At initial evaluation, the most common hematologic features were bicytopenia (20%) and aplastic anemia (16%); three patients received hematopoietic stem cell transplantation. Among patients with a suspicion of an IBMFS, the most common physical abnormality was minor craniofacial features in 83% of patients and neurodevelopmental disorders in 52%. The specific suspicions that we built were DBA (31%), SDS (18%), DC (14%), TAR (4%), and SCN (4%), whereas 27% of cases remained as undefined IBMFS. SDS, TAR, and SCN were more commonly suspected at an earlier age (<1 year), followed by DBA (2 years) and DC (5 years). Conclusions: Thorough examination of reported clinical data allowed us to highly suspect a specific IBMFS in approximately 70% of patients; however, an important number of patients remained with suspicion of an undefined IBMFS. Implementation of NGS and telomere length measurement are forthcoming measures to improve IBMFS diagnosis in Mexico.

TGFß pathway is required for viable gestation of Fanconi anemia embryos.

Overexpression of the TGFß pathway impairs the proliferation of the hematopoietic stem and progenitor cells (HSPCs) pool in Fanconi anemia (FA). TGFß promotes the expression of NHEJ genes, known to function in a low-fidelity DNA repair pathway, and pharmacological inhibition of TGFß signaling rescues FA HSPCs. Here, we demonstrate that genetic disruption of Smad3, a transducer of the canonical TGFß pathway, modifies the phenotype of FA mouse models deficient for Fancd2. We observed that the TGFß and NHEJ pathway genes are overexpressed during the embryogenesis of Fancd2-/- mice and that the Fancd2-/-Smad3-/- double knockout (DKO) mice undergo high levels of embryonic lethality due to loss of the TGFß-NHEJ axis. Fancd2-deficient embryos acquire extensive genomic instability during gestation which is not reversed by Smad3 inactivation. Strikingly, the few DKO survivors have activated the non-canonical TGFß-ERK pathway, ensuring expression of NHEJ genes during embryogenesis and improved survival. Activation of the TGFß-NHEJ axis was critical for the survival of the few Fancd2-/-Smad3-/- DKO newborn mice but had detrimental consequences for these surviving mice, such as enhanced genomic instability and ineffective hematopoiesis.

Fanconi anemia and dyskeratosis congenita/telomere biology disorders: Two inherited bone marrow failure syndromes with genomic instability.

Inherited bone marrow failure syndromes (IBMFS) are a complex and heterogeneous group of genetic diseases. To date, at least 13 IBMFS have been characterized. Their pathophysiology is associated with germline pathogenic variants in genes that affect hematopoiesis. A couple of these diseases also have genomic instability, Fanconi anemia due to DNA damage repair deficiency and dyskeratosis congenita/telomere biology disorders as a result of an alteration in telomere maintenance. Patients can have extramedullary manifestations, including cancer and functional or structural physical abnormalities. Furthermore, the phenotypic spectrum varies from cryptic features to patients with significantly evident manifestations. These diseases require a high index of suspicion and should be considered in any patient with abnormal hematopoiesis, even if extramedullary manifestations are not evident. This review describes the disrupted cellular processes that lead to the affected maintenance of the genome structure, contrasting the dysmorphological and oncological phenotypes of Fanconi anemia and dyskeratosis congenita/telomere biology disorders. Through a dysmorphological analysis, we describe the phenotypic features that allow to make the differential diagnosis and the early identification of patients, even before the onset of hematological or oncological manifestations. From the oncological perspective, we analyzed the spectrum and risks of cancers in patients and carriers.

Fanconi Anemia Patients from an Indigenous Community in Mexico Carry a New Founder Pathogenic Variant in FANCG.

Fanconi anemia (FA) is a rare genetic disorder caused by pathogenic variants (PV) in at least 22 genes, which cooperate in the Fanconi anemia/Breast Cancer (FA/BRCA) pathway to maintain genome stability. PV in FANCA, FANCC, and FANCG account for most cases (~90%). This study evaluated the chromosomal, molecular, and physical phenotypic findings of a novel founder FANCG PV, identified in three patients with FA from the Mixe community of Oaxaca, Mexico. All patients presented chromosomal instability and a homozygous PV, FANCG: c.511-3_511-2delCA, identified by next-generation sequencing analysis. Bioinformatic predictions suggest that this deletion disrupts a splice acceptor site promoting the exon 5 skipping. Analysis of Cytoscan 750 K arrays for haplotyping and global ancestry supported the Mexican origin and founder effect of the variant, reaffirming the high frequency of founder PV in FANCG. The degree of bone marrow failure and physical findings (described through the acronyms VACTERL-H and PHENOS) were used to depict the phenotype of the patients. Despite having a similar frequency of chromosomal aberrations and genetic constitution, the phenotype showed a wide spectrum of severity. The identification of a founder PV could help for a systematic and accurate genetic screening of patients with FA suspicion in this population.

Large-scale topological disruption of chromosome territories 9 and 22 is associated with nonresponse to treatment in CML.

Chronic myeloid leukemia (CML) is a myeloproliferative neoplasm defined by the presence of t(9;22) translocation whose origin has been associated with the tridimensional genome organization. This rearrangement leads to the fusion of BCR and ABL1 genes giving rise to a chimeric protein with constitutive kinase activity. Imatinib, a tyrosine kinase inhibitor (TKI), is used as a first-line treatment for CML, though ~40% of CML patients do not respond. Here, using structured illumination microscopy (SIM) and 3D reconstruction, we studied the 3D organization patterns of the ABL1 and BCR genes, and their chromosome territories (CTs) CT9 and CT22, in CD34+ cells from CML patients that responded or not to TKI. We found that TKI resistance in CML is associated with high levels of structural disruption of CT9 and CT22 in CD34+ cells, increased CT volumes (especially for CT22), intermingling between CT9 and CT22, and an open-chromatin epigenetic mark in CT22. Altogether our results suggest that large-scale disruption of CT9 and CT22 correlates with the clinical response of CML patients, which could be translated into a potential prognostic marker of response to treatment in this disease and provide novel insights into the mechanisms underlying resistance to TKI in CML.

Unravelling complex mosaicism of sex chromosomes in a patient with primary amenorrhea through cytogenetic analysis on urothelial cells.

OBJECTIVE: Sex chromosome mosaicism remains challenging in the study of disorders of sex development (DSD). Aneuploid cells in the developing gonad play a major role in sex determination. Therefore, it is necessary acknowledge their presence by different methods. Our aim was to stand out the utility of urothelial cells for unravelling complex and hidden cell lines in DSD patients. CASE REPORT: Herein we report on a 19-year-old female with primary amenorrhea, short stature without ambiguous external genitalia. She had a 45,X/46, XY karyotype in leukocytes. Interphase FISH revealed hidden 45,X/47,XYY/47,XXY/46,XY/46, XX mosaicism in leukocytes and urothelial cells. CONCLUSION: These findings highlight the importance of investigating sex chromosome mosaicism in other tissues. Of particular interest in cases of DSD are the cells from the urinary epithelium, which may reflect the cell composition of the urogenital ridge, the analysis of these cells should be considered within the clinical assessment of DSD patients.

Heterogeneity and Clonal Evolution of Acquired PARP Inhibitor Resistance in TP53- and BRCA1-Deficient Cells.

Homologous recombination (HR)-deficient cancers are sensitive to poly-ADP ribose polymerase inhibitors (PARPi), which have shown clinical efficacy in the treatment of high-grade serous cancers (HGSC). However, the majority of patients will relapse, and acquired PARPi resistance is emerging as a pressing clinical problem. Here we generated seven single-cell clones with acquired PARPi resistance derived from a PARPi-sensitive TP53 -/- and BRCA1 -/- epithelial cell line generated using CRISPR/Cas9. These clones showed diverse resistance mechanisms, and some clones presented with multiple mechanisms of resistance at the same time. Genomic analysis of the clones revealed unique transcriptional and mutational profiles and increased genomic instability in comparison with a PARPi-sensitive cell line. Clonal evolutionary analyses suggested that acquired PARPi resistance arose via clonal selection from an intrinsically unstable and heterogenous cell population in the sensitive cell line, which contained preexisting drug-tolerant cells. Similarly, clonal and spatial heterogeneity in tumor biopsies from a clinical patient with BRCA1-mutant HGSC with acquired PARPi resistance was observed. In an imaging-based drug screening, the clones showed heterogenous responses to targeted therapeutic agents, indicating that not all PARPi-resistant clones can be targeted with just one therapy. Furthermore, PARPi-resistant clones showed mechanism-dependent vulnerabilities to the selected agents, demonstrating that a deeper understanding on the mechanisms of resistance could lead to improved targeting and biomarkers for HGSC with acquired PARPi resistance. SIGNIFICANCE: This study shows that BRCA1-deficient cells can give rise to multiple genomically and functionally heterogenous PARPi-resistant clones, which are associated with various vulnerabilities that can be targeted in a mechanism-specific manner.

MYC Promotes Bone Marrow Stem Cell Dysfunction in Fanconi Anemia.

Bone marrow failure (BMF) in Fanconi anemia (FA) patients results from dysfunctional hematopoietic stem and progenitor cells (HSPCs). To identify determinants of BMF, we performed single-cell transcriptome profiling of primary HSPCs from FA patients. In addition to overexpression of p53 and TGF-ß pathway genes, we identified high levels of MYC expression. We correspondingly observed coexistence of distinct HSPC subpopulations expressing high levels of TP53 or MYC in FA bone marrow (BM). Inhibiting MYC expression with the BET bromodomain inhibitor (+)-JQ1 reduced the clonogenic potential of FA patient HSPCs but rescued physiological and genotoxic stress in HSPCs from FA mice, showing that MYC promotes proliferation while increasing DNA damage. MYC-high HSPCs showed significant downregulation of cell adhesion genes, consistent with enhanced egress of FA HSPCs from bone marrow to peripheral blood. We speculate that MYC overexpression impairs HSPC function in FA patients and contributes to exhaustion in FA bone marrow.

Inhibition of TGFß1 and TGFß3 promotes hematopoiesis in Fanconi anemia.

Fanconi anemia (FA) is a chromosome instability syndrome with congenital abnormalities, cancer predisposition and bone marrow failure (BMF). Although hematopoietic stem and progenitor cell (HSPC) transplantation is the recommended therapy, new therapies are needed for FA patients without suitable donors. BMF in FA is caused, at least in part, by a hyperactive growth-suppressive transforming growth factor ß (TGFß) pathway, regulated by the TGFß1, TGFß2, and TGFß3 ligands. Accordingly, the TGFß pathway is an attractive therapeutic target for FA. While inhibition of TGFß1 and TGFß3 promotes blood cell expansion, inhibition of TGFß2 is known to suppress hematopoiesis. Here, we report the effects of AVID200, a potent TGFß1- and TGFß3-specific inhibitor, on FA hematopoiesis. AVID200 promoted the survival of murine FA HSPCs in vitro. AVID200 also promoted in vitro the survival of human HSPCs from patients with FA, with the strongest effect in patients progressing to severe aplastic anemia or myelodysplastic syndrome (MDS). Previous studies have indicated that the toxic upregulation of the nonhomologous end-joining (NHEJ) pathway accounts, at least in part, for the poor growth of FA HSPCs. AVID200 downregulated the expression of NHEJ-related genes and reduced DNA damage in primary FA HSPC in vitro and in in vivo models. Collectively, AVID200 exhibits activity in FA mouse and human preclinical models. AVID200 may therefore provide a therapeutic approach to improving BMF in FA.

Chromosome Instability in Fanconi Anemia: From Breaks to Phenotypic Consequences.

Fanconi anemia (FA), a chromosomal instability syndrome, is caused by inherited pathogenic variants in any of 22 FANC genes, which cooperate in the FA/BRCA pathway. This pathway regulates the repair of DNA interstrand crosslinks (ICLs) through homologous recombination. In FA proper repair of ICLs is impaired and accumulation of toxic DNA double strand breaks occurs. To repair this type of DNA damage, FA cells activate alternative error-prone DNA repair pathways, which may lead to the formation of gross structural chromosome aberrations of which radial figures are the hallmark of FA, and their segregation during cell division are the origin of subsequent aberrations such as translocations, dicentrics and acentric fragments. The deficiency in DNA repair has pleiotropic consequences in the phenotype of patients with FA, including developmental alterations, bone marrow failure and an extreme risk to develop cancer. The mechanisms leading to the physical abnormalities during embryonic development have not been clearly elucidated, however FA has features of premature aging with chronic inflammation mediated by pro-inflammatory cytokines, which results in tissue attrition, selection of malignant clones and cancer onset. Moreover, chromosomal instability and cell death are not exclusive of the somatic compartment, they also affect germinal cells, as evidenced by the infertility observed in patients with FA.

Meiotic susceptibility for induction of sperm with chromosomal aberrations in patients receiving combination chemotherapy for Hodgkin lymphoma.

Improvements in survival rates with gonad-sparing protocols for childhood and adolescence cancer have increased the optimism of survivors to become parents after treatment. Findings in rodents indicate that chromosomal aberrations can be induced in male germ cells by genotoxic exposures and transmitted to offspring and future generations with effects on development, fertility and health. Thus, there is a need for effective technologies to identify human sperm carrying chromosomal aberrations to assess the germ-line risks, especially for cancer survivors who have received genotoxic therapies. The time-dependent changes in the burden of sperm carrying structural chromosomal aberrations were assessed for the first time in a cancer setting, using the AM8 sperm FISH protocol which simultaneously detects abnormalities in chromosomal structure and number in sperm. Nine Hodgkin lymphoma (HL) patients provided 20 semen samples before, during, and after NOVP therapy (Novantrone, Oncovin, Velban and Prednisone) and radiation therapy that produced scattered gonadal doses from <0.05 to 0.6 Gy. Late meiosis was found to be the most sensitive to NOVP treatment for the production of sperm with chromosomal abnormalities, both in structure and number. Earlier stages of spermatogenesis were less sensitive and there was no evidence that therapy-exposed stem cells resulted in increased frequencies of sperm with abnormalities in chromosomal structure or number. This indicates that NOVP therapy may increase the risks for paternal transmission of chromosomal structural aberrations for sperm produced 32 to 45 days after a treatment with these drugs and implies that there are no excess risks for pregnancies conceived more than 6 months after this therapy. This clinical evaluation of the AM8 sperm FISH protocol indicates that it is a promising tool for assessing an individual's burden of sperm carrying chromosomal structural aberrations as well as aneuploidies after cancer therapy, with broad applications in other clinical and environmental situations that may pose aneugenic or clastogenic risks to human spermatogenesis.

Non-classical 1p36 deletion in a patient with Duane retraction syndrome: case report and literature review.

BACKGROUND: Monosomy of 1p36 is considered the most common terminal microdeletion syndrome. It is characterized by intellectual disability, growth retardation, seizures, congenital anomalies, and distinctive facial features that are absent when the deletion is proximal, beyond the 1p36.32 region. In patients with proximal deletions, little is known about the associated phenotype, since only a few cases have been reported in the literature. Ocular manifestations in patients with classical 1p36 monosomy are frequent and include strabismus, myopia, hypermetropia, and nystagmus. However, as of today only one patient with 1p36 deletion and Duane retraction syndrome (DRS) has been reported. CASE PRESENTATION: We describe a patient with intellectual disability, facial dysmorphism, and bilateral Duane retraction syndrome (DRS) type 1. Array CGH showed a 7.2 Mb de novo deletion from 1p36.31 to 1p36.21. DISCUSSION: Our patient displayed DRS, which is not part of the classical phenotype and is not a common clinical feature in 1p36 deletion syndrome; we hypothesized that this could be associated with the overlapping deletion between the distal and proximal 1p36 regions. DRS is one of the Congenital Cranial Dysinnervation Disorders, and a genetic basis for the syndrome has been extensively reported. The HES3 gene is located at 1p36.31 and could be associated with oculomotor alterations, including DRS, since this gene is involved in the development of the 3rd cranial nerve and the 6th cranial nerve's nucleus. We propose that oculomotor anomalies, including DRS, could be related to proximal 1p36 deletion, warranting a detailed ophthalmologic evaluation of these patients.