CNTNAP2) gene, whereas the microduplications at 15q13.3 and Xp22.33 involved the cholinergic receptor nicotinic α 7 subunit (CHRNA7) and the cytokine receptor-like factor 2 (CRLF2) genes, respectively. Here, we describe a female patient harbouring three CNVs whose additive contribution could be responsible for her clinical phenotypes.Copy number variations (CNVs) play a key role in the pathogenesis of several diseases, including a wide range of neurodevelopmental disorders. Here, we describe the detection of three CNVs simultaneously in a female patient with evidence of severe myoclonic epilepsy, microcephaly, hypertelorism, dimorphisms as well as severe psychomotor delay and intellectual disability. Array-CGH analysis revealed a ~240 kb microdeletion at the 7q35 inherited from her father, a ∼538 kb microduplication at the 15q13.3 region and a ∼178 kb microduplication at Xp22.33 region, both transmitted from her mother. The microdeletion in 7q35 was included within an intragenic region of the contactin associated protein-like 2 ( Copy number variations (CNVs) are deletions or duplications of genomic DNA larger than 1 kilobase . RecurreCNVs may be inherited from a parent or arise from de novo in an individual . CNVs caCNTNAP2  7q35(146236230_146475758)×1 pat), involving partially 69) gene A, 15q13.69) gene B and mic69) gene C, includSybr Green qPCR assay confirmed CMA data in our patient and demonstrated the paternal segregation of 7q35 microdeletion, whereas 15q13.3 and PAR1 microduplications were inherited from her mother with hypothyroidism. The healthy brother of the proband was a carrier of 15q13.3 and Xp22.33 microduplications; both inherited from his mother . Here, we report on a patient presenting with severe myoclonic epilepsy, microcephaly, hypertelorism, dysmorphisms as well as severe psychomotor delay and intellectual disability. CMA of the proband identified three different CNVs, a paternal 7q35 microdeletion in combination with two maternal microduplications at 15q13.3 and Xp22.33. CNTNAP2. This gene encodes for a protein of the neurexin superfamily, known as contactin-associated protein 2 (Caspr2), which is a transmembrane protein involved in cell–cell interactions between neurons and glial cells and plays a crucial role in the axonal differentiation and guidance [The 7q35 microdeletion encompasses partially the intron 1 and the entire exon 2 of guidance . CNTNAP2 contains two important regulatory regions for the transcription factors Storkhead box 1A (STOX1A) and Forkhead box P1 (FOXP1) [CNTNAP2 are essential for the correct control of CNTNAP2 expression; a deletion within intron 1 covering the FOXP2 binding sites has been observed in patients suffering from language delay and autism [CNTNAP2 in combination with other chromosomal rearrangements has been observed in a patient with speech delay and ASD [It has been shown that intron 1 of  (FOXP1) . These td autism  and bipod autism . A simil and ASD .CNTNAP2 dysfunction and neurodevelopmental disorders. It has been described a patient with non-specific dysmorphic features, speech problems and learning disability having an intragenic deletion which disrupts the reading frame of the CNTNAP2 [CNTNAP2 gene in a subject with Alzheimer’s disease (AD) [CNTNAP2 were observed in certain subjects with epilepsy, autism, bipolar disorder, schizophrenia and ADHD, indicating that CNTNAP2 was involved in several neurologic or psychiatric diseases [Other genetic studies support the link between  CNTNAP2 ,30. Impoase (AD) . In addidiseases .CNTNAP2 in the development of neurological diseases. Toma et al., in their large comprehensive study, showed that multiple classes of DNA variations such as SNPs, de novo variants and CNVs of CNTNAP2 are unlikely to play a role in the development of psychiatric or neurological disorders [Conflicting results have been described in the literature regarding the role played by isorders . CNTNAP2 (chr7:146203548-146334635) is not only present in affected individuals but also in an unaffected relative. Therefore, it was observed that this CNV deletion is unlikely to be highly penetrant and did not segregate with bipolar disorder in the above-described family [CNTNAP2 deletions may have incomplete penetrance, as a consequence of the variable expression of the CNTNAP2 allele [In addition, by studying a large family with bipolar disorder, they showed that a CNV deletion that removes the FOXP2 transcription binding site in intron 1 of d family . The lim2 allele . CNTNAP2 does not segregate with the disease, indicating that heterozygous CNTNAP2 deletions are not always fully penetrant [In addition, by studying an extended family, Toma C. et al. observed that a deletion which removes the FOXP2 binding site in intron 1 of enetrant .The chromosome 15q13 region has been observed to be a hotspot for CNVs because it contains low copy repeat elements (LCRs), which promote non-allelic homologous recombination (NAHR) resulting in chromosomal microduplications and microdeletions ,17, TourCHRNA7 gene, which codes for α7 nicotinic acetylcholine receptor (α7nAChRs) in the brain [The 15q13.3 microduplication encompasses the CHRNA7 dosage sensitivity was suggested as the cause of several of the neuropsychiatric and neurodevelopmental phenotypes detected in patients harbouring 15q13.3 CNVs [Although it has been shown that deletions at 15q13.3 in patients with neuropsychiatric phenotypes manifest incomplete penetrance (80%)  and much3.3 CNVs . 15q13.3 duplication have been shown to have incomplete penetrance in two patients with childhood-onset schizophrenia . FurtherCRLF2) gene mapped at the PAR1 on the short arm of the X and Y chromosomes (Xp22.33/Yp11.32) [Cytokine receptor-like factor 2 (mes Xp22./Yp11.32 Yp11.32) . The actYp11.32) . It has Yp11.32) . CurrentYp11.32) .CRFL2 were not yet described. CRLF2 copy number gain and therefore dysregulation of CRLF2 expression has been described only in patients with acute lymphoblastic leukaemia (ALL) [CRLF2 and other genes such as SHOX, CSF2RA and IL3RA associated with abnormality of the nervous system were annotated in the database of chromosomal imbalance and phenotype in humans (DECIPHER n. 289637 and n. 294106). Considering these findings, CRLF2 dysregulation could affect neuronal signalling and have an additive role in the development of the brain disorders observed in the proband, but further studies are needed to confirm this hypothesis.Until now, patients with neurodevelopmental phenotypes having microduplications at Xp22.33 involving only ia (ALL) . Two dupTo our knowledge, the concomitant presence of these three private CNVs in a patient has not previously been reported.The girl’s father is a carrier of 7q35 microdeletion, and her mother was found to have microduplication of 15q13.3 and Xp22.33, the same as the proband’s brother, but without neurodevelopmental phenotypes. This suggests that each of the CNVs alone described above are not pathogenic enough to induce clinical manifestations, whereas the simultaneous occurrence of these three CNVs  may induce the brain disorders observed in our patient.The two-hit model could be an explanation for phenotypic differences between probands and their carrier parents ,40. ThisCNTNAP2  [We do not exclude that microcephaly, severe psychomotor delay and dysmorphic signs observed in the proband could be due to the fact that proband’s parents are third cousins. In support of this hypothesis, Rodenas-Cuadrado P. et al. observed facial dysmorphisms and psychomotor problems in a case report in which the proband’s parents were consanguineous (first degree cousins) carrying a 203 kb deletion encompassing exons 2–3 of the 914.175) . It is essential to underline that the interpretation of the clinical significance of small CNVs (∼500 kb), such as those observed in this patient, is very challenging because their role in the etiopathogenesis of several neurological seems to be associated to different factors, among which multifactorial effects or epigenetic alteration modifying gene expression . ConsequThe main limitations of this study are the absence of direct sequencing and whole exome/genome sequencing for the proband and her parents. Sanger sequencing could give us important information regarding the identified variants, whereas whole exome/genome sequencing can be used to exclude other genetic factors that can contribute to the observed phenotypes. In addition, since family history showed that proband’s parents are third cousins, it is possible that other genetic factors than those described by us could be involved in the development of the observed phenotype. CNTNAP2, CHRNA7 and CRLF2 genes in neurodevelopmental disorders.Importantly, future functional studies are needed to better understand the potential role of In conclusion, we hypothesise that the simultaneous presence of these three CNVs has an additive effect on phenotype, and together with other environmental or genetic factors, could be responsible for the clinical manifestations of the affected female described here.


It is a known fact that road traffic accidents are responsible for nearly 1.3 million deaths and another 50 million injuries in a year around the globe. Depending on the economic development of the country, victim category may change. In countries like Sri Lanka Vulnerable Road Users (VRU) are more susceptible for road traffic accidents. According to police records pedestrian involved accidents were top the list among other victim categories up to 2015. At present motor cycle riders are recorded as the highest number of victims due to road traffic accidents and pedestrians are at the second place. This paper is about pedestrians involved road traffic accidents reported to Kandy Police station in Sri Lanka to investigate and report a comparison of accidents recorded. Data Collection By law it is mandatory to report all the road traffic accidents to the police station concerned. For this study, road traffic accidents reported to Kandy police station during the period 29.08.2017 to 31.12.2018 were considered. Kandy is the second largest city in Sri Lanka and Kandy has nearly 10% of total traffic related incidents reporting compared to rest of the Country. As explained by Ming Sun et al it is very important pay detailed information about the pedestrian involved accidents to find an acceptable solution to ensure their safety. As a start, summary of accident categories are summarized in the table 1. Table 1: Summary of victim categories reported to Kandy Police Station Severity Gender of the Victim Female Male Grand Total Pedestrian Others Pedestrian Others Fatal 13 2 10 10 35 Grievous 30 19 36 63 148 Grand Total 43 21 46 73 183 According to the table 1, it is evident that pedestrians are highly vulnerable to road traffic accidents in the study area concerned. Further, Table 2 is representation of the same data set in percentages. Table 2 : Percentages of road traffic accidents for Pedestrians and others Pedestrian Others Pedestrian Others Fatal 37.14% 5.71% 28.57% 28.57% 100.00% Grievous 20.27% 12.84% 24.32% 42.57% 100.00% It is important to check whether the accidents are prominent in weekdays or weekends. Table 3 is a summary of accidents summarized according to the day of the week; Table 3 Accident variation according to the day of the week Pedestrian Involved accident Other Accidents Weekdays 70 70 Weekends 19 23 Accident / day (Weekday) 14 14 Accident/ Day (Weekend) 9.5 11.5 According to the dataset considered, there is no difference of accidents per day for weekdays for pedestrian involved and others. But for Weekends, there is a difference of accidents per day on weekends. This may be due to more pedestrians travel during weekdays due to official commitments, schooling etc. Table4. Vehicles involved in Accidents Vehicle Category Victim Category Pedestrian others Motor cycle 31 17 Three wheelers 9 37 Other Vehicles 49 39 Grand Total 89 93 According to the table 4, Motor Cycles and Three wheelers are responsible for most of the Pedestrian involved accidents. Since Motor cycles and three wheelers also categorized as vulnerable road users similar to pedestrians, they should be educated about the importance of sharing facilities with their fellow road users. Table 5 Accident locations Female Male Pedestrian Others Pedestrian Others Fatal Pedestrian crossing 6 0 2 0 others 7 2 8 10 Grievous Pedestrian crossing 9 0 11 1 Others 21 19 25 62 Grand Total 43 21 46 73 as per the information collected and analyzed, pedestrian involved accidents are high at pedestrians crossings compared to other locations. It is a known fact that pedestrian movements, especially across roads are higher at pedestrian crossings, but making such places safer also an important responsibility of the authorities.Pedestrian involved accidents are at a very high percentage. This may be because of low property damage and minor injury relate accidents also considered. Due to various uncertainties and possible underreporting, only fatal accidents and accidents categories as grievous are considered. According to the dataset considered, pedestrians are at high risk on roads compared to other road users. Main culprits in making pedestrian related accidents are other groups of vulnerable road users namely Motorcycle riders and Three-wheeler drivers. Pedestrian related accidents are more prominent during weekends compared to other victim categories.Traffic Accidents, Pedestrian accidents, Vulnerable road users


In the original article, there was an error. We have identified four sentences where the words “increase” and “decrease” were mistakenly transposed. These four transpositions do not affect the integrity of the data and were the result of an error in translating the sign of the effect sizes from analyses to text. As a result of correcting these four transpositions, two sentences in the discussion contextualizing direction of change required modification.the Abstract sentences #9 and 11, the Results subsectionFKBP5 Change from Before to after MBSR Treatment sentence #1, the Discussionsubsection FKBP5 sentence paragraph 1sentence #1, paragraph 2 sentence #5, andparagraph 3 sentence #2.A correction has been made to Abstract sentence #9: There was a significant time x responder group interaction for methylation in FKBP5 intron 7 bin 2  whereby responders had an increase in methylation and non-responders had a decrease in methylation from before to after treatment in this region.Abstract sentence #11: Increases in FKBP5 methylation after treatment in responders as compared to decreases in non-responders suggest that effective meditation intervention may be associated with stress-related pathways at the molecular level.Results>Methylation of FKBP5>Change from Before to after MBSR Treatment>sentence #1: There was a significant time x responder group interaction for methylation in FKBP5 intron 7 bin 2  whereby responders had an increase in methylation and non-responders had a decrease in methylation from before to after treatment in this region. Discussion>FKBP5>sentence #1: Analyses of FKBP5 revealed increases in intron 7 methylation after treatment in responders while non-responders had decreases in methylation within a specific FKBP5 CpG site of bin 2.Discussion>FKBP5>Paragraph #2>sentence #4: Additionally, consistent with our findings is that despite examining a different regulator region of FKBP5 than our study (exon 1 promoter vs. intron 7 GRE in our study) both of these investigations showed that changes in methylation were related to better symptom response with corresponding measures of increased gene expression identified in the study of PTSD response to exposure therapy (24).Discussion>FKBP5>Paragraph #3>sentence #2: Collectively our findings as well as those of others are consistent with the hypothesis that non-pharmacologic interventions may facilitate stress reduction through the regulation of FKBP5 to feedback on glucocorticoid hyperactivity to reduce stress.The authors apologize for this error and state that this does not change the scientific conclusions of the article in any way. The original article has been updated.


At the molecular level, the circadian clock is regulated by a time delayed transcriptional-translational feedback loop in which the core proteins interact with each other rhythmically to drive daily biological rhythms. The C-terminal domain of a key clock protein PER2 (PER2c) plays a critically important role in the loop, not only for its interaction with the binding partner CRY proteins but also for the CRY/PER complex’s translocation from the cytosol to the nucleus. Previous circular dichroism (CD) spectroscopic studies have shown that mouse PER2c (mPER2c) is less structured in solution by itself but folded into stable secondary structures upon interaction with mouse CRYs. To understand the stability and folding of human PER2c (hPER2c), we expressed and purified hPER2c. Three oligomerization forms of recombinant hPER2c were identified and thoroughly characterized through a combination of biochemical and biophysical techniques. Different to mPER2c, both thermal unfolding DLS and CD analyses suggested that all forms of hPER2c have very stable secondary structures in solution by themselves with melting temperatures higher than the physiological body temperature, indicating that hPER2c does not require CRY to fold. Furthermore, we examined the effects of EDTA, salt concentration, and a reducing agent on hPER2c folding and oligomerization. The ability of hPER2c forming oligomers reflects the potential role of hPER2c in the assembly of circadian rhythm core protein complexes. Per and Cry genes. After transcription and translation with time delays, PER and CRY proteins accumulate in the cytosol and form complexes that translocate into the nucleus to inhibit the function of the CLOCK/BMAL1 complex. Thus, PERs and CRYs regulate their own transcriptions to generate a negative feedback loop  = θobs/cnl where, c stands for the concentration of protein in moles, n for the number of residues and l for the path length of the cuvette. Furthermore, thermal denaturation studies were carried out by heating each sample from 5°C to 90°C with 5°C intervals, and the ellipticities were measured using the same parameter settings described above. At each temperature, the sample was equilibrated for 3 minutes prior to the CD measurements. The thermal unfolding profile of each sample was characterized using the mean residue ellipticity minima at 208nm ([θ]208) to determine Tm values by fitting the Boltzmann sigmoid equation to [θ]208 using Origin . The spectrum was further analyzed using BeStSel software to determine secondary structure content [http://pcddb.cryst.bbk.ac.uk/) having accession ID from CD0006240000 to CD0006242000.Far-UV CD measurement of the recombinant hPER2c was conducted using a J-1500 CD spectrophotometer (JASCO) connected to a Peltier temperature controller. Elution fractions of recombinant hPER2c from SEC were concentrated to approximately 15μM in the same SEC buffer . For each measurement, 400μl of sample was added in a 0.1cm path length rectangular quartz cuvette . The CD ellipticities  with sequence coverage of 68%. I-TASSER server provided the five best homology models of hPER2c with C-scores (confidence score)  of -2.33Recombinant hPER2c was purified to high purity by affinity chromatography and SEC  to excluThe size of all four forms of hPER2c was further confirmed by TEM . The hPEThe CD spectra of all four hPER2c forms were analyzed by BeStSel in order to determine their secondary structure compositions . The norPrevious studies speculate that the mPER2c domain gains secondary structure upon interacting with mCRY1 . The CD The thermal stability of hPER2c dimer, 20mer, and 40mer were analyzed by DLS . The hyd208) for all three forms of hPER2c were plotted against temperature and fitted with the Boltzmann sigmoid equation using Origin , suggesting that the 20mer is likely to convert into the metastable transition state under physiological temperature. Fitting of ellipticity values of hPER2c from this transition state to a completely unfolded state resulted a second Tm of 87.3°C (standard error 2.20°C), indicating that the high stability of the transition state far beyond physiological temperature 2+ ion waresidues , suggestrization .Using multiple analytical techniques including SEC, DLS, and TEM, three different and stable forms of recombinant hPER2c were observed with molecular weights close to 40mer, 20mer, and dimer. These different forms of hPER2c were then further characterized in terms of thermal stability and secondary structure composition using DLS and CD. Thermal stability studies from both CD and DLS show that the hPER2c 40mer has a higher stability than 20mer. Our study here reveals that, under physiological temperature, hPER2c 20mer is likely to adapt to a metastable state . ImportaMouse PER2c was suggested to be largely unstructured and becomes highly ordered once it interacts with CRY1 . In thisIt has been speculated that mammalian PER2 could form a homodimer with the rich coil-coil structure near the C-terminal domain . In our E.coli, where a reducing environment prevents the formation of the disulfide bonds. We speculated that the dimeric hPER2c could be an artificial product introduced during harvesting/purification process in vitro, where the reducing potential wasn’t sufficient to prevent the formation of the disulfide bonds. The dimer of hPER2c with disulfide bonds should not exist in an eukaryotic cytosol which also has a reducing environment. Based on these observations, without the disulfide bonds, the hPER2c will form large oligomers. This is consistent to the observation that the hPER2c 40mer and 20mer are produced in the high concentration in our expression system.To investigate whether disulfide bonds involve in stabilities of all three forms of hPER2c, they were treated with 20mM DTT for up to 36hrs. No effects on 40mer and 20mer were observed. However, both DLS, SEC and TEM showed the appearance of oligomers and aggregated forms after DTT treatment of the hPER2c dimer . The levAs mentioned above, both CD and DLS thermal stability studies show that both 40mer and 20mer have high stability with melting temperatures. Furthermore, our secondary structural analyses show that both hPER2c 40mer and 20mer contained more helices and less strands when compared to the dimeric hPER2c. Overall, hPER2c 40mer has higher percentage of helices and better stability than 20mer. We speculate these two oligomer forms were stably formed with helical coil-coiled structures. Wheel plot analysis of the longest helix in hPER2c modeled structure (residue 1158 to 1175) showed a unique amphipathic property  with oneIt is noteworthy that the oligomerization of hPER2c only results in two different oligomeric forms with distinct molecular weights close to 40mer and 20mer instead of a continuous size distribution. In a recent study on the assembly products of the core circadian clock proteins in mouse liver cell, large cIn conclusion, we identified and characterized three different forms of recombinant hPER2c, 40mer, 20mer, and dimer. Our study provides experimental evidences to clarify the long-standing speculation about whether or not hPER2c indeed requires hCRYs to facilitate folding into a stable structure. Unlike the structure of mPER2c published before, our data clearly shows that hPER2c has very stable secondary structure and does not need to interact with other protein to fold. Despite the high homology between hPER2c and mPER2c, our results indicate significant differences in their thermal stabilities. The ability of hPER2c to form oligomers reflects its potentials in assembly of circadian protein complexes. The molecular mechanism of forming distinct oligomers with discrete sizes is unknown. High resolution structural determination of these forms of hPER2c is needed to fully understand the mechanisms of hPER2c oligomerization and its roles in the circadian rhythm.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S1 Fig(A) DLS profile of hPER2c 20mer measured as the protein was being concentrated. (B) DLS profile of hPER2c dimer measured as the protein was being concentrated.(TIF)Click here for additional data file.S2 Fig(TIF)Click here for additional data file.S3 FigThe effect of the salt on the stability and polymerization of hPER2c 20mer (A) and dimer (B).(TIF)Click here for additional data file.S4 FigThe effect of the DTT and EDTA on the polymerization of hPER2c 40mer (A and B), 20mer(C and D) and dimer (E and F).(TIF)Click here for additional data file.S5 FigAliphatic residues are marked with blue squares; Hydrophilic residues are marked with red diamonds; Positively charged residues with black octagons. This wheel map is generated by EMBOSS pepwheel.(TIF)Click here for additional data file.


After this article  and its Specifically:In Fig 1B, the pCEFL-GST and pCEFL-GST+MG132 panels appear to report the same image.○Fig 2D VEGF in  appears ○Fig 2E VEGF in  appears ○Fig 2E β-actin in  appears Similarities were noted between the following western blot results reported in Fig 2 of  and Fig In response to the concerns about similarities between results reported in  and 3],,3], the PLOS ONE Editors retract this article.In light of the unresolved concerns and in line with the corresponding author’s request, the YX agreed with retraction. The other authors either could not be reached or did not respond directly.


Focal Cortical Dysplasias (FCD) are localized malformative brain lesions in epilepsy. FCD3a associated with hippocampal sclerosis, affects the superficial cortex and is presumed to have an ‘acquired’ rather than developmental origin. Precursor cells may arise outside neurogenic zones including cortical layer I. Our aim was to characterise subsets of glial progenitor cells in the superficial cortical layers, known to be involved in gliosis and gliogenesis and that could distinguish FCD3a from other subtypes.Using immunohistochemistry we quantified the density of glial progenitor subsets in superficial cortex layers using markers against PAX6, GFAP, Olig2 and PDGFRβ and proliferation marker MCM2 in ten FCD3a cases compared to 18 other FCD types and 11 non-FCD controls.Glial progenitor cells types were present in the cortical layer I and II in all FCD groups. GFAP cells frequently expressed PAX6 and significantly higher GFAP/PAX6 than GFAP/MCM2 cell densities were identified in the FCD3a group (p < 0.05). Olig2 cell densities were significantly higher in FCD3b than FCD3a (p = 0.002) and significantly higher GFAP/MCM2 compared to PDGFRβ/MCM2 cell densities were identified in both FCD3b and FCD2 groups. There was no correlation between cell densities and the age of patients at surgery and between cortical regions.Immature and proliferative glial populations across FCD variants reflect reactive cell types and differences may provide insight into underlying pathomechanisms. Higher PAX6 expression in astroglial cells in FCD3a may indicate a switch to astrocytic maturation and enhanced superficial gliosis. Higher Olig2 and GFAP/MCM2 densities in FCD3b may reflect margins of the tumour infiltration zone rather than true cortical dysplasia. FCD3a principally involves the superficial cortical layers with abnormal clustering of neurones in layer II, reduction of neurones in the lower part of layer II/III and accompanying laminar gliosis . This FCThe superficial cortical layers are the last to form during development with complete maturation extending into post-natal period. Experimental studies show deficient Robo1-mediated signalling, that regulates normal neuronal migration to the superficial layers, results in abnormal distribution of neurones in layer II and III during the post-natal stages , reminisWe hypothesised that glial progenitor cell types are integral to both the development and phenotype of FCD3a. Our aim was to evaluate this using paired-box transcription factor (PAX6), which has developmental regulatory roles in differentiation and proliferation of astrocytes , Olig2 t239 surgical cases were selected from the archives of the Epilepsy Society Brain and Tissue Bank which included ten representative cases of FCD3a, eighteen cases with other FCD subtypes , seven cases with ILAE type I HS and no dysplasia and four non-epilepsy controls ; FCD subtypes were determined using current ILAE criteria  Table 1Table 1. Immunohistochemistry was carried out on 5 μm formalin-fixed paraffin-embedded brain sections using anti-NeuN , anti-MCM2 , anti-PAX6 , anti-GFAP  and anti–PDGFRβ  as described in previous studies . Chromog3Qualitative assessment of immunolabelling with NeuN labelling confirmed the subtype of FCD including the typical clustering of neurones in layer II that defines FCD3a as distinct from other FCDs and normal cortex B-C. PAX6Quantitative evaluation showed variability in labelling between cases . For sin4We observed a range of glial progenitor cell types in cortical layer I and II common to FCD types but with differences that could reflect their underlying aetiology. Our aim was to compare FCD3a which primarily involves the superficial cortex to other ‘pan-cortical’ FCD and one finding was higher PAX6/GFAP than GFAP/MCM2 co-labelled cells in FCD3a. PAX6 is a transcription factor and stem cell marker, primarily expressed in radial glia during development with roles in cortical patterning . In the PDGFRβ is known to be expressed in brain parenchymal cells other than pericytes, including NG-2 cells, and show dynamic changes in number, proliferation and distribution following seizures and cortical injury . In the 5Our findings highlight differences in glial regenerative populations between dysplasia types of relevance to their divergent underlying aetiologies and potentially to their pathophysiology , warrantThe authors report no declarations of interest.


Aldehyde dehydrogenase 1A3 (ALDH1A3) has been implicated in the survival and proliferation of prostate cancer cells.We retrospectively reviewed our patients with advanced disease on adjuvant hormonal therapy after prostatectomy. Time to castration resistance stage was documented. And Immunohistochemistry analysis for ALDH1A3 was performed for those patient samples on tissue microarray. Bioinformatics anslysis was used for RNA sequencing data of both primary prostate cancer and metastatic castration resistance prostate cancer (mCRPC) from online datasets. Crispr-Cas9 was used to knock out ALDH1A3 in prostate cancer luminal cells, and morphologic analysis as well as the Gene Set Enrichment Analysis (GSEA) were facilitated to discover the mechanisms of the resistance phenotype.We found that the patients with ALDH1A3 low expression had shorter time to progression to castration resistance compared with those of higher expression group on adjuvant hormonal therapy after radical prostatectomy. The ALDH1A3 knockout cells gradually acquired resistance to androgen deprivation therapy, a few cells have been found in knockout group showing as that the spindle-like luminal cells in charcoal stripped medium. Furthermore, PI3K pathway activation has been confirmed by Western blot. The PI3K pathway inhibitor BEZ235 has been demonstrated that the acquired ADT resistance by ALDH1A3 down regulation could be rescued by PI3K pathway inhibitor.These results suggested a novel function for ALDH1A3 in development of mCRPC, and indicated PI3K pathway inhibitor has the potential in the treatment of a subgroup of mCRPC patients. Androgen deprivation therapy (ADT) is the standard of care for advanced prostate cancer or progression after localized definitive treatment. However, most patients eventually progress to a condition known as castration-resistant prostate cancer (CRPC), characterized by lack of response to ADT. Despite the several treatment options for this stage of disease, the impact on overall survival is less than optimal and, most importantly, there is no reliable biomarker to predict the response of the treatment or resistance. As a result, no standard guidance is available to optimally sequence approved treatments for individual patients. Since 2005, the next-generation sequencing (NGS) technologies  have madThe aldehyde dehydrogenase family 1 member A3 (ALDH1A3) catalyzes the oxidation of retinal to the pleiotropic factor retinoic acid using nicotinamide adenine dinucleotide (NAD+). The level of ALDHs enzymatic activity has been regarded as a cancer stem cell (CSC) marker and seems to correlate with tumor aggressiveness  which haWe found, surprisingly, that ALDH1A3 was down regulated in metastatic castration resistant prostate cancer from previous sequencing data . CombingThe protocol to generate the tissue microarrays (TMAs) in this cohort has been described in our previous report . We retrThree datasets .The RNA sequencing data were downloaded from SU2C database. The median value of RPKM for ALDH1A3 was used as cut-off value, any sample which is higher than the median value was determined as ALDH1A3escribed . The GenThe human prostate cancer cell line  were purchased from the Cell Bank Type Culture Collection of the Chinese Academy of Sciences  and maintained in RPMI medium with 10% fetal bovine serum within a humidified atmosphere containing 5% CO2 at 37 °C.http://crispr.mit.edu/), targeting the first exon. The sequence of the guide is as follows—ALDH1A3: 1- AGTTATGGCTACCACCAACG; 2-TAGTCTGCGGCGCACCGGCT; green fluorescent protein (GFP): GGCGAGGAGCTGTTCACCG. Then, we ligated the guide to the LentiCrispr-V2 system followed by Sanger sequencing validation [We designed the guide RNA for ALDH1A3 from , Phospho-Akt  and glyceraldehyde 3-phosphate dehydrogenase  were used in Western Blot assay in accordance with the manufacturer’s instructions.p < 0.05 was considered to be statistically significant. All the statistical calculations were performed using GraphPad Prism v6.0 software .Differences in vitro experiment like cell numbers between groups were subjected to Student’s t test. Our earlier work has demonstrated that ALDH1A3 highly expressed in human prostate, which had a strong correlation with primary prostate cancer luminal signature and could be a potential biomarker of AR signaling pathway. Then we moved on to investigate its expression in the castration resistant prostate cancer. We retrospectively reviewed 79 patients with advanced disease who underwent radical prostatectomy followed by adjuvant hormonal therapy in our center. It was indicated that negative expression of ALDH1A3 predicted shorter time progression to castration resistance on adjuvant hormonal therapy . The ALDH1A3 ranking the most significant changes in the whole list confirmed the results. The Gene Set Enrichment Analysis (GSEA) finally correlated ALDH1A3 signature with several biologic event. We found the ALDH1A3 signature had significant positive correlation with ERG signature and prostate cancer luminal signature, respectively   Fig. a, b, andion Fig. c, d.FigBased on the above results, we aimed to investigate the mechanisms of negative expression of ALDH1A3 in mCRPC samples. We designed small guide RNA targeting the functional exon of ALDH1A3 to knock out this gene on cell level to see the phenotype changes. After validation of the knock out efficiency Fig. , we pickBased on the above GSEA results, the ALDH1A3 signature negatively correlated with PI3K-AKT-mTOR signaling pathway. We speculate that the ALDH1A3 loss could activate the PI3K pathway. In order to validate this hypothesis, we did Western blot assay to test the PI3K pathway activation to compare the ALDH1A3 wild type cells and knockout cells. The blotting showed that both in LnCaP and VCaP cells, phospho-AKT had been elevated in ALDH1A3 knockout group Fig. , 5. NextOur previous study has demonstrated that ALDH1A3 is specifically expressed in luminal compartment in human prostate epitheliums. From the TCGA data of 333 primary prostate cancer, ALDH1A3 correlated with AR signaling pathway and corresponding luminal signature. It is also suggested that ALDH1A3 has a potential to be a predictor of survival in primary prostate cancer patients. In present study, with our single center follow up database, we performed IHC analysis on tissue microarray for patients with advanced disease upon adjuvant hormonal therapy after radical prostatectomy. The results showed that ALDH1A3 low-expression patients indicated shorter time to progression to castration resistance. The phenotype that we showed at the beginning has implications to understand the mechanisms of this prostate specific gene in the development of cancer progression. From the metastatic prostate cancer samples database, the RNA sequencing data demonstrated that ALDH1A3 was down regulated in mCRPC group compared with the primary prostate cancer. Next-generation sequencing (NGS) analysis has made it possible to reclassify different subtypes in a specific cancer by molecular changes. It indeed could provide benefits for clinical practice in oncology, such as diagnosis, prognosis, and treatment decisions. For example, patients with cancers of unknown primary (CUP), traditionally, are generally assumed to have a poor prognosis with a treatment in cytotoxic chemotherapy guided by histologic features and the pattern of metastatic spread. A new study showed that NGS may provide an opportunity for CUP patients to benefit from individualized therapies according to the targetable genomic alterations identified by tumor molecular profiling . In thatThanks to the RNA sequencing data, we found that the PI3K pathway signature had been highly correlated with ALDH1A3 signature. Then we speculated the PI3K signaling pathway activation might be due to ADT resistance. And we also confirmed this hypothesis by showing the up-regulation of phospho-AKT upon ALDH1A3 knockout. Furthermore, PI3K signaling pathway inhibitor BEZ235 could rescue the ADT resistance following ALDH1A3 knockout. The PI3K/AKT/mTOR pathway is altered in almost 50% of mCRPC through either PTEN inactivation or/and aberrant activation in PIK3CA/B . It’s beIn conclusion, the NGS provides multiple new opportunities and tools to accelerate and facilitate the entire process of drug testing toward accelerated drug positioning and approval for precise and personalized medicine. It also allows researchers to discover some hidden pattern of the complex cancer. Such strategies include the development of inhibitors with a higher potency against their intended target, like ADT and Abiraterone, and the use of combination therapies incorporating inhibitors of parallel or alternative signaling pathways mediating acquired resistance, like the mTOR inhibitor BEZ235 in the treatment of PI3K-AKT-mTOR pathway activation. In this paper, we’ve investigated alterations in the targeted gene leading to ADT resistance to mCRPC. Based on the RNA sequencing and experimental results, we found that PI3K pathway alteration or activation might be the cause of the resistance. We, then, rescued the ADT resistance by PI3K pathway inhibitor-BEZ235. The acquired resistance to ADT therapy by some patients with low level of ALDH1A3 could be overcome by combination therapy with PI3K pathway inhibitor, which will provide a new potential approach to the treatment of mCRPC.Additional file 1 : Table S1: A differential expression gene list as ALDH1A3 signature.Additional file 2 : Figure S1: Original data of western blot (ALDH1A3) in Fig. ot ALDH1A in Fig. Additional file 3 : Figure S2: Original data of western blot (GAPDH) in Fig. Additional file 4 : Figure S3: Original data of western blot (ALDH1A3) in Fig. Additional file 5 : Figure S4: Original data of western blot (p-AKT) in Fig. Additional file 6 : Figure S5: Original data of western blot (GAPDH) in Fig.


CHK2), in which gene mutations convey up to twofold higher risk for breast cancer but do not increase lung cancer risk. We have investigated the role of CHK2 and the related kinase checkpoint protein 1 (CHK1) in cell cycle regulation in primary breast and lung primary epithelial cells. At the molecular level, CHK1 activity was higher in lung cells, whereas CHK2 was more active in breast cells. Inhibition of CHK1 profoundly disrupted the cell cycle profile in both lung and breast cells, whereas breast cells were more sensitive toward inhibition of CHK2. Finally, we provide evidence that breast cells require CHK2 to induce a G2–M cell cycle arrest in response of DNA damage, whereas lung cells can partially compensate for the loss of CHK2. Our results provide an explanation as to why CHK2 germline mutations predispose for breast cancer but not for lung cancer.Cancer is a life-threatening disease that affects one in three people. Although most cases are sporadic, cancer risk can be increased by genetic factors. It remains unknown why certain genes predispose for specific forms of cancer only, such as checkpoint protein 2 ( BRCA1) and 2 (BRCA2) strongly predispose for breast and ovarian cancer, whereas germline mutations in the DNA repair genes MSH2, MSH6, and MLH1 are associated with hereditary nonpolyposis colon cancer.Cancer is a disease that can arise in virtually any tissue. Most cases are the result of mutations that occur by chance. However, germline variants can affect cancer risk too. Many cancer predisposition genes (CPGs) have key roles in DNA repair, cell cycle control, and cell survival pathways, which are necessary to maintain genomic integrity. Surprisingly, despite their role in basic cellular programs, CPGs appear to affect cancer development across tissues differently. For instance, mutations of the DNA repair genes breast cancer protein 1 -checkpoint protein 2 (CHK2) pathway, which is activated in response to DNA DSBs, is more active in primary breast than in lung cells4. Interestingly, both ATM and CHK2 germline mutations predispose for breast cancer6.Why mutations in DNA repair genes predispose for specific cancer types is an outstanding mystery. Since many breast CPGs are involved in the repair of DNA double-strand breaks (DSBs)BRCA1, which regulates many processes, confer a 10–20% higher risk than BRCA2 mutations, which functions exclusively in DSB repair8. In addition, BRCA1 mutation carriers develop breast cancer at a younger age than BRCA2 mutation carriers9. Both BRCA1 and CHK2 play roles in cell cycle control11. Since a dysregulated cell cycle can lead to genetic errors and genomic instability, uncontrolled cell division is one of the hallmarks of cancer12. It is therefore possible that mutations in BRCA1 and CHK2 contribute to cancer development by deregulation of the cell cycle.However, DNA repair proteins may contribute to breast cancer risk in additional ways. For instance, mutations in 13. Second, several breast CPGs are known, whereas the genetic component of lung tumorigenesis appears to be very small14. We observed that breast and lung cells have a different cell cycle distribution, which is reflected in differential CHK1 and CHK2 activity. We provide evidence that breast cells depend on CHK2 to induce a G2 cell cycle arrest in response to DSBs, whereas lung cells appear to have compensatory mechanisms. These findings may help to explain why CHK2 germline mutations predispose for breast cancer but not for lung cancer.To understand differences in tissue-specific cancer risk, we focused on primary breast and lung cells for two reasons. First, breast and lung cancer are among the most common kinds of cancer, suggesting that they have a high cancer risk4. Interestingly, CHK1 and CHK2 also play roles in cell cycle regulation: CHK1 is required for checkpoints throughout the cell cycle, whereas CHK2 is mostly active during the G1 phase. We therefore set out to compare the cell cycle profile of breast and lung primary cells.We previously observed that the functionally related CHK1 and CHK2 play tissue-specific roles in the DNA damage response in primary breast and lung cells4. Consistent with the slow population doubling times, the majority of these cells were in G0–G1 phase P21 Fig. 16. In aging Fig. .These results suggest that the differential activity of CHK1 and CHK2 in primary breast and lung cells affects cell cycle regulation.17.CHK1 and CHK2 are known to play key roles in linking DNA damage signaling to cell cycle control. We wondered whether the role of CHK1 and CHK2 in inducing a DNA damage cell cycle checkpoint was different in both cell types. To test this, we made use of the DNA DSB-inducing agent doxorubicin, which is known to induce a G2–M arrest4 and activate CHK1 and CHK2. In both cell types, 16-h exposure to doxorubicin increased the G2–M fraction . Since the CDC2/Cyclin B complex is a better substrate for tyrosine kinase Wee129 than CDC2 alone, we hypothesize that the drop in Cyclin B levels may be responsible for this decrease. Thus the initial inactivation of CDC2 triggers an early G2–M arrest, whereas the drop in Cyclin B1 levels at a later stage may maintain G2–M phase arrest. The mechanism responsible for the loss of Cyclin B1 may be the upregulation of P5318.Interestingly, we previously observed that CHK1 is preferentially upregulated after DNA damage in lung cells, whereas CHK2 activation was stronger in breast cells after DNA DSBs30. Concurringly, sporadic mutations of CHK1 and CHK2 have been found in most types of cancer. In addition, germline mutations of CHK2 appear to predispose for certain types of cancer. People who harbor truncating CHK2 mutations  have an approximately twofold increased risk of developing breast cancer32. Carriers also have an increased likelihood to develop prostate34 and colon cancer37, but, intriguingly, no increased risk of lung cancer39.Cell cycle checkpoints are important to prevent transmission of damaged DNA to the daughter cells, and hence CHK1 and CHK2 can protect cells against cancerCHK2 predisposes for breast cancer but not for lung cancer. Since mice harboring a CHK2*1100delC mutation did not show the tissue-specific bias observed in humans40, a better disease model is needed to understand CHK2-mediated cancer predisposition. The comparative analysis of healthy primary epithelial cells may provide further insights into the relation between loss of CHK2 and tissue-specific cancer development.Considering the pivotal importance of cell cycle arrests to prevent genomic instability, our data may provide an explanation for why loss of Supplementary Information


Hair follicles cycle through periods of growth (anagen), regression (catagen), rest (telogen), and release (exogen). Telogen is further divided into refractory and competent telogen based on expression of bone morphogenetic protein 4 (BMP4) and wingless-related MMTV integration site 7A (WNT7A). During refractory telogen hair follicle stem cells (HFSC) are inhibited. Retinoic acid synthesis proteins localized to the hair follicle and this localization pattern changed throughout the hair cycle. In addition, excess retinyl esters arrested hair follicles in telogen. The purpose of this study was to further define these hair cycle changes. BMP4 and WNT7A expression was also used to distinguish refractory from competent telogen in C57BL/6J mice fed different levels of retinyl esters from two previous studies. These two studies produced opposite results; and differed in the amount of retinyl esters the dams consumed and the age of the mice when the different diet began. There were a greater percentage of hair follicles in refractory telogen both when mice were bred on an unpurified diet containing copious levels of retinyl esters (study 1) and consumed excess levels of retinyl esters starting at 12 weeks of age, as well as when mice were bred on a purified diet containing adequate levels of retinyl esters (study 2) and remained on this diet at 6 weeks of age. WNT7A expression was consistent with these results. Next, the localization of vitamin A metabolism proteins in the two stages of telogen was examined. Keratin 6 (KRT6) and cellular retinoic acid binding protein 2 (CRABP2) localized almost exclusively to refractory telogen hair follicles in study 1. However, KRT6 and CRABP2 localized to both competent and refractory telogen hair follicles in mice fed adequate and high levels of retinyl esters in study 2. In mice bred and fed an unpurified diet retinol dehydrogenase SDR16C5, retinal dehydrogenase 2 (ALDH1A2), and cytochrome p450 26B1 (CYP26B1), enzymes and proteins involved in RA metabolism, localized to BMP4 positive refractory telogen hair follicles. This suggests that vitamin A may contribute to the inhibition of HFSC during refractory telogen in a dose dependent manner. The hair follicle has a self-cycling ability. It goes through a regeneration phase (anagen), a degenerative phase (catagen) involving the apoptosis and loss of the lower two-thirds of the hair follicle, a resting phase (telogen), and a release phase (exogen) . The traRetinoids is a general term for both synthetic and natural forms of vitamin A. The dietary form of vitamin A is retinyl esters. Retinyl esters are important for healthy hair ; since bThe active form of vitamin A is retinoic acid (RA). RA is synthesized at or near the site of action , by a feDgat1tm1Far null mice also altered the hair cycle by both lengthening the first anagen and shortening the second telogen  reduced zig-zag hairs, but did not block hair development. Recently, reduced retinal synthesis in Del(4Sdr16c5-Sdr16c6)1Nyk double null mice altered the hair cycle (In a previous study in C57BL/6J (B6) mice, retinyl esters dose dependently altered the hair cycle . Feeding telogen . These eethality . Conditiir cycle . These mir cycle . In contThe Jackson Laboratory  Institutional Animal Care and Use Committee approved all procedures. C57BL/6J  mice  were used.B6 Dams and female pups were fed the NIH 31 diet  throughout this study. Only females were used because they fight less, which reduces wounds. Wounds by themselves will initiate anagen, which would interfere with this study . Mice wen = 8), 28 (n = 9), or 56 (n = 10) IU retinyl acetate/g diet  for 16 weeks  until 12 weeks of age, and then switched to an American Institute of Nutrition (AIN)93 maintenance diet containing 4 (16 weeks  (Everts 16 weeks . All mic16 weeks . In addi16 weeks . The fir16 weeks  and reduull mice .n = 7), 28 (n = 10), or 56 (n = 6) IU retinyl acetate/g diet  for 12 weeks . Second generation female pups were used in this study . Six-wee12 weeks . At 6 weImmunohistochemistry was performed in serial sections as described previously . PrimaryBone morphogenetic protein 4 positive and negative telogen hair germs and dermal cells were counted from mice fed various levels of vitamin A in studies 1 and 2. The percent of hair follicles in refractory telogen was calculated in two steps. First, the number of BMP4 positive telogen hair germs was divided by the total number of telogen hair follicles to get the fraction of telogen hair germs in refractory telogen. Second, this fraction was multiplied by the percentage of telogen follicles calculated in the original diet studies  because Co-immunofluorescence was performed using a two-antibody co-localization system in frozen dorsal skin sections as previously described . PrimaryImmunohistochemistry was first performed with antibodies against KRT6 and CRABP2 using AEC+ (red) as described above with additional blocking steps using the streptavidin, biotin blocking kit . IHC was then performed with the Vector mouse anti-BMP4 antibody using the Mouse on Mouse ImmPRESS Peroxidase kit with some modifications . First, we doubled the concentration of blocking reagent and incubated the slides for 2 h. Second, we diluted the ImmPRESS reagent 1:1 in the provided 2.5% normal horse serum. Slides were then treated with DAB Eq (brown) for 10 min, counterstained and mounted as described above. The number of hair follicles with positive KRT6 or CRABP2 plus or minus dermal BMP4 was counted. The percent of positive KRT6 or CRABP2 hair follicles with dermal BMP4 was then calculated separately from the percent of KRT6 or CRABP2 hair follicles without dermal BMP4. An average of 26 hair follicles (range of 2–54) were scored per mouse. Four to seven mice per diet were analyzed. Images were taken with the Nikon Eclipse i80 microscope with the DS-Ri2 camera . Images were sized, cropped, composite figures were created, and whole images were adjusted for brightness, contrast, and color balance in Adobe Photoshop CS3. Unaltered images are in post-hoc test when significant treatments effects were seen. When there was a significant interaction between study and diet a one-way ANOVA was performed on the interaction variable followed by Tukey post-hoc test. When equal variances were not seen, each study was analyzed by Kruskal–Wallis followed by Mann–Whitney U test. P value < 0.05 is defined as statistical significance.Equal variance was first confirmed using Levene’s test of equality of error variances using SPSS version 24 . Comparison among experimental groups was analyzed by univariate analysis of variance using a 2 × 3 full factorial model followed by Tukey Mice from study 1  on the 5Next, we attempted to determine the localization pattern of vitamin A metabolism proteins during telogen using BMP4 to mark refractory telogen. Dorsal skin was collected from B6 mice fed an unpurified diet at 70–101 days of age and IHC performed in serial sections with antibodies against BMP4 (Abcam), KRT6, SDR16C5, RDH10, ALDH1A2, CRABP2, and CYP26B1. Using the Abcam antibody, all proteins localized to BMP4 positive telogen hair follicles , 3. SDR1This study was done to better define the impact retinyl esters have on the hair cycle. In mice bred on an unpurified diet, excess  levels of retinyl esters lead to a greater percentage of hair follicles in refractory telogen. Regardless of retinyl ester level fed, KRT6 and CRABP2 localized to the inner bulge cells almost exclusively in refractory telogen in these mice. SDR16C5, ALDH1A2, and CYP26B1 also localized to the hair follicle at greater intensities during refractory telogen in mice bred and fed an unpurified diet. In contrast, when mice were bred on a purified diet containing recommended  levels of retinyl esters the percent of hair follicles in refractory telogen decreased as retinyl ester levels increased. In addition, KRT6 and CRABP2 localized to the inner bulge cells during both competent and refractory telogen in adult mice fed recommended (4IU) and high (28IU) levels of retinyl esters. These data suggest that vitamin A may regulate HFSC in a dose and time dependent manner.tm1BlhKrt1-15crePGR)22Cot/J/Foxc1 null mice identified increased Aldh1a2 expression. Assay for transposase accessible chromatin then sequencing (ATAC-seq) analysis of the promoters of differentially expressed genes revealed a SMAD binding motif in the promoter of Aldh1a2, but not NFATC1 or FOXC1 binding sites. This suggests that BMP signaling may increase ALDH1A2 and RA synthesis during refractory telogen to contribute to HFSC quiescence.Bone morphogenetic protein 2, BMP4, and BMP6 inhibit HFSCs during refractory telogen . BMP2 anCyp26b1tm1Hh null mice decreased Lhx2 at embryonic day E16.5 and E18.5 along with altering several other HFSC regulatory genes and blocked hair follicle development (tm2(cre)Wrst;Cyp26b1tm1HhEn1 null mice. The finding that CYP26B1 localized to the HFSC and inner bulge in addition to the dermis in this study suggests that reduced RA may be critical within the HFSC and/or inner bulge cells but not the dermal papilla. CRABP2 shuttles RA to both RARs and CYP26B1 (Del(4Sdr16c5-Sdr16c6)1Nyk double null mice and excess RA in Dgat1tm1Far null mice; and an increase of hair follicles in refractory telogen following excess retinyl esters in study 1, reduced retinyl esters in study 2, and pharmacological doses of synthetic retinoids in the clinic.Retinoic acid may also regulate HFSC quiescence. Excess RA in the elopment . However CYP26B1 . This suRetinoic acid synthesis proteins localizing to the KRT6 positive inner bulge cells during telogen is similar to what was seen in the inner cell layer and companion layer during anagen . ALDH1A3Cyp26b1 expression decreased in these inner bulge cells in late exogen verses early exogen 22Cot/J, Foxc1 null mice and FGF18 injections revealed opposite effects based on the stage of the hair cycle the follicle was in at the time , arresting hair follicles in refractory telogen is a good thing. This is because CCCA in mice only occurs when hair follicles are in anagen . No miceThe original contributions presented in the study are included in the article/The animal study was reviewed and approved by The Jackson Laboratory  Institutional Animal Care and Use Committee.HE and JS conceptualized, acquired funding, provided resources, provided supervision, and reviewed and edited the manuscript. HE administered the project and curated the data. LS, CV, EH, and HE performed the analyses. LS wrote the original draft. CV wrote the first draft of the revision. NK provided resources. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


In the article , there w"The cohort was validated against a set of nearly 60,000 laboratory records matched manually by a team of research assistants who found that the approach had 6.3% overmatching (laboratories that were matched should not have been) and 1.4% undermatching (laboratories that should have been matched were not) [22]."The last sentence reads:"The cohort was validated against a set of nearly 60,000 laboratory records matched manually by a team of research assistants who found that the approach had 1.4% overmatching (laboratory records that were matched should not have been) and 6.3% undermatching (laboratory records that should have been matched were not) [22]."The last sentence should have read:


Transcription factor 4 (TCF4) intron 3 is the main cause of Fuchs’ endothelial corneal dystrophy (FECD) and may confer an increased risk of developing bipolar disorder (BD). Usage of alternative 5′ exons for transcribing the human TCF4 gene results in numerous TCF4 transcripts which encode for at least 18 N-terminally different protein isoforms that vary in their function and transactivation capability. Here we studied the TCF4 region containing the CTG TNR and characterized the transcription initiation sites of the nearby downstream 5′ exons 4a, 4b and 4c. We demonstrate that these exons are linked to alternative promoters and show that the CTG TNR expansion decreases the activity of the nearby downstream TCF4 promoters in primary cultured neurons. We confirm this finding using two RNA sequencing (RNA-seq) datasets of corneal endothelium from FECD patients with expanded CTG TNR in the TCF4 gene. Furthermore, we report an increase in the expression of various other TCF4 transcripts in FECD, possibly indicating a compensatory mechanism. We conclude that the CTG TNR affects TCF4 expression in a transcript-specific manner both in neurons and in the cornea.The CTG trinucleotide repeat (TNR) expansion in  TCF4 is expressed in almost every tissue type in human5. In the brain TCF4 expression peaks during late embryonic development and continues at a relatively high level during postnatal development7. Notably, transcription from TCF4 gene can begin at multiple mutually exclusive 5′ exons leading to transcripts with varying composition of functional protein domains which modulate the ability of TCF4 to regulate transcription8.Transcription factor 4 (TCF4) is a basic helix-loop-helix transcription factor that plays a vital role in the development of the nervous and immune system11. In addition, mutations in the gene regions included only in the longer isoforms of TCF4 have been associated with intellectual disability12. Alterations in TCF4 expression levels have been described in patients with depression13. An expansion of a CTG TNR, located in an alternative promoter region between TCF4 exons 3 and 4 (known as CTG 18.1), upstream of TCF4 5′ exons 4a, 4b and 4c causes Fuchs' endothelial corneal dystrophy (FECD) and has been linked to Bipolar disorder (BD)15.TCF4 gene is implicated in susceptibility to schizophrenia, and mutations in TCF4 cause Pitt-Hopkins syndrome, a rare developmental disorder characterized by severe motor and mental retardation16. Out of the many genetic mutations associated with FECD the CTG TNR expansion in TCF4 is thought to be one of the major factors in the development of the disease, as the presence of a TCF4 allele with 50 or more CTG TNR-s confers an increased risk of developing the disease17. BD is a psychiatric disorder that affects up to 1% of the global population, causing severe mood alterations in affected patients18. It has been shown that a TCF4 CTG TNR expansion of over 40 CTG TNR-s is frequent in a subset of patients with a severe type of BD and that the repeat expansion in TCF4 may increase vulnerability to BD15.FECD is an ocular disorder associated with corneal edema and vision disruption having a varying prevalence between different populations affecting about 4% of people over 40 in the United StatesTCF4 CTG TNR expansion and the mRNA levels of TCF4 transcripts spanning the TCF4 CTG TNR region have produced contradictory results. A study by Foja et al.19 reported that TCF4 CTG TNR expansion is connected with a reduction in the levels of TCF4 transcripts beginning in the proximity of the CTG TNR, whereas a study by Okumura et al.20 has reported that TCF4 CTG TNR expansion is connected with an increase in overall TCF4 levels and in the levels of TCF4 transcripts beginning in proximity of the CTG TNR. Two other studies22 have reported no effect of the CTG TNR expansion on total TCF4 mRNA levels.Studies on the connection between the TCF4 CTG TNR expansion affects the levels of TCF4 transcripts and total TCF4 levels. Here, we hypothesized that the TCF4 CTG TNR region contains functional promoters that regulate the transcription of nearby 5′ exons and that the activity of these promoters is altered by the length of TCF4 CTG TNR expansion. For this, we first characterized the TCF4 alternative promoter region containing the CTG TNR by identifying transcription start sites (TSS) and describing the splicing of nearby 5′ exons 4a, 4b, and 4c. We then used luciferase reporter assay to investigate whether the CTG TNR expansion could influence TCF4 expression in primary neurons by affecting the ability of surrounding regulatory regions to promote transcription. Furthermore, RNA-seq data from corneal tissue of FECD patients with an expanded TCF4 CTG TNR was analyzed to determine the expression levels of TCF4 transcripts beginning both proximal and distal to the CTG TNR. Collectively, our results demonstrate that the CTG TNR expansion differentially modulates the activity of TCF4 promoters.It is currently unknown whether the TCF4 5′ exons 4a, 4b and 4c, which are located between internal exons 3 and 4  spanning the TCF4 CTG TNR region from just downstream of exon 3 into 5′ exon 4a and a longer sequence (TCF4 p4abc) spanning the entire TCF4 CTG TNR from just downstream of exon 3 to inside exon 4 . The luciferase reporter assay revealed that an extended CTG TNR with a length of 54, 67/70 or 144 repeats significantly reduced the activity of the promoter for both TCF4 p4a and p4abc . Our analysis revealed that FECD patients with an expanded CTG TNR displayed reduced levels of transcripts containing 8. The major TCF4 transcripts comprising of 5′ exons 3b, 3c and 3d encode for isoform TCF4-B when spliced to internal exon 3; transcripts with exons 3b and 3d encode for isoform TCF4-C when spliced to exon 4; transcripts with exons 8a, 8bII and 8cII encode for isoform TCF4-D when spliced to exon 8; transcripts with exons 10a, 10b and 10c spliced to exon 10 encode for isoforms TCF4-A, TCF4-I and TCF4-H, respectively  and Fuchs’ dystrophy (> 50)15. Soliman et al. has shown that the severity of FECD correlates with the length of the CTG TNR in TCF4 as patients with a CTG TNR expansion exhibited a more severe form of FECD, but the mechanism underlying this phenotype remains unknown30. It is important to note that our determination of TSS-s by 5′ RACE and reporter experiments were done using human cerebellar RNA and rat cultured cortical neurons, respectively. Therefore, it would be interesting to conduct similar experiments in human corneal endothelial cells. This would help to translate our findings between different cell types and further validate the effect of the CTG TNR expansion on transcription also in FECD patients.Previous studies have indicated that the CTG TNR expansion in intron 3 of 28 revealed that the levels of TCF4 transcripts containing alternative 5′ exons 4aI and 4aIII were reduced in the corneal endothelial cells of FECD patients with an expanded CTG TNR. These results support our findings that the TCF4 CTG TNR expansion reduces the activity of proximal downstream promoters linked to these 5′ exons also in human corneal endothelium. Interestingly, an increase in the levels of TCF4 transcripts encoded by downstream alternative 5′ exons distal to the CTG TNR was also noted, which may indicate a compensatory mechanism to rescue the levels of TCF4 protein arising from the deficit of transcripts encoding TCF4-C. This compensation phenomenon needs to be considered when studying TCF4 expression levels in FECD and other diseases connected with the TCF4 intronic CTG TNR and could explain why different research groups have published contradictory results concerning changes in TCF4 levels when studying FECD22. Our results indicate that the levels of TCF4 transcripts change bidirectionally in response to an expanded CTG TNR—transcripts beginning near the repeat region decline just as Foja et al. reported19 while certain transcripts beginning downstream of the repeat region increase as reported here, and mask the decrease of long TCF4 transcripts. As the expression of different TCF4 transcripts decline and rise simultaneously the overall TCF4 levels may not change significantly in FECD as has been reported by Mootha et al.21 and Ołdak et al.22. In contrast, Okumura et al.20 found an increase in total TCF4 expression levels which is also evident in our RNA-seq analysis as we saw a slight rise in TCF4 expression when measuring the expression of internal exons present in all TCF4 transcripts (exons 10–21). Our results illustrate the importance of the exact transcript measured when studying TCF4 expression levels. The original RNA-seq study by Chu and others concluded that the TCF4 CTG TNR expansion increases the stability and thus the amount of expanded CTG repeat-including intronic RNAs in the corneal endothelium and causes comprehensive changes in splicing. No alterations in the overall expression of mature TCF4 mRNA was noted28. The study by Nikitina et al. was a data article and no conclusions were made27.Detailed analysis of previously published FECD RNA-seq datasets31. This can cause an accumulation of RNA polymerases in the CTG TNR and dissociation of the polymerase, leading to a decrease of full-length TCF4 transcripts beginning from upstream of the CTG repeat. An increase in the expression of 5´ exons spliced to exon 3 may be due to preferential splicing of transcripts to the exon before the CTG TNR (exon 3), as changes in splicing have been described before in diseases associated with repeat expansion31.Interestingly, we also detected an increase in the expression of 5′ exons spliced to exon 3 encoding for TCF4-B in patients with FECD, showing that almost all the TCF4 promoters far upstream from the CTG TNR had increased activity due to the repeat expansion. However, the increase in upstream promoter activity did not reflect in the levels of transcripts containing exons 3 and 4. It is plausible that the CTG repeat expansion could regulate transcriptional elongation of RNA polymerase by slowing down the polymerase in the CTG TNR region8. Currently FECD research focuses mainly on the CTG TNR and missplicing of longer TCF4 transcripts in FECD17, and little research has been done to analyze the expression of all the TCF4 transcripts in FECD. Our detailed analysis provides new insight into FECD as we show that the CTG TNR directly modulates the expression of TCF4 which may be among the underlying causes for the development of the disease. Since TCF4 mRNAs detected in the present study are expressed virtually in all tissues, with high levels in the fetal and postnatal brain4, there may also be a similar correlation between the CTG TNR length and the expression levels of TCF4 transcripts in vivo in the brain which could predispose development of BD. However, it should be noted that the link between the CTG TNR expansion in TCF4 and BD has only been shown once and has not been reported by newer studies.We have previously shown that TCF4 protein isoforms can be divided into longer and shorter isoforms which vary in their function and transactivation capabilityn TNR containing TCF4 mRNAs are the cause of Fuchs' corneal dystrophy33. According to this mechanism, the TNR-carrying RNAs cluster RNA binding proteins, interfering with the splicing of various mRNAs. Of note, antisense therapy using Fuchs' dystrophy ex vivo cell models leads to inhibition of RNA foci and mis-splicing in Fuchs' dystrophy35. Since the CTG TNR is located in the intron between exons 3 and 4, this repeat is not included in the fully mature TCF4 mRNA4, but the CTG TNR is still included in the pre-mRNA of transcripts initiated at the upstream promoters .Strong evidence has also been provided in support of a mechanism in which the toxic (CUG)29 and unstable TNR-s may occur in both coding and noncoding regions, including promoters, introns and untranslated regions (UTR) of genes36. Among noncoding TNR-s, one of the most studied is the TNR repeat (CGG) located in the 5′ UTR of Fragile X Mental Retardation (FMR1) gene. This TNR causes hypermethylation and silencing or increases in the expression level of the gene, depending on TNR length37. TNR diseases with TNR in the promoter region of the affected gene have been less studied. Recently, an intronic polymorphic CGG repeat in a conserved alternative promoter of the AFF3 gene, an autosomal homolog of the X-linked AFF2/FMR2 gene, was shown to lead to hypermethylation of the promoter and transcriptional silencing of AFF3 expression in the brain38. However, the effect of TNR on promoter activity using transient expression analysis of promoters linked to TNR was not studied. Research on Friedreich ataxia, which is caused by an expansion of the intronic TNR (GAA) in the FXN gene, has revealed reduced expression of the gene in patient derived cell lines39. A hexamer repeat expansion (GGGGCC) located in the 5′ regulatory region of the C9ORF72 gene, causing hereditary amyotrophic lateral sclerosis, has been shown to reduce the ability of the surrounding region to promote the expression of a reporter protein in human kidney and neuroblastoma cell lines40. Overall, these results indicate that expansion of TNR can alter the expression of the nearby genes. This is in agreement with our results showing that the expression levels of different TCF4 transcripts are altered in FECD due to the CTG TNR expansion.Repeat expansions have been associated with more than 40 diseasesTCF4 transcripts in FECD has displayed varying results. Analyzing only total TCF4 levels or levels of certain TCF4 transcripts can produce misleading results due to the complexity of the TCF4 gene and its regulation. The current study shows that the TCF4 CTG trinucleotide repeat expansion modulates the activity of nearby TCF4 promoters in a length dependent manner—an expanded CTG TNR causes reduction in promoter activity. Analysis of RNA-seq datasets revealed that the expression levels of the many TCF4 transcripts are increased or decreased simultaneously in the cornea of FECD patients. Further work is needed to elucidate the exact mechanism how this repeat region affects TCF4 transcription and whether the changed TCF4 levels contribute to the development of FECD and BD.Taken together, our results help to explain why previous research on the levels of Human postmortem tissues were used to obtain DNA and RNA samples. All protocols using human tissue samples were approved by Tallinn Committee for Medical Studies, National Institute for Health Development (Permit Number 402). All experiments were performed in accordance with relevant guidelines and regulations.TCF4 gene fragments were screened from human DNA samples for the TCF4 CTG TNR length and fragments with the desired CTG TNR length were amplified by PCR from 20 ng of genomic DNA in a 20 μl mixture using 0.4 units of Phusion Hot Start II (Thermo Scientific) and primer p4a_p4abc_F paired with the p4abc_R or p4a_R primer (Supplementary Table TCF4 p4abc) and the shorter (TCF4 p4a) sequence of the TCF4 gene  were acquired from human genomic DNA by PCR. A sixth synthetic DNA segment with 144 CTG repeats was ordered from GenScript. All the generated constructs were verified by sequencing as the length of TNR tended to be unstable in bacteria when producing plasmids . All experiments were performed in accordance with the relevant guidelines and regulations.41. Neurons grown 6 days in vitro were transfected with 180 ng firefly reporter construct and 20 ng pGL4.83[hRlucP/PGK1/Puro] as described previously4 for 4 h on a plate shaker using Lipofectamine 2000  with a reagent to DNA ratio 3:1. Two days after transfection neurons were lysed in 50 μl Passive Lysis Buffer (Promega) and luciferase reporter assay was performed using the Dual-Glo Luciferase Assay System (Promega) according to manufacturer’s protocol. Luciferase signals were measured using the GENios Pro microtiter plate reader (Tecan). For analysis, the signals were first normalized to the signal of the Renilla luciferase and then normalized to the respective ratio in cells transfected with the 11 repeat CTG construct. One-way repeated-measures analysis of variance (ANOVA) with Greenhouse–Geisser correction followed by Dunnett’s post hoc test was used to determine the statistical significance compared to the luciferase signals from the 11 repeat CTG construct group.Prenatal rat cortical neurons were cultured as described previouslyTotal RNA from post-mortem adult human cerebellum was treated with Turbo DNase (Thermo Fisher Scientific) according to the supplier’s protocol. 5′ RACE analysis was carried out on human cerebellar RNA using the GeneRacer Kit (Thermo Fisher Scientific) according to manufacturer’s protocol with primers outlined in Supplementary Tables 20 and a random hexamer primer mixture  according to the manufacturer’s protocol. A negative control (− RT) was also included where Superscript III reverse transcriptase was not added. After cDNA synthesis, PCR was performed in 20 μl using 3 units of Hot FirePol (Solis Biodyne) and primers listed in Supplementary Table For RT-PCR, cDNA was synthesized from human cerebellar RNA using 100 units of SuperScript III reverse transcriptase (Thermo Fisher Scientific) with oligo(dT)42 was used to locate potential TCF4 TSS-s. Both predicted TSS-s  and total counts of CAGE reads for the reverse strand (encoding for TCF4) were visualized in UCSC Genome Browser together with the EST-s from GenBank (accessed at 10.07.2020) in the area surrounding the TCF4 CTG TNR region . The FANTOM5 data can be accessed at https://fantom.gsc.riken.jp/5/datahub/hg19/reads/ctssTotalCounts.rev.bw.Cap Analysis of Gene Expression (CAGE) data from the Functional Annotation of the Mammalian Genome project phase 5 (FANTOM5)27 and SRP23860928) using prefetch tool (version 2.10.0) from the SRA toolkit. Reads in fastq format were extracted using fasterq-dump. Adapter and quality trimming was done using BBDuk (part of bbmap version 38.79) using the following parameters: ktrim = r k = 23 mink = 11 hdist = 1 tbo qtrim = lr trimq = 10 minlen = 100 (minlen = 85 for data from PRJNA524323). Reads were mapped to hg19 genome  using STAR aligner (version 2.7.3a) with default parameters. To increase sensitivity for unannotated splice junctions, splice junctions obtained from the 1st pass were combined (per dataset) and filtered as follows: junctions on mitochondrial DNA and non-canonical intron motifs were removed; only junctions supported by at least 6 reads in the whole dataset were kept. The filtered junctions were added to the 2nd pass mapping using STAR. Intron-spanning reads were quantified using FeatureCounts (version 2.0.0) with the following parameters: -p -B -C -s 2 -J. To count reads from TCF4 extended exons (exons 4c and 7bII), reads crossing a region 2 bp 5′ from the internal exon  were quantified using FeatureCounts and a custom-made saf file. Splice junctions in the TCF4 region showing less than 4 reads for the whole dataset were discarded, the rest of the splice junctions associated with TCF4 were manually curated and annotated according to Sepp et al.4. A custom R script was used to quantify the expression of different TCF4 splice variants. Reads crossing the indicated splice junctions were normalized using the number of all splice-junction crossing reads in the respective samples. Then, data summed by the Exon column (see Supplementary Table TCF4 internal exons and transcripts encoding different TCF4 protein isoforms. The annotated splice junction table for quantifying different TCF4 splice sites and transcripts encoding different isoforms is shown in Supplementary Table Raw RNA-seq data from corneal endothelium of FECD patients and controls (see Supplementary Table Supplementary Information 1.Supplementary Table S4.