DNA methylation has recently been identified as a mediator between in utero famine exposure and a range of metabolic and psychiatric traits. However, genome-wide analyses are scarce and cross-sectional analyses are hampered by many potential confounding factors. Moreover, causal relations are hard to identify due to the lack of controlled experimental designs. In the current study, we therefore combined a comprehensive assessment of genome-wide DNA methylation differences in people exposed to the great Chinese famine in utero with an in vitro study in which we deprived fibroblasts of nutrition.We compared whole blood DNA methylation differences between 25 individuals in utero exposed to famine and 54 healthy control individuals using the HumanMethylation450 platform. In vitro, we analyzed DNA methylation changes in 10 fibroblast cultures that were nutritionally deprived for 72 h by withholding fetal bovine serum.ENO2, ZNF226, CCDC51, and TMA7) that were related to famine exposure in both analyses. Pathway analysis with data from both Chinese famine samples and fibroblasts highlighted the nervous system and neurogenesis pathways as the most affected by nutritional deprivation.We identified three differentially methylated regions (DMRs) in four genes  contains supplementary material, which is available to authorized users. DNA methylation is one of the epigenetic mechanisms that plays an important role in the cellular responses to detrimental environmental influences that are involved in the etiology of many diseases . StudiesA seminal example of the impact of in utero exposure to nutritional deprivation is the cohort study on offspring from mothers that were pregnant during the Dutch hunger winter during the Second World War, which was intense and well-documented but with brief duration . This stWhile the Dutch famine is the most extensively studied famine in the literature, the Chinese great famine 1959–1961) was one of the largest famines recorded around the world and had more severe consequences resulting in an estimated 30 million deaths . The off9–1961 waSince a cross-sectional population-based study is subject to residual confounding and does not allow the examination of the direct effect of nutritional deprivation, we subsequently performed an in vitro study of human fibroblasts before and after exposure to nutritional deprivation. By combining the result of a genome-wide methylation approach of both studies, we aim to provide an unbiased investigation of DNA methylation changes induced by nutritional deprivation.The sample of Chinese famine is part of our previous study and has been described in more detail elsewhere . In shor2 at 37 °C . After reaching 70–80% confluence, the supernatant was removed and the cells were washed three times with phosphate buffered saline (PBS) . Next, one of the T25 flasks from each donor was cultured in the non-famine condition with Minimum Essential Medium (MEM) (Gibco®) supported with 15% FBS, while the other T25 flasks were cultured in only Minimum Essential Medium (MEM) as famine condition. After 72 h, cells were harvested from each flask and stored as cell pellet for DNA isolation.The in vitro fibroblast experiment was described in more detail previously . In shorDNA from the Chinese famine samples was extracted from whole blood using the Gentra Puregene Kit . Fibroblast cell pellets were used for DNA isolation according to the manufacturer’s instructions . The DNA concentration and quality were examined using NanoDrop . Bisulfite conversion of each DNA sample was conducted according to the manufacturer’s instructions of the Zymo EZ DNA MethylationTM Kit . Quality and quantity of the bisulphite treated single stranded DNA was examined using NanoDrop.M value calculation, and cell-type proportion estimates were obtained using the minfi package (version 1.10.2) in Bioconductor [n = 279 probes) or a detection p value larger than 0.001 in at least 5% of the samples (n = 2125 probes). Non-autosomal or cross-hybridizing probes were discarded as were loci with SNPs of minor allele frequency larger than 1% within 1 bp of the primer [One hundred and fifty nanograms of bisulfite-converted DNA from the Chinese famine study was used to quantify genome-wide patterns of DNA methylation using the Illumina Infinium HumanMethylation450 BeadChip. Genome-wide DNA methylation levels of fibroblasts were obtained using Illumina HumanMethylation EPIC BeadChip arrays. For the Chinese famine samples, intensity readouts, beta and onductor . Probes e primer . None ofe primer , and 397e primer . Moreoveples n = 25 probesn = 80), including DNA samples of other studies that included brain and blood DNA. However, normalization was conducted for the fibroblast samples only. No mismatches between methylation-predicted sex and actual gender were present nor were there samples with outliers on mean of methylated and unmethylated channels. Probes were removed if they failed quality control  or a bead count < 3 for > 10% of samples (n = 68)), were non-specific [The quality control for fibroblasts was performed in a similar workflow as the Chinese famine samples but adjusted to the newer EPIC methylation beadchip. The dataset was pre-processed in R version 3.3.1 with the meffil package  using fuspecific , or wereβ value, ranging from 0 (unmethylated cytosine) to 1 (completely methylated cytosine), but analyses were performed using M values , for better statistical validity [For both the Chinese sample and fibroblast samples, the level (percentage) of methylation is expressed as a validity . To examWe performed Gene Set Enrichment Analysis (GSEA) for the nominal significant CpGs that overlapped from the Chinese famine and fibroblasts samples. SetRank tool was chosen in the current study for GSEA analysis since it could eliminate many false positive hits , especiap values were calculated from all potential DMRs with the same number of CpGs throughout the genome. From the fit of the actual identified DMR in this distribution, an empirical p value was derived. The probability of finding the number of overlapping DMRs that we presented from all potential matches was established. All these analyses were based on 10,000 permutations.The significance level of the identified DMRs was confirmed by permutation analysis whereby Statistical analyses were carried out using R . Analysip value < 0.001) as a logical result from the larger number of loci on the methylation array of the fibroblast experiment. However, the odds of identification as nominal significant was also significantly larger in the famine study  most likely as a result of a larger power. Analysis of DMRs in the Chinese famine cohort identified 613 different methylated regions (DMRs) and 1080 DMRs in fibroblast samples. Among these significant DMRs, three DMRs were similarly associated (significant and same direction of effect) in both samples. The three replicated DMRs are all hypomethylated in relation to famine exposure and highlight four gene promoters: DMR1, enolase 2 (ENO2) , and DMR2, zinc finger protein 226 (ZNF226) . DMR3 is related to 2 gene promoters: coiled-coil domain containing 51 (CCDC51) and translation machinery associated 7 homolog (TMA7) . The study from Hannon et al. [ENO2 gene were all significantly correlated between blood and four brain regions: the prefrontal cortex (PFC), entorhinal cortex (EC), superior temporal gyrus (STG), and cerebellum (CER). Other loci with significant correlation between blood and brain were cg19331658 in ZNF226 and cg26094714 in CCD51/TM7 that were correlated with PFC, cg18912645 in ENO2 and cg12370248 and cg15853329 in CCD51/TM7 that were correlated with EC, and blood cg19720347 methylation in ENO2 that was correlated with CER. Table p value of p = 0.099, although combined p values of both analyses remain significant (p = 0.0016). Additional file Analysis of single CpG methylation did not identify significant differences after adjustment for multiple testing due to insufficient power. The QQ plot indicated the analysis was underpowered to detect genome-wide differentially methylated probes  are sensitive to nutritional deprivation.ENO2, ZNF226, CCDC51, and TMA7) that are consistently hypomethylated in relation to nutrition deprivation in both Chinese famine and fibroblast in vitro samples (Table S-adenosyl methionine (SAMe) which is dependent on nutrients such as folate, vitamin B1, B6, and B12. Genes identified in the current study have a wide range of functions, but the involvement of the gene ENO2 is one of the most interesting findings. ENO2 is abundantly expressed neurons and peripheral neuroendocrine tissue [ENO2 promotes cell proliferation, glycolysis, and glucocorticoid resistance [ENO2 serves as a biochemical marker for tumors derived from neuronal and peripheral neuroendocrine tissues [ENO2 is found to be expressed higher in the brain of schizophrenia (SCZ) patient as compared to controls and may affect glucose metabolism in SCZ patients [ENO2 hypermethylation in autism alongside with decreased transcription and translation of this gene [ENO2 (cg08003732) has a similar DNA methylation pattern in blood and brain tissue.Analysis of the overlapping DMRs from Chinese famine samples and in vitro fibroblast samples identified three DMRs in four gene promoters  and a further 6 genes were verified in mass spectrometry-based EpiTYPER assay . Althougetically . AnotherThe pathways most commonly related to malnutrition exposure are in the nervous system and neurogenesis, specifically, positive regulation of nervous system development in the GO pathway analysis  that are suggested by pathway analyses can provide new leads to understand the pathways from nutrition deprivation to disease.Using an unbiased genome-wide approach, the current study examined the association between DNA methylation and severe nutritional deprivation in two unique samples separately (Chinese famine and in vitro fibroblasts) and leads to the identification of DMRs that were consistently hypomethylated in both samples. The three DMRs in the four gene promoters Additional file 1:p value distribution for the regression of DNA methylation and Chinese famine samples. B. QQ plot of the p value distribution for the paired t test of DNA methylation and fibroblast in vitro samples. (PDF 58 kb)A. QQ plot of the Additional file 2:p value of each locus associated with nutritional deprivation in Chinese and fibroblast samples. (XLSX 4646 kb)Nominally associated loci of Chinese and fibroblast samples. The file contains the loci ID and Additional file 3:Permutation analysis of DMRs. The file contains the results of the permutation analysis. (DOCX 14 kb)


Alzheimer’s Disease (AD) is the most common form of dementia in the elderly. Due to the facts that biological causes of AD are complex in addition to increasing rates of AD worldwide, a deeper understanding of AD etiology is required for AD treatment and diagnosis.To identify molecular pathological alterations in AD brains, GSE36980 series containing microarray data samples from temporal cortex, frontal cortex and hippocampus were downloaded from Gene Expression Omnibus (GEO) database and valid gene symbols were subjected to building a gene co-expression network by a bioinformatics tool known as differential regulation from differential co-expression (DCGL) software package. Then, a network-driven integrative analysis was performed to find significant genes and underlying biological terms.A total of 17088 unique genes were parsed into three independent differential co-expression networks. As a result, a small number of differentially co-regulated genes mostly in frontal and hippocampus lobs were detected as potential biomarkers related to AD brains. Ultimately differentially co-regulated genes were enriched in biological terms including response to lipid and fatty acid and pathways mainly signaling pathway such as G-protein signaling pathway and glutamate receptor groups II and III. By conducting co-expression analysis, our study identified multiple genes that may play an important role in the pathogenesis of AD.The study aimed to provide a systematic understanding of the potential relationships among these genes and it is hoped that it could aid in AD biomarker discovery. Aging causes an increasing susceptibility to cognitive performances due to a developing neurodegeneration leading to neurologic disorders, such as dementia. More than 20 million people worldwide suffer from dementia, and this number is expected to exceed 80 million by 2040 because of the rapid increase in the numbers of the elderly Rewiring of the biological networks to detect co-regulated and co-expressed units will help to facilitate looking into network’s components and depicting the relationships between interconnected genes. Gene co-expression networks enable us to highlight molecular mechanisms underlying diseases CEL files for GSE36980 series were downloaded from the GEO (http://www.ncbi.nlm.nih.gov/geo/) database and normalized with RMA method by using the Linear Models for Microarray Data (limma) R package. The main reason for selecting and exploiting this dataset is that GSE36980 series cover interspecies transcriptome analysis of various regions in gray matter in postmortem brains suiting the goal of dissecting pathological alterations in AD in several brain areas. Moreover, a number of researches have previously used these series and therefore would be able to compare the findings. After removing ambiguous probes, the extracted probe IDs were transformed into gene symbols. This data consists of a total of 79 samples Y = Next, DCGs and DCLs were extracted from DCp and DCe values previously calculated by DCp and DCe functions. DCp filters co-expression values of a pair of genes were assessed in control and treatment conditions. The DC of a given gene is calculated with the following equation:If the resulting DCGs and DCLs coincide with a TF, they will be referred to as a DRG and DRL, respectively. The DRGs and DRLs were scrutinized by DR-sort function in Differential Regulation Analysis (DRA) module. In fact, DRA module identifies potential TF as upstream regulators of DCGs and DCLs http://pantherdb.org/) to underlying pathways . Afterward, using 2918 unique genes, co-expression analysis was performed on temporal cortex, frontal cortex, and hippocampus datasets separately. Expression based filter removes genes whose mean expression between experiments is lower than the median of this value for all genes and variance based filter removes genes that are not significantly variable than the median gene PAX5 transcription factor and genes including ARID1A, CDC42 and LPPR4 were highlighted as the most important units within the genes network with more interconnected links . There were 199 significant TFs in the results of TED analysis and 35 significant TFs in TDD analysis. 35 TFs that were significant in both of these two analysis results were chosen (Supplementary file 2). DRA analysis yielded 7 DRGs and 33 DRLs. DRGs were not only differentially co-expressed but also differentially co-regulated with 35 mentioned TFs. Then, a network of DRGs and DRLs was visualized using Cytoscape 3.4.0. Based on ed links .PAX5 and IKZF1 as TFs and genes including GRIK3, MAGI3, PRRX1 and DCAF6 were found as highlighted nodes with more connectivity.In frontal cortex datasets, 628 DCGs and 166256 DCLs were summarized to 20 DRGs and 164 DRLs (Supplementary file 3). There were 199 significant TFs in TED analysis result and 135 significant TFs in TDD analysis result from which 135 TFs were chosen that were significant in both TED and TDD results (Supplementary file 3). In the inferred network, PAX5, ARNT, GATA1, EGR3 and IKZF1 TFs and genes including KCNK1, CACHD1, FABP3 and CHRNB2 showed highlighted roles as network nodes.According to hippocampus datasets, 670 DCGs and 56264 DCLs were summarized to 16 DRGs and 43 DRLs (Supplementary file 4). There were 199 common and significant TFs in TED and TDD analysis which were used for inferring differential co-expression network with DRGs. There was more connectivity in hippocampus network than the other two networks. DEGS1) and fatty acid binding protein 3 (FABP3) in hippocampus and lipid phosphate phosphatase-related protein type 4 (LPPR4) in temporal cortex datasets may fairly explain the relationship between brain damages happening in these regions and lipid metabolism. DEGS1 encodes a member of the membrane fatty acid desaturase family which is shown to interfere in AD via lipid rafts FABP are elevated in a significant proportion of patients with various neurodegenerative diseases including AD LPPR4 acts as phospholipid dephosphorylate involving axonogenesis. The control of ion flow across the lipid membrane is essential for many cellular functions, including neuronal excitability and dysfunction of conveying ions through lipid bilayers involved in multiple neurologic diseases via ionotropic glutamate receptors (iGluR) and metabo-tropic glutamate receptors (mGluR), both of which have been implicated in AD GRIK3) and voltage-dependent R-type calcium channel subunit alpha-1E (CACNA1E) in frontal cortex datasets may be biologically relevant with the mentioned pathways in AD brain areas. Concordantly, a significant change in the expression of the GRIK3 gene was detected in a patient diagnosed with severe developmental delay CDC42. CDC42 has been linked to neuronal diseases like Alzheimer and Parkinson’s disease through its role in cytoskeletal organization CNTN2, KCNK1 and S100A1 were found common in frontal cortex and hippocampus datasets. S100A1 encodes for calmodulin signaling molecules. Increased levels of calmodulin have been reported in the hippocampus of AD model mice KCNK1, recent genetic studies suggest a central role for neuroinflammation. KCNK1 is a voltage-gated potassium channel upregulated by activated microglia and a mediator in amyloid-mediated microglial priming, additionally reactive oxygen species production that was shown to be related with autoimmunity Aging is believed to be one of the most important non-modifiable risk factors of cognitive diseases that lead unequivocally to a number of detrimental changes in the neural system, increasing neuromorbidity and mortality. AD, as a progressive neurodegenerative disorder with no effective treatment options, is typically characterized by the presence of amyloid-beta plaques and hyper phosphorylated paired helical filament tau protein-rich neurofibrillary tangles These findings probably show that hippocampus and frontal cortices might deeply play a role in AD by mediating with conveying ions. Their obtained DRGs participated in vital processes like signaling, ion transportation and homeostasis. However, these processes mostly signal pathways somehow shared with temporal cortex implying the role of signal molecules within and between brain areas in neurologic dysfunctions. Concordantly, a comprehensive study has been already carried out on GSE36980 series to examine the alteration in the expression of diabetes-related genes in AD brains where they illustrated that hippocampi of AD brains have the most significant alteration in gene expression profile et alet alet alWith a glance at The purpose of the study was to explore the molecular mechanism in the development of AD, and a comparison of AD in three regions of the brain was done. Therefore, in the frame of network reconstruction and data mining approaches, a small number of possible genes and TFs were identified that their interplay could lead to neural dysfunctions toward AD. However, one should be cautious regarding small sample size while by utilizing more adequate samples, the results would be more reliable evidences.An expected outcome of such a work would possibly shed light on the bridges between AD-associated brain damage in transcriptome level and presenting crucial evidence in clinical diagnosis and treatment.


Gastric emptying is impaired in patients with gastroparesis whereas it is either unchanged or accelerated in obese individuals. The goal of the current study was to identify changes in gene expression in the stomach muscularis that may be contributing to altered gastric motility in idiopathic gastroparesis and obesity.Quantitative real time RT-PCR and whole transcriptome sequencing were used to compare the transcriptomes of lean individuals, obese individuals and either lean or obese individuals with idiopathic gastroparesis.Obesity leads to an increase in mRNAs associated with muscle contractility whereas idiopathic gastroparesis leads to a decrease in mRNAs associated with PDGF BB signaling. Both obesity and idiopathic gastroparesis were also associated with similar alterations in pathways associated with inflammation.Our findings show that obesity and idiopathic gastroparesis result in overlapping but distinct changes in the gastric muscularis transcriptome. Increased expression of mRNAs encoding smooth muscle contractile proteins may be contributing to the increased gastric motility observed in obese subjects, whereas decreased PDGF BB signaling may be contributing to the impaired motility seen in subjects with idiopathic gastroparesis.The online version of this article (10.1186/s12920-019-0550-3) contains supplementary material, which is available to authorized users. These cells transmit inhibitory neural signals to gastric smooth muscle cells [+ cells without a change in ICC [Gastroparesis defined as delayed gastric emptying in the absence of a physical obstruction, is estimated to occur in about 1.8% of the adult population, with the majority of cases being in women . Patient disease –7. ICC a disease , 8. The le cells . Using qe in ICC . Recentle in ICC . These oe in ICC , 12.2) have increased rates of gastric emptying as compared to low BMI subjects (BMI 18-25 kg/m2) [In our recent studies, and in other studies published by the NIDDK Gastroparesis Clinical Research Consortium, control biopsies have been obtained from patients undergoing bariatric weight loss surgery , 13–15. 5 kg/m2) , 17. ThiAll procedures were conducted under protocol approved by the Indiana University Institutional Review Board. Informed consents were obtained from control subjects and patients with idiopathic gastroparesis. Idiopathic gastroparesis was defined as patients with symptoms of gastroparesis ≥3 months with delayed gastric emptying by 4-h gastric scintigraphy or the presence of a gastric bezoar with undigested foods by upper endoscopy after an overnight fast. Gastric scintigraphy was performed following consumption of a low-fat egg-white meal with imaging at 0, 2 and 4 h as described previously , 19. AbnBiopsies from the low BMI control group (no reported diabetes or gastroparesis) were obtained from organ transplant donors at the time of organ transplant. Biopsies from the high BMI control group were obtained from non-diabetic individuals without gastroparesis symptoms that were undergoing bariatric weight loss surgery . Surgical full-thickness biopsies from the idiopathic gastroparesis patients were obtained at the time of routine pathological analysis in patients with severe symptoms or at the time of gastric stimulator placement. In each case (controls and gastroparesis patients), the biopsy was taken about 10 cm from the pylorus on the anterior aspect midway between the greater and lesser curvatures of the stomach. All biopsies were obtained using linear cutting staplers. For sleeve gastrectomy subjects the sample was obtained after the stomach was removed from the abdomen, for all other subjects the samples were obtained laparoscopically. Tissues were collected into ice cold DMEM (Dullbeco’s modified eagle media) or RNAlater (Ambion) and immediately transferred to the investigator’s laboratory. Both the submucosal and mucosal layers were carefully removed from the muscularis by dissection under a binocular microscope, the muscularis was then flash frozen in liquid nitrogen and ground to a powder under liquid nitrogen prior to storage at − 80°. A small amount of frozen muscle tissue powder (~ 150 mg) was homogenized in Trizol (Invitrogen) and a Purelink Micro RNA isolation kit was used to isolate total RNA. Contaminating DNA was removed by on-column DNase treatment. The integrity and purity of the isolated RNA was determined using an Agilent Bioanalyzer. Samples used had an RNA integrity number of > 7. Levels of ATP4A and gastrin mRNA were analyzed in all samples by qRT-PCR to screen for mucosal contamination. Only samples which had levels of expression of these markers that were less than 100-fold lower than the levels present in mucosa were utilized for further analysis.The concentration and quality of total RNA samples was first assessed using Agilent 2100 Bioanalyzer. A RIN (RNA Integrity Number) of 7 or higher was required to pass the quality control. Then 200 ng of RNA per sample was used to prepare a dual-indexed strand-specific cDNA library using KAPA mRNA Hyperprep Kit (Roche). The resulting libraries were assessed for their quantity and size distribution using Qubit and Agilent 2100 Bioanalyzer. Two hundred pico molar pooled libraries were utilized per flowcell for clustering amplification on cBot using HiSeq 3000/4000 PE Cluster Kit and sequenced with 2.75 bp paired-end configuration on HiSeq4000 (Illumina) using HiSeq 3000/4000 PE SBS Kit. A Phred quality score (Q score) was used to measure the quality of sequencing. More than 90% of the sequencing reads reached Q30 . The sequencing was performed in the Center for Medical Genomics (CMG) at Indiana University School of Medicine, a sequencing core facility of the Indiana CTSI.p-values using the Benjamini-Hochberg procedure. Complete RNA-sequencing data is available from the NCBI GEO database, accession number GSE129398.The sequencing data were first assessed using FastQC  for quality control. Then all sequenced libraries were mapped to the human genome (UCSC hg38) using STAR RNA-seq aligner  with theqRT-PCR. 500 ng of RNA was used in reverse transcription reactions . Real time PCR was performed using Sybr Green (Roche). Levels of mRNA expression were normalized to expression of TATA binding protein (TBP) as an internal control and are expressed relative to the mean values seen in samples from control low BMI patients. The primers used for qRT-PCR are listed in Additional file Significant differences between groups were identified using one way Anova analysis with a Kruskal-Wallis test and a Duns Multiple Comparison post-test.n = 9), high BMI control bariatric surgery patients  and low BMI subjects with idiopathic gastroparesis . The clinical data of the subjects used are summarized in Additional file To determine if obesity is affecting the transcriptome of the stomach muscularis externa we obtained full thickness biopsy samples from transplant donors at the time of organ harvest and reevaluated the previously reported mRNA changes described in idiopathic gastroparesis subjects . TissuesIn contrast to our previously reported decreases in expression of mRNAs encoding some contractile proteins in idiopathic gastroparesis subjects compared to high BMI control subjects , when con = 6), high BMI control bariatric surgery patients  and low or high BMI subjects with gastroparesis . To visualize the overall differences in mRNA expression between each of the groups of samples we performed a heat map analysis on all genes that were found to be significantly different between any group of samples as determined by an ANOVA analysis with an FDR cutoff of < 0.05. For the heat map analysis sample grouping was assessed using a complete linkage analysis together with a Pearson distance analysis (Heatmapper). This analysis clearly showed that the samples obtained from control patients with a low BMI clustered away from both the samples from control patients with high BMI and the samples from subjects with idiopathic gastroparesis Additional file 2:Figure S2. Comparison of the changes in mRNA expression observed in the current study to those previously reported by Grover et al. (JPG 490 kb)Additional file 3:Figure S3. qRT-PCR verification of additional genes with altered expression. (JPG 2440 kb)Additional file 4:Table S1. Primers used for qRT-PCR assays. (DOCX 169 kb)Additional file 5:Table S2. Clinical summary of subjects studied. The table lists the subjects used in the study, their BMI, age, sex and 4-h gastric retention. ND-not determined. (XLSX 37 kb)Additional file 6:Table S3. RNA- sequencing data. The table shows the list of samples used for RNA-sequencing, mapping statistics for each sample and differential gene expression analysis either with Y-chromosome encoded genes filtered out  or when samples were only compared between subjects of the same sex . (XLSX 30240 kb)Additional file 7:Table S4. Summary of changes in expression of predicted PDGF BB target genes obtained from RNA-sequencing data. PDGF BB target genes that were altered in the Low BMI gastroparesis subjects relative to the low BMI control subjects with a FDR < 0.05 are indicated, together with the predicted PDGF BB target genes identified in the previously published Idiopathic Gastroparesis cohort [s cohort .


Primary lymphoma that arises de novo from the central nervous system (CNS) is most commonly a non-Hodgkin's B-cell lymphoma and by definition lacks the presence of disease outside the CNS. It demonstrates a characteristic imaging appearance on computed tomography (CT) and magnetic resonance imaging (MRI) exams related to its inherent hypercellularity. On CT, primary CNS lymphoma (PCNSL) demonstrates a hyperdense appearance; on MRI, it commonly demonstrates restricted water diffusion on diffusion-weighted sequences and homogeneous enhancement on post-contrast sequences. We present a case of primary CNS lymphoma in an immunocompetent patient with progressive necrosis and loss of restricted diffusion on diffusion-weighted imaging (DWI) with an atypical enhancement pattern. We further provide a review of the literature regarding the CT and MRI appearance of primary CNS lymphoma and discuss the role of immune status in determining the imaging characteristics of this disease process. Lymphoma may occur as a de novo primary central nervous system (CNS) lymphoma (PCNSL) with absence of disease outside the CNS or as a secondary CNS lymphoma (SCNSL) related to systemic disease and CNS metastasis -2. Both Whereas SCNSL typically involves the skull and dura, PCNSL much more commonly involves the brain parenchyma and has a characteristic imaging appearance in the immunocompetent patient population related to the tumor’s inherent hypercellularity, high nuclear/cytoplasmic ratio, and disruption of the blood-brain barrier. On non-contrast computed tomography (CT), PCNSL is typically hyperdense. When magnetic resonance imaging (MRI) is performed, PCNSL demonstrates diffusion restriction on diffusion-weighted imaging (DWI) and avid enhancement with gadolinium administration. PCNSL has a predilection for the deep structures of the brain and typically demonstrates a periventricular or perivascular location. Two-thirds of tumors present as a solitary mass. The presence of necrosis and hemorrhage with PCNSL is uncommon unless the patient is immunocompromised ,4-5.A 50-year-old male with a past medical history of hypertension and heavy alcohol use in the past (reportedly abstinent for the preceding four months) presented with one month of recurrent and progressive dizziness and worsening memory impairment. The patient also endorsed anomic aphasia (word finding difficulty), acalculia , and confusion. Neurologic exam demonstrated 3+ hyperactive lower extremity reflexes, a positive Babinski sign bilaterally, decreased lower extremity vibratory sensation, and conjugate nystagmus on lateral gaze with horizontal diplopia. The remaining neurologic exam was unremarkable. 9/L and a normal thiamine level; remaining laboratory findings were within normal limits. A lumbar puncture with cerebrospinal fluid (CSF) analysis was unremarkable. Non-contrast head CT followed by serial MRI examinations of the brain without and with intravenous (IV) gadolinium was performed for further assessment.Based on presenting signs and symptoms, the patient was tentatively diagnosed with Wernicke-Korsakoff syndrome. Laboratory testing, including comprehensive metabolic panel, complete blood count, thyroid stimulating hormone, HIV, and thiamine level were performed. Laboratory findings revealed an elevated white blood cell count of 12.6 × 10Initial non-contrast CT of the head demonstrated a well-demarcated area of slightly hyperdense attenuation with surrounding edema centered in the bilateral parietal lobes and splenium of the corpus callosum extending across the midline. There was no hemorrhage or mass effect Figure .Based on the head CT findings and clinical presentation, a follow-up pre and post-contrast brain MRI was performed and demonstrated a mass in the subcortical white matter in the bilateral parietal lobes and splenium of the corpus callosum with extension across the midline and surrounding vasogenic edema. The mass was isointense on T2-weighted and fluid-attenuated inversion recovery (FLAIR) sequences. DWI with a B-value of 1000 and corresponding apparent diffusion coefficient (ADC) map demonstrated mild diffusion restriction. The post-contrast T1-weighted sequence demonstrated discontinuous areas of homogeneous enhancement. No associated mass effect, volume loss, or hemorrhage was present  was raised based on the follow-up imaging. The patient subsequently underwent a left occipital-approach stereotactic biopsy of the lesion. The specimen yielded sheets of large lymphocytes with open chromatin, nucleoli and high nuclear to cytoplasmic ratio consistent with diffuse large B-cell lymphoma. Inpatient treatment with chemotherapy was initiated. Post treatment brain MRI demonstrated a positive response to therapy with decreased volume of the lesion and resolution of the peripheral enhancement Figure .This case is unique for several reasons. Serial follow-up brain MRI exams demonstrate intercurrent lack of hypercellularity associated with the tumor as no restricted water diffusion on DWI was present at the three and five month intervals. A study by Haldorsen et al.reviewed the CT and MRI findings of 76 patients with biopsy-proven PCNSL and found 100% of their patients demonstrated either isodense or hyperdense attenuation of the tumor relative to white matter on non-contrast head CT .al.describe homogeneous contrast enhancement on MRI in 90% of patients in the non-AIDS population [The serial follow-up MRI exams performed in this patient who refused biopsy and surgical treatment also demonstrated an increasingly necrotic appearance of the tumor on T2-weighted imaging with internal areas of hyperintense signal intensity within the corpus callosum and biparietal deep white matter. This imaging appearance on MRI has been observed in AIDS-related PCNSL with many of the lesions exhibiting necrotic regions and more commonly a multifocal or disseminated appearance ,5,9. SubAtypical MRI appearance of PCNSL  in an immunocompetent patient is rare in the USA. This presentation is more common among patient with tumors who are positive for EBV -10. EBV-Given the imaging appearance on follow-up brain MRI exams, the question of whether this lesion could represent tumefactive demyelination (TDL) was also raised. Differentiating PCNSL from TDL may be extremely difficult in some patients due to the variability of clinical presentation and imaging findings . The lymHistologically, differentiating demyelination from PCNSL may also be challenging due to the fact the PCNSL may present in association with steroid-responsive multifocal demyelinating sentinel lesions, which histologically are indistinguishable from multiple sclerosis (MS), containing predominantly T-cell infiltrates and a few B cells ,11. Addi−6 mm2/s was the best indicator for differentiating TDL from atypical PCNSL (a sensitivity of 81% and specificity of 89%) [A few studies have tried to differentiate TDLs from PCNSLs using advanced MR imaging techniques. Two studies demonstrated a significantly higher ADC minimum in TDL than in PCNSL with one study demonstrating that ADC minimum with a threshold of 556 × 10 of 89%) -18.Another study showed a lower choline/N-acetylaspartate (NAA) ratio in TDLs than in PCNSLs, with a threshold for differentiation of 1.73 yielding sensitivity of 89% and specificity of 76%. In addition, non-contrast CT hypoattenuation of MRI enhancing regions was observed in 93% of TDL cases, but only 4% of primary brain tumors . One stuWe present an unusual case of PCNSL in an immunocompetent patient demonstrating a progressive, atypical imaging appearance on MRI. Findings help reinforce the protean appearance of PCNSL, which may occasionally lack the characteristic hypercellular imaging appearance on CT and MRI exams especially if left untreated. Review of the literature suggests the presence of necrosis and peripheral enhancement on MRI to be uncommon in the immunocompetent population and more commonly associated with EBV-positive tumors or in HIV positive patients. The lack of the typical imaging appearance of PCNSL should not dissuade one from the diagnosis, especially for a lesion located in the deep white matter or corpus callosum.


In an Essay, Mark Tenforde and colleagues advocate continued provision of baseline CD4 cell count testing in HIV care in low- and middle-income countries. Antiretroviral therapy (ART) has substantially decreased HIV morbidity and mortality in high-income as well as low- and middle-income countries (LMICs). Several randomized trials have demonstrated benefits from starting ART regardless of CD4 count  1–3]; t–3; t1–3]In the “treat all” era, the challenge is targeting effective prophylaxis against the bacterial, mycobacterial, and fungal pathogens that are the major causes of death in these late presenters. Such prophylaxis saves lives (see below) but also costs money and raises the potential risk of antimicrobial resistance if used indiscriminately. Further, when prophylaxis is provided conditional on a diagnostic test result, unless truly point-of-care, there is the potential for delays in ART initiation, which may substantially increase mortality risk.Specific tests that have evidence supporting their use in reducing mortality and are recommended to target treatment/prophylaxis to patients with HIV and low CD4 cell counts include tuberculosis (TB) smear, Xpert MTB/RIF and urinary lipoarabinomannan (LAM) for TB, and cryptococcal antigen (CrAg) to detect cryptococcal infection . TheoretCD4 has consistently been found to be a key predictor of mortality after ART initiation , strongeGiven the limited ability of clinical assessment alone to identify patients with advanced HIV disease and the evidence for mortality benefit of enhanced OI screening in patients with advanced disease , we suggCD4 testing remains important for staging HIV pre-ART, as it is the only way to identify the population at highest risk of death. However, even where immediate CD4 testing is unavailable, particularly in sub-Saharan Africa (SSA), all algorithms should still begin with intensive case finding for TB. Same-day screening with either smear or Xpert MTB/RIF should ideally be performed, but absence of such testing should not be a barrier to prompt ART initiation with isoniazid and cotrimoxazole (preferably as a fixed-dose-combination with pyridoxine ) when clThe TEMPRANO trial, which had a predominance of patients with early HIV (CD4 > 350 cells/μL), demonstrated the added benefit of 6 months’ isoniazid prophylaxis to ART (35% reduction in death or severe HIV-related illness), as did the REMEMBER trial in participants with advanced HIV disease (CD4 < 50 cells/μL); in both trials, mortality rates in the isoniazid treatment arm were lower than historical controls ,9. Of noSeveral assays (including Alere’s PIMA and Becton Dickinson’s FACSPresto) are commercially available for rapid CD4 testing. If rapid CD4 testing is available and results are <100 cells/μL, where other tests are not available, low-dose (100 mg/kg) fluconazole and a 5-day course of azithromycin (500 mg/day) along with isoniazid/cotrimoxazole/pyridoxine (as a late-presenters “package”) decreased mortality risk by 27% (3% absolute risk reduction) in the REALITY trial [Where rapid CD4 testing is available and additional tests for OIs can be performed, the challenge is balancing the excess mortality risks associated with potential delays to ART while waiting for test results against benefits of targeting prophylaxis more narrowly. In hospitalized patients with advanced HIV, urinary LAM testing followed by 4-drug anti-TB treatment for positives decreased 6-month mortality ,17. LAM Immunosuppressed patients also benefit from CrAg screening and preemptive therapy with fluconazole if positive, when bundled with ART initiation. The REMSTART trial, which included participants with CD4 < 200 cells/μL, found that CrAg screening coupled with community-based ART support decreased mortality by 28% (4% absolute reduction) . The IMMp = 0.03) in patients receiving enhanced prophylaxis including azithromycin; this difference may have been due in part to reductions in bacterial sepsis. Among those dying from unknown causes, baseline CrAg prevalence was very low, suggesting that these were not due to undiagnosed cryptococcosis [Based on the REALITY trial , 5 days coccosis .Baseline CD4 testing is still important to guide the diagnostic evaluation for common OIs and prophylaxis in patients newly diagnosed with HIV. Conventional laboratory-based CD4 testing using flow cytometry requires substantial infrastructure and technical expertise and may lead to significant delays in obtaining actionable results, with a systematic review estimating a mean time of 10.5 days from the time a CD4 test was conducted to the time the result was received, versus 0.1 days with point-of-care CD4 testing . SeveralBaseline CD4 is an essential part of HIV care, and implementation research is needed to better streamline this and other new point-of-care tests for OIs to make them practical to perform in underresourced centers. We present a resource-based public health approach according to diagnostic test availability that could decrease early mortality after ART initiation and would be practical to implement. Our approach does not allow “the best to be the enemy of the good.” Even the most resource-constrained settings can immediately implement interventions that have the potential to save thousands of lives, and further refinement can be offered in settings where rapid screening for common OIs is feasible. An optimal approach requires that pre-ART CD4 (preferably as a simple point-of-care threshold test) continues to be available. We believe this provides a pragmatic algorithm to avoid delaying ART for the most immunosuppressed patients who are at the highest risk of dying.


The presence of supernumerary centrosomes is prevalent in cancer, where they promote the formation of transient multipolar mitotic spindles. Active clustering of supernumerary centrosomes enables the formation of a functional bipolar spindle that is competent to complete a bipolar division. Disruption of spindle pole clustering in cancer cells promotes multipolar division and generation of non-proliferative daughter cells with compromised viability. Hence molecular pathways required for spindle pole clustering in cells with supernumerary centrosomes, but dispensable in normal cells, are promising therapeutic targets. Here we demonstrate that Aurora A kinase activity is required for spindle pole clustering in cells with extra centrosomes. While cells with two centrosomes are ultimately able to build a bipolar spindle and proceed through a normal cell division in the presence of Aurora A inhibition, cells with supernumerary centrosomes form multipolar and disorganized spindles that are not competent for chromosome segregation. Instead, following a prolonged mitosis, these cells experience catastrophic divisions that result in grossly aneuploid, and non-proliferative daughter cells. Aurora A inhibition in a panel of Acute Myeloid Leukemia cancer cells has a similarly disparate impact on cells with supernumerary centrosomes, suggesting that centrosome number and spindle polarity may serve as predictive biomarkers for response to therapeutic approaches that target Aurora A kinase function. The formation of a mitotic spindle with two spindle poles is integral to proper chromosome segregation. During mitosis a pair of centrosomes, the microtubule nucleating and organizing centers of the cell, serve as focal points at which molecular motors and crosslinker proteins cluster microtubules (reviewed in  and 2]))2]). In Aurora A (AurA) is a serine/threonine kinase that is important for proper bipolar spindle formation. Localized to the centrosome during interphase and to spindle poles during mitosis, AurA phosphorylates protein targets to regulate centrosome maturation and spindle assembly such that loss of AurA kinase–dependent phosphorylation of key mitotic targets disrupts spindle formation , 17. SomHere we show that the response to AurA kinase inhibition is influenced by centrosome number and the capacity to form multipolar spindles during mitosis. As described previously, inhibition of AurA kinase activity in cells with normal centrosome content transiently disrupts mitotic spindle formation –29. HoweTo explore the relationship between spindle polarity and response to AurA inhibition we utilized two experimental systems to manipulate centrosome number. First, we induced supernumerary centrosomes using a previously well-characterized human epithelial cell line carrying a doxycycline-regulated PLK4 expression construct (indPLK4) . PLK4 isAlisertib (MLN8237) is an orally bioavailable inhibitor of AurA kinase that is ∼200 fold more selective for AurA than the closely related Aurora B . PharmacTo assess how mitotic cells with excess centrosomes respond to AurA inhibition, both control and indPLK4 RPE-1 cells, and HCT116 cells ± Cyto B were treated with inhibitor for 16 hours, followed by immunofluorescence imaging. This duration of treatment was sufficient to limit each cell to one mitotic event in the presence of AurA inhibition. Consistent with previous reports, we find that cells with two centrosomes exhibit an increase in acentrosomal and disorganized mitotic spindle poles following exposure to any one of four specific inhibitors of AurA kinase activity: alisertib, MLN8054 (MLN), Aurora A inhibitor 1 (AA1), and MK-5108 (MK/VX-689)  22]. Ne. Ne22]. Following Aurora A inhibition, cells with supernumerary centrosomes form multipolar and disorganized spindles similarly to control cells. In these cells centrosomes are present at the majority of excess spindle poles Figure  and 1D aCells that are unable to form a bipolar spindle are expected to accumulate in mitosis. However, FACs analysis of cellular DNA content, together with imaging-based assessment of mitotic enrichment indicate that the 4N (G2/M) population of cells is not significantly changed and mitotic cells do not surpass ∼10% of the cell population following short term (16–24 h) AurA inhibition . TogetheAurora A is known to function in both centrosome maturation and spindle assembly pathways and long term inhibition or RNAi-based depletion strategies compromise both processes. Therefore, to assess the role of AurA specifically in spindle bipolarity in cells with excess centrosomes, while limiting confounding effects of AurA inhibition on centrosome maturation, we performed live cell imaging on cells that entered mitosis within the first 30 minutes of drug-induced AurA inhibition (ie after centrosome maturation). These cells were then followed though mitotic exit and for the next 36 hours to assess cell fate. Images of RPE-1 cells expressing an RFP-tagged histone (RFP-H2B) to enable monitoring of chromosome movement and cell cycle progression were captured every 5 minutes for the duration of the experiment. Untreated RPE-1 cells progressed from nuclear envelope breakdown (NEB) to anaphase onset in ∼20 minutes. Consistent with our fixed cell analysis, following a prolonged mitosis ∼85% of cells with extra centrosomes (indPLK4) ultimately exit mitosis with a bipolar division Figure , while tConsistent with a high incidence of bipolar anaphases seen in fixed cell imaging Figure , followiFollowing completion of mitosis, each cell was tracked to monitor cell cycle re-entry. indPLK4 cells that were able to achieve a bipolar division were just as likely as their counterparts with normal centrosome content to proceed through a subsequent cell cycle and enter into a second mitosis Figure .in situ hybridization (FISH)- based approaches using centromere-targeted enumeration probes for individual chromosomes similarly demonstrate that while short term induction of PLK4 (<2 cell cycles) results in a 2-fold increase in aneuploid cells, indPLK4 cell populations exhibit a 4-fold increase in aneuploid cells and a >6-fold increase in tetraploid cells following AurA inhibition  cell lines. AML is a cancer context where AurA kinase is often amplified, and where its inhibition is actively being exploited in preclinical and clinical approaches –37. WestWhile FISH-based analysis of chromosome copy number indicates that all 4 AML cell lines have a modal chromosome copy number near 4 , immunofOur data support a model whereby the presence of extra centrosomes, acquired either through over-duplication or a previous failed cytokinesis, can alter the response of cells to inhibitors of mitotic spindle assembly and subsequently impact the efficacy of such drugs. We find that following AurA inhibition, cells with supernumerary centrosomes exhibit multipolar and disorganized spindles that cannot be resolved prior to mitotic exit. Instead, such cells are subject to abortive mitoses where cytokinesis is attempted in the absence of metaphase alignment, and anaphase chromosome segregation is lacking. Daughter cells that result from such catastrophic mitoses are multinucleated and exhibit dramatic changes in chromosome content. Importantly, such progeny are not competent for continued proliferation. In contrast, following AurA inhibition, cells with normal centrosome number ultimately overcome a transient disruption of spindle geometry to form bipolar spindles and complete near-normal cell division. Progeny from these divisions remain proliferative and proceed through the cell cycle to enter a second mitosis with frequency and timing comparable to control cells. In this way, we propose that a differential response to AurA inhibition in cancer cells may stem not from a change in the initial impact on spindle assembly, but instead from an essential and cancer-specific dependence on AurA-driven dynamics of spindle-pole clustering activity in cells with supernumerary centrosomes.Proteins that are required for centrosome clustering are often overexpressed in human cancers with extra centrosomes (reviewed in: ). IndeedTogether with cell biological evidence that inhibition of AurA compromises mitotic progression, an increased AurA expression profile in cancer supports that AurA is a clinically relevant drug target . PharmacCells were grown in DMEM medium (RPE-1) or RPMI 1640 medium  supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin and maintained at 37° C with 5% CO2. For experiments where PLK4 expression was induced, media was supplemented with 2 μg/mL Doxycycline for 36 hours. Alternatively, cells were treated with 30 μM Cytochalasin B for 16 h to induce cytokinesis failure and generate tetraploid cells with double the normal centrosome number . Drug trTo assess cell cycle differences and response to mitotic inhibitors, cells were treated with 100 ng/mL Nocodazole (to induce mitotic arrest) and/or 100 nM alisertib for 16–18 hours, fixed and stained with propidium iodide, and processed for FACs analysis as previously described . To asseCells were prepared and fixed as described previously  and α-saFor immunofluorescence imaging, cells were cultured on glass coverslips (RPE-1), or spun onto coverslips by centrifugation at 1000 RPM for 3 minutes (AML cell lines) prior to fixation for 10 min in ice-cold methanol, blocked in TBS-BSA , and stained for α-tubulin , and/or centrin-2 (Santa Cruz) as indicated. DNA was detected with 0.2 μg/mL DAPI (Sigma), and coverslips mounted with Prolong Antifade Gold mounting medium (Molecular Probes). Cells were plated in 12 well imaging bottom tissue culture dishes for live cell imaging. Images of fixed and live cells were captured with a Zyla sCMOS camera mounted on a Nikon Ti-E microscope with a 60x Plan Apo oil immersion objective, or a 20x CFI Plan Fluor objective. A minimum of 50 mitotic cells or 1000 interphase cells were analyzed per condition for each of 3 biological replicates. Live cell images were captured at 10 coordinates per condition at 5 min intervals for 24 hours, for each of 3 biological replicates. 100 individual cells were tracked through mitosis and analyzed for each condition and replicate. Error bars throughout represent standard error unless otherwise indicated. Pairwise analyses were performed using a one-way ANOVA to determine statistical significance.


C9orf72), progranulin (GRN), and microtubule-associated protein tau (MAPT) genes, with mutations more rarely seen in a number of other genes. This review will discuss the recent updates in the field of genetic FTD. Age at symptom onset in genetic FTD is variable with recently identified genetic modifiers including TMEM106B (in GRN carriers particularly) and a polymorphism at a locus containing two overlapping genes LOC101929163 and C6orf10 (in C9orf72 carriers). Behavioural variant FTD (bvFTD) is the most common diagnosis in each of the genetic groups, although in C9orf72 carriers amyotrophic lateral sclerosis either alone, or with bvFTD, is also common. An atypical neuropsychiatric presentation is also seen in C9orf72 carriers and family members of carriers are at greater risk of psychiatric disorders including schizophrenia and autistic spectrum disorders. Large natural history studies of presymptomatic genetic FTD are now underway both in Europe/Canada (GENFI—the Genetic FTD Initiative) and in the US (ARTFL/LEFFTDS study), collaborating together under the banner of the FTD Prevention Initiative (FPI). These studies are taking forward the validation of cognitive, imaging and fluid biomarkers that aim to robustly measure disease onset, staging and progression in genetic FTD. Grey matter changes on MRI and hypometabolism on FDG-PET are seen at least 10 years before symptom onset with white matter abnormalities seen earlier, but the pattern and exact timing of changes differ between different genetic groups. In contrast, tau PET has yet to show promise in genetic FTD. Three key fluid biomarkers have been identified so far that are likely to be helpful in clinical trials—CSF or blood neurofilament light chain levels , CSF or blood progranulin levels (in GRN carriers) and CSF poly(GP) dipeptide repeat protein levels (in C9orf72 carriers). Increased knowledge about genetic FTD has led to more clinical presymptomatic genetic testing but this has not yet been mirrored in the development of either an accepted FTD-specific testing protocol or provision of appropriate psychological support mechanisms for those living through the at-risk phase. This will become even more relevant as disease-modifying therapy trials start in each of the genetic groups over the next few years.Frontotemporal dementia (FTD) is a highly heritable group of neurodegenerative disorders, with around 30% of patients having a strong family history. The majority of that heritability is accounted for by autosomal dominant mutations in the chromosome 9 open reading frame 72 ( Frontotemporal dementia (FTD) is a heterogeneous neurodegenerative disorder presenting with distinct changes in behaviour, language and motor function. Despite often being considered as a rare disease, FTD is probably the most common form of dementia experienced in people under the age of 60, with an estimated lifetime risk of 1 in 742 . People FTD is a highly heritable disorder but almost uniquely within the neurodegenerative disease spectrum, it is neither purely genetic  nor a mainly sporadic condition (like Alzheimer’s disease) Fig. . The extGRN), microtubule-associated protein tau (MAPT) and chromosome 9 open reading frame 72 (C9orf72) [GRN mutations [C9orf72 seems to be the most common worldwide cause of genetic FTD, followed by GRN and then MAPT. A list of pathogenic and other variants in these genes has been collated online in the AD&FTD Mutation Database (http://www.molgen.ua.ac.be/FTDmutations): 79 GRN and 45 MAPT pathogenic variants are currently described. However, a Pubmed search of mutations reported over the last 5 years in these genes identifies a further 35 GRN and 18 MAPT pathogenic variants not included in that database, i.e. 114 GRN and 63 MAPT mutations currently identified in total. This number excludes the majority of missense variants in GRN, many of which may be risk factors for Alzheimer’s disease rather than a Mendelian cause of FTD, although identifying pathogenicity is not always easy [The majority of the heritability of FTD is accounted for by autosomal dominant mutations in three genes: progranulin (C9orf72) , 13. Eacutations , 15). Ovays easy .VCP (2004), CHMP2B (2005), TARDBP (2008), FUS (2009), SQSTM1 (2012), CHCHD10 (2014), TBK1 (2015), OPTN (2015), CCNF (2016), TIA1 (2017). Cumulatively, they account for < 5% of all FTD, with most only found in a small number of families across the world. Recent studies have identified TBK1 as probably the fourth most common genetic cause overall of FTD, accounting for between 1 and 2% of all cases . Whilst MAPT mutations are fully penetrant in most cases, both GRN [C9orf72 [GRN and C9orf72 carriers, TMEM106B has been identified as a genetic modifier, the association being stronger with GRN than with C9orf72 [GRN may well be related to carrying the risk allele, with homozygous carriers of the protective allele rarely found in symptomatic GRN carriers, suggesting that this may be a factor in age-related penetrance [GRN carriers, GFRA2, did not seem to affect age at onset [C9orf72 carriers identified a locus on chromosome six containing two overlapping genes (LOC101929163 and C6orf10) in which a polymorphism at rs9357140 was associated with age of onset: median age of onset in GG carriers was 6 years earlier than AA carriers [C9orf72 repeat expansion length remains unclear, with no definitive evidence of an association with age of onset [MAPT group, although a recent study suggested that ApoE ε4 carriers had a lower age at onset in tauopathies including MAPT mutations [Age at symptom onset is variable in each of the genetic forms of FTD, with intrafamilial variability (even within the same generation) of at least a decade in some families . This is a natural history study with detailed phenotyping of both presymptomatic and symptomatic mutation carriers [http://www.genfi.org.uk/fpi.html), aiming to share information and inform future clinical trial design.Until recently, clinical studies of genetic FTD have been small and single centre. However, the Genetic FTD Initiative (GENFI) started recruiting in 2012 and now encompasses 25 centres across Europe and Canada (carriers . In the Much of the work being performed in these studies (and in other single centre investigations) over the last few years has aimed to develop validated biomarkers that robustly measure disease onset, staging and progression Fig. . The folMAPT, GRN and C9orf72 carriers [MAPT carriers have both naming and episodic memory difficulties presymptomatically [GRN carriers [Neuropsychometric measures are abnormal in presymptomatic carriers around 5 years prior to expected symptom onset . Whilst carriers . A numbeatically –34, consatically . As mentcarriers .Validated measures of psychiatric symptoms or functional decline are limited in genetic FTD. The Neuropsychiatric Inventory (NPI) has been the most studied, although was not designed with FTD in mind, and does not include all relevant psychiatric symptoms that are seen in FTD . The CamGrey matter atrophy and hypometabolism both appear to occur at least 10 years before symptom onset in genetic FTD, whilst white matter tract abnormalities are seen earlier . HoweverMAPT carriers, atrophy is present about 15 years prior to symptom onset in the anterior and medial temporal lobes, orbitofrontal lobe and insula [GRN carriers, presymptomatic atrophy can be observed in frontal, parietal, and insular cortex as well as the striatum around 10 years prior to symptom onset [GRN carriers commonly have a very asymmetrical pattern of brain atrophy, and this asymmetry can be observed around 5 years prior to onset [C9orf72 mutation carriers appear to have earlier grey matter volume loss than the other two groups, before the age of 40 [In presymptomatic d insula , 38, whiom onset , 38. Symto onset . C9orf72ge of 40 , and potge of 40 . This apge of 40 , 38, 39.MAPT mutation carriers had involvement of CA1-4, C9orf72 expansion carriers CA4, CA1 and the dentate gyrus, and GRN mutation carriers the presubiculum and subiculum [Volumetric MRI studies of genetic FTD have particularly highlighted the importance of subcortical structures in the pathogenesis of FTD, and more recent work using novel postprocessing techniques has aimed to study the subregions within these structures, e.g. there are differential patterns of atrophy within hippocampal subregions in the different genetic groups: ubiculum .GRN mutation carriers during the symptomatic period  comparecarriers .GRN and MAPT mutation carriers appeared to fall mainly into a single group, there were two distinct patterns of disease progression for C9orf72 expansion carriers—it remains unclear pathophysiologically what differs between these two groups.Few studies have investigated disease staging of genetic FTD. One novel machine-learning methodology combining subtyping and staging identified genetic FTD subtypes and their stages over time from structural T1-weighted imaging alone . InteresGRN mutations [A number of studies have now shown that white matter hyperintensities  are characteristic of utations , 45. Thiutations .Patterns of hypometabolism commonly mirror the pattern of grey matter atrophy in genetic FTD –51, withMAPT mutation carriers have alterations in the uncinate fasciculus and parahippocampal cingulum, while GRN mutation carriers show involvement of the anterior and posterior internal capsule [C9orf72 expansion carriers have earlier white matter tract pathology, which occurs in posterior tracts such as the posterior thalamic radiation, the posterior corona radiata and the splenium of the corpus callosum [C9orf72 expansion carriers [Changes in white matter integrity are commonly measured with diffusion tensor imaging (DTI), although newer techniques such as neurite orientation dispersion and density imaging (NODDI) have recently been developed. Studies in genetic FTD suggest that changes can be observed as far back as 30 years prior to symptom onset . As with capsule . Presympcallosum , 53. A scarriers .C9orf72 expansion carriers [MAPT mutation carriers [GRN mutation carriers [There have been fewer investigations of functional connectivity but small studies implicate particularly the salience network and a medial pulvinar thalamus-seeded network in presymptomatic carriers , the defcarriers  and a frcarriers –58.MAPT mutations (V337M and R406W) are associated with PHF-tau and have shown strong binding with the AV1451 tracer [C9orf72 expansions, where the major pathology is TDP-43 [Studies of novel radioligands developed to bind tau protein have so far not proven to be particularly helpful in FTD, binding much more strongly to paired helical filament (PHF)-tau found mainly in Alzheimer’s disease than to other forms of tau found in the primary tauopathies. However, two particular 1 tracer –61. Unfos TDP-43 .The fluid biomarker field in genetic FTD has yet to identify many robust measures, e.g. neither CSF nor blood assays of tau or TDP-43 are yet to yield FTD-specific markers. However, recent work has identified three markers which will play an important role in forthcoming trials: neurofilament light chain (NfL), progranulin and poly(GP) dipeptide repeat proteins (DPRs).C9orf72-associated ALS and lowest in MAPT mutation carriers [Increased NfL levels (both in CSF and blood) reflect axonal damage and appear to be a measure of disease intensity, and predict progression and survival in genetic FTD , 64. Levcarriers .GRN mutations [r = 0.54: GRN carriers.Low serum, plasma or CSF progranulin levels have almost perfect sensitivity and specificity for detecting pathogenic utations , 67. LevC9orf72 expansion carriers both presymptomatically and symptomatically [Increased poly(GP) levels have been identified in the CSF of atically –70. One GRN mutation carriers but not the other genetic groups [A particular focus of biomarker research in genetic FTD is the development of markers of neuroinflammation. CHIT1 and YKL-40 are microglial markers that appear to be raised in symptomatic genetic FTD  with litc groups .C9orf72 expansions become pathogenic (as intermediate length expansions are not clearly causative of disease [TBK1 and we have identified mutations in these genes by exome sequencing in those with negative NGS and C9orf72 sequencing [GRN mutation) [Testing in symptomatic patients with dementia has changed in recent years. Next-generation sequencing (NGS) panels are now available to test multiple genes at the same time—these have identified mutations causative of FTD pathology not just in those with an FTD clinical syndrome . Issues  disease ); and whquencing . Howeverquencing . In the utation) , 76.Fig.Once a causal mutation has been established in a symptomatic relative, the option of predictive genetic testing can be raised with at-risk family members. While potential treatments for FTD are still lacking, appropriate clinical care for presymptomatic populations is integral. The genetic counselling and support systems in place lag far behind those seen in other neurodegenerative disorders. Whilst in practice the HD predictive genetic testing protocol is currently used as the gold standard , there ahttp://www.raredementiasupport.org/fftd/). Specific interventions at an appropriate time such as cognitive behaviour therapy or mindfulness have yet to be trialled.The HD predictive guidelines stress the importance of psychological evaluation in presymptomatic carriers, with others suggesting that psychological assessment is a necessary process for identifying an individual’s risk of experiencing an adverse psychological reaction to presymptomatic testing . There rMAPT mutations [GRN carriers [MAPT mutations) [GRN mutations include modification of proteins such as sortilin and HDAC that lead to increased GRN levels [There are currently no disease-modifying therapies for genetic FTD but trials are now underway or planned in each of the three main genetic FTD groups. Antisense oligonucleotide therapy shows promise for both C9orf72 expansions  and MAPTutations , whilst carriers , 85, alttations) , and othN levels , 88.Much has been learnt about genetic FTD in the last decade, with the majority of autosomal dominant FTD now accounted for. The development of collaborative international multicentre natural history studies in GENFI and ARTFL/LEFFTDS has brought together researchers and families, and has helped to set the background for clinical trials that are now getting started and being planned. An associated support network for those living at-risk of genetic FTD is important and there is work to be done in improving this; but with the advent of specific gene-targeted therapeutics, there is hope in the community for the future outlook.


Mammalian phenotypes are shaped by numerous genome variants, many of which may regulate gene transcription or RNA splicing. To identify variants with regulatory functions in cattle, an important economic and model species, we used sequence variants to map a type of expression quantitative trait loci (expression QTLs) that are associated with variations in the RNA splicing, i.e., sQTLs. To further the understanding of regulatory variants, sQTLs were compare with other two types of expression QTLs, 1) variants associated with variations in gene expression, i.e., geQTLs and 2) variants associated with variations in exon expression, i.e., eeQTLs, in different tissues.cis additive genetic variance in different tissues. Thus, an effective but simple-to-implement meta-analysis combining information from three tissues is introduced to increase power to detect and validate sQTLs. sQTLs and eeQTLs together were more enriched for variants associated with cattle complex traits, compared to geQTLs. Several putative causal mutations were identified, including an sQTL at Chr6:87392580 within the 5th exon of kappa casein (CSN3) associated with milk production traits.Using whole genome and RNA sequence data from four tissues of over 200 cattle, sQTLs identified using exon inclusion ratios were verified by matching their effects on adjacent intron excision ratios. sQTLs contained the highest percentage of variants that are within the intronic region of genes and contained the lowest percentage of variants that are within intergenic regions, compared to eeQTLs and geQTLs. Many geQTLs and sQTLs are also detected as eeQTLs. Many expression QTLs, including sQTLs, were significant in all four tissues and had a similar effect in each tissue. To verify such expression QTL sharing between tissues, variants surrounding (±1 Mb) the exon or gene were used to build local genomic relationship matrices (LGRM) and estimated genetic correlations between tissues. For many exons, the splicing and expression level was determined by the same Using novel analytical approaches, we report the first identification of numerous bovine sQTLs which are extensively shared between multiple tissue types. The significant overlaps between bovine sQTLs and complex traits QTL highlight the contribution of regulatory mutations to phenotypic variations.The online version of this article (10.1186/s12864-018-4902-8) contains supplementary material, which is available to authorized users. Cattle are an important source of meat and dairy products for humans worldwide. Also, cattle can be used as clinical models to study genetic causes of human diseases . To imprMany of the sequence variants that are associated with complex traits (quantitative trait loci or QTL) are not coding variants and are presumed to influence the regulation of gene expression, that is to be expression QTLs . An exprAfter transcription, RNA is spliced by intron removal and exon ligation to create various mature transcripts. An expression QTL associated with the changes in the expression ratio of an exon to the gene implies that it alters RNA splicing. This type of expression QTL is then defined as a splicing QTL or sQTL, which have been studied in humans by inferring the individual splicing ratio from RNA sequence data . More reKnowledge of large mammal regulatory mutations is limited mainly to humans, where there have been multiple studies reporting on expression QTLs , 11, 12.In total, we analysed 378 transcriptomes of 19 tissue types from 214 cattle generated from four experiments covering major dairy and beef cattle breeds  were significantly associated with tissue or breed (FDR < 0.1). To verify the significantly spliced exons, we imposed a requirement that at least one adjacent intron had an excision ratio , 24 thatCSN3 [Using data from all experiments Table , there wCSN3 , 26, wheThe mapping of sQTLs was based on data from 312 transcriptomes generated from experiments III and IV, including white blood cells, milk cells, liver and muscle tissues , which as described above was differentially spliced in Holstein and Jersey cattle . This predicted that the expression and inclusion ratio of the 5th exon would be significantly higher in the Holstein cattle than Jersey cattle, which was in line with the observations in Fig. In the milk cell transcriptome, the fifth exon of tle Fig. , and wasMany more significant eeQTLs than sQTLs were detected in all tissues studied Table . In whitFigure n > =105, Table n < = 41) and low sequencing depth, the largest absolute amount of SNP sharing was between eeQTLs and geQTLs, followed by the amount of SNP sharing between sQTLs and eeQTLs  to test their effect , eeQTLs (expression level of the exon) and geQTLs (expression level of the gene) between tissues, 598 had genetic correlations significantly (p < 0.05) different from 0, out of which 561 had genetic correlations insignificantly (p > = 0.05) different from 1. That is, in many cases, the variation in exon expression in white blood cells and milk cells was associated with the same cis polymorphism (s).The correlation between estimated SNP effects on gene splicing and expression in different tissues are lower in magnitude than the true correlation between SNP effects, because the effects are estimated with error and these errors are independent between tissues. To estimate the true correlation, we computed the genetic correlation between SNP effects in two different tissues by GREMLene Fig. . LGRM diDDX19B, CTSD and EFF1A1  on the expression levels of the 2nd exon in the milk cells and a similar but less significant effect (p < 1e-4) on the 2nd exon expression in white blood cells  of at yield , 30) hadp < 0.05) in all of white blood cells, milk cells and muscle transcriptomes, their standardised effects  in each transcriptome were simply combined and tested for significance against a χ2 distribution with 1 degree of freedom. Overall, the multi-transcriptome meta-analysis based on summary statistics substantially increased the power of expression QTLs detection  associated with more than one of 24 dairy traits  and withe 6 Fig. . eeQTLs e 6 Fig.  and was e 6 Fig. . eeQTLs e 6 Fig. . Of the e 6 Fig. .Fig. 7SiMGST1, where effects of milk cell eeQTLs were highly significantly associated with their effects on milk fat yield [MGST1  in multiple tissues centred around RNA splicing events, using a large number of RNA and whole genome sequence data from an important domestic animal species. Overall, differential splicing between tissues is ubiquitous and between breeds is common. Differential splicing between individuals due to SNPs (sQTLs) occurs for many genes and is enriched with cattle complex trait QTL. Within each tissue, all cis expression QTLs types showed significant overlap. Most geQTLs and sQTLs were detected as eeQTLs indicating that the exon expression can be altered by changing the expression of the whole gene or by changing the splicing. However, an sQTL was likely to be an eeQTLs and, to a lesser extent, geQTLs. Between tissues, while all QTLs types showed significant overlap between white blood cells and milk cells and between liver and muscle, the strongest cross-tissue sharing appeared to be at the exon level (sQTLs and eeQTLs). This is supported by many significant tissue pair genetic correlations. Such cross-tissue expression QTL sharing allowed the multi-transcriptome meta-analysis of expression QTL effects which substantially increases power to detect significant expression QTLs.CAPN1 is also a SNP chip based significant pleiotropic SNP for 16 beef cattle traits  with the variant Chr6:87390576 which has been suggested as a putative causal variant for effects on milk protein yield and percentage [In the milk cell transcriptome, a significant sQTL  and had highest concentrations of intronic SNPs Fig. . In humaWithin each studied bovine tissue, the largest amount of overlap between expression QTL types was found either between exon expression eeQTLs and sQTLs or between eeQTLs and geQTLs Fig. . FurtherA hypothesis to explain these results is that mutations in regulatory DNA may increase the expression of one or more transcripts from a gene. If they increase expression of one transcript then they may be detected as an eeQTL for the exons in that transcript, as a sQTL for exons spliced out of that transcript or as a geQTL if this transcript forms a large part of the total transcription from the gene. Thus, there is expected to be overlap between eeQTLs, geQTLs and sQTLs, but at least sQTLs and eeQTLs should overlap and this is what we found , the combined effects of all types of expression QTLs of the three tissues from different experiments were highly significant   and ther6  and thp-values. The genetic correlation between expression QTLs in different tissues was often indistinguishable from 1.0 indicating that many expression QTLs operate in a similar way across tissues. Consequently, combining results from several tissues using the multi-transcriptome meta-analysis increased power to detect all three types of expression QTLs. The potential of exon-level QTLs information was demonstrated by the identification of several strong candidates of putative causal mutations for complex traits: sQTL Chr6:87392580 within CSN3 for milk production and eeQTL Chr5:93945738 within MGST1 for milk fat yield.We found that eeQTLs overlapped with both geQTLs, due to polymorphisms affecting the level of expression of the whole gene, and with sQTLs, due to polymorphisms affecting the exon usage within the gene. sQTLs tended to be closer to the transcription start sites more often located in introns than geQTLs. We found the largest number of sQTLs in white blood cells probably because the power to find them was greatest in this dataset. However, many of the sQTLs found in other tissues were also detected in blood cells and many sQTLs found in blood could be detected in other tissues at higher semitendinosus muscle and 35 liver from Angus bulls was previously described by [https://www.encodeproject.org/about/experiment-guidelines/) biological replicates were favoured over technical replicates for experiments II–IV. However Chamberlain et al. [For Experiment I, the sampling of 18 tissues from one lactating Holstein cow followed procedures described by Chamberlain et al. . For Expribed by , 43. As n et al.  assessedFor Experiment I, RNA extraction and sequencing followed the procedures described by Chamberlain et al. . For ExpExperiments III and IV had whole genome sequence genotypes imputed from the SNP chip genotypes using FImpute  based onhttp://davidaknowles.github.io/leafcutter/). Those exons and introns with ratio values < 0.001 were removed and the remaining ratio values were transformed to log2 scale, then underwent exon/intron –wise quantile normalisation and individual –wise zscore standardisation [Gene count data were generated by Python package HTSeq  using dedisation .Both exon inclusions and intron excisions were analysed and used in combination for gene differential splicing for (1) the overall tissue effects and (2) the breed effects. Primarily, differential splicing was defined for the gene containing exons whose variation in inclusion ratios were significantly (FDR < 0.1) associated with the tissue or breed variable. To be called as significantly spliced exons, they were required to have at least one adjacent intron whose variation in excision ratios were also significantly (FDR < 0.1) associated with the tissue or breed variable. The tissue effects were analysed in a linear mixed model in lme4  in R as:y = exon inclusion or intron excision ratios, bi = the animal random effects (i = 214), xj = the experiments (j = 4), tk = tissue type (k = 19), e = random residual term. The fitting of the animal random effects accounted for the fact that only one animal was used in experiment I. The P values of F tests were calculated using Satterthwaite approximation implemented in lmerTest [Where lmerTest . The brey = exon inclusion or intron excision ratios in the milk cell transcriptome, breedl = breeds . p values of exons/introns for the tissue effects in Eq.  cis splicing QTLs was expected to satisfy two conditions simultaneously: (1) a SNP, within or up to ±1 Mb away from the exon, was significantly (FDR < 0.1) associated with the variation in the exon inclusion ratio and (2) the same SNP was significantly (FDR < 0.1) associated with at least one event of the excision of the intron next to the same exon at the same significance level. Both individual exon inclusion and intron excision values were used as phenotype to map associated QTLs with widely used  Matrix eTo compare cis sQTLs with exon expression cis eeQTLs and cis gene expression geQTLs, the expression count data were normalised by voom  estimatihttp://www.ensembl.org) and the absolute difference between the position of a SNP and the transcription start site of the gene were calculated for the SNP with significant cis effects. The SNP functional categories were generated using predictions from Ensembl Variant Effect Predictor [The gene transcription start site coordinates were downloaded from Ensembl  implemented in GeneOverlap [The significance of overlaps were compared with the expected number using the Fisher’s exact test  and of tr2, varlg(tr2) and the local additive genetic covariance between t1 and t2, covlg using GREML [cis expression QTLs defined in this study  and allowed the estimation of genetic correlationThe cross-tissue sharing of SNPs were confirmed by bivariate GREML analysis using GCTA . For an ng GREML . This apGenetic correlations were also tested for their significance of being different from 0 or 1, by fixing the correlation value to 0 and 1 using GCTA .t values were calculated from single-transcriptome results of white blood cells (t1), milk cells (t2) and muscle (t3). The significance of multi-transcriptome effects of an expression QTL was tested by χ2 distribution with 1 degree of freedom:The validation based on expression QTL effect commonality across tissues was conducted by comparing the combined expression QTL effects from white blood cells (experiment III), milk cells (experiment III) and muscle (experiment IV) transcriptomes with their effects in the liver transcriptome (experiment IV). The standardised expression QTL effects, b/se, signed N = the number of studied tissues (N = 3 in this case) where the original SNP t values were estimated. Provided the individual t-values followed a t-distribution under the null hypothesis, the properties of the average t value in the current study was a simple mathematical result which approximated the chi square distribution with 1 degree of freedom, the null hypothesis of which was that the SNP does not have any significant associations in any of the three tissue types. Previously, the concept of meta-analysis combining SNP t values estimated from different datasets has been also applied to analyse multiple quantitative phenotypic traits in large animals to increase power  with the third tissue from a different experiment with relatively weak power . The single tissue left to be compared with was liver, a tissue which also appeared to show weak power and was from experiment IV. These choices intended to create enough differences for the meta-analysis to combine the SNP effects and for the combined SNP effects to be compared with the SNP effects in the single tissue.wer Additional file 2:Figure S1. Gene coverage uniformity plots for each tissue type for a) all genes, b) 500 highest expressed genes and c) 500 lowest expressed genes, as determined by Qualimap 2 [Figure S2. Splicing junction saturation analysis for each tissue type for a) all splicing junctions, b) known splicing junctions and c) novel splicing junctions. Figure S3. The distribution of concordance between imputed and RNA sequence genotypes. Figure S4. Sample principal components clustering based on exon expression. Figure S5. Correlation of sQTLs (splicing quantitative trait loci), eeQTLs (exon expression QTLs) and geQTLs (gene expression QTLs) effects  between tissues. Figure S6. Sharing of sQTLs (splicing quantitative trait loci), eeQTLs (exon expression QTLs) and geQTLs (gene expression QTLs) in paired tissues at p < 0.05 level. Figure S7. Manhattan plot of eeQTLs for the MGST1 gene. (PDF 737 kb)Additional file 3:Table S1. RNA-seq reads mapped to different bovine genome origins. (XLSX 14 kb)Additional file 4:Table S2. Splicing junction annotation analysis using BAM files generated by different alignment software. (XLSX 13 kb)Additional file 5:Table S3. 2 × 2 tables for the overlap between exon and intron analyses. (XLSX 15 kb)Additional file 6:Table S4. Genes that display significant differential splicing cross tissues. (XLSX 18699 kb)Additional file 7:Table S5. Genes that display significant differential splicing between breeds. (XLSX 38 kb)Additional file 8:Table S6. Summary of significant cis splicing sQTLs (within 1 Mb distance to the exon). (XLSX 14902 kb)Additional file 9:Table S7. Summary table for count values of sQTLs and eeQTLs in white blood and milk cells. (XLSX 9 kb)Additional file 10:Table S8. SNP overlap between expression QTLs and SNPs with pleiotropic effects on conventional traits of dairy cattle. (XLSX 38 kb)Additional file 11:Table S9. SNP overlap between expression QTLs and SNPs with pleiotropic effects on conventional traits of beef cattle. (XLSX 20 kb)Additional file 12:Table S10. SNP overlap between blood eeQTLs and putative causal variants identified by Littlejohn et al. [n et al. . (XLSX 2


Uncontrolled blood pressure remains an urgent issue in clinical practice worldwide. This study aimed to compare the characteristics and effectiveness of hypertension control in family medicine pratice in the first treatment year, in relation to the geographical position, socio-economic standard, and access to medical services and public pharmacies in urban, rural and island environments .A historical cohort study included 213 patients diagnosed from 2008 to 2014 with essential arterial hypertension (AH) and without related complications or diabetes mellitus. Each patient was followed up for 365 days from the visit when the diagnosis of hypertension was ascertained. Normotension was defined as arterial pressure < 140/90 mmHg. The annual cost of drugs prescribed for treating newly diagnosed hypertensive patient and the total price for defined daily dose per patient were also evaluated.P = 0.981). Higher initial systolic blood pressure was a positive predictive prognostic factor on achieveing normotension (odds ratio (OR) 0.96, 95% confidence interval 0.95–0.98). ACE inhibitors were the most commonly prescribed antihypertensive agents in monotherapy (35.1%), as well as considering overall prescriptions (25.2%). Calcium channel blockers were the most commonly prescribed initial BP-lowering single agents in urban areas (28.6%), whereas angiotensin-converting enzyme inhibitors were more common in rural (28.0%) and island areas (22.7%) (P = 0.037). The median annual antihypertensive drug cost was 169.4 (95% CI 151.5–201.8) Croatian kunas and was similar across the study sites.More than half patients achieved normotension within a year from the initial diagnosis in all family medicine practices (57.3%), without significant differences among the three geographic regions  contains supplementary material, which is available to authorized users. High systolic blood pressure (BP) was one of the most frequent Level 3 risk factors for global burden of disease according to the systematic analysis published in 2016 –10. The This historical cohort study included patients of either sex, aged ≥18, whose arterial hypertension was first diagnosed from 2008 to 2014. According to clinical practice guidelines for the managament of AH valid in the time of our research, participants with the mean systolic arterial pressure ≥ 140 mmHg and/or diastolic arterial pressure ≥ 90 mmHg were classified as hypertensive –19, and Hypertensive patients were not included in the study if they had 1) AH diagnosed in the period before 2008, 2) incomplete medical records for the studied period, and 3) serious acute and chronic diseases . Since various abnormalities present in diabetes mellitus and arterial hypertension have significant pathogenetic contribution to the development and exacerbation of each other , 28, andThe data were collected from family medicine offices files. Included practices were long-standing and led by a single-physician, who had individual contracts with the mandatory national Croatian Health Insurance Fund (CHIF). Family medicine practices in Croatia provide health care for an average 1840 patients, with minimum 1275 and maximum 2125 patients allowed .Each patient was followed for 1 year after the initial diagnosis. The urban area included the city of Split and the rural area included the town of Imotski and municipalities Lećevica, Muć, and Runovići . Southern Croatian islands of Brač  and Korčula (town of Korčula) represented the island environment (Table The primary outcome measures were: 1) rate of achieving normotension after one-year treatment, 2) mean arterial pressure (MAP) before and after one-year treatment, and 3) complications of treatment, including cardiovascular complications and side effects of medications. The achieved blood pressure control was defined as the proportion of participants whose average systolic and diastolic blood pressure 365 days after the initial diagnosis  was less than 140/90 mmHg. The secondary outcome measures were: 1) choice of initial treatment, 2) diagnosis by a physician or a medical specialist , 3) length of sick leave days, 4) full or partial coverage of prescribed medications by the national insurance fund, CHIF, 5) total price for defined daily dose (DDD) of all prescribed medications per each patient, and 6) total annual cost of prescribed drugs for treating one newly diagnosed hypertensive patient.CHIF provides the mandatory health insurance in Croatia, with the obligatory health insurance contributions of employed citizens as the major source of its revenue . This puTo estimate the cost of antihypertensive drugs prescribed during 1 year, original drug packacking prices (including VAT) and prices for defined daily dose (DDD) were taken from the Basic and the Supplementary List, which were valid during data extraction, both in force since 30 May 2015 until 19 July 2015 for the Basic List and until 19 August 2015 for the Supplementary List . The totWe also recorded whether the initial antihypertensive treatment focused on lifestyle changes (Dietary Approaches to Stop Hypertension)  without 2 test. Patients’ age, MAP before and after one-year treatment, number of visits to the family medicine office and sick leave days were compared according to the patients’ place of residence, using analysis of variance (ANOVA) with the post-hoc Scheffé test. Kruskal-Wallis test and post-hoc Mann-Whitney U test were used for the median comparison of the total annual number of prescribed medications per patient and the total annual cost of drugs prescribed for treating one newly diagnosed hypertensive patient by a physician. Mann-Whitney and χ2 test were used to determine which variables were significantly different in controlled and uncontrolled hypertensive patients; those variables were further analysed by logistic regression. P-values less than 0.05 were considered statistically significant. All statistical analyses were performed using MedCalc, version 12.5 .The data were presented as a median or a mean, with 95% confidence interval (CI), depending on the data distribution; the normality of distribution was checked using the Kolmogorov-Smirnov tests. For the comparison of categorical variables we used χn = 41, 64.1%), where patients had significantly higher prevalence of dyslipidemia, the most common comorbidity  and the highest rate of patients with only primary education  from urban, 78 (36.6%) from rural and 71 (33.3%) from island environment of South Croatia  compared to the rural  and island areas  . Analysis of the outcome variables in logistic regression model indicated higher initial systolic blood pressure as a positive predictive prognostic factor on achieveing normotension .The patients in the whole sample showed a significant reduction in systolic and diastolic blood pressure . There were no differences in the participants with average systolic and diastolic blood pressure lower than 140/90 mmHg: 37 (57.8%) in urban, 45 (57.7%) in rural and 40 (56.3%) in island environment (P < 0.001). Individual physicians did not differ in prescribing medications from two Croatian National Medicine Reimbursement Lists  Croatian kunas. Only a single physician from the rural area had significantly higher annual cost than the others , whereas angiotensin-converting enzyme inhibitors (ACEI) were more common in rural  and island areas .Pharmacotherapy approaches to the first-line treatment differed in urban, rural and island environment , β-blockers ranked second , followed by calcium channel blockers  and thiazide-like diuretics .Within the first year of therapy, a total of 77 patients (39.1%) out of 197 patients who received pharmacological therapy received monotherapy, whereas 120 patients , followed by β-adrenergic receptor antagonists , calcium channel blockers  and diuretics  and ramipril  were the most widely prescribed. Thiazide-like diuretics, chlortalidone  and indapamide , were the most commonly used among single-agent diuretics. Thiazide diuretics were prescribed only in fixed-dose combinations that were available on the Croatian drug market. Among fixed-dose combinations, the most frequently applied in the first year of treatment was lisinopril in combination with hydrochlorothiazide .In the case of overall pattern of all prescribed antihypertensive agents , ACEI were again the most commonly prescribed  and bisoprolol  and similar in different geographical settings: in the second largest city in Croatia (Split) and its more geographically isolated hinterland and islands, regardless of socioeconomic inequalities , 38, accIn an indirect manner, these findings correspond to a recent study in this region, which found that general health status in this area did not differ with respect to the geographical and socioeconomic factors . It is iThe second important finding of our study was that the different antihypertensive drugs and treatment choices led to similar effectiveness in BP control. ACE inhibitors were the most widely prescribed antihypertensive single drug therapy in the first-line in rural (28.0%) and island areas (22.7%), whilst calcium channel blockers predominated in urban setting (28.6%). The choice of initial treatment was in line with the 2013 guidelines of the European Society of Hypertension (ESH) and European Society of Cardiology (ESC), which recommended the following five pharmacotherapeutic groups for the initiation and maintenance of antihypertensive treatment – diuretics, β-blockers, calcium channel blockers, ACE inhibitors and angiotensin receptor II blockers, as monotherapy or combined polytherapy . HoweverDiuretics are the first-line therapy choice for most hypertensive patients according to the recommendations of the Seventh Joint National Committee (JNC 7) guidelines  as well Since better BP reduction is often achieved by combining drugs from two different classes in comparison with doubling the dose of one drug  and withTaking into account that we found only a single physician from rural area with significantly higher total annual cost of prescribed drugs for treating one newly diagnosed hypertensive patient, it is difficult to make judgements on differences in the costs of AH treatment across different regions. However, comparing the highest annual average antihypertensive drugs cost in this study  with the cost of €258 during follow-up period of 36 ± 3 months in French general practices , it coulPrimary care is an important interface between the patients and the health care system, shown to be associated with improved health outcomes . The resThe key strenght of this study lies in its comprehensive analysis of the effectiveness of newly diagnosed arterial hypertension treatment in primary care setting. It provides insight into the impact of primary care in the achieving control of uncomplicated essential hypertension in the real-life socio-economic conditions. The major limitation of the study is its retrospective character. Furthermore, due to the inability to access two Croatian National Medicine Reimbursement Lists valid in time periods that involved one-year following intervals, original drug packaging prices were taken from the Croatian National Medicine Reimbursement Lists which were valid during the data extraction, in force since May 2015 . FinallyTo conclude, there is a remarkable lack of association between socioeconomic conditions and uncomplicated arterial hypertension control in primary care setting. Different antihypertensive prescribing patterns seem do not have an impact on the effectiveness in blood pressure control after the one-year treatment. Family physicians seem to be able, without any intervention by medical specialists, to achieve normotension in newly diagnosed patients with arterial hypertension, emphasizing their key role in dealing with this major public health problem.Additional file 1:Table S1. General characteristics of patients according to their blood pressure control after one-year treatment. Table S2. Prescribed pharmacological subgroups for patients with and without achieved blood pressure control after one-year treatment. Table S3. Prescribed generic names for patients with and without achieved blood pressure control after one-year treatment. (DOCX 68 kb)