Castanea caused by Cryphonectria parasitica, which has ravaged across American chestnut and most of European chestnut since the early twentieth century. Interestingly, the Chinese chestnut is strongly resistant to chestnut blight, shedding light on restoring the ecological status of Castanea plants severely affected by chestnut blight. To better explore the early defense of Chinese chestnut elicited in response to C. parasitica, the early stage of infection process of C. parasitica was observed and RNA sequencing-based transcriptomic profiling of responses of the chestnut blight-resistant wild resource ‘HBY-1’ at 0, 3 and 9 h after C. parasitica inoculation was performed.Chestnut blight, one of the most serious branch diseases in C. parasitica, which was the basis of further study on transcriptional activation of Chinese chestnut in response to chestnut blight in the early stage. In the transcriptome analysis, a total of 283 differentially expressed genes were identified between T9 h and Mock9 h, and these DEGs were mainly divided into two clusters, one of which was metabolism-related pathways including biosynthesis of secondary metabolites, phenylpropanoid biosynthesis, amino sugar and nucleotide sugar metabolism, and photosynthesis; the other was related to plant-pathogen interaction and MAPK signal transduction. Meanwhile, the two clusters of pathways could be connected through junction among phosphatidylinositol signaling system, phytohormone signaling pathway and α-Linolenic acid metabolism pathway. It is worth noting that genes associated with JA biosynthesis and metabolic pathway were significantly up-regulated, revealing that the entire JA metabolic pathway was activated in Chinese chestnut at the early stage of chestnut blight infection.First, we found that 9 h was a critical period for Chinese chestnut infected by C. parasitica and observed the morphological changes of Chinese chestnut wounds at the early stage of infection. In response to chestnut blight, the plant hormone and MAPK signal transduction pathways, plant-pathogen interaction pathways and metabolism-related pathways were activated at the early stage. JA biosynthesis and metabolic pathway may be particularly involved in the Chinese chestnut resistance to chestnut blight. These results contributes to verifying the key genes involved in the resistance of Chinese chestnut to C. parasitica.We identified the important infection nodes of The online version contains supplementary material available at 10.1186/s12870-023-04072-7. Castanea is an economically important tree in the world [Castanea plants are recognized, and there are significant differences in the resistance to chestnut blight among Castanea species [Cryphonectria parasitica (Murr.) Barr., causing an overwhelming crisis in European chestnut production and an endangered state of American chestnut throughout North America. However, Castanea plants of East Asia suffered less damage, especially Chinese chestnut and Japanese chestnut show significant resistance to chestnut blight [he world . At pres species . Since tt blight , 3. TherC. parasitica also revealed that due to the widespread introduction of Japanese chestnut in North America, C. parasitica had widely spread into North America [C. parasitica introduced from North America was discovered in northern Italy in 1938, resulting in their widespread death [C. parasitica virulence and has allowed the European chestnuts to remain commercially viable in European [C. crenata, C. henryi, C. mollissima and C. seguinii) are generally resistant to C. parasitica, with strong immune activity. The reason is possibly co-evolution with C. parasitica in history [C. parasitica commonly develop superficial lesions or ulcers on parts of the trunk and survive with burl at the infestation [C. sativa) and American (C. dentata and C. pumila) species are highly susceptible to this pathogen. Once infected, they are killed [C. parasitica, and the incidence rate even in China is as high as 60% in recent years, especially in chestnut orchards in southern China due to improper management and continuous environmental changes [Chestnut blight is one of the world-famous forest diseases and is a typical example of plant disease that destroyed a dominant tree species . The pat America . In Euroad death , 8. Luck history . Resistaestation , 13. In e killed , 12. But changes . The inf changes .C. parasitica virulence was discovered and widely applied in production across Europe [TGA1, RGA3, PBS1, RD19a, DAZ-associated protein 1-like, Proteasome subunit alpha type-7, etc. facilitating further study on blight resistance [C. dentata plants with overexpression of OxO enhancing resistance to chestnut blight [C. parasitica, not to mention how the Chinese chestnut mediates immunity in response to this stress. In this study, the critical period of infection (CPI) of Chinese chestnut responding to C. parasitica was confirmed based on observation of the infection process. The transcripts of the inoculated and non-inoculated pathogen were compared to identify DEGs. It will provide insight into the resistance of Castanea plants to chestnut blight by exploring the disease resistance characteristics of the Chinese chestnut.To restrict the further expansion of chestnut blight, researchers had been seeking various ways in the past few decades. A naturally occurring double-stranded RNA (dsRNA) hypovirus that can reduce s Europe , 16. Sins Europe , 18. Sims Europe –22. Amons Europe . Some res Europe . The seqsistance . The SUNt blight , 26. ChiCryphonectria parasitica, the virulent strain ‘EP155’ was used to explore the infection process on a resistant wild Chinese chestnut resource ‘HBY-1’. The infection locations of C. parasitica and morphological contrast of the bark tissue structure for inoculated and non-inoculated branches were observed with electron microscopy (EM) at 3, 6, 9 and 12 hours, respectively. . Six hours after inoculation, the fungus could be observed in the epidermis and the wounds , which provided an important time node for us to further study the molecular mechanism of Chinese chestnut response from the beginning of infection to the time of structural changes in plant epidermal cells.Therefore, during the period of 0-6 h, the on Table . Hence, C. parasitica, the result revealed that the morphology of epidermal cells at the infection site kept a whole shape at 3–6 hours after infection, which was a stable period for the proliferation of C. parasitica. 9 h of infection was a key time point that the C. parasitica began to invade the intercellular space. At this time, it was also the time point that Chinese chestnut had an obvious stress response to the C. parasitica, and the cells at the infected wound of Chinese chestnut were degraded. Therefore, 0, 3 and 9 hours were selected as the three key periods for analyzing the response mechanism of chestnut to the parasitism of C. parasitica. In this experiment, total RNA was extracted from Chinese chestnut branches at 0 hour (without a scratch), 3 and 9 hours after non-inoculation (scratch), 3 and 9 hours after inoculation (scratch and inoculation), and 15 cDNA libraries were constructed . After the raw data were removed and trimmed, we obtained 17–32 million clean reads for each library. The Q20 and Q30 were > 97.61% and > 93.12%, respectively. In addition, the GC contents were 43.57–44.84% among all samples , and the DEGs were distributed in pathways including biosynthesis of secondary metabolites, MAPK signaling pathway, phosphatidylinositol signaling system, phenylpropane biosynthesis and plant-pathogen interaction and so on  were extracted according to their expression levels in our sequenced samples and identified at Mock3 h and Mock9 h, and we found 1390 genes (762 up-regulated genes/628 down-regulated genes) and 1668 genes (626 up-regulated genes/1042 down-regulated genes), respectively  at this stage, which is consistent with the appearance of apoptosis at the wound site in microscopic observations. Simultaneously, the PR and EDS disease process genes were also activated, and the sensitivity of Chinese chestnut to C. parasitica was enhanced, which in turn stimulated a stronger autoimmune response. Unfortunately, the resistance gene (R gene) related to the second layer of defense (ETI) was not found in the DEGs. We speculated that due to the relatively short infection time, the ETI response of Chinese chestnut may not be activated during the 9 h inoculation process, and the R gene remained silent.When plants are stressed by pathogens, plants often respond through the innate immune system, metabolism, and phytohormone-mediated defense responses. Among them, the innate immunity of plants mainly includes PAMP-triggered immunity (PTI) and effector-triggered immunity (ETI). Therefore, in order to characterize the innate immune response of Chinese chestnut at the early stage, we focused on the genes of the pathways involved in plant-pathogen interaction and MAPK signaling pathway. Subsequently, we further annotated the DEGs of the relevant pathways at 3 h and 9 h after inoculation in the KEGG databases. At 3 h after inoculation, a total of 13 genes were differentially expressed in the MAPK signaling pathway, including 4 WRKY, 2 CALM, 2 FRK, 2 ERF, 2 PYL and 1 MYC. In the plant-pathogen interaction pathway, a total of 16 genes were differentially expressed, including 2 CALM, 4 CML, 3 CPK, 1 CNGC, and 2 FRK genes, revealing that the cationic channel was activated and CaC. parasitica. The genes of other enriched metabolic pathways, such as amino sugar and nucleotide sugar metabolism pathways, α-linolenic acid metabolism pathway and phenylpropanoid biosynthesis pathway, were all up-regulated. Among them, 9 genes were enriched in amino sugar and nucleotide sugar metabolism pathways , 2 DEGs (CMHBY206784 and CMHBY208616), and 9 DEGs  and 3 DEGs  in the biosynthesis pathways of gibberellins (GAs), abscisic acid (ABA), ethylene (ET)) and brassinosteroids (BR) were identified. Furthermore, it is worth noting that most of the key genes on the Jasmonic acid (JA) metabolic pathway exhibited differential expression patterns, including hydroperoxide dehydratase (AOS), acyl-CoA oxidase (ACX), alpha-dioxygenase (DOX), 12-oxophytodienoic acid reductase (OPR), jasmonate O-methyltransferase (JMT), phospholipase A (DAD), JAR, enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase (MFP), etc. The results of expression thermogram showed that except for CMHBY231087 and CMHBY220949, the expression of the other genes decreased significantly  and RBOHD genes besides the genes mentioned above which were consistent with the observation of the microstructure. The MAPK cascade also plays a central role in defense signaling against pathogens [C. parasitica where the Ca2+ regulation signal and MAPK pathway are required, laying a foundation for its further defense response.Plants are generally attacked by a variety of microbial pathogens throughout the whole life cycle . Despiteathogens . 9 h aftC. parasitica strains growth to some extent [CsCHI3 [C. parasitica.The co-evolutional characteristic of plants and pathogens is an intense competition between penetrating and strengthening cell walls. Pathogens degrade plant cell walls through synthesizing proteases such as cellulase to infect the host. Simultaneously, to resist damage from pathogens, plants strengthen cell walls via a reverse way like synthesizing enzyme inhibitors and accumulating callose and lignin . Likewise extent . Recent  [CsCHI3 . HoweverC. parasitica. In addition, it is worth noting that more genes were significantly enriched in the linolenic acid metabolism pathway which is the key pathway of JA biosynthesis. JA and its derivatives are involved in a variety of plant stress responses, especially in plant defenses against insects and necrotrophic pathogens [C. parasitica, but also displays that the mechanism among these hormones is very complicated during this response. It remains to be investigated how these three signalings pathways interact with each other and if another hormone involves.Previous research has shown that salicylic acid (SA), jasmonic acid (JA), ethylene (ET), brassinosteroids (BR), gibberellins (GAs) and abscisic acid (ABA) were the main hormones involved in plant disease resistance. Of them, SA, JA and ET have been known as plant disease-resistant hormones so far –45. Afteathogens , 50. Subathogens . These pC. parasitica via observation of the morphological wound changes infected at the early stage in Chinese chestnut. Then, the transcriptome datasets of inoculation and non-inoculation were designed and sequenced enclosing the key early disease resistance-related genes and pathways. This analysis displayed that JA, SA, ET and MAPK signaling pathways and plant-pathogen interaction pathways were mediated at 9 h after inoculation by C. parasitica. Importantly, the expression pattern of genes related to JA biosynthesis, phenylpropanoid biosynthesis, and chitinase biosynthesis resist the stress of C. parasitica. All these differentially expressed genes contribute to our more extensive and deeper understanding of the disease defense of Chinese chestnut and facilitate future breeding of chestnut blight resistant resources. These results provide important references for further understanding the defense of Chinese chestnut resistance to chestnut blight.In summary, we firstly determined 9 h representing the important infection nodes of C. parasitica preserved in the College of Plant Science and Technology, Beijing University of Agriculture. The wild Chinese chestnut material in this experiment was grafted at the Chestnut Technology Experiment and Extension Station of Huairou District in 2015. Meanwhile, to reduce the errors caused by the environment and the growth state of branches, some criteria had been established, (1) the test branches can be healthy and non-damaged; (2) the test branches are biennial branch with a uniform diameter; (3) the branches of the mock group and treatment group were derived from the same branch group of a tree. Based on this, we preferentially selected a healthy biennial branch larger than 18 cm and sterilized spraying 75% ethanol. Then, the branch was aliquoted 2 groups of branches: one was Mock group, the other was Treatment(T) group. Subsequently, 5 mm wound were created by a scalpel where its depth reached to the cambium and its intervals were 3 cm. Meanwhile, creating the agar plugs with C. parasitica with a cork borer (diameter 6 mm) around the actively growing border of the fungal colony. After wounding, an inoculated agar plug was immediately placed directly on each wound as a treatment group and a non-inoculated agar plug (without C. parasitica) was also placed on each wound as a Mock group, and each group was repeated 3 times. The branches with agar plugs were then placed in a plastic tray lined with slightly damp paper towels. Trays were stored in an incubator with a temperature of 28 °C, a humidity of 70%, and illumination of 16 h/d.Inoculations were done primarily with the strain of Samples for microscopic observation were collected at 0 h, 3 h, 6 h, 9 h, and 12 h after inoculation, and then stored in fixative at 4 °C. The Chinese chestnut branch semi-thin slices were prepared as per Hao et al. (2022) .C. parasitica were cut into small cylindrical pieces with a thickness of 0.5–1 mm. Cuts were placed into a 2 ml centrifuge tube filled with 1 mL fixative solution . The samples were evacuated until there were no bubbles in the solution soaking the smples and the material sinked to the bottom of the centrifuge tube. They were stored in the refrigerator at 4 °C for 2 days. They were rerinsed with PBS buffer solution pH = 7.4 with 5–6 times, for 30 minutes each time. The cleaned chestnut branch samples were dehydrated by ethanol solutions with different concentration gradients  for 30 min, and was dehydrated by 100% ethanol twice. After dehydration, they were replaced with tert-butanol three times for 30 min each time at room temperature. Placed the replaced sample into a 2 ml centrifuge tube, then added an appropriate amount of tert-butanol, when tert-butanol just covered them,the samples were chilled at − 20 °C for 15 minutes. The samples were dried in freeze-dryer until tert-butyl alcohol was vanished. Samples were adhered to the sample table with conductive adhesive and then placed in a scanning electron microscope for observation and photography after powdered gold spraying.The inoculated parts of branches infected with All bark tissues treated with EP155 for 3, 9 hours and tissues treated without EP155 for 0,3, 9 hours were sampled for RNA-Seq with three independent biological replicates for each. Total RNA was extracted using an E.Z.N.A.® Plant RNA Kit  and the quantity and quality of RNA were evaluated by a NanoDrop spectrophotometer . Subsequently, the RNA samples were subjected to strict quality control, mainly through the Agilent 2100 bioanalyzer: precise detection of RNA integrity. The mRNA was enriched by magnetic beads with Oligo (dT) for RNA-Seq. The constructed library was sequenced with Illumina Hi-Seq6000 .https://www.bioinformatics.babraham.ac.uk/projects/fastqc/). Using HISAT2 software to quickly and accurately align the quality control data with the reference genome of chestnut (https://www.ncbi.nlm.nih.gov/bioproject/PRJNA527178), and then using StringTie software to assemble new transcripts. After transcript assembly, we annotated and counted the new transcripts in five major databases including nonredundant (nr), Swiss-Prot, GO, Pfam and KEGG.Firstly, Fast-QC was used for quality control analysis of RNA-SEQ sequences that were generated by Illumina sequencing platform  ≥ 1.5 were considered to be differentially expressed. The Gene trend analysis, KEGG enrichment analyses of DEGs and pathway network analysis were performed using the OmicShare tools, a free online platform for data analysis (http://www.kegg.jp/kegg/path- way.html and https://www.omicshare.com/tools) [The analysis of DEGs was performed using the DESeq. The m/tools) –54.Actin as the internal control. Each condition of qRT-PCR has three biological replicates, and the relative expression levels of each gene were calculated. To further prove the role of JA metabolic pathway in chestnut resistance to chestnut blight, qRT-PCR also was performed for the genes related to this pathway  using a known Chinese chestnut cultivar ‘Yanhong’ with great disease resistance. Specific primers designed according to the selected JMT, OPCL, DAD and JAR sequences are listed in the attached file .To verify the accuracy of the screened genes, the total RNA of bark tissues was extracted at 3 h and 9 h after inoculation and at 0 h, 3 h and 9 h without inoculation, and the 20 differential expression genes were analyzed by qRT-PCR after reverse transcription  and non-inoculated (Mock) wounds of branches at 3 h, 6 h, 9 h and 12 h through stereomicroscopy.Additional file 2: Table. S1. Data quality assessment of each sample.Additional file 3: Fig. S2. Differentially expressed genes (DEGs) at different time points.Additional file 4: Fig. S3. Differentially expressed genes (DEGs) at different trend characteristics at Mock0h-T3 h-T9 h. a: The genes showing an upward trend were annotated in KEGG; b: The genes showing an downward trend were annotated in KEGG.Additional file 5: Table S2. The gene information of different trends of Plant-pathogen interaction, Plant hormone signal transduction and MAPK signaling pathway.Additional file 6: Fig. S4. The related genes expression thermogram of plant hormone signal transduction pathway.Additional file 7: Table S3. The primer information of 20 genes for qRT-PCR.


Glyma.16g181900, Glyma.16g182200, and Glyma.16g182300. The genetic variation in Glyma.16g182200, encoding glycosylphosphatidylinositol-anchored protein (GPI-AP), caused a critical structure disruption on the active site of the protein. This structural variation of GPI-AP may change various properties of the ion channels which are the targets of pyrethroid insecticide including etofenprox. This is the first study that identifies the candidate gene and develops SNP markers associated with PE. This study would provide genomic information to understand the mechanism of phytotoxicity in soybean and functionally characterize the responsive gene. Phytotoxicity is caused by the interaction between plants and a chemical substance, which can cause critical damage to plants. Understanding the molecular mechanism underlying plant-chemical interactions is important for managing pests in crop fields and avoiding plant phytotoxicity by insecticides. The genomic region responsible for sensitivity to phytotoxicity of etofenprox (PE), controlled by a single dominant gene, was detected by constructing high density genetic map using recombination inbred lines (RILs) in soybean. The genomic region of ~ 80 kbp containing nine genes was identified on chromosome 16 using a high-throughput single nucleotide polymorphism (SNP) genotyping system using two different RIL populations. Through resequencing data of 31 genotypes, nonsynonymous SNPs were identified in  Glycine max (L.) Merr.] have shifted from foliage-feeding coleopteran and lepidopteran pests to sap-sucking hemipteran pests during the last few decades1. Various stink bugs and soybean aphid species are the main hemipteran pests that damage soybean plants by sucking the juice from plant tissues2. Significant losses in soybean yield, quality, and germination have been caused by stink bug feeding3. The soybean aphid can cause up to 58% yield loss, and a $2.4 billion loss has been estimated annually in the US4.Because of global warming, the major pests of soybean [9. The symptoms of phytotoxicity differ, including leaf speckling, leaf margin necrosis (browning) or chlorosis (yellowing), brown or yellow leaf spots, leaf cupping or twisting, plant stunting, and plant death10.Although synthetic pyrethroid insecticides have been widely used to manage insects, they might also harm plants. Phytotoxicity is defined as a harmful effect on various physiological processes. In various crop species including soybean, the several studies on interactions between plant physiology and pesticides have been reported11. Voltage-gated sodium channels are the most effective targets for the neurotoxic effects of pyrethroids, while voltage-gated chloride, and calcium channels work as secondary sites of action for a subset of pyrethroids12. Although the insecticide of etofenprox has been widely used in soybean fields to manage pests, few physiological and genetic studies of interactions between plants and pesticides have been reported. In a previous study, we firstly reported a novel trait, sensitivity to phytotoxicity of etofenprox (PE) in soybean, and it was illustrated that it was controlled by a single dominant gene through genetic analysis13. Only a limited number of genotypes, Danbaek and Kwangan, indicated leaf shrinkage due to the etofenprox application13. PE in Danbaek and Kwangan was regulated by the same gene inherited from a common ancestor, Tohoku 6913.The insecticidal mode of action of pyrethroids, which derive etofenprox, relies on their ability to bind to voltage-gated sodium channels, disrupting insects’ nerve system18. For example, single genes could be associated with flowering (Glyma.10g221500) and pod shattering (Glyma.16g141600), based on high throughput single nucleotide polymorphisms (SNPs) data in soybean16. Using genotypic data of Korean soybean collection comprising 1957 domesticated and 1079 wild accessions, domestication and evolutionary history were studied17. Through Axiom SoyaSNP array data of 2782 soybean collections, the core collection was developed for further genome-wide association study (GWAS)18.Because research tools have been rapidly developed, especially high-throughput genotyping systems , genomic research of soybean has been widely reportedThe main goal of this study was to identify candidate genes responsible for sensitivity to PE using genomic approaches in soybean. To achieve this goal, a high saturated genetic map was constructed using a recombinant-inbred line (RIL) population, and a validation study was used with different RIL populations using TaqMan based SNP marker. Furthermore, whole-genome resequencing data from diverse soybean germplasm was used to develop SNPs in candidate genes associated with PE.13. In this study, two RIL populations were used to identify candidate genes and validate the results of genetic mapping. Among the 113 RIL populations derived from Daepung (insensitive) × Danbaek (sensitive), 79 lines showed sensitivity to PE and 34 lines showed insensitivity to PE. Among the 138 RIL population derived from 5002T (insensitive) × Kwangan (sensitive), 83 and 55 lines showed sensitivity and insensitivity to PE, respectively. Both populations exhibited discrete phenotypic distribution indicating PE is a qualitative trait.The sensitivity of PE was identified as a qualitative trait regulated by a single dominant geneT gene  was located in this region. To construct chromosome 13 of the genetic map, 881 SNPs out of 1953, were used  was 20 Kbp apart from this region , nine gene models were located with the candidate genomic region of ~ 80 kbp region related to PE21  and white (43), gray (76), and brown (37), respectively. The responsive genes, T and W1, for the colors of pubescence and flower could be identified using the constructed genetic map.Through the 180K Axiom SoyaSNP assay, the high-density genetic map was constructed for the Daepung × Danbaek RIL population. In the population, the colors of flower and pubescence, which had been reported to be controlled by single genes previouslyhttps://soybase.org/gb2/gbrowse/glyma.Wm82.gnm4/ accessed on 18 January 2021). Using high-depth resequencing data on 31 soybean germplasm, three candidate genes, Glyma.16g181900, Glyma.16g182200 and Glyma.16g182300 had amino acid substitutions between genotypes with insensitivity (29 genotypes) and sensitivity (Danbaek and Kwangan) to PE. Glyma.16g182200 is the most promising candidate gene associated with sensitivity to PE, Therefore, accurately evaluated phenotypes, high-density genetic map, and genomic information from diverse germplasm would be essential for successful genetic mapping to identify candidate gene(s) of a target trait.The candidate region (~ 80 kbp) conferring PE was narrowed down from (kbp) to ~ 80 kbp using the second RIL population (5002T × Kwangan) through TaqMan based SNP assay. The genomic region contains nine annotated gene models on chromosome 16 based on the Williams 82 genome assembly . In wild-type, trichomes have a stalk and three or four branches. In contrast, in zwichel (zwi) mutants, the trichomes have a shortened stalk and only two branches resulting in leaf senescence, similar to the morphological symptoms induced by PE22. Glyma.16g182300 encodes cleavage and polyadenylation specificity factor 100 (CPSF 100), a homologue of AT5G23880.1 in Arabidopsis. In Arabidopsis, CPSF100 mutant showed early flowering phenotypes induced by modified RNA processing of flowering control locus A enhancer of flowering25. Besides flowering time, the CPSF protein family is involved in various functions in biological processes such as environmental response and amino acid metabolism27. A variant of this gene might also affect the responses of plants to environmental stresses at the level of transcription, causing sensitivity to PE.Arabidopsis homologous gene, AT5G23890.1, Arabidopsis orthologue of Glyma.16g182200, encodes glycosylphosphatidylinositol (GPI)—anchored protein (GPI-AP), allowing various proteins to associate with membrane lipid bilayers and anchor on the external surface of the plasma membrane28. The GPI oligosaccharide structure is ubiquitous among eukaryotes with a common minimal backbone comprising three mannoses, one non-N-acetylated glucosamine, and an inositol phospholipid, which covalently links the carboxyl terminus (C terminus) of GPI-Aps to the lipid bilayer29. GPI-Aps include diverse families, such as cell wall structure proteins, proteases, enzymes, and lipid transfer proteins. They are involved in several functional processes, including cell wall composition, cell wall component synthesis, polar cell expansion, stress responses, hormone signaling responses, and pathogen responses in Arabidopsis28.12. GPI-Aps have been reported to play roles in ion channels32.Voltage-gated sodium channels are important for initiating and propagating the action potential of neurons. Because of their essential roles in electrical signaling, sodium channels are primary targets of synthetic insecticides, including etofenprox. These sodium channel neurotoxins bind to the receptor on the sodium channel, changing different channel properties, including ion selectivity, ion conductance and/or channel opening and closing33. A recent crystal structure analysis of TEX101 provides evidence that this molecule has two LU domains, which function as a regulator of ion channels34. This structural feature holds great promise for elucidating the actual interactions between this molecule and a group of molecules associated with the ion channels35. In zebrafish, voltage-gated sodium channels are not located at the cell surface in the mutant of a subunit of GPI transamidase, which is crucial for membrane anchoring of GPI-anchored proteins. The biogenesis of GPI-anchored proteins is necessary for cell surface expression of sodium channels of neurons in zebrafish36.TEX101, a germ cell-specific GPI-AP, belongs to the lymphocyte antigen 6 (Ly6)/urokinase-type plasminogen activator receptor (uPAR)-(LU) protein superfamilyhttps://alphafold.ebi.ac.uk/)  Fig. 37. Compapes Fig. B,C. TakeGlyma.16g182200, encodes GPI-AP, is the most promising causal single gene regulating PE in soybean. Further functional characterization would be required through RNA expression analysis or cloning to transformation of the candidate gene. Identification of the responsive gene will improve our understanding of the basic mechanism of sensitivity to PE in soybean.The SNP markers and high-resolution genetic map identified in the this study will facilitate MAS of PE in soybean. PE-sensitive genotype specific SNP markers were identified. Among the three candidate genes, 38 and Danbaek39. Daepung showed insensitivity to etofenprox and Danbaek showed sensitivity to etofenprox13. After artificial crossing in 2012, F1 progeny was advanced using single seed descent in the Dankook University greenhouse resulting in 119 F5:8 lines used for this study in 2019.Two recombinant inbred line (RIL) populations were used in the present study. The first population of RILs was developed from a cross between Daepung40 showing insensitivity to etofenprox and Kwangan41 showing sensitivity to etofenprox, in the experimental field of Kyungpook National University. 5002T was developed for high yield with maturity group V in the southern USA in 200240 and Kwangan was developed for high-protein13. This population was used to confirm genetic mapping and inheritance of sensitivity to PE with a different genetic background. Furthermore, from these two RIL populations, SNP markers associated with sensitivity to etofenprox were developed.The second 138 RIL population was derived from a cross between 5002T1 stage13. Distilled water was used as a negative control. A 10 mL was applied to each plant in each application and the phenotype (sensitivity/insensitivity) was evaluated by presence/absence of leaf shrinkage a week after the final treatment. The treatments were conducted with three biological replications13.The RILs were sowed using a mixture of horticultural soil and the nursery bed soil at the ratio of 3: 1 in 50 deep cell seed trays, 55 × 27 × 12 cm (Wide × Length × Height). Etofenprox 20% EC 1000 × dilution was foliar sprayed four times in two weeks at VW1 and T genes, respectively, previously reported20. Three qualitative phenotypic data  were recorded by two types, reference allele or alternative allele.Flower color and pubescence color were also scored in two RIL populations for detecting 42 with the following modifications : an incubation time of 90 min, re-suspension of the DNA pellet in 500 μL 1 × TE, and no RNase A treatment. First, all DNA was quantified using an ND-1000 Spectrophotometer and diluted to 100 ng µL−1 for further study.Genomic DNA from each line and the parents of two RIL populations was extracted from young trifoliate leaves using the cetyltrimethylammonium bromide method14 in the Deapung × Danbaek RIL population. Genomic DNA from the lines and parents was hybridized into the Affymetrix GeneTitan array system and then scanned using GeneTitan Scanner  following the manufacturer’s protocol. SNP genotype analysis was conducted based on Axiom Genotyping Solution Data Analysis User Guide (http://www.affymetrix.com)43.For the SNP genotyping, Axiom 180K SoyaSNP assay  was used44 according to the following parameters: binning by segregation distort 0.01, grouping by a 3.0 logarithm of odds threshold, nnTwoOpt  for ordering algorithm45, and rippling by the sum of adjacent recombination. The kosambi mapping function was used for the genetic map construction46. The genotype information of the unmapped SNP marker is used to narrow down the mapping region. Redundant markers were removed for map construction.The genetic map was constructed using the QTL ICIMapping software ver. 4.147.To validate the result of genetic mapping from the Deapung × Danbaek RIL population, the probes for the TaqMan assay were designed based on the twelve SNPs originating from Axiom 180K SoyaSNP assay  Table . The ext21. To confirm the sequences variation, diverse soybean germplasm, totally 31 soybean germplasm, including 25 parents for the Korean soybean nested association mapping populations, were used 37. The previously identified nonsynonymous substitution was generated using the mutation function in Swiss PDB viewer 4.1.0 and energy minimization was conducted to obtain predicted protein structure in Danbaek. Both the predicted protein structures of Daepung and Danbaek were visualized and compared using USCF Chimera X software49.The protein structure of the candidate gene was obtained from the AlphaFold Protein Structure Database (https://www.ars-grin.gov). All the experiments carried out on plants in this study followed relevant institutional, national, and international guidelines and legislation.The soybean cultivars ‘Danbaek’, ‘Kwangan’ and ‘Daepung’ were provided by National Agrobiodiversity Center in Jeonju, Korea. The soybean cultivar ‘5002T’ was provided by USDA Germplasm Resources Information Network (GRIN) database (Supplementary Table S1.Supplementary Information.


Crohn's disease (CD) is a potentially debilitating condition that burdens Italian healthcare substantially. The symptomatic management relies on prompt therapy adjustment to reduce flares and follow-up diagnostic inputs to maximise remission. Capsule endoscopy (CE) has introduced advantages in CD diagnostics, allowing the direct inspection of the entire gastrointestinal mucosa. The diagnostic procedure is comparable in effort to standard ileocolonoscopy (IC) but requires no anaesthesia. Whether CE follow-up improves clinical outcomes remains to be defined.To provide a preliminary evaluation of CE in terms of clinical outcomes with respect to the standard of care ileocolonoscopy/MRE in Italy.This retrospective analysis utilises anonymised, monocentric data from the S. Orsola-Malpighi Hospital IBD database in Bologna, Italy, collected between 1999 and 2019. Out of 421 adult patient records, 100 were included in the analysis . The CE represented the intervention arm, whereas ileocolonoscopy/magnetic resonance enterography was the standard of care. The use of biologics, symptomatology course, and surgery were the outcomes.The two techniques performed similarly overall. In general, no significant difference emerged in the use of biologics. The use of biologics appears reduced in the CE group, only in L4 patients after the first follow-up year. Similarly, surgery was seemingly less frequent among L4 patients in the CE group. No difference was found between groups in flare occurrence and duration. CE patients might have experienced longer and earlier first remissions, but no long-term difference persisted.The CE group showed an apparent reduction in biologics and surgery, limiting to L4 diagnoses. More extensive, prospective, multicentre, randomised studies must corroborate these preliminary findings. Crohn's disease (CD) is a potentially debilitating, idiopathic chronic inflammatory disorder of the gastrointestinal tract (GI)  and a coFrom the patients' perspective, CD has a detrimental impact on quality of life, scoring between 0.52 and 0.76 on the EuroQol 5D scale for severe and milder forms, respectively , 9. LongThe diagnosis and continued surveillance of CD have relied on markers such as C-reactive protein (CRP) and faecal calprotectin , instrumental assessment by cross-sectional imaging, and direct visual mucosal inspection by ileocolonoscopy (IC), depending on the disease location and comorbidities. However, molecular markers and cross-sectional imaging have limited efficacy: The global magnetic resonance index of activity (MaRIA) and fCal values correlate moderately with endoscopic activity and do not correlate with each other . One in Direct imaging of the small bowel became possible with the introduction of capsule endoscopy (CE), currently included in the ECCO Guidelines recommendations , 23, 24.The management of CD articulates through early symptomatic treatment with corticosteroids to achieve rapid remission and long-term maintenance therapy to prevent flares and reduce the risk of complications , 36, 37.However, the practical clinical implications of CE diagnostics and follow-up in guiding the course of treatment and remission have yet to be comprehensively characterised, i.e., whether CE-based diagnostics and follow-up have any real, practical benefit compared to IC/MRE in CD management. From this perspective, this study aims to provide a preliminary, retrospective, and monocentric gauge of the impact of CE on clinical outcomes in Italy.Retrospective, single-centre data were extracted from the IBD registry of the Azienda Ospedaliero-Universitaria Policlinico S. Orsola-Malpighi Hospital, Bologna, Italy. Data included limited patients' demographics  in 6-month intervals over a five-year follow-up. An IC/MRE diagnosis qualified as the standard of care (SOC), whereas CE (Medtronic PillCam™ SB2/PillCam2) was the intervention group. Diagnoses in both arms were grouped per location according to the Vienna classification . No patiData were pseudonymised by the data controllers (CC and DG) before transmission to the data processor (Coreva Scientific) and handled according to the General Data Protection Regulation (GDPR). The Sant'Orsola-Malpighi Hospital Ethics Committee reviewed and approved the study (approbation number 173/2017/O/OssN).The analysis included CD patients aged 18 or older with at least five years of available follow-up data from entry in the database. Patients who entered the database before 1999, missing diagnosed CD location or initial CD activity index (CDAI), were excluded. Patients with a history or symptoms of suspected strictures were ineligible for inclusion in the CE group and assigned to SOC.none for CDAI < 150, mild for 150 ≤ CDAI < 220, moderate for 220 ≤ CDAI < 450 and severe for CDAI ≥ 450. Remission was defined as two consecutive asymptomatic intervals . A flare was defined as the recurrence of moderate or severe symptoms (CDAI ≥ 220) following at least an asymptomatic (CDAI < 150) or mildly symptomatic year (150 ≤ CDAI < 220). Gaps in the CDAI data were excluded as missing data. Prescription data did not include the specific medication consistently and, therefore, did thus not allow stratifying per type of biologics.The outcomes investigated were the incidence of surgery, evolution of symptomatology in terms of CDAI score, and biologics therapy . Symptomatology was classified as The preparation for CE included a clear liquid diet for 24 h plus 12 h fasting and receiving 2 L of polyethylene glycol (PEG) solution 2–8 h before capsule ingestion (PillCam SB2/PillCam2). An additional fluid bolus was given after 2 h from capsule ingestion to facilitate transit through the small bowel (SB). All images were reviewed using the RAPID 8 software . SB was divided into three main segments: proximal, medial, and distal. Mucosal inflammation was quantified using the Lewis Score (LS) , 43. UsiExaminations were performed with a single oral contrast preparation consisting of 1500 ml PEG solution ingested gradually for 45 min. MRE was carried out with an Intera 1.5 T MR system with a 5-element Synbody coil . Patients were examined supine. The protocol contained the sequences Cor T2 , and axial T1W , with discontinuous breath-hold before and after contrast. Gadodiamid 0.1 mmol/kg  was given intravenously, and hyoscinbutyl-bromide 20 mg  was administered to reduce peristalsis during the procedure. CTE was performed with a 64-slice CT system  using: CARE-Dose: on, 120 kV, up to 150 mA; rotation time, 0.5 s; pitch, 1.5; collimation, 0.6 mm; increment 2. Contrast-enhanced CT scanning in the portal phase was performed after intravenous 100 ml iomeprol  300 mg/ml using an automatic injector OptiVantage DH  at an injection rate of 4 ml/s. Patients with a body weight greater than 80 kg received 150 ml iomeprol at the same injection rate. All images were evaluated by using an Impax PACS workstation  with two Coronis monitors  . CST readers identified active CD by segmental mural hyperenhancement, mural stratification, increased density in perienteric fat, sinus tract, or fistula. Fibrofatty proliferation and luminal narrowing without hyperenhancement were considered to represent inactive CD. Small bowel distension was rated on a two-point scale: sufficient  or poor  for each examined segment. The image quality was rated as good , sufficient , or poor . A bowel wall of 4 mm or more measured perpendicular to the bowel surface was considered thickened. Small bowel stenosis was defined as a change in bowel calibre with dilatation of the proximal segment above 2.5 cm and/or a collapse of the distal segment. MRE was interpreted at the time of examination by two experienced gastrointestinal radiologists. All SOC patients received MRE at least once a year.a priori power calculation. Medians and interquartile ranges are provided for demographic data and outcomes, where applicable. Inference testing was performed using non-parametric tests . A group matching algorithm was run using the R “MatchIt” package v3.0.2. Matching was performed over sex, age, disease duration, disease location, initial CDAI score, surgery history, and gastrointestinal bleeding.Data were processed using R v4.0.2 without After applying exclusion criteria to 421 patient records, 325 were included in the study: 270 were diagnosed by IC/MRE (SOC group) and 55 by CE . After matching the groups, fifty patients per arm were ultimately included in the analysis. There was no statistically significant distinction between the groups' demographics after matching . SubsequThe use of biologics in the two groups is summarised in The reliance on surgery in the SOC and CE groups is summarised in Capsule endoscopy has entered the field of IBD diagnoses and offers a competitive alternative to traditional IC/MRE imaging. Beyond indicators of increased sensitivity, technical performance, and improved patient acceptance, all of which are not to be overlooked, we questioned whether CE diagnostics alone brings tangible benefits to clinical outcomes. A substantial equivalence emerged between the two diagnostic approaches in the continued surveillance of CD patients. In general, neither the treatment choice nor the prognosis appeared to depend on the diagnostic approach. Nonetheless, despite the restrictions imposed by the unicentric, retrospective nature of the study and the small population size, some tendencies worthy of future investigations emerged. A preliminary, qualitative consideration concerns the apparent dichotomy in the course of biologics use among L4 patients. Although biologics were initiated at approximately the same time in the two arms, the cumulative incidence of biologics progressed slower among L4 patients in the CE group from the first year of follow-up. A similar reduction in the overall incidence of surgery is observed among L4 patients in the CE group throughout follow-up. A hypothesis to explain the decreased use of biologics and surgery in the CE group, notably among L4 patients, could be a superior clinical efficacy of CE in continued vigilance, i.e., CE might allow for a prompter and more flexible therapy remodulation , in partAny definitive conclusions necessitate a larger sample. Nonetheless, this hypothesis is in line with our recent experience in the proactive use of CE in CD patient management, including active CD  and evidIn this retrospective, matched cohort analysis, we observed a reduced use of biologics and surgery in patients with L4 disease in the CE group. In addition, the CE group also showed greater incidence and duration of remission independent of disease location for patients symptomatic at baseline. More extensive, prospective, multicentre, randomised studies are required to corroborate these preliminary findings.


MTARC1 and GPAM, underscoring them as potential drug targets. We leveraged messenger RNA expression, splicing and predicted coding effects to identify 16 putative causal genes, of which many are implicated in lipid metabolism. We analyzed levels of 4,907 plasma proteins in 35,559 Icelanders and 1,459 proteins in 47,151 UK Biobank participants, identifying multiple proteins involved in disease pathogenesis. We show that proteomics can discriminate between NAFL and cirrhosis. The present study provides insights into the development of noninvasive evaluation of NAFL and new therapeutic options.Nonalcoholic fatty liver (NAFL) and its sequelae are growing health problems. We performed a genome-wide association study of NAFL, cirrhosis and hepatocellular carcinoma, and integrated the findings with expression and proteomic data. For NAFL, we utilized 9,491 clinical cases and proton density fat fraction extracted from 36,116 liver magnetic resonance images. We identified 18 sequence variants associated with NAFL and 4 with cirrhosis, and found rare, protective, predicted loss-of-function variants in  MTARC1 and GPAM. Plasma proteomic analyses provide insight into proteins involved in disease pathogenesis.Genome-wide analyses identify loci associated with nonalcoholic fatty liver disease, including rare, protective loss-of-function variants in  NAFL, the first stage of NAFLD, can progress to nonalcoholic steatohepatitis (NASH)5. Furthermore, some patients with NASH develop liver cirrhosis and hepatocellular carcinoma (HCC). Obesity, metabolic syndrome, diabetes and hypertension are recognized risk factors for NAFL and NASH7, and NASH-related cirrhosis is the second most common indication for liver transplantation in the United States of America9. Several sequence variants have been associated with liver enzymes12, cirrhosis13 and NAFLD16, including missense variants in PNPLA3 (ref. 17), TM6SF2 (ref. 18), GCKR (ref. 19) and MTARC1 (ref. 20). Furthermore, a protein-truncating variant in HSD17B13 has been associated with NASH and fibrosis but not with steatosis21.NAFL disease (NAFLD), when >5% of the liver is fat with no identifiable secondary cause, is estimated to affect 25% of the world’s population24. Magnetic resonance imaging (MRI)-derived proton density fat fraction (PDFF) provides accurate liver fat quantification, but liver biopsy is essential for NASH diagnosis and staging. However, biopsy involves sampling variability and a risk of complications25. Currently, no pharmacological therapy has been approved for NASH. The identification of potential drug targets and biomarkers to monitor disease progression and treatment response is therefore paramount.It is challenging to both diagnose and stage NAFLD. Although liver enzymes are commonly elevated, they are nonspecific and poor predictors of progressionWe performed genome-wide association studies (GWASs) of PDFF, NAFL, cirrhosis and HCC, and a combined GWAS of PDFF and NAFL. To further characterize the risk variants, we tested them for association with clinical traits, mRNA expression in various tissues and circulating protein levels in large datasets from Iceland and the UK Biobank (UKB). We further investigated whether plasma proteins discriminate between disease stages.r2 = 0.96) with 3,869 PDFF measurements calculated by others using LiverMultiScan26  using each individual’s first available MRI measurement. We also meta-analyzed GWASs on clinically diagnosed NAFL 27, code K76.0), including 785 cases from Iceland (deCODE genetics), 5,921 from the UK (UKB), 2,134 from the USA (Intermountain INSPIRE and HerediGene registries) and 651 from Finland (FinnGen), for a total of 9,491 NAFL cases and 876,210 controls. We combined the summary-level GWAS PDFF data and the NAFL ICD-10 code meta-analysis data to maximize power to detect associations using multitrait analysis28.We performed a GWAS on the PDFF estimates . The variants associated with the combined PDFF and NAFL at genome-wide significance (GWS) were nominally significant for both phenotypes (P < 0.05). Furthermore, their effects on the two phenotypes were comparable  = 1.47 for NAFL). This variant has been reported as being associated with NAFLD29. Two of the eighteen variants had greater effect on PDFF in men than in women, p.Ile43Val in GPAM  and p.Glu167Lys in TM6SF2 .We identified 18 independent sequence variants at 17 loci in the combined GWAS (Table APOH (encoding β2-glycoprotein 1) . APOH is highly expressed in the liver31 and p.Cys325Gly has been associated with liver enzymes16. A low-frequency (MAF = 1.32%) missense variant, p.Asn252Lys, in PNPLA2, the closest homolog of PNPLA3 (ref. 32), is also associated with NAFL . Homozygous mutations in PNPLA2 have been associated with neutral lipid storage disease and fatty liver is among its features33. PNPLA2 p.Asn252Lys has been associated with increased waist:hip ratio (WHR) and high-density lipoprotein-cholesterol levels35 but not with NAFL. Adjusting for the WHR does not affect the association . A common NAFL-associated variant, rs6955582[A], intronic in GUSB  correlates with a missense variant in GUSB, p.Leu649Pro . An intronic variant (MAF = 9.23%) in TOR1B, rs7029757[A]  is also associated with NAFL. The rs7029757[A] has been reported as associating with alanine aminotransferase (ALT) levels and cirrhosis but not NAFL13.The NAFL variants include a low-frequency  = 2.96%) missense variant, p.Cys325Gly, in ncases UK = 2,301, ncases Iceland = 691, ncases USA = 392, ncases Finland = 1,425, ncontrols = 967,898) and 861 HCC cases  using ICD-10 codes. Four variants were associated with cirrhosis (Table P > 0.19) are a splice-region variant in HSD17B13, rs72613567[TA], which has been reported as being associated with NASH21, and a missense variant p.Glu366Lys in SERPINA1, which causes α1-antitrypsin deficiency36.We also performed meta-GWASs on 4,809 all-cause cirrhosis  and p.Cys282Tyr in HFE (homeostatic iron regulator). These two variants are associated with cirrhosis through alcohol consumption (ADH1B)37 and hemochromatosis (HFE)38, rather than solely through hepatic fat. Similarly, the effects of the 18 variants on HCC were proportional to the PDFF effects  and adipose tissue (n = 770), and publicly available data in the Genotype-Tissue Expression project . Fourteen lead associations were with missense variants or variants in high linkage disequilibrium  with a missense variant: in PNPLA3, APOE, GCKR, GPAM, PNPLA2, TMC4, MTARC1, APOH, ADH1B, HFE, ERLIN1, GUSB and two independent variants in TM6SF2. The missense variants in GUSB, TM6SF2, PNPLA3 and TMC4 are associated with expression levels of the corresponding gene (cis-eQTLs) in various tissues, and the missense variant in TMC4 is also associated with liver expression of MBOAT7. Loss of MBOAT7 has been associated with NAFLD41. These variants are top eQTLs, that is, they are either the strongest association at their loci with expression levels of these genes or highly correlated to it (r2 > 0.8). The variants in TOR1B, HSD17B13 and GUSB associate with splicing in whole blood as top sQTLs (Supplementary Table TOR1B (torsin 1b), is associated with cryptic splicing ) or splicing QTLs (sQTLs). For this analysis, we used annotation of 46.5 million sequence variants tested in the GWAS and measured mRNA levels using inhouse RNA-sequencing (RNA-seq) data from whole blood , heart failure  and higher levels of lipoprotein (a) (Lp(a); P = 2.2 × 10−56, effect = 0.10 s.d.) . We compared variant associations with alcoholic liver disease (ALD) diagnoses and NAFL diagnoses  than of NAFL .We tested the 20 NAFL and cirrhosis variants for association with 51 phenotypes related to liver function or NAFLD, using data from Iceland, the UK, Denmark (CHB-CVDC/DBDS) and available meta-analyses  and p.Thr189GlyfsTer5 in GPAM (MAF = 0.11%)  , suggesting that p.Thr189GlyfsTer5 decreases NAFL risk through decreased GPAM function. Although neither of these pLOFs is associated with NAFL or cirrhosis, they give information on whether previously reported associations are gain or loss of function. MTARC1’s p.Arg305Ter is located in the gene’s last exon, and we found no evidence of nonsense-mediated decay in our RNA data , which is associated with increased triglyceride levels , similar to the NAFL risk-increasing allele of the GCKR common missense variant p.Leu446Pro.Among the 20 variants with GWS associations with NAFL and cirrhosis, 14 are common or low-frequency missense variants MAF < 3%). Although these variants implicate probable relevant genes, it is not clear whether loss or gain of function of the encoded proteins reduces or increases disease risk. To investigate this, we looked for associations with rare predicted loss-of-function (pLOF) variants in candidate causal genes at these loci, using data from Iceland and UK. We tested 47 pLOF variants for associations with the same traits that we detected with the lead variant at the locus and found two rare pLOF variant associations in Iceland: p.Arg305Ter in %. AlthouHSD17B13 and p.Ile148Met in PNPLA3 interact in their effects on liver enzymes21. We replicated the interaction between rs72613567[TA] in HSD17B13 and p.Ile148Met in PNPLA3 (P = 9.0 × 10−15 and 2.9 × 10−12 on aspartate transaminase (AST) and ALT, respectively) .rs72613567[TA] in r2 = 0.32), with only 2% of individuals having high PDFF (>5%) and low BMI (<25 kg m−2) compared with 19% with high PDFF and high BMI (≥25 kg m−2) . Missense variants in PNPLA3, TM6SF2, APOE and GUSB interacted with BMI to affect PDFF  were correlated (r2 = 0.90), as were BMI measurements (r2 = 0.60) , with direction of change in the two measures that on average were the same .Among participants in the UKB, 2,795 had 2 liver MR images taken 2–3 years apart. Consecutive PDFF measurement were correlated  with NAFL  and 948 with cirrhosis  in either Iceland or UKB.To gain insight into proteins affecting the pathogenesis of NAFL or cirrhosis and search for relevant biomarkers, we analyzed protein levels measured with 4,907 aptamers by SomaScantrans, using either Iceland (SomaScan) or UKB (Olink), as top protein QTLs (pQTLs) , of which 26 were associated with both SomaScan and Olink measurements. Levels of ten proteins were associated with five or more variants   showing the most significant association in Iceland (SomaScan)  and keratin 18 (KRT18) in UKB (Olink) . The rs72613567[TA] in HSD17B13 was associated most significantly with levels of the DnaJ B member homolog subfamily 9 protein (DNAJB9) in Iceland (SomaScan) and carboxylesterase 3 (CES3) in UKB (Olink). Missense variants in MTARC1 (p.Thr165Ala) and GPAM  were both associated with levels of group XIIB secretory phospholipase A2-like protein (PLA2GXIIB) as measured in Iceland (SomaScan), and inactive Cα-formylglycine-generating enzyme (SUMF2), hypoxia-upregulated protein 1 (HYOU1) and acid sphingomyelinase-like phosphodiesterase 3a (SMPDL3A), as measured in the UKB (Olink). PLA2GXIIB is highly expressed in the liver and the NAFL protective allele of these variants was associated with lower protein levels . Moreover, the rare pLOF variants in Iceland in GPAM and MTARC1 were both associated with lower PLA2GXIIB protein levels . We note that other NAFL variants were associated nominally with PLA2GXIIB, some with an opposite direction of effects to the LOF variants in GPAM and MTARC1  were proportional to their effect on PDFF  and, second, for insulin-like growth factor-binding protein (IGFBP) 2 . IGFBP2 levels were elevated in individuals with cirrhosis but reduced in NAFL compared with population controls. Using UKB (Olink), the most significant difference between NAFL and cirrhosis was, first, for thrombospondin 2  and, second, for IGFBP7 . Levels of THBS2, IGFBP7 and IGFPB2 are measured with both SomaScan and Olink and are associated in both datasets. For NAFL compared with the population, aminoacylase-1 (ACY1) showed the strongest association in both Iceland and UKB . In both Iceland (SomaScan) and UKB (Olink), the models trained with the plasma proteome outperformed other models in discriminating between NAFL and cirrhosis, NAFL and the population, and cirrhosis and the population Fig. –17.Fig. 52.We identified 18 sequence variants that are associated with NAFL and two additional variants that are associated with cirrhosis and not PDFF. The NAFL variants affect the risk of cirrhosis and HCC proportional to their effects on PDFF, supporting a causal relationship between hepatic fat accumulation and these diseasesPNPLA3 interacts with rs72613567[TA] in HSD17B13 and BMI but not with age. Longitudinal PDFF measures suggest that p.Ile148Met carriers are more susceptible to change in BMI than noncarriers, in line with previous studies suggesting that the p.Ile148Met genotype increases sensitivity to the beneficial effects of dietary interventions and bariatric surgery56.The NAFLD variants are associated with other phenotypes, with variable patterns of association. One interpretation of this is that perturbations of more than one biochemical pathway lead to NAFLD. The strongest NAFL-associated variant, p.Ile148Met, in 58. For example, variants at TM6SF2 exert their effects on hepatic lipid concentration by reducing TM6SF2 function, causing reduced secretion of triglyceride-rich lipoproteins59. Several variants affect NAFL and blood lipids in the same direction, including p.Leu446Pro in GCKR, which also decreases glycated hemoglobin and type 2 diabetes risk. This is consistent with evidence that the variant increases hepatic glucose metabolism and de novo lipogenesis60. The p.His48Arg in ADH1B is associated with less risk of having an NAFL diagnosis. The variant associates with less alcohol consumption and reduces the risk of ALD substantially more than the risk of NAFL. The amount of consumed alcohol  used to distinguish between NAFL and ALD is quite arbitrary61. Therefore, it is likely that the association of the variant with NAFL diagnosis is driven by its effect on alcohol consumption below the ALD diagnosis threshold. None of the other NAFL variants was associated with a significantly greater effect on ALD than NAFL.Sixteen NAFL-associated variants were coding, five were eQTLs and three sQTLs, and many implicated genes were involved in lipid metabolism, reinforcing the notion of its importance as a primary process in NAFL pathogenesisGPAM and MTARC1, which both encode mitochondrial enzymes and are highly expressed in liver and adipose tissue31. GPAM encodes glycerol-3-phosphate acyltransferase 1, which catalyzes the first step of triglyceride synthesis. Gpam knockout mice have lower hepatic triglyceride content and overexpression has the opposite effect62. The results for MTARC1 are in line with previous reports of the effects of missense and LOF variants in MTARC1, but the mechanism explaining the associations was less clear20. The similarity in their associations with other traits indicates that mutual or similar pathways explain the MTARC1 and GPAM associations. Furthermore, both missense variants affect plasma levels of PLA2GXIIB protein. We identified rare pLOF variants in both genes affecting the same traits and reducing liver enzymes and total cholesterol levels. This suggests that GPAM and MTARC1 have an etiological role and inhibition of GPAM and MTARC1 could be therapeutic for NAFL or NASH, with a favorable effect on the metabolic profile. The lack of associations with increased risk of a large set of diseases is reassuring with regard to treatment safety.The NAFL associations include missense variants in APOH and GUSB. APOH is highly and almost exclusively expressed in the liver31. The other trait associations of p.Cys325Gly in APOH strongly suggest a role in lipid metabolism64. Furthermore, the variant has been reported in GWASs of coronary artery disease65 and Lp(a) levels64. We replicated the Lp(a) association and observed associations with an increased risk of atrial fibrillation and heart failure. Our results indicate a role for GUSB in the etiology of NAFL. The missense variant p.Leu649Pro in GUSB associates with both NAFL and RNA expression levels of GUSB. Furthermore, seven NAFL variants associate with GUSB plasma protein levels. GUSB encodes β-glucuronidase, a lysosomal enzyme involved in the breakdown of glycosaminoglycans66.Among associations with NAFL are missense variants in MTARC1 and GPAM are associated with levels of PLA2GXIIB in plasma. PLA2GXIIB is highly expressed in the liver and knockout mice have severe hepatosteatosis67. However, our proteomic analysis does not support an etiological role for PLA2GXIIB because NAFL variants affect plasma PLA2GXIIB levels with effect directions inconsistent with their NAFL effects. Evidence suggests that PLA2GXIIB is a mediator of hepatic lipoprotein secretion and its inhibition results in decreased levels of plasma lipids68. Thus, PLA2GXIIB may mediate variant effects on circulating lipids without directly affecting hepatic fat and thus could serve as a biomarker of drug target engagement.Plasma proteome analysis revealed that missense and pLOF variants in 2. We designed models including plasma proteins that outperformed a model trained on liver enzymes and GRSs in discriminating between NAFL and cirrhosis diagnoses. Thus, levels of plasma proteins have the potential to serve as a noninvasive tool for use in the diagnosis and monitoring of disease, whereas GRSs are associated with a lifetime risk of disease. THBS2 was elevated in individuals with cirrhosis compared with NAFL and the population, and ACY1 in individuals with NAFL compared with the general population. Intrahepatic THBS2 expression levels have previously been shown to correlate with fibrosis in patients with NAFLD69. The association of IGFBP2 and IGFBP7 with cirrhosis and NAFL is consistent with previous studies of NASH progression70. Both proteins bind insulin-like growth factors (IGFs) and modulate their availability72. IGFs are mainly produced in the liver, and IGFBP2 and IGFBP7 elevation may reflect imbalances in the IGF system due to liver damage73. However, an etiological role has been suggested for IGFBP7, which may contribute to hepatic fibrogenesis74 and act as a tumor suppressor in HCC75. Levels of SHBG are also associated with cirrhosis compared with NAFL, consistent with previous reports of a positive correlation with advanced fibrosis in NASH70. There are conflicting epidemiological studies about whether NAFLD is associated with increased or decreased levels of SHBG. In line with this, many NAFL variants are associated with SHBG plasma levels with inconsistent directions of effect compared with their effect on hepatic fat content76.Diagnosis and monitoring of complications in patients with NAFLD are challenging77.A limitation of the present study is the lack of data enabling a more detailed phenotype stratification, in particular histological data for disease staging. Furthermore, information on other causes of liver disease, such as alcohol consumption, is limited. Our approach is, however, in line with the recent opinion to not base the disease diagnosis on a criterion of exclusion of other diseases, such as ALDIn conclusion, we used multiomics data to shed light on the genetic basis of NAFLD. Analysis of pLOF variants, blood RNA expression and plasma proteomics data pointed to causative genes and whether changes in their functions contribute to the pathogenesis. We demonstrated the diverse effects of NAFL risk alleles on other diseases and traits, including blood lipids and proteins, and showed that plasma proteomics has the potential to stage NAFLD.78. The present study has collected extensive phenotypic and genotypic information on participants, including ICD-10-coded diagnoses from inpatient and outpatient hospital episodes and abdominal MRI through its imaging study79. Genotype imputation data were available for 487,409 participants (May 2017 release), of which 408,658 were included because they self-reported as white80. The UKB resource was used under application no. 56270. All phenotype and genotype data were collected following an informed consent obtained from all participants. The North West Research Ethics Committee reviewed and approved UKB’s scientific protocol and operational procedures (REC reference no.: 06/MRE08/65).The UKB project is a large prospective cohort study of ~500,000 individuals from across the United Kingdom, aged between 40 and 69 years at recruitment81 and their untyped close relatives based on genealogy. Sequencing was done using GAIIx, HiSeq, HiSeqX and NovaSeq Illumina technology. SNPs and insertions/deletions (indels) were identified, and their genotypes were called using joint calling with Graphtyper84. All participants who donated blood signed an informed consent. The personal identities of the participants and biological samples were encrypted by a third-party system. The study was approved by the Icelandic Data Protection Authority and the National Bioethics Committee of Iceland (no. VSN-20-182).The Icelandic deCODE genetics study is based on WGS data from 49,708 Icelanders participating in various research projects at deCODE genetics. Variants identified through WGS were imputed into 155,250 chip-genotyped Icelanders using long-range phasinghttps://www.finngen.fi/en/access_results) and methods have been documented (https://finngen.gitbook.io/documentation). The FinnGen database consists of samples collected from the Finnish biobanks and phenotype data collected at the national health registers. The Coordinating Ethics Committee of the Helsinki and Uusimaa Hospital District evaluated and approved the FinnGen research project. The project complies with existing legislation . The official data controller of the present study is the University of Helsinki.FinnGen summary statistics, including fatty liver disease and cirrhosis, were imported in December 2020 from a source available to researchers  and the Danish Data Protection Agency (P-2019-93). For binary phenotypes, the control group included blood donors from the Danish Blood Donor Study (DBDS) , approved by the Danish Data Protection Agency (P-2019-99) and the Scientific Ethical Committee system (NVC 1700407)44. Chip typing and genotype imputation of CHB-CVDC and DBDS were performed at deCODE genetics using a north European sequencing panel of 15,576 individuals .The Copenhagen Hospital Biobank Cardiovascular Study (CHB-CVDC) was used to acquire secondary cardiovascular phenotypes. CHB-CVDC involves a targeted selection of patients with cardiovascular disease from the CHB, a biobank based on patient blood samples drawn in Danish hospitalsn = 8,288) and genotyped using Illumina GSA chips . Samples were filtered on a 98% variant yield. Over 245 million high-quality sequence variants and indels, to a mean depth of at least 20×, were identified using Graphtyper82. Quality-controlled chip genotype data were phased using Shapeit 4 (ref. 85). A phased haplotype reference panel was prepared from the sequence variants using the long-range phased chip genotype data. The haplotype reference panel variants were then imputed into the chip-genotyped samples using inhouse tools and methods described previously84. In the US association analysis, samples assigned <93% CEU ancestry (northern European from Utah) were excluded. We adjusted for sex, age and the first 20 principal components. Phenotypic data were based on ICD-10 code diagnoses of individuals. The Intermountain Healthcare Institutional Review Board approved the study and all participants provided written informed consent before enrollment.The samples from the USA (Intermountain dataset) were whole-genome studied using NovaSeq Illumina technology  = 27 ms, time to echo (TE) =  ms, bandwidth = 710 Hz, flip angle (FA) = 20%, voxel size = 2.5 × 2.5 × 6.9 mm3 and a 160 × 160 acquisition matrix. The IDEAL sequence used TR = 14 ms, TE =  ms, bandwidth = 1,565 Hz, FA = 5%, voxel size = 1.719 × 1.719 × 10.0 mm3 and a 256 × 232 acquisition matrix.The MR images used for calculating PDFF for 36,116 individuals were collected as a part of the UKB abdominal protocol, which, in turn, is part of the UKB imaging enhancementn = 8,448) or IDEAL . We implemented the PDFF estimation methods using a tailored Python code. For the GRE acquisition, we used a three-point Dixon method87 to compute a PDFF map using the second, fourth and sixth echoes. Eight 25-voxel rectangular regions of interest (ROIs) were defined within the liver and we computed the mean and s.d. of the PDFF maps over those ROIs. The reported PDFF was the ROI with the lowest s.d. By choosing the lowest s.d., we avoided ROIs with water–fat swaps. For the IDEAL acquisition, we assumed the following signal model88 for each voxel:ρw and ρf are the water and fat components, respectively, Δf is the chemical shift of fat with respect to water, φ quantifies B0 field inhomogeneity, ti the ith echo time. The parameters of interest, ρw, ρf and 89:We used two different approaches for calculating the PDFF from the liver MR images depending on whether the acquisition was GRE  from electronic health records. To analyze NAFL complications, we defined an all-cause cirrhosis phenotype based on cirrhosis- and fibrosis-related ICD-10 codes  and used ICD-10 code C22.0 for HCC. Analyzing this cirrhosis/fibrosis phenotype has previously been shown to increase power to detect associations compared with subtypes of nonalcoholic and alcoholic cirrhosisNAFL-associated variants were tested for association with other phenotypes from deCODE genetics, UKB, FinnGen, CHB-CVDC/DBDS or other publicly available data sources, which contain extensive medical information on various traits. A total of 51 phenotypes was chosen for the analysis , 3.5 × 10−8 for variants with moderate impact , 3.2 × 10−9 for low-impact variants , 1.6 × 10−9 for other variants in DNase I hypersensitivity sites and 5.3 × 10−10 for all other variants.We used logistic regression to test for association between sequence variants and binary phenotypes assuming an additive genetic model. For deCODE genetics, the model included the following covariates: sex, county of birth, current age or age at death (first- and second-order terms included), blood sample availability for the individual and an indicator function for the overlap of the lifetime of the individual with the time span of the phenotype collection. In CHB-CVDC/DBDS, the covariates were sex, age and 20 principal components to adjust for population stratification and blood sample availability. In the UKB study, 40 principal components were used to adjust for population stratification, and age and sex were included as covariates in the logistic regression model. When analyzing PDFF, BMI was included as a covariate in the analysis to increase power of associations. We used a linear mixed model implemented in BOLT-LMM to test for association between sequence variants and quantitative traits. The measurements used were adjusted for sex, year of birth and age at measurement (when available), and these were subsequently standardized to have a normal distribution. For the meta-analysis of summary-level statistics from different populations, we used a fixed-effects inverse variance method based on effect estimates and s.e.m. With the aim of studying NAFL, we combined summary-level data from the GWAS of PDFF and the meta-analysis of GWAS using ICD-10-code-based NAFL with multitrait analysis of genome-wide association summary statistics (MTAG)β0, β1 and β2 of the secondary variant in each group separately, where the quantitative trait was standardized to a normal distribution. We estimated the interaction between the primary and secondary variants by considering the following model:b and γ are the unknown parameters to be estimated. To assess the significance of the interaction parameter γ, we applied a likelihood ratio test, comparing our model with the null model: β0 = β1 = β2 .To test whether a specific primary variant is affecting the mean effect of another secondary variant in a given quantitative trait , we split the population into three groups based on the genotype of the primary variant, denoted by n = 17,846) and subcutaneous adipose tissue (n = 750). We isolated RNA using Chemagic Total RNA Kit special (PerkinElmer) in whole blood and RNAzol RT in adipose tissue, according to the manufacturer’s protocol . The concentration and quality of the RNA were determined using an Agilent 2100 Bioanalyzer (Agilent Technologies). RNA was prepared and sequenced on the Illumina HiSeq 2500 and Illumina Novaseq systems according to the manufacturer’s recommendation.RNA-seq analysis was performed on whole blood  was tested using linear regression assuming an additive genetic model and quantile-normalized percentage-spliced-in values (PSI) of each splice junction, adjusting for measurements of sequencing artefacts, demography variables, and 15 leave-one-chromosome-out principal components of the quantile-normalized PSI matrix. All variants with MAF > 0.2% within 30 kb of each LeafCutter cluster were tested, and the strongest association for each splice junction with P < 1 × 10−8 was called a top cis-sQTL.Quantification of alternative RNA splicing in whole blood was done using LeafCutter50. All plasma samples were measured with the SomaScan v.4 assay  containing 4,907 aptamers, providing a measurement of relative binding of the plasma sample to each of the aptamers in relative fluorescence units (r.f.u.). When testing for association between protein levels and disease, logistic regression was used with age and sex as covariates. The date of diagnosis was not available and the analysis was therefore not adjusted for the time from diagnosis. A pQTL association is considered to be in cis if the associated variant is located no more than 1 Mb from the transcription start site of the gene that encodes the measured protein, and in trans otherwise. A pQTL was considered to be significant if identified in the previous study (P < 1.8 × 10−9). A top pQTL is the top (most significantly associated) variant per megabase bin.Plasma samples were collected from Icelanders through two main projects: the Icelandic Cancer Project  and various genetic programs at deCODE genetics, Reykjavík, Iceland (48%). The samples collected at deCODE genetics were mainly collected through the population-based deCODE Health study. The average participant age was 55 years (s.d. = 17 years) and 57% were women. In the case of repeated samples for an individual, we selected one of them at random. This left measurements for 39,155 individuals. Of these, 35,559 Icelanders were used in the protein GWASs, because they also had genotype informationhttps://www.olink.com/products-services/explore) at Olink’s facilities in Uppsala, Sweden. Measurements with the Olink Explore platform use the NPX values recommended by the manufacturer, which include normalization. When testing for associations with sequence variants, the protein levels were standardized to a normal distribution.For a subset of 54,265 individuals in the UKB study , the levels of 1,472 proteins were measured with the Olink Explore 1536 platform as a part of the UKB–Pharma Proteomics Project (UKB application no. 65851). A large majority of the samples were randomly selected across the UKB. The UKB plasma samples were measured using the Olink Explore 1536 platform  and PANTHER protein classes. The analysis was performed with the PANTHER v.16.0 tool98 using Fisher’s exact test and false discovery rate (FDR) correction. The associated pQTLs were tested against a reference list that included all measured SomaAmers . Pathway analysis was performed for all variants associating with more than two proteins.The 99 was used to select relevant features. The selected proteins were preprocessed with Yeo–Johnson power transforms100 and then scaled to zero mean and unit variance (estimated from the training set) before being fed to the logistic regression model. The elastic net λ and α parameters were tuned using grid search to minimize tenfold crossvalidated average precision. Model performance was evaluated by considering the mean and s.e.m. of the receiver operating characteristic (ROC) area under the curve (AUC) of 1,000 repeated, tenfold-stratified crossvalidation runs.To examine how effective circulating protein levels (SomaScan and Olink panels) are at discriminating liver disease stages, we trained a logistic regression model with an elastic net penalty on protein levels to classify between individuals diagnosed with cirrhosis and NAFL. To test how this model compares with one trained on liver enzymes, an additional logistic regression model was exclusively trained on age, sex, BMI, ALT, AST and γ-glutamyl transferase. Genetic risk scores for NAFL and cirrhosis from the identified genetic variants were constructed to compare with the protein-based prediction. We created NAFL and cirrhosis GRSs by calculating the sum-of-effect alleles of the NAFLD variants weighted by their cirrhosis and NAFL GWAS effects. In addition, logistic regression models combining the protein scores, liver enzymes and GRSs were trained. In that case, the protein scores were calculated using out-of-fold predictions (with tenfold). The SomaScan analysis was performed on 181 individuals with NAFL and 73 with cirrhosis, and the Olink analysis was performed on 610 individuals with NAFL and 262 with cirrhosis. Models trained to discriminate between the presence of disease diagnosis and population were trained, respectively, on an additional set of 20,619 individuals (SomaScan) and 38,018 individuals (Olink) without cirrhosis and NAFL. The SomaScan measurements were log(transformed) to reduce the effect of outliers. However, we found that this was not necessary for the Olink data. BorutaFurther information on research design is available in the Any methods, additional references, Nature Research reporting summaries, source data, supplementary information, acknowledgements, peer review information; details of author contributions and competing interests; and statements of data and code availability are available at 10.1038/s41588-022-01199-5.Supplementary InformationSupplementary Figs. 1–15 and Supplementary Note.Reporting SummaryPeer Review FileSupplementary Table 1Supplementary Tables 1–17.


BRCA2 pathogenic variant carriers are associated with prostate cancer risk. Ashkenazi Jewish people are at higher risk of breast cancer due to the high prevalence of specific founder germline BRCA1/2 variants. The distribution of these variants (BRCA1 vs. BRCA2) in Ashkenazi men with prostate cancer is not clear. This systematic review and meta-analysis indicates that germline BRCA1 variants are higher in the Ashkenazi Jewish ethnicity in comparison to non-Ashkenazi men. Instead, BRCA2 variants present a similar distribution between the two considered groups. Germline BRCA2 in men with prostate cancer, due to the presence of a strong association with this gene. Some ethnicities present disparities in genetic distribution for the relation with specific founder variants. Ashkenazi Jewish people are, importantly, at high risk of breast cancer for their inherited cluster with germline BRCA1/2 variants. However, in Ashkenazi men with prostate cancer, the prevalence of BRCA1 and/or BRCA2 is not well defined. We assessed the frequency of these variants in Ashkenazi vs. non-Ashkenazi men with prostate cancer. Materials and Methods: In accord with the Preferred Reporting Items for Systematic reviews and Meta-Analyses (PRISMA) statement, we revised all germline BRCA variants reported in MEDLINE from 1996 to 2021 in Ashkenazi and non-Ashkenazi men with prostate cancer. Results: Thirty-five original studies were selected for the analysis. Among populations from Israel and North America, Ashkenazi Jewish men presented higher prevalence of BRCA1 variants  and a lower prevalence of BRCA2 variants  in comparison to the non-Ashkenazi population. Conclusions: Since germline BRCA1 variants are more prevalent and BRCA2 variants are less prevalent in PCa patients of Ashkenazi Jewish ethnicity in comparison to non-Ashkenazi patients, prostate cancer genetic screening in Ashkenazi men should not be restricted to the BRCA2 gene.Background and aims: International guidelines recommend testing  Prostate cancer (PCa) is the second most commonly diagnosed cancer in men, accounting for 15% of all male cancers ,3.Many factors have been associated with the risk of PCa or as being important for disease progression . ObesityBRCA2 mutations and PCa risk. However, there are no standardized measures for risk containing in asymptomatic high-risk individuals  AND 1996/01:2021/12[EDAT]”. We also reviewed references and citations of key articles and previous meta-analyses on the association between BRCA1/2 variants and PCa using Scopus. The analysis was limited to studies involving subjects affected by PCa screened for germline BRCA variants  statement . We revivariants . This stBRCA1/2 and PCa were considered. Case series, editorials, case reports, and congress abstracts were excluded. Titles and abstracts were screened by two authors to determine whether studies met the eligibility criteria for full-text assessment, and a sample of 25% was screened independently by another author as a quality assurance process. The full-text assessment was conducted by two authors with consultation from a third author if required. Full-text publications in English were considered. Case series, editorials, case reports, and congress abstracts were excluded from the evidence synthesis.Original studies that contained information on both germline BRCA1 and BRCA2 variants, and number of tested PCa.Data extraction was performed by one author (PM), with another independent extraction by one of two other authors (GC and AC). Any disagreements were resolved by discussion and consensus. The following data were extracted using predefined cells: first author, publication date, country, screening interval, ethnicity, number of identified BRCA1/2 variants in PCa from the published reports. We calculated 95% confidence intervals (CIs) using the Clopper–Pearson Exact binomial method. For the meta-analysis, we used random-effects models, with maximum likelihood estimates and the Freeman–Tukey double arcsine transform method to stabilize variances. Heterogeneity between studies was assessed using the Cochran Q test. The percentage of total variation across studies due to heterogeneity was evaluated by the I2 measure. We produced forest plots including the study specifics and the overall prevalence estimates. Probability of publication bias was assessed using funnel plots with the Begg’s test. Statistical analyses were performed using the R package meta. All p-values were two-sided.For each study, we first estimated the prevalence of BRCA1 and 24,633 for BRCA2. Three studies comprised heterogeneous populations , and also from other countries . No significant difference was observed in the prevalence of BRCA1 variants among non-Ashkenazi patients from Israel and North America, and non-Ashkenazi patients from other regions (p = 0.50) (We identified 46  = 0.50) . BRCA2 variant status, Ashkenazi (Israel and North America) patients had a lower prevalence of variants, in comparison to the similar non-Ashkenazi population . Regarding BRCA1 and BRCA2 groups (p < 0.01), both (Studies from non-Ashkenazi populations had a significant heterogeneity in 1), both .BRCA1/2 variants reported in the respective original studies. When a variant’s nomenclature was available in the text, we revised the pathogenic role of these alterations according to the most recent ClinVar classification  vs. Non-Ashkenazi : −p = 0.006Ashkenazi  vs. Non-Ashkenazi (Other region): −p = 0.50Non-Ashkenazi  vs. Non-Ashkenazi (Other region): −Test for subgroup differences:−p = 0.08Ashkenazi  vs. Non-Ashkenazi : −p = 0.86Ashkenazi  vs. Non-Ashkenazi (Other region): −p = 0.08Non-Ashkenazi  vs. Non-Ashkenazi (Other region): Test for subgroup differences: p = 0.22 and p = 0.39, respectively, for BRCA1 and BRCA2 by Begg’s test)  .BRCA1/2 variants among PCa Ashkenazi and non-Ashkenazi patients. We identified a higher prevalence of germline BRCA1 variants in Ashkenazi Jewish patients in comparison to the non-Ashkenazi population, while no significant difference of the prevalence of germline BRCA2 variants was observed between the two populations. This study presents a different distribution of germline CDH1 germline pathogenic variants for gastric cancer risk, as well as in Ashkenazy Jewish and Hakka Chinese populations with BRCA variants for BC risk . In general, BRCA2 carriers are at a two to five times higher risk of PCa compared to men in the general population. In contrast, BRCA1 variants are at most associated with a moderate PCa risk [BRCA2 mutations are prevalent in non-Ashkenazi populations. Disparity in Pca incidence and mortality across different countries and geographical areas might, at least in part, be related to differences in race, ethnicity, and genetic causes . About 1.1–0.2%) ,52,53. Ahenotype . A studyhenotype  estimatePCa risk . In our BRCA2 genetic test starting at age 40 years in cases of a personal history of metastatic, node-positive, biochemically recurrent, Gleason ≥8, or Gleason 7 disease with a strong family history of BRCA-related cancers. Additional men with PCa were referred if their personal and/or family history was suspicious for hereditary cancer predisposition, such as Lynch Syndrome [BRCA1 is not routinely included in PCa genetic screening but only “considered”, due to the lower prevalence in men with PCa. Our results demonstrate a similar prevalence of BRCA1 and BRCA2 in PCa Ashkenazi Jewish patients. This suggests “recommending” both genes in their genetic testing to inform decision-making for active surveillance. The National Comprehensive Cancer Network (NCCN) recommends a Syndrome . HoweverBRCA1/2 variant frequency could be affected by research activity, publication bias or access to medical care, and some data could be not retrievable from MEDLINE. However, we assume that this potential bias is very low. Data collection was performed independently by two authors. The germline BRCA1 and BRCA2 due to the lack of information about healthy carriers. However, our first aim was to assess only the prevalence of BRCA variants between two different populations. We were not able to quantify the real PCa risk in BRCA1/2 “hotspot” points such as 185delAG, 5382insC, 6174delT, and not all the entire coding regions. We can consider this approach as a “safe procedure” because founder variants are classically identified at a higher (significant) frequency in these populations. Some studies reported in this systematic review performed genetic screening in Ashkenazi Jewish patients only considering the BRCA variants reported in these studies. Some of these studies did not specify if these variants are variants of unknown significance (VUS) or pathogenetic [BRCA variants. To provide additional information for further studies, when variants’ nomenclature was available in the original study, we have revised these alterations in accord with the most recent ClinVar classification.We collected all germline ogenetic ,40,41,46ogenetic ,34,38 inBRCA1/2 variants in men with PCa, with a prevalence of BRCA2 (1–3%) [BRCA1 and BRCA2 occur in PCa Ashkenazi with a similar prevalence; moreover, the prevalence of germline founder variants presents a different frequency in PCa in comparison to BC, in particular with 6176delT (BRCA2) and 185delAG (BRCA1) variants. This should be considered for active surveillance in Ashkenazi men. Ashkenazi Jewish ancestry was associated with 2 (1–3%) ,26,30. T


Subclinical anthracycline therapy related cardiac dysfunction (ATRCD) can be detected with speckle tracking echocardiographic image (STE), which is not widely available in Uganda. We aimed to investigate the role of the two conventional echocardiographic parameters [mitral annular plane systolic excursion (MAPSE) and mitral annular peak systolic tissue Doppler velocity (S’)] on diagnosing subclinical ATRCD.207 cancer patients who underwent anthracycline based chemotherapy were recruited at baseline and followed up until 6 months after ending anthracycline therapy. Comprehensive echocardiographic data were collected at each visit. Global longitudinal strain (GLS) by STE was used as the gold standard diagnostic test to define the case of subclinical ATRCD. Data of the 200 patients who had no evidence of clinical ATRCD were analyzed. One hundred and seventy-two (86.0%) were female, with a median age of 42 years and 47 (23.5%) patients were diagnosed with subclinical ATRCD at the end of anthracycline therapy by GLS criteria. The area under the curve (AUC), cutoff point, sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of reduction of MAPSE (ΔMAPSE) were 0.6736 , ≥ 2 mm, 74.5% , 54.9% , 33.7% , and 87.5% . The AUC, cutoff point, sensitivity, specificity, PPV, and NPV of reduction of S’ (ΔS’) were 0.6018 , ≥ 0.5 cm/s, 61.7% , 52.7% , 29.0% , and 76.1% . When ΔMAPSE and ΔS’ are used as parallel test, the net sensitivity and specificity is 89.4% and 28.8%, respectively, the net PPV and NPV is 27.8% and 90.0%, respectively.The ΔMAPSE and ΔS’ showed fairly good accuracy, sensitivity and NPV to detect subclinical ATRCD in Ugandan cancer patients. These conventional echocardiographic parameters may serve as screening tools for detecting subclinical ATRCD in resource limited settings. Anthracyclines is a group highly effective antineoplastic agents used to treat many types of malignancies. Anthracycline therapy related cardiac dysfunction (ATRCD) is the most common and well-recognized chemotherapy induced cardiovascular side effect results from this chemotherapy . It is dLeft ventricular Global Longitudinal Strain (GLS) obtained by Speckle Tracking Echocardiography (STE) is currently the most accepted diagnostic tool for subclinical ATRCD . It is aWhen STE technology is not available or not feasible, the assessment of LV longitudinal function by simple conventional ultrasound parameters, namely mitral annular plane systolic excursion (MAPSE) by M-mode echocardiography, or mitral annular peak systolic velocity (S’) by pulsed wave tissue Doppler image (TDI), could be potentially useful tools to provide additional information to LVEF in the evaluation of LV systolic function –9. MAPSEWe performed a sub analysis of the SATRACD  study, which is a cancer cohort who underwent anthracycline based chemotherapy. The SATRACD study design, patient recruitment, and data acquisition have been descripted in details in a published methodology paper . In brieAmerican Society of Echocardiography and the European Association of Cardiovascular Imaging criteria  were useDiagnostic criteria for subclinical ATRCD:•LVEF ≥ 50% and a relative percentage decrease of GLS ≥ 15%, compared with baseline.Diagnostic criteria for clinical ATRCD:•Decrease in LVEF of > 10 percentage points, to a value < 50%, compared with baseline.®  transthoracic echocardiographic machine was used to acquire the cardiac images by two cardiologists with a 1.5–4.6 MHz transducer (M5Sc). All the images were stored and analyzed by a single observer. LVEF was assessed by 2D method (Simpson biplane), from apical 4- and 2-chamber views, calculated by “automatic EF” function. Further manual adjustment was done whenever necessary. MAPSE was measured by M-Mode at the apical 4-chamber view. Septal and lateral MAPSE were obtained and mean value was calculated . LV global and regional longitudinal systolic strain were measured through 3 different views . Manual adjustment was attempted, in the case of unsatisfied endocardial tracking. All the regional longitudinal strains were demonstrated on a Bull’s eye diagram, while GLS was calculated as the mean of the regional longitudinal strains.We randomly selected 10 patients in order to assess intra and inter-observer reliability. The intraclass correlation coefficients for intra and inter-observer reliability were 0.91 and 0.89, respectively for MAPSE, 0.91 and 0.90, respectively for S’, 0.91 and 0.93, respectively for LV GLS assessments.The study was approved by School of Medicine Ethics and Research Committee, College of Health Sciences Makerere University (REC REF 2018–081), Uganda National Council of Science and Technology (HS220ES) and Faculty of Health Sciences Human Research Ethics Committee, University of Cape Town (HREC 054/2020sa).A written copy of the consent was obtained from all participants. Any participants’ question regarding the research was answered prior to signing of the consent form.SD) or medium (interquartile range) and categorical variables as frequencies and percentages. Paired t-test and Wilcoxon signed rank test were used to compare dependent continuous variables, where appropriate. Pearson correlation was used to assess the correlation between GLS and MAPSE/S’. Receiver operating characteristic (ROC) analysis was used to assess the accuracy of the two conventional echocardiographic parameters on diagnosing subclinical ATRCD. GLS measured by STE was used as the gold standard diagnostic test to define the case. Empirical cutpoint estimation was done using nearest to  method. A two-sided p-value < 0.05 was considered statistically significant for all analyses.We analyzed the data using STATA v14 . All continuous variables were expressed as a mean ± standard deviation  age of 42 (20–69) years, with 172 (86.0%) were female. Patients’ baseline characteristics are showed in r = 0.376, p < 0.001 and r = 0.233, p < 0.001, respectively) . At the end of the anthracycline therapy, LVEF, GLS, MAPSE and S’ showed significant reduction compared to baseline measurements  . This courements . There wIn the present study we assessed the role of conventional echocardiographic parameters (MAPSE and S’) on detecting subclinical ATRCD. Our results showed fairly good accuracy of both tests.Subclinical ATRCD has been defined as a relatively reduction of GLS ≥ 15% compare to baseline . GLS meaLeft ventricular longitudinal function can be reduced despite of a normal LVEF, and this has been demonstrated using several echocardiographic parameters including GLS by STE –17, MAPSAs a novel echocardiographic modality, GLS has several advantages over MAPSE and S’ such as its relative independence from translation, tethering and the angle of incidence . Howeverr = 0.376) compared to that between GLS and S’(r = 0.233) was also observed by Peverill and Cheng  , 28 and Several studies demonstrated the usefulness of S’ in detecting subclinical LV systolic dysfunction in deferent clinical settings. Study published by Vinereanu et al. in 2003 and Raafat et al. in 2018 suggested measurement of S’ may be a more sensitive marker of subclinical changes in LV performance in diabetes than assessment of global function by LVEF , 31. HowPrevious studies have also demonstrated the role of MAPSE in various cardiovascular diseases. Reduced MAPSE can be used as a sensitive early marker of LV systolic dysfunction in hypertension , diabeteWe found ΔMAPSE ≥ 2 mm was able to detect subclinical ATRCD with AUC of 0.6736, sensitivity of 74.47% and specificity of 54.9%.It is to be noted our results showed both ΔMAPSE ≥ 2 mm and ΔS’ ≥ 0.5 cm/s had a higher NPV (87.5 and 76.1%) than PPV (33.7 and 29.0%), which underlining these parameters can serve as a screening test for subclinical ATRCD when GLS by STE is not available or feasible. The sensitivity and NPV are further improved when the two test are used as simultaneous  test.Our study is so far the largest anthracycline therapy cohort in the country to investigate the accuracy of conventional echocardiographic parameters to detect subclinical ATRCD. Nevertheless, it needed to note a major limitation of MAPSE/S’ is they are not able to show the LV regional longitudinal function, while GLS by STE can easily demonstrate this information by the bull’s eye diagram , 42. BesMAPSE and S’ have a significant correlation with absolute GLS. The ΔMAPSE and ΔS’ showed fairly good accuracy, sensitivity and NPV to detect subclinical ATRCD in Ugandan cancer patients. These simple conventional echocardiographic parameters may serve as screening tools for detecting subclinical ATRCD in resource limited settings where GLS by STI is not available or not feasible. Both test maybe measured simultaneously whenever possible in order to improve the sensitivity and NPV.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by the School of Medicine Ethics and Research Committee, College of Health Sciences Makerere University. The patients/participants provided their written informed consent to participate in this study.WZ, KS, FA, EO, and JK contributed to conception and design of the study. WZ and IS organized the database. WZ, EL, and JN performed the statistical analysis. WZ wrote the first draft of the manuscript. All authors contributed to manuscript revision, read, and approved the submitted version.