LMNA gene, and are implicated in fundamental processes such as nuclear structure, gene expression, and signal transduction. LMNA mutations predominantly affect mesoderm-derived cell lineages in diseases collectively termed as laminopathies that include dilated cardiomyopathy with conduction defects, different forms of muscular dystrophies, and premature aging syndromes as Hutchinson-Gilford Progeria Syndrome. At present, our understanding of the molecular mechanisms regulating tissue-specific manifestations of laminopathies are still limited.Intermediate filament proteins that construct the nuclear lamina of a cell include the Lamin A/C proteins encoded by the LMNA splice-site mutation (c.357-2A > G) in an affected family with cardiac disease, we conducted deep RNA sequencing and pathway analysis for nine fibroblast samples obtained from three patients with cardiomyopathy, three unaffected family members, and three unrelated, unaffected individuals. We validated our findings by quantitative PCR and protein studies.To gain deeper insight into the molecular mechanism of a novel IGFBP5) in patient samples. Pathway analysis showed involvement of the ERK/MAPK signaling pathway consistent with previous studies. We found no significant differences in gene expression for Lamin A/C and B-type lamins between the groups. In mutant fibroblasts, RNA-seq confirmed that only the LMNA wild type allele predominately was expressed, and Western Blot showed normal Lamin A/C protein levels.We identified eight significantly differentially expressed genes between the mutant and non-mutant fibroblasts, that included downregulated insulin growth factor binding factor protein 5 (IGFBP5 may contribute in maintaining signaling pathway homeostasis, which may lead to the absence of notable molecular and structural abnormalities in unaffected tissues such as fibroblasts. Compensatory mechanisms from other nuclear membrane proteins were not found. Our results also demonstrate that only one copy of the wild type allele is sufficient for normal levels of Lamin A/C protein to maintain physiological function in an unaffected cell type. This suggests that affected cell types such as cardiac tissues may be more sensitive to haploinsufficiency of Lamin A/C. These results provide insight into the molecular mechanism of disease with a possible explanation for the tissue specificity of LMNA-related dilated cardiomyopathy. LMNA) gene encodes for Lamin A and Lamin C proteins that, along with Lamin B1 and Lamin B2, form an intricate intermediate filament protein meshwork termed the nuclear lamina (NL) and play important roles in maintaining nuclear structure and stability and in fundamental nuclear functions [LMNA gene. Lamin A is produced from all twelve exons of the gene, while Lamin C is the product of only ten exons. In vivo, Lamin A is produced as prelamin A and undergoes extensive post-translational processing of the C-terminus to become mature Lamin A protein while Lamin C is produced as mature protein [The Lamin A/C  with conduction defects [LMNA-related DCM with conduction defects is one of the most common forms of inherited dilated cardiomyopathy, second only to DCM associated with mutations in sarcomere protein genes [LMNA mutation [LMNA mutations give rise to diseases limited to cardiac tissues include the mechanical defect and the gene expression hypotheses [LMNA mutation compromises the structural integrity of a cell, subsequently causing the cells to be more prone to necrosis leading to diseases [LMNA mutation impairs the NL structure which leads to aberrant epigenetic modification, abnormalities in signaling transduction and ultimately affecting proper gene expression [LMNA-related cardiomyopathy.Although Lamin A/C is expressed in most differentiated cell types,  defects –20 and d defects –23 and L defects , to seve defects , 26. LMNin genes , 28, witmutation –31. Currpotheses . The mecdiseases . The genpression , 34. As LMNA c.357-2A > G (p.N120Lfs*5), in a multigenerational family with DCM, heart failure, and sudden death [LMNA gene is associated with altered expression of genes that played a significant role in the nuclear lamina structure and function. By deep RNA-seq of patient and control fibroblasts, we evaluated expression of Lamins A and C, tested for aberrant splicing of LMNA transcripts, and altered ratio of LMNA transcript variants. We also sought to determine any compensatory mechanism by Lamin B1 and/or Lamin B2 in response to the mutation.We recently identified a novel splice-site mutation in the Lamin A/C gene, LMNA mutation or other genes and pathways influencing the observed phenotype. Surprisingly, from the RNA-seq data we did not find any significant differential expression for the Lamin isoforms between the sample groups. However, we found plausible genes and pathways that may contribute to the cardiomyopathy phenotype that we observe in this family. Our study highlighted tissue specificity as a major feature of the novel heterozygous LMNA splice-site mutation as evidenced by the lack of changes in nuclear lamina gene expression and absence of aberrant splicing events in patient fibroblasts. The study conducted here laid the foundation for future disease modeling studies in cardiomyocytes to elucidate on the molecular mechanism of the novel LMNA splice-site mutation.We further expanded the analyses to examine the most significantly differentially expressed genes across the genome, and determine potential pathways affected by the LMNA splice-site mutation and three unaffected individuals (Control 1–3) who do not have the LMNA splice-site mutation. To serve as Unrelated Controls (U), fibroblast cells from three healthy individuals were obtained from biorepositories . To confirm the LMNA genotype, genomic DNA (gDNA) was extracted from Patient, Control, and Unrelated Control fibroblasts and evaluated by Sanger sequencing of the 12 LMNA exons as previously described [LMNA genotype for each sample was also confirmed by examining the RNA-seq data.Nine primary fibroblast cell lines were cultured from skin biopsies Table  as previTotal RNA from Patient, Control, and Unrelated Control fibroblasts (at passage 7) was isolated and quantified as described . RNA-seqp-value ≤0.05. DE of isoforms was provided by cufflinks/cuffdiff2 software to distinguish between the expression of Lamin isoforms. From this list of significantly DE genes, we further filtered the list to include only DE genes that were expressed similarly between the Unrelated and Patient groups and between the Control and Patient groups. Our rationale to filter the genes using these parameters was to find genes that potentially were affected by the LMNA splice-site mutation. We reasoned that DE genes that were shared between Control and Unrelated groups were due to intrinsic gene expression differences and not due to the mutation. By looking only at genes that were at the intersection of Unrelated vs. Patient and Control vs. Patient groups, we narrowed down the candidate genes to those that potentially were affected by the mutation.RNA-seq data was first examined for quality using FastQC software . Reads wHeatmaps of the gene expression in FPKM values of the most highly DE genes were constructed with cummeRbund . HeatmapACTB) was used as the housekeeping gene. Statistical analysis was performed using One-Way ANOVA followed by Tukey post hoc test to determine statistical significance between groups. P values less than 0.05 were considered statistically significant. Primer sequences used for validation are provided in Additional File Quantitative PCR (qPCR) assays were performed to validate RNA-seq results for Lamin A, Lamin C, Lamin B1, Lamin B2, and IGFBP5. cDNA was synthesized using QuantiTect Reverse Transcription Kit (Qiagen) from Unrelated, Control and Patient RNA samples. 100 ng/μl cDNA was used as the reaction template with 10 μM of pre-designed Kicqstart forward and reverse primer pairs  specific to Lamin A and Lamin C, 10 μM of independently designed primer pairs for Lamin B1, Lamin B2, and IGFBP5 , and SYBR green dye along with the necessary reaction components . The reaction was run in three technical replicates for each sample. Comparative Ct (ΔCt) method was used to determine relative quantitative gene expression (QGE) , 43. BetPatient, Control and Unrelated Control fibroblasts were grown to confluency, harvested using TrypLE Select 1X , and lysed using cold RIPA buffer supplemented with protease inhibitors cocktail . Protein concentration was quantified using Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific). 50 μg of total protein lysate along with protein ladder were run on a Bolt™ 4–12% Bis-Tris Plus Gels (Thermo Fisher Scientific) under denaturing conditions followed by wet transfer. Target protein was detected using a primary antibody for Lamin A/C , at 4 °C overnight followed by incubation with secondary antibody conjugated to HRP  for one hour at room temperature. Beta Actin was used as loading control. Protein visualization was conducted using iBright FL1000 Imaging System and relative quantification of protein bands was performed using ImageJ .LMNA-related cardiomyopathy. Differences between the gene expression in the pooled sample groups  were observed by plotting the top DE genes LMNA isofMMP3) [KRT18) [IGFBP5. This gene was of particular interest due the known effects of Lamin A/C on the IGF pathway in different forms of laminopathies [IGFBP5 mRNA level to be downregulated in Lmna −/− mouse embryonic fibroblasts, compared to Lmna +/+ mouse embryonic fibroblasts [IGFBP5 gene   and for  [KRT18) . In addiopathies . Previouroblasts . We alsoene Fig. c. The qPLMNA splice-site mutation. At present, the molecular mechanisms regulating tissue-specific manifestations of laminopathies are still unidentified. However, multiple studies have shown that LMNA mutations cause nuclear envelope defects in mouse and human LMNA mutant fibroblasts [LMNA-related diseases, upregulation of the ERK/MAPK pathway also is documented [Here we report gene expression profiling using RNA-seq on fibroblasts from a family bearing a pathogenic roblasts –51 and droblasts , 10. In cumented , 53. TakLMNA mutant and normal human fibroblasts. Among eight significantly DE genes was the IGFBP5 gene that encodes for an IGF binding protein that stabilizes IGF cytokines to promote or inhibit its signaling cascade and subsequent IGF function in cell culture [IGFBP5, were connected through the ERK/MAPK pathway. Our RNA-seq data showed a significant reduction in IGFBP5 gene expression in patient samples compared to control (7-fold reduction) and unrelated samples (9-fold reduction). This reduction may potentially impact the signaling pathways downstream to IGFBP5 binding. IGFBP5 protein translocates to the nucleus and interacts with Early growth response-1 transcription factor in human primary lung fibroblast to induce extracellular matrix production by activating the ERK/MAPK pathway, independent of the IGF pathway [Consistent with this, our gene expression profiling uncovered genes involved in signal transduction that are differentially expressed between  culture . Aside f culture –58. Over culture . These o culture . Subsequ pathway . Since wLMNA mutant fibroblasts, the significant reduction of IGFBP5 expression may contribute to maintaining homeostasis of the ERK/MAPK signaling pathway and its downstream effects, masking the LMNA mutation phenotype. The postulation is supported by the observations of normal baseline ERK1/2 activity in patient fibroblasts [LMNA mutant cardiomyocytes which leads to laminopathy phenotype [With these findings, we hypothesize that in unaffected tissues such as roblasts . In conthenotype , 62. Toghenotype .FOXC2), whose overexpression induces hyperphosphorylation of ERK1/2 in cancer cells and is associated with aberrant cell proliferation [FOXC2 mRNA reduction by shRNA silencing has been shown to downregulate ERK/MAPK activity [IGFBP5 and FOXC2 gene expression may contribute to the compensatory mechanism that maintains the normal activity of ERK/MAPK pathway and absence of observable phenotypes in fibroblasts. While the current study was performed on an unaffected tissue, we were able to gain insight on the possible signaling pathways and molecular mechanisms involved in the tissue specificity of laminopathies and LMNA-related cardiomyopathy.Another DE gene on the list, Forkhead Box C2  with no aberrantly spliced products from the mutant allele in patient fibroblasts.Previously, we showed that there was monoallelic expression of NA decay . In suppLMNA transcript levels, ratio of Lamin A to Lamin C transcripts, or expression of other NL-genes, LMNB1 and LMNB2 between the Patient group and two control groups. These results are in line with our previous structural studies that found no significant difference in nuclear abnormalities and suggests that there were no compensatory mechanisms at the transcription level for the other proteins that construct the nuclear lamina [We also found that there were no significant changes in r lamina .LMNA mutation at the splice site of intron 5 showed reduced LMNA expression with the mutant LMNA allele expressed at lower level compared to the normal allele [Our results are in contrast to previous studies in patient fibroblasts that showed decreased Lamin A/C expression and altered ratio of Lamin A and Lamin C expression , 66. Real allele . In a diLMNA mutations have similar Lamin A/C protein levels compared to control fibroblasts [LMNA mutations do not result in significantly observable phenotypes such as nuclear abnormalities or reduced protein level [Numerous studies have reported that differential gene expression does not correlate directly with changes in protein expression –70. Muchroblasts . Additioin level , 71–73.LMNA allele in response to the degradation of the mutant allele or that expression of only one copy of the wild type allele is sufficient for normal levels of Lamin A/C protein to maintain normal physiological function in unaffected cell types. Other potential molecular mechanisms, such as epigenomic alterations specific to patient cardiac tissue [Here, we performed validation studies for Lamin A/C protein expression using Western Blot to resolve these discrepancies. We found that there was no significant differences in Lamin A/C protein expression in mutant fibroblasts. This contrasts the decreased or absent levels of Lamin A/C protein detected previously in patient cardiac tissues , 75. Thuc tissue , 76, tisc tissue , 77, or c tissue , may conWe obtained in total nine dermal fibroblast samples from affected, related unaffected, and unrelated individuals to perform RNA-seq. Though we age-matched and gender-matched the patients to controls to the best of our abilities, we found significant variability between samples within groups. While age is thought to be a factor that can influence gene expression , recent Epigenomic modifications play a role in modulating gene expression –83. We pLMNA splice-site mutation and heart disease. Among these eight genes, we identified IGFBP5 as one of the candidate genes affected by LMNA mutation and propose a possible molecular mechanism that explains the lack of significant molecular and structural phenotype in unaffected tissues. The identification of the ERK/MAPK pathway connecting the candidate genes together supports published studies that implicate this pathway in the development of LMNA-related cardiomyopathy. Our results for normal Lamin A/C protein and LMNA gene expression from only the wild type allele in mutant fibroblasts also suggest that cardiac tissues are more sensitive to decreases in Lamin A/C protein.Using deep RNA-seq and pathway analysis, we identified eight differentially expressed genes in fibroblasts from an affected family with a novel LMNA splice-site mutation. Thus, our results indicate the necessity to develop a model system to study this disease in vitro. To that end, we will generate patient-specific induced pluripotent stem cells lines that will be differentiated to cardiomyocytes for molecular studies to confirm the proposed tissue-specific mechanism of disease.We are aware of the limitations of our studies as presented here. The variation of gene expression within and between sample groups introduced confounding factors to our analysis and our use of an unaffected tissue (fibroblasts) instead of affected tissue (cardiomyocytes) result in a partial explanation of the molecular mechanisms of this particular Additional file 1: Table S1. Sequencing statistics. Table S2. qPCR primer sequences. Table S3. RNA-seq expression values for LMNA, LMNC, and IGFBP5.Additional file 2: Figure S1. IGV screenshots showing expression of LMNA transcripts at exons 1, 2 and 3. Figure S2. Lamin B1 and Lamin B2 heatmap and qPCR validation. Figure S3. Original Lamin A/C Western Blot.


Fanconi anemia (FA) is a DNA repair disorder resulting from mutations in genes encoding for FA DNA repair complex components and is characterized by variable congenital abnormalities, bone marrow failure (BMF), and high incidences of malignancies. FA mosaicism arises from reversion or other compensatory mutations in hematopoietic cells and may be associated with BMF reversal and decreased blood cell sensitivity to DNA-damaging agents (clastogens); this sensitivity is a phenotypic and diagnostic hallmark of FA. Uncertainty regarding the clinical significance of FA mosaicism persists; in some cases, patients have survived multiple decades without BMF or hematologic malignancy, and in others hematologic failure occurred despite the presence of clastogen-resistant cell populations. Assessment of mosaicism is further complicated because clinical evaluation is frequently based on clastogen resistance in lymphocytes, which may arise from reversion events both in lymphoid-specific lineages and in more pluripotent hematopoietic stem/progenitor cells (HSPCs). In this review, we describe diagnostic methods and outcomes in published mosaicism series, including the substantial intervals (1–6 years) over which blood counts normalized, and the relatively favorable clinical course in cases where clastogen resistance was demonstrated in bone marrow progenitors. We also analyzed published FA mosaic cases with emphasis on long-term clinical outcomes when blood count normalization was identified. Blood count normalization in FA mosaicism likely arises from reversion events in long-term primitive HSPCs and is associated with low incidences of BMF or hematologic malignancy. These observations have ramifications for current investigational therapeutic programs in FA intended to enable gene correction in long-term repopulating HSPCs.The online version of this article (10.1007/s00277-020-03954-2) contains supplementary material, which is available to authorized users. Fanconi anemia (FA) is a deoxyribonucleic acid (DNA) repair disorder resulting from mutations in genes encoding for protein components of the FA/BRCA DNA repair pathway. More than 20 identified proteins interact as components of the FA core, FANCI, FANCD2 and downstream complexes. In conjunction with other DNA repair elements, FA proteins facilitate repair of interstrand crosslinks (ICLs) and other forms of DNA damage , 2. FA pFA is characterized by frequent bone marrow failure (BMF), myeloid malignancies, and epithelial cancers , 6. The Somatic mosaicism in FA (hereafter referred to as mosaicism) arises from reversion or other compensatory mutations in hematopoietic stem cell/progenitor cells (HSPCs) from which arise a population of bone marrow and blood cells with a functional DNA repair capacity. Patients in whom a reversion in a pathogenic FA mutation has occurred frequently have two distinct blood cell populations. One population is sensitive to DNA-damaging agents (mitomycin-C [MMC] or diepoxybutane [DEB]) and consistent with an FA diagnosis and another population resistant to these DNA-damaging (or clastogenic) agents – hence the “mosaic” label indicating the coexistence of phenotypically distinct cellular components –10. The FANC gene (FANCC) in 1992 [Mosaicism in FA patients was noted prior to initial identification of the first  in 1992 , with in in 1992 , 13. Sev in 1992 , 14, 15. in 1992 . The mecReversion mutations and resultant mosaicism have been reported in Bloom syndrome, X-linked and adenosine deaminase severe combined immunodeficiency (X-SCID and ADA-SCID), Diamond Blackfan anemia (DBA), Wiskott-Aldrich syndrome, epidermolysis bullosa, tyrosinemia, and dyskeratosis congenita (DC) –25. The In the abovementioned diseases, mosaicism has been described as “natural gene therapy” , 28, andThe presence of a revertant FA hematopoietic cell population has often been associated with a favorable hematologic course in FA patients. However, the presence of clastogen-resistant blood and marrow cells may not uniformly be associated with long-term BMF- or AML-free survival. Gregory and colleagues reported a male patient with hematologic stability during the initial 9–17 years of life and a stable population of DEB-resistant peripheral blood lymphocytes (61–74%). Analysis of bone marrow progenitors at age 15 indicated multilineage presence of the revertant mutation . At ages 15 and 17, this patient nonetheless developed evidence of an 11q deletion in a population of non-revertant bone marrow cells – a cytogenetic abnormality potentially associated with myeloid malignancy . The proDiagnosis of mosaicism in FA is contingent on demonstration of a population of blood and/or bone marrow cells that display resistance to concentrations of DNA-damaging agents that are typically toxic to FA hematopoietic cells. Diagnostic challenges persist because mosaicism may be restricted to specific cell lineages and evaluation of multiple blood and marrow lineages is frequently not clinically feasible. The percentage of resistant cells within a single lineage may not be indicative of the overall hematologic milieu and  may not provide clinically meaningful prognostic information.A critical component of an FA diagnosis and of potential FA mosaicism involves evaluation of cultured T lymphocytes in the presence of DNA-damaging agents, most frequently DEB or MMC. Lymphocytes from FA patients frequently display marked chromosomal instability  in the presence of clastogen concentrations that result in minimal abnormalities in non-FA cells , 10. SomGene sequencing has become an increasingly valuable and utilized modality for the confirmation of an FA diagnosis; however, sequencing of distinct blood and marrow progenitor cell populations has been performed infrequently and continues to be a predominantly research-focused modality , 37. Evan = 5–14 per publication) [FANCA, FANCC, and FANCD2); there is limited published information on FA mosaicism arising from mutations in downstream or more recently discovered FA genes/proteins [FA mosaicism has largely been reported in cases arising from more frequently observed mutations in FA core and ubiquitination complex genes  has been reported, with a limited number of studies showing correlation between the percentages of resistant PB cells and clinical outcomes.  .FA mosaicism has been frequently but not universally associated with stable or increasing hematologic parameters over prolonged follow-up.In the limited settings where normalization of blood counts has been observed over time, increases in blood lineages have been reported over a 1- to 6-year interval (most frequently over 2–3 years), and multilineage increases have been observed in non-contemporaneous  sequences as indicated in Table In the limited settings where normalization of blood counts has been observed over time, platelet normalization has at times been the most indolent and incomplete relative to other lineages, with some increases resulting in levels below normal limits but not requiring clinical intervention, as indicated in Table Evaluation of MMC resistance in bone marrow CFCs has been undertaken infrequently. In the one series in which BM MMC resistance was evaluated, 5 of 5 patients with documented MMC resistance were alive and without hematologic complications at last follow-up  and 2 of 4 patients in whom marrow CFCs were sensitive to MMC died at ages 11 and 18 of BMF and unspecified complications .In addition to the two illustrative and deeply investigated FA mosaic cases described previously, several additional series published over a 16-year period (1997–2013) have detailed the clinical course and essential laboratory correlates in FA mosaic patients. These include 4 publications and a published doctoral thesis, describing outcomes in FA mosaic patients (range: ication) , 38, 39.An additional investigation based on these and other published FA mosaic cases is described in a subsequent section of this review. We undertook this literature-based investigation with an intent of identifying a subset of FA mosaic patients most likely to harbor reversion mutations in long-term repopulating HSCs.Relatively little has been reported regarding the incidence or clinical significance of hypomorphic mutations in FA, in which the impacted protein is expressed with reduced quantity and/or function. Hypomorphic mutations have been described in FANCD2 and were not associated with any reduction in the typically aggressive hematologic manifestations associated with this complementation group; the incidence of mosaicism in FANCD2 also appears comparable to that in FANCA or other core complex FA groups .FANCA hypomorphic mutations were reported, in which mutations resulted in FANCA proteins that localized to cytoplasm and facilitated more normalized mitochondrial function (relative to cells lacking FANCA), with peripheral blood-derived lymphoblast sensitivity comparable to non-mosaic FA patients. The clinical course of this cohort ranged from indolent (n = 3) to severe (n = 2), with the majority of patients (n = 6) encountering moderate single-, bi-, or trilineage cytopenias and receiving allogeneic hematopoietic transplant at ages 9–21 [Recently, a cohort of 11 Sicilian patients with ges 9–21 . It is lges 9–21 .The clinical significance of FA mosaicism remains uncertain more than 20 years after this phenomenon was initially reported. We sought a comprehensive literature-based understanding of FA mosaicism with emphasis on cases most likely emanating from reversion mutations in long-term repopulating HSC populations. Focusing on the minority of FA mosaic cases in which normalization of all peripheral blood lineages was reported, we identified a population of FA mosaic patients in whom there appears to be high likelihood of multi-decade BMF-free and leukemia-free survival.Our objectives were to identify FA mosaicism cases reported in the medical literature for which there was information regarding individual patient clinical outcomes and to further identify the subset of FA mosaic cases in which there was normalization of all peripheral blood lineages (because such cases likely result from reversion mutations in long-term repopulating HSC populations) and to determine clinical course in this clinical subpopulation. In particular, we sought to quantify the incidences of BMF, hematologic malignancy, and other adverse outcomes in FA mosaic patients, including those with blood count normalization and those with no or incomplete normalization.3, hemoglobin to at least 9 g/dL, and platelets to at least 50,000/mm3 after previously lower levels or maintenance of these levels over multiple intervals in settings where earlier counts were not available or presence of these levels at a single timepoint with prior/subsequent hematologic stability reported by the investigator or blood counts normalized or within normal range per investigator without precise values specified.We identified published FA mosaic cases via PubMed searches, reference citations, and a publicly available doctoral thesis on this specific condition. A database was constructed from cases in which there was sufficient clinical information enabling evaluation of age at FA and mosaic diagnosis, duration of follow-up and development of BMF, malignancy, allogeneic hematopoietic stem cell transplant (HSCT), survival, and cause of death. Blood count normalization was defined as improvement in absolute neutrophil count (ANC) to at least 1000/mmn = 123 patients) contained clinical information. A subset of 37 patients (30%) had normalization of blood counts (including 19 for whom detailed blood count information over multiple timepoints was reported). Fifteen FA mosaic patients were reported in whom no or incomplete normalization  was described. Blood count information was not reported in 71 patients. The subgroup of 37 FA mosaic patients with normalized blood counts were reported from investigators in Spain (n = 13), Germany (n = 10), France (n = 8), the USA (n = 5), and Japan (n = 1). Complementation groups of patients in this cohort were Group A (n = 24), Group B (n = 1), Group C (n = 1), Group E (n = 1), Group T (n = 1), and not reported/known (n = 9). Publications detailing these patients are shown in Supplemental Table Figure The clinical outcomes for the 37 FA mosaic patients with normalized blood counts are depicted in Fig. p < 0.0001, Fisher’s Exact Test, 2-sided) and more likely to remain alive (p = 0.0034) relative to FA mosaics without normalization. A lower percentage of mosaic patients with normalization developed hematologic malignancy (3% vs. 14%), but this difference was not statistically significant. The incidences of solid organ malignancies were similar between these subgroups.FA mosaic patients with blood count normalization were less likely to develop BMF  of patients whose peripheral lymphocyte assessments indicate resistance to DNA-damaging agents via standard testing; the potential for reporting bias is such that the actual incidence may be lower; it is likely that fewer than 5% of all FA patients experience blood count normalization as a result of reversion/compensatory mutations. FA mosaicism with normalized blood counts is associated with very limited incidences of BMF or hematologic malignancy, limited requirement for allogeneic HSCT, and relatively limited mortality during the initial 2–4 decades of life. Solid tumors arise during the 3rd and 4th decades of life in FA mosaic patients regardless of blood count normalization, as is the case with non-mosaic FA patients. Two of four FA patients with normalized blood counts who developed solid tumors had a second malignancy diagnosed within several years of the initial cancer diagnosis; solid tumors were fatal in at least 2 of 4 reported patients.These findings indicate that there is a population of FA mosaic patients with very limited risk of BMF or hematologic malignancy, likely as a result of reversion mutations in primitive HSPC populations. Limitations for conclusive analysis result from the absence of bone marrow-based genetic or mitomycin-resistance evaluation in the overwhelming majority of FA mosaic cases. Analysis is also limited because sequential chronologic assessment of peripheral blood or bone marrow clastogen resistance has been performed infrequently. It is very likely that FA mosaicism with normalized blood counts is the result of mutations in long-term HSPCs, but it is not known what percentage of HSPC with reversion events is required to enable stable or normalized peripheral blood cell counts , 37. PreClinical trials of autologous gene therapy in FA have been conducted during the prior decades, with most recent results indicating genetic correction and reversal of sensitivity to DNA-damaging agents at levels resembling those seen in FA mosaic patients , 50, 51.In summary, FA mosaicism is a condition in which varied and frequently divergent degrees of resistance to DNA damage are present within the hematopoietic system of an individual patient, or (more rarely) the entire hematopoietic system displays evidence of normal or near-normal resistance to DNA damage despite clastogen sensitivity in non-hematopoietic tissues. These findings are at times accompanied by normalized or stable peripheral blood counts, with some patients surviving into adulthood without bone marrow failure or hematologic malignancy. Some degree of mosaicism is likely present in 15–20% of FA patients, although the incidence of reversion mutations in primitive HSCs resulting in sustained hematologic stability likely occurs in fewer than 5% , 15, 38.ESM 1(DOCX 21.9 kb)


Our statistical findings provide a new pathway to characterizing metal plasticity and towards comprehension of the sample size effects that limit the mechanical reliability in small-scale structures.Plastic deformation in crystalline materials consists of an ensemble of collective dislocation glide processes, which lead to strain burst emissions in micro-scale samples. To unravel the combined role of crystalline structure, sample size and temperature on these processes, we performed a comprehensive set of strict displacement-controlled micropillar compression experiments in conjunction with large-scale molecular dynamics and physics-based discrete dislocation dynamics simulations. The results indicate that plastic strain bursts consist of numerous individual dislocation glide events, which span over minuscule time intervals. The size distributions of these events exhibit a gradual transition from an incipient power-law slip regime (spanning  This arguably led to the interpretation in that the size distribution of the dislocation bursts fulfils a mean-field depinning phase transition paradigm characterized by a critical 14. Truncation of intermittent responses is a key feature in the scaling of physical phenomena ranging from earthquakes to neural activity15. In crystal plasticity, 13. Alternatively, in the advent of self-organization processes of the dislocation structure during straining, dislocation glide may occur at a fixed criticality level. The slip distribution then becomes independent of the applied stress, leading to the hypothesis of self-organized critically (SOC)18. Along these lines, it may be argued that the self-similar scaling of the dislocation cells arising at large shear strains during single crystal deformation may tentatively result in SOC.Crystal plasticity is characterized by the onset of intermittent dislocation glide events or avalanches, which lead to prominent strain burst emissions when the sample size is reduced to a few micrometers. The emission probability, 18. While the applied stress and dislocation density both fluctuate during subsequent avalanche emissions, a net increase in dislocation density prevails due to the storage of the mobile dislocations within the steady network, yielding a net positive slope 19. This conception is markedly affected by the sample size. In the smaller micrometer and submicrometer-sized free-standing crystals, plasticity is governed by the scarcity of dislocation sources and by the onset of a single-ended dislocation source arrangement pinned at the sample bulk that intermittently produces dislocation avalanches. The term confined plasticity is then coined to embrace the notion in that the formation of the dislocation network is drastically affected by the limited sample size. Under these conditions, surface annihilation of mobile dislocations becomes a fundamental mechanism that prevents dislocation network development27.The full-fledged evolutionary character of the dislocation network becomes manifest during plastic straining of macroscopic material samples, where the mobile dislocations are intermittently pinned by immobile forest counterparts through junction forming processes30.A transition to bulk-like plasticity may occur in micrometer-sized samples where the incipient dislocation structure evolves into a heavily entangled (dense) network containing a myriad of dislocation sources that activate at large applied stresses. Dislocation multiplication thus outweighs surface annihilation. Bulk-like plasticity also develops in larger microcrystals with mildly entangled networks, where the interaction of the mobile dislocations with the forest arrangement already hinders significant surface annihilation. Since the dislocation density may approach similar values as in macroscopic scales, plastic straining occurs at similar levels of applied stresses18. Tentatively, STC would then prevail under vanishing hardening  and body-centered cubic (BCC) dislocation glide mechanisms in micro-scale crystals deforming under confined and bulk-like plasticity.Computational simulations have shown that the interaction between mobile and forest dislocations affects the avalanche distributions. The seminal mean-field understating to this result is that with the increasing dislocation entanglement that occurs upon the onset of subsequent strain bursts, the distance to the critical driving stress remains constant which results in the attainment of an intermittent, steady-state response in the context of SOC. The cut-off slip size then becomes independent of the applied stressHere, we investigate the dislocation mechanisms and statistical features of the individual dislocation glide events which were concealed in the previous investigations of strain bursts in micro-scale sample sizes. A comprehensive set of strict displacement-controlled microcrystal compression experiments and physics-based simulations was performed to gain access to these individual intermittent events, spanning over few nanoseconds, which are regarded as the fundamental constituent elements to dislocation-mediated plasticity. We show that the size distribution of the individual dislocation glide events is characterized by a transition from an incipient power-law slip regime to a large avalanche domain. This transition is a fingerprint of the dislocation glide phenomenology developing in FCC and BCC microcrystals, which governs the onset of confined and bulk-like plasticity in micrometer-sized samples along with the competition between STC and SOC.31.Our microcrystal compression experiments, performed under strict displacement control, enabled the identification of individual dislocation glide events. These are the constituent plastic intermittencies which occur within the strain bursts observed in experiments driven with conventional dynamically-reactive testing systems . The cylindrical pillar-like microcrystals used in our compression experiments were focused ion beam (FIB) milled from [111]-oriented Cu and Ta single crystals, a [100]-oriented Al single crystal, and a [100]-oriented W single crystal. Pillar-like rectangular microcrystals with square cross-sections were also FIB-milled from a [100]-oriented Cu single crystal and a [123]-oriented Al surface. Beam current in the Ga source was reduced to less than 200 pA in all final machining steps to reduce damage. The cylindrical Al microcrystals had a diameter 32. The applied strain rates, 33.The compression experiments were performed using a modified Alemnis nanoindenter  fitted inside the high vacuum chamber of a Zeiss DMS 962 scanning electron microscope (SEM). This system is capable of operating under strict, intrinsic displacement control, thus maintaining indenter tip position during sudden avalanche emissions . The nanoindenter was custom modified to enable testing at high temperatures with the sample and indenter temperature matched to minimize any influence of thermal expansion drift on applied displacementsand non-periodic boundary conditions; where the dislocation density, 35. All simulations were carried out with the LAMMPS code36 under an NPT ensemble, where the uniaxial stress 37 and Sects. 6 and 7 in the Supplementary Material). Upon application of periodic boundary conditions to the simulation cells, the stress–strain curves were taken to represent bulk material responses. The freestanding MD simulations were then exclusively employed to assess slip trace formation at the surface. Details of the strengthening provided by the incepted networks (see Ref.38) are given in Sect. 6 of the Supplementary Material.Molecular dynamics (MD) simulations were conducted by uniaxially compressing cuboidal [110]-oriented Al and cuboidal [100]-oriented Ta simulation cells with periodic 39 to capture confined plasticity responses where dislocation annihilation occurs at the free surfaces. All dislocation reactions are planar in this in-house version of the open source code, further incorporating a set of atomistically-informed, physics-based cross-slip mechanisms, the details of which are described in30. The assumed elastic material properties matched those of FCC Ni (Young's modulus Eν = 0.31). The stress–strain curves were obtained by uniaxially compressing rectangular computational cells with a square cross-section under a nominal strain rate control of −1 . Since the long-range image field resulting from the traction-free boundary conditions give rise to relatively small corrections in the current simulation cells41, this image field is ignored here.The 3D DDD simulations were conducted with an in-house version of the open source code ParaDis42. This process is described through43, the following correlation between the slip magnitudes and the shear strain area swept by the expansion of the unzipped three-dimensional dislocation segments along with the revolving of the surface truncated single-ended dislocation sources in the active slip plane.Representative stress–strain curves under true displacement control in conjunction with the associated surface slip traces are shown in Fig. mentclass2pt{minimmentclass2pt{minimThe avalanche size distribution was investigated by recourse to the complementary cumulative distribution function, An important finding from the current MD and DDD simulations is that microscale plasticity is essentially characterized by the onset of individual avalanche events associated with recurrent stress drops Since the MD simulations indicate that the propagation time frame is release rate is 20 times greater than the elastic energy storage rate prior to the onset of the intermittency. The ratio between these elastic energy rates is reduced to 4 within the small slip domain, while it approaches unity for Finally, the MD simulations illustrate the energetics of the current individual slip events. It is found that within the large avalanche domain, the elastic energy 4, the distribution of the individual dislocation glide events is in better accord with the currently employed relation . Along these lines, the statistical scatter of the power-law exponent 44, see Sect. 3 in the Supplementary Material.Master slip distributions as a function of microcrystal orientation, size and crystalline structure are shown in Figs. 45, detailed investigations in FCC crystals have revealed the intricate connection between a mean, deterministic value of the dislocation swept distance 47. In the light of these investigations, we find 14 m−2 and 13 m−2 which develop under the applied shear stresses of 18.The background to the scaling of the slip distributions stems from Eq. , where iinto Eq.  yields tFigure mentclass2pt{minimThe dislocation mechanisms underlying the above master slip distribution are shown in Fig. ume Fig. a–d. In tWhile the above observations concern bulk-like responses, the DDD simulations incorporate the role of the free surfaces in avalanche propagation at dislocation density levels rendering similar entclass1pt{minimaage Fig. j.The general slip distribution function in Eq.  is maintmentclass2pt{minimThe following discussion concerns the scaling of the slip distributions as a function of the microsample size. In this sense, bulk-like microcrystal plasticity is known to fulfill a classic continuum mechanics description where the deformed state is prescribed by the shear strain entclass1pt{minimaentclass1pt{minima14 to 1015 m-2 where bulk-like microcrystal plasticity develops.Equations  and 3)  3) prescentclass1pt{minimaFigure entclass1pt{minimaThe slip distributions from a second set of experiments in Cu microcrystals, tested as a function of the characteristic sample size ure Fig. , are keymentclass2pt{minimmentclass2pt{minim-2; As the sample diameter decreases further towards submicrometer sizes (i.e. Assessment of the correlation between the magnitude of the slip events and the applied shear stresses in the active slip systems is finally illustrated in Fig. entclass1pt{minimaexperiment does not provide a significant number of slip events within a window of stress so as to enable assertion of the possible stress-binning of function Along the above analyses, it is noted that a single displacement-controlled The slip distributions in BCC microcrystals, shown in Fig. mentclass2pt{minim27 at applied stresses reaching the distinctive large Peierls barrier50. Plasticity thus sets-in at D 51 leads to the wavy slip trace emissions observed in the W microcrystals (see Fig. In submicrometer-sized pillars deformed at RT, plastic deformation proceeds through single-ended source operationmentclass2pt{minimmentclass2pt{minimmentclass2pt{minim51 is consistent with the observed wavy glide in the W microcrystals (3 μm When mentclass2pt{minim52. The MD simulations with free-standing cells that contain similar entangled dislocation networks then predict the predominance of nanoscale, wavy slip features as the meandering mobile dislocation segments released from the dislocation structure intercept the surface (Fig. in the absence of cross-kinking. The development of planar, localized glide has been observed in bulk BCC crystals53 which also exhibit the smooth stress–strain curves in Fig. A highly dense dislocation network may also develop in the larger microcrystals (mentclass2pt{minimlarge applied stresses in Fig. smaller stresses. It is noted that the larger smallerConcerning the statistical analysis of the slip distributions, Fig. mentclass2pt{minim51, as well as the marked rise in the velocity of the mobile dislocations. The large avalanche domain is then associated with the gliding of several dislocation segments along predominantly edge sides, a feature that is facilitated by the enhanced mobility of the lateral, partnering screws. The edge segments thus sweep across The MD simulations reveal that irrespective of the testing temperature, the incipient slip domain is governed by the gliding of individual screw and edge dislocation segments over a mean free path length cf. Fig.  and S3. smaller W microcrystals (large applied stresses (larger W microcrystals (smaller applied stresses (The influence of sample size on the slip distribution is less clear at elevated temperatures Fig. a, where sed Fig. b. To ratmentclass2pt{minimOur RT experiments finally show that function cf. Fig. d,e, so tSTC Fig. c.Prior investigations of plastic intermittencies involved the large strain bursts triggered with dynamically-reactive testing systems. Under these driving conditions, the microcrystal is rapidly compressed as a function of the system’s dynamic response, which ultimately governs the termination of the instability and its overall size. We argue that a dynamically-induced plastic instability or strain burst is comprised of many of the currently measured individual dislocation avalanche events, propagating over minuscule time scales, which are ubiquitous to crystal plasticity. Our investigation provides in-depth statistical and mechanistic understandings to these individual ultrafast dislocation avalanche events, extending our knowledge of intermittent plasticity.8. It is comprised of multiple dislocation segments that simultaneously glide at numerous locations in the microcrystal without significant dislocation density build-up.Our experiments and simulations consistently show that the size distribution of the individual dislocation avalanche events is characterized by a power-law, incipient slip regime which spans over up to 2 decades of plastic slip sizes (vis-à-vis the onset of collective dislocation glide, dislocation interaction and surface dislocation annihilation mechanisms—in intermittent crystal plasticity. This provides insight into the role of the underlying slip phenomenology upon the dislocation avalanche distribution59.A large avalanche domain with reduced emission probability sets-in upon the truncation of the incipient slip regime at the cut-off slip size n in Eq. . It is s45. In the absence of a marked increase of the dislocation density decrease if plastic straining involved pronounced dislocation storage at increasing applied stresses (increase in Bulk-like plasticity in FCC Al and Cu microcrystals is characterized by a homogeneous slip trace distribution along with sample barreling. Large avalanche propagation then results in the storage of dislocation segments through junction forming processes. In continuum microcrystal plasticity analyses, the cut-off slip higher60 and moderate Pure FCC microcrystals that deform under confined plasticity exhibit greater values of the cut-off slip Larger values of the cut-off slip smaller BCC microcrystals, where plasticity is triggered through single-ended source operation under the large shear stress level surpassing the Peierls barrier of the screw segments. This indicates an increase of the product The slip distributions in BCC microcrystals are governed by the sluggish glide of the screw dislocation segments. In spite of the markedly different BCC dislocation glide phenomenology, it is found that the range of The following are the salient conclusions from our work:Supplementary Information.Supplementary Video 1.Supplementary Video 2.Supplementary Video 3.


To compare the diagnostic accuracy (ACC) in the detection of acute posterior circulation strokes between qualitative evaluation of software-generated colour maps and automatic assessment of CT perfusion (CTP) parameters.Were retrospectively collected 50 patients suspected of acute posterior circulation stroke who underwent to CTP  within 24 h after symptom onset between January 2016 and December 2018. The Posterior circulation-Acute Stroke Prognosis Early CT Score (pc-ASPECTS) was used for quantifying the extent of ischaemic areas on non-contrast (NC)CT and colour-coded maps generated by CTP4 (GE) and RAPID (iSchemia View) software. Final pc-ASPECTS was calculated on follow-up NCCT and/or MRI (Philips Intera 3.0 T or Philips Achieva Ingenia 1.5 T). RAPID software also elaborated automatic quantitative mismatch maps.By qualitative evaluation of colour-coded maps, MTT-CTP4D and Tmax-RAPID showed the highest sensitivity (SE)  and ACC  compared with the other perfusion parameters . Baseline NCCT and CBF provided by RAPID quantitative perfusion mismatch maps had the lowest SE  and ACC . CBF and Tmax assessment provided by quantitative RAPID perfusion mismatch maps showed significant lower SE and ACC than qualitative evaluation. No significant differences were found between the pc-ASPECTSs assessed on colour-coded MTT and Tmax maps neither between the scores assessed on colour-coded CBV-CTP4D and CBF-RAPID maps.Qualitative analysis of colour-coded maps resulted more sensitive and accurate in the detection of ischaemic changes than automatic quantitative analysis. Posterior circulation (PC) stroke accounts for 20–25% of ischaemic strokes and is characterised by a wide range of clinical features that make its clinical diagnosis challenging –12. In eVisual inspection of CTP colour-coded maps can be an effective way to discriminate areas of core infarct and penumbra and may be enough to guide therapeutic choice. It is rapid and simple to use but its strengths may be affected by variability in post-processing, broad range of imaging and computational approaches, selection of parametric maps, expertise skills and the generally qualitative nature of such approaches .On the other side, quantitative CTP analysis has reported to be efficient in demonstrating acute ischaemia, distinguishing salvageable from unsalvageable ischaemic tissue and predicting therapeutic outcome, although protocols and guidelines for quantitative thresholds vary –33. DiffIn this context, the recent introduction of a fast, vendor- and operator-independent computational tool working by totally automated lesion segmentation and pixel-wise parametric thresholding for semi-quantitation (RApid processing of PerfusIon and Diffusion [RAPID]) seems to overcome the limits of qualitative approaches to CTP imaging in acute stroke , 35–37. The diagnostic value of CTP in the posterior circulation has been little investigated with few evidences of its reliability and accuracy; moreover, the diagnostic effects of RAPID introduction in the evaluation of infratentorial ischaemic changes have not yet been clarified , 38–40. The aim of this study was to examine and compare the diagnostic accuracy (ACC) in the detection of acute posterior circulation strokes between qualitative evaluation of software-generated colour maps  and automatic assessment of CTP data by RAPID software.According to the approval of the local ethical committee , imaging data were retrospectively collected from a prospective database of 1498 consecutive patients admitted to the stroke unit of our hospital between January 2016 and December 2018. Written informed consent was waived for this study. All patients were initially evaluated by vascular neurologist in the emergency setting with initiation of an institutional stroke protocol facilitating expedited triage, imaging, interpretation and treatment when appropriate. Patients were enrolled regardless of therapy. The inclusion criteria for this study were (1) suspected acute posterior circulation ischaemic stroke as defined in the Oxfordshire classification ; (2) mulOut of all patients who underwent multimodal CT due to suspected acute stroke, were selected control patients who fulfilled the same inclusion and exclusion criteria as our case cohort but did not show an acute posterior circulation ischaemia on follow-up CT/MRI scan neither change in at least two perfusion parameters in the same location.Patients underwent an institutional stroke imaging protocol including NCCT, CTA and CTP at admission. CT protocol was conducted on a 40-mm, 64-detector row clinical system . NCCT helical scans were performed from the skull base to the vertex . CTAs were conducted from the aortic arch to the top of frontal sinus  after intravenous administration of 60–70 mL iodinated contrast medium injected at 4 mL/s and followed by 50 mL saline flush.CTP scans  consisted of a continuous 65 s acquisition that started 7 s after administration of 50 mL of iodinated contrast medium injected into an antecubital vein at 4 mL/s and followed by 50 mL saline flush; 400 images were reconstructed covering 80 mm. CTP coverage was lowered to cover the cerebellum to the occipital lobes, including all three levels of the pc-ASPECTS.Follow-up imaging consisted in NCCT. If clinically indicated, MRI was performed either soon after the CTP at the admission or later as follow-up control. MRI exams were achieved using two scanners  including diffusion-weighted imaging (DWI) and fluid-attenuated inversion recovery in the transverse plane.Raw CTP data were initially processed by a commercially available delay-insensitive deconvolution software . All patients were obtained CBF, CBV and MTT maps after positioning a region of interest manually in correspondence to a main arterial vessel and a vein. Then, raw CTP data was sent from scanner to a networked computer running fully automated RAPID software  which generated CBF, CBV, MTT, Tmax maps and lesion segmentation and then sent processed images to institutional Picture and Achieving System (PACS). The software also generated a colour perfusion mismatch map for each patient based on the mismatch model Tmax–CBF, providing a comparison between brain regions with substantial reductions in CBF (rCBF < 30%) in pink representing the ischaemic core and regions with significant hypoperfusion in green as reflected by delays in contrast arrival times (Tmax > 6.0 s). The difference between these volumes (mismatch volume) as well as the ratio between these volumes (mismatch ratio) was automatically calculated.The neuroimaging data was independently reviewed by two board-certified neuroradiologists  blinded to patient clinical information using the institutional PACS; any discrepancies were resolved through a consensus discussion in a separate session.For NCCT, early ischaemic changes were assessed; an area was considered ischaemic if there was parenchymal hypoattenuation with or without cortical swelling of the brain , 43. AreFor CTP, a qualitative evaluation of perfusion deficit was initially performed on colour-coded CBV, CBF and MTT maps generated by CTP4D software. Regions with colorimetric asymmetries on CTP maps in comparison with non-affected regions  were rated as abnormal. A visible qualitative focal reduction in CBF or CBV and a focal increase MTT on colour-coded maps was rated as abnormal and evaluated with pc-ASPECTS. When a perfusion deficit was suspected in the maps, it was compared with the NCCT scan to exclude false positive caused by beam hardening artefacts, chronic infarct or overlapping with liquoral spaces .In the same way, in a separate scoring session, colour-coded CBF and Tmax maps automatically generated by RAPID software were then visually evaluated assessing pc-ASPECTS for each map. For each patient, the quantitative colour perfusion mismatch map was also evaluated: brain regions with reductions in CBF (< 30%) appeared in pink suggesting ischaemic core while regions with significant increase of Tmax (> 6.0 s) indicating hypoperfusion appeared in green; the abnormal areas identified on perfusion mismatch maps were compared with perfusion changes location on colour-coded maps to assess anatomic consistency in order to exclude artefacts.PCT images showing no alterations in the same location in at least two visually evaluated perfusion parameter maps neither in automatic maps were considered negative .Standard follow-up imaging consisted in NCCT for all patients which was used to determine the presence of an infarct. Final pc-ASPECTS was calculated on follow-up NCCT and/or on MRI if performed.Follow-up images were compared with PCT studies for anatomic correlation. The initial NCCT and PCT data was then compared with the follow-up NCCT data or MRI data if available, which was used as criterion standard to calculate sensitivity (SE), specificity (SP) and diagnostic ACC of the abnormalities assessed on NCCT scan and CTP maps for the detection of posterior circulation ischaemic stroke.For each patient, NCCT and PCT maps showing no ischaemic changes despite follow-up imaging that confirmed the stroke were considered as false negative. PCT maps showing alterations out of the territory of the affected arterial vessel  were considered as false positive. NCCT and CTP ischaemic changes were considered true positives if there was an anatomic vascular correspondence to findings on follow-up imaging. NCCT and PCT images showing no alterations in patients whose follow-up imaging did not confirm the stroke were considered as true negative.the correspondence between the infarcted area (core) assessed with qualitative evaluation of both colour-coded CTP4D maps and RAPID maps and the ischaemic lesion confirmed on direct follow-up CT or MRI;the correspondence among the perfusion changes identified with the qualitative analysis of colour-coded CTP4D maps, colour-coded RAPID maps and the lesions spotted in the RAPID software-generated colour perfusion mismatch map;the comparison of the respective ASPECTSs evaluated on NCCT, colour-coded CTP4D maps and colour-coded RAPID maps with the final ASPECTSs assessed on direct follow-up CT or MRI.Main evaluations were:Q tests were used to compare differences of percentages among three or more dependent variables, under the consideration of the null hypothesis that there was no difference between the variables. A value of p < 0.05 was considered statistically significant. When the Cochran’s Q test was positive , then a minimum required difference for a significant difference between two proportions was calculated using the minimum required differences method with Bonferroni p value corrected for multiple comparisons according to Sheskin. The McNemar’s exact test was used to test the difference between two paired proportions. Additional statistical tests were performed on stroke patient parameters using the Friedman’s ANOVA test. Particularly, if Friedman’s ANOVA test was positive , a Wilcoxon signed rank post hoc test was performed to individualise significant differences between two diagnostic parameters.The statistical analysis was performed by Matlab statistical toolbox version 2008  for Windows at 32 bit. SE, SP and ACC for selected parameters were computed with correspondence confidence intervals (CIs) at 95%. The multiple comparison Cochran’s Data are expressed as mean value ± standard deviation (SD) for continuous variables and as absolute number or percentage for categorical variables.± 72.6 h and 76.6 ± 98.8 h. Out of the 28 patients who undergone MRI, 14 patients (50%) underwent DWI study within 8 h after multimodal CT study at admission. Vessel occlusion was located in one of more of the posterior circulation branches involving extracranial vertebral artery in 9 patients, intracranial vertebral artery in 5 patients, basilar artery in 15 patients, posterior cerebral artery in 22 patients and superior cerebellar artery in 7 patients. Inside local stroke protocol multimodal neuroimaging  was used for decision making about inclusion/exclusion criteria for revascularization therapies as intravenous thrombolysis and mechanical thrombectomy. In our study, 26 patients had intravenous thrombolysis, 6 patients underwent to thrombectomy/thromboaspiration, 7 patients had intravenous thrombolysis and underwent to thrombectomy/thromboaspiration while 5 patients were not treated. Negative controls were not treated.Among 69 patients meeting inclusion criteria, a total of 50 patients respected the exclusion criteria. Six out of the 50 patients did not show ischaemic core at follow-up imaging neither alteration of at least two perfusion parameters in the same location and were grouped as negative controls. All patients underwent to follow-up NCCT and 28 of them also underwent DWI-MRI. Mean time from multimodal CT study at admission to follow-up CT study and MRI were, respectively, 34.2 Table Table 1 and CBF1), and automatic quantitative Tmax and CBF RAPID software-generated perfusion mismatch map (Tmax2 and CBF2), were compared.SE, SP and diagnostic ACC of qualitative evaluation on baseline NCCT, colour-coded CTP4D maps , colour-coded Tmax and CBF RAPID maps , while NCCT at baseline and CBF2 provided by RAPID quantitative perfusion mismatch maps had the lowest values . NCCT at baseline and CBF2 provided by RAPID quantitative perfusion mismatch maps also showed false negative cases in most patients variously located in one or more pc-ASPECTS territories. Automatic Tmax2 evaluation provided by RAPID overlooked 11 ischaemic lesions which mostly (8/11) were lacunar infarcts involving thalamus (4/8) or brainstem (4/8) while the remaining were larger lesions involving occipital lobe an cerebellum (3/11) . For CBV and CBF parameters, the number of false negative cases located in one or more pc-ASPECTS regions probably reflected the potentially salvageable tissue. However, it is relevant that automatic assessment of CBF2 provided by RAPID mismatch map resulted negative for core extent in most (41/44) of patients, among them 20 were lacunar infarcts involving thalamus (15/20) and brainstem (5/20).The sensitivity of qualitative evaluation of colour-coded MTT map and colour-coded Tmax1 maps and automatic evaluation of Tmax2 provided by RAPID quantitative perfusion mismatch maps were characterised by some false positive cases showing perfusion disturbance in anterior circulation territory.About specificity, there was a significant difference among parameters but post hoc test did not confirm significant differences considering all pairwise comparison. However, qualitative evaluation of MTT and Tmax1 showed the highest values  while CT at baseline and CBF2 provided by RAPID quantitative perfusion mismatch maps had the lowest values . Between MTT and Tmax1 map, there was no significant difference  in terms of diagnostic accuracy.About diagnostic accuracy, MTT and Tmax1 and CBF2 parameters  and between Tmax1 and Tmax2 parameters   Table .Table 3C2 assessment provided by quantitative RAPID perfusion mismatch maps showed significant lowest sensitivity (6.8%) in comparison with qualitative evaluations of both colour-coded CBF-CTP4D and CBF1-RAPID maps ; no significant difference of sensitivity was found between qualitative evaluations of colour-coded CBF-CTP4D and CBF1-RAPID maps . Analogous results were observed for diagnostic accuracy, while there were no significant differences of specificity among CBF, CBF1 and CBF2 parameters .CBF1-RAPID maps showed significant higher sensitivity and accuracy than quantitative assessment of Tmax2 automatically provided by RAPID perfusion mismatch maps  while about specificity, there were no significant difference .Qualitative evaluation of colour-coded Tmax1 and CBF1 maps was established by the exact count approach: MTT, 0.82; CBV, 0.88; CBF, 0.86; Tmax1, 0.84; CBF1, 0.8.Interobserver agreement for visual evaluation and ASPECT scoring of MTT, CBV, CBF, Tmax1 and CBF1 maps generated by RAPID software and the reference standard imaging (follow-up NCCT or MRI)] in stroke patients were analysed by the Friedman’s ANOVA test and pairwise Wilcoxon signed-rank post hoc test  neither between MTT (colour-coded CTP4D) and Tmax1 (colour-coded Rapid). No significant differences were even found between CBV  and CBF1  values. NCCT at baseline, instead, significantly differed from all the other parameters.Pc-ASPECTSs assessed for each qualitative imaging evaluation [NCCT at baseline, visually evaluated colour-coded MTT, CBV and CBF maps generated by CTP4D software, visually evaluated colour-coded TmaxBrain imaging plays a key role in the evaluation of patients suspected of ischaemic stroke allowing to reach a confident diagnosis of infratentorial stroke , 12. MRIWhile the infarcted core typically shows a uniform decrease in CBF and CBV, the penumbra is characterised by a reduction of the CBF value with a normal or even increased CBV due to the vasodilation of the precapillary arteries and venous obstruction , 19. SucPrior studies have shown that ischaemic core presents > 70% reduction in CBF (rCBF < 0.3) in comparison with the mean CBF of normally perfused brain parenchyma while a Tmax delay of > 6 s is a good predictor of critically hypoperfused tissue that is at risk to progress to infarction in the absence of timely reperfusion; RAPID mismatch maps in our study were automatically elaborated according to these thresholds , 28–33.Concordant to other studies, our findings confirmed that CTP in PC allows to detect significantly more ischaemic strokes than NCCT , 5, 39. p > 0.05) and ACC  despite a superiority of Tmax as described in literature [Perfusion change in the posterior circulation has previously been described to be most frequent and pronounced on MTT and Tmax maps , 5, 38. terature .2 (provided by RAPID perfusion mismatch maps) presented significant lower sensitivity and accuracy compared with qualitative visual evaluation of Tmax1 maps. Automatic Tmax2 evaluation, qualitative evaluation of MTT and Tmax1 overlooked ischaemic changes mainly located in pons-midbrain regions and thalamus.Automatic quantitative assessment of Tmax1 had low specificity for perfusion deficits [Despite potential shortcomings due to beam hardening artefacts, previous studies investigating the specificity of CTP in the detection of posterior circulation infarcts demonstrated specificity higher than 90% , 17. In deficits .The detection of the ischaemic core is most relevant for the prediction of the functional outcome of stroke patients and core extent is described to be better delineated on CBF compared with CBV maps because CBV parameter may underestimate core extent , 38.p > 0.05).In agreement with these evidences, in our study, qualitative evaluation of CTP4D maps showed lower sensitivity and accuracy of CBV parameter compared with CBF ones although without significant difference  and accuracy . Instead, the automatic quantitative assessment of CBF2 (provided by RAPID perfusion mismatch maps) presented significant lower sensitivity and accuracy as compared with qualitative visual evaluation of both CTP4D and RAPID colour-coded CBF maps. CBV, CBF, CBF1 and CBF2 parameters presented the highest specificity among all assessed CTP parameters without false-positive cases; this is a critical point to not erroneously exclude patients from therapeutic intervention.CTP maps, especially CBF abnormalities, are described to significantly differ among commercial software even when using identical source data ; however2 evaluation  was characterised by significant low sensitivity and accuracy values resulting less adequate even than NCCT for detecting ischaemic lesions in PC regions. Indeed, automatic RAPID assessment did not identify ischaemic lesions visually recognised on NCCT in 10 patients   Fig. . Only inThe determination of pc-ASPECTs on colour-coded perfusion maps does not require any volumetric software and can be applied to NCCT as well to colour-coded perfusion maps .In our study, the pc-ASPECTSs assessed by NCCT were mainly higher and significantly different from the corresponding final pc-ASPECTs evaluated on control CT/MRI scan and NCCT presented the lowest rate of scores equal to the final ones. These findings are clearly related to the low SE of NCCT which can depict only the ischaemic core and not perfusion changes at risk of infarction.1 maps resulting MTT and Tmax1 the most efficient parameters to evaluate the patients’ status. This finding can be interpreted as the result of the progression of ischaemic hypoperfusion to necrosis in most of our patients. Moreover, MTT and Tmax1 maps led to assign lower ASPECTS significantly more  and higher ASPECTS significantly less  frequently than the other parameters; a lower score than final one reflected the presence of hypoperfusion not progressed to necrosis while a higher score than final one can be interpreted as a worsening of ischaemic damage and/or as an overlooked ischaemic change. In four patients with midbrain infarct, all perfusion colour-coded maps were scored 10 and the automatic quantitative assessment did not identify these lesions.We found no significant differences among the scores assessed on control CT/MRI scan, colour-coded MTT and Tmax1 maps neither between the scores assessed on colour-coded CBV and CBF1 maps; according to this, in our study, the mismatch models MTT-CBV and Tmax1-CBF could be considered comparable being not different the determination of hypoperfusion by MTT and Tmax1 neither different the assessment of core extent by CBV and CBF1.Interesting was that no significant differences were found between the scores assessed on colour-coded MTT and TmaxA possible limitation of this study is the retrospective design. Intravenous thrombolysis and thrombectomy may also have influenced SE and SP of perfusion maps because they can lead to temporary reperfusion in the potentially salvageable penumbra. Furthermore, the use of 64-slice CT did not allow the study of the whole parenchyma and a careful positioning of the slab was necessary although resulting not always adequate. This could have influenced the SE of CTP maps.In this study, CTP showed good diagnostic accuracy in the identification of acute vascular ischaemic lesions of the PC and the infarct detection of CTP was significantly higher than NCCT , 39. The1 as well as between CBV and CBF1 suggesting the equivalence of both mismatch models (MTT-CBV and Tmax-CBF) commonly applied in acute setting with implications for treatment decision-making.Independently to the software employed, qualitative analysis of colour-coded maps resulted more sensitive in the detection of ischaemic changes than automatic quantitative analysis. RAPID software-generated mismatch maps overlooked and underestimated the extent of the ischaemic core in the major part of the patients as compared with the qualitative analysis . In our For these reasons, the multimodal evaluation of the whole neuroradiological examination  must be underlined .Given the advantages of a more rapid and operator independence elaboration of perfusion maps and their easier interpretation, this opens the potential for automatic software implementation and optimization of perfusion parameters’ thresholds for the evaluation of posterior circulation ischaemia , 35.


Neurofibromatosis type 1 (NF1) is characterized by major and minor cutaneous findings, whose recognition plays a key role in the early diagnosis of the disease. The disease affects multiple systems and clinical manifestation has a wide range of variability. Symptoms and clinical signs may occur over the lifetime, and the complications are very diverse. Although significant progress has been made in understanding the pathophysiology of the disease, no specific treatment has been defined. Multidisciplinary approach is required to provide optimum care for the patients. The aim of this paper is to provide the clinician with a complete guide of skin findings of NF1.Neurofibromatosis type 1 (NF1) is a complex autosomal dominant disorder associated with germline mutations in the NF1 tumor suppressor gene. NF1 belongs to a class of congenital anomaly syndromes called RASopathies, a group of rare genetic conditions caused by mutations in the Ras/mitogen-activated protein kinase pathway. Generally, NF1 patients present with dermatologic manifestations. In this review the main features of café-au-lait macules, freckling, neurofibromas, juvenile xanthogranuloma, nevus anemicus and other cutaneous findings will be discussed. Neurofibromatosis type 1 (NF1) is a complex autosomal dominant disorder associated with germline mutations in the NF1 tumor suppressor gene . GeneralNF1 gene is needed in order to be effective. Half of the cases are familial, but the remaining half arise from de novo NF1 mutations. Descendants of an NF1 patient are at a 50% risk of inheriting the altered NF1 gene; however, the disease manifestations vary extremely even within the same family members. In addition, somatic mutations in NF1 during fetal development also cause phenotype diversity, known as mosaic NF1 [NF1 is inherited in an autosomal dominant pattern, and a single copy of a mutated or deleted saic NF1 .NF1 gene encodes neurofibromin protein which interacts with the Ras-specific guanosine triphosphatase (GTPase), a family of proteins involved in cellular signal transduction, controlling cell proliferation. Overactive Ras-GTPase signals lead to tumor growth. Neurofibromin has the role of downregulating the RAS/MAPK signaling pathway and suppressing cell overgrowth. Therefore, the NF1 gene is considered to be a tumor suppressor gene and has been associated with benign and malignant tumors in many different areas from nerve cells to myeloid cells [id cells .NF1 gene, which has been identified so far as having more than 3000 pathological allele variants, is a large gene covering 275 kb of genomic DNA and containing almost 60 constitutive exons located at 17q11.2 [NF1 gene [A diagnosis of NF1 is clinically established by the presence of two or more of the following diagnostic criteria as formulated by the National Institute of Health (NIH) in 1988: six or more cafe-au-lait macules (CALMs) (>5 mm in children or >15 mm adults), two or more cutaneous neurofibromas (CNFs) or one plexiform neurofibroma (PNF), axillary or inguinal freckling, optic nerve glioma (OPG), two or more Lisch nodules, distinctive bony dysplasia , and first degree relative with NF1 . AlthougNF1 gene .NF1 gene with nonfamilial facial dysmorphism, a lower IQ and possibly increased incidence of malignant peripheral nerve sheath tumors (MPNSTs). Codeletion of SUZ12, especially, within the common NF1-microdeletion has been identified to be a significant alteration for the malignant transformation [p.Arg1809 [NF1 gene identified as a single amino acid NF1 deletion (c.2970–2972 delAAT) has been related to mild form of NF1 disease without visible CNFs or PNFs [The clinical presentation of NF1 may be multifactorial. While biallelic inactivation is necessary for the development of some clinical findings such as CALMs and neurofibromas, some manifest in haploinsufficiency of NF1. Additional genetic alterations also affect the disease course as in the malignant transformation of peripheral nerve sheath. Even though clear genotype–phenotype correlations, which are hard to establish in NF1, there are some important relations identified. One study investigating mutation and phenotype trends found that NF1 was more prevalent in patients with truncating/splicing mutations and large deletions than in those with missense mutations. They also reported more items of NF1 in patients with truncating/splicing mutations . Anotherormation . Noonan- or PNFs . Koczkow or PNFs . FurtherNF1 gene. It occurs usually with no antecedent family history. NF1 in mosaic forms can be present as generalized, segmental, or gonadal [NF1 gene in either the Schwann cells of neurofibromas or in the melanocytes of CALMs in individuals with different types of segmental disease [Mosaic NF1 is caused by postzygotic somatic mutation in the  gonadal . Early s gonadal . Maerten disease . While i disease   . CALMs mAnother type of CALM which has irregular jagged margins (“coast of Maine”) is mostly solitary and larger; arrangement in a blaschkoid pattern is suggestive of sporadic cases or other genetic disorders. Additionally, it is extraordinary for new sporadic CALMs to occur after the age of 6 .Histological findings are basal hyperpigmentation and sometimes giant melanosomes (macromelanasomes), although diagnosis is clinical and pathology rarely necessary. Recently, mutation of both alleles of the NF1 gene has been detected in the melanocytes of NF1-CALM while normal skin has a germline mutation . CALMs iFreckling in skinfold is one of the seven cardinal diagnostic features of NF1. They are described as small (1–4 mm), clustered pigmented macules located mostly in axillary and inguinal regions (Crowe sign) . It is cLisch nodules are benign iris hamartomas which are seen as densely pigmented flat or slightly elevated lesions during the slit-lamp examination. These lesions are the most common NF1 feature in adults. They appear earlier than neurofibromas and multiple Lisch nodules are specific to NF1. They do not have any ophthalmologic complication, no association has been reported with clinical severity, and therefore no specific treatment is needed. However, periodical examination to detect these benign hamartomas may be useful to confirm the diagnosis of young NF1 patients .Neurofibromatosis is associated with many kinds of tumors but the neurofibromas are the most prevalent benign tumors found in the affected individual, hence the appellation neurofibromatosis. They also have a negative impact on the life of NF1 patients. Different variants have been described related to NF1; CNFs or dermal neurofibromas, subcutaneous neurofibromas and PNFs ,30. WhilThousands of discrete CNFs, fleshy, pink to brown pedunculated or sessile papulonodules, depict the most common dermatological finding in NF1. They may be soft or firm in texture ranging from few millimeters to few centimeters located anywhere along the course of peripheral nerves. “Buttonhole sign” describes the pathogonomic invagination of the lesion with pressure. Subcutaneous neurofibromas arise as nodules in deep dermis or the subcutaneous layer, usually presenting a less obvious appearance than CNFs. Both cutaneous and subcutaneous neurofibromas may initially appear as slightly raised discolored skin patches and most typically they become visible in adolescence. They may grow and increase in number with age; acceleration may occur during pregnancy  Figure .Blue red macules and pseudoatrophic macules are bizarre variants of cutaneous neurofibroma. If there is the predominance of wide vessels in neurofibromatous tissue located in papillary dermis, the lesion may appear as blue-red macules. Pseudoatrophic macules occur as a result of neurofibromatous tissue proliferation damaging collagen in reticular dermis. These two presentations are less frequent types of neurofibromas and they fulfill the diagnostic criterion of NF1 .Physical removal appears to be the most effective and practical treatment for CNFs as there is no gold standard approach. Surgical removal, laser ablation, modified biopsy removal, photocoagulation, electrodessication and radiofrequency ablation are the modalities that have been developed. Camouflage and moisturizers may also provide some relief for patients . In a prPNFs are specific for NF1 and they affect between 30% and 50% of patients ,37. PNFsNF1 gene [NF1 gene. Spinal NF1 is characterized by multiple bilaterally located spinal and paraspinal neurofibromas with very few dermal manifestations of classical NF1. This form should be differentiated due to life-threatening massive neoplastic burden [PNFs are associated with increased morbidity and mortality in NF1. Distortion of limbs or head, soft tissue and bone deformities, degeneration of airway or spinal cord and neurological deficits may occur due to invasion . Spinal NF1 gene . As we mc burden . Althougc burden . The RAFc burden .MPNSTs are aggressive sarcomas which may develop in 3–15% of patients with NF1 . They moNF1 gene in Schwann cells seems to be an important initiating factor in the development of MPNST, genomic studies show that the malignant degeneration arises as a result of some additional alterations. Somatic loss-of-function mutations or deletions of the Polycomb repressive complex 2 (PRC2) its core subunits (EED or SUZ12) have been described in NF-1-associated MPNSTs. PRC2 plays a role in chromatin compaction, cell proliferation, differentiation and stem-cell plasticity. A cooperative role of TP53, epidermal growth factor receptor (EGFR) inactivation has also been proposed to contribute MNPST transformation. CDKN2A loss has also been linked to early steps of MPNST [Although the inactivation of the loss of biallelicity of the of MPNST .While high-grade MPNST often metastasizes to bone and lung, low-grade tumors have a better prognosis. The main therapy remains a complete surgical excision with negative margins . DiscussMetastatic disease remains challenging due to a lack of promising therapies. Chemotherapy is known to be the standard approach. Biological therapies combining MEK/mTOR inhibition are still under investigation in preclinical and clinical trials. Pexidartinib (tyrosine kinase inhibitor) and sirolimus (mTOR inhibitor) combination contributed to stabilize the disease in a phase I study . AdditioIn terms of the future direction of the treatments, gene-based therapies targeting PRC2, the Janus Kinase (JAK) pathway and HIPPO pathway are presented as a new hope. EZH2 inhibitors—such as tazemetostat for the PRC2 complex, Tyrosine Kinase 2 (TYK2) inhibitors targeting the JAK/STAT pathway and verteporfin inhibiting the HIPPO pathway—could be considered in the combination treatments .NF1 is one of the most common heritable brain tumor predisposition disorders, in which affected individuals develop low-grade gliomas classified as WHO grade I gliomas (pilocytic astrocytomas). They are generally indolent neoplasms usually occurring before the age of 8 located within the optic pathway . OPGs ocAlthough prognostic features are not clear, clinical experiences show that girls experience visual impairment more than boys. Additionally, postchiasmatic optic pathway-located OPGs and tumors that occur before the age of 2 appear to be more aggressive. After diagnosis, performing an eye examination and visual assessment with MRI is requested every 3 months in the first year for follow up.Chemotherapy remains a mainstay treatment. Surgery seems to be problematic due to the location of these tumors and radiation is known to be a trigger for secondary malignancies along with vasculopathy. mTOR and MEK inhibitors for the treatment of optic pathway gliomas are still in progress .Nevus anemicus (NA) is a congenital, cutaneous anomaly characterized by hypopigmented, well-defined patches with limited vascular supply. While rubbing or heat causes redness of normal skin, the NA lesion remains pale. The vascular structure of the lesion is found to be normal but the excessive response to catecholamines causes vasoconstriction. NA is also seen in the normal population, but prospective studies suggest that prevalence of NA in NF1 patients is much higher—around 50%, affecting both sexes equally ,60,61. TJuvenile xanthogranuloma (JXG) is the most common form of non-Langerhans-cell histiocytosis characterized by yellowish-pink dome-shaped granulomatous tumors often located on the upper side of the body. It may be solitary or multiple and may start as a pink macule. The estimated prevalence is around 15–35% in NF1 children. JXG may be present as early as CALM, therefore it was suggested as a potential sign for the diagnosis of NF1. The lesion may disappear spontaneously ,63. JuveNF1 gene or a “second hit” that occurs specifically within the α-smooth muscle actin-positive cells of the glomus body has been added to an NF1-related tumor spectrum [Glomus tumor is a benign neoplasm that arises from the neuromyoarterial glomus body or the Suquet–Hoyer canal. Lately, the discovery of biallelic inactivation of the spectrum . It is ospectrum . There aspectrum ,71.NF1 patients have about four-fold higher risk of neoplasms  through NF1 alteration comes along with an increased risk of developing malignant conditions such as MNPST, leukemia, rhabdomyosarcoma and adrenal gland tumors. Pheochromocytoma is a rare benign adrenal medulla tumor that is capable of produce excessive amounts of catecholamine resulting in tachycardia, hypertension, sweating, and flushing. The overall incidence is low—0.1–5.7% of patients with NF develop pheochromocytoma—mostly between the third and fourth decade of life. However, the specific alteration responsible for the association is still unknown. It is usually solitary and benign but long-term follow-up is recommended. Surgical removal is the standard approach for the treatment .Rhabdomyosarcoma (RMS) is the most common soft-tissue sarcoma in children. Sporadic RMS is more common, but RASopathies and disorders with TP53 alterations such as NF1 have also been associated with RMS. Around 1% of patients with NF1 develop RMS, specifically embryonal-type due to RAS activation in this form. RMS in NF1 cases seems to have early onset favoring the pelvic location. Careful clinical examination and attention to any sign pointing out possible RMS are important for early diagnosis. NF1-associated RMS requires the same treatment protocol as the sporadic cases .Recently, several reports have depicted the relationship between gastrointestinal stromal tumors (GISTs) and NF1. NF1-associated-GISTs tend to occur at a younger age in distal locations of the gastrointestinal tract. They generally have lower mitotic indexes and do not show typical KIT/platelet-derived growth factor receptor alpha mutations .NF1 is a genetic disorder which needs a collaborative and interdisciplinary approach to understand and manage the condition. The future direction of therapies seems to direct cell type-specific growth control pathways with mutation-directed therapeutics in a personalized manner.


Screening the polar bear genome identified only 13 numts. Genomic analyses of two additional ursine bears and giant panda indicate that all except one of the discovered numts entered the bear lineage at least 14 million years ago. However, short read genome assemblies might lead to an under-representation of numts or other repetitive sequences. Our findings suggest low integration rates of numts in bears and a loss of the original polar bear mitochondrial genome.Phylogenetic analyses of nuclear and mitochondrial genomes indicate that polar bears captured the brown bear mitochondrial genome 160,000 years ago, leading to an extinction of the original polar bear mitochondrial genome. However, mitochondrial DNA occasionally integrates into the nuclear genome, forming pseudogenes called  Polar and brown bears are two well-recognized species, which differ in their morphology and ecology Nowak . Recent numts    genome while 1859 copies (2093 kb) are found in opossum (Monodelphis domestica) . If indeed, at 160 kya, the polar bear mt genome was replaced by the brown bear mt genome, all polar bear numts that entered the nuclear genome between the divergence of both species (∼600 kya) and the mitochondrial capture event (∼160 kya) are genomic ‘fossils’ of the original polar bear mtDNA , which represent 57% of the mt genome and represent mainly the NADH1 to COII region. Both rRNA genes as well as NADH4 and NADH5 are only partially covered  (Supplementary Data 1). Fourteen of the 22 identified numts form clusters consisting of two or three numt fragments localized on the same scaffold (Supplementary Table 2). For each cluster, the scaffold localization, the gene order and orientation suggest that these are eroded fragments from single longer ancient integrations. Merging the consecutive fragments in a cluster reduced the total number to 13 numts (Supplementary Table 2). Aligning the genomic polar bear numt loci to orthologous sequences from the giant panda (Ailuropoda melanoleuca) . Numts were fragmented by interspersed transposable elements  or expanding short tandem repeats (loci 6 and 11). Some of these interspersed sequences were only found in polar bear or giant panda, causing different inter-fragment distances (Supplementary Table 3). A full LINE1-1_Ame transposable element was inserted in locus 10 in the giant panda genome. Nearly all identified polar bear numt sequences seem to have been inserted prior to the evolution of Ursidae and no numts representing ancestral or recent polar bear mtDNA have entered the nuclear genome  caller Lumpy , brown and polar bear, which gives a minimum age of 3 My  and rat only have between 6-39 kb numts in their respective genomes, which is among the lowest numbers of numts in mammals  and thus facilitate their incorporation into genome assemblies (Sohn & Nam numts in Ursidae.However, another important reason for the lack of recent numts create problems for population and phylogenetic analyses of mt genes, if they are PCR amplified instead of the mt genes (Bensasson et al. Recently inserted numts. The majority of the identified numt insertions have a minimum age of 14 My. A low numt insertion frequency has been reported for rodents and might be common in other mammalian groups. Thus, a low number of numts in bear genomes is not unreasonable, but an artefact from whole genome assembly algorithms cannot be excluded until further de novo assembled bear genomes become available. Utilizing long-read high-throughput technologies like PacBio or Nanopore might yield further insight into the fate of numts in Ursidae or other taxonomic groups with suspected numt depletion.The current polar bear genome assembly lacks recently inserted Click here for additional data file.