Interestingly, these pathways were inactivated in the late TPs after sublethal exposure in concordance with a delayed deleterious effect. Modulated transcription of a variety of collagen types, laminin, and peptidase genes underlay the modulated functions of these hematologically important pathways. Several other SDTGs related to platelet and leukocyte development and functions were identified. These results outlined genetic determinants that were crucial to clinically documented radiation-induced hematological and skin damage with potential countermeasure applications.The lack of an easy and fast radiation-exposure testing method with a dosimetric ability complicates triage and treatment in response to a nuclear detonation, radioactive material release, or clandestine exposure. The potential of transcriptomics in radiation diagnosis and prognosis were assessed here using wet skin (blood/skin) biopsies obtained at hour 2 and days 4, 7, 21, and 28 from a mouse radiation model. Analysis of significantly differentially transcribed genes (SDTG;  Exposure to ionizing radiation (IR) results in immediate, short-, and long-term injuries based on the intensity of the exposure ,2. The oRealizing these challenges, the Biomedical Advanced Research and Development Authority (BARDA) is supporting the development of four products for the measurement of an absorbed radiation dose. One or more of these products is anticipated to aid in improving the outcome of care ensuing a nuclear or radiation occurrence . The proAcute radiation syndrome (ARS) manifests as a compiled multi-tissue and multi-system injury with hematopoietic damage that is central to victim survival in the rescuable dose range (2–6 Gy) where correction of the hematocytopenia improves patients’ survivability. Therefore, early detection of radiation-induced damages to hematopoiesis and a better understanding of the underlying mechanisms postulate an important therapy intervention site for outcome mitigation. Genomic changes in blood samples irradiated in ex vivo ,29,30,31All animals in the study were handled according to facility standard operating procedures under the animal care and use program accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC) and Animal Welfare Assurance through the Public Health Service (PHS). All performed animal work was reviewed and approved by the MedStar Health Research Institute’s Institutional Animal Care and Use Committee (IACUC-2010-021).Male C57BL/6 mice were purchased from The Jackson Laboratory  at eight weeks old. The mice were acclimated for one week before initiating IR exposures and specimen collection. All animal work and sample collection were completed before animals reached 14 weeks of age. Animals were housed at a density of five animals per cage under standard housing conditions of food, temperature, water, and 12/12 h of light/dark cycles.Mice in groups of five were placed in a round container split similarly to a pie into five equally-sized triangular compartments where in each house there was a mouse with its head pointing to the center of the container, which was covered with the same vented lid. The container was placed under a linear accelerator  with its field size set at 32 cm × 32 cm to ensure coverage of the whole container. Based on the desired dose, monitor units (MU) were calculated and delivered half from the anterior and half from the posterior (standard AP/PA technique). The energy of the bean used to deliver the dose was 6 MV photons, which was run at a dose rate of 600 MU per minute. Machine output was calibrated following the TG51 protocol . The mic2 biopsy was collected. Biopsy sites were closed using prolene sutures . Animals exposed to 0, 1, 3, 6 Gy survived the full experiment time course and did not show signs of pain or distress after biopsy or during housing. Mice exposed to 20 Gy showed decreased activities by post-exposure day 6 and developed signs of distress, lethargy, and dehydration by day 7. Mice in this exposure group were euthanized on the same day when morbidity signs were observed per humane endpoint criteria defined in the IACUC-approved study protocol. Mice in the sham group were transported to the same radiation facility as the mice in the radiation groups, transferred to the radiation table, and returned to the housing facility with no radiation exposure. At designed time points, sham animals were anesthetized, shaved, and biopsied following the exact procedure of irradiated groups. At the end of the experiment time course, euthanasia was performed via exsanguination using cardiac puncture under anesthesia. Death was confirmed by lack of pedal and corneal reflexes and opening of the thoracic cavity to ensure lack of heartbeat.Animals returned to the housing facility and skin biopsies were collected from each animal at day 0 , day 4 (d4), day 7 (d7), day 21 (d21), and day 28 (d28) post-irradiation. Briefly, the animals’ dorsa were shaved using standard veterinary clippers and a 1 cmTotal RNA was isolated from liquid nitrogen flash-frozen biopsies after thorough grinding in a cold mortar and pestle. Each grounded biopsy was transferred into a 1.5 mL tube containing 1 mL TRIzol reagent  and RNA was isolated following the manufacturer’s protocol. Concentrations and quality of yielded RNA were assessed using NanoDrop 1000  and the Agilent 2200 Tapestation system . Isolated materials were aliquoted and stored at −80 °C until further use.25–200 nanograms of RNA was used following Agilent’s two-color array workflow utilizing the Two-Color Low Input Quick Amp Labeling Kit, Two-Color RNA Spike-In Kit, Gene Expression Hybridization Kit, and Gene Expression Wash Buffer Kit  following all manufacturer’s instructions. Briefly, samples and purchased reference RNA (Agilent Technologies) were reverse transcribed and labeled with Cy-5 and Cy-3 dyes respectively. All samples were then purified using the RNeasy Mini Kit  and quantified on Nanodrop. Labeled cDNAs were simultaneously hybridized for 17 h at 65 °C on Agilent 4 × 44 K Whole Mouse Genome Microarray Kit (GPL7202: Agilent-014868) then slides were washed .p < 0.05 were deemed significant. Further narrowing in the selection of significantly differentiated genes was performed on an excel sheet of all elements of the array by sorting based on the FC selection of elements where FC > abs 2 and p-value < 0.05 only. These lists were crossed with lists of annotated SDTG lists obtained after loading to ingenuity pathway analysis . Only genes that were common to both lists were included in all subsequent analyses. Top pathways were reported based on abs z scores from IPA. Lists of genes in the reported pathways were obtained from IPA with no further processing of additional cutoffs. Analyses were performed comparing results of significantly differentially regulated genes after exposure to different doses of X-rays over several time points.Arrays were immediately scanned using an Agilent G2505C Scanner . Images were processed using Agilent’s default Feature Extraction software v11.0.1.1 and analyzed using custom R scripts to obtain lists of probe sets differentially expressed. Minimum information about a microarray experiment (MIAME)-compliant intensity, quality, and normalized ratio data for this series of experiments have been deposited in the gene expression omnibus (GEO) database maintained by the National Center for Biotechnology Information (accession no. GSE185149). Uncentered Pearson clustering was done with tools developed by the Division of Computational Bioscience of the Center for Information Technology and the Cancer Genetics Branch of the National Human Genome Research Institute at the NIH. Fold change of gene expression was calculated by normalization of the transcriptomes at different doses of X-ray exposure over all time points to that of the sham-treated animals. Changes in gene expression at Benjamini–Hochberg FDR adjusted Five groups of mice each consisting of five animals were used in the study. Each mouse group received 1, 3, 6, or 20 Gy of whole-body X-ray radiation and the fifth group followed the exact steps of exposure protocol without radiation for use as a reference. Skin biopsies were collected from each mouse at h2, d4, d7, d21, and d28 post-exposure. Transcriptomes in each biopsy were examined using microarrays. Mice exposed to 20 Gy did not complete the study time course and were euthanized by d7 following the humane endpoint defined by the IACUC-approved study protocol underscoring the lethality of the 20 Gy IR dose . All anip-value ≤ 0.05 and FC ≥ 2) after lethal exposure was performed. The initial analysis included data from biopsies collected at h2, d4, and d7. Filtering the identified pathway using stringent statistical cutoffs  identified three pathways that played a critical role in hematopoiesis and hematological response among the top of functionally altered pathways. The glycoprotein 6 (GP-VI) signaling pathway, the dendritic cell maturation pathway, and the intrinsic prothrombin activation pathway were at the top of the list of affected pathways  after irradiation was described in a separate paper (in review) and showed a larger number of predominantly downregulated SDTGs after exposure to a lethal dose (20 Gy). This trend was reversed after exposure to sublethal doses  where the numbers of SDTGs were less and were primarily upregulated. To examine whether transcriptomics could identify genetic modulations underlying the clinically reported hematological alterations induced by radiation and pathway enrichment analysis of the SDTGs  after sublethal IR dose exposure showed that all three pathways that play important roles in hematological responses were among the top five identified pathways in support of transcriptomics application in detecting radiation-induced hematological injury . While tThe GP6 signaling and the prothrombin signaling pathways contain many common genes. Significant modulation in the transcription of several collagens, laminin β 1, and calmodulin-like 5 genes introduced the GP6 signaling pathway as a main modulated pathway by IR . SimilarTranscription modulations in many genes encoding interleukins and heat shock proteins among other genes  introducLILRB3), the heme binding protein 2 (HEBP2), the hemicentin 2 (HMCN2), the hemoglobin subunit β (HBB), and the hemoglobin subunit α 1 and 2 (HBA1/HBA2) genes were all downregulated sharply at all TPs after exposure to a lethal dose. Many of these genes were upregulated at different TPs after exposure to sublethal doses  after a lethal dose and in several later TPs after sublethal IR doses would translate to a disruption in the coagulation signaling and contribute to coagulopathies and other cutaneous symptoms reported in affected patients [The downregulation of serin protease kallikrein -related peptidases 3,5-8  and the erythroid differentiation regulator (Erdr1) were among the few upregulated genes in response to lethal IR exposure. These two genes encode proteins that modulate erythrocyte adhesion properties [HBA1 and 2), hemoglobin subunit β (HBB), hemopexin (HPX), heme binding protein 2 (HEBP2), and hemojuvelin BMP co-receptor (HJV) were downregulated at all TPs after lethal IR exposure and sporadically at TPs of sublethal exposures including early TPs. These genes encode subunits of hemoglobin and other accessory proteins that play important roles in blood coagulation, platelet aggregation, nitric oxide transport, oxygen transport, peroxide catabolism, hemopoiesis, glutathione metabolism, bicarbonate transport, the collapse of mitochondria membrane potential, calcium transport, positive regulation of transcription from RNA polymerase II promoter, and iron ion homeostasis. The differential transcription of these sets of genes and corresponding functional damages provide new insights into the clinically observed dysfunctions after irradiation [SLP1) and plasminogen (PLG), were upregulated after lethal IR dose and late TPs  after large sublethal doses. The PLG gene encodes an inactive form of a serine protease that cleaves many extracellular matrices and blood clot components [LILRB3) and the platelet-derived growth factor-like (PDGFL) were downregulated after the lethal IR dose and upregulated at d4 after 6 Gy exposure. The LILRB3 protein is thought to ameliorate inflammatory responses and limit autoreactivity by binding to MHC class I molecules on antigen-presenting cells and might confer a survival advantage when upregulated, and the PDGFL protein has tumor suppression properties. Correcting hematological damages after sublethal IR exposures are possible and are likely to improve survival rates, however, lethal IR doses resulting in immediate and long-term damages to different tissues, including the substantial bone marrow suppression that prevents recovery, is still a challenging task because they are associated with the long-lived free radicals and reactive oxygen species with an abundance of pro-inflammatory cytokines/chemokines [Several other genes that play important roles in the function and development of erythrocytes were identified. The erythroblast membrane-associated protein , keratinocyte chemoattractant (KC), and controlling hematological damages after sublethal doses to improve survival after exposure to sublethal range doses , targeti


Formerly, extensive procedures such as the Thompson procedure  were proposed to achieve cure for multifocal duodenopancreatic neuroendocrine tumors (dpNETs) in patients with multiple endocrine neoplasia type 1 (MEN1).The current study observed a severe complication (Clavien–Dindo grade ≥ 3) in nearly two-thirds of patients.To improve patient care, future perspectives include optimization of equivocal surgical indications to facilitate improved preoperative patient selection as well as a reduction of the burden of complications once major surgery is performed.First, more specific and mainly tumor-based risk factors are needed to improve patient selection and to abandon the “one size fits all” treatment principle; For example, recent clinical data imply that a shift from 2 to 3 cm as surgical cutoff might yield similar oncological outcomes for pNETs, and one might therefore consider a prolonged wait-and-scan policy in some patients.Second, centralization of care will likely improve patient outcomes in MEN1. Preoperative multidisciplinary and ideally multicenter team discussions will enable decision-making regarding the most complex patients with MEN1 within expert teams. When major surgery is considered, centralization of surgery within “high-volume” teams consisting of endocrine and hepatopancreatobiliary surgeons with vast experience in MEN1-related dpNETs should not be influenced by geography or travel distances and should potentially not be limited by national borders. Aims of these teams should be to reduce the incidence as well as the impact of complications. Whether patients with MEN1-related dpNETs have an increased risk of complications as compared with patients with sporadic dpNETs should be investigated in further studies to optimize referral patterns.


ALMS1 was prioritized for functional genomic and mechanistic analysis. Loss of function mutations of ALMS1 have been implicated in Alstrom syndrome (AS) [OMIM 203800], a rare recessive ciliopathy that has been associated with cardiomyopathy. The variant of interest results in a frameshift introducing a premature stop codon. RNA-seq of the proband’s dermal fibroblasts confirmed the impact of the novel ALMS1 variant on RNA-seq reads and revealed dysregulated cellular signaling and function, including the induction of epithelial mesenchymal transition (EMT) and activation of TGFβ signaling. ALMS1 loss enhanced cellular migration in patient fibroblasts as well as neonatal cardiac fibroblasts, while ALMS1-depleted cardiomyocytes exhibited enhanced proliferation activity. Herein, we present the unique pathological features of pEFE compared to DCM and utilize integrated genomic analysis to elucidate the molecular impact of a novel mutation in ALMS1 gene in an AS case. Our report provides insights into pEFE etiology and suggests, for the first time to our knowledge, ciliopathy as a potential underlying mechanism for this poorly understood and incurable form of neonatal cardiomyopathy.Among neonatal cardiomyopathies, primary endocardial fibroelastosis (pEFE) remains a mysterious disease of the endomyocardium that is poorly genetically characterized, affecting 1/5000 live births and accounting for 25% of the entire pediatric dilated cardiomyopathy (DCM) with a devastating course and grave prognosis. To investigate the potential genetic contribution to pEFE, we performed integrative genomic analysis, using whole exome sequencing (WES) and RNA-seq in a female infant with confirmed pathological diagnosis of pEFE. Within regions of homozygosity in the proband genome, WES analysis revealed novel parent-transmitted homozygous mutations affecting three genes with known roles in cilia assembly or function. Among them, a novel homozygous variant [c.1943delA] of uncertain significance in Primary endocardial fibroelastosis (pEFE) is a rare form of neonatal cardiomyopathy that occurs in 1/5000 live births with significant consequences but unknown etiology.Integrated genomics analysis (whole exome sequencing and RNA sequencing) elucidates novel genetic contribution to pEFE etiology.In this case, the cardiac manifestation in Alstrom syndrome is pEFE. To our knowledge, this report provides the first evidence linking ciliopathy to pEFE etiology.Infants with pEFE should be examined for syndromic features of Alstrom syndrome.Our findings lead to a better understanding of the molecular mechanisms of pEFE, paving the way to potential diagnostic and therapeutic applications.The online version contains supplementary material available at 10.1007/s00109-021-02112-z. Cardiomyopathy is a heterogenous group of disorders mainly characterized by abnormal ventricular hypertrophy or dilation leading to ventricular dysfunction , 2. InheNeonatal cardiomyopathies are multifactorial disorders that manifest early after birth as an isolated trait or as a part of a syndromic picture . SyndromPrimary endocardial fibroelastosis (pEFE) is a rare form of neonatal DCM that occurs in 1/5000 live births with significant consequences but unknown etiology –11. It iTAZ gene, which encodes a mitochondrial targeted acyltransferase known as tafazzin, have been implicated in an X-linked form of pEFE in association with features of BARTH syndrome [OMIM 300394] , an extremely rare, autosomal recessive, Mendelian disorder with a prevalence of less than 1 per million in the general population  and introduction of a premature stop codon in pulation –22. As apulation –20, or lpulation , 22. How disease . To our Please refer to All human studies were conducted in accordance with regulation of the University of California Los Angeles (UCLA) Institutional Review Board (IRB). Subjects provided written informed consent to participate in this study. Electronic medical record, family pedigree, and specimen collection were acquired through the UCLA Congenital Heart Defect (CHD) BioCore  followint test  and ANOVA with post hoc Kruskal–Wallis were used for comparing 2 groups and more than 2 groups, respectively; P value less than or equal to 0.05 was considered significant, unless specified otherwise. The correlation of gene expression for each mRNA/trait pair was calculated using Pearson’s correlation and Benjamini–Hochberg correction methods. A Benjamini-Hochberg-adjusted correlation P value less than or equal to 0.05 was considered significant.Quantified results are presented as mean ± SEM. Student’s All human studies were conducted in accordance with regulation of the University of California Los Angeles (UCLA) Institutional Review Board (IRB). Subjects provided written informed consent to participate in this study. Electronic medical record, family pedigree, and specimen collection were acquired through the UCLA Congenital Heart Defect (CHD) BioCore  followinThe pEFE proband is a female infant of healthy parents of Hispanic descent, who presented with profound congestive heart failure at 6 weeks of life. The prenatal course and delivery history were uneventful. Newborn screening, infectious work-up, and comprehensive metabolic panel revealed no abnormalities Fig. A, B. pEFPathological examination was performed by an expert anatomic pathologist. A gross image of the explanted pEFE heart is shown in Fig. TAZ , the 1000 Genomes database , or ExAC (http://exac.broadinstitute.org/). To investigate the genetic contribution to pEFE etiology, we performed WES on our pEFE proband and her parents combined with RNA-seq analysis of pEFE proband dermal fibroblasts  and is predicted to be deleterious. The novel homozygous single nucleotide deletion in DNAH8  results in a frame shift [p.Phe2385fs] that causes a premature termination of translation leading to a truncated protein. However, DNAH17 and DNAH8 mutations have been clinically implicated in spermatogenic failure [OMIM: 618643] [ 618643]  and haveALMS1 encodes the centrosome and basal body-associated protein 1, which functions in the formation and maintenance of cilia and in microtubule organization  is a single nucleotide deletion, which results in a frameshift [p.Val649fs] and introduction of a premature stop codon. Mutations in ALMS1 are known causal of AS [OMIM 203800]  –17, a ra 203800] –20, suggIntegrative Genomics Viewer (IGV) analysis confirmed the zygosity of the variants Fig. B.ALMS1 is the candidate causal gene, we set out first to confirm the impact of these variants on RNA expression. We isolated total RNAs from pEFE proband dermal fibroblasts derived from a punch skin biopsy of the pEFE proband and from hNDFs as a control, and performed paired-end RNA-seq as we previously described   . Likewisfoz/foz] . ConsistCilia play essential roles throughout cardiac development. During cardiogenesis, primary cilia are present on the majority of heart cell types acting as cellular sensors for signal transduction that control cardiac myocytes and fibroblast differentiation –39. DeleALMS1 variant in pEFE, the availability of dermal fibroblasts derived from the pEFE proband allowed us to examine the broader impact at the molecular and cellular levels by performing unbiased transcriptome analysis of the patient cells that harbor the ALMS1 [c.1943delA] mutation, using hNDFs as control. EFE is a specialized form of cardiac fibrosis that involves excessive fibroblast-mediated deposition of elastin (ELN) and collagen fibers, predominantly, in the subendocardium layer of the left ventricle [ALMS1-deficient fibroblasts from the pEFE proband exhibited significant upregulation of several ECM constituents compared to hNDF Supplementary file2 (PDF 162 KB)Supplementary file3 (PDF 226 KB)Below is the link to the electronic supplementary material.


Apolipoprotein (APOE) ε2 or ε4 (APOE2 or APOE4) dosages with cognitive change may differ across racial groups.Associations of APOE2 and APOE4 dosages were the two main exposures, while v1 and annual rate of change in cognitive performance (between v1 and v2) on 11 test scores were the main outcomes of interest (v1: 2004–2009 and v2: 2009–2013). Mixed-effects linear regression models were conducted adjusting for socio-demographic, lifestyle, and health-related potential confounders. Race (African American vs. White) and sex within racial groups were main effect modifiers.Longitudinal data on 1770 middle-aged White and African American adults was compiled from the Healthy Aging in Neighborhoods of Diversity across the Life Span (HANDLS 2004-2013) study. APOE4 allelic dosage was associated with faster decline on a test of verbal memory among Whites only , but not among African Americans. In contrast, among African American women, APOE4 dosage was linked to slower decline on a test of attention , while no association was detected among African American men. APOE2 and APOE4 dosages showed inconsistent results in other domains of cognition overall and across racial groups that did not survive correction for multiple testing.Upon adjustment for multiple testing and potential confounders, APOE4 dosage was associated with faster decline on a test of verbal memory among Whites only, while exhibiting a potential protective effect among African American women in the domain of attention. Further longitudinal studies are needed to replicate our race and sex-specific findings.In conclusion, The online version contains supplementary material available at 10.1186/s13195-021-00855-y. APOE4) and the risk for age-related cognitive decline is growing; APOE4 status  is among the most well-established genetic risk factors for late onset Alzheimer’s disease (AD) and for age-related cognitive decline ). Genotyping of these variants was achieved with the Taqman Assays (Applied Biosystems Assay-On-Demand part numbers C__3084793_20 and C__904973_10) on a 7900HT Sequence Detection System (Applied Biosystems). Detailed information on assay procedures are available elsewhere .Other important associations that were detected in race-stratified models Table  prior to1 or decline over time, after adjustment for multiple testing.Findings from the secondary analyses, stratified by sex, are shown in Table SAPOE2 dosage vs. change in BTA over time) was more pronounced in women . Specifically, among White women, APOE2 allelic dosage was directly associated with annual rate of change in BTA, reflecting a protective effect , an association not detected among White men. Among African Americans, several other associations between APOE2 or APOE4 dosages and cognitive performance over time differed between men and women. Most notably, APOE4 dosage was directly associated with annual rate of change in BTA, suggestive of improvement over time in the domain of attention, among African American women  and passing correction for multiple testing, although that relationship was not detected among men (p > 0.10). The contrast in BTA trajectories across APOE4 dosages based on Model 2 between African American women and men is displayed in Fig. Sex differences in associations of APOE2 or APOE4 allelic dosages with cognitive performance over time were noted within each racial group , though these associations did not survive correction for multiple testing. They were also markedly attenuated in Model 2, mainly due to adjustment for current smoking and drug use status and HEI-2010 total score. Our key findings were not altered when a sensitivity analysis was conducted with all eligible participants, including those that died within 12 months of follow-up .Finally, among African American men (unlike among women), both APOE2 and APOE4 dosages with cognitive performance over time, particularly longitudinal change in test scores spanning key domains of cognition in sample of middle-aged urban adults. We observed several key findings. Upon correction for multiple testing, socio-demographic, lifestyle, and health-related potential confounders, APOE4 allelic dosage was associated with faster decline on a test of verbal memory among Whites only , but not among African Americans. In contrast, among African American women, a higher APOE4 dosage (from 0 to 2) was linked to slower decline on a test of attention , while no such association was detected among African American men with respect to APOE4 dosage and change in BTA over time. APOE2 and APOE4 dosages showed inconsistent results in other domains of cognition overall and across racial groups that did not survive correction for multiple testing.Our present work is among few longitudinal studies to examine racial differences in the associations of APOE genotype and cognitive health has also reported heterogeneity in the magnitude of these associations across racial/ethnic groups and depending on the phenotype under study. For example, several studies [Prior work evaluating associations between  studies –4, 15 ha studies , the aut studies . Moreove studies . This fi studies . In the  studies , genotyp studies . Unlike APOE, and cognitive decline. Our main findings were not affected by introducing lifestyle or health-related factors into the models. Other secondary findings that did not survive correction for multiple testing were affected by inclusion of factors such as smoking and diet quality. Future studies should uncover how these lifestyle and health-related factors play a role in the association between APOE and various neuro-cognitive outcomes.In response to the consistent finding that the ε4 allele may be detrimental to cognitive health according to previous studies , investAPOE genotypes or allele dosages with various cognitive outcomes were mostly studied among individuals of European ancestry [APOE4(+) status  predicted dementia significantly , with nonsignificant sex differences, women had significantly stronger positive associations than men between APOE4(+) status and impairment or decline on the California Verbal Learning Test  and on Verbal Fluency Test-Categories [APOE4 carriers have faster rates of memory decline than their male counterparts among MCI individuals [APOE4 dosage’s association with cognitive decline  being stronger among White women vs. White men, and sex differences as such were not detected in the total sample. Such lack of heterogeneity by sex was found in at least one other comparable prospective cohort study [Sex differences in the association between ancestry , 6. In ort study .APOE genotype has long been associated with cardiovascular disease, dementia, and Alzheimer’s disease [APOE genotype and neurobiological markers of aging at various stages of the life course [APOE vary with respect to their affinity for binding to serum cholesterol which, in turn, influences the extent to which individuals can metabolize dietary fat in the blood [APOE3 homozygotes [APOE genotype was linked to higher levels of serum triglycerides which may exacerbate cerebrovascular disease, when compared with ε3/ε3 [APOE2 allelic dosage and decline on global mental status. However, an in-depth analysis of the association between APOE genotypes with serum lipid trajectories over time may be needed, which in turn can be studied in relation to markers of neurodegeneration. The putative protective effect of APOE4 dosage among African American women on performance in the domain of attention over time requires replication and deserves further study with respect to underlying mechanisms. Specifically, the difference between African American men and women in this association may be the result of biological or hormonal sex differences, as it could be caused by socioenvironmental factors related to gender. Finally, a recent study by Morris et al. found that lower CSF concentrations of total tau and phosphorylated tau181 in African American individuals may reflect a significant race by APOE4 dosage interaction, suggesting a differential effect of this Alzheimer risk variant in African American individuals compared with white individuals [In terms of biological plausibility,  disease . More ree course . The thrhe blood . Individozygotes ; an assoozygotes . Neverthth ε3/ε3 . In factth ε3/ε3 . This maividuals . Neverthividuals . DespiteAPOE2 or APOE4 dosages as an ordinal variable  as opposed to studying genotypes using a common referent approach . This improved statistical power in our models, although it is worth noting that for some race × sex analyses, the APOE dosage may be representing a move from “0” to “1” dosage only, given the lack of availability for the “2” category. Thus, our main focus was on the stratification by race only.The strengths of our study include its large sample size allowing us to detect small effects, both cross-sectional and longitudinal, within the context of mixed-effects linear regression models with outcomes being 11 test scores spanning various domains of cognition, in a socioeconomically and racially diverse sample of middle-aged urban adults. Given the possibility of small effect being detected, some of the main findings were presented in terms of effect size, using standardized regression coefficients, in addition to showing their statistical significance. The larger sample size and adequate balance by race and sex also allowed testing for effect modification by both of these socio-demographic factors. Our analysis adjusted for important potential confounders, corrected for multiple testing, adjusted for selection bias due to missingness in outcome data using a 2-stage Heckman selection model, included multiple imputation of covariates, and formally tested effect modification by race and sex. Our results can be extrapolated to many African American and White middle-aged urban adult populations, as HANDLS is representative of 14 urban settings across the USA . DespiteAPOE ε4 allele dosage and cognitive decline in the memory domain among Whites, while among African American women, APOE ε4 allele dosage had a potential protective effect on the domain of attention over time. Such domain-specific inconsistencies have been reported in other studies, particularly when comparing the effect of APOE4 allele status or dosage on memory to that on other more crystallized domains of cognition . APOE2 dosage appeared to have less consistent associations with various domains of cognition across racial groups. Further longitudinal studies are needed to replicate our race and sex-specific findings.In conclusion, we found putative adverse associations between the Additional file 1.Additional file 2:Figure S1. Standard deviations (SD) of cognitive performance test scores over period of follow-up for each of 11 cognitive tests, in eligible sample , HANDLS 2004-2013a. Abbreviations: AF = Animal Fluency; BTA = Brief Test of Attention; BVRT = Benton Visual Retention Test; CDT = Clock Drawing Test; CVLT-DFR = California Verbal Learning Test-Delayed Free Recall; CVLT-List A = California Verbal Learning Test-List A; DS-B=Digits Span-Backward; DS-F=Digits Span-Forward; HANDLS = Healthy Aging in Neighborhood of Diversity across the Lifespan; MMSE = Mini-Mental State Examination; SD=Standard Deviation; TRAILS A = Trailmaking Test, Part A; TRAILS B = Trailmaking Test, part B. a SD computed for each of 11 cognitive test performances over v1 and/or v2 for eligible sample . The SD is used to contextualize the effects obtained in mixed-effects linear regression models as a proportion of the SD.


ABCB5, is a chemoresistance mediator expressed on specific cell subsets in colorectal cancer (CRC) and other malignancies. This study evaluated the molecular signature expression and its clinical relevance of DTCs in bone marrow from patients with colon cancer.Disseminated tumor cells (DTCs) are critically involved in tumor relapse and survival in several invasive tumors. We previously showed that the ATP-binding cassette (ABC) transporter, ABCB5 in comparison to CD133 (molecule for identifying cancer initiating cells), Lgr5  as well as Cytokeratin (CK) 20  in these cells was evaluated.This study included 49 consecutive patients (UICC stage I-IV) that underwent curatively intended or palliative surgery for CRC. We analyzed cells from bone marrow aspirates obtained before surgery and derived from patients that had completed minimally a 5-year follow-up. The gene expression of ABCB5 and Lgr5 and to lesser extent CD133 and CK20 genes were significantly expressed in the analyzed cells from bone marrow aspirates while only ABCB5 and Lgr5 were significantly negative associated with tumor progress and overall survival.Bone marrow analysis showed differential expression between the analyzed genes. ABCB5 and Lgr5 in bone marrow negatively influenced patient survival pointing to a specific chemo resistant and pluripotent cell subgroup of DTCs in the bone marrow. ABCB5 like Lgr5 positive cells seem to be involved in limited tumor related patient survival, suggesting that ABCB5- and Lgr5-positive cells may be relevant for specific clinical intervention strategiesOverexpression of  After application of Oxaliplatin, 5-FU or Cyclophosphamide this cell fraction was enriched in vitro and in vivo pointing to a specific role in chemo resistance [(Lgr5) as a Wnt target gene is expressed on normal intestinal stem cells and in human colon CICs [ABCB5, a novel human multidrug resistance mediator recently shown to be expressed by cells of melanocytic lineage and additional cancers responsible for resistance to chemotherapy in vitro [ABCB5 identifies CICs in malignant melanoma that correlate with clinical disease progression and that can be specifically targeted to abrogate tumor growth [ABCB5 is expressed at higher levels in clinical CRC compared to healthy colonic mucosa, and that the ABCB5-positive tumor cell population was significantly induced following 5-Fluorouracil therapy in CRC patients’ tissue [ABCB5, CD133, Lgr5, and CK20 expression in bone marrow cells from patients with CRC and with completed 5-year follow up. This could lead to more directed therapies which specifically target therapy resistant tumor cells in CRC patients.Colorectal cancer (CRC) is the second most commonly diagnosed type of cancer and the second most common cause of cancer-related death in developed countries . The 5-yobserved . The disobserved ,12 that el cells . Its expel cells –16. Untisistance ,18. Not sistance . It is ilon CICs . Anotherin vitro ,23. Subsr growth . Recentl’ tissue , demonstConsecutive patients undergoing elective or palliative surgery for primary colorectal cancer (n=670) at the University Hospital of Wuerzburg were prospectively enrolled from July 2003 until June 2008 in this study. Of these, 49 patients with consent for bone marrow sample aspiration were included in this study. Patients with secondary carcinoma of other origin were excluded. With a median follow-up period of 58 months (range 4–141), all patients were followed-up regularly at 3 to 6-month intervals in accordance with the guidelines of the German tumor centers (completeness index of 0.96) [After the induction of general anesthesia prior to primary tumor surgery, bone marrow samples (7–12 mL) were obtained by aspiration from both iliac crests. After density centrifugation through Ficoll-Paque  mononuclear cells were harvested and washed twice in phosphate-buffered saline. Cell pellets were snap-frozen in liquid nitrogen and stored at −80°C until further use.RNA extraction from bone marrow samples were lysed in Trizol reagent  according to the manufacturer’s recommendations. cDNA synthesis was carried out by using iScript cDNA Synthesis Kit  as described by our group .ABCB5, Lgr5, CD133, and CK20 gene expression was analyzed in bone marrow samples (n=49) from colon cancer patients and healthy control individuals (n=20) by RT-qPCR assay. Total RNA (1 μg) was incubated with DNase I and reverse transcribed with oligo(dT) with SuperScript II RT-PCR (Promega). Reverse transcriptase product (100 ng) was amplified by primer pairs specific for Lgr5 (QT00027720), CD133 (QT00075586) and ABCB5  and 5V-GCTGAGGAATCCACCCAATCT-3V, reverse) which were purchased from Qiagen . Beta-actin was used as a normalizing control. Relative gene expression was measured with the GeneAmp 7000 Sequence Detection System (Applied Biosystems). The average value of both duplicates was used for calculation of relative transcript quantity according to the ΔCt method, as described previously [−ΔΔCt. Upregulated gene expression, described as positive expression in ABCB5, Lgr5, CD133, and CK20 in bone marrow was defined on the definition of ≥ 1.5 fold difference compared to the gene expression determined in controls.eviously . ResultsABCB5, Lgr5, CD133, and CK20 determined by quantitative Real Time PCR analysis were examined using univariate analysis. Statistical differences in univariate analysis were calculated using the Log-rank test. A p value<0.05 was considered statistically significant._The data collected were analyzed using SPSS for Windows version 12.0 . Differences between groups were analyzed using Chi square or Fisher’s Exact test. The calculation of survival rates and overall survival were performed using the Kaplan-Meier method. Disease free survival was calculated from the date of surgery to the date of tumor relapse, and overall survival from the date of surgery to the date of the patient’s death. Patients without evidence of disease recurrence were censored at the date of last contact, as a small number of patients who were lost to follow-up. Factors such as age, UlCC-stage (pTNM), and gene expression for th UICC TNM staging system, 3, 3, 33 and 10 patients had stage I, II, III and IV cancers, respectively. Among the analyzed patients 82% and 90% of the individuals significantly expressed ABCB5 and Lgr5, while 71% and 59% showed upregulated expression for CD133 and CK20, suggestive for the existence of DTCs in this compartment (The patient and tumor characteristics are presented in partment  and 2.ABCB5 and Lgr5 in the bone marrow was dependent on tumor grading (both p=0.01) and T-category . Significantly increased expression was shown in most of the samples for ABCB5 and Lgr5 and to lesser extent for CD133 and CK20  was significantly upregulated when compared to patients from Group III that represented long-term survivors without any tumor relapse . In addition, most patients with a cluster of genes for ABCB5, Lgr5, and CD133 were derived from group I that developed metastases and/or died tumor related. However, only co-expression of the two genes ABCB5 and Lgr5 but not of CD133 correlated with patient prognosis .Each transcript was tested in its dependency from gender, age, differentiation, T-, N-, and M-category, and UICC-stage. Transcript-positive patients were compared to transcript-negative patients. Expression of  disease ; Group I relapse . PatientABCB5, Lgr5, CD133, and CK20 gene expression in bone marrow collected from patients with colon cancer was further quantified and tested for their influence on overall survival in the patients. Patients overexpressing ABCB5 and Lgr5 but not CD133 nor CK20 in their bone marrow showed a worse overall survival compared to biomarker-negative patients  (ctively) .ABCB5 and Lgr5 when detected in the bone marrow significantly influenced overall survival. Thus, it implicates a subset of specific tumor cells migrating from the primary tumor during tumor progression into the bone marrow and becoming part of future tumor recurrence. That means that dissemination of therapy-refractory cancer cells into a ‘niche’ within the bone marrow would enable the tumor to progress at later time after primary surgery when such cells re-start to proliferate.The prognosis of patients with colorectal cancer is largely determined by the occurrence of distant metastases and the fact of chemo resistance during tumor progression. Relapse is mainly due to clinically occult micro metastasis present but not detectable in secondary organs as well as compartments like peripheral blood and bone marrow at the time of primary diagnosis. Bone marrow of patients after R0-resection of solid tumors are currently discussed to be a common homing site of remaining disseminated and circulating tumor cells. Beside the ongoing discussion of the source of tumor cells responsible for tumor relapse after primary cure, recent evidence suggests a specific side population of cells with a distinct self-renewal capacity and chemo resistance, which is gaining increasing interest in the field of DTCs. Thus, it seems to be possible that such a subset of chemo resistant cells with self-renewal capacity within a ‘specific niche’ as the bone marrow may be a major source for tumor maintenance. Moreover, this tumor cell dissemination can be an early event during tumor progression. Recent evidence has suggested that a specific subpopulation of chemo resistant cells may be a major source of tumor maintenance. Nevertheless, there is no clear evidence so far for such a cell subset derived from the primary tumor. This may be of highly relevance for adjuvant therapies when detected even before its complete resection. Our findings suggest that a subset of therapy-refractory cancer cells may have prognostic significance. Gene signature expression of these cells was identified in the bone marrow of patients with CRC. This indicates such a subset of DTCs with putative self-renewal and chemo resistant characteristics among DTCs and its prognostic significance. mRNA expression for CK20 in CRC and other gastrointestinal cancers. However, in the majority these studies were based on a single marker level like CK20 that detects differentiated circulating tumor cells [Other studies in the past have analyzed gene expression levels solely from peripheral blood indicating circulating tumor cells within this compartment. Most of them demonstrated significant impact on tumor recurrence or survival for patients positive for individual markers like or cells ,28.CD133 is one of the most frequently analyzed putative markers for CICs in patients with CRC, pancreatic cancer or hepatocellular carcinoma [CD133 expression profiles in peripheral blood were associated with decreased cumulative survival in patients in a metastasized tumor stage [CD133 was an independent prognostic factor [CD133 being a universal marker for tumor initiating cells as CD133− cells were also capable of tumorigenesis [arcinoma –35. Clinor stage . Anotherigenesis ,38. Inteigenesis Lgr5 [Lgr5 expression in selected UICC stage IV patients was associated with a poor prognosis [Lgr5 did not influence overall survival nor relapse probability in bone marrow although its expression significantly correlated with CK20 expression. However, more recent evidence from a meta-analysis points to an association between Lgr5 expression and prognosis [ABCB5, in CICs has been characterized in previous studies on human melanoma [ABCB5 expression was significantly correlated with resistance to 5-FU chemotherapy in a xenotransplantation model of CRC [ABCB5 expression was also correlated with resistance to doxorubicin, camptothecin, and 5-FU in malignant melanoma and hepatocellular carcinoma. Moreover, we found overexpression of ABCB5 in a subset of 5-FU-refractory cells in primary tumor specimens resected from patients with rectal cancer after neoadjuvant radio chemotherapy. Those results pointed to the clinical significance of ABCB5 in CRC. We could show that ABCB5 expression in the bone marrow from patients with CRC was strongly dependent on tumor grading and wall infiltration. It negatively influenced overall survival of the analyzed patients when detected in their bone marrow, indicating a cofactor for recurrent disease in these patients.Intestinal stem cells and a subpopulation of colon cancer cells were shown to express Lgr5 –43. It wrognosis . Expressrognosis . This sumelanoma ,23 and hmelanoma . Recentll of CRC . ABCB5 eABCB5 and Lgr5 as gene profile for cancer cells with specific characteristics for chemo resistance and self-renewal in the bone marrow of patients with CRC. These findings indicate that tumor cell migration can occur early during tumor progression, dependent on differentiation (grading) of the primary tumor (poorly versus well differentiated cancer cells), and that a subset of these migrating cells with specific self-renewal capacity and chemo resistance has significant negative influence on overall survival. This sheds light on prognostic significance of cancer initiating cell dissemination with future therapeutic implications in colorectal cancer.Our data demonstrate a molecular signature expression for


ZFX (n = 8/98), RANK (n = 5/98), and others. MDS derived MSCs display higher mutational burdens, increased replicative stress, senescence, inflammatory gene expression, and distinct mutational signatures as compared to healthy MSCs. However, validation experiments in serial culture passages, chronological BM aspirations and backtracking of high confidence mutations by re-sequencing primary sorted MDS MSCs indicate that the discovered mutations are secondary to in vitro expansion but not present in primary BM. Thus, we here report that there is no evidence for clonal mutations in the BM stroma of MDS patients.The bone marrow (BM) stroma in myeloid neoplasms is altered and it is hypothesized that this cell compartment may also harbor clonal somatically acquired mutations. By exome sequencing of in vitro expanded mesenchymal stromal cells (MSCs) from n = 98 patients with myelodysplastic syndrome (MDS) and n = 28 healthy controls we show that these cells accumulate recurrent mutations in genes such as  Bone marrow-derived mesenchymal stroma cells (MSCs) in myeloid neoplasia have been hypothesized to carry somatic mutations and contribute to pathogenesis. Here the authors analyse ex-vivo cultures and primary MSCs derived from patients with myelodysplastic syndromes, finding functional alterations but no evidence of clonal mutations. An active role of the BM niche in MDS but also other myeloid neoplasms such as MPN or AML has been demonstrated in several experimental approaches9. In patient-derived xenograft experiments, we have previously shown that primary MDS samples were dependent on the interaction with BM-derived mesenchymal stroma cells (MSCs) for their propagation10. This led to the hypothesis that MDS hematopoietic cells may re-educate their BM niche to create a supportive environment for their preferential growth. In support of this hypothesis, several studies have described aberrant gene expression and epigenetic profiles of MDS-derived MSCs14. The selective introduction of mutations to non-hematopoietic cellular compartments of the BM niche such as osteolineage cells17 was able to disrupt hematopoiesis, induce MDS-like diseases, and control leukemic propagation18. Inflammatory programs mediated by S100A family proteins7 and Toll-like receptor 4 (TLR4)19 in mesenchymal precursors have further been shown to drive genotoxic stress in hematopoietic stem- and progenitor cells, proposing a mechanism of accumulation of genetic mutations in hematopoietic cells. Moreover, especially aging-related changes in the BM stroma are hypothesized to confer growth advantages for putatively pre-malignant hematopoietic subclones carrying typical mutations of early clonal hematopoiesis of indeterminate potential (CHIP)20 such as DNMT3A21.While there has been substantial progress in the identification of the molecular landscape and pathogenesis of myelodysplastic syndrome (MDS)25. However, little validation has been performed so far to address whether such molecular lesions were not merely clonal outgrowths resulting from the strenuous and massively expansive cell culture procedures.An open question in the field is, therefore, whether there may also be acquired mutations in the BM stroma compartment of MDS patients, which act as causative or contributing pathogenic factors in the disease. Previous studies have identified chromosomal aberrations in ex vivo expanded MSC cultures from MDS and AML patients26.We and others have previously used MDS-MSCs as highly purified germline controls for genomic profiling of the MDS hematopoietic clonesn = 98 MDS patients and n = 28 healthy age-matched individuals to interrogate the question of acquired mutations in MSCs from MDS patients. While recurrent mutations can be detected in ex-vivo expanded MDS MSCs, these are not reproducible in serial BM aspirations from the same patients and do not remain stable in serial culture passages. Ultimately, high confidence mutations detectable in ex vivo MDS MSC cultures cannot be backtracked by targeted re-sequencing of primary sorted BM MSCs from the same patients. While MDS MSCs are biologically and functionally altered in comparison to healthy MSCs, our data indicate that there is no evidence for clonal somatically acquired mutations in non-hematopoietic BM stromal cells in MDS patients in vivo.In this work, we present comprehensive data from whole-exome sequencing of in vitro expanded MSCs from 26, we have used in vitro cultured MSCs as germline controls for exome sequencing of paired hematopoietic cell fractions. In order to address the question of whether MDS patient-derived MSCs also carry acquired molecular lesions, we took advantage of this data by performing a reversed bioinformatics approach using the corresponding hematopoietic fraction as a germline control for the MSC exome sequencing data. With this approach, we analyzed whole-exome sequencing results of MSCs derived from a total of n = 98 MDS patients  in the MDS MSCs , LRP2 , or MUC16 , of which recurrence increased further up to n = 60 when removing the 10% VAF cutoff.The initial whole-exome sequencing dataset of 98 MSC samples produced a predominantly low-level mutational spectrum with a median variant allele frequency (VAF) of 5.3% in all called mutations Fig. . By addiSCs Fig. . Of noteSCs Fig. . In ordeZFX) , and RANK  or affected a splice site, suggesting loss of function mutations  . Also, other genes detected in the MDS cohort such as MUC16 or LPR2 were frequently mutated in the healthy cohort. All recurrently mutated genes in the healthy control cohort were summarized in Supplemental Data RANK or ZFX) were not found to be mutated in the healthy control group.To evaluate whether the acquisition of such mutations in cultured BM MSCs of MDS patients was possibly associated with myeloid neoplasia we also performed exome-sequencing on MSC cultures in a control cohort of 05) Fig. . Notably05) Fig. , Table 1n = 120 mutations in n = 117 genes in n = 12 samples . Notably, all mutations in ZFX and RANK were confirmed by TDS. Next, we exemplarily validated the strategy of exome sequencing MSCs versus BM MNCs as germline control compared to another potential germline specimen such as sorted CD3-positive T-cells from peripheral blood or buccal swab DNA from the same patient  of a total of ent Fig. . We obseios Fig. .Fig. 2Va7, we assessed these phenotypes in our MSC cultures. Thereby, we found markers for genotoxic stress such as frequency of γH2AX foci28 or phosphorylation of replication protein A (RPA)29 to be significantly increased in MDS-derived MSCs as compared to healthy MSCs  and observed higher expression of inflammatory markers of SASP in MDS MSCs  of MDS cases , Filamin B , Tenascin C . Yet, as predicted by MutSigCV, such mutations most likely resemble non-specific mutational hits (p > 0.2) and were only considered to have low to moderate impact on protein structure.Finally, since RANK is one of the central regulators of bone morphogenesisSCs Fig. . These dr = 0.25, p = 0.03). Moreover, primary MDS MSCs that were cultured for an additional passage (P2) showed an increased number of mutations with VAFs >10% .Since most of the detected acquired mutations in the MSCs were subclonal, we interrogated their dependency on culture parameters to address to which extent mutations in MSCs were possibly a result of selection during in vitro culture. We found a moderately positive correlation of the number of mutations with the duration of in vitro expansion of MDS-derived MSCs , most mutational clusters were characterized by low VAFs (<25%) and exclusiveness for either the early or the later passage . Since low cell numbers in this range are even challenging to re-sequence in a targeted amplicon-based NGS approach, we have previously verified that we could technically quantify low VAF SNPs in samples with low cell number input material26. In addition, we analyzed DNA from a less stringent isolation strategy comprising CD45−, CD235a−, CD271+/−, CD31+/− cells, therefore also including the CD31+ endothelial cell population . Moreover, several genetic mouse models have repeatedly shown proof of principle data that isolated genetic manipulation of BM niche components could induce MDS-like or myeloproliferative diseases and influence propagation of malignant hematopoietic clones46. Therefore, we here aimed to clarify a long-standing question, as to whether primary MSCs of MDS patients carry somatically acquired mutations with clonal relevance in vivo.The scientific background and rationale for this investigation were an increasing body of evidence suggesting the pathogenesis of MDS and other myeloid neoplasms such as MPN or AML not only takes place in the hematopoietic compartment but also in the BM microenvironment. This conception is supported by a multitude of different studies and approaches describing altered phenotypes of bone marrow stroma components of malignant myeloid disease such as features of increased senescence and impaired proliferation capacity47. However, it has never been entirely ruled out that such molecular lesions were not secondary to the highly expansive in vitro cultures, which are necessary to obtain these cells.Several lines of work have addressed this hypothesis before and revealed chromosomal and mutational events in MSCs derived from MDS or AML patients18. However, from the few studies that have performed molecular analyses on such highly purified and scarce cells it has become clear, that so far, it has not been successful to reproducibly isolate enough of these cells to perform robust explorative whole-genome or exome sequencing on a representative cohort. Therefore, also in our study, we have made the compromise to begin analysis on in vitro expanded MSCs of MDS patients. To this end, we have analyzed a representative cohort of n = 98 MDS and myeloid neoplasia derived BM MSCs as well as n = 28 healthy controls and used their own paired hematopoietic cells as germline controls. By standard bioinformatic evaluation, accounting for LOH in the hematopoietic germline fraction, we detected a large number of acquired mutations in genomes of ex vivo expanded MDS MSCs and even enrichment of recurrent events with frequencies of up to 19%. Interestingly, MDS MSCs clearly presented with more mutations and a higher mutational burden than the healthy control group suggesting that MDS or myeloid neoplasia-derived MSCs may be functionally altered in comparison to healthy MSCs. We performed several lines of characterization experiments with the MSCs from our study demonstrating that MDS-derived MSCs had indicators for higher replicative stress, increased senescence, and increased levels of inflammatory IL-6 as compared to healthy MSCs. Moreover, MDS MSCs had significantly different COSMIC mutational acquisition signatures35, which possibly resulted from mutational processes specific to in vitro cultivation48. Together with previously published data of increased levels of reactive oxygen species (ROS) in the BM niche of myeloid neoplasms53, these results corroborate the above-described notion that MSCs in myeloid neoplasia are molecularly and functionally altered. The observed replicative stress, activation by inflammation, and ROS may induce higher cell division rates and increased baseline genomic instability of MSCs derived from myeloid diseases and therefore explain higher disease-associated mutagenesis.The ideal experiment to address the question of acquired mutations in BM stroma of MDS patients would therefore be explorative sequencing performed in sorted primary non-hematopoietic cells from primary bone marrow samples. However, to date, there is still uncertainty on the immunophenotypic profiles the target cells for this experiment should have. Human BM-derived mesenchymal stem cells have previously been defined as CD45−, CD235a−, CD31−, and CD271+, CD146+, CD105+TTN27, which is known to be frequently mutated in explorative WES studies and most likely does not represent a clonal driver mutation. Therefore, we hypothesized that these events were largely secondary due to expansion in culture rather than true driver lesions originating from clonal mutations in the non-hematopoietic BM compartment. To follow this hypothesis we performed additional experiments that asked the question whether patient individual profiles detected in standard MSCs cultures (P1) were also measurable in the earliest possible in vitro culture passages (P0) and whether they remained stable in measurements of serial culture passages. Both approaches revealed that most mutational events occurred in minor clone sizes and were largely not even stably passed on from P0 to P1, precluding them from the potential discovery of real driver mutations. Nevertheless, this was not necessarily applicable to all mutations. For instance, ZFX stood out as a possible true mutational driver event due to its recurrence (n = 8 cases), its higher VAF of up to ~35%, and its stable detectability during serial cultures. The same was true for other high confidence candidate mutations such as DCHS1 or RANK55, of which the latter also had a functional impact on in vitro osteogenic capacity in our hands, which was in line with previous work57. Due to the putatively higher relevance of these mutations, we attempted to backtrack them in the primary BM of the patients, in which they were detected. These high confidence candidate mutations were neither detectable via TDS in the unselected whole bone marrow samples, nor in the primary sorted and non-expanded CD45-, CD235a-, CD31- and CD271 + cells as well as the less stringently sorted CD45−, CD235a−, CD271+/−, CD31+/− fractions. We have previously confirmed the technical validity of our applied amplicon-based deep re-sequencing approach on low DNA amounts from low cell number samples by serial dilution spike-in experiments26. As an alternative method, we also re-cultured the original MSC samples by CFU-F assays and performed genotyping of the candidate mutations in single colony sequencing. Thereby, the top recurrent candidate mutations could also not be detected. From these results, we finally concluded that there were no relevant clonal mutations in the BM stroma fractions of these MDS patients and that even the high confidence mutations detected in serial MSC culture samples rather reflect outgrowth of particularly fit clones originating probably of single cells selected by the culture conditions. This is also in line with previous approaches using lentiviral barcoding, which have shown that culture expansion of MSCs is associated with massive clonal selection and loss of clonal complexity58. Since previous mechanistic experiments in murine models had shown proof of concept that ectopic molecular alterations of the BM niche could induce MDS like phenotypes, we also interrogated, whether we could detect stromal alterations known to induce MDS in murine models such as PTPN1117, β-catenin16, DICER15, Sbds15, or RARγ59 in MDS MSCs but found no relevant mutations in these genes in our data.Most of the detected mutational events in MSCs had low-level VAFs and were mostly accumulated in sites with higher mutability during the in vitro expansion such as, e.g., 60. Furthermore, from our data, we cannot exclude the possibility that on a confined local level of the bone marrow there may be clusters of clonally mutated stroma cells.While we ruled out the evidence for acquired clonal mutations in the stroma compartment of MDS patients, the possibility for other molecular differences such as increased inflammatory status, epigenetic, transcriptional, senescence, and disturbed niche interaction of MDS derived MSCs, of course, remainNevertheless, collectively this comprehensive analysis leaves little doubt that if acquired mutations in the stroma of MDS patients play a role in MDS disease initiation at all, then at such a low clonal and possibly locally confined level, that they are not detectable with currently feasible sample acquisition and methodology. In our current study, we discovered no evidence for acquired mutations as disease initiators for MDS.n = 98 MDS and MDS associated myeloid neoplasia cases, who were treated at the Department of Hematology and Oncology of the Medical Faculty Mannheim, Heidelberg University, Germany . As healthy controls, a cohort of n = 28 healthy BM samples was acquired from patients undergoing hip replacement surgery . These healthy donor samples all had normal blood counts, absence of active or prior malignancy, and other confounding co-morbidity. All experiments were performed after written informed consent in accordance with the Declaration of Helsinki and approved by the Medical Ethics Committee of the Medical Faculty Mannheim. Detailed clinical characteristics of patients and healthy donors are provided in Table The study cohort consisted of MSCs were expanded adherently on plastic dishes by seeding 100 µm-filtered full BM fragments and additionally seeding 5 × 10e6 mononuclear cells in StemMACS MSC Expansion Medium XF (Miltenyi Biotec) in T25 flasks (P0). After 2 days, the medium was changed and non-adhesive cells were removed. Cells were then further expanded with weekly medium changes. At 80% confluency, this initial culture was trypsinized and split into 2–4 T75 flasks, corresponding to P1 with yields of approximately 60,000 cells per flask. Of note, the in vitro culture was carried out for the shortest possible period of time to obtain sufficient cell numbers for bulk DNA isolation. Cells were harvested before senescence or confluence. The median time of in vitro expansion before DNA preparation was 34 days, : 22–50 d).n = 45 MSC exome sequence data sets of this cohort was previously used as germline control for mutational analyses of BM in a prior study26. The additional n = 43 cases were sequenced de novo for the current study. To interrogate somatically acquired mutations in MSCs we reversed the bioinformatic settings and defined the hematopoietic cells as germline controls, which is described in detail in the supplemental methods section. Median coverage of exome sequencing was 81× (range 19–140×) for the MDS MSCs and 164× (range: 118×–215×) for the healthy cohort.A subset of 27 from maf files after vcf2maf (https://github.com/mskcc/vcf2maf) conversion from the mskcc repository. We used publicly available context files for coverage, covariates, and mutational dictionaries. Mutational signatures were extracted using the vcfs generated previously according to Maura et al.61 using the R (v3.6.3) MutationalPattern (v1.10) package62. For extraction of COSMIC signatures whichSignatures from the deconstructSigs63 (v1.9) were applied to vcfs with context normalization and default trinucleotide counts.Mutational significance was calculated with MutSigCV (v1.4)n = 5 MDS samples and n = 3 healthy sequenced in a previous study10 were combined with de novo sequenced samples from an additional n = 3 MDS patients and n = 3 healthy donors in the current study. All samples were prepared equally and finally added up to a combined cohort of n = 8 MDS samples and n = 6 healthy samples. In brief, 500 ng RNA was subjected to the Illumina Stranded TruSeq RNA protocol and sequenced to a median of 94 million reads per sample. The bioinformatic analysis consisted of mapping hisat2 v2.04 to hg38 and cufflinks—cuffdiff v2.2.1 for transcript assembly and differentially expressed gene analysis according to the vignette with default settings.For RNA sequencing analyses, polyA libraries from ®−, CD45−, CD235a−, CD31−, CD271+  on a BD FACS Melody sorting device. In addition, a second FACS strategy  was employed to enrich non-hematopoietic cells. Cells were directly sorted into Qiagen ALT lysis buffer and the whole genome amplified with the Qiagen repliG Kit in the majority of cases. For validation with targeted deep sequencing, single PCRs surrounding mutational sites (Primers in Supplemental Data Validation of high confidence candidate mutations was carried out in the original samples as well as in primary, non-expanded MSCs, which were FACS sorted from viably frozen BM specimen of the corresponding patient samples, in whose MSC cultures the mutations were detected. Viable cells were stained and sorted for the parameters SytoxFurther information on research design is available in the Supplemental InformationPeer review fileDescription of supplementary filesSupplemental Data1Supplemental Data2Supplemental Data3Supplemental Data4Supplemental Data5Reporting Summary


SDHA, SDHB, SDHC, SDHD) or NF1. However, despite this high heritability, familial clustering of wtGIST is extremely rare. Here, we report a mother–son diad who developed wtGIST at age 66 and 34 years, respectively. Comprehensive genetic testing revealed germline truncating variants in both SDHA (c.1534C>T (p.Arg512*)) and PALB2 (c.3113G>A (p.Trp1038*)) in both affected individuals. The mother also developed breast ductal carcinoma in-situ at age 70 years. Immunohistochemistry and molecular analysis of the wtGISTs revealed loss of SDHB expression and loss of the wild-type SDHA allele in tumour material. No allele loss was detected at PALB2 suggesting that wtGIST tumourigenesis was principally driven by succinate dehydrogenase deficiency. However, we speculate that the presence of multilocus inherited neoplasia alleles syndrome (MINAS) in this family might have contributed to the highly unusual occurrence of familial wtGIST. Systematic reporting of tumour risks and phenotypes in individuals with MINAS will facilitate the clinical interpretation of the significance of this diagnosis, which is becoming more frequent as strategies for genetic testing for hereditary cancer becomes more comprehensive.Gastrointestinal stromal tumour (GIST) is a mesenchymal neoplasm arising in the gastrointestinal tract. A rare subset of GISTs are classified as wild-type GIST (wtGIST) and these are frequently associated with germline variants that affect the function of cancer predisposition genes such as the succinate dehydrogenase subunit genes ( KIT or PDGFRA in most cases but in 15% of adult cases and 85% of paediatric instances, no such driver is identified and the tumour is termed a wild-type GIST (wtGIST) [Gastrointestinal stromal tumour (GIST) is a mesenchymal neoplasm arising in the gastrointestinal tract, most commonly occurring in the stomach or small intestine with around 10% in other locations [(wtGIST) , 3.SDHA, SDHB, SDHC or SDHD (SDHX genes) or somatic SDHC promoter hypermethylation. Whereas only 3–4% of non-wtGISTs occur in association with a causative germline variant , one may be detected in as many as half of individuals with dSDH-wtGIST [SDHX gene variants associated with dSDH-wtGIST differs from that seen in the tumour types most commonly associated with dSDH (phaeochromocytoma and paranglioma (PPGL) and head and neck paraganglioma) in that germline SDHA variants are frequent with wtGIST but an infrequent cause of PPGL or head and neck paraganglioma [SDHA function have previously been reported [Amongst wtGISTs, more than three-quarters show evidence of succinate dehydrogenase (SDH) enzyme deficiency (known as dSDH-wtGIST), generally detectable by tumour immunostaining that reveals loss of SDHB expression , 4. dSDHH-wtGIST , 8. The anglioma , 9. Despreported , series reported ) wtGISTsSDHA variant was detected. In addition, a germline truncating PALB2 variant was also found meaning that two cancer predisposition gene variants with implications for genetic counselling were present in the same individual. This situation that has previously been termed multilocus inherited neoplasia alleles syndrome (MINAS). Whilst there is little to indicate that the PALB2 variant contributed to GIST tumourigenesis, we speculate that this unusual finding of may have led to increased penetrance in this family.Here we describe a rare example of familial dSDH-wtGIST where a germline truncating https://databases.lovd.nl/shared/diseases/04296 . Both affected individuals gave written informed consent for research investigations and the research was approved by the relevant Research Ethics Committees (South Birmingham REC and East of England—Cambridge South REC).The family was ascertained via a clinical genetics centre after a mother and son presented with wtGIST. Information relating to these individuals has been submitted to the Leiden Open Variant Database and can be found at SDHB immunohistochemistry was performed on 3-μm sections of FFPE tissue mounted on adhesive slides. The staining was performed on a fully automated BOND III IHC and ISH stainer system . The SDHB primary antibody rabbit polyclonal  was used. The Primary antibody binding to tissue sections was visualized using BOND Polymer Refine Detection system .WGS and bioinformatic processing to produce variant call format (VCF) files was performed on samples from study participants as part of, and according to protocols devised by, the NIHR BioResource Rare Diseases study . DNA LibVariants occurring in genes included in a list of 83 cancer predisposition genes were filtered and assessed according to a protocol described previously .Slides were prepared from formalin-fixed paraffin-embedded (FFPE) tumour blocks by the Human Research Tissue Bank, Cambridge University Hospitals. Following de-paraffinisation, slides were reviewed by a pathologist to mark selected tissue and tumour dissection was performed by colleagues in the Department of Haematology and Oncology diagnostic services, Cambridge University Hospitals. DNA was subsequently extracted from resulting tissue.SDHA region of interest. An Illumina MiSeq instrument was used for sequencing. Alignment was performed with Burrows-Wheeler Aligner [Library preparation was undertaken in the Stratified Medicine Core Laboratory using a custom AmpliSeq panel  that included the  Aligner  and resu Aligner .PALB2 variant identified in the corresponding blood DNA according to standard protocols .DNA extracted from tumours was also subject to Sanger sequencing for a SDHA and NM_024675.3 for PALB2.Variants in this article are described based on transcripts NM_004168.4 for A 66-year-old female presented with symptoms of satiety and was endoscopically investigated, revealing a 35 mm gastric GIST. She went on to undergo a distal gastrectomy, at which time she was identified as having liver metastases. Further treatment was with imatinib. Subsequently, her son also developed a gastric GIST at the age of 34 years, which was detected as an incidental finding following imaging performed due to trauma. The 53 mm tumour was removed laparoscopically. Histology and immunohistochemistry of both GISTs showed a mixed epithelioid growth pattern and loss of SDHB staining on immunohistochemistry, indicating deficiency of a component of the succinate dehydrogenase (SDH) multiprotein complex Fig. . There wSDHB, SDHC, SDHD, KIT and PDGFRA genes in the son’s sample did not reveal a causative constitutional variant and both individuals were recruited to research studies to try and elicit the cause. At the time of investigation (2014), analysis for SDHA variants or SDHC epimutations was not included in the diagnostic workflow. Following recruitment, the mother was also diagnosed with ductal carcinoma in situ of the breast at the age of 70 years.The family was referred for Clinical Genetic assessment and initial routine analysis of SDHA (c.1534C>T (p.Arg512*)) in both individuals. This variant has previously been reported as associated with GISTs and paragangliomas in the germline heterozygous state [−5 (seven occurrences) with a maximum allele frequency of 9.3 × 10−5 in non-Finnish Europeans.WGS on DNA extracted from blood was performed on a research basis. Variant filtering based on a list of 83 cancer predisposition genes and variant assessment according to American College of Medical Genetics criteria revealed a nonsense variant in us state , 17 and us state  with patus state  is 4.9 ×PALB2 c.3113 G>A (p.Trp1038*) in blood from both participants. PALB2 is a breast cancer predisposition gene and this is a frequently identified variant with >10 ClinVar entries with pathogenic assertion, mostly with reference to breast cancer predisposition. Overall allele frequency in gnomAD is 2.1 × 10−5 (two occurrences) with a maximum allele frequency of 3.1 × 10−5 in non-Finnish Europeans.Variant filtering and assessment also identified SDHA variant being causal and also investigate whether there was any evidence for the PALB2 variant contributing to tumourigenesis.Loss of heterozygosity (LOH) analysis was performed on DNA from both tumours to support the role of the SDHA wild-type allele was confirmed with a panel-based sequencing assay  and methods section) where variant allele fraction (VAF) was 0.42 in the blood sample from the mother and 0.92 in her tumour sample. The son’s samples also showed some evidence of LOH with VAF’s of 0.57 in blood and 0.85 in tumour . PHD normally promotes hypoxia-inducible-factor 1-alpha (HIF1) breakdown through hydroxylation and reduction in this function leads to a pseudo-hypoxic state with associated upregulation of a range of genes that can promote neoplasia (e.g. IGF-1 and VEGF). Succinate accumulation also leads to oncogenic aberrant methylation via inhibition of TET enzymes [carriers . It enco enzymes  and has  enzymes . Alterna enzymes , 22.SDHA, like other SDHX genes, can predispose to the development of PPGL but penetrance for these tumours is estimated to be under 10% from studies of relatives of probands diagnosed with PPGL and analysis of population databases [SDHA variant carriers ascertained by genetic testing for PPGL [SDHA germline variant carriers found through agnostic cancer genetic testing [Germline variants in atabases –25. Penefor PPGL  and 95 cfor PPGL  each sho testing  and four testing .PALB2 encodes a protein that interacts with BRCA2 to execute double-stranded DNA repair through homologous recombination (HR) and was identified as a breast cancer predisposition gene in 2007 [PALB2 is widely expressed at RNA and protein level, including in the gastrointestinal tract [ in 2007 . Penetra in 2007 . PALB2 ial tract  but no aPALB2 can cause Fanconi Anaemia associated with DNA repair deficiency. In individuals with monoallelic variants, a ‘second hit’ at the cellular level can lead to HR DNA repair deficiency and increased repair through the error-prone alternative mechanism of non-homologous end joining (NHEJ) [BRCA1 and BRCA2 (due to HR deficiency) but has also been demonstrated in breast [PALB2 truncating variants. Around two-thirds of breast cancers from carriers of function compromising PALB2 variants have a second hit observed as either loss of the wild-type allele or a second somatic variant. Such biallelic deficits correlate closely with evidence of HR deficiency in tumours but tumours without an observable second hit can also occasionally have observable deficiency [Constitutional biallelic variants in g (NHEJ) . These cg (NHEJ) . One sign breast  and pancn breast  cancers ficiency , 35, sugPALB2 variant contributing to increased penetrance as opposed to being an incidental finding. Tumour studies in both affected individuals supported the primary tumourigenic role of the SDHA variant with histology typical of SDH deficiency, loss of SDHB immunostaining (which indicates loss of a SDH complex component and is an indirect measure of SDHA loss) and evidence for loss of the wild-type SDHA allele on somatic sequencing. LOH analysis for PALB2 did not show evidence for loss of the wild-type allele. The PALB2 variant therefore, may have contributed to the breast cancer occurring in the mother  but not the wtGISTs in this family. Preservation of the wild-type PALB2 allele in tumour studies supports that notion. However, absence of LOH in the context of a tumour suppressor gene does not always imply absent contribution as alternative mechanisms may disrupt the wild-type allele such as epimutation, structural variation not resulting in copy number loss, or a missense variant not assessed as contributory to tumourigenesis. More extensive tumour studies could potentially have been revealing in delineating the contribution of the PALB2 variant in these GISTs. IHC was useful to demonstrate the role of the SDHA variant but no equivalent assay was available for PALB2. Whole-genome sequencing was not performed due to inadequate DNA from FFPE tissue but may have revealed evidence of the HR deficient mutational signature that is characteristic of PALB2 related cancers. Interestingly, SDH deficiency has also been reported to suppress DNA repair by HR [Given that familial wtGIST is rare, we performed further tumour studies to investigate whether there was evidence for the ir by HR  so that SDHA associated MINAS. The term describes the scenario in which an individual harbours clinically significant germline variants affecting more than one cancer predisposition gene and was suggested to facilitate information sharing about such cases [To our knowledge, this is the first case of ch cases . This ocBRCA1/BRCA2 variant combination) but some cases show severe manifestations  and cases with a protective effect are less likely to be investigated or reported.A key question following identification of a MINAS case is the nature of the resultant phenotype. Feasible effects include synergy between the variants to produce a more severe or penetrant phenotype, an independent manifestation where each variant leads to cancer risks equivalent to a scenario where the other is not present, and a protective effect due to synthetic lethality. We previously reviewed MINAS cases reported in the literature and generally observed evidence for an independent mechanism  at https://databases.lovd.nl/shared/diseases/04296.Our studies suggest an independent action of the two variants where the Supplemental MaterialConsortium group