Loxodonta africana) is classified as endangered by the International Union for Conservation of Nature (IUCN), in some isolated habitats in southern Africa, contraception is of major interest due to local overpopulation. GnRH vaccination has been promoted as a non-invasive contraceptive measure for population management of overabundant wildlife. We tested the efficacy of this treatment for fertility control in elephant bulls.Although the African elephant (® (1000 μg GnRH conjugate) intramuscularly after the first examination, followed by a booster six weeks later and thereafter every 5–7 months. In a retrospective study group , one examination was performed on bulls which had been treated with GnRH vaccine for 5–11 years.In total, 17 male African elephants that were treated with a GnRH vaccine were examined in two groups. In the prospective study group 1 , semen quality, the testes, seminal vesicles, ampullae and prostate, which were all measured by means of transrectal ultrasound, and faecal androgen metabolite concentrations were monitored over a three-year period. Each bull in the prospective study received 5 ml of Improvac6/ml, total motility: 0–90%), while after 5–8 injections, only 50% of these bulls produced ejaculates with a small number of immotile spermatozoa. The ejaculates of group 2 bulls (vaccinated >8 times) were devoid of spermatozoa. Faecal androgen metabolite concentrations measured in captive males decreased significantly after the fourth vaccination. None of the males entered musth during the treatment period.In all bulls of group 1, testicular and accessory sex gland sizes decreased significantly after the third vaccination. In six males examined prior to vaccination and again after more than five vaccinations, the testis size was reduced by 57.5%. Mean testicular height and length decreased from 13.3 ± 2.6 cm x 15.2 ± 2.8 cm at the beginning to 7.6 ± 2.1 cm x 10.2 ± 1.8 cm at the end of the study. Post pubertal bulls  examined prior to vaccination produced ejaculates with viable spermatozoa (volume: 8–175 ml, sperm concentration: 410-4000x10Our results showed a marked decrease in semen quality, testicle and secondary sex gland sizes following repeated GnRH vaccinations. After 2–4 years of continuous treatment every 5–7 months, the effects were similar to surgical castration. Gonadotrophin releasing hormone (GnRH) is synthesized in specialized neuroendocrine cells in the hypothalamus and transported to the anterior pituitary via the hypothalamic-pituitary portal system, where it induces synthesis and release of the gonadotrophic hormones, follicle stimulating hormone (FSH) and luteinizing hormone (LH) . Both goOdocoileus virginianus, [Bison bison, [Sus scrofa, [Loxodonta africana) and Asian elephant (Elephas maximus) bulls [Commercial GnRH vaccines have been designed for domestic animals and tested in both sexes of pigs , cattle inianus, ), bison n bison, ) and wil scrofa, ) with mos) bulls –14. The s) bulls . Indepens) bulls  and a fes) bulls  suggest s) bulls  and Afris) bulls  and aftes) bulls . Only ons) bulls  and noneAlthough African elephant populations are under pressure, in southern Africa fertility control is warranted as capacities in smaller, fenced game reserves are exceeded. Due to the intra-abdominal location of the testes, surgical castration is impractical and expensive in these large animals, especially when conducted in the wild. Laparoscopic vasectomy has also been performed, but requires specialized operators and expensive equipment and is not without risks for the animal . VasectoCaptive (n = 13) and wild (n = 4) African elephant bulls were used for the study and examined between March 2011 and April 2014 . In the The treatment protocol for both groups consisted of a primary immunization, followed by a first booster after 6 weeks, and thereafter boosters every 5–7 months. In group 1, animals were treated over the examination period of two to three years, in group 2 for 5–11 years. The vaccine was administered by deep intramuscular injection into the muscle mass of the hind leg. In captive elephants, this was mostly delivered by hand injection and occasionally with a dart gun. Wild bulls were injected by hand under anaesthesia (first injection) or remotely with a dart gun from a helicopter, in which case the gluteal muscle mass was targeted. Each immunization consisted of 5 ml Improvac®  containing 1000 μg GnRH-protein conjugate.In all but one captive bull procedures were carried out under standing sedation with medetomidine and butorphanol, as previously described . One capPrior to ultrasound examination and semen collection of standing or recumbent bulls, faeces were removed manually after which the rectum was flushed with water using a hosepipe. Trans-rectal ultrasound examinations were performed as previously described ,23. A poSix to ten second long ultrasound video recordings, each of the ampullae, prostate, seminal vesicles and testes, were made for retrospective analysis. Frozen images were used to measure the length and height of each organ using the largest dimensions in each case. The diameter was calculated as a mean of both values for statistical analyses. Since accessory sex glands are mostly asymmetrical or amorphous-shaped organs, the circumference was also measured by circumscribing the organ. The outlined 2-D images were then used to calculate the respective areas for statistical analyses. Since testes and accessory sex glands are paired organs, the means of the combined measurements were calculated.Two semen collection methods were initially used and then consistently applied in subsequent examinations of individual bulls. Semen collection of bulls under standing sedation was performed by trans-rectal, manual massage of the prostate and ampullae of the vas deferentia as previously described for trained  or sedatet al. [The method used for electro-ejaculation of one captive and four wild immobilised bulls was similar to the one described by Howard et al. . A portaet al. . In our ®)  version 4.1.0.0 or 5.4.0.0 at 50 frames per second. Chambered Leja® slides  with a defined volume (3 μl) and depth (20 μm) were loaded by micropipette for automated analysis. All disposables such as slides and pipette tips used were maintained at 37°C. Where sperm concentration was too high for automated analysis, the semen was diluted in Ham’s F10 culture medium . At least 500 spermatozoa were analyzed for motility with SCA for each elephant. The best quality semen fraction was used to report total motility, progressive motility, sperm concentration and sperm morphology for each bull. Semen smears were processed and stained with SpermBlue®  according to van der Horst & Maree [Total volume was recorded after adding all fractions together. Ejaculatory fractions of each bull were assessed individually for the presence of spermatozoa. An aliquot of each fraction was immediately assessed for total and progressive motility and semen smears were made. Sperm concentration and percentage motile sperm were determined using automated image analysis techniques. This analysis involved a Nikon Eclipse 50i microscope  with a temperature controlled stage at 37°C  and fitted with a Basler 312fc digital camera . Sperm concentration and motility were assessed using a 10x negative phase objective and analysed with the Sperm Class Analyser  and conducted at the Endocrine Research Laboratory at the Faculty of Veterinary Science, University of Pretoria.Faecal samples were collected within 6 hours of defecation from the 11 captive bulls twice monthly and one week prior to, during and one week after each examination. Faecal matter was thoroughly mixed during collection and stored at -20°C until further processing. Frozen faecal samples were lyophilized, pulverized, and put through a nylon sieve to separate course material . A weighFor the ultrasound related data, descriptive statistics were calculated, histograms were plotted, and the Anderson-Darling test for normality was performed  to assess the normality assumption. Data were transformed using the natural logarithm to improve the distributional form prior to statistical analysis. The number of GnRH vaccinations was transformed into an ordinal variable for statistical modelling based on the following criteria: no GnRH vaccinations, 2 vaccinations, 3 vaccinations, 4 vaccinations, and 5 or more vaccinations. Correlations between quantitative data were assessed by calculating Spearman’s rho. The effect of multiple GnRH vaccinations was estimated within a general linear modelling approach to adjust for the repeated sampling of individual elephants. Elephant age was dichotomized based on less than 20 years of age and height was dichotomized based on the median of the data (287 cm). A random effect term was included for elephant and a first order autoregressive correlation structure was used to adjust for repeated measurements. Models included a fixed effects for number of previous GnRH vaccinations, age, height at the shoulder (used to approximate body weight by formulas described previously), and time since first vaccination. Post-hoc pairwise comparisons were adjusted using the Bonferroni method. Statistical analyses were performed in commercially available software  and results interpreted at the 5% level of significance. Values are presented as mean ± standard deviation (SD), unless stated otherwise.For assessment of the faecal androgen metabolite concentration in group 1, one-way repeated measure ANOVA was applied.In this study, it was possible to monitor 11 bulls treated with GnRH (Improvac®) over a three-year period. Another six elephants  that had previously been treated with Improvac® more than eight times were examined once only .All bulls were healthy and in good body condition. Application of the vaccine by dart gun or hand-injection proved equally effective. Only four of the 84 (3.4%) Improvac® injections resulted in side-effects. These consisted of local swelling  and/or dDimensions of testes, seminal vesicles, ampullae and prostate at the beginning (0–2 vaccinations) and end of the study (≥5 vaccinations) are shown in Negative correlations between age and size-adjusted organ area at the time of examination and number of vaccinations were found for the testes  was visible in the ampullae of all mature animals . The fluPenis extrusion of elephants in standing sedation was sometimes spontaneous and probably as a result of the sedative drugs. If not, extrusion occurred during the cleaning of the rectum and trans-rectal massaging. In most cases, continuous trans-rectal massaging of the underlying organs resulted in the development of a fully extended penis. In two cases, urine dribbling was observed prior to massaging. When the urethra was stimulated in the correct area, however, urine dribbling ceased and a semen sample was obtained. Urine contamination of some fractions was recorded in four of 31 manual collections. One (bull # 5) of the seven males assessed prior to treatment was 8 years old and, according to the appearance of reproductive organs, low testosterone levels and absence of spermatozoa in the ejaculate, was assessed as prepubertal. This male was therefore not incorporated in the semen evaluation over time.In untreated males, transrectal massage prior to the first vaccination produced ejaculate volumes of 8–50 ml. Electro-ejaculation of the immobilised naïve wild bulls was highly successful and produced ejaculate volumes of 43 ml (bull #11) and 175 ml (bull #10) prior to vaccination. Electro-ejaculation of wild bulls on longstanding treatment  produced only drops of sperm-free seminal plasma.6/ml at this stage. Here, however, no sperm motility was recorded and 43% detached heads were noted. A consistent finding in all bulls was head-tail separation, which involved as much as 53–85% of spermatozoa after three vaccinations . The high variability in individual fAM level detected between untreated bulls can probably be attributed to the different age classes and social ranks. A significant reduction in fAM concentrations was seen after four vaccinations  after more than four vaccination in 50% of elephant bulls. The remaining bulls had only few abnormal and immotile spermatozoa present in their ejaculates. All these findings on sperm alteration are consistent with those in mature stallions immunized with a GnRH vaccine . BreedinCervus dama) resulted in abscess and/or granuloma formation in nearly all animals after the first immunization [Although other studies did not report side effects in elephants treated with GnRH vaccines –18, we nnization . Thus, sA very important question, particularly for some species or circumstances, is the reversibility of GnRH vaccines. We did not plan to test reversibility of the GnRH vaccine in the current study, but it certainly needs to be investigated. The loss of testosterone at a young age may lead to retarded development of the penis, testicles and accessory sex glands in elephants , 5. In aAn alerting finding was the ultrasonographic appearance of the testes following a number of GnRH vaccinations . The losIn summary, the complete absence of spermatozoa in ejaculates of long-term treated bulls, baseline fAM concentrations and a lack of sexual interest suggest a castration-like effect as a result of GnRH vaccination.Although our initial results show an impressive effect already within 1.5 years of vaccination with Improvac®, more research is needed to determine optimal treatment intervals, reversibility of the treatment, and effects in juvenile versus adult African elephant bulls. Furthermore, the effects on social, reproductive and hierarchical behaviours, possible demographic impacts and effects on bone metabolism need to be investigated.The GnRH vaccine Improvac®, originally developed for the pig industry, appears to have a potent effect on African elephant bulls. Regular boosters at 5–7 month intervals are sufficient to down-regulate testicular size, testosterone levels and spermatogenesis in male elephants. If elephants are treated continuously for more than 2–3 years (4–8 times), irreversible effects are possible. For the use as a contraceptive measure in wild elephant bulls, single-shot, long-acting vaccines would be more practical. On the other hand this may affect reversibility.


This retrospective, multicenter study aimed to reveal risk predictors for mortality in the intensive care unit (ICU) as well as survival after ICU discharge in patients with acute myeloid leukemia (AML) requiring treatment in the ICU.Multivariate analysis of data for 187 adults with AML treated in the ICU in one institution revealed the following as independent prognostic factors for death in the ICU: arterial oxygen partial pressure below 72 mmHg, active AML and systemic inflammatory response syndrome upon ICU admission, and need for hemodialysis and mechanical ventilation in the ICU. Based on these variables, we developed an ICU mortality score and validated the score in an independent cohort of 264 patients treated in the ICU in three additional tertiary hospitals. Compared with the Simplified Acute Physiology Score (SAPS) II, the Logistic Organ Dysfunction (LOD) score, and the Sequential Organ Failure Assessment (SOFA) score, our score yielded a better prediction of ICU mortality in the receiver operator characteristics (ROC) analysis . Factors predicting decreased survival after ICU discharge were as follows: relapse or refractory disease, previous allogeneic stem cell transplantation, time between hospital admission and ICU admission, time spent in ICU, impaired diuresis, Glasgow Coma Scale <8 and hematocrit of ≥25% at ICU admission. Based on these factors, an ICU survival score was created and used for risk stratification into three risk groups. This stratification discriminated distinct survival rates after ICU discharge.Our data emphasize that although individual risks differ widely depending on the patient and disease status, a substantial portion of critically ill patients with AML benefit from intensive care. AML is the most common type of acute leukemia in adults, accounting for approximately 2.8% of all cancer worldwide . WithoutThe decision to admit a patient to the ICU is often an ethical dilemma and is based on the individual clinician’s decision, which is loosely based on established scores  –20. BecaThe objective of this study was to establish and validate risk factors associated with mortality during and after ICU treatment based on a large and multicenter cohort and to establish potential risk scores.The AML in ICU score was established in a cohort of 187 adults with AML admitted to the ICU at the University Hospital of Muenster, representing all patients with a diagnosis of AML admitted to the ICU between 11/2004 and 09/2011. Data were collected retrospectively from patient records and follow-up physicians. Prior to analysis, the data were anonymized and de-identified. Approval for this investigation was obtained from the Ethics Board of Westfalian Wilhelms-University Muenster and the Physicians Chamber of Westfalia-Lippe, Germany (2015-688-f-S).Validation was performed on a cohort of 264 patients with AML admitted to the ICU at the University Hospital of Grosshadern in Munich, the University Hospital of Cologne and the Municipal Hospital of Augsburg  between 01/2004 and 02/2010.Intensive induction treatment consisted of any of the following: “7+3” ; “TAD” ; “HAM” ; and “S-HAM” . With the exception of dose-dense S-HAM induction, patients <60 years of age routinely received two induction courses, whereas patients aged 60+ received a second induction course only in case of persisting bone marrow blasts on day 15 after the start of treatment. Postremission treatment consisted of high-dose cytarabine in case of “7+3” or “7+GO” induction . Postremission treatment also consisted of TAD consolidation followed by prolonged monthly maintenance therapy in case of “TAD(-HAM)”, “HAM(-HAM)” or “S-HAM” induction or was followed by autologous SCT in younger patients. Details of the treatment protocols have been published elsewhere –23. AccoOnly disease status was included in the analysis. Owing to the broad heterogeneity of the applied protocols, a detailed analysis of chemotherapy regimen related to death in the ICU and survival after ICU discharge could not reasonably be performed. (Re)induction protocols are applied in active disease or at primary diagnosis, and consolidation regimens are administered in cases of complete remission. Owing to this strong correlation, information about chemotherapy protocols is already included in the variable disease status.Complete remission was defined as hematological recovery with at least 1,000 neutrophils per μl, at least 100,000 platelets per μl, and < 5% bone marrow blasts. ICU mortality was defined as death at any time during the course of the ICU stay. Overall survival after ICU discharge was survival from the day of ICU discharge until death from any cause, and censoring of patients known to be alive at the time of last follow-up was performed. The term mortality, when given as a percentage, was defined as the number of deaths per number of ICU stays observed.Arterial oxygen partial pressure (paO2) was evaluated at the time of ICU admission, irrespective of oxygen supply. Active AML at the time of ICU admission included 1, patients with primary diagnosed AML; 2, patients with relapsed AML before or during reinduction therapy or before evaluation of the remission status at the time of ICU admission; and 3, patients with persisting disease after induction or reinduction therapy at the last evaluation of disease status preceding ICU admission. Advanced AML at the time of ICU discharge was defined as refractory or relapsed AML status. Serve infections as reason for ICU admission required at least two of the following criteria: temperature >38°C (100.4°F) or <36°C (96.8°F), tachycardia (>90 bpm), tachypnea >20/min or clinically proved infectious complications . To avoid affections by the AML and/or associated therapy, leukocyte count was not considered. Days until ICU admission were the days between hospital admission and ICU admission. The Glasgow Coma Scale (GCS) was applied as previously described . The SimCorrelations with ICU mortality were evaluated using the Chi-square test for categorical data and the Mann-Whitney U test for continuous variables. Only variables with less than 10% missing values in the training cohort were considered. Missing values were replaced by the median value of the variable. Continuous variables were categorized where appropriate . VariablSurvival analyses were performed by Kaplan Meier estimates. Correlations with survival were evaluated by the log-rank test; parameters with p<0.1 in the log-rank test were evaluated in multivariate Cox regression analysis with stepwise backward selection and a threshold value of 0.05 for inclusion and 0.1 for exclusion. Unless otherwise stated, the significance level was alpha = 0.05 in all analyses.All statistical analyses were performed in cooperation with the Institute of Biostatistics and Clinical Research of the University Hospital of Muenster, Germany, and were computed with IBM SPSS Statistics for Windows, Version 22.0 .de novo versus secondary), time interval between hospital admission and ICU admission, mean arterial pressure at ICU admission, diuresis and GCS at the time of ICU admission were not available for the validation cohort.Patient characteristics are presented in An overview of all parameters selected for analysis and their classification is presented in Independent prognostic factors for ICU mortality in a multivariate model, displayed in Formula 1:Formula 2:Coding of parameters used in formula 1:Hypoxemia: paO2 <72 mmHg at admission = 1; paO2 ≥72 mmHg at admission = 0Active AML on ICU: relapsed, refractory, newly diagnosed AML = 1; remission = 0Severe infection: reason for ICU admission was infection with two of the following criteria  = 1; other reason for ICU admission = 0Hemodialysis: need for hemodialysis = 1; no need for hemodialysis = 0Mechanical ventilation: need for mechanical ventilation = 1; no need for mechanical ventilation = 0When applying our score to the validation group, predicted ICU mortality had an AUC in the ROC of 0.841 (95% CI: 0.784–0.897), compared with an AUC of 0.730 (95% CI: 0.661–0.799) for the SAPSII Score, an AUC of 0.773 (95% CI: 0.696–0.851) for the LOD score, and 0.783 for the SOFA score (95% CI: 0.714–0.864). In quanSeventy-nine patients (42%) in the training cohort survived their ICU stay. Formula 3:Coding of parameters used in formula 3:Advanced disease: relapsed or refractory = 1; newly diagnosed or in remission = 0alloSCT: Previous alloSCT = 1; no history of alloSCT = 0Days in hospital before ICU admission: Number of days between hospital admission and ICU admissionDays spent in ICU: Number of days between ICU admission and ICU dischargeDecreased urine production: urine production <1,000 ml/24 hours at ICU admission = 1; urine production >1,000 ml/24 hours at ICU admission = 0Decreased GCS: GCS <8 at ICU admission = 1; GCS ≥8 at ICU admission = 0Decreased hematocrit: hematocrit of <25% at ICU admission = 1; hematocrit of ≥25% at ICU admission = 0Stratifying the ICU survivors into three risk groups according to the risk score calculated using formula 3 revealed marked differences in survival after ICU discharge. Patients with the lowest risk  displayed one-year survival after ICU discharge of 100%. Patients with intermediate risk  exhibited 1-year survival of 82% (95% CI: 68–97%), and patients with the highest risk  exhibited 1-year survival of 42% (95% CI: 22–63%) .Applying the same stratification to the validation group (median follow-up of 1.4 years) revealed comparable differences in survival from the time of ICU discharge, although the survival rates were lower. One-year survival after ICU discharge was 69% (95% CI: 55–81%) in the 59 patients with the lowest risk, 51% (95% CI: 41–60%) in the group of 131 patients with intermediate risk, and 19% (95% CI: 4–33%) in the 42 patients with high risk .Despite encouraging survival rates of ICU survivors compared to non-ICU patients , assumedet al. published a report stating that neither SAPS II nor APACHE II are sufficiently accurate to be used in the routine management of cancer patients requiring ICU treatment [Sculier reatment . They evBased on the data for 451 patients with AML receiving available intensive care, the largest cohort of AML patients analyzed to date, we were able to specifically analyze prognosis in this defined patient population. Several risk predictors for ICU outcome as well as subsequent survival were identified, and we established a score predicting ICU mortality in patients with AML. This score outperformed the established SAPS II, LOD, and SOFA scores in the training and the validation cohort with respect to the area under the curve in the ROC analysis, regardless of hospital, treating physician, or treatment. Although the potential to discriminate was higher in the training cohort it was still evident in the validation cohort. The ability of our score to accurately predict ICU mortality in this independent cohort supports the reliability of the score. The results for patients with a low mortality risk may encourage clinicians to initiate or extend intensive care to AML patients. However, the decision to pursue ICU treatment for an AML patient requires an interdisciplinary approach that includes hematologists, intensive care physicians, and consideration of the patients’ wishes and expectations. Thus, such a decision can never be based solely on the results of a score.Originally all three scores, the SAPS II, LOD, and SOFA score, were generated on the basis of the “worst” values in the first 24-hour period after admission,28,29. TPrevious studies have defined single parameters predicting ICU or hospital mortality in cohorts including or comprising patients with AML, such as use of mechanical ventilation, low fibrinogen , use of The mortality rate in the ICU was 58% in the training cohort and 36% in the validation cohort. The reason for this discrepancy is unclear because the differences in the baseline characteristics  were notIn addition to predictors of ICU mortality, we also identified prognostic factors for ICU survival by AML patients. Not surprisingly, advanced disease status was a strong negative prognostic factor for survival, and impaired immune responses to pathogens, particularly in the early phase after allogeneic SCT, and severe acute and extensive chronic graft versus host disease are clearly associated with infectious complications .et al. reported that fewer days in the hospital before ICU admission was associated with improved hospital survival [Days spent in the hospital before ICU admission negatively influenced outcomes after ICU discharge, whereas days spent in the ICU before ICU discharge had an opposite prognostic influence on future survival. Azoulay survival . HoweverAlthough a low hematocrit value does have a positive influence on the prognosis of ICU patients , we idenELN low risk was significantly associated with survival after ICU discharge, whereas ELN high risk exhibited only borderline significance. However, in contrast to the intention of ELN risk classification, these cohorts do not represent homogeneous populations of patients with untreated, newly diagnosed disease. The significant influences of disease status and previous allotransplant on prognosis after ICU discharge suggest that ELN risk is diluted by disease status.Several limitations must be addressed. Our survival index distinguished three separate prognostic groups in the test as well as in the validation cohort, but patient survival was inferior in the validation cohort compared to the training cohort in every risk category. Missing values in the validation cohort  and imbalances with respect to the proportion of remission status and lower paO2 see  are possOur study indicates promising survival rates for patients with AML requiring intensive care treatment. Based on data from a large multicenter cohort, we identified and validated relevant risk predictors, which provided a basis for two scores distinguishing between survival differences both in the ICU as well as after ICU discharge. However, while these scores might aid the prognostication of patients with AML treated in the ICU, decisions about initiating or pursuing intensive treatment must not rely solely on the results of these scores.This study should encourage further prospective analyses.S1 Table(DOCX)Click here for additional data file.


Neurons arise in very specific regions of the neural tube, controlled by components of the Notch signalling pathway, proneural genes, and other bHLH transcription factors. How these specific neuronal areas in the brain are generated during development is just beginning to be elucidated. Notably, the critical role of proneural genes during differentiation of the neuronal populations that give rise to the early axon scaffold in the developing brain is not understood. The regulation of their downstream effectors remains poorly defined.This study provides the first overview of the spatiotemporal expression of proneural genes in the neuronal populations of the early axon scaffold in both chick and mouse. Overexpression studies and mutant mice have identified a number of specific neuronal genes that are targets of proneural transcription factors in these neuronal populations.Together, these results improve our understanding of the molecular mechanisms involved in differentiation of the first neuronal populations in the brain.The online version of this article (doi:10.1186/s13064-016-0077-7) contains supplementary material, which is available to authorized users. In the embryonic rostral brain, the first neurons differentiate in very specific domains and project axons to give rise to the early axon scaffold. This is an evolutionary conserved structure, formed from longitudinal, transversal and commissural axon tracts that act as a scaffold for the guidance of later axons , 57, 59.Tagln3), Chromogranin A (Chga) and Contactin 2 (Cntn2) were identified and introduced to a network of downstream proneural targets genes [Nhlh1 and Stathmin 2 (Stmn2), revealed interesting patterns overlapping with the first neuronal populations of the early axon scaffold in the developing chick brain [In all neuronal tissue, expression of specific neuronal transcription factors needs to be tightly controlled to ensure the correct patterning of neuronal populations both temporally and spatially . This pats genes . Analysick brain .Nhlh1, Tagln3, Chga, Cntn2 and Stmn2), which are likely to play an essential role in the differentiation of these neuronal populations.Identifying gene regulatory networks are essential for understanding the molecular cascades involved in subtype specification of neurons. Here, we describe the molecular cascade implicating Notch signalling, proneural genes and downstream targets at the level of the first neuronal populations that give rise to the early axon scaffold in both chick and mouse embryos. We identified several target genes that are known neuronal markers  eggs were obtained from E.A.R.L. Les Bruyères (France). Eggs were incubated in a humidified incubator at 38 °C until the required developmental stages described according to Hamburger and Hamilton [Fertilised chicken  added at 0.2% to facilitate visualisation of the DNA solution. The DNA solution was injected into the rostral neural tube of chick embryos at Hamburger and Hamilton stage (HH) 10-11, using a nanoinjector (Drummond Scientific). Electrodes were placed either side of the neural tube, targeting the mesencephalon. Five pulses of 15 V/50 ms were applied, using a square wave pulse electroporator . After electroporation, the eggs were sealed and incubated for a further 24 h.The pCAGGS-IRES-nuclearGFP (pCIG) plasmid was used for control experiments. The overexpression constructs for rat  plasmid . The expin situ hybridisation or immunohistochemistry. Anti-sense probes were generated either from plasmids cloned as previously described [in situ hybridisation has been previously described [All embryos were fixed in 4% PFA/PBS at 4 °C overnight, rinsed and processed for whole-mount RNA escribed  or plasmescribed . For douescribed .Nhlh1, Tagln3, Chga, Cntn2 and Stmn2 were analysed between E8.5 and E10.5   and trochlear (IV) motor neurons also differentiated at the ventral midline. As the nucleus of the oculomotor nerve (nIII) was not easily identifiable from the nMLF and nTPC at E10.5. Therefore,  neurons  to distiIII Fig. . Tagln3,IV) Fig. .Chga was not expressed along the dorsal midline of the mesencephalon in the mouse . Hes5 was downregulated, but expression was not completely lost throughout the RBPj mutant brain . There was also expression along the dorsal and ventral rhombencephalon, the locus coeruleus (LC), the pretectum (Ptec) and the prethalamus (Pth) . Although Ascl1 expression was upregulated, the neuronal populations remained identifiable. This showed that Notch signalling negatively regulates neurogenesis and that lateral inhibition involving Ascl1 was implicated in the differentiation of the neuronal populations of the early axon scaffold tracts in mouse brain.In the control embryos, TPC Fig. '; n = 10th) Fig. . ExpressAscl1 expression in some regions of these RBPj mutant brains, such as, the Pth and nTPC. As Ascl1 should be expressed in these populations already, this suggested there was already a developmental delay in these mutant embryos . Both genes were upregulated throughout the neuronal populations that give rise to the early axon scaffold tracts, which genetically confirmed expression of these genes was regulated by the Notch pathway  and Neurog3 was only expressed in the developing hypothalamus [As proneural genes are essential transcription factors for neurogenesis , we wantcl1 Fig. . Other pthalamus , 52.Fig.Ascl1 was first expressed in the brain from E8 along the dorsal midline of the mesencephalon [Neurog1 was also first expressed along the dorsal midline of the mesencephalon, slightly later at E8.5 , and then at HH14 the nTPC . These results confirmed that the Ascl1 construct used here had the ability to induce neurogenesis in cells that were not yet destined to become neurons. Eventually neurons in this region will become tectobular forming the ventral commissure [Ascl1, however, their ability to follow the correct path was affected.Previously, upregulation of  neurons , 15, 24.ons Fig. . After 2mmissure . While HAscl1 and Neurog2 and analysed the effect on expression of the neuronal markers Nhlh1, Tagln3, Chga and Stmn2.To establish a possible specificity of the proneural gene for one of the neuronal markers, we electroporated Nhlh1, Tagln3, Chga and Stmn2 was observed in cells expressing the control plasmid and each gene was normally expressed within the early neuronal populations  or mouse Neurog2 (minimum n = 3 for each gene) were overexpressed, cells that ectopically expressed the proneural gene, also expressed the markers Nhlh1  or Sox10  were upregulated. Together, these results suggested that both ASCL1 and NEUROG2 were able to regulate the same neuron specific genes tested here.In embryos electroporated with the pCIG control plasmid (n ≥ 3), no ectopic expression of ons Fig. , e, i, mlh1 Fig. , d, Taglln3 Fig. , Chga . Surprisingly, Ascl1 null mutant embryos still expressed Tagln3 in all of the neuronal populations at E10 . Chga expression was also downregulated in the cda and in the LC . Indeed, while proneural genes are expressed in complementary regions, there are numerous studies that show they able to compensate for each other [Neurog2 has the capacity to rescue the development of Ascl1-dependent neurons [Ascl1 null mutant embryos, the expression of Tagln3 is not downregulated in neuronal populations expressing more than one proneural gene. This suggests there is compensation of other proneural genes in these populations. However, Tagln3 expression is not downregulated in the nVI where Ascl1 is the exclusively expressed proneural gene is unexpected. Other known proneural genes, Neurog1, Neurog2, Neurog3 and Atoh1 seem to be not expressed in the nVI. What is regulating Tagln3 here is yet to be determined.This study shows that regulating expression of the target genes analysed here is not specific to either the overexpression of ch other , 37, 45. neurons , 37. It Ascl1 and Neurog2 genes should be informative in answering this question.The highly conserved expression patterns of the proneural genes in the early ventral forebrain argue against a model of stochastic induction. An important selection pressure may exist to maintain this complementary proneural gene expression within the chick and mouse brains. We still have to determine why these neuronal target genes are expressed in some populations but not others, especially if these genes can be regulated by any proneural gene. It has been demonstrated, that these proneural genes are not always functionally equivalent and this capacity appears to vary in different regions of the nervous system . How theQuestions still remain, including what is controlling the specification of the individual neuronal populations that give rise to the early axon scaffold tracts and other early populations.Sax1 could play a role in specifying the nMLF subtypes [Sax1 results in an enlargement of the nMLF area [If proneural genes can regulate the same target genes, we still need to determine the specific genes or combination of genes (in a cascade) that regulate identity of each individual neuronal population of the early axon scaffold. Although a single proneural gene is sufficient to induce neuronal features, the additional expression of other factors is necessary to generate specific identity, for example, in fibroblasts , 54. Thusubtypes , as gainInitially, a critical step is the establishment of morphogen gradients controlling the distinct sets of transcription factors resulting in the establishment of progenitor domains . Such a The organisation of the brain is more complex and harbours a greater diversity of neurons compared with the spinal cord. However, to our knowledge, no study investigating the specification of the neuronal populations that give rise to the early axon scaffold in any mutant mouse models has been done. Our present study gives essential tools to explore more accurately the formation of these neuronal populations in mutant models. This will provide a better understanding of how these early neurons differentiate in a specific territory with a specific identity.


Microfabrication offers opportunities to study surface concepts focused to reduce bacterial adhesion on implants using human minimally invasive rapid screening (hMIRS). Wide information is available about cell/biomaterial interactions using eukaryotic and prokaryotic cells on surfaces of dental materials with different topographies, but studies using human being are still limited.  To evaluate a synergy of microfabrication and hMIRS to study the bacterial adhesion on micropatterned surfaces for dental materials.  Micropatterned and flat surfaces on biomedical PDMS disks were produced by soft lithography. The hMIRS approach was used to evaluate the total oral bacterial adhesion on PDMS surfaces placed in the oral cavity of five volunteers . After 24 h, the disks were analyzed using MTT assay and light microscopy. µm. Overall, bacterial adhesion did not significantly differ between the flat and micropatterned surfaces. However, individual analysis of two subjects showed greater bacterial adhesion on the micropatterned surfaces than on the flat surfaces.  In the present pilot study, microwell structures were microfabricated on the PDMS surface via soft lithography with a spacing of 5  Microfabrication and hMIRS might be implemented to study the cell/biomaterial interactions for dental materials. The formation of oral biofilms on natural materials  and restorative biomaterials promotes the development of diseases, such as dental caries, periodontitis, and peri-implantitis , 2. MicrThe microenvironment plays a decisive role in the formation of the oral biofilm on dental materials due to direct or indirect interactions . FactorsSoft lithography allows the production of random or ordered surfaces in a controlled manner. Such surfaces modulate the cellular response , specifiThe aim of the present study was to evaluate a synergy of microfabrication and hMIRS to study the bacterial adhesion on micropatterned surfaces for dental materials.This pilot study had approval from the ethical committee of the Universidad Cooperativa de Colombia, Medellín, Colombia. Five subjects between 18 and 35 years of age were invited, who met the following inclusion criteria: no antibiotic treatment during the 3 months prior to the test, no use of orthodontic appliances, nonsmoker, non-dental student, and have signed informed consent.µm diameter, 5 µm height, and 5 µm spacing) needed for the biofilm study [Microfabricated surfaces were obtained using biomedical PDMS (FDA approved) processed by soft lithography technology, which included the manufacturing of a master model by UV photolithography for two different purposes: (a) to test pattern transferability using raised and recessed features of different sizes, and (b) to fabricate the test geometries . The disks were incubated in a solution of 3--2,5-diphenyltetrazolium bromide  at 0.5 mg/ml in saline solution for 2 hours at 37°C. Quantification of the area covered by the biofilm was performed on 6 random micrographs obtained by optical microscopy through an ad hoc digital image-processing strategy that included binarization, edge detection, segmentation, and pixel counting using ImageJ 1.51 g , 18.A comparison of the area covered by biofilm was performed for the two evaluated surfaces with a nonparametric Wilcoxon rank-sum test blocked by the subject using R software.µm.μm well structure  is observed with no defects.p=0.06). Patient-to-patient comparison between the micropatterned surfaces and the flat surfaces did not show statistically significant differences for subjects 2, 3, and 4  and pillars (168 nm diameter and 100 nm high) and found that increasing the height of the topography reduced epithelial cell adhesion. In addition, cells on pillars had a smaller area of covering compared to flat surfaces.Andersson et al.  studied Dalby et al.  evaluateStaphylococcus aureus when compared to smooth surfaces. Their results showed that the topography inhibited the development of biofilm. Such response was attributed to the fact that the protruding features of the surfaces provide a physical barrier that prevents the expansion of small colonies of bacteria. Hochbaum and Aizenberg demonstrated bacterial ordering and orientated attachment on the single-cell level induced by nanometer scale periodic surface features [μm or an increase in the free energy of the surface will facilitate the formation of biofilms in restorative materials.In contrast, bacterial studies on micropatterned surfaces are limited. Chung et al.  developefeatures . The posfeatures  concludeμm and hMIRS in terms of limited contact duration (24 h) and in contact with uncompromised oral mucosa. General comparisons showed no statistically significant difference in bacterial adhesion between flat and microfabricated surfaces on PDMS. These results might be explained by antiadhesive barrier as a consequence of high hydrophobicity in the biomedical PDMS used, which could be reduced with increased exposure time, but this would be opposite to the rapid screening model described. Modified Essix retainer was used for a period of 24 hours, which matched the in vivo human studies reported in the literature [μm, values that are similar to those reported in 24-hour studies [The present pilot study used a surface model of microwells with a diameter, depth, and interspacing of 5 terature , 29, 30.terature  evaluate studies , 29, 30.In the present pilot study, the sample size used was similar to that in in vivo human studies, which had sample sizes ranging from 5 to 10 subjects , 29, 30.


Eurytemora affinis. Failure to recognize the high frequency of these donor sites compromised the modeling of genes in this newly sequenced genome, including 10 conserved ionotropic glutamate receptor (GluR) family genes curated herein. These introns appear to have been acquired recently, along with many additional idiosyncratic introns. Their high frequency implies the evolution of modified intron donor splice site recognition in this copepod.The noncanonical 5′ intron donor splice sites GA and GG are exceedingly rare in described eukaryotic genomes; however, they are present in ∼12% of introns in the genome of the copepod  The canonical sequence for 5′ intron donor splice sites in eukaryotes has an obligate G to start the intron followed by U, or occasionally C, as part of a larger consensus splice site sequence, with DNA sequence AG/GTAAGT . For exaEurytemora affinis, one of the first copepod genomes to be sequenced. Among others, the authors reported nine Ionotropic Receptor (IR) genes and five members of the ionotropic GluR family, from which the IRs evolved . RNAseq reads spanning each intron were either obtained from the i5k genome browser if there was RNAseq mapped across an intron, or from the Short Read Archive (SRA) at the National Center for Biotechnology Information (NCBI) using BLASTN searches with flanking exon sequences as queries and default parameters. Exons missing in assembly gaps were recreated using raw genome reads from the SRA, using the RNAseq reads obtained above as queries. Models were compared with the protein sequences reported by e.g., EaffNMDAR2-1 comp31752_c0_seq1 CUFF.1933.3). They also commonly span both GA and GG donor introns as well as misassembled exons and exons missing from the assembly . Sequence logos for exon–intron junctions were built at http://weblogo.berkeley.edu/logo.cgi.Gene models were built manually in the text editor TEXTWRANGLER using genomic sequences from the assembly published in File S1, as are the nucleotide sequences surrounding the 36 noncanonical intron 5′ splice sites.The sequences of the 10 focus proteins are presented in IR8a, 21a, 25a, 76b, and 93a) and the five GluR members  were successfully extended by employing RNAseq and genome reads to full-length genes (sequences available in File S1). Full-length status was confirmed by comparison with orthologs from other insects , and a tick and mite , because their mapping algorithm would not accept such unusual noncanonical splice donors, and the genes were only partially modeled by the MAKER pipeline that the authors employed for similar reasons, in addition to misassembled and missing exons. These are all long genes (19–123 kb) with large numbers of mostly short exons (19–47), providing a reasonably large sample of 297 introns . The sequences of these 36 noncanonical donors are provided in File S1, and sequence logos of them and the canonical GT and rare GC donors are shown in The five conserved members of the IR family , indicating that this phenomenon is likely to be genome-wide. These 26 genes, while not a random sample, contain 623 introns interrupting their coding sequences, of which 54 have GA donors and 10 have GG donors , frequencies comparable with those of the 10 focus genes above. GA and GG donors have not been reported at anything like 10–12% of introns in any other eukaryote genome. Their recognition will greatly improve gene modeling in this copepod genome, but would require acceptance of only G, instead of GY, as the obligate 5′ intron donor splice site nucleotide.Once this pattern of absence of spliced RNAseq reads in the genome browser and likely GA or GG donor sites was recognized, many of these introns could be seen in a wide variety of large genes with deep RNAseq coverage in the genome browser. Full-length models were built in the Apollo browser for 26 such large genes containing GA and GG donors and encoding a wide variety of proteins (https://www.hgsc.bcm.edu/arthropods/i5k) has also sequenced the genome of another copepod, Tigriopus californicus (available in the i5k Workspace@NAL genome browser), and a genome sequence for the related T. kingsejongensis has been published  and D. pulex (e.g., Eurytemora, along with many additional idiosyncratic introns with canonical donors. For example, of the 33 introns in IR25a in E. affinis, nine are shared with other arthropods and the remaining 24, including the six GA or GG donors, are idiosyncratic to it. It appears that this copepod underwent an explosion of intron gains, including those with noncanonical donors.The i5k pilot consortium (Alternative splicing of the GA donor sites in the vertebrate fibroblast growth factor receptor 1–3 genes is a complicated process involving a nearby consensus sequence (www.g3journal.org/lookup/suppl/doi:10.1534/g3.117.300189/-/DC1.Supplemental material is available online at Click here for additional data file.Click here for additional data file.Click here for additional data file.


Chinese cabbage buds were soaked with Ethyl methanesulfonate (EMS) to induce mutagenesis. The influence of different EMS concentrations and treatment durations on microspore development, embryo production rate and seedling rate were evaluated in five Chinese cabbage genotypes. Mutations in four color-related genes were identified using high resolution melting (HRM) curves of their PCR products. The greatest embryo production and seedling rates were observed when buds were treated with 0.03 to 0.1% EMS for 5 to 10 min, while EMS concentrations greater than 0.1% were lethal to the microspores. In total, 142 mutants with distinct variations in leaf shape, leaf color, corolla size, flower color, bolting time and downy mildew resistance were identified from 475 microspore culture derived Doubled Haploids. Our results demonstrate that microspore derived Doubled Haploids from EMS soaked buds represents an efficient approach to rapidly generate homozygous Chinese cabbage mutants. Naturally occurring mutations represent a major force driving evolution; however, they occur at an extremely slow rate. Artificially induced genetic variations represent important supplementary variation in plant breeding programs complementary to sources from natural origins . TreatinArabidopsis thaliana in 2000 (Targeting Induced Loci Lesions in Genomes (TILLING) is a reverse genetic approach that allows directed identification of mutations in specific genes. Since TILLING was originally developed in  in 2000 , it has  in 2000 , wheat  is an important vegetable crop in Asia, with numerous uses in fresh and processed food products and with a high nutritional value  generated by treatment with EMS and ethyl nitrosourea  were grown in a greenhouse on an experimental farm at Hebei Agricultural University in Baoding, China in January 2012. After 2 months of vernalization at low temperatures (3–10°C), six seedlings of each genotype were transplanted in soil and cultivated normally.Inflorescences of each genotype were collected after the first one to three flowers had opened. Flower buds ranging from 2.0 to 2.5 mm in length with middle uninucleate or early binucleate stage microspores were selected . Approxi5 mL-1 in NLN13 medium containing active carbon at a concentration of 0.05 to 0.1 mg/mL. The suspension was poured into Petri dishes (60 mm diameter) for culturing (3 mL/dish). The cultures were incubated at 30°C for 24 h in the dark and then transferred to 25°C in the dark.Microspores were cultured according to the protocol described by During microspore culturing, the embryonic development and regeneration were evaluated using an inverted microscope (Olympus CK40). Swollen cells were observed under a 10X ocular after exposure to a 24-h heat shock. Dividing cells and cell clusters were measured under a 10X ocular following incubation in the dark for 2 to 3 days. After an additional incubation in the dark for 7 to 14 days, the shapes of the embryo were examined under a 10X ocular. The total number of embryos for each genotype was calculated as the embryo production (average rate of embryo production = embryo number/buds number).After 20 to 25 days of culture, embryos were counted for each genotype. The cotyledonary embryos were gradually introduced to light over 48 to 72 h. Next, embryos with green cotyledons were rooted on 1/2 MS medium. The cultures were grown in a growth chamber and maintained at 25°C using a photoperiod of 14 h light and 10 h darkness. Eight weeks after the embryos were transferred to regeneration medium, plantlets with 5 to 6 true leaves were transplanted to soil in the greenhouse. The seedling rate of the different genotypes was counted (average seedling rate = plantlet number/embryo number).Compared with wild type (at least 10 plants of each wild type), phenotypic variations including leaf shape, leaf color, corolla size, flower color, bolting resistance during the vegetative growth period  and reproductive period were investigated. Leaf shape was recorded as long ellipse, long inverted ellipse, broad ellipse, close round and round at vegetative stage. Leaf color was scored by eye as green, yellow-green, light yellow and light white at rosette, heading and reproductive stage. At reproductive stage, flower color was scored by eye as white and yellowish color, in which at least 10 flowers of each plant were phenotyped. Corolla size was measured by ruler and compared with wild types. Bolting time was recorded when the first flower bud appeared. Downy mildew tolerance was measured after touch inoculation by contaminated leaves. The downy mildew symptoms were evaluated when 2–3 leaves were contaminated.Ploidy levels  were identified by flow cytometry.Sixteen primers were designed using Primer 5 to amplify 100 to 400 bp regions within four genes related to the flower color pathway (Supplementary Table -1. LightScanner software (Version 2.0) was used for data analysis according to Following PCR, each sample was transferred to a 96-well LightScanner System  and overlaid with mineral oil. The samples were heated from 70°C up to 96°C using a ramp rate of 0.10°C sThe genes were amplified from genomic DNA of the HRM-identified mutants and wild type plants 85-1) using on one side primers used for HRM screening, combined with opposite strand primers to obtain a fragment of approximately 800 bp . After culturing in the dark for 2 to 3 days, the swollen microspores initiated the first division . Cell clusters  appeared after 5 days of culture. After 7 to 14 days, visible embryos with different shapes  were observed. Cotyledon embryos  appeared after 16 to 20 days of culture.The cytology of microspore embryogenesis is illustrated in Figure 1J), however, small cotyledonal embryos appeared following 24 to 26 days of culture. When cultured in the light, some of the cotyledonal embryos did not become green but turned brown and died , producing albino seedlings . With increasing EMS concentration and duration of exposure in all genotypes, the number of embryos produced clearly decreased .In general, embryo development was delayed after EMS treatment and spherical embryo’s were observed after 10 days of culture with slow growth. Development of most globular embryos was halted , 15 plants with flower color variation , one plant with tolerance to downy mildew , six plants with corolla size variation and eight plants with bolting time variation were observed. Leaf shape of the wild type ‘A03’ was ellipse, while the blade tip of mutants was slightly pointed; flower color was changed from white to yellowish.In total, 475 regenerated plants were obtained from EMS treated buds of the five genotypes (0.10% for 5 min). Some typical phenotypic variations among the 142 diploid mutants obtained by EMS treatment are shown in To demonstrate that HRM is suitable for efficient mutant identification in the TILLING lines, four target genes related to flower color formation were selected for the HRM analyses . Seven out of the 142 plants were considered to possess mutations in any of the four flower color genes, including five diploid mutants, one haploid mutant and one tetraploid plant.HRM analysis revealed individual microspore derived plants with mutations in any of these four genes (Table 3). Compared to wild type, all of the five diploid mutant plants possessed mutations, in the sequenced genes including six single nucleotide polymorphisms, one deletion (one base) and one insertion mutant. Four of these diploid mutant plants displayed changes in flower color, among which three  turned yellow at the late stage and one (11-6) was yellow at all growth stages, confirming these mutations.PCR amplification of the putative mutant genes and DNA sequencing were used to confirm the sequence variations of the five diploid mutants identified by HRM  and used to screen pools of DNA samples from 8 plants using HRM technology, resulting in 83 point mutations. These results demonstrate that HRM is also a high-throughput technique for screening larger mutant libraries induced by EMS.Using HRM screening, five mutants with flower color were obtained from the microspore culture plantlets derived from EMS bud mutagenesis. In combination with validation of mutants by direct sequencing of PCR fragments, HRM was shown to be useful for EMS mutant screening as a low cost, easy to conduct, high throughput method. The traditional platform of Conformation Sensitive Capillary Electrophoresis endonuclease-Li-Cor , despiteB. napus . In B. r studies , one ChiIn summary, in the present study, we provide evidence that microsporogenesis from buds of Chinese cabbage soaked in an appropriate concentration of EMS is an efficient way to generate mutantions which can be identified by high-throughput HRM screening.The study was approved by Hebei Agricultural University, China. All provided written informed consent.YL got mutants by EMS treatment and contributed to writing; SD and AG did microspore culture and help in writing; ML, YW, SL, YZ, and SW did HRM and PCR, and helped in the discussion and drafted the manuscript; SX, XC, and XL did field experiment and performed the analysis; GB helped writing and language editing; JZ and SS designed the study and supervised the whole experiment and help in writing.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.


C3 gene variants, in two uveitis entities. Eight haplotype-tagging SNPs of C3 gene were genotyped in 141 anterior uveitis (AU), 158 non-infectious intermediate and posterior uveitis (NIPU) and 293 controls. The results showed that none of the tagging SNPs had a significant association with uveitis (P > 0.05), either in the global uveitis or subtypes. Although rs428453 showed a nominal association with NIPU subtype in the recessive model (P = 0.042), the P value could not withstand the Bonferroni correction (Pcorr > 0.05). Stratification analyses according to HLA-B27 status and correlation analysis still did not find any significant interactions or genetic markers regarding AU. Logistic regression analysis also revealed no gender-related epistatic effects of C3 on uveitis. Two haplotype blocks were defined across the C3 locus but neither of them was significantly associated with uveitis or subtypes. This study shows no significant association of the C3 gene with uveitis, suggesting C3 confers either no or limited risk for uveitis susceptibility.Uveitis is a devastating ocular disease that causes blindness. Our previous studies have achieved great advancements in depicting the genetic profiles of uveitis regarding complement pathway genes. This study aimed to provide additional insights into this interest by testing the “central” factor of the complement system,  Uveitis can be frequently classified into anterior uveitis (AU), intermediate uveitis (IU), posterior uveitis (PU), and panuveitis according to the anatomical location of the inflammation3. AU, which refers to inflammation of the iris and ciliary body, is the most common form found in clinics. Although less common than AU, non-infectious intermediate and posterior uveitis (NIPU) typically is either idiopathic and comprises many well-defined uveitic ocular conditions or associated with systemic underlying autoimmune disorders, including Vogt–Koyanagi–Harada disease (VKH), Behçet’s disease (BD), and sarcoidosis4.Uveitis is a group of heterogeneous ocular inflammatory diseases with complex phenotypes, which is considered as a substantial visual impairment as well as an important socio-economic problem, being the fourth cause of blindness worldwide5. Moreover, genetic factors in the initiation and development of uveitis have been recognized for a few decades11.Although the exact pathogenesis of uveitis remains unclear, it is generally accepted as an inflammatory condition and mainly mediated by various endogenous immunological mechanisms12. Notably, in recent years, accumulating studies have provided increasing evidence that complement is involved in the pathogenesis of uveitis. These studies revealed that activation of the complement system is critical for the development of experimental autoimmune anterior uveitis (EAAU), conversely, depletion of the host’s complement system could result in complete inhibition of EAAU14. In addition, several key components and regulators in the complement system have been implicated in the development of uveitis models and other autoimmune diseases. There is a well-established concept that most immune-related disorders share a certain percentage of their genetic component, implying that some pathogenesis may be influenced by common pathways15. Evidence from the above studies led us to explore the genetic impact of complement genes on uveitis susceptibility. In our lab, several complement pathway genes have been extensively investigated in two different uveitis entities in our study cohort, AU and NIPU. These genes included complement factor H (CFH), complement factor B (CFB), complement factor I (CFI), Component 1 inhibitor (C1INH), and complement component 5 (C5)23.The complement system is a key component of innate immunity and plays an important role in modulating various immune and inflammatory responses. The activation of the complement system occurs along three routes - the classical, alternative, and lectin pathwaysCFH polymorphisms for the first time. CFH-rs1065489 TT genotype was identified as a clinical marker associated with higher uveitis recurrent frequency while interactions with human leukocyte antigens (HLA)-B27 status was also observed21. Interestingly, similar associations of CFH as described in AU were also found with NIPU patients in our study cohort, suggesting that these two uveitis entities shared general genetic background although presenting different clinical phenotypes19. We further demonstrated that genetic variants of CFB and CFI, in the same complement alternative pathway, are risk factors for both AU and NIPU patients. Moreover, a significant joint-effect among these genes, as well as genotype and phenotype correlations was observed23. In addition, parallel studies also demonstrated that genetic variants involved in the alternative pathway, CFH and CFB, as well as C5, considered as the “downstream” complement regulator, were also associated with type 2 diabetic retinopathy (T2DM), viewed from the perspective of inflammation22. These results further consolidate the concept that these different immune-mediated diseases may be influenced by common pathways and shared many genetic similarities. Apart from this, we also investigated C1INH gene, with a view to elucidating the involvement of the classic pathway in uveitis pathogenesis. The results showed no significant associations with either AU or NIPU patients. Nevertheless, previous reports from our laboratory have successfully established a genetic profile of complement pathway genes in uveitis susceptibility.CFH is a key regulator involved in the complement alternative pathway, and we identified a gender-specific association between AU and C3 has been shown to ameliorate the incidence and severity of EAU14. Additionally, variations in the C3 gene have been associated with several inflammatory diseases, such as age-related macular degeneration (AMD), polypoidal choroidal vasculopathy (PCV), systemic lupus erythematosus (SLE), and inflammatory bowel disease26. So far however, little is known about the genetic profile of C3 in uveitis. Together with our previous studies and others, we herein aimed to explore whether C3 gene variants are involved in the genetic predisposition to uveitis.Complement component 3 (C3) is the “central” component of the complement cascade and is involved in all three pathways. Genetic deficiency of The study protocol was approved by Ethnic Committee on Human Research, Harbin Medical University. All the procedures were conducted according to the tenets of the Declaration of Helsinki. Informed consent was obtained from all study subjects after an explanation of the nature of the study.A total of 299 uveitis patients and 293 control subjects aged ≥55 years without major eye diseases or any systemic immune-related disorders were recruited. The patients were given detailed clinical and ophthalmic assessments, including ocular tonometry, corrected visual acuity, slit-lamp microscopy, and fundus examinations. Clinical information and demographic conditions of the patients were also documented.27. All AU patients were recruited during the active phase of uveitis and followed for at least two years after recruitment. The intermediate and posterior uveitis comprise a group of ocular disorders. Considerring that they may share an underlying immune etiology, we combined IU and PU to investigate the genetic impact of complement factors on the whole uveitis susceptibility. Patients were categorized into three specific subtypes: IU, VKH and Behçet’s disease. All IU patients had IU in isolation without posterior uveitis or panuveitis, while VKH and Behçet’s disease patients had either panuveitis or posterior uveitis. Patients with uveitis secondary to ocular or systemic infections were excluded from the study.The definition of AU was based on the Standardization Uveitis Nomenclature (SUN) classificationC3 region were obtained from HapMap Project database for the Han Chinese population (http://hapmap.ncbi.nlm.nih.gov/). Eight SNPs were selected by the tagger-pairwise method with R square and MAF values greater than 0.80 and 0.10 respectively. This set of 8 SNPs captured 96% of alleles in the C3 locus with MAF larger than 0.1 and a mean r2 of 0.97. All SNPs were genotyped by TaqMan SNP Genotyping Assays  in the Light Cycler 480 Genotyping Master  according to manufacturers’ protocols. All PCR amplifications were performed with the following thermal cycling conditions: 95 °C for 10 minutes followed by 40 cycles of 92 °C for 15 seconds, and 62 °C for 1.5 minutes. The HLA-B27 allele was detected by nested polymerase chain reaction (nPCR).Haplotype tagging SNPs across χ2 test for genotype frequencies of all SNPs in the control group. Allelic or genotype distribution of each SNP was evaluated using the chi-square test or Fisher exact test . Dominant and recessive models were applied to investigate the disease association with regard to the minor allele. The odds ratio (OR) and corresponding 95% confidence interval (CI) were also estimated. Pairwise linkage disequilibrium  and EM-based haplotype association analysis were performed by Haploview (ver. 4.2). Logistic regression analysis was performed to adjust the effect of SNPs with gender. We stratified the study subjects according to subtype, gender and performed association analysis of the SNPs in each gender stratum, P < 0.05 was considered statistically significant. P values were corrected by Bonferroni test for multiple comparisons , or permutation test in Haploview software.Hardy-Weinberg Equilibrium (HWE) was tested by P = 0.097) compared with controls. Therefore, gender was adjusted in the following association analysis by using logistic regression. Among AU patients, 55 (39.0%) were HLA-B27-positive and 86 (61.0%) were HLA-B27-negative.In our study, a total of 299 uveitis patients were recruited; which were grouped into AU 141, 47.2%) and NIPU  subtypes listed below. NIPU, as a mixed disease entity, is comprised of 51 (32.3%) VKH, 45 (28.5%) IU and 62 (39.2%) BD. The details of clinical subtypes, age, and sex distribution in the uveitis group and controls are shown in Table 41, 47.2%P = 0.042), which was obvious in the female subgroup (P = 0.005). Among NIPU patients, the mean age of VKH was significantly higher than that of IU in general and male subgroups in particular . The mean age of the control individuals was significantly greater than that of all uveitis patients as expected . This is because we purposely recruited subjects older than 55 as control, so as to largely reduce the confounding effects from younger subjects.The mean age of the AU was significantly greater than that in NIPU patients (P > 0.05). The allelic frequencies of all C3 variants were generally closer, and not significantly different among groups, suggesting that none of the 8 SNPs had allelic association with either global uveitis or its subtypes  or CFB rs1048709 (data not shown). Furthermore, the logistic regression analysis revealed that none of the C3 variants were significantly associated with uveitis and its two sub-clinical entities after being adjusted for gender and SNP-gender interaction . In addition, gender independence and SNP-gender effects did not provide further information  cells, age of onset, and the presence of posterior synechiae (PS) and keratic precipitates (KP), was also applied. However, we still did not find any associations of is Table  nor any PU Table .Table 5CC3 locus by using these 8 SNPs, which defined two haplotype blocks in total uveitis entity and its two subtypes . To the best of our knowledge, this is the first study examining the association of C3 polymorphisms in two different clinical uveitis entities but with similar genetic background, AU and NIPU. Our results demonstrated that there was no significant association of the C3 gene with uveitis.Based on the crucial role innate immune mediator C3 plays in EAAU and EAU models, we explored the potential association of the 28. The genetic impact of several complement pathway genes on susceptibility to uveitis was extensively investigated. Our results provided novel findings that complement genes play a crucial role in the development of uveitis. Of these, variants in CFH, CFB and CFI involved in the alternative complement pathway, were identified as genetic risk factors for uveitis and specific subtypes. As described above, CFH-rs1065489 and CFB-rs1048709 were identified as clinical markers associated with a specific disease phenotype. Moreover, joint effects between their risk genotypes conferring a strongly increased risk to uveitis were also found23. The molecular mechanism might be affecting the binding affinity with C3b and subsequently disturbing the activation of the alternative pathway C3-convertase (C3bBb). C3 is the central component of complement and all the complement activation pathways converge at C3. On activation, C3 can break into C3a and C3b fragment that is called anaphylatoxin, which shows proinflammatory and immunoregulatory actions29. Genetic deficiency of C3 has been shown to ameliorate in the incidence and severity of EAU14. Additionally, C3-deficient mice have been shown to have an impaired ability to produce Th2 cytokines, the latter has been implicated in pathogenesis and regulation of EAU in animals31. On the other hand, there are a number of studies showing genetic association of the C3 gene with multiple inflammatory diseases that share similarities with uveitis26.The complement system belongs to the groups of sensing exogenous and endogenous danger-associated molecular patterns and has been implicated in the pathogenesis of uveitisC3 could be a good candidate gene for the genetic susceptibility to uveitis. However, in the present study, we failed to identify any significant associations of SNPs or haplotypes with uveitis. Considering the shared genetic component between AU and NIPU identified in our previous studies, both of these subtypes were recruited in the present study, which may help to consolidate our findings and identify more specific associations. In the analysis, multiple comparisons of the allelic and genotype frequency of C3 variants were performed among different groups: global uveitis patients, AU, NIPU and controls, as well as within NIPU subtypes. The non-significant results identified in this study suggest that the C3 gene may confer either no or limited risk for uveitis susceptibility, whether in AU or NIPU patients.Taking all of the above into account, C3 variants and any subsets, although the sample size within subsets was relatively small. Gender differences in the genetic profiles were reported in uveitis and PCV, in the study of Liu et al., C3 rs17030 showed a male-specific association with PCV, and suggested that the C3 gene is likely to be a risk factor for the male predominance of the disease32. Similarly, our previous studies also found gender may influence the association of complement genes with uveitis19. However, no SNP-gender interactions in C3 were observed in the present study. Further analysis was also performed to determine the linkage of C3 with other established significant findings, in which no gene*gene interaction was detected. The data strengthen the view that C3 is unlikely to have a major contribution to uveitis. The genetic alterations in the complement system may be located in “upstream” cascade.Next, several implementation-specific analyses have been performed. Regarding AU, we failed to find any genetic interactions with HLA-B27 status or any clinical markers. Stratification analysis according to NIPU entity was also applied to look for specific disease-association wherein significant associations were not observed between C3 locus, which encoded a “central” factor of the cascade. However, our results did not show any significant associations of C3 gene variants with uveitis or any subtypes. Conclusively, this data further enriches our growing understanding of uveitis genetics, and clarifies the specific roles of each complement pathway in ocular inflammatory diseases. Further replication studies are required to clarify the current situation because of the limited samples in this study.Given the well-established genetic profile of complement genes in uveitis, significant linkage evidence points to the C3 is a large gene consisting of 41 exons and containing hundreds of SNPs. We analyzed 8 tag-SNPs to narrow down the regions and aimed to capture the majority of common genetic variations of this gene and they may not sufficiently reflect the disease risk of unexamined variants, as some identified functional SNPs conferring susceptibility to immune-related diseases were not investigated in this study due to less MAF. Second, although we focused extensively on common variants in C3 itself here, it is possible that variants in other genes important in downstream signaling of the active C3 fragments may also be significant . It is also possible that epistatic effects of variants in other genes within the complement system  may contribute further to uveitis susceptibility35. Last but not least, the results so far failed to identify any significant associations with any subtypes of uveitis due to either a true lack of association or small sample sizes with insufficient statistical power to detect weak associations. Thus, these results should be interpreted cautiously.A number of limitations within the current study need to be discussed. First, C3 gene do not contribute greatly to uveitis susceptibility. The evidence presented so far suggests that genetic and immunologic investigations in uveitis should be focused on the “upstream” process of the complement system. Meanwhile, further studies to identify the rare and causal variants that focus on the possible effects on gene function will further elucidate the role of C3 in ocular inflammatory disorders.In summary, our results demonstrated that common variants in the


Multiple primary melanoma (MPM) is a rare condition, whose genetic basis has not yet been clarified. Only 8-12% of MPM are due to germline mutations of CDKN2A. However, other genes  have been demonstrated to be involved in predisposition to this pathology.To our knowledge, this is the first family study based on two siblings with the rare coexistence of MPM and oculocutaneous albinism (OCA), an autosomal recessive disease characterized by the absence or decrease in pigmentation in the skin, hair, and eyes.In this study, we evaluated genes involved in melanoma predisposition , pathogenesis , skin/hair pigmentation  and in immune pathways (CTLA4) to individuate alterations able to explain the rare onset of MPM and OCA in indexes and the transmission in their pedigree.From the analysis of the pedigree, we were able to identify a “protective” haplotype with respect to MPM, including MGMT p.I174V alteration. The second generation offspring is under strict follow up as some of them have a higher risk of developing MPM according to our model. The occurrence of multiple primary melanoma (MPM) is quite rare, with an estimated incidence ranging from 0.2%-8.6% . A genetMelanoma predisposition is related to germline mutations in several high and medium penetrance genes. CDKN2A gene mutations have been widely reported as the most common cause of inherited susceptibility to melanoma . An incrAlthough about 50% of the fM cases might be attributed to variants in CDKN2A and CDK4, a larger number of genes should be investigated.Recent studies, performed by two different groups ,7, identMGMT is a crucial gene in DNA repair pathways, and its loss is also associated with an increased risk of melanoma development .MC1R and MITF are considered moderate-risk genes for melanoma susceptibility –11. The The association between melanoma and BRCA1/2 mutations remains an open question which needs clarifications. BRCA1 mutations have not been significantly associated with melanoma. In contrast, suspected BRCA2 mutation carriers have shown an increased risk (2-5 times) of developing melanoma compared with the general population .The RAS/RAF/MEK/ERK and PI3K/AKT signalling pathways are strongly involved in the pathogenesis of cutaneous melanoma (CM). The RAS/RAF/MEK/ERK pathway, activated in >80% of all CM by BRAF or NRAS mutations, has been shown to promote cell proliferation, cell survival and tumor metastasis. The inactivation of PTEN is present in 40–60% of sporadic melanomas, which in turn negatively regulates the PI3K/AKT pathway conferring invasive and stem cell like features to melanoma cells.The study of immune checkpoints is another interesting field in melanoma research. CTLA4 is expressed on T cells acting as a negative regulator and inhibiting host immune response to melanoma. Furthermore, an association between CTLA4 gene polymorphisms and skin autoimmune disease like vitiligo has been firmly established  as well To our knowledge, this is the first family study based on two siblings with the rare coexistence of MPM and oculocutaneous albinism (OCA), an autosomal recessive disease characterized by the absence or decrease in pigmentation in the skin, hair, and eyes. OCA patients have a higher incidence of squamous cell carcinomas than the general population, but melanomas are rare and usually amelanotic.In this study, we evaluated genes involved in melanoma predisposition , pathogenesis , skin/hair pigmentation  and in immune pathways (CTLA4) to individuate alterations able to explain the rare onset of MPM and OCA in indexes. Moreover, differences with three other siblings and the possible transmission of these alterations to their relatives have been analyzed.Ten nanograms of DNA were processed according to the manufacturer’s protocol. In all samples an adequate library for subsequent sequencing was obtained, and at least one alteration of the genes included in our Next Generation Sequencing (NGS) custom panel was shown in each analyzed specimen.Variant calling was carried out by the variant caller of Torrent Suite and a custom pipeline . The custom pipeline showed 89% and 94% sensitivity and specificity respectively for single nucleotide variant calling. Thus, we considered variants called by both methods. Regarding indels, hard filtering of our custom pipeline needs improvement given the high discordance rate.strong RHC  or weak RHC  variants. Moreover, in one sample, a not yet classified MC1R variant was found (p.Phe235Leu). All CTLA4 altered cases showed p.Thr17Ala SNP. Instead, the PIK3CA alterations more frequently individuated were p.Glu707Lys and p.Ile391Met.Of the custom panel genes, three of them  did not reveal any type of alterations. The most altered genes resulted MC1R (94%), CTLA4 (65%), MGMT and PIK3CA (both 53%). In fact, all samples except one showed MC1R missense variants classified as As regards the BRAF gene, we did not report any mutations, but only a synonymous variant (p.Gly643=). Furthermore, in this study, MITF, KIT and RB1 resulted to be genes with few variants yet to be classified.Interestingly, when we consider the indexes and their three healthy siblings, it is possible to note that the MC1R genetic pattern, including the missense variant p.Ile155Thr and the synonymous alteration p.Thr314=, is present in all 4 siblings with OCA, explaining the feasible relation with albinism. On the contrary, the indexes did not show MGMT p.Ile174Val, found in the other three healthy siblings, probably demonstrating that the absence of this alteration may be related to the onset of melanoma. BRCA1/2 variants were searched in the two indexes finding no mutation.The full length coding sequence and the UTR regions of CDKN2A gene and the Italian founder mutation p.Ser270Asn of POT1 were analyzed by Sanger Sequencing in all indexes and relatives, evidencing two SNPs in 3′UTR region of CDKN2A (c. 500 C>G and c.540C>T) in 41% of all cases. Only one specimen showed a variant known to predispose to melanoma p.Ala148Thr of CDKN2A. Mutational results are summarized in pedigree shown in Figure The two index cases and their siblings were investigated for germinal MGMT promoter methylation status. No blood sample resulted to be methylated.As MGMT p.Ile174Val alteration was not detected in the index cases, we hypothesized a “protective ” role with respect to melanoma onset of this alteration, primarily to exclude an impairment of the protein function. Annovar predictions were obtained from 5 prediction scores , 3 conservation scores  and 3 ensemble scores , as shown in Table MPM onset is a rare and poorly investigated condition. Family history and the presence of atypical nevi are the two factors most related to MPM. Frequently, CDKN2A and CDK4 are able to explain approximately 50% of fM, and therefore several genes have to be further investigated to better elucidate mechanisms leading to MPM occurrence. Considering the extremely rare coexistence of MPM and OCA, we decided to analyze in the two indexes affected by both conditions, over CDKN2A and CDK4 alterations, a pattern of other genes involved in melanoma predisposition and pathogenesis, in skin/hair pigmentation and in immune pathways. We aimed to individuate possible alterations able to explain the onset of MPM in the OCA indexes, the differences with other three healthy siblings and the possible transmission of these alterations to the relatives.It was not possible to genetically characterize the grandfather, but neither him nor his wife were affected by albinism. Thus we hypothesized that both of them were healthy carriers of predisposing alterations. In such a context, it was possible to distinguish the genetic pattern of the two indexes affected both by MPM and OCA and of their siblings affected only by the latter. The second generation was also analyzed. In particular, none of the offspring was affected by OCA, which is an autosomal recessive disease. Therefore, as the second generation is unaffected by both pathologies, it was not been considered for further analyses. However, the second generation offspring is under strict follow-up for risk of melanoma.As regards the most investigated CDKN2A gene, the indexes showed a common SNP in the 3′UTR, c.540 C>T, present in 33% of relatives. No relation of this SNP with melanoma risk has been reported . On the Another important result of our study concerns the same genetic pattern of the investigated family, probably related to the albinism condition. In fact the indexes and the healthy OCA siblings shared the same MC1R alterations . On the other hand the only sister without MPM and OCA did not show any alteration in MC1R. This gene is a central control point in skin and hair pigmentation. Previous studies reported an association between MC1R and OCA2 variants in determining a modification of the OCA2 phenotype ,19. ThesInterestingly, our findings revealed a new gene as a probable player in MPM pathogenesis. Indeed, the NGS results evidenced the absence of the MGMT p.Ile174Val variant in the two indexes compared to the other siblings. MGMT was shown to have a crucial role in the DNA repair pathway. The MGMT gene, located in chromosome band 10q26, has previously been associated with several cancer types where its epigenetic silencing has been described, such as glioblastoma, colorectal cancer and gastric cancer . In addiIn conclusion, given that the indexes affected by MPM did not carry MGMT p.Ile174Val,and in the light of its neutral role, we hypothesized that the haplotype found in healthy siblings could have a protective role. Moreover, our findings highlighted a possible involvement of MC1R in the occurrence of albinism. The second generation offspring is under strict follow up as some of them have a higher risk of developing multiple primary melanoma according to our model.The index cases were two siblings  affected by both OCA and MPM pathologies.in situ melanoma of the neck region when he was 57 years old, and a second primary nodular melanoma in the right arm four years later. The latest one was a pT1a melanoma with a Breslow thickness of 0.8 mm. Moreover, this index was surgically treated for a basal cell carcinoma and underwent stringent follow-up for the presence of atypical nevi. The primary melanoma sample revealed 11% of somatic MGMT promoter methylation.The female index developed a first primary melanoma in the left leg when she was 57 years old. It was a pT1a superficial spreading melanoma with a Breslow thickness of 0.17 mm, radically removed by surgery. Two years later, she developed a second primary melanoma in the left arm. It was a pT2b ulcerated nodular melanoma with a Breslow thickness of 2 mm associated with metastases in 2 out of 17 left axillar lymphnodes. The genetic analysis of this primary melanoma sample revealed the absence of BRAF mutations, the presence of p.Q61L NRAS mutation and a 40% of somatic MGMT promoter methylation. This index then underwent a regular follow-up. To date, the female index has developed brain and lung metastases and has begun chemo-immunotherapy with a schedule including fotemustine and ipilimumab in an ongoing clinical trial. The male index developed a first primary Regarding the first generation, the remaining siblings  were enrolled, two of whom had OCA but not melanomas. Moreover, the mother of the indexes, the spouses and the offspring of the entire first generation were enrolled. No relative showed either albinism or melanoma.The study was approved by the local Ethics Committee of the IRCCS “Giovanni Paolo II” of Bari  and was performed in accordance with the international standards of Good Clinical Practice. All indexes and their relatives signed informed consent and blood samples from all of them were obtained.DNA was isolated from blood samples using the QIAamp DNA Blood Midi Kit (Qiagen) according to the manufacturer’s instructions. The extracted DNA from each sample was quantified by Nanodrop and Qubit methods.Eleven  of the 15 genes to investigate were analyzed by a custom panel previously developed by our group . As regavariant caller v5.0” program. In order to eliminate errors in base calling, the Somatic-High Stringency parameters setting was used to generate the final variant calling. Filtered variants were annotated using the Ion Reporter software v5.0 (Termo Fisher Scientific). Mutations were visually examined using Integrative Genomics Viewer (IGV) software (http://www.broadinstitute.org/igv).Data from the PGM runs were processed initially using the Ion Torrent platform-specific pipeline software Torrent Suite to generate sequence reads, trim adapter sequences, and filter and remove poor signal-profile reads. Initial variant calling from the Ion AmpliSeq sequencing data was generated using Torrent Suite Software v5.0 with a plug-in “https://www.broadinstitute.org/gatk/) [http://bio-bwa.sourceforge.net/) for sequence alignment [Data from the NGS custom panel were also analyzed by a custom pipeline to verify their reliability. In detail, we followed Toolkit for Genome Analysis   recommenlignment  and GATKlignment ,30.Mutational screening of exons 1α, 1β, 2 and 3, including the exon-intron boundaries  of CDKN2A, and of the POT1 region including the p.Ser270Asn mutation, was performed by PCR and direct sequencing on the 3500 Genetic Analyzer (Thermo Fisher Scientific). In detail, PCR amplification of the regions of interest was performed in a Gene-Amp PCR System 9700 (Thermo Fisher Scientific) using the primers listed in Table PCR amplification conditions were as follows: 95°C for 7 min, 35 cycles of 94°C for 1 min, Tm°C for 1 min, and 72°C for 1 min, followed by a final extension step at 72°C for 7 min.Promoter methylation status of the MGMT gene was conducted with a PCR-based method using DNA treated with bisulfite followed by a real-time pyrosequencing that targeted CpG islands (EpigenDxInc). This method allowed us to quantify methylation at multiple CpG sites individually. Pyrosequencing was performed using PyroGold Q96 SQA Reagents and the Pyro Q-CpG software on a PyroMark ID pyrosequencer (Qiagen) as per the manufacturer’s recommendation. The sequencing results were analyzed using the PSQ PyroMark software (Qiagen). As controls, CpGenome Universal Methylated DNA  and DNA from FFPE non-tumorous skin tissue (negative methylation control) were included in the assay, as well as a reaction without any template DNA (non-template control). All tumour and control specimens were measured in triplicates.The Blastp alignment tool  was used