Abstract The Fanconi anaemia (FA) DNA repair pathway is an emerging target for precision cancer therapy. Using a high-throughput FANCD2-monoubiquitination assay, we identified a class of small molecules, including MSG010, that inhibit the FA E3 ligase complex in vitro . Because these molecules, and the metabolite, palmitoyl-CoA, are known to engage allosteric drug and metabolite (ADaM) binding site in AMP-activated kinase (AMPK), we hypothesised that a similar pocket exists within the FA complex. Supporting this, long-chain, but not short-chain, fatty acyl-CoA molecules inhibited the FA E3 ligase complex activity, and sequence analysis revealed similarity between the AMPK ADaM site and a WD40 repeat in the FA subunit FANCX. Targeted mutagenesis of this FANCX region disrupted E3 ligase activity or abolished inhibition by MSG010, suggesting the presence of an ADaM-like site in FANCX. Moreover, MSG010 preferentially killed BRCA1 -deficient cells in vitro . These findings identify a putative small-molecule binding site in the FA pathway that may be developed further to test for exploitation as anticancer therapeutics.

Synthetic lethality describes a genetic relationship where the loss of two genes results in cell death, but the loss of one of those genes does not. Drugs used for precision oncology can exploit synthetic lethal relationships; the best described are PARP inhibitors which preferentially kill BRCA1-deficient tumours preferentially over BRCA1-proficient cells. New synthetic lethal targets are often discovered using genetic screens, such as CRISPR knockout screens. Here, we present a competitive co-culture assay that can be used to analyse drugs or gene knockouts with synthetic lethal effects. We generated new BRCA1 isogenic cell line pairs from both a triple-negative breast cancer cell line (SUM149) and adapted pre-existing non-cancerous BRCA1 isogenic pair (RPE). Each cell line of the isogenic pair was transformed with its own fluorescent reporter. The two-coloured cell lines of the isogenic pair were then grown together in the same vessel to create a more competitive environment compared to when grown separately. We used four PARP inhibitors to validate the ability to detect synthetic lethality in BRCA1-deficient cancer cells. The readout of the assay was performed by counting the fluorescently coloured cells after drug treatment using flow cytometry. We observed preferential targeting of BRCA1-deficient cells, by PARPi, at relative concentrations that broadly reflect clinical dosing. Further we reveal subtle differences between PARPi resistant lines compared to BRCA1-proficient cells. Here, we demonstrate the validation and potential use of the competitive assay, which could be extended to validating novel genetic relationships and adapted for live cell imaging.

Fanconi anemia is a rare inherited bone marrow failure syndrome caused by inactivation of genes in the Fanconi anemia/BRCA DNA repair pathway. We report a patient with X-linked Fanconi anemia, and atypical physical features whose genetic diagnosis was initially inconclusive. Over time, his bone marrow karyotype shifted from diploid (46,XY) to triploid (69,XXY). The triploid cells lacked the Fanconi anemia cellular phenotype, enabling sustained hematopoiesis and providing an unexpected route to phenotypic rescue. Genomic analysis indicated early post-zygotic incorporation of the second polar body as the triploid origin. These findings suggest that the selective advantage of restored DNA repair in hematopoietic stem cells, outweigh the potentially deleterious effects of triploidy. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement W.C. receives a fellowship related to this work from the Victorian Cancer Agency (MCRF21006) and funding from the National Health and Medical Research Council (GNT2030115). SVI receives Operational Infrastructure Support from the Victorian State Government. The funding bodies played no role in the research. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: The Human Research Ethics Committee of the Royal Childrens Hospital, Melbourne, Australia gave ethical approval for this work. Study participants provided written informed consent. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes All data produced in the present study are available upon reasonable request to the authors

Premature ovarian insufficiency is a common form of female infertility affecting up to 4% of women and characterised by amenorrhea with elevated gonadotropin before the age of 40. Oocytes require controlled DNA breakage and repair for homologous recombination and the maintenance of oocyte integrity. Biallelic disruption of the DNA damage repair gene, Fanconi anemia complementation group A (FANCA), is a common cause of Fanconi anaemia, a syndrome characterised by bone marrow failure, cancer predisposition, physical anomalies and POI. There is ongoing dispute about the role of heterozygous FANCA variants in POI pathogenesis, with insufficient evidence supporting causation. Here, we have identified biallelic FANCA variants in French sisters presenting with POI, including a novel missense variant of uncertain significance and a likely pathogenic deletion that initially evaded detection. Functional studies indicated no discernible effect on DNA damage sensitivity in patient lymphoblasts. These novel FANCA variants add evidence that heterozygous loss of one allele is insufficient to cause DNA damage sensitivity and POI. We propose that intragenic deletions, that are relatively common in FANCA, may be missed without careful analysis, and could explain the presumed causation of heterozygous variants. Accurate variant curation is critical to optimise patient care and outcomes.

Agents that induce DNA damage can cure some cancers. However, the side effects of chemotherapy are severe because of the indiscriminate action of DNA-damaging agents on both healthy and cancerous cells. DNA repair pathway inhibition provides a less toxic and targeted alternative to chemotherapy. A compelling DNA repair target is the Fanconi anemia (FA) E3 ligase core complex due to its critical-and likely singular-role in the efficient removal of specific DNA lesions. FA pathway inactivation has been demonstrated to specifically kill some types of cancer cells without the addition of exogenous DNA damage, including cells that lack BRCA1, BRCA2, ATM, or functionally related genes. In this perspective, we discuss the genetic and biochemical evidence in support of the FA core complex as a compelling drug target for cancer therapy. In particular, we discuss the genetic, biochemical, and structural data that could rapidly advance our capacity to identify and implement the use of FA core complex inhibitors in the clinic.

Background: The IgE- and IgG(4)-binding patterns of the major fish allergen parvalbumins are not clearly understood. IgE antibody-binding to parvalbumin from Asian seabass, Lat c 1.01, is implicated in up to 90 % of allergic reactions, although the region of IgE or IgG(4) epitopes are unknown. In the present study, we characterized the specific IgE- and IgG(4)-binding regions of Lat c 1.01 using serum from pediatric and adult patients with clinically-confirmed fish allergy. Methods: A comparative investigation of patient IgE- and IgG4-binding to recombinant Lat c 1.01 was performed by immunoblotting and indirect ELISA using serum from 15 children and eight adults with clinically confirmed IgE-mediated reactions to fish. The IgE- and IgG(4)-binding regions of Lat c 1.01 were determined by inhibition ELISA using seven overlapping peptides spanning the entire 10(2) amino acid sequence. Elucidated IgE-binding regions were modelled and compared to known antibody-binding regions of parvalbumins from five other fish species. Results: Ninety five percent (22/23) patients demonstrated IgE-binding to rLat c 1.01, while fewer patients (10/15 children and 7/8 adults) demonstrated robust IgG(4) binding when determined by immunoblots. IgE-binding for both cohorts was significantly higher compared to IgG(4)-binding by ELISA. All patients in this study presented individual IgE and IgG(4) epitope-recognition profiles. In addition to these patient-specific antibody binding sites, general IgE epitopes were also identified at the C- and N-terminal regions of this major fish allergen. Conclusions and Clinical relevance: Our findings demonstrate two specific IgE epitopes on parvalbumin from Asian seabass, while IgG(4) binding is much lower and patient specific. This study highlights the importance of advancement in epitope analysis regardless of the age group for diagnostics and immunotherapies for fish allergy.

Site-specific conjugation of ubiquitin onto a range of DNA repair proteins regulates their critical functions in the DNA damage response. Biochemical and structural characterization of these functions are limited by an absence of tools for the purification of DNA repair proteins in purely the ubiquitinated form. To overcome this barrier, we designed a ubiquitin fusion protein that is N-terminally biotinylated and can be conjugated by E3 RING ligases onto various substrates. Biotin affinity purification of modified proteins, followed by cleavage of the affinity tag leads to release of natively-mono-ubiquitinated substrates. As proof-of-principle, we applied this method to several substrates of mono-ubiquitination in the Fanconi anemia (FA)-BRCA pathway of DNA interstrand crosslink repair. These include the FANCI:FANCD2 complex, the PCNA trimer and BRCA1 modified nucleosomes. This method provides a simple approach to study the role of mono-ubiquitination in DNA repair or any other mono-ubiquitination signaling pathways.

DNA inter-strand crosslinks (ICLs) threaten genomic stability by creating a physical barrier to DNA replication and transcription. ICLs can be caused by endogenous reactive metabolites or from chemotherapeutics. ICL repair in humans depends heavily on the Fanconi Anaemia (FA) pathway. A key signalling step of the FA pathway is the mono-ubiquitination of Fanconi Anaemia Complementation Group D2 (FANCD2), which is achieved by the multi-subunit E3 ligase complex. FANCD2 mono-ubiquitination leads to the recruitment of DNA repair proteins to the site of the ICL. The loss of FANCD2 mono-ubiquitination is a common clinical feature of FA patient cells. Therefore, molecules that restore FANCD2 mono-ubiquitination could lead to a potential drug for the management of FA. On the other hand, in some cancers, FANCD2 mono-ubiquitination has been shown to be essential for cell survival. Therefore, inhibition of FANCD2 mono-ubiquitination represents a possible therapeutic strategy for cancer specific killing. We transferred an 11-protein FANCD2 mono-ubiquitination assay to a high-throughput format. We screened 9,067 compounds for both activation and inhibition of the E3 ligase complex. The use of orthogonal assays revealed that candidate compounds acted via non-specific mechanisms. However, our high-throughput biochemical assays demonstrate the feasibility of using sophisticated and robust biochemistry to screen for small molecules that modulate a key step in the FA pathway. The future identification of FA pathway modulators is anticipated to guide future medicinal chemistry projects with drug leads for human disease.

FANCI:FANCD2 monoubiquitination is a critical event for replication fork stabilization by the Fanconi anemia (FA) DNA repair pathway. It has been proposed that at stalled replication forks, monoubiquitinated-FANCD2 serves to recruit DNA repair proteins that contain ubiquitin-binding motifs. Here, we have reconstituted the FA pathway in vitro to study functional consequences of FANCI:FANCD2 monoubiquitination. We report that monoubiquitination does not promote any specific exogenous protein:protein interactions, but instead stabilizes FANCI:FANCD2 heterodimers on dsDNA. This clamping requires monoubiquitination of only the FANCD2 subunit. We further show using electron microscopy that purified monoubiquitinated FANCI:FANCD2 forms filament-like arrays on long dsDNA. Our results reveal how monoubiquitinated FANCI:FANCD2, defective in many cancer types and all cases of FA, is activated upon DNA binding.

SummaryBackgroundFish is a well‐recognized cause of food allergy and anaphylaxis. The evolutionary and taxonomic diversity of the various consumed fish species pose a challenge in the identification and characterization of the major fish allergens critical for reliable diagnostics. Globally, fish is a rising cause of food allergy complicated by a large under‐investigated variety of species as well as increasing global tourism and trade. This is the first comprehensive study on allergen profiles of heat‐processed fish from Vietnam.ObjectiveThe aim of this study was to identify the major heat‐stable allergens from frequently exported Asia‐Pacific freshwater and marine fish and to characterize the major allergen parvalbumin (PV) from one of the most consumed and exported fish species from Asia, the Indian mackerel (Rastrelliger kanagurta).MethodsHeated protein extracts from 33 fish species were separated by gel electrophoresis. PV isoforms were identified by immunoblotting utilizing 3 different PV‐specific monoclonal and polyclonal antibodies and further characterized by mass spectrometry. IgE reactivity was investigated using sera from 21 patients with confirmed fish allergy.ResultsHeat‐stable IgE‐reactive PVs, with up to 5 isoforms per species, were identified in all 33 analysed fish species. In the Indian mackerel, 7 PV isoforms were identified by 2D‐gel electrophoresis combined with mass spectrometric analyses. The amino acid sequence deduced from cDNA of the most expressed isoform showed a high identity (>90%) to PVs from 2 other mackerel species.Conclusions and Clinical RelevanceDifferent PVs were identified as the major heat‐stable allergens in all 33 analysed freshwater and marine fish species from Vietnam, many of which are exported world‐wide and 21 species that have never been investigated before. The Indian mackerel PV represents a novel fish allergen, now officially registered as Ras k 1. Improved diagnostics for fish allergy against Asia‐Pacific species should be developed with focus on PV.

Lupine belongs to the genus Lupinus and includes three species commonly consumed by humans. The Lupinus genus is closely related to other legumes, such as peanuts, soya, chickpeas, peas, lentils and beans. However, the consumption of lupine (and related legumes) can cause severe allergenic reactions. Therefore, reliable analytical detection methods are required for the analysis of food samples. In this study three commercially available ELISA test kits were analyzed for the detection capability of three common lupine species, as well as cross-reactivity to related legumes. All three ELISA test kits could detect the lupine species, though with different sensitivities. Cross-reactivity varied for the ELISA test kits and all showed some cross-reactivity to related legume samples analyzed.

SummaryAllergy to bony fish is common and probably increasing world‐wide. The major heat‐stable pan‐fish allergen, parvalbumin (PV), has been identified and characterized for numerous fish species. In contrast, there are very few reports of allergic reactions to cartilaginous fish despite widespread consumption. The molecular basis for this seemingly low clinical cross‐reactivity between these two fish groups has not been elucidated. PV consists of two distinct protein lineages, α and β. The α‐lineage of this protein is predominant in muscle tissue of cartilaginous fish (Chondrichthyes), while β‐PV is abundant in muscle tissue of bony fish (Osteichthyes). The low incidence of allergic reactions to ingested rays and sharks is likely due to the lack of molecular similarity, resulting in reduced immunological cross‐reactivity between the two PV lineages. Structurally and physiologically, both protein lineages are very similar; however, the amino acid homology is very low with 47–54%. Furthermore, PV from ancient fish species such as the coelacanth demonstrates 62% sequence homology to leopard shark α‐PV and 70% to carp β‐PV. This indicates the extent of conservation of the PV isoforms lineages across millennia. This review highlights prevalence data on fish allergy and sensitization to fish, and details the molecular diversity of the two protein lineages of the major fish allergen PV among different fish groups, emphasizing the immunological and clinical differences in allergenicity.

Fish allergy is a common food allergy, with prevalence rates in the general population ranging between 0.2% and 2.3%. In both adults and children fish ranks in the top eight foods known to cause IgE mediated food allergy. Fish allergy is rarely outgrown and individuals with fish allergy may be allergic to some but not all species of fish. Whilst fish allergy occurs around the world, the characterization of allergenic components of individual species of fish has been largely confined to Northern hemisphere and European fish species. To date allergy to commonly consumed fish in the Asian-Pacific region including barramundi (Asian seabass; Lates calcarifer) have been less well investigated. The aim of this study was to identify and characterize allergenic proteins from barramundi in both fish allergic adult and pediatric patients. Serum from 17 fish allergic adults and children from Australia were characterized by immunoblotting and enzyme linked immunosorbent assays (ELISA) against raw and heated barramundi. Molecular analysis of identified allergens included genetic sequencing and generation of recombinant isoallergens. Two novel parvalbumin isoforms of the β-type were identified as the only allergens in barramundi and subsequently designated as Lat c 1.0101 and Lat c 1.0201 by the International Union of Immunological Societies. These two isoallergens do not differ in their ability to bind IgE antibodies, but are differentially expressed in barramundi tissue. This study characterized two novel heat stable parvalbumin allergens from barramundi, with differential IgE binding capacity between adults and pediatric patients.

Globally, the rising consumption of fish and its derivatives, due to its nutritional value and divergence of international cuisines, has led to an increase in reports of adverse reactions to fish. Reactions to fish are not only mediated by the immune system causing allergies, but are often caused by various toxins and parasites including ciguatera and Anisakis. Allergic reactions to fish can be serious and life threatening and children usually do not outgrow this type of food allergy. The route of exposure is not only restricted to ingestion but include manual handling and inhalation of cooking vapors in the domestic and occupational environment. Prevalence rates of self-reported fish allergy range from 0.2 to 2.29 % in the general population, but can reach up to 8 % among fish processing workers. Fish allergy seems to vary with geographical eating habits, type of fish processing, and fish species exposure. The major fish allergen characterized is parvalbumin in addition to several less well-known allergens. This contemporary review discusses interesting and new findings in the area of fish allergy including demographics, novel allergens identified, immunological mechanisms of sensitization, and innovative approaches in diagnosing and managing this life-long disease.

Fish are the largest and most diverse group of vertebrates. Fish are also a part of the eight food groups that cause the majority of IgE mediated food reactions. Detection tools for fish allergens are however limited due to the great diversity of fish species, despite fish allergy and its major allergen parvalbumin being well documented. The most commonly studied fish are frequently consumed in North America and Europe. However, much less is known about fish allergens in the Australasian region although fish is widely consumed in this region. A comprehensive phylogenetic analysis was performed of known parvalbumin amino acid sequences to determine possible candidate antigens for new cross-reactive antibodies to be used to detect most fish parvalbumins. Polyclonal rabbit antibodies were raised against parvalbumins from frequently consumed barramundi (Lates calcarifer), basa (Pangasius bocourti), pilchard (Sardinops sagax) and Atlantic salmon (Salmo salar). These were evaluated for cross-reactivity against a panel of 45 fish extracts (raw, heated and canned fish). Anti-barramundi parvalbumin proved to be the most cross-reactive antibody, detecting 87.5% of the 40 species analyzed, followed by anti-pilchard and anti-basa antibody. In contrast the anti-salmon antibody was very specific and only reacted to salmonidae and a few other fish. All analyzed fish species, except mahi mahi, swordfish, yellowfin tuna and all 5 canned fish had parvalbumin detected in raw extracts. However antibody reactivity to many fish was heat liable or susceptible to denaturation, demonstrating that some parvalbumins have most likely conformational epitopes, which lose antibody reactivity after heat treatment. We have demonstrated the generation of highly cross-reactive anti-parvalbumin antibodies that could be used for the detection of allergenic fish parvalbumin in contaminated food products. This cross-reactivity study thus shows processing of fish, especially canning, can have on impact on antibody recognition by ELISA, possibly similar to IgE-binding in vivo.