Corneal stromal and/or penetrating ocular injuries from pencils and pencil lead are more common in childhood and may lead to intraocular infection or severe intraocular sterile inflammatory reaction. Herein we report 3 children with ocular trauma due to pencil lead injuries. The first case had corneal stromal injury caused by a pencil. In the second case, a pencil perforated the cornea and contacted the iris. In the third case, pencil lead perforated both the cornea and iris and reached the vitreous through the lens zonules. Intracameral triamcinolone (2 mg/0.05 mL) was injected after the pencil lead was removed from the eyeball. Topical anti-inflammatory and cycloplegic drops were prescribed. In conclusion, corneal and especially penetrating ocular injuries from pencil lead may have a good prognosis with the use of appropriate anti-inflammatory and prophylactic antibiotic treatment and follow-up. Although it has been reported that the carbon particles from pencil lead may remain dormant in the eye without inducing inflammation for long periods of time,3 they have also been reported to cause severe endophthalmitis5 or endothelial dysfunction and corneal edema.6 In this report we share three cases of pencil lead injury, one with corneal stromal injury and two with intraocular penetrating injuries.Organic intraocular foreign bodies generally cause serious inflammatory reaction and infection. The inflammatory reaction induced by inorganic foreign bodies is related to the composition of the object.An 8-year-old boy was admitted to our urgent ophthalmology clinic the same day of a pencil injury to his left eye. His visual acuity was 20/20 bilaterally. Slit-lamp examination of the left eye revealed intact corneal epithelium and intrastromal silver-gray carbon particles in the inferonasal quadrant of the cornea. The conjunctiva was mildly hyperemic. There was no sign of anterior chamber reaction. Anterior segment examination of the right eye was normal. Intraocular pressure measured by applanation tonometry was 14 mmHg in the right and 17 mmHg in the left eye. Fundus examination was normal in both eyes. The patient was administered moxifloxacin ophthalmic drops  4 times a day for 3 weeks and preservative-free artificial tears  4 times a day for 4 weeks. No signs of inflammatory reaction or injection were observed in follow-up examinations at 1 week and 1, 4, and 6 months  and 1B.A 13-year-old patient presented after being poked in the right eye by a pencil. The patient’s visual acuity was counting fingers (CF) from 0.5 m in the right eye and 20/20 in the left eye. On slit-lamp examination, a fragment of pencil lead was found lodged in the inferonasal (at 5 o’clock) paracentral cornea of the right eye. Seidel test was negative. Anterior chamber examination revealed +2 cells in the right eye and was normal in the left eye. No signs of pathology were observed in fundus examination of either eye. The patient was admitted for surgical removal of the foreign body. The pencil lead fragment lodged in the cornea was removed using forceps and viscoelastic to support the anterior chamber. The corneal laceration was closed with 4 sutures using 10/0 nylon suture. After the procedure, 2 mg/0.05 mL triamcinolone and 0.25 mg/0.05 mL moxifloxacin were injected into the anterior chamber. On postoperative day 1, treatment was initiated with moxifloxacin drops . Dexamethasone ophthalmic drops  were started at 8 times a day for 5 days, then reduced to 6/day for 1 week, then reduced to 4/day for 1 week and tapered by 1 application/day each week until discontinuation. On postoperative day 3, visual acuity on Snellen chart was 20/25 (with or without correction). No inflammatory reaction was observed on anterior segment examination. The corneal sutures were removed at 3-month follow-up. At 7 months, the patient’s visual acuity was 20/20 bilaterally . CornealA 15-year-old male patient whose right eye was stabbed with pencil lead was referred from outside the city to our clinic on the second day after the trauma. His visual acuity was CF from 0.5 m on the right and 20/20 on the left. Slit-lamp examination revealed pencil lead in the right temporal cornea (between 8-9 o’clock) that had perforated the cornea and iris near the limbus. Cyclitic membrane was noted around the pupillary region and hypopyon was present . The lef9 The first reported case of intracorneal carbon particles was presented by Jeng et al.2 The patient presented due to a chemical injury to the right eye and silver-gray crystalline opacities were observed in the corneal stroma. It was learned that the patient had sustained a pencil injury to the left eye 8 years earlier. However, the patient’s medical records indicated that the injury had in fact been to the right eye. Slit-lamp examination revealed intact corneal epithelium and silver carbon particles in the inferonasal stroma. This demonstrated that carbon particles in the corneal stroma were well tolerated in the long term. Philip et al.3 reported a case in which intracorneal carbon particles were observed during routine eye examination in a patient who had sustained a pencil injury to the same eye 3 years earlier. Slit-lamp examination of the right eye revealed anterior stromal scar, though no signs of previous or current inflammation were detected in the intraocular structures.Pencil lead is made of a mixture of carbon, clay and animal fat and is surrounded by a wooden sheath. The main component, carbon, is known to usually remain inert in the eye. However, potential toxicity due to the other components is controversial.5 In another case with a history of pencil injury, a suspected conjunctival melanoma was excised and the histopathologic report indicated granulomatous reaction due to carbon particles.10There are also reports in the literature of pencil lead causing severe inflammatory reaction and endophthalmitis. One reported case underwent corneal suturation and lens extraction following a pencil injury to the right eye. Pencil lead fragments were noted in the vitreous and on the second postoperative day the patient developed endophthalmitis. Although bacterial endophthalmitis was suspected based on clinical findings, a vitreal sample taken during pars plana vitrectomy was culture negative. It was proposed that the wood and aluminum found in pencils may have caused a severe inflammatory reaction.9 No anterior chamber inflammation occurred during the 1-month follow-up period . A case reported by Gül et al.8 presented with severely reduced vision (CF at 2 m) following ocular trauma by pencil. On slit-lamp examination, corneal perforation and fragments of pencil lead were observed at the wound site. The +4 anterior chamber reaction observed preoperatively continued after corneal suturation and foreign body extraction. Examination on the same day revealed linear carbon accumulation on the endothelial surface, and a pencil lead fragment was visible on the lens after pupil dilation. With hourly steroid therapy, the anterior chamber reaction resolved and the endothelial accumulation and material on the lens disappeared. Han et al.6 reported a patient with a pencil injury 12 years earlier who presented with stromal keratitis. Antiviral and anti-inflammatory therapy was initiated for a preliminary diagnosis of herpetic stromal keratitis. The patient showed improvement of clinical findings, but at 3-month follow-up, a previously unnoticed foreign body was observed at the anterior chamber angle. The authors believed that the previously inert pencil lead fragment came into contact with the endothelium when it moved, thus triggering an inflammatory reaction. Pencil lead perforated the cornea in our second case and in the third case it perforated both the cornea and iris, reaching the vitreous through the zonules. To prevent a possible inflammatory reaction induced by pencil lead in these patients, triamcinolone was injected into the anterior chamber at the end of surgery, which may be considered possibly beneficial in such cases.Another patient who had sustained a pencil injury to the left eye 4 months earlier presented to an ophthalmologist with a complaint of pain in the left eye for 2 days. Examination revealed a full-thickness corneal scar, a small area of iris atrophy, and a black foreign body resembling pencil lead in the anterior chamber. No inflammatory reaction was observed in the anterior chamber and surgery was performed to remove the foreign body.It can be concluded that carbon particles in the cornea are well tolerated in the long term, and that a good prognosis can be achieved in cases of intraocular pencil lead injury with anti-inflammatory therapy, prophylactic antibiotic therapy, and monitoring.Informed Consent: It was taken.Peer-review: Externally peer-reviewed.


Interactions between environment and genetics may contribute to multiple sclerosis (MS) development. We investigated whether the previously observed interaction between smoking and HLA genotype in the Swedish population could be replicated, refined and extended to include other populations. We used six independent case–control studies from five different countries . A pooled analysis was performed for replication of previous observations . Refined detailed analyses were carried out by combining the genetically similar populations from the Nordic studies . In both the pooled analyses and in the combined Nordic material, interactions were observed between HLA-DRB*15 and absence of HLA-A*02 and between smoking and each of the genetic risk factors. Two way interactions were observed between each combination of the three variables, invariant over categories of the third. Further, there was also a three way interaction between the risk factors. The difference in MS risk between the extremes was considerable; smokers carrying HLA-DRB1*15 and lacking HLA-A*02 had a 13-fold increased risk compared with never smokers without these genetic risk factors . The risk of MS associated with HLA genotypes is strongly influenced by smoking status and vice versa. Since the function of HLA molecules is to present peptide antigens to T cells, the demonstrated interactions strongly suggest that smoking alters MS risk through actions on adaptive immunity.The online version of this article (doi:10.1007/s10654-017-0250-2) contains supplementary material, which is available to authorized users. Multiple sclerosis (MS) arises from a combination of a complex genetic predisposition and environmental factors. The strongest genetic associations with MS are located within the human leukocyte antigen (HLA) complex , 2 whereFor each study a more detailed description of study design and data collection is presented in supplementary table 1, and a flow chart presenting the falling-off in each study is presented in Table The first Swedish study was based on EIMS which is an ongoing population-based case–control study, comprising the Swedish general population aged 16–70 years. Incident cases of MS were recruited from neurology clinics, including all university hospitals in Sweden. All cases fulfilled the McDonald criteria . For eacThe second Swedish study was Genes and Environment in Multiple Sclerosis (GEMS) in which prevalent cases, distinct from those in EIMS, were identified from the Swedish National MS-registry  and contPatients fulfilling the McDonald criteria were recruited from Neurology units in Danish hospitals between October 2009 and December 2014. The control group comprised healthy white Danish blood donors residing in the area of Copenhagen. The controls were matched to the cases by gender and age in 5-year age strata when included in the present study. Informed consent was obtained from all participants and the study was approved by the local Ethics Committee. The part of the Danish study used in this report comprised 1474 prevalent cases and 3469 controls  using electronic medical records. Controls were randomly selected from KPNC members and were individually matched to cases on gender, birth date, race/ethnicity, and zip code of the case residence. The study protocol was approved by the Institutional Review Boards of the KP Division of Research and the University of California, Berkeley. The part of the KPNC study used in this report comprised 1013 prevalent cases and 794 controls.For each case in all studies but the Serbian and American studies, the time of the initial appearance of MS symptoms was used as an estimate of the disease onset, and the year in which this occurred was defined as the index year. The corresponding controls were given the same index year. Subjects who smoked regularly during the index year were defined as smokers whereas those who had never smoked before or during the index year were defined as never smokers. We have previously demonstrated that the increased risk of developing MS associated with smoking slowly abates after smoking cessation. A decade after stopping smoking, there is no longer an association between smoking and MS risk . TherefoSubjects in all studies were genotyped for HLA-DRB1*15 and HLA-A*02 alleles. Detailed information regarding genotyping in each study is presented in supplementary table 1. The HLA-DRB1*15 allele is associated with an increased risk of developing MS with an OR around 3, and the subjects were classified according to the carriage of any HLA-DRB1*15 allele versus no carriage. The HLA-A*02 allele has protective association to MS. Absence of HLA-A*02 is thus a risk factor of developing the disease, and the participants were classified according to no carriage of any HLA-A*02 allele versus carriage.Subjects with different genotypes and smoking habits were compared with regard to MS risk, by calculating odds ratios (OR) with 95% confidence intervals (CI) using logistic regression models. When controls are frequency matched to cases, the matching variables should be included in an unconditional logistic model, whereas conditional logistic regression, in which each matched set forms a stratum, should be used when controls are individually matched to cases. However, losses may be substantial when data contain incomplete matched sets. Data from an individual matched case–control study may also be analyzed with unconditional logistic regression as long as the variables used to form the match are included in the model.In all studies but the Serbian and American studies, we performed both matched and unmatched analyses of the data. However, only the results from the unmatched analyses are presented since these were in close agreement with those from the matched analyses but had higher precision in terms of more narrow confidence intervals. The Serbian data was analyzed using only unconditional logistic regression whereas conditional logistic regression was used when analyzing the American study.3), the total attributable proportion (TotAP3) and the total synergy index (TotSI3);i+, Xi− denote the presence (Xi = 1) or absence (Xi = 0) of Xi, i = 1, 2, 3, and RR the relative risk for the development of the disease, given the combination of the presence or absence of the risk factors X1, X2, … X3, as compared to their absence .We investigated the gene–gene interaction between HLA-DRB1*15 and absence of HLA-A*02 as well as the interactions between each of these genetic risk factors and smoking. The potential interactions were analysed using departure from additivity of effects , 19 and  absence . These mWhen the results from all six studies were pooled together, study specific results (adjusted for age and gender) were pooled together by utilising a model allowing for random effects. When the Nordic studies were combined into one, the analyses were adjusted for age, gender and study.In EIMS and GEMS, the interaction analyses were further adjusted for a large number of potential confounding variables described in supplementary table 2. Adjustment for these factors had minor influence on the results. The analyses were conducted using Statistical Analysis System (SAS) version 9.2 and Stata Statistical Software, release 11 .The increased MS risk associated with smoking was similar in all studies. HLA-DRB1*15 increased the risk of MS in all studies, whereas HLA-A*02 was associated with a decreased MS risk in all studies but the Serbian study (supplementary table 3). In the pooled analyses there was an interaction between HLA-DRB*15 and absence of HLA-A*02 and between smoking and each of the genetic risk factors (Table By combining the Nordic datasets (6265 cases and 8401 controls), we show that interactions take place between both HLA-DRB1*15 and HLA-A*02 regardless of smoking status .An interaction between smoking and absence of HLA-A*02 was observed only in the Nordic studies. No interaction between smoking and absence of HLA-A*02 was observed in the Serbian or American cohort. This could be due to chance, differences in life style habits or in genetic background. Notably, the HLA-A*02 protective association to MS vary considerably between different populations , 22.Since use of oral tobacco in the form of moist snuff is not associated with increased risk of MS , the criThere are several potential mechanisms that can be involved in a casual role of smoking. Smoke-induced lung-irritation causes increased pro-inflammatory cell activation and induces post-translational modifications of proteins in the lungs –28. AlteThe specific interactions may shed light on potential disease mechanisms for MS. Preferences in peptide binding by allelic variants of class II molecules are likely to be critical for the HLA class II influences on autoimmune diseases . The intApproximately 20% of all MS cases in Sweden has been estimated to be attributable to active or passive smoking, whereas 41% of the cases among those carrying HLA-DRB1*15 but lacking HLA-A*02 were attributable to smoking . From a In all studies, information regarding smoking habits was collected retrospectively. The EIMS study was designed as a case–control study with incident cases in order to minimize recall bias, whereas the other studies used prevalent cases. The questionnaires in all Nordic studies contained a wide range of questions regarding many potential environmental risk factors and no section in the questionnaires was given prime focus. Furthermore, in the Nordic studies as well as in the American study, exposure information from cases and controls was obtained in an identical way from cases and controls.A potential selection bias may arise when recruiting cases and controls. Considering the structure of the public health care system in Sweden, which provides equal free of charge access to medical services for all citizens, it is likely that almost all cases of MS are referred to neurological units. However, some cases may not have been asked to participate in EIMS due to the attending neurologists’ lack of time. In GEMS and the Norwegian study, cases were identified through MS registers in each country and these registers may not have covered all prevalent cases. In the Danish study, the majority of cases were randomly recruited by neurologists from hospitals with MS Centers in Denmark and the cases may not be representative for the general MS population. However, the recruitment of cases was probably not affected by smoking or HLA genotype.In the Swedish studies, the problem of selection bias was minimized by the population-based design and even though there was a relatively high proportion of non-responders among the controls, this bias is probably modest because the prevalence of life style factors, such as smoking and alcohol consumption, among the controls was consistent with that of the general population in similar ages . The disFurthermore, there were no significant differences with respect to age, gender, or smoking habits between those who provided a blood sample and those who did not, indicating that selection bias did not take place in this step. We consider it unlikely that our main finding of an interaction between MS risk HLA genotypes and exposure to smoking would be affected by bias to a large extent, especially since such a bias would then depend on HLA types.In the Danish study, the control group comprised healthy white Danish blood donors and in the Norwegian study, controls were selected from the National Bone Marrow Donor Registry. In these studies, a selection bias towards a more than normally healthy group may therefore have occurred. However, educational level and autoimmune co-morbidity were similar among Norwegian cases and controls, indicating that the groups were comparable with regard to socioeconomic status and thus probably also general . The proIn conclusion, an interaction between smoking and HLA-DRB1*15 was observed in all cohorts, whereas an interaction between smoking and absence of HLA-A*02 was observed only in the Nordic cohorts. The combined Nordic studies allowed detailed analyses of the interaction between the three studied factors, and in addition to significant two way interactions between each combination of the three variables, invariant over categories of the third, there was a significant three way interaction between smoking, HLA-DRB1*15, and absence of HLA-A*02. We hypothesize that smoke-induced lung-irritation may trigger autoaggressive T cells in the lungs or post-translationally modify peptides that are cross-reactive with CNS antigens, promoting a CNS-directed autoaggressive immunity that results in MS.Supplementary material 1 (DOC 39 kb)Supplementary material 2 (DOC 26 kb)Supplementary material 3 (DOC 26 kb)Supplementary material 4 (DOC 54 kb)Below is the link to the electronic supplementary material.


To perform a cross-sectional study to determine whether plasma neurofilament light chain (NfL) concentration is elevated in patients with Charcot-Marie-Tooth disease (CMT) and if it correlates with disease severity.Blood samples were collected from 75 patients with CMT and 67 age-matched healthy controls over a 1-year period. Disease severity was measured using the Rasch modified CMT Examination and neuropathy scores. Plasma NfL concentration was measured using an in-house-developed Simoa assay.p < 0.0001) and correlated with disease severity as measured using the Rasch modified CMT examination  and neuropathy  scores. Concentrations were also significantly higher when subdividing patients by genetic subtype  or into demyelinating or axonal forms compared to healthy controls.Plasma NfL concentration was significantly higher in patients with CMT (median 26.0 pg/mL) compared to healthy controls (median 14.6 pg/mL, There are currently no validated blood biomarkers for peripheral neuropathy. The significantly raised plasma NfL concentration in patients with CMT and its correlation with disease severity suggest that plasma NfL holds promise as a biomarker of disease activity, not only for inherited neuropathies but for peripheral neuropathy in general. There is therefore a need for a biomarker that reflects peripheral nerve damage and can be used to assess response to treatment.Peripheral neuropathy affects an estimated 10 million people in the European Union (7 million in the United States) and represents a substantial public health burden.3 Damage to nerves leads to the release of these proteins into the CSF or plasma, as demonstrated by the 20-year-old observation that NfL concentration is increased in the CSF of patients with amyotrophic lateral sclerosis (ALS).4 Increased NfL concentrations have been reported in CSF, and more recently in plasma, in neurodegenerative diseases of the CNS; for example, frontotemporal dementia (FTD), multiple sclerosis (MS), and Alzheimer disease (AD).5–8Neurofilaments are the major cytoskeletal proteins of neurons in both the CNS and peripheral nervous system and form a lattice comprising neurofilament light (NfL), medium, and heavy (NfH) chains.9 in patients with both demyelinating and axonal forms of inherited neuropathy (Charcot-Marie-Tooth disease [CMT]), a group of diseases in which patients experience slowly progressive axonal degeneration at a constant rate.10 A blood biomarker of disease activity is likely to be of value in future treatment trials in CMT, a disease that often shows only minimal progression during the usual 1- to 2-year period of a clinical trial and in which current clinical outcome measures are insensitive in detecting disease progression during such a time period.11In contrast to diseases of the CNS, biomarkers of peripheral nerve damage are less well-developed. In this study, we sought to determine the biomarker potential of plasma NfL in peripheral neuropathy by measuring the plasma NfL concentration using ultrasensitive Simoa immunoassay technology,12 (the weighted CMTES), was recorded at the same time as plasma was collected. The weighted CMTES is a validated outcome measure for assessing the severity of CMT. It is a composite score that includes the patient's symptoms and examination findings.11 All patients underwent nerve conduction studies to confirm the presence of a neuropathy; however, a weighted CMT neuropathy score (CMTNS)  was only included if a nerve conduction study had been performed within 18 months of the blood sample.Blood samples were collected prospectively, with informed consent, from all patients with CMT with a genetically confirmed diagnosis attending the inherited neuropathy clinic at the National Hospital for Neurology and Neurosurgery, London, between January 2011 and May 2013. Patients with other neurologic diseases were excluded as determined by review of the clinical notes and a questionnaire. The disease severity, as measured using the Rasch modified CMT examination score, version 2Blood samples were obtained from healthy relatives of patients attending the inherited neuropathy clinic and staff at the UCL Institute of Neurology and from the relatives of patients with ALS recruited as part of a separate study. Healthy controls were excluded if they had coexistent neurologic disease as determined by a symptom and medical history–based questionnaire.All participants were evaluated in outpatient clinics and blood samples were taken and processed within 1 hour. Whenever possible, a repeat blood sample was taken after 1 year. Blood was collected into EDTA-containing tubes and centrifuged at 20°C at 3,500 rpm for 10 minutes. Plasma was then aliquoted and stored at −80°C.This study was approved by The National Hospital for Neurology and Neurosurgery Research Ethics Committee/Central London REC 3 09/H0716/61 and the East London and the City Research Ethics Committee 1 (09/H0703/27). Written informed consent was obtained from all participants in the study.7 Briefly, paramagnetic carboxylated beads  were coated with a mouse anti-NfL antibody  and incubated for 35 minutes with sample and a biotinylated mouse anti-NfL antibody  in a Simoa HD-1 instrument (Quanterix). The bead-conjugated immunocomplex was thoroughly washed before incubation with streptavidin-conjugated β-galactosidase (Quanterix). After additional washes, resorufin β-d-galactopyranoside (Quanterix) was added and the immunocomplex was applied to a multiwell array designed to enable imaging of every single bead. The average number of enzymes per bead (AEB) of samples was interpolated onto the calibrator curve constructed by AEB measurements on bovine NfL (UmanDiagnostics) serially diluted in assay diluent. Samples were analyzed blind and in duplicate using one batch of reagents. The average repeatability coefficient of variation of a sample with the mean concentration 14.3 pg/mL was 7.5% and the interassay coefficient of variation was 12%, and for a sample with a mean concentration of 129.5 pg/mL this was 5.3% and 10.2%, respectively. The limit of detection, determined as the mean blank signal +3 SD for the Simoa NfL assay, was 0.3 pg/mL, and the lower limit of quantification (LLOQ), determined as the mean blank signal +10 SD, was 2.7 pg/mL when compensated for a 4-fold sample dilution. All samples analyzed were above the LLOQ.Plasma sample NfL concentration was determined using the in-house Simoa NfL assay, which has been described in detail previously.U test, and correlations were assessed using Spearman and Pearson correlation coefficients. Fisher r to z transformation was used to compare correlation coefficients. Multiple linear regression was performed using SPSS to model the relationship between plasma NfL (dependent variable) and age and weighted CMTES score (independent variables).Statistical analysis was performed using SPSS version 23.00  and GraphPad Prism 5.0 . A normal probability plot of the residuals of plasma NfL concentration revealed a skewed distribution of the data points. Plasma NfL concentration was therefore compared using a 2-sided Mann-Whitney SPTLC1 and SPTLC2) and 67 healthy controls were enrolled into the study. There was no significant difference in the mean age of the 2 groups (p = 0.73). There were significantly more women in the control (69%) than the CMT cohort ; however, this is unlikely to have influenced the comparison between the 2 cohorts as there was no significant difference in plasma NfL concentration between male and female controls . The CMT cohort comprised 10 different subtypes of CMT. The commonest subgroup was CMT1A (n = 31), followed by SPTLC1 (n = 20) and CMTX1 (n = 11). Hereditary sensory neuropathy due to mutations in SPTLC1 was disproportionately represented in this study as samples were collected from patients recruited into a natural history study of HSN1 that was running concurrently. Sixty-four percent of patients with CMT had CMT1 (demyelinating neuropathy) vs 36% with CMT2 , which is reflective of the relative prevalence of these disease subtypes in the general population.13 All patients included in the study had a weighted CMTES calculated; however, only 30 (40%) had a neurophysiologic examination within 18 months of sample collection and for whom a weighted CMTNS was calculated.A total of 75 patients with CMT ; Plasma NfL concentration correlated with age and this was more pronounced in controls than in patients with CMT . The outlier is a patient with CMT1A and a weighted CMTES score of 12 but a very high plasma NfL concentration of 81.0 pg/mL. A retrospective clinical review of this patient (6 years after sample collection) did not reveal an alternative cause for the high concentration. The correlation between plasma NfL concentration and disease severity remained significant when correlated against the weighted CMTNS , although this correlation could only be performed for the 40% of patients who had a weighted CMTNS calculated (p = 0.012). When plasma NfL concentration was predicted using a multiple linear regression model with age and the weighted CMTES as independent variables, it was found that disease severity as measured using the weighted CMTES was a significant predictor , whereas age was not . The overall model fit was R2 = 0.17. There was no significant correlation between plasma NfL concentration and the ulnar compound muscle action potential (CMAP) .The weighted CMTES and CMTNS are disease severity scales for peripheral neuropathy that have been modified to create a linear scale. The CMTES includes a symptom and examination score; the CMTNS in addition includes neurophysiologic data. A comparison of the weighted CMTES and plasma NfL concentration revealed a significant correlation between NfL concentration and disease severity (lculated . If the t test p = 0.74) or controls  when comparing baseline with 1-year follow-up samples (p = 0.5).On a selected number of patients with CMT (n = 9) and controls (n = 13), plasma samples were collected at baseline and after 1 year. There was no significant difference in plasma NfL concentration in patients with CMT . This suggests a role for plasma NfL in monitoring disease activity may be extended to multiple forms of CMT. Further studies are therefore warranted to discover if these results can be replicated for other common forms of CMT such as CMT1B and CMT4C. The observation of raised plasma NfL in individual patients with these subtypes in this study is encouraging.5–14 Therefore, plasma NfL concentration will not by itself be useful for diagnosis; however, it may provide a dynamic measure of axonal damage and serve as a biomarker of disease activity for future clinical trials and monitoring response to treatment, especially in the many conditions like CMT where neuropathy is the only neurologic manifestation with no CNS involvement and therefore any change in NfL levels would reflect neuropathy changes alone. As neurofilaments are almost exclusively expressed in neurons, there are likely to be few non-neurologic confounding factors; however, neurofilaments are expressed in T lymphocytes and theoretically the concentration may be falsely elevated in T-cell proliferative disorders.11Increased plasma NfL concentration is not specific to CMT as a similar increase has been reported in several other neurologic disorders such as ALS, MS, FTD, and AD.15 This is unsurprising and reflects similar paradoxical results in cohorts of patients with ALS16 and may reflect the formation of NfH aggregates resulting in falsely low levels.17 In this study, we were also unable to detect a correlation between the ulnar CMAP and plasma NfL concentration. One possible explanation may be that our sample population included many individuals with mild CMT (CMTES <10) in whom the ulnar CMAP may be normal.We have previously measured plasma NfH concentration in the same cohort of patients and controls and were unable to detect a difference or a correlation with disease severity.18 There are, however, several limitations to the score, including a quasi-linear scale and a ceiling effect for severe patients.18 A blood biomarker of axonal damage and thereby disease activity is likely to be of considerable value in future treatment trials in CMT and other peripheral neuropathies that show only minimal progression during the usual 1- to 2-year period of a clinical trial. To date there have been 4 large randomized clinical trials in patients with the commonest form of CMT, CMT1A, comparing ascorbic acid to placebo.19–22 All 4 trials failed to show a therapeutic benefit of ascorbic acid, but perhaps equally as important, the first version of the CMTNS, which was used as the primary outcome measure in 3 of the 4 trials, was unable to detect disease progression over a 1-year period.18 This is important, as treatments for neurodegenerative diseases such as CMT are at best likely to stop progression. Encouragingly, quantitative MRI has been shown to be sensitive to detecting an increase in the fat fraction of muscle in patients with CMT1A over a 1-year period, suggesting its suitability as a primary outcome measure for future treatment trials.23 The sensitivity of muscle MRI, however, is dependent on the choice of muscle, whereby severely affected and unaffected muscles are susceptible to both ceiling and floor effects. A similar floor effect is also seen with nerve conduction studies, where in a proportion of patients with moderate to severe peripheral neuropathy, the nerves are so severely damaged that no electrical response can be recorded. In this regard, a blood biomarker has the advantage of reflecting damage to all nerves and is likely to be more sensitive to multifocal peripheral nerve diseases such as vasculitis.Our observation that plasma NfL concentration correlates with disease severity in CMT suggests that plasma NfL may also show promise as a biomarker of disease activity. The CMT neuropathy and examination scores are clinical outcome measures based on a patient's symptoms, clinical examination, and neurophysiology and are currently the gold standard for measuring disease severity for patients with CMT.24 This finding also suggests that plasma NfL concentration may show promise as a biomarker of disease activity in other demyelinating peripheral neuropathies such as Guillain-Barré syndrome and chronic inflammatory demyelinating polyneuropathy.Although neurofilaments are axonal cytoskeletal proteins, plasma concentration was similarly raised in both axonal and demyelinating forms of CMT. This is perhaps unsurprising in view of the importance of Schwann cell axonal interaction in neuronal maintenance and previous observations that the degree of disability in demyelinating CMT1A is due to the degree of axonal loss rather than the degree of conduction velocity slowing (a surrogate marker of demyelination).We collected follow-up samples from 9 individuals with CMT at 1 year, revealing an intrasubject variability of plasma NfL concentration of 16.4%. As one would expect for a genetic disease such as CMT in which there is constant but slow progression, implying a constant level of peripheral axonal degeneration, we could not detect a change in plasma NfL concentration over 1 year. The plasma concentration of NfL is likely to be dependent on the rate of production , the volume of distribution, and the half-life. A successful treatment for peripheral neuropathy should lead to a reduction in axonal degeneration and a decrease in plasma NfL concentration. Further studies will be required to assess the sensitivity of plasma NfL for detecting individual temporal changes in axonal degeneration over time.In this study, we have shown that plasma NfL concentration is sensitive to detecting peripheral axonal damage in CMT, that it correlates with disease severity, and that there is little intrasubject variability. We have not demonstrated the responsiveness of plasma NfL concentration to change in the rate of axonal degeneration, a vital step in assessing the suitability of plasma NfL concentration in monitoring response to therapy in peripheral neuropathy and as a biomarker for treatment trials in CMT. Further studies addressing this point are currently in progress.


Neurospora crassa, blocking the function of the standard mitochondrial electron transport chain results in the induction of an alternative oxidase (AOX). AOX transfers electrons directly from ubiquinol to molecular oxygen. AOX serves as a model of retrograde regulation since it is encoded by a nuclear gene that is regulated in response to signals from mitochondria. The N. crassa transcription factors AOD2 and AOD5 are necessary for the expression of the AOX gene. To gain insight into the mechanism by which these factors function, and to determine if they have roles in the expression of additional genes in N. crassa, we constructed strains expressing only tagged versions of the proteins. Cell fractionation experiments showed that both proteins are localized to the nucleus under both AOX inducing and noninducing conditions. Furthermore, chromatin immunoprecipitation and high throughput sequencing (ChIP-seq) analysis revealed that the proteins are bound to the promoter region of the AOX gene under both conditions. ChIP-seq also showed that the transcription factors bind to the upstream regions of a number of genes that are involved in energy production and metabolism. Dependence on AOD2 and AOD5 for the expression of several of these genes was verified by quantitative PCR. The majority of ChIP-seq peaks observed were enriched for both AOD2 and AOD5. However, we also observed occasional sites where one factor appeared to bind preferentially. The most striking of these was a conserved sequence that bound large amounts of AOD2 but little AOD5. This sequence was found within a 310 bp repeat unit that occurs at several locations in the genome.In  Proper mitochondrial function requires coordinated expression between the mitochondrial and nuclear genomes. Although there is considerable variation in the coding capacity of mitochondrial DNA among different groups of eukaryotes  of mitochondria contains four large enzyme complexes as well as two smaller electron carriers, ubiquinone and cytochrome make ATP . Many orIn most fungi, AOX is undetectable, or present at very low levels, under standard laboratory growth conditions. However, inhibition of the sETC results in induction of AOX. This may occur via the action of chemical inhibitors, such as antimycin A or cyanide, which affect specific complexes of the sETC, or by mutations that decrease the function of sETC components  consisting of two CGG triplets separated by seven nucleotides, found in the promoter region of the AOX-encoding aod-1 gene  and A. nidulans  are also known to be required for the expression of phosphoenolpyruvate carboxykinase (PEPCK) and fructose-1,6-bisphosphatase (FBP), two enzymes which are crucial for the process of gluconeogenesis , which showed that the proteins bind to the promoters of their target genes in both inducing or noninducing growth conditions. The ChIP-seq study also showed a wider involvement of the factors in controlling aspects of cell metabolism outside of AOX and gluconeogenesis.Here, we further define the mechanism of regulation that AOD2 and AOD5 play in N. crassa strains was as described . Finally, they were examined for their ability to produce wild-type levels of AOX when grown in the presence of Cm. The isolates described in For localization studies and ChIP-seq analysis, strains were developed that expressed only tagged versions of either AOD2 or AOD5. The tags were either triple myc or triple hemagglutinin (HA). Tags were inserted at either the N- or C-terminus of the proteins . Genes eaod-2 aod-5 mutant strain DX-13  was followed. However, after pelleting the mitochondria by centrifugation, 1 ml of the supernatant was processed further. The supernatant sample was centrifuged at 48,000 rpm  in a Beckman TLA-55 rotor for 1.5 hr at 4°. The pellet was suspended in 150 μl SEMP buffer  as the microsomal or PMP fraction, whereas the supernatant was saved as the cytosolic fraction.To isolate cytosol and microsomes  the mitochondrial isolation procedure . The suspension was gently rocked for 2 hr at 4°. Insoluble material was removed by centrifugation at 13,000 rpm  for 30 min at 4° in a Sorvall Biofuge fresco centrifuge. The supernatant containing salt-extracted proteins was loaded onto a desalting column  which was placed into a fresh 1.5 ml Eppendorf tube. The desalting column was centrifuged at 1500 × g (4000 rpm) for 2 min at 4° in a Sorvall Biofuge fresco centrifuge. The flow-through was immediately used in coimmunoprecipitation experiments with the Pierce ProFoundTM HA or c-Myc Tag IP/Co-IP kit (Thermo Scientific).To extract nuclear proteins, purified nuclei (100 μg protein) were suspended in 60 μl of suspension buffer [25 mM sucrose, 50 mM Tris-HCl (pH 7.5), 5 mM MgClChIP-seq was performed on eight separate samples. Strain AOD2-C-HA-8  was grown in both the presence and absence of Cm. ChIP was done on both samples using an antibody against the HA tag. As controls, wild-type cells (NCN251) were also grown in both the presence and absence of Cm. ChIP was also done on these samples using the same antibody to the HA tag. Similarly, strain AOD5-C-Myc-4  was grown in the presence and absence of Cm and ChIP was performed on both samples using antibody against the myc tag. Again, control wild-type cultures were grown in the presence and absence of Cm and ChIP was performed using the same antibody against the myc tag. This resulted in four data sets, when controls were subtracted from experimental results.8 conidia in 100 ml Vogel’s medium, and were grown for 12 hr at 30° in the absence of Cm, or 14 hr in the presence of Cm. The final isolated ChIP-seq DNA was further treated using a Covaris S2 ultrasonicator to obtain DNA fragments with an average size of 200 bp for use in library construction. The sequence data for determination of binding sites of AOD2 following growth in the presence of Cm were obtained using an Illumina HiScan SQ with read lengths of 51 nucleotides. All other data were obtained using an Illumina HiSeq2000 with read lengths of 101 nucleotides. Covaris treatment, library construction, and DNA sequencing were performed by Delta Genomics .The protocol for ChIP was as described (N. crassa Broad Institute Assembly 10 with BWA using default parameters (https://github.com/taoliu/MACS/). The q value cutoff was set to 0.05. However, after selecting those peaks in each data set with fourfold or higher enrichment over the corresponding control, the highest q value in the data sets was 0.0022. The online program “Venny” was used to determine peaks in common to different data sets (http://ascobase.cgrb.oregonstate.edu/cgi-bin/gb2/gbrowse/ncrassa_public/). Functional Catalogue (FunCat) analysis of genes  or 14 hr (in the presence of Cm). RNA was isolated using the QIAGEN  RNeasy Plant Mini Kit. RNA integrity was measured using an Agilent 2100 Bioanalyzer system and the Agilent RNA 6000 Nano kit (Agilent Technologies). Only those samples that had an RNA integrity number value of >9 were used for cDNA synthesis. First-strand cDNA was synthesized using SuperScript III Reverse Transcriptase (Invitrogen). Reactions were performed using KAPA SYBR Fast qPCR reagents (KAPA Biosystems) and the StepOnePlus qPCR system . Data were analyzed using StepOne software (Applied Biosystems). The β-tubulin gene was the internal control for ΔΔCt quantification. The qPCR data for each gene examined were obtained from four biological replicates, each with three technical replicates.N. crassa  or absence (AOX-noninducing conditions) of Cm. Cells were harvested and various subcellular fractions were isolated. Analysis of strains expressing a C-terminal HA-tagged AOD2 (AOD2-HA) and a C-terminal myc-tagged AOD5 (AOD5-myc) revealed that both proteins were only detectable in the nuclear fraction and their localization was not influenced by growth in the presence or absence of Cm .Although ce of Cm . The levin vitro findings have shown that protein fragments containing the DNA-binding domains of AOD2 and AOD5 act as a heterodimer when binding DNA. To demonstrate that the two full-length proteins associated in vivo, we performed pull-down experiments using proteins extracted from nuclei isolated from a strain expressing differentially tagged versions of both proteins (AOD2-myc and HA-AOD5). We found that either tagged protein would coimmunoprecipitate the other , which is a preferred nitrogen source for Neurospora . Larger increases were seen following growth in acetate and ethanol. Growth in glycerol as the carbon source had virtually no effect on the level of PEPCK transcript, probably because glycerol enters the gluconeogenic pathway after the action of PEPCK. The mRNA for FBP   . The peaCU07953) . The mosCU07953) . Though aod-2, and Δaod-5 cells, each grown in the presence and absence of Cm. Eleven of the genes examined showed greater than twofold decreased expression in cells lacking either AOD2 or AOD5 in both of the growth conditions . Conceivably, unknown conditions may elicit a response in these genes which is mediated by AOD2 and AOD5, with or without contributions from other protein factors.Examination by qPCR of the expression of 13 genes from Unexpectedly, five genes showed less than twofold change in expression in the absence of AOD2 or AOD5, but did show two to fourfold increases in expression when cells were grown in the presence of Cm, regardless of whether AOD2 or AOD5 was present . Two of aod-2 Δaod-5 double mutant strain and examined transcript levels for certain genes requiring AOD2 and AOD5 for expression. There was virtually no change in transcript levels when the single mutants were compared with the double mutants , found 170 bp upstream of the predicted aod-1 start codon, bound AOD2 and AOD5 only when both proteins were present, suggesting that they bind as a heterodimer. The element was also required for maximal expression of the aod-1 gene in N. crassa . Of those 54, 23 were found to be in seven classes that were over-represented in the sample, relative to known genes in the N. crassa genome, with 11 of the 23 genes listed in two or more functional categories (Table S1). The seven classes fall into three of the main FunCat categories: energy, metabolism, and cellular transport. Eleven of the genes found in the FunCat analysis were among those analyzed by qPCR for expression differences in the absence of AOD2 or AOD5. Seven were found to have altered expression while no effects were observed for four others. However, the appearance of these genes in functionally enriched categories argues that the binding of the transcription factors may be significant, but that proper conditions for altering expression have not yet been discovered.The 98 genes in P. anserina microarray study that was not found in any of the groupings described here using the criteria chosen. Six genes showed no differences greater than twofold in any pairwise comparisons . One of the genes in this category was NCU04797, which encodes N. crassa FBP. Nine of the genes examined showed an over twofold expression difference in at least one pairwise comparison of the different conditions examined. Of those, one  shows an increase in the absence of AOD2 when grown in the presence of Cm  that contain the originally identified 14 bp sequence. The inverted repeats may have functional significance since they are more highly conserved than the region between them (The upstream regions of the genes listed in identity . There aeen them .Neurospora genome are subject to a mutagenic process termed repeat induced point mutation (RIP) .Repeated sequences in the on (RIP) . Althougon (RIP) . It appeon (RIP) . Of the Figure S6), as compared with most “typical” peak sets observed in this study, such as for NCU07953 (AOD1) and NCU06940  . The variable amount of AOD5 bound at the other repeat sites is also shown by the fact that five of these peak sets were identified in the original group of 70 that had greater than fourfold enrichment in all four experiments. Another characteristic of the 10 sites that bind predominantly AOD2 is that the AOD2 peak is usually divided into two summits. The AOD2 peak summits at these sequences are always found associated with all or a portion of the highly conserved 14 bp sequence  . One obvsequence  that occsequence .Figure S2). NCU02128 was found to depend on AOD2 but not AOD5 for expression , and it was identified in the group of 65 peaks common to all four experiments. An AIM sequence is also associated with the summit where AOD5 is most abundant . For NCU04392 and NCU09144, small increases in transcription were seen in strains lacking AOD5 , and it is difficult to imagine a negative regulatory role for AOD5 at these promoters. An alternative explanation might be that if AOD5 is not present, more AOD2 is available for binding and affecting transcription.The qPCR analyses in strains lacking AOD2 or AOD5 that were performed during the investigation of genes described above, included eight genes associated with the nine peak sets occurring in the repeated regions that have at least one gene in the proper orientation. Genes NCU03409, NCU05202, and NCU05203 showed no changes greater than twofold . Furthermore, no AIM sequences were found to be associated with these peak sets. All three of these genes were examined by qPCR in the strains lacking AOD2 or AOD5, and no changes greater than twofold were observed .Visual inspection of the ChIP-seq data also revealed two additional peak sets (NCU01053 and NCU01224/NCU01225) with an excess of AOD2 binding over AOD5, as compared with the more typical peak sets. These genes are not associated with the repeat sequence, they do not have the conserved 14 bp sequence in their upstream regions, and they do not exhibit the “double summit” pattern of AOD2 binding seen at the repeat sites , while that of FBP was not.The orthologs of AOD2 and AOD5 in aod-1 mRNA level was not significantly affected by growth in poor carbon sources, but was induced ∼18-fold by growth in the presence of Cm. Despite these differing patterns of expression for PEPCK and AOX, both inductions required AOD2 . Similar observations were made in P. anserina where the expression of AOX and several other genes was seen to be much higher during growth in the presence of antimycin A compared with when cultures were grown using acetate as a carbon source. However, the opposite was observed for the expression of the genes encoding PEPCK and FBP  in low nitrogen-containing medium, but sevenfold in nitrogen-rich Vogel’s medium. Similarly, Cm induced AOX 18-fold in low nitrogen-containing medium, but ∼30-fold in Vogel’s medium. This suggests that the general nutritional status of a culture may also influence the regulation. Taken together, these data strongly suggest that different signals arising from various conditions of growth can differentially affect the transcription of a number of genes in an AOD2- and AOD5-dependent fashion. Whether the interpretation of different signals can be achieved by AOD2 and AOD5 alone, or if additional factors are required, is not known.aod-2 and aod-5 grew at a reduced rate compared with wild-type cells, even when sucrose was the carbon source. This suggested that the proteins may play roles in regulating other pathways, in addition to AOX and gluconeogenesis. The results of the ChIP-seq experiments, where a large number of peaks were identified by MACS2 analysis in each of the four experiments, supported this idea. As described in the Results, we chose to focus on peak sets that fulfilled various criteria, leaving 98 genes as candidates for being controlled by AOD2 and AOD5. Applying these criteria may result in the exclusion of some genes that are legitimate targets, but should help minimize the number of false positives. Inspection of the peaks revealed a wide array of binding characteristics with respect to size and shape of peaks. This may reflect different binding efficiencies which could be due to sequence variation, involvement of additional binding factors, or state of chromatin in different regions of the growing mycelium.The growth analysis also revealed that deletion mutants of aod-1, were regulated in a positive fashion that required both AOD2 and AOD5 , points to a complex mechanism of transcriptional control involving AOD2, AOD5, other factors, and possibly different signals resulting from growth in the presence of Cm.An additional five genes examined by qPCR showed no effect on transcript levels when AOD2 or AOD5 were missing, but did show increased transcript levels in the presence of Cm . The facMany genes examined during our validation process failed to show differences in expression in the absence of either protein or in the presence or absence of Cm. Some of these may simply represent false positives, which can be frequent in ChIP-seq studies , but not RSE2 (the AOD2 ortholog), results in reduced fertility . However, no influence of either AOD2 or AOD5 on transcript levels of the gene could be seen during growth in standard conditions or in the presence of Cm.Twelve peak sets were identified as binding larger amounts of AOD2 compared with AOD5. Two of these sets had no obvious defining characteristics. However, 10 of the sets were associated with the 310 bp repeat region. In nine of these 10 cases, the repeat region is in a position that corresponds to the upstream region of one or both of the flanking genes. Seven of these genes were examined by qPCR for effects on expression due to Cm and loss of AOD2 or AOD5. A variety of effects were observed so that for the conditions tested it cannot be concluded that AOD2 is required for expression of genes at all these sites. Only one of the 11 repeated regions with >75% identity found in the genome was not associated with AOD2 binding. It is possible that the arrangement of chromatin, or other bound proteins at this locus under the conditions tested, prevents access for AOD2.Neurospora species available at FungiDB (http://fungidb.org/fungidb/) revealed that N. tetrasperma contained four sequences with high identity to the entire 310 bp region. N. discreta had no sites related to the entire 310 bp sequence but did have two smaller sites with high similarity to portions of the inverted repeat region. Thus, the sequence may have originated in an ancestral species, but is only being maintained in N. crassa. A consensus sequence was identified at the summit of the AOD2 binding sites, but further work is required to determine if it is actually a binding site for the protein.The origin of the 310 bp repeat regions and the relevance of AOD2 binding within them are not apparent. BLAST searches of other N. crassa  in our data set that is categorized simply as a pentatricopeptide repeat protein in the database, but has now been shown to be involved in the assembly of respiratory complexes in S. cerevisiae, the production of enzymes necessary for the synthesis of α-ketoglutarate are under the control of the classic retrograde response (RTG) system originally discovered in this organism. Thus, rtg mutants in S. cerevisiae are glutamate auxotrophs in conditions where respiratory activity is low  of the four genes encoding these proteins were present in the group of 65 peaks common to all four ChIP-seq experiments. The peaks associated with NCU02475 were quite robust and qPCR analysis confirmed that AOD2 and AOD5 were required for optimal expression. Peak sets associated with the other two genes were very small. Expression of NCU08877 was not affected by the absence of AOD2 or AOD5. NCU06424 expression was not examined. However, all three sets of peaks were associated with AIM sequences at their summits. Both glutamate and glycine are required for the synthesis of glutathione. Glutathione is an important molecule that provides a response to various cellular stresses, including reactive oxygen species in mitochondria (The glycine enzymes identified in the current study are named for the glycine cleavage system, but the system is also known as glycine synthase and can act in the reverse direction to synthesize glycine . The comA. nidulans (It has been suggested that malate is an inducing molecule for the AOD2 and AOD5 orthologs in In conclusion, our data show an involvement of AOD2 and AOD5 in the expression of several genes that play a role in energy metabolism and various metabolic pathways. The factors appear to act predominantly in a positive fashion, but examples of repressing activity were also seen. The majority of genes appear to bind the transcription factors as a heterodimer, but rare cases of preferential binding of one or the other were also seen. The mechanism of function of the proteins is complex, allowing different responses to different conditions.www.g3journal.org/lookup/suppl/doi:10.1534/g3.116.035402/-/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.Click here for additional data file.Click here for additional data file.Click here for additional data file.Click here for additional data file.Click here for additional data file.Click here for additional data file.Click here for additional data file.Click here for additional data file.Click here for additional data file.Click here for additional data file.Click here for additional data file.Click here for additional data file.Click here for additional data file.Click here for additional data file.Click here for additional data file.Click here for additional data file.Click here for additional data file.