Hereditary spastic paraplegia (HSP) is one of the most genetically heterogeneous neurological disorders. HSP is classified as pure when only a spastic weakness of the lower extremities is present. Complex HSP comes with additional neurological or systemic abnormalities. Complex HSP with skeletal abnormalities is rare and mostly seen in autosomal recessive HSP. Autosomal dominant (AD) complex HSP with skeletal abnormalities are consistently seen only in SPG9 (spastic gait type 9). In this paper, we report a kindred condition with AD HSP among four living affected individuals who had progressive, adult onset spastic paraparesis that was associated with a distal arthrogryposis (DA) in every affected individual. They also had episodes of rhabdomyolysis without any clinical signs of myopathy. Exhaustive genetic analysis including targeted sequencing of known HSP and DA genes and whole exome sequencing did not identify the disease-causing gene. It excluded all known HSP and DA genes. We propose that this is a novel genetic type of complex AD HSP. Elucidation of a genetic cause of this type of HSP will further contribute to our understanding of axonal degeneration and skeletal abnormalities. Hereditary spastic paraplegia (HSP), which is also known as Strumpell-Lorraine disease, is a clinically and genetically heterogeneous group of neurodegenerative disorders characterized by progressive spasticity and weakness of the lower extremities ,2,3. AltEven though it is now accepted that many patients with an apparently pure HSP commonly have additional neurologic problems including dementia in SPG4 (spastic gait 4) and polyneuropathy in several types of autosomal dominant (AD) HSP, this clinical classification remains a useful clinical concept ,5,6. ExtSPG20/spartin gene, is a prototypical complicated HSP with skeletal abnormalities including short stature, foot deformities, and kyphoscoliosis. These are cardinal features of this entity [PNPLA6 gene-related group of disorders known as SPG39 [Troyer syndrome, which is caused by homozygous mutations of the s entity ,8. Spartas SPG39 .ERLIN2 gene, SPG28 with biallelic DDHD1 (Phoshatidic acid-preferring phospholipase A1) mutations, and SPG43 with mutations in the C19ORF12 gene among others [Multiple congenital contractures affecting at least two different body segments are also known as arthrogryposis and they can be seen in more than 300 heterogeneous conditions . Many tiClinically isolated distal arthrogryposes are a group of AD disorders that mainly involve the distal parts of the hands and feet including pes equinovarus without a primary muscle disease or any other syndromic findings. The prototypic distal arthrogryposis is type 1, which is characterized largely by camptodactyly and clubfoot ,12. The TPM2), fast troponin T (TNNT3), fast troponin I (TNNI2), embryonic myosin heavy chain (MYH3), fetal myosin heavy chain (MYH8), and slow myosin binding protein C (MYBPC1) [TNNI2, which may have more prominent myopathic changes and an elevation of serum creatine kinase (CPK) [Autosomal dominant distal arthrogryposes are typically caused by mutations in different sarcomeric proteins including β-tropomyosin ((MYBPC1) ,14. MuscIn this paper, we report a kindred condition with AD HSP among four affected individuals who had progressive, adult onset spastic paraparesis that was associated with a congenital distal arthrogryposis in every affected individual and episodic rhabdomyolysis without any other signs of myopathy. Extensive genetic evaluation including whole exome sequencing did not identify any causative mutations. We propose that this is a novel type of complex AD HSP.We identified a small 2-generational kindred condition with four affected living individuals through a proband , SPG3A (atlastin 1), NIPA1, REEP1, and KIF5A genes were analyzed as candidate genes for HSP. β-tropomyosin (TPM2), fast troponin T (TNNT3), fast troponin I (TNNI2), embryonic myosin heavy chain (MYH3), fetal myosin heavy chain (MYH8), and the slow myosin binding protein C (MYBPC1) were included as candidate genes of distal arthrogryposis. Clinical genetic analysis also included an analysis of copy number variation (CNV) using the multiplex ligation-dependent robe amplification (MLPA) assay to detect possible deletions or duplication. This was performed using a commercial laboratory. Subjects III/1 and III/2 were analyzed.The phenotype of each individual was determined as definitely affected or unaffected before genetic analysis. DNA was available from all affected living subjects and it was isolated using standard procedures. The first phase of genetic analysis was focused on the exclusion of candidate genes for AD HSP and AD distal arthrogryposis type 1. Coding exons of analyzed genes were amplified using the polymerase chain reaction with published or custom-designed intron-based primers and conditions. Polymerase chain reaction products were purified through Sephadex G-50 columns  and sequenced using an ABI PRISM dRhodamine Terminator Cycle Sequencing Ready Reaction and the ABI PRISM 3100 Genetic Analyzer , which was described previously . Each exXT All Exon Kit 51 Mb . Enriched DNA fragments were sequenced on the Illumina's HiSeq 2500 platform, which were paired-end 100–125 bp reads. On average, more than 95% of exons were covered at >20×. The percentage of exome coverage was based on exons targeted by the 51 Mb All Exon v4 Kit , which incorporates the Consensus Coding Sequence (CCDS), the NCBI Reference Sequence (RefSeq), and GENCODE annotations. Sequence reads (FASTQ) from Illumina were mapped to the human reference genome (NCBI build 37.1).The second stage of genetic analysis was performed with whole exome sequencing (WES) of three subjects  using Illumina HiSeq 2500 platform . Genomic DNA was sheared to yield 100–450 base pair (bp) fragments, which was described before . In-soluVariants were annotated to RefSeq gene definitions using ANNOVAR software . DamaginAll included subjects (four living and one deceased) had a history of congenital distal arthrogryposis that was more prominent in the hands . The sevMotor and psychosocial development was normal in every affected subject and they acquired all milestones without any delays. All affected individuals had gait with a tendency for tiptoeing and this was attributed to reduced dorsiflexion of ankles caused by feet deformities. This has resolved by five years of age and they did not have any signs of early spasticity and their gait was later reported as normal. First, neurological problems were reported in the third decade of life and were described as episodes of weakness and muscle pain including passing very dark urine. Medical records were available for subjects III/1 and III/2 and they had documented episodes of rhabdomyolysis with CPK elevations in the range of 20,000 U/L to 50,000 U/L. Some episodes were provoked by more strenuous activities but also unprovoked episodes were reported in every affected individual. The baseline CPK levels between the episodes of rhabdomyolysis were normal.Changes in gait were reported during the third or fourth decade of life. At the ages of 30 years, 39 years, 27 years, and 32 years, subjects I/1, II/1, II/2, III/1, and III/2, respectively, were reported to have an onset of tripping and stiffness. They had signs of otherwise isolated spastic paraparesis with a reduced vibratory sensation. No signs of spasticity were present in the upper extremities. Their gait was spastic with a typical scissoring and no signs of ataxia. They had a tendency to walk on tiptoes and this was associated with reduced passive dorsoflexion in ankles. No other neurologic abnormalities were present and they all had a normal proximal strength and a mild distal weakness attributed to arthrogryposis.Proband underwent additional clinical testing and his repeated electromyography with nerve conduction studies did not show any signs of myopathy or neuropathy. An MRI of the brain and C spine was unremarkable. A skeletal survey did not identify any other bone abnormalities other than hand and foot deformities. A muscle biopsy was also considered normal and he did not have any specific abnormalities with a normal, random pattern of fiber types and no evidence of group atrophy or fiber type grouping. There were no “ragged red” fibers, target fibers, or abnormal inclusions. An occasional and very mild variation in fiber sizes was observed, but this was considered non-specific. Enzymatic testing of the muscle tissue focusing on the myoglobinuria profile was also within normal limits. Baseline CPK levels were within normal values.Sequencing of candidate genes associated with AD HSP and AD artrogryposis did not reveal any disease-causing mutations. We were also able to exclude the SPG9 locus because of a random distribution of reconstructed haplotypes. CNV MLPA analysis did not detect any deletions or novel duplications that were considered pathogenic. WES identified 20,856 variants for subject II/2, 20,789, and 20,047 for subject II/3. 960 variants that were non-synonymous or potentially affected splice sites were selected for further analysis. No definite disease-causing mutations were identified in the kindred condition and we identified more than 40 putative non-coding and coding changes that would segregate with the disease (data not shown).HSP has emerged as one of the most genetically diverse syndromes with nearly 70 disease-causing genes identified so far ,2. The eWe propose that this type of HSP represents a novel type of AD complex HSP. Affected individuals exhibited congenital arthrogryposis, which was followed by the development of recurrent episodes of myoglobinuria without the evidence of myopathy. Later they experienced adult onset of a spastic gait. Even though the likelihood of coexisting separate conditions cannot be fully excluded without any proven unifying genetic cause, this is somewhat unlikely because we observed a complete co-segregation of these diverse phenotypic features in all analyzed patients. Furthermore, the likelihood that WES would not identify either of two or more causative genes, if one assumes a coexistence of several independent phenotypic features, is also quite low. The overlap of three different phenotypic features, which include arthrogryposis, spastic paraplegia, and recurrent rhabdomyolysis, may also suggest a contiguous gene syndrome caused by deletions of several genes. However, clinical analysis of copy number variation did not identify any large deletions that could account for this mechanism. An additional possibility is a biogenic inheritance, but the pattern of transmission in the kindred condition is more consistent with a straightforward AD mode. This would be only the third type of AD complex HSP where musculoskeletal abnormalities are a constant feature of the clinical phenotype.ALDH18A1) gene exhibit spastic paraplegia associated with short stature and bone dysplasia, gastroesophageal reflux, hiatal hernia, and cataracts [ZFYVE27/protrudin gene [ZFYVE27/protrudin mutation p.G191V have been challenged and it was suggested that it may actually represent a benign single nucleotide polymorphism [Musculoskeletal problems are relatively rare in AD HSP . Patientataracts ,26. Thisataracts . Anotherdin gene . Only onmorphism .Pathogenesis of HSP is very diverse but the abnormalities of intracellular transport have emerged as one of the cardinal mechanisms of distal axonal degeneration ,31,32. WWe describe a novel type of AD complex HSP with skeletal abnormalities and episodes of rhabdomyolysis without any clinical evidence of myopathy. Our extensive genetic analysis including WES has not identified a causative gene but the additional analysis is ongoing. Identification of new kindreds with the same phenotype should facilitate the discovery of a genetic cause of this type of HSP.


IL6) may contribute to the placenta’s inflammatory response, thus predisposing some pregnancies to aCA. These genetic variants may modulate molecular processes such as DNA methylation and gene expression, and in turn might affect susceptibility to aCA. Currently, there is remarkably little research on the role of fetal  genetic variation in aCA. We aimed to explore the associations between genetic variants in candidate immune-system genes and susceptibility towards inflammatory responses in the placenta, which is linked to a strong inflammatory response in the newborn.Acute chorioamnionitis (aCA), inflammation of the placenta and fetal membranes, is a frequently reported lesion in preterm deliveries. Genetic variants in innate immune system genes such as Interleukin-6  were collected for this study. Samples were genotyped at 55 ancestry informative markers (AIMs) and 16 additional single nucleotide polymorphisms (SNPs) in 12 candidate innate immune system genes using the Sequenom iPLEX Gold Assay. Publicly available datasets were used to obtain DNA methylation  and gene expression data .IL6 placental allele frequencies were associated with aCA  with the CC genotype specifically implicated . In a subset of the placental samples , we showed that the IL6 SNP (rs1800796) was associated with differential DNA methylation in five IL6-related CpG sites , where individuals with a CC genotype showed higher DNA methylation levels than individuals carrying the GG genotype. Using two publicly available datasets, we observed that the DNA methylation levels at cg01770232 negatively correlated with IL6 gene expression in the placenta .Differences in IL6 SNP (rs1800796), which is largely limited to East Asian populations, is associated with the presence of aCA. This SNP was associated with increased DNA methylation at a nearby MEPC2 binding site, which was also associated with decreased expression of IL6 in the placenta. Decreased expression of IL6 may increase vulnerability to microbial infection. Additional studies are required to confirm this association in Asian populations with larger sample sizes.We demonstrated that the minor C allele at the The online version of this article (10.1186/s12881-019-0768-0) contains supplementary material, which is available to authorized users. Preterm birth (PTB) refers to all births occurring before 37 weeks of gestation, as defined by the World Health Organization . PTB occGenetic susceptibility for aCA can be hypothesized based on: i) high heritability estimates of PTB (15–30%) , 9, ii) TLRs) is associated with sPTB [TLR genes has been shown to modulate immune responses during parturition, and thus confer an altered risk of preterm delivery [IL6) have also been associated with intrauterine infection and/or inflammation in sPTB [IL6 in maternal serum, cervical secretions and amniotic fluid are associated with sPTB [EBF1, EEFSEC, and AGTR2 genes were replicated in an independent cohort of > 8000 women, none of the identified genes had been previously identified in sPTB or known to have a direct role in inflammatory mechanisms [Studies investigating candidate genes have reported that maternal and fetal genetic variation in Toll-like receptors . The study cohort is based on an ongoing collection of samples for our Epigenetics in Pregnancy study (EPIC), and overlaps with samples described in previous publications related to placental DNA methylation –58. MostPlacentas were collected from pregnancies delivered at the Children’s & Women’s Health Centre, Vancouver, Canada. Placentas from 72 aCA cases were selected based on a diagnosis of aCA determined by pathological examination of the placenta and associated membranes using consensus histological criteria . AnotherDemographic characteristics of the study cohort is presented in Table Further, the aCA cases and the non-aCA cases were sampled from a single urban population (Vancouver) and delivered at a single centre, BC Children’s & Women’s Health Centre which is located in a high socio-economic status neighborhood. Of the documented observations for maternal smoking status (80/269), almost all (79/80) identified themselves as non-smokers. Additionally, the most reproducible finding between maternal smoking and altered DNA methylation is observed at sites linked with AHRR and CYP1A1. We therefore tested for differences at these sites and did not observe altered DNA methylation associated with our pathology at these sites in our study cohort (data not shown).Chorionic villus samples were obtained from the fetal side of the placenta at multiple distinct sites (cotyledons). Samples were thoroughly washed with phosphate-buffered saline to avoid maternal blood and amniotic fluid contamination. Placental DNA was extracted by a standard salting out procedure, modified from Miller et al. . A NanoDSNPs in 12 innate immune system genes were chosen based on published findings of an association with any of chorioamnionitis –50, placGenetic association studies are prone to population stratification if case and control groups are not matched by ancestry. Because ancestry was largely unknown in the study cohort, we also selected 55 ancestry informative marker (AIM) SNPs to genotype in our population as previously described . These An = 1), and ii) removal of SNPs with call rate < 90%  and 1 aCA candidate SNP (rs4986790). After primary quality control, each gene involving multiple SNPs was investigated for linkage disequilibrium (LD) to determine if the SNPs were independent of one another. Strong evidence for LD was observed for two SNP pairs (rs1800795–rs1800796 in IL6 and rs1800896–rs2222202 in IL10) with D’ = 0.99. Haplotype analysis was performed for these two SNP pairs to investigate whether carriers of a specific haplotype had increased susceptibility for aCA. The 15 SNPs were also tested for Hardy-Weinberg Equilibrium (HWE) to detect genotyping error and/or population stratification.All samples were genotyped using the Sequenom iPlex Gold platform at the Génome Québec Innovation Centre . Primary quality control of the genotype data comprised of the following steps i) removal of samples with call rate < 90%  used as ancestry reference populations, as described in Del Gobbo et al. (2018) [k-means clustering, with k = 3, was used to cluster samples into three groups of common ancestry.After initial quality control, 50 AIM SNPs were used to infer ancestry in 269 placental villus DNA samples. Ancestry was described using the top 3 coordinates derived from a multidimensional scaling (MDS) analysis of AIM SNP genotypes of the placental villus samples and . (2018) . Using tn = 67). Some of these  were assessed on the Illumina Infinium HumanMethylation450 BeadChip  [n = 42) were assessed on the Illumina Infinium HumanMethylationEPIC BeadChip  [minfi package [sva package [To investigate whether candidate SNPs were associated with altered DNA methylation, we used our previously obtained DNA methylation microarray data that was available for a subset of placental (chorionic villus) samples n = . Some ofG sites) . Probes G sites) . To acco package . Princip package . DNA met package .Further, we took advantage of publicly available datasets to determine whether DNA methylation levels correlated with gene expression at the associated gene. Our group has previously published gene expression data (GSE44711)  on a setp-values for Table https://www.snpstats.net/start.htm) [http://osse.bii.a-star.edu.sg/index.php).All statistical analyses were performed using R version 3.4.1. art.htm) . Comparip > 0.05, Fig. p < 0.01). Because rs1800796, rs1800795 and rs1554973 deviated from HWE, we next investigated whether the frequencies of these genetic variants were associated with ancestry. Significant differences in the distribution of the two ancestry MDS coordinates between the SNP genotypes was observed . .To investigate whether placental  genetic variation may lead to an increased susceptibility to developing aCA, we genotyped 16 SNPs within 12 innate immune system genes among our study cohort samples . We limited our analysis to SNPs previously implicated in aCA or related phenomenon. Given our small sample size, we estimated that our study had 50% power to detect differences in allele frequencies associated with aCA, at a IL6 rs1800796 SNP is known to be strongly correlated with ancestry. Based on 1000 Genomes Project data, the C allele is common in individuals of East Asian (70–80%), and rare in individuals of European ancestry (3–5%) (https://www.ncbi.nlm.nih.gov/variation/tools/1000genomes/). We also found that the genotype frequencies of this SNP varied between different ancestries in our study population  that were largely of East Asian ancestry . Specifically, 8 of the 12 (67%) cases of aCA in cluster 3 had the “CC” genotype as compared to 10 of 29 (34%) non-CA cases. In our study cohort, rs1800796 was in nearly complete LD with rs1800795 (D’ = 0.99); however, unlike rs1800796, rs1800795 was polymorphic in individuals of European ancestry .The IL6 and DNA methylation of the CpGs in the promoter region of IL6 as a mechanism by which genetic variants may modulate disease risk [IL6 SNP rs1800796 was associated with differential DNA methylation in IL6-related CpG sites (n = 8 CpGs) in a subset chorionic villus samples for which DNA methylation microarray data were available (n = 67). Modest (r > 0.5) to strong (r > 0.7) correlations were observed between β values across most of the CpG sites , cg02335517 , cg07998387 , cg13104385 ; and cg0526589 , whereby homozygous C samples showed significant hypermethylation compared to homozygous G samples   . This allele was linked to increased DNA methylation, at both an upstream regulatory region and within the gene body, and associated with decreased expression of IL6.In our study sample, we were able to confirm that the C allele in the Interleukin-6 is a pleiotropic cytokine with a wide range of biological functions . PrimariIL6 SNPs rs1800796 and rs1800795 were previously associated with increased incidence of aCA and development of sepsis in children in a study undertaken in Finland [PGR locus, specifically the PGR SNP rs11224580, that significantly modulates PGR expression in the ovary has been shown to be common in East Asians compared to individuals of European and African ancestry [IL6) and rs11224580 (PGR) exhibit extreme population-specific allelic variation, these genomic loci are likely to have undergone positive selection that is specific to Asian populations [The  Finland . In the  Finland –87; howe Finland , 88, 89.ancestry , but in ancestry . Becauseulations , 91.IL6 expression in the placenta. As IL6 mediates a protective immune response against microbial infections [IL6 expression observed in the placenta may predispose an individual to infection, by preventing an appropriate innate immune response to microbes.In addition to genetic variation, circulating levels of IL6 in the serum and amniotic fluid may influence the risk for aCA. Although placental trophoblast cells have the capacity to synthesize IL6 , 93, thefections , 95–98, IL6 in modulating innate immune response has been well-elucidated, molecular mechanisms such as DNA methylation underlying the genetic regulation of IL6 transcription have rarely been investigated [IL6 genetic variants in modulating DNA methylation patterns in aCA-affected placentas. We identified that carriers of the rs1800796 C allele had increased placental DNA methylation levels at multiple IL6-related CpGs, most significantly at cg01770232, which is located at an upstream enhancer, compared to individuals with IL6 G allele. Although this CpG has been described as linked to an mQTL in blood, we showed that this relationship also exists in the placenta. Further, we observed that aCA cases were more methylated than the non-aCA cases at cg01770232, cg07998387, and cg02335517  [IL6 repression in pancreatic adenocarcinoma cell lines is facilitated by binding of methyl-CpG-binding protein (MeCP2) and H3meK9 to the methylated CpGs spanning from positions − 666 to − 426 relative from the transcription start site of IL6. Interestingly, rs1800796 and cg01770232 is located at position 572 and 662 respectively, and it is therefore possible that rs1800796 alters the binding of MeCP2 and H3meK9 to cg01770232, thereby affecting IL6 expression and DNA methylation, though this has not been tested in placental tissue. Further, the IL6 upstream region contains several (A/T)> 4 motifs adjacent to the methylated CpGs including cg01770232, shown to mediate high-affinity MeCP2 binding [IL6 may contribute towards disease pathogenesis in aCA.The DNA methylation patterns in the placenta may imply a primary phenomenon where increased DNA methylation at cg01770232 is associated with an increased risk of developing aCA in individuals carrying the . (2009)  demonstr binding . UnderstIL6 rs1800796 polymorphism, and confirmed that DNA methylation changes correlated with changes in IL6 gene expression, we could not examine whether IL6 protein levels in maternal blood would reflect placental DNA methylation and expression. Further, potential confounding factors for our study, such as socio-economic status, maternal smoking status, maternal alcohol use, and PPROM status, were not documented for all the cases in our study cohort and thus not accounted for in statistical analyses. Finally, our results only highlight the biological significance of placental  genetic variants in aCA, but this does not exclude the role of maternal genetic factors in altering disease risk to aCA, given that maternal genetic effects have been shown as important contributors to PTB [Overall, the present study has limitations. Our sample size was relatively small, especially among the genetically homogenous subpopulations; therefore the findings of this study should be evaluated in larger subpopulations of different ancestries. In particular, the lack of an association between the other genetic variants we investigated and aCA might be a result of our small sample size. Although we utilized publicly available datasets to investigate functional consequences of the s to PTB .IL6 SNP (rs1800796), which is largely limited to individuals of East Asian ancestry, is associated with aCA. Placentas with the CC genotype exhibited increased DNA methylation at multiple CpG sites upstream and within IL6, as compared to those of GG genotype. This increased DNA methylation was associated with lower expression of IL6 and with aCA status. While overexpression of IL6 is associated with various inflammatory conditions, this can be a consequence of infection; whereas innate IL6 deficiency can lead to impaired immunity against microbial infection. Taken together, we conclude that IL6 genetic polymorphisms may influence susceptibility to aCA by affecting the risk of acute infection. Larger samples sizes are needed to confirm these findings.Our findings suggest that a placental  CC genotype at the Additional file 1:Figure S1. Shows the association of SNP genotypes  with ancestry in study cohort. Figure S2. Shows the correlation of β values across eight IL6-related CpGs. Figure S3. Shows differential methylation of IL6-related CpGs based on IL6 genotype status (rs1800796). Figure S4. Shows altered DNA methylation at IL6-related CpGs is associated with aCA status. Figure S5. Shows the correlation between placental DNA methylation and gene expression at IL6 locus. Figure S6. Shows no association of IL6 expression with gestational age, fetal sex and preeclampsia status. (DOCX 1205 kb)Additional file 2:Table S1. Table listing the allele counts and frequencies of the SNPs used in the statistical analyses. Table S2. Table listing the counts per genotype for IL6 SNPs based on the three clusters. (XLSX 20 kb)


Lactobacillus and Alistipes, that could be implicated in the management of endothelial and inflammatory dysfunctions associated with CVD, and to unravel the role of nutrition in the modulation of those bacteria.The vascular dysfunction is the primary event in the occurrence of cardio-vascular risk, and no treatment exists until now. We tested for the first time the hypothesis that chitin-glucan (CG) - an insoluble fibre with prebiotic properties- and polyphenol-rich pomegranate peel extract (PPE) can improve endothelial and inflammatory disorders in a mouse model of cardiovascular disease (CVD), namely by modulating the gut microbiota. Male Apolipoprotein E knock-out (ApoE−/−) mice fed a high fat (HF) diet developed a significant endothelial dysfunction attested by atherosclerotic plaques and increasing abundance of caveolin-1 in aorta. The supplementation with CG + PPE in the HF diet reduced inflammatory markers both in the liver and in the visceral adipose tissue together with a reduction of hepatic triglycerides. In addition, it increased the activating form of endothelial NO-synthase in mesenteric arteries and the heme-nitrosylated haemoglobin (Hb-NO) blood levels as compared with HF fed ApoE−/− mice, suggesting a higher capacity of mesenteric arteries to produce nitric oxide (NO). This study allows to pinpoint gut bacteria, namely  Given the growing prevalence of obesity, type 2 diabetes and metabolic syndrome, it is estimated that the number of people with chronic cardiometabolic disease will continue to grow in the next two decades2. Obese individuals that develop atherosclerotic CVDs are often characterized by a low-grade chronic inflammation. It has been hypothesized that changes in inflammatory status partly explain the individual differences in cardiovascular risk2. Endothelial dysfunction is an early key marker of CVDs reflecting the integrated effects of risk factors on the vascular system3. It comes from the incapacity of endothelial cells to equilibrate synthesis and release of damaging or protective mediators, among which nitric oxide (NO) is the most important. The main feature of endothelial dysfunction is the inability of endothelium to promote vasodilation in response to agonist or shear forces4.Despite the enormous progress made in the diagnosis and treatment of cardiovascular diseases (CVDs) over the last twenty years, CVDs remain the leading cause of disability and death in Western countries5. Endothelium-dependent relaxation is reduced in response to acetylcholine in 13-months-old ApoE−/− mice compared to wild-type mice6. However, this is not observed in youngest mice, suggesting that vascular disorders occurring in ApoE deficiency depend on age. Obesity are associated to altered vascular functions, with impaired vessel reactivity and lower endothelium-dependent relaxation. Interestingly, high fat (HF) diet feeding accelerates the process of atherosclerosis in ApoE−/− mice7. In addition, HF diets participate to worsening metabolic and vascular phenotypes which negatively influences the evolution of CVD in this murine model9.Apolipoprotein E (ApoE) is a key mediator for transport and reuptake of cholesterol. ApoE knock-out mice (ApoE−/−) are proposed as a model of atherosclerosis and endothelial dysfunction10. The gut microbiota can be considered as a “microbial organ” under the influence of the diet and host factors. Hence, the gut microbiota and its activity are intricately intertwined with the physiology and pathophysiology of the host13. Obesity in humans has been associated with the composition and function of the gut microbiota, but only a few studies focus on the role of gut microbiota in the CVDs1.The pathophysiology of CVDs is complex and involves multiple organs, establishing distant communications via bloodstream. Recently, a growing body of evidence has emerged regarding the critical role of gut microbiota in CVDs and metabolic disorders14. For years, we and others have demonstrated that dietary fibre supplementation modulates the composition of the gut microbiota, and eventually improves host physiology16. This is particularly the case for inulin-type fructans (ITF), which are classified as prebiotics. Prebiotics are ‘substrates that are selectively utilized by host microorganisms conferring a health benefit’17. Whether CVD and atherosclerosis can be successfully treated by fibre targeting the microbiota is still not clear. ITF-fed ApoE −/− mice have been shown to have reduced cholesterol and atherosclerotic lesions compared to control mice. However, the mechanism was unexplored18. Recently, we demonstrated that ITF improve endothelial dysfunction in ApoE−/− mice fed a n-3 PUFA-depleted diet and developing steatosis. We found that ITF-induced changes in gut microbiota linked endogenous GLP-1 production with an improved endothelial function19. If proven in humans, those studies suggest that prebiotics might be proposed as a novel approach in the prevention of metabolic disorders-related CVDs.Modulation of the diet with specific dietary components could be used to prevent atherosclerosis. Dietary fibre is thought to have beneficial effects on health because it is considered to reduce the risk of CVD. In humans, this effect could be attributed to a decrease in plasma LDLin vitro SHIME microbiota simulator20. In a previous study, we have highlighted beneficial effects of this insoluble fibre on the development of obesity and associated diabetes and hepatic steatosis in mice, through a mechanism related to the restoration of the composition and/or the activity of gut bacteria21.Chitin-glucan (CG) has been shown to modulate the human gut microbiota in the In the present study, we have tested the prebiotic potency of chitin-glucan, an insoluble dietary fibre, alone or in combination with a pomegranate peel extract (PPE) rich in polyphenols in a model of accelerated atherosclerosis in ApoE−/− mice fed a high fat diet during 8 weeks.The HF diet significantly increased body weight gain and the development of epididymal, visceral and subcutaneous adipose tissues of ApoE−/− mice as compared with control diet Fig. . The fatIt is interesting to note that ApoE−/− mice upon HF feeding and treated with CG or CG + PPE exhibited the higher cecal tissue and cecal content weight compared to HF group; this effect reached statistical significance for the cecal tissue from the mice treated with the combination Table .The HF diet did not significantly affect lipid content, cholesterol content and triglyceride content of the liver tissue from the ApoE−/− mice compared to CT group as shown in Fig. Fas), a key enzyme involved in lipogenesis and carnitine palmitoyl transferase-1 (Cpt1), a marker of fatty acid oxidation. None of the dietary treatments significantly affect Fas or Cpt1 expression (data not shown).In addition, we measured the expression of two hepatic genes regulating lipid metabolism: fatty acid synthase , the lipopolysaccharide binding protein (LBP), a key pattern recognition receptor (Toll-like Receptor-4 (TLR4)), one of the most potent chemokines identified for monocytes recruitment (monocyte chemotactic protein-1 (MCP1)), two important proinflammatory cytokines  and interleukin-1β (Il1β)) and the gene coding for cyclooxygenase (COX-2) that produces prostaglandin E2, a regulator of inflammation, both in the liver and the visceral adipose tissues24. Several markers of macrophage infiltration and/or inflammation were induced in the visceral adipose tissue (p < 0.05 ANOVA for Mcp1) and in the liver  of ApoE−/− mice due to HF feeding as compared to CT group . In line with a potential reduced endothelial function25, aorta showed increased abundance of caveolin-1 , the negative allosteric regulator of endothelial NO synthase (eNOS)  in conductance (thoracic) versus resistance (mesenteric) arteries  Fig. . We perfies Fig. . Interesies Fig. .Figure 4Mesenteric arteries segments were mounted in wire myograph in order to evaluate endothelial function. Resting parameters revealed no difference  between the four groups concerning the basal tone, the maximal contraction after a KCl challenge and the mean arterial diameter . The endothelium-dependent relaxation was studied upon acetylcholine addition in the incubation medium on pre-constricted arteries in the presence of a KCl-enriched solution and in the presence of a cyclooxygenase inhibitor (indomethacin) or a NO synthase inhibitor (L-NAME). Mesenteric arteries isolated from HF treated ApoE−/− mice relaxed less in response to acetylcholine than vessels obtained from ApoE−/− mice fed a control diet, suggesting the presence of endothelial dysfunction in HF fed mice whatever the supplementation Fig. . In contAkkermansia and Alistipes among other bacterial genera were significantly different between the dietary treatments at the p-value. In addition to the sequencing of the cecal microbiota, we analyzed some specific bacteria with a complementary quantitative approach by using qPCR method on an educated guess basis, meaning that we selected bacteria for which previous reports have shown a link between these bacteria and inflammation/metabolic disorders: (1) several studies demonstrated that specific Bifidobacterium or Lactobacillus strains alone or in combination, decreased the metabolic alterations (decrease of body weight and fat mass gain) together with a reduction of the inflammatory events, occurring in the liver and/or the adipose tissue in diet-induced obesity models27; (2) a recent paper demonstrated that Roseburia interacted with dietary plant polysaccharides to lower systemic inflammation and ameliorated atherosclerosis in germ-free apolipoprotein E-deficient mice colonized with synthetic microbial communities28. Moreover, our previous work showed that CG had metabolic interest that could be related to the increase in Roseburia spp. in a mouse model of HF induced-obesity21; (3) Alistipes were positively associated with HF-induced obesity in several independent studies31; (4) finally, Akkermansia is a bacterium capable of reducing adipose tissue inflammation and dyslipidaemia in obese mice33. The Fig. Alistipes spp. and Lactobacillus spp in the cecal content due to HF feeding was counteracted by the CG + PPE supplementation  during 17 weeks promotes obesity and accelerates atherosclerosis in ApoE−/− mice7. This effect was accompanied by the development of a metabolic syndrome phenotype characterized by a low level of inflammation and atherosclerotic lesion7. In the present study, we show that HF feeding  induces obesity, hypercholesterolemia, inflammatory disorders and endothelial dysfunction in young (10-wk-old at the beginning of the treatment) ApoE−/− mice fed a HF diet for only 8 weeks. We then used this model to test the health benefits of original nutrients in the management of endothelial and inflammatory dysfunctions associated with CVD.The nutritional quality of diets underlies or exacerbates several chronic pathologies, including CVD37. Chitin-glucan (CG), a natural component of the cell wall of microscopic fungi has been approved as a novel food ingredient after a positive safety evaluation in 2010 by the European Food Safety Authority38. The CG used in the present study is a high-purity biopolymer extracted from the mycelium of Aspergillus niger. This insoluble fiber is composed of two types of polysaccharide chains, namely chitin (poly-N-acetyl-D-glucosamine) and beta--D-glucan  linkage). We previously demonstrated its beneficial effects on the development of obesity and associated diabetes and hepatic steatosis in HF-fed mice, through a mechanism related to the restoration of the composition and/or the activity of gut bacteria [33]. On the other side, health benefits of pomegranate fruit and/or juice consumption have recently received considerable scientific focus. The PPE used in this study is a source of polyphenol (40%), with punicalagin and ellagic acid levels reaching 10% and 2%, respectively. Our previous findings support that pomegranate extract constitutes a promising food ingredient to control atherogenic and inflammatory disorders associated with diet-induced obesity since it alleviated tissue inflammation and hypercholesterolaemia in HF diet-induced obese mice23. In line with our previous results, here we observe that CG + PPE reduces several proinflammatory parameters such as the expression of the chemokine MCP-1 (involved in the recruitment of inflammatory cells inside the tissue) in both the visceral and liver tissues and the expression of the proinflammatory cytokines IL-1β and TNFα in the liver.The health-promoting effect of prebiotic fibre and plant extracts is subject to interesting developments39. In the present study, HF feeding did not induce higher lipid accumulation in the liver of ApoE−/− mice than the low fat diet. This is probably due to the short-term dietary treatment investigated here. Importantly, the supplementation with CG + PPE reduced significantly hepatic triglycerides, one of the critical features of NAFLD. Further studies are needed to unravel the mechanism behind the lower hepatic fat accumulation since two key genes in the liver that regulate lipid metabolism (FAS and CPT-1) were not affected by the dietary treatments.The presence of hepatic steatosis has previously been reported for ApoE−/− mice fed a HF diet and the model has been proposed to study features of nonalcoholic fatty liver disease (NAFLD) on cardiovascular events40. Eight weeks of HF-diet were able to accelerate the process of endothelial dysfunction in aorta namely by increasing abundance of caveolin-1, a negative allosteric regulator of eNOS leading to endothelial dysfunction exacerbation and associated CVD41. However, the dietary combination did not affect this parameter in the aorta, the major representative conductance vessel of the macrocirculation. We used western blotting to assess the activation of eNOS through analysis of its phosphorylated forms (p-eNOSser1177) both in conductance (thoracic) and resistance (mesenteric) arteries. We observed that the activated form of eNOS was a third higher in mesenteric arteries due to supplementation with CG + PPE compared to HF ApoE−/− mice, suggesting that the innovative dietary combination activated the NOS/NO pathway in the resistance arteries representing the microcirculation near the gut. Furthermore, we investigated the relaxation profile of mesenteric arteries. We demonstrated that HF feeding for 8 weeks accelerated the process of endothelial dysfunction in resistance mesenteric arteries in ApoE−/− genotype, as already shown in previous studies43. We concluded that prostanoids do not play an major role in this early stage of endothelial dysfunction, based on the lack of effect of indomethacin, a non-selective cyclooxygenase inhibitor. In most cases, endothelial dysfunction is related to a reduced NO availability in mice6. Accordingly, impairment of endothelial function due to HF feeding is mainly dependent on a reduced NO bioavailability in ApoE−/− mice as L-NAME (a NOS inhibitor), counteracted the vasorelaxation of mesenteric arteries from all mice and repealed the differences between genotypes, independently of the dietary conditions. In contrast, resting parameters  and contraction profile  of mesenteric arteries were not affected by the HF treatment whatever the dietary supplementations. In our experimental setting, CG + PPE supplementation did not improve the NO-dependent endothelium relaxation measured in mesenteric arteries. Of more relevance for human CVD, the effects of CG + PPE supplementation were not restricted to the phosphorylated form of eNOS in enteric vascular tree since this dietary treatment also increased the circulating levels of Hb-NO in peripheral blood at distance from the mesenteric bed. Altogether, our results demonstrate that, besides interesting hepatic events highlighted above, the supplementation with CG and PPE for 8 wk improved the endothelial function with consequences on systemic NO bioavailability in HF-fed ApoE−/− mice. It was previously shown that pomegranate extract administrated alone during 30 days enhanced endothelium-dependent artery relaxation although it concerned coronary arteries isolated from perfused hearts of spontaneously hypertensive ovariectomized rats44. Furthermore, they demonstrated in the same study that the treatment with their extract inhibited p-eNOS Thr495 without affecting p-eNOS Ser1177, improved lipid profile and was able to prevent the reduction in plasma nitrite levels spontaneously induced by castration.Metabolic diseases such as NAFLD are associated with macro- and microcirculation damage, initiating an impairment of endothelium-dependent relaxation12. Establishing the causal and consequential actions of the gut microbiota in driving obesity and related-metabolic diseases has been challenging. Interestingly, Stepankova et al.45 reported that germ-free ApoE−/− mice developed more aortic atherosclerotic plaques compared with conventionally raised ApoE−/− mice fed the same low standard cholesterol diet. Along the same lines, compared to germ-free mice, the levels of cholesterol and triglycerides in plasma of conventionally raised mice are reduced, whereas they are increased in adipose tissue and liver46. Both findings support the hypothesis of a protective ability for the gut microbiota in CVD and in the management of plasma levels of cholesterol and lipids10. We have previously shown that the improvement of vascular dysfunction by ITF was linked to an activation of the NOS/NO pathway, which could be dependent on events occurring at the gut microbiota level . Although dietary treatment induced clusterization due to CG and CG + PPE supplementation, our targeted microbiota characterization did not reveal any changes in bacterial diversity or relative abundance of bacteria whatever the taxonomical level considered. However, the CG and PPE counteracted the effect of the HF feeding on the abundance of Alistipes spp and Lactobacillus spp present in the cecal content of mice only when these compounds were combined. Recently, Moschen and colleagues identified Alistipes spp. as one of the top ten most abundant genera associated with human colorectal carcinoma and provide experimental evidence that Alistipes potently induces inflammation in colitis-prone Il10−/− mice47. In line with this finding, Alistipes were positively associated with HF-induced obesity in several independent studies31. The depletion of Alistipes by CG + PPE in the current study is particularly relevant, considering that in both studies, they demonstrated that transmissible microbial and metabolomic remodeling by dietary fiber improved metabolic homeostasis30. Our results indicated that Lactobacillus spp. and Akkermansia muciniphila are other bacteria susceptible to dietary manipulation. It has been reported that members of the Lactobacillus, Akkermansia muciniphila and Bifidobacterium genera may play a critical role as anti-obesity drivers in experimental models and humans49. One study linked specific species of Lactobacillus (L. reuteri) with obesity in humans50 whereas many studies tested for their ability to affect positively obesity and risk factors associated with CVD51. Here, the Lactobacillus genus increased upon HF feeding whereas the supplementation with CG + PPE blunted this effect. The study of Fak and Backhed demonstrated that bacterial strains of the same Lactobacillus species showed different effects on adiposity and insulin sensitivity in ApoE−/− mice, illustrating the complexity of host bacterial cross-talk and the importance of strain specificity52. In this context, it would be of interest to further characterize the identity and the functionality of the Lactobacillus strains affected by the dietary treatments. Although Akkermansia muciniphila is a bacterium capable of reducing fat mass development, insulin resistance, metabolic endotoxemia, adipose tissue inflammation and dyslipidaemia in obese mice and in a pilot study conducted in humans33, we observed a decreased abundance of this bacteria in the cecal content of ApoE−/− mice treated with the combination CG + PPE. Our previous work showed that CG had metabolic interest that could be dependent on the increase in Roseburia spp. in a mouse model of HF induced-obesity21 but their level was not impacted the dietary treatments in the model of HF-fed ApoE−/− mice. Therefore, we proposed that neither Akkermansia nor Roseburia spp. were involved in the metabolic and anti-inflammatory effects of the combined treatment (CG + PPE) observed in the present study. These data also clearly support the notion that we should not generalize the effects of dietary fibers and polyphenols on Akkermansia, as discussed in a recent review, predicting which fibers or polyphenols are the most suited to maximize beneficial health effects is highly dependent on the health situation, the individual and its microbiota53Gut microbiota plays a crucial role in the control of host intestinal functions and management of NAFLD, through the release and/or biotransformation of metabolites  which regulate gut endocrine functionLactobacillus and Alistipes could be implicated in the management of metabolic and inflammatory dysfunctions associated with CVD. The beneficial effects were observed only when CG was associated with PPE in the diet, confirming a previous study that highlighted the need to consider the interactions among bioactive food components when evaluating potential prebiotic effects of dietary fiber54.In conclusion, our study demonstrated that food supplementation with CG and PPE limited triglyceride accumulation in the liver and exerted anti-inflammatory effects both in the liver and in the visceral adipose tissue of ApoE−/− mice fed a HF diet. In addition, the combination improved endothelial function in mesenteric arteries through a higher production and availability of NO in this model. Gut bacteria such as All experiments were performed in strict accordance with relevant guidelines and regulations for the care and use of animals and in accordance with the EU directive. All mouse experiments were approved by and performed in accordance with the guidelines of the local ethics committee for animal care of the Health Sector of the Université catholique de Louvain under the specific agreement numbers 2017/UCL/MD/005. Housing conditions were as specified by the Belgian Law of 29 May 2013, on the protection of laboratory animals (Agreement LA 1230314). Every effort was made to minimize animal pain, suffering, and distress and to reduce the number of animals used.Apoetm1Unc/J from Charles River Laboratories, L’Arbresle, France) were housed two or three mice per cage  and with a 12 h light/dark cycle at 22 °C, diet and water were ad libitum. Mice were fed a low fat (10 kJ%), no sucrose, purified ingredient diet  or a high fat (HF) diet  for 8 weeks. HF-fed mice were supplemented with or without 5% CG  and a combination of 5% CG and 0.5% PPE . CG used in the study was derived from the cell walls of the mycelium of Aspergillus niger, in which two types of polysaccharide chains, i.e., chitin (poly N-acetyl-D-glucosamine) and β-D-glucan, are associated. PPE extract  used in the study was derived from pomegranate peel in which polyphenol content reached 40%  and the concentrations of punicalagin and ellagic acid were 10 and 2% (ultra-performance liquid chromatography method with diode array detection), respectively.Nine weeks old male and ApoE−/− mice  and one for other parameters . Body composition was assessed every two weeks by using 7.5 MHz time domain-NMR .55.After a total of 8 weeks of dietary treatment, mice were anaesthetized in postprandial state . Second and third order mesenteric arteries were rapidly removed and carefully isolated from visceral adipose and/or connective tissues as previously describedAfter a total of 8 weeks of dietary treatment and a 6-h period of fasting, mice were anesthetized with isoflurane  and blood samples were harvested. We collected venous blood (0.2 mL) from the right ventricle, immediately frozen in heparinized calibrated tube in liquid nitrogen. Venous blood samples were also collected and centrifuged  for further analysis. Liver, aorta, adipose tissues and caecum were carefully dissected, weighted and immersed in liquid nitrogen before storage at −80 °C; pieces of liver and aorta were embedded in OCT compound and frozen in nitrogen cooled isopentane for histology.55.The level of circulating heme-nitrosylated haemoglobin (Hb-NO) was assayed in whole blood of mice from the electron paramagnetic resonance (EPR) signal of 5-coordinate-α-Hb-NO as previously described56.Plasma triglycerides, total cholesterol and non-esterified fatty acid levels were analyzed in venous blood using kits . Alanine aminotransferase (ALAT) levels were measured in the serum as a marker of liver damage using the ALAT/GPT kit (DiaSys Diagnostic and Systems). Plasma concentrations of cardiovascular analytes  and of cytokines  were determined using two multiplex immunoassay kits ). Triglycerides and cholesterol were measured in the liver tissue after extraction with chloroform–methanol according to the Folch method, as previously describedSections of aortic arch, previously isolated from connective tissues, were frozen in embedding medium . Sections of artic arch of 5 µm were cut at 3 different levels for each sample. Haematoxylin-eosin stained sections were digitalized at a 20x magnification using a SCN400 slide scanner . Frozen sections of the main liver lobe (embedding in Tissue-tek) were sliced and stained with the Oil Red O. Haematoxylin-eosin stained sections were digitalized at a 20x magnification using a SCN400 slide scanner . The quantification of positive area/tissue area was determined by thresholding for the Oil Red O signal .56. Ribosomal 7 protein L19 (RPL19) RNA was the housekeeping gene. Sequences of the primers are given in Supplementary File −ΔΔCT method.Total RNA from tissues was extracted with TriPure reagent . cDNA was obtained by reverse transcription of 1 μg total RNA . Real-time qPCRs were performed using Mesa Fast qPCRTM  for detection as previously described19. Gels were analysed and quantified by ImageQuantTM TL instrument .Equal amount proteins from thoracic aorta and mesenteric arteries were separated by SDS/PAGE and transferred to nitrocellulose membrane, blocked with 5% non-fat dry milk or bovine serum albumin (BSA) in tris-buffered saline tween-20. The membranes were incubated overnight at 4 °C with antibodies as previously described57 and detailed in Supplementary File Bifidobacterium spp., Roseburia spp., Akkermansia muciniphila and Lactobacillus spp. quantification by qPCR, primers are detailed in Supplementary File 33. For Alistipes spp., method was detailed in Supplementary File Genomic DNA was extracted from the cecal content using a QIAamp DNA Stool Mini Kit  according to the manufacturer’s instructions, including a bead-beating step. 16S rDNA profiling, targeting V1-V3 hypervariable region and sequenced on Illumina MiSeq were performed as described previouslyData are presented as mean ± SEM. Within-groups variances were compared using a Bartlett’s test. If variances were significantly different between groups, values were normalized by Log-transformation before proceeding to the analysis. Dixon’s Q-test was performed to statistically reject outliers (95% confidence level). Differences between groups were assessed using one-way ANOVA, followed by the Tukey post hoc test. For cecal bacteria, we used the non-parametric Kruskal-Wallis test. Data with different superscript letters were significantly different (p ≤ 0.05) according to the post hoc ANOVA statistical analysis. Grouped analyses were assessed by two-way ANOVA. Statistical analyses were performed using GraphPad Prism version 7.04 for windows.Supplementary File S1Supplementary Dataset 1


Although perianal fistulas occur commonly in pediatric Crohn’s disease (CD), evaluations of health services have been limited since no validated claims-based methods exist for identifying cases.To develop and validate accurate case definitions for perianal fistulas among pediatric patients with CD from administrative claims.Retrospective cohort study in which we developed and tested candidate case definitions for perianal fistula. Patients (age 5–21 years between 2005–2012) with CD enrolled in Michigan Medicaid with healthcare at University of Michigan were identified via claims. Medical records were obtained from all identified patients, whose entire records were abstracted. Medical record evidence for perianal fistula was considered the “gold standard” against which candidate case definitions were compared. The reference case definition of perianal fistula (ICD9 565.1) and candidate case definitions were evaluated.Of 843 patients identified via claims, 274 (33%) met CD criteria for inclusion. The true perianal fistula rate among CD patients was 18% (n = 49). The top-performing candidate case definition identified 15% (n = 42), had sensitivity of 77.6%, specificity of 98.2%, positive predictive value (PPV) 90.5%, negative predictive value (NPV) 95.3%, and area under receiver operator characteristic curve (ROC) of 0.88. In contrast, the reference case definition identified 9% (n = 26), sensitivity 51.0%, specificity 99.6%, PPV 96.2%, NPV 90.3%, and had an area under ROC of 0.75.We demonstrated that it is feasible to use administrative claims data to accurately identify pediatric patients with perianal fistula complications. Claims-based case definitions were found to be highly accurate through medical record review, providing a high degree of confidence for future studies where chart review is not feasible. These claims-based methods can be applied to claims data in other settings for the evaluation of health services utilization as well as to assess the comparative effectiveness of prevention and treatment strategies. Gastrointestinal fistulas are abnormal connections between the bowel and adjacent structures such as the skin, bladder, vagina, fallopian tubes, or to other loops of bowel, and have significant negative impact on their quality of life.–3 FistulDespite the profound effects on quality of life and health care costs, very little is known about the epidemiology of fistulas due to Crohn’s disease. Prospective adult studies found that perianal fistulas commonly develop among 15% within 5 years and 23% within 10 years after Crohn’s disease diagnosis. Onset ofWhile these studies provide some important initial insights, population-based methods to assess the prevalence of fistulizing Crohn’s disease among children and related outcomes do not exist. The few population-based studies of perianal fistulas that have been conducted in claims-based data were not validated with medical record review and therefore their accuracy is unknown., 15, 16 We developed and tested alternative claims-based methods for identifying perianal fistula complications of pediatric patients with Crohn’s disease among a statewide population of children enrolled in Michigan Medicaid. We validated our claims-based methods among the sub-population of pediatric patients with Crohn’s disease at the University of Michigan who were enrolled in the Michigan Medicaid program during the 2005–2012 study period. This study was approved by the institutional review board (IRB) at the University of Michigan (HUM00079109) and the Michigan Department of Health and Human Services (MDHHS). The IRBs granted a waiver of consent in order allow the linkage of claims data to the electronic medical records and to permit review of the medical records necessary for comparison with claims data. While permission was granted to allow medical record validation, these data cannot be made publicly available due to patient privacy considerations. All aggregate data used in this study are reported in the manuscript, tables and figures.Previous claims-based studies of perianal fistula in Crohn’s disease have previously used a single diagnosis code (ICD-9 diagnosis = 565.1) to identify fistula cases, although the accuracy of this method is unknown. For the Our candidate case definitions were based upon a review of the medical literature in which we summarized the range of approaches to evaluation and treatment of perianal fistulas among patients with Crohn’s disease. These approaches ultimately served as the basis for identification of cases based upon the procedure and medication codes reported in claims data. In total, our review yielded 99 candidate strategies for which we identified the relevant diagnosis, procedure and medication codes that could be used with administrative claims data to identify perianal fistula cases . DetaileWe considered a broad range of evaluation strategies to identify potential cases of perianal fistulas. Patients with Crohn’s disease who have suspected or identified perianal fistulas and/or abscesses are most commonly evaluated by either cross sectional imaging (computed tomography [CT] or magnetic resonance imaging [MRI]) or endoscopic ultrasound (EUS).–23 HistoWe also considered a diverse array of potential treatment options in our approach to identify potential perianal fistula cases. The literature supports treating patients with perianal fistula with aggressive medical therapy for Crohn’s disease including immunomodulator and/or anti-tumor necrosis alpha (TNFα) therapy., 24–26 TWe acquired all Michigan Medicaid administrative claims for pediatric patients 5–21 years with Crohn’s disease between January 1, 2005 and December 31, 2012. As an initial screening, potentially eligible patients were required to have at least three encounters with diagnosis of IBD. This method has previously been validated by medical record review with a high degree of sensitivity, specificity, and negative predictive value (NPV), though it has a low positive predictive value (PPV)., 31, 32 We used Medicaid claims to classify perianal fistula cases based upon codes indicating the evaluation and/or treatment of Crohn’s disease. Our general approach to claims classification built upon our prior experiences with classifying chronic disease cases using administrative claims.–38 All cWe reviewed the entire medical record during the study timeframe for each patient, including all inpatient and outpatient clinician notes. Diagnostic information was sought from descriptions of physical examination, recorded diagnoses, endoscopy reports, imaging reports, operative reports, etc. The diagnosis of Crohn’s disease was confirmed according to the Porto criteria, guidelines for diagnosis that include endoscopic evaluation with biopsy in addition to evaluation of the small bowel with medical imaging. Crohn’s During medical record review, a patient was considered to have a perianal fistula if either 1) a perianal fistula and/or 2) a perianal abscess was documented. For the purposes of this study, a perianal abscess without explicit identification of a fistula tract was still considered as having a perianal fistula., 39 PiloOur candidate claims-based case definitions for perianal fistulizing Crohn’s disease were evaluated using our sample of medical records as the gold standard. Though the medical record was abstracted for the entire study timeframe, the analyses were limited to periods of Medicaid enrollment. Perianal fistulas documented outside of the time period of Medicaid enrollment were not included as outcomes. Following an approach successfully employed in a prior study, measures of performance were calculated for each candidate case definition, including sensitivity, specificity, PPV, NPV, and area under the receiver operating characteristic (ROC) curve.A total of 843 patients at the University of Michigan were identified in Michigan Medicaid claims as having at least 3 claims for IBD during the study period , all of Among the 274 patients with claims reporting Crohn’s disease, 26 (9%) had at least one claim for a perianal fistula, and 32 (12%) had claims for perirectal abscess. Lesions other than fistula or abscess, such as a perianal skin tag, or hemorrhoid were reported among 16 patients (6%). A subset of patients (39%) had missing data for immunomodulators and antibiotics. Among those with complete medication data (N = 166), 108 (64%) with at least one claim for an immunomodulator, and 101 (61%) for an antibiotic. Among all 274 patients, 156 (57%) with at least one claim for an anti-TNFα medication. Overall, 10 patients (4%) had seton procedures, 7 (3%) had fistula closure procedures, 3 (1%) had excision of a perianal lesion and 2 (1%) had hemorrhoidectomy. Imaging procedures included 120 (44%) CT, 106 (39%) MRI, 22 (8%) fluoroscopic procedures, and no EUS procedures.Using the medical record as the gold standard, the true fistula rate among Crohn’s disease patients identified through claims was 18% (N = 49). Among the 274 patients with Crohn’s disease, the reference case definition for perianal fistula (one ICD-9 diagnosis code = 565.1) identified 26 patients (9%) with perianal fistula, 1 of whom was determined to be false positives through medical record review. This reference case definition had a sensitivity of 51.0%, specificity 99.6%, PPV 96.2%, NPV 90.3%, and area under the ROC curve of 0.75.Measures of performance of the candidate definitions varied widely among the 99 candidate case definitions tested . SensitiThe overall top performing case definition (J3) identified 42 patients (15%), of whom 4 were determined to be false positives through medical record review. This definition had a sensitivity of 77.6%, specificity 98.2%, PPV 90.5%, NPV 95.3%, and area under the ROC curve of 0.88.We developed and evaluated novel alternative administrative claims-based methods to identify perianal fistula cases among pediatric patients with Crohn’s disease. We conducted a medical record review to evaluate the accuracy of an administrative claims-based case definition that has been used in prior studies., 15, 16 We found that several candidate definitions outperformed our reference case definition, which reflects a claims-based case definition used in prior studies. The most accurate case definition (J3) required codes indicating the presence of any perianal lesion  or genital lesion (fistula or abscess) or any relevant surgical procedure . This combination produced the most accurate result and identified an additional 88% of cases above that of the reference case definition in our study population. While several of our case definitions performed well, no case definition accurately identified all cases. Even our highest performing measures had some false negatives (affecting sensitivity) as well as false positives (affecting specificity). However, the highest-performing candidate case definitions identified fewer false negatives and false positives then the reference case definition, realizing an overall improvement in accuracy compared to prior claims-based methods.Although no previous study has assessed the accuracy of claims definitions, administrative claims-based studies of perianal fistula have been performed among patients with Crohn’s disease. Prior claims-based studies have identified the prevalence of perianal fistulas ranging from 4–6%., 16 The Our findings demonstrate that accurate claims-based methods for identifying perianal fistulizing disease among Crohn’s disease patients are feasible. We found several candidate measures that performed well, based on diagnosis, procedure, and medication codes readily available in administrative claims data. These claims-based case definitions can be broadly applied to administrative data widely available to commercial and Medicaid health plans. In contrast, prospective observational studies of perianal fistulas are difficult and time-consuming to conduct due to the requirement of performing perianal examination on patients who may not have active perianal complaints., 43, 46 While our claims-based alternative case definitions outperformed the reference case definition, there are still important limitations to acknowledge. First, the case definition of requiring a minimum of 3 Crohn’s disease claims used for identifying patients with Crohn’s disease is imperfect, and was likely to have identified patients without Crohn’s disease., 32 HoweOur claims-based method for identifying perianal disease among children with Crohn’s disease provides a validated method to identify patients for studies of prevention and treatment of perianal fistulas among children with Crohn’s disease. The high specificity observed in our best performing measures provides a high degree of confidence that cases identified using these methods are actually true cases of perianal fistulizing disease. Given the detrimental impact on quality of life and high cost-burden, interventions aimed at improving the quality care of patients with perianal fistula should now be feasible with these new methods.–6, 15 CoWe demonstrated that it is feasible to use administrative claims data to accurately identify pediatric patients with perianal fistula complications. Claims-based case definitions were found to be highly accurate through medical record review, providing a high degree of confidence for future studies where chart review is not feasible. These claims-based methods can be applied to claims data in other settings for the evaluation of health services utilization as well as to assess the comparative effectiveness of prevention and treatment strategies.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.S5 Table(XLS)Click here for additional data file.S1 Checklist(DOCX)Click here for additional data file.


We present clinically relevant alterations identified by OncoPanel in two patients with recurrent LGSOC: a patient with a BRAF V600E mutation who derived clinical benefit from BRAF inhibitor vemurafenib, and a patient with progressive disease after durable response to hormonal therapy whose recurrent tumor harbored an ESR1 mutation associated with resistance to antiestrogen therapy. These cases suggest that patients with recurrent LGSOC may benefit from targeted sequencing to inform selection of targeted agents and, more broadly, to identify rational targeted agents and combinations to treat recurrent disease.We describe two patients with LGSOC whose clinical management was informed by targeted-panel next-generation sequencing (NGS) performed in our institution. The OncoPanel test at Dana-Farber Cancer Institute consists of targeted NGS of formalin-fixed tumor samples covering exons of > 300 cancer-associated genes, plus intronic regions of genes involved in somatic rearrangements.BRAF c.1799T>A (p.V600E) mutation (Appendix), she started vemurafenib at 480 mg twice daily, which was dose reduced to 240 mg twice daily because of cutaneous toxicity. Her cancer antigen 125 level, which was 193 at initiation of vemurafenib, decreased to 12. A CT scan 1 year after initiation of vemurafenib showed improved retroperitoneal adenopathy and no new sites of disease  mutation (Appendix). She continued endocrine therapy.The patient presented at age 36 years with pelvic pain during in vitro fertilization treatments. A CT scan revealed a fluid-filled adnexal structure, and she underwent surgical exploration. Intraoperative biopsies revealed an incidental serous borderline tumor of the ovary. She underwent complete surgical cytoreduction, with pathology showing stage III invasive serous borderline tumor and one area suggestive for LG serous carcinoma. Two years later, she developed recurrent disease and underwent secondary cytoreduction of metastatic serous carcinoma at multiple sites in the abdomen and pelvis, followed by platinum-based chemotherapy. At subsequent disease progression, she started hormonal therapy with anastrozole, with leuprolide ovarian suppression. During endocrine therapy, she maintained stable low-volume peritoneal disease for almost 5 years . HoweverTP53 mutations, few other somatic driver mutations, extensive copy number variations and aneuploidy, and alterations in HRR genes.15 In contrast, LGSOCs have more stable genomes, with fewer copy number aberrations, but harbor driver mutations in specific oncogenic pathways.16 Both serous borderline tumors and LGSOCs have frequent alterations in the RAS/mitogen-activated protein kinase (MAPK) pathway, including mutually exclusive hotspot KRAS mutations (G12D and G12V) and BRAF mutations (V600E).22NRAS mutations also cooperate with EIFAX to drive proliferation of LGSOC.24 Whole-exome sequencing of LGSOC shows a low mutation rate and rare recurrent mutations in other genes.24 MAPK pathway mutations in LGSOC led to a clinical trial of the MEK1/2 inhibitor selumetinib, which showed promising activity.25 Despite these findings, targeted therapies for LGSOC have not yet been integrated into clinical practice.Recent genomic profiling of LGSOC suggested several potential targetable pathways and highlighted dramatic differences in the genomes of LGSOC and HGSOC. HGSOCs have near 100% frequency of BRAF V600E mutation. BRAF V600E mutations are present in 35% of serous borderline cancers or LGSOCs18 and are associated with better prognosis and decreased likelihood of requiring systemic therapy.18 Despite the low prevalence of BRAF V600E mutations in patients with recurrent disease requiring treatment, this case highlights that this mutation may correlate with sensitivity to BRAF inhibitors, such as vemurafenib. Two previous patient cases of LGSOC with BRAF V600E mutations and sustained response to vemurafenib were described.27 One patient was treated in a basket trial of solid tumors with BRAF V600E mutations and responded for at least 12 months.26 A second patient, who had experienced progression throughout chemotherapy, hormonal therapy, and antiangiogenic therapy, had a BRAF V600E mutation on a recurrence biopsy and was treated with vemurafenib, achieving clinical and radiographic responses.27 Despite dose reduction because of skin rash, the patient continued to receive vemurafenib with an ongoing partial response for nearly 2 years.27 It is notable that both this patient and our patient seemed to respond to significantly lower doses of vemurafenib than the US Food and Drug Administration–approved dose in melanoma , suggesting that LGSOC tumors with BRAF V600E mutations may be more sensitive to RAF inhibition. Given that not all solid tumors with BRAF V600E mutations respond to RAF inhibition , these cases illustrate that recurrent LGSOC with a BRAF V600E mutation may derive clinical benefit from treatment with vemurafenib or other RAF inhibitors, arguing for routine assessment of these mutations in recurrent LGSOC. Our case further highlights that although skin toxicities can be considerable, dose reduction can be both efficacious and tolerable. In addition to BRAF V600E, the high frequency of other alterations in the RAS/MAPK pathway may render LGSOC sensitive to different targeted inhibitors of the pathway. Selumetinib showed a 15% response rate in recurrent LGSOC in a phase II trial.25 An exceptional responder in this trial had a response to selumetinib of > 5 years and had a deletion in MAP2K1 (encoding MEK1), which has oncogenic activity.22 Another patient with LGSOC and a KRAS G12D mutation had a response of > 7 years to selumetinib.29We present two cases of LGSOC in which targeted NGS was informative for patient management. The patient in case 1 had recurrent LGSOC and experienced progression throughout chemotherapy, hormonal therapy, and antiangiogenic therapy. Her tumor harbored a ESR1 Y537S mutation. Although ESR1 mutations in the ligand-binding domain (most commonly D538G and Y537C/S/N) have been reported in breast cancers treated with hormonal therapy, this is to our knowledge the first ESR1 mutation reported in LGSOC.35ESR1 mutations are rare in primary, untreated breast cancers but prevalent in metastatic breast cancers resistant to hormonal therapy.36 Structural alterations conferred by these activating mutations, including Y537S, result in ligand-independent activation of the estrogen receptor and resistance to endocrine antagonists.37 It is reasonable to surmise that this mutation contributed to tumor progression during AI therapy. Because the remainder of her disease remained stable with anastrozole, the patient continued treatment with the same AI after resection of the resistant lesion. However, in patients who develop progressive disease with ESR1 mutations, it may be possible to select hormonal agents with alternative mechanisms of action downstream of the activated estrogen receptor and bypass the resistance mechanism. For instance, the oral selective estrogen receptor degrader (SERD) AZD9496 potently binds and downregulates D538G and Y537S ESR1 proteins in vitro and was effective against breast cancer xenografts with Y537S and other ESR1 mutations, whereas the US Food and Drug Administration–approved SERD fulvestrant had only a partial effect against Y537S.39 Combinations of antiestrogen agents such as AIs or SERDs with targeted therapies such as mammalian target of rapamycin inhibitors  and CDK4/6 inhibitors  may also overcome resistance associated with ESR1 mutations. In the PALOMA3  trial of fulvestrant plus palbociclib versus placebo in AI-resistant patients, the benefit of addition of palbociclib to fulvestrant was seen irrespective of specific ESR1 mutation.40 In BOLERO2  trial of exemestane plus everolimus, the benefit of everolimus was evident in tumors with an ESR1 D538G mutation but was not clear in Y537S ESR1–mutated tumors because of low numbers.42ESR1 mutations can be detected by targeted sequencing in tumors and cell-free DNA, indicating that clinical testing for these resistance mutations is feasible.42Our second case of recurrent LGSOC highlights that targeted NGS can elucidate the mechanism of resistance in a patient with excellent response to hormonal therapy and may help tailor future therapy. This patient had a sustained response to hormonal therapy for almost 5 years until she had an isolated recurrent lesion that harbored an Tumor heterogeneity is an important challenge in interpreting targetable mutations. In our cases, only a recurrent lesion underwent OncoPanel testing. It is unknown whether each mutation of interest was already present in the primary tumor or acquired in the recurrent tumor. Furthermore, multiple metastatic sites might harbor different driver or resistance mutations; the mutation status of the other residual disease deposits was not tested in these patients. On a practical level, both patients maintained stable low-volume disease with the selected targeted therapy, and identifying different mutations in other lesions might not necessitate a change in management as long as the patients remain asymptomatic.BRAF V600E by targeted sequencing in LGSOC, as well as the possibility of meaningful clinical response to even low doses of vemurafenib in patients with LGSOC with a BRAF V600E mutation. In the second case, targeted sequencing helped elucidate the mechanism of resistance in a patient with LGSOC with prolonged response to AI therapy, which could be relevant for selection of additional therapy to overcome resistance. Both cases support that targeted sequencing may be a valuable tool for the clinical management of patients with LGSOC.In conclusion, our first case highlights the potential utility of testing for


We use microfluidic chambers to achieve compartmentalization and established high throughput live organelle imaging at standardized distal and proximal axonal readout sites in iPSC-derived spinal motor neuron cultures from human amyotrophic lateral sclerosis patients to study trafficking phenotypes of potential disease relevance. Our semi-automated pipeline of organelle tracking with FIJI and KNIME yields quantitative, multiparametric high content phenotypic signatures of organelle morphology and their trafficking in axons. We provide here the resultant large datasets to enable systemic signature interrogations for comprehensive and predictive disease modelling, mechanistic dissection and secondary hit validation . Due to the nearly complete coverage of analysed motility events, our quantitative method yields a bias-free statistical power superior over common analyses of a handful of manual kymographs.Neurodegenerative diseases pose a complex field with various neuronal subtypes and distinct differentially affected intra-neuronal compartments. Modelling of neurodegeneration requires faithful  Such perturbed transport logistics detrimentally impact on various essential cellular processes like signal transmission, gene regulation, nutritional metabolism, proteasome function, energy supply and distal protein biosynthesis, eventually leading to compromised axonal pathfinding, diminished neuronal plasticity and neurodegeneration1–8.The role of neurite trafficking defects in the pathophysiology of neurodegenerative diseases is a hotly debated issue9. One important hallmark is the retrograde retraction and dying back of the axon from the neuromuscular junction10–12. In our recent publication6, we recapitulated these dying back processes in vitro using iPSC-derived spinal motor neurons from familiar ALS patients carrying a mutation in FUS, eventually resulting in neurodegeneration. Moreover, we observed severe distal trafficking defects of mitochondria and lysosomes in axons clearly preceding the onset of first structural damage, i.e. signs of dying back. Remarkably, the early rescue of these distal organelle trafficking defects through treatment with promising drug candidates completely abrogated the premature die back and neurodegeneration of ALS neurons, thereby suggesting that early restoration of premanifest disease hallmarks provides novel therapeutic intervention points in ALS. As for other mutated genes causing ALS, we recently observed trafficking defects in TDP43 mutants13 distinct from FUS, i.e. a more ‘global’ phenotype occurring in the proximal axon parts as well that developed more slowly during the aging of MNs in culture with a distinct response to rescue candidates3.Amyotrophic lateral sclerosis (ALS) is a fatal, incurable motor neuron disease characterised by muscle denervation, demyelination and loss of spinal and cortical motor neurons (MNs) causing progressive muscle paralysis6, we used organelle mean speed and track displacement as analytical live imaging readout parameters to document distal trafficking defects in axons. However, for accurate disease modelling we wished to evolve our original method further for a more ambitious disease profiling. Therefore, we established standardized live readout positions at distal and proximal positions in axons (versus retrograde). We verified the absence of dendrites in our readout windows by further immunostainings for MAP2 and SMI32 . By analysing the acquired movies in a semi-automated pipeline with the embedded FIJI Track Mate plugin for dynamic organelle tracking and particle analyser to model static organelle morphology, we achieved an analytical coverage of nearly all organelles  yielding multiple phenotypic descriptors of high sensitivity. These comprised nine dynamic tracking parameters (6), we obtained a comprehensive phenotypic HC signature of 40 quantitative parameters expressed as Z-score deviations from proximal wild type control baseline  impacted on the resultant HC signature’s tracking or morphology part  chemical disruption of microtubules (nocodazole), ATP production (oligomycin A) or a combination of mitochondria fusion promotor and fission inhibitor  muogy part , and finrameters . These fe states , 4d, 4e.18. This circumstance can easily lead to a bias and erratic results when only a handful of manually selected kymograph profiles are analysed – a still common practice to unravel trafficking patterns in neurites19.To summarize, because more subtle phenotypes may occur only in minor fractions of the overall organelle population, such accurate and comprehensive multiparametrization requires high throughput organelle tracking in compartmentalized neuronal cultures that enable us to reveal the distal trafficking phenotypes as opposed to the physiological state at proximal sites , 3, 4. F20.We provide here the resultant parameter tables of the dynamic organelle tracking  and static morphological analysis  along with the calculated Z-scores  for the interested reader to test any alternative HC profiling and clustering algorithm. Moreover, we have deposited all movie raw data as 16-bit TIFF image stacks  for analysis with any other object recognition and tracking algorithmGeneral remark: the following sub headers of the methods section mirror the structure of the schematic workflow shown in We choose two different monogenetic ALS causing gene models and respective control to validate the power of our algorithm.6. The subsequent differentiation to neuronal progenitor cells (NPC) and further maturation to spinal motor neurons (MN) was performed as described21. The coating and assembly of MFCs to prepare for the seeding of MNs was performed as described6. MNs were eventually seeded for final maturation into one site  per channel over 2 min  with 115 ms exposure time as follows: Lysotracker Red  and Mitotracker Deep Red . Dual channel imaging was achieved sequentially by fast switching between both laser lines and emission filters using a motorized filter wheel to eliminate any crosstalk between both trackers.To track lysosomes and mitochondria, cells were double-stained with live cell dyes 50 nM Lysotracker Red DND-99 (Molecular Probes Cat. No. L-7528) and 50 nM Mitotracker Deep Red FM (Molecular Probes Cat. No. M22426). Trackers were added directly to culture supernatants and incubated for 1 h at 37 °C. Live imaging was then performed , step 5 Our live set up (at 100x magnification and using the Andor camera as described above) covers in its viewing field at each readout position 2 channels in parallel, each with 117.53 μm of their entire length (900 μm) from either the distal exit or from the proximal entry.6, we have observed a remarkable remote mode of action for some compounds which we added exclusively to the proximal soma site. Specifically, when we added the PARG23 inhibitor Gallotannin (PARGi) exclusively to the proximal site of FUS MNs, all trafficking defects at the distal axon were reverted back to normal Ctrl-like motility. Vice versa, PARP123 inhibitor ABT-888 (PARP1i) exclusively added to the proximal site of Ctrl MN mimicked all distal trafficking defects of FUS MN axons. Since these alterations at the distal readout occurred in the physical local absence of PARGi or PARP1i, we postulated a long range nucleo23 - axonal crosstalk. In order to investigate these remote modes of action in more detail and to validate our organelle tracking and morphology analysis  and thereby motor proteins (dyneins and kinesins) for distal organelle motility, control experiment for technical validation of our HC tracking method  was dissolved in water to obtain a 30 mM stock and ABT-888 (Santa Cruz Biotechnology Cat.# sc-202901) was dissolved in DMSO to obtain a 20 mg/ml stock, nocodazole (Sigma-Aldrich Cat.# M1404) and oligomycin A (Sigma-Aldrich Cat.# 75351), M1 fusion promoter (Merk Cat.# 475859) and Mdivi-1 fission inhibitor (Merk Cat.# 4758564) were dissolved in DMSO to obtain a 10 mM stock, respectively. Final concentrations were as follows: 30 μM for PARGi, 2 μg/ml for PARP1i, 10 μM for nocodazole, oligomycin A, M1 and Mdivi-1, respectively. DMSO was used for Mock controls (0.1%). PARGi and PARP1i were added to distal or proximal site in MFCs 72 h prior to imaging , step 5 versus the proximal HC response upon site-specific compound addition, we aimed to clearly distinguish between local and remote action as well as carefully assessing the leakiness of the MFC’s microgroove barrier.To summarize, by comparing the distal Original file containers generated by the microscope software were opened in FIJI and channels for Mito- and Lysotracker separated. Individual movies were saved as 16 bit TIFF image stack files and subsequently annotated with information regarding the experimental conditions , step 6,Movies sorted into the hierarchical folder tree  were opened individually and movement of organelles was analysed , step 7aThe movies were automatically loaded consecutively from the parental folder  into FIJI with our custom-tailored morphology macro , step 7bThe results were saved as one CSV – file per movie in the folder where the movie was stored , step 8.The ‘Konstanz Information Miner’ (KNIME) was used to process, filter and group the data , step 9 From both final parameter result tables , step 9 To obtain the final phenotypic HC signatures, Z-scores were plotted for each condition (e.g. FUS Mock) over all parameters in the following order:For Mitotracker at the distal readout, parameter # 1-10:Track DurationTrack DisplacementTrack Mean SpeedTrack Max SpeedTrack Min SpeedTrack Median SpeedTrack SD SpeedTrack LengthRatio Antero-/Retrograde TracksMitochondrial Aspect Ratio (ratio of major/minor radius of fitted ellipse)The same parameter set applied for Mitotracker at the proximal readout, resulting in parameter # 11-20.The same parameter set applied for Lysotracker at the distal readout, resulting in parameter # 21-30, except that the aspect ratio was replaced by the Feret diameter as size measure  as lysosomes always appeared of globular morphology.The same parameter set applied for Lysotracker at the proximal readout, resulting in parameter # 31-40.Z-scores.xlsx, Data Citation 1, provides all Z-scores used to plot the phenotypic HC signatures shown in Finally, to validate our batched movie analysis, we sought to obtain an estimate of the intra- vs inter-experimental variability. To this end, we compared the variabilities of (i) the organelle population of one channel vs the other within a given movie, (ii) one movie vs another within a given experiment, and (iii) one movie of experiment 1 vs another of experiment 2. The resultant pairwise Z-scores were marginal (mostly less than 10%) as compared to our stringent significance threshold  and can be obtained from All movie raw data (16-bit TIFF image stacks) were sorted into the hierarchical folder tree and are deposited in figshare .The XLSX result table of dynamic tracking parameters is available in figshare as Dynamic Tracking Parameters.xlsx, Data Citation 1. For details on how it was generated, refer to Methods, subsections ‘Handling of meta data throughout the work flow’, ‘Automated object recognition and dynamic tracking with the FIJI Track Mate plugin’, ‘Data mining in CSV result files and assembly of final XLSX result tables with KNIME’. On the left, highlighted in blue, Dynamic Tracking Parameters.xlsx, Data Citation 1, shows selected meta data information for each individual track in 4 columns as specified by the file path. Further layers of information could easily be added by further folder levels and configuring the respective nodes of the KNIME workflow. The blue part defines the experimental condition of each track and provides full transparency from the original movie file via the individual track (mid part in black showing filename and Track ID) to the final outcome of the tracking analysis in FIJI Track Mate, i.e. the parameters on the right highlighted in brown. Parameters returned by Track Mate but not further considered are greyed out on the far right.The XLSX result table of static morphological parameters is available in figshare as Static Morphology Parameters.xlsx, Data Citation 1. Same generation and layout as Dynamic Tracking Parameters.xlsx, Data Citation 1, except that the FIJI Morphology macro was used, for details refer to Methods, subsection ‘Automated object recognition and static morphology analysis with the FIJI Morphology macro’.The XLSX Z-score table used to plot all Z-score signatures as shown in The illustration of the axon–specificity in our readout windows is available in figshare as Axon Specificity.jpg, Data Citation 1.The illustration of the object recognition and tracking analysis is available in figshare as Tracking Illustration.jpg, Data Citation 1.versus proximal) changes in organelle motility and shape, we tested the Z-score HC signature response in Ctrl MNs upon addition of compounds knowingly impacting on trafficking and mitochondria morphology  oligomycin A to block mitochondrial ATP production through complex V ATP synthase inhibition25, which leads to slowing down of dyneins and kinesins and (iii) a mixture of M1 fusion promoter15 and Mdivi-1 fission inhibitor14 to cause an extended elongation of mitochondria. Following common statistical standards, we regarded Z-score values of 1 only as theoretical border line significance and considered a more stringent minimum of 5 as clearly significant deviation from Ctrl conditions  of the signature, both types of organelle responded mostly in a similar fashion to nocodazole and oligomycin A treatment , disruption of their motility through nocodazole or oligomycin A impacts relatively more on their track displacement  whereas physiological mitochondria exhibit a major stationary fraction anyway18.As for organelle - specific features in the mitochondrial (left part) versus oligomycin A (indirect energy deprivation of motor proteins). For example, nocodazole had no impact on lysosomal mean speed, led to increased min speed and decreased track length  15 and Mdivi-1 fission inhibitor14  to compare site – specific phenotypes . Oligomy6 and TDP43 mutants3 characterized by a reduction in track mean speed and displacement. These trafficking perturbations became already apparent in the maximum intensity projections of the movie raw data, particularly for FUS at the distal site . However, in this study we took the whole multiparametric TDP43 signature into account and obtained a more accurate refinement of our original finding by uncovering a global phenotype with a profound distal emphasis  or rescuing (PARGi) the distal FUS phenotype was further confirmed by hierarchical clustering . Two maiFurther refined clustering with partial signatures   yielded To summarize, our multiparametric HC profiling was very sensitive in detecting remote drug actions through axons across the MFC microgroove barrier against local modi of actions. Therefore, our method provides a powerful analytical tool to (i) detect the effects of compounds/genetic interrogation on the full content signature thus allowing better predictability of partial vs. complete effects and (ii) further interrogate the intriguing nucleo – axonal crosstalk with more compounds and RNAi reagents for mechanistic dissection.How to cite this article: Pal, A. et al. High content organelle trafficking enables disease state profiling as powerful tool for disease modelling. Sci. Data. 5:180241 doi: 10.1038/sdata.2018.241 (2018).Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.