Detection of SARS-CoV-2 infections to date has relied heavily on RT-PCR testing. However, limited test availability, high false-negative rates, and the existence of asymptomatic or sub-clinical infections have resulted in an under-counting of the true prevalence of SARS-CoV-2. Here, we show how influenza-like illness (ILI) outpatient surveillance data can be used to estimate the prevalence of SARS-CoV-2. We found a surge of non-influenza ILI above the seasonal average in March 2020 and showed that this surge correlated with COVID-19 case counts across states. If 1/3 of patients infected with SARS-CoV-2 in the US sought care, this ILI surge would have corresponded to more than 8.7 million new SARS-CoV-2 infections across the US during the three-week period from March 8 to March 28, 2020. Combining excess ILI counts with the date of onset of community transmission in the US, we also show that the early epidemic in the US was unlikely to have been doubling slower than every 4 days. Together these results suggest a conceptual model for the COVID-19 epidemic in the US characterized by rapid spread across the US with over 80% infected patients remaining undetected. We emphasize the importance of testing these findings with seroprevalence data and discuss the broader potential to use syndromic surveillance for early detection and understanding of emerging infectious diseases. The ongoing severe acute respiratory syndrome–coronavirus 2 (SARS-CoV-2) pandemic continues to cause substantial morbidity and mortality around the world  yet test negative for influenza . The US-wide ILI surge appeared to peak during the week starting on March 15 and subsequently decreased in numerous states the following week; notable exceptions are New York and New Jersey, two of the states that were the hardest hit by the epidemic, which had not started a decline by the week ending March 28.We identified excess ILI cases by first subtracting cases due to influenza and then subtracting the seasonal signal of non-influenza ILI . Our appWe estimated the ILI surge in each US state as an increase in the proportion of outpatients with ILI in that state compared to all outpatient visits in that state. Consequently, changes in the care-seeking behavior of individuals with ILI or of non-ILI during this time period could each affect our estimates of disease prevalence during the ILI surge. If patients with mild ILI were more likely seek medical care during the month of March 2020 than in prior years, then our estimates of COVID-19 prevalence based on the ILI surge would be falsely elevated. Additionally, if non-ILI patients were less likely to seek medical care during the month of March compared to prior years, then this too could falsely elevate our estimates of COVID-19 prevalence based on the ILI surge. Although ILINet does not provide information to ascertain care seeking behavior, we were able to obtain syndromic surveillance data from New York City’s emergency departments, which provided up to date information on care seeking behavior of both ILI and non-ILI conditions . This observation suggests that patients with mild ILI presented less often to hospital emergency departments. Such a decrease in care-seeking behavior for mild ILI, if similar across the US, could deflate the estimated size of the ILI surge in the later weeks of March by a factor of approximately 3.If non-ILI patients were less likely to seek medical care, then we would expect that the number of patients complaining of other symptoms not typically associated with COVID-19 (for example vomiting) would also decrease compared to prior years. In the month of March, the daily number of patients presenting with vomiting decreased by as much as a factor of 3 compared to the baseline rate in prior years (fig. S3B). Assuming that all non-ILI conditions were similarly decreased during March, this would suggest that our estimates of the ILI surge could be inflated by as much as a factor of 3. This assumption is conservative as it assumes that even individuals with severe conditions (such as severe trauma) would avoid seeking health-care in response to COVID-19 at the same rate as those with more mild conditions such as vomiting. However, the potential 3-fold decreased care-seeking behavior for non-ILI conditions cancels out the potential 3-fold decreased care-seeking behavior of mild ILI, suggesting that our estimates of prevalence based on the ILI surge may be insensitive to recent changes in care-seeking behavior (fig. S3C). Overall these estimates suggest a conceptual model in which health care utilization for both mild ILI and non-ILI conditions declined at similar rates as COVID-19 increased in the US.To estimate the proportion and magnitude of the March 2020 US ILI surge attributable to SARS-CoV-2 infections, we made the following three assumptions: (1) that the patient population reported by sentinel providers is representative of their state each week; (2) that changes in care-seeking behavior of ILI patients is occurring at a similar rate as that of other non-ILI patients; and (3) that the total number of patients in the US who require medical care over the course of a year has not substantially changed since 2018. Our first assumption is common and underlies prior studies which have used ILI to estimate influenza prevalence . This estimate likely represents an underestimate given that the majority of passengers were over 60 years old, a demographic thought to have a lower asymptomatic rate than younger individuals (We define the syndromic case detection rate as the number of confirmed COVID-19 cases in a week divided by the size of the ILI surge that week. The syndromic case detection rate varied by state and over time (fig. S5). Our estimated syndromic case detection rates increased over the month of March; this was expected given increases in testing capacity across the US since the February 28 detection of community transmission in Washington State. For the week ending March 14, COVID-19 cases in the states with the highest estimated syndromic case detection rate  only captured approximately 1% of ILI surges in those states. In the last week of the month ending on March 28, the syndromic case detection rate across the US increased to 12.5% .The true prevalence of SARS-CoV-2 is unknown at the time of this writing. However, if we assume the excess non-influenza ILI is almost entirely due to SARS-CoV-2, an assumption that becomes more valid as SARS-CoV-2 becomes more prevalent, we can use the excess non-influenza ILI to define lower bounds on the exponential growth rate of the US SARS-CoV-2 epidemic. By estimating the number of patients visiting clinics for COVID-19 in the US in March, we can also identify the mutual dependence of exponential growth rates, the rate of sub-clinical infections, and the time between the onset of infectiousness and a patient reporting as ILI . Using sIn keeping with our sub-4 day doubling times, we found that across the entire US, new deaths due to COVID-19 doubled every 3.01 days over the month of March . If there was only a 1-day lag from onset of infectiousness to presentation with ILI and the entirety of the first week of the US ILI surge is comprised of patients with COVID-19, then an epidemic starting January 15th and growing at the rate of deaths in the US would imply a 12% clinical rate . A four-Faster growth rates require lower clinical rates to explain the ILI surge. Epidemic curves growing at the rate of deaths in Italy, doubling every 2.65 days, could better match the curvature of the ILI surge by peaking around mid to late March, but would imply a clinical rate of 4.7% the second week of March with a 4-day lag between onset and recorded as ILI . If the Last, estimating the infection fatality rate from the ILI surge requires knowing the clinical rate and the delay from clinical presentation with ILI to death. If patients present with ILI at the onset of their illness, exhibit a 16 day median lag between onset and death  suggests that between March 8 to March 28 other monitored respiratory viruses were at low prevalence .Throughout our methods, we use Since 2010 the CDC has maintained ILINet for weekly influenza surveillance. Each week approximately 2,600 enrolled providers distributed throughout all 50 states as well as Puerto Rico, the District of Columbia, and the US Virgin Islands, report the total number of patient encounters https://worldpopulationreview.com/states/. The number of primary care providers in each state per 100,000 residents https://github.com/nytimes/covid-19-data. This dataset contains the daily cumulative confirmed case count for COVID-19 for each state l. The dataset of deaths in Italy was downloaded from https://github.com/pcm-dpc/COVID-19 on April 6, 2020.In addition to ILINet data, we downloaded US State population data for the 2020 year was downloaded from Within the ILINet dataset, New York City and New York were summed into a combined New York variable representing both New York City and the surrounding state. Due to incomplete data in one or more of the data-sources described above the Virgin Islands, Puerto Rico, The Commonwealth of the Northern Mariana Islands, and Florida were excluded from subsequent analysis. In addition, to match the weekly reporting of ILI from ILINet, daily cumulative confirmed COVID-19 cases were converted to weekly counts of new cases byTo subtract influenza signal from Mean imputation based on neighboring states was used to address missing values in laboratory influenza quantification. To assess the impact of this model for extracting non-influenza ILI signal, we calculated COVID-19 prevalence without first removing signal from influenza, we found little change in our prevalence estimates (fig. S7). This likely reflects that influenza also demonstrates strong seasonal patterns that can be addressed as discussed below.We identified ILI surges inbasset from the R package stray . Still, we used the GP-derived estimates throughout this paper due to their better accounting for the known binomial count and week-to-week correlation structure of ILI-causing pathogen prevalence.To exclude variation attributable to unseasonably high rates of other ILI causing viruses (such as the outbreak of RSV in Washington state in November-December 2019) we only investigated i has COVID-19 as estimated from ILINet data. Let i and let i. We translated the inferred proportion of individuals with ILI due to COVID-19 to the state level by considering the average number of patients seen across all providers in the state in a 5-day work-week. In addition, we added a discount factor λ=0.55 to calibrate these estimates with prior reports regarding the total number of outpatient visits per year . A plot of the fatality rate by lag for raw and unadjusted ILI surges revealed a large range of fatality rates compatible with the ILI surge and highly sensitive to the estimate of lag and clinical rates. One study  reflecting empirical estimates of a median 5 days from exposure to symptom onset with 4.2-6 day 95% credible interval (I(t) grew with a specified exponential growth rate early in the epidemic. We ran a total of 2,000 simulations for each of the two growth rate distributions  analyzed. Growth rates were drawn at random from a normal distribution with standard deviation of 0.1 and centered on The following SEIR modelsI(t) for that day, and a weekly pool of infected patients was computed by a moving sum over the number of infected individuals every day for the past week, Each simulation was initialized with Defining


As the coronavirus disease 2019 (COVID-19) pandemic unfolds, laboratory services have been identified as key to its containment. This article outlines the laboratory organisation and management and control interventions in Niger.Centre de Recherche Médicale et Sanitaire was designated as the reference centre for COVID-19 testing. The national plan for COVID-19 testing is being gradually adopted in other regions of the country in response to the rapidly evolving COVID-19 emergency and to ensure a more rapid turn-around time.The capitol city of Niger, Niamey, adopted a ‘National COVID-19 Emergency Preparedness and Response Plan’ to strengthen the preparedness of the country for the detection of severe acute respiratory syndrome coronavirus-2. Laboratory training and diagnostic capacity building were supported by existing active clinical and research laboratories for more rapid and practicable responses. The National Reference Laboratory for Respiratory Viruses located at the After the decentralisation of COVID-19 testing to other regions of the country, turn-around times were reduced from 48–72 h to 12–24 h. Reducing turn-around times allowed Niger to reduce the length of patients’ stays in hospitals and isolation facilities. Shortages in testing capacity must be anticipated and addressed. In an effort to reduce risk of shortages and increase availability of reagents and consumables, Niamey diversified real-time reverse transcriptase–polymerase chain reaction kits for severe acute respiratory syndrome coronavirus-2 detection.Continued investment in training programmes and laboratory strategy is needed in order to strengthen Niger’s laboratory capacity against the outbreak. Coronaviridae and the genus Betacoronavirus.3 Worldwide, real-time reverse transcriptase–polymerase chain reaction (rRT-PCR) is the gold standard method used to detect the presence of SARS-CoV-2 RNA in clinical specimens.In December 2019, a new viral respiratory infection emerged. The infection was first detected and reported in the Chinese province of Hubei and has since spread globally, affecting people and socio-economic development in both developing and developed countries. The disease, later named the coronavirus disease 2019 (COVID-19) and declared a global pandemic by the World Health Organization, is caused by a species of coronavirus known as severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), which belongs to the family 4 These figures make Africa the continent least affected by this pandemic, with the most significantly affected countries in the region being South Africa (70 038 cases), Nigeria (16 085 cases), Ghana (11 422 cases), and Algeria (10 919 cases).4As of 15 June 2020, 175 503 laboratory-confirmed COVID-19 cases were reported in Africa, with 4111 deaths.5The index case in the Republic of Niger was detected on 19 March 2020. This was an imported case involving a 36-year old man who arrived by road from Burkina Faso. Since the official declaration of the index case, a total of 980 cases have been confirmed as of 15 June 2020.As this pandemic unfolds, laboratory services have been identified as key to containment efforts. This article outlines the laboratory organisation and management and control interventions in Niger.This article followed all ethical standards for research without direct contact with human or animal subjects.https://tinyurl.com/y2x3vpmt) to strengthen Niger’s preparedness for the detection of SARS-CoV-2. This plan details the procedures for containment, contact tracing and screening based on the risk of spread. The plan is structured around five major strategic axes including reinforcement of coordination, strengthening of epidemiological surveillance, strengthening of health services capacities, reinforcement of risk communication and community engagement, and, lastly, the creation of isolation facilities.Severe acute respiratory syndrome coronavirus-2 is an emerging respiratory pathogen spreading rapidly through the general population. Niamey, the capitol city of Niger, adopted the ‘National COVID-19 Emergency Preparedness and Response Plan’  to enable expedited building and implementation of SARS-CoV-2 testing capacity in the eight regions of the country in tune with the expansion of the pandemic. The Laboratory and Research Team, which is charged with the responsibility of improving laboratory capacity and capability, is structured into three groups comprising a pre-analytical group , an analytical group (rRT-PCR testing) and a post-analytical group .Centre de Recherche Médicale et Sanitaire (CERMES) was designated as the reference centre for COVID-19 testing. The Veterinary Research Laboratory ‘Laboratoire Central de l’Elevage’, the National Reference Laboratory for HIV and Tuberculosis, the National Hospital of Niamey and the Research Institute for Sustainable Development supported the laboratory response by providing equipment for PCR techniques. Moreover, seven laboratory technicians from these centres, who have had extensive training on PCR techniques, were mobilised by the Ministry of Health, in addition to the CERMES technicians, to support the response by performing the rRT-PCR testing at CERMES, which received and analysed samples collected from across the country. As part of COVID-19 response, Niamey and its partners provided logistical air support for sample transport from remote regions such as Diffa, Zinder, Maradi, Tahoua and Agadez.For more rapid and practicable responses, laboratory diagnostic capacity building is being supported by existing active clinical and research laboratories. The National Reference Laboratory for Respiratory Viruses located at the Gradually, the national plan on COVID-19 testing is being adopted in other regions of the country in response to the rapidly evolving COVID-19 emergency and to ensure a more rapid turn-around time (TAT). This adoption involves gradual decentralisation of the SARS-CoV-2 RNA rRT-PCR assays to three regions, Tahoua, Maradi and Zinder, located 550, 662 and 891 kilometres from Niamey . The choThe Tahoua laboratory analysed samples collected from the Tahoua and Agadez regions. Samples collected from the Zinder and Diffa regions were analysed in Zinder, whereas the Maradi laboratory analysed samples collected in the Maradi region. Samples collected from the Niamey, Dosso and Tillabery regions were analysed at CERMES. Logistics and testing capacity are handled by CERMES, which reported the availability of the reagents and materials needed to sample patients and to perform the rRT-PCR on a weekly basis to the Ministry of Health.6 The interim guidance from the World Health Organization suggests that non-propagative processing and handling of specimens containing SARS-CoV-2 must be performed in standard biosafety level-2 laboratories.8 Consequently, Niamey leveraged the safe practices and techniques, safety equipment and appropriate facility design of CERMES to reduce or eliminate exposure of laboratory workers to SARS-CoV-2-containing materials according to the World Health Organization requirements.7 Before the decentralisation of SARS-CoV-2 RNA rRT-PCR assays to other regions, samples collected from across the country were sent to CERMES. Samples from remote regions such as Diffa, Zinder and Agadez were transported using flights in accordance with category B transportation regulations.9 In an effort to help close contact management, sample collection was performed during home visits.Severe acute respiratory syndrome coronavirus-2 is classified as a Risk Group 3 human pathogen, similar to Middle East respiratory syndrome coronavirus and severe acute respiratory syndrome coronavirus.® Viral RNA Mini Kit (250)  or Nucleic Acid Isolation or Purification Reagent  according to the manufacturer’s instructions. Two teams of four laboratory technicians were dedicated to RNA extraction at CERMES. These teams were often overworked due to the large volume of samples handled.Ribonucleic acid extraction from samples was performed manually using QIAamp10 Two target genes, the open reading frame1ab (ORF1ab) and nucleocapsid protein (N), were simultaneously amplified. A cycle threshold value lower than 40 was defined as a positive test result, and a cycle threshold value of 40 or more was defined as a negative outcome according to the manufacturer’s protocol. Alternatively, rRT-PCR targeting the RdRp gene of the SARS-CoV-2 or SARS-like coronavirus was performed using the LightMix® Modular SARS-CoV-2 RdRp . Positive samples for RdRp gene were confirmed by performance of rRT-PCR for the detection of the E gene using LightMix® SarbecoV E gene plus equine arteritis virus control . Given the extraordinary demand for reagents and consumables, risk of supply shortages became the main issue. Niamey experienced a shortage of RNA extraction kits and this led to the prioritisation of the testing of vulnerable people, health professionals and patients requiring hospitalisation.Qualitative rRT-PCR assays were performed using the nucleic acid testing kit .As of 15 June 2020, a total of 5386 samples had been tested for SARS-CoV-2 in Niger. Of these, 980 (18.2%) were confirmed positive . All tesIn March and April 2020, before the decentralisation of SARS-CoV-2 RNA rRT-PCR assays to other regions, the number of samples received exceeded the capacity of the single centralised laboratory earmarked for the testing. Consequently, TATs were between 48 h and 72 h. This TAT length often created a disconnect between clinicians and laboratory staff. Indeed, according to the clinicians, the TAT of 48–72 h delayed treatment and increased patients’ length of stay, particularly in isolation facilities where patients are often asymptomatic. After decentralisation, on 15 June, 2020, the TAT had decreased significantly to 12 h at Maradi and between 12 h and 24 h at Tahoua, Zinder and CERMES, Niamey.Experience gained in the management of previous outbreaks  helped Niamey to build a quick response to the COVID-19 pandemic. However, additional control efforts are needed to improve the laboratory strategy and response against COVID-19 in the country. Firstly, a well-coordinated laboratory strategy and operational plan is needed in order to address the shortcomings of the existing plan. Moreover, considering the importance of improved SARS-CoV-2 laboratory capacity, the country should provide extensive training to laboratory technicians in preparation for rapid expansion of laboratory diagnostic capacity. Secondly, laboratory TAT is critical in determining the success of both the laboratory response programme and the management of patients. Reducing the TAT allowed reductions in patients’ length of stay in hospitals and isolation facilities. Thirdly, communication between clinical and laboratory staff needs to be improved in order to ensure that laboratory results are understandable to clinicians.11Fourthly, the shortages in testing capacity need to be anticipated and addressed. If capacity is exceeded, priority should be given to the testing of vulnerable patients, health professionals and patients requiring hospitalisation. Niamey diversified rRT-PCR kits for SARS-CoV-2 detection to reduce the risk of shortages and increase the availability of reagents. It has been demonstrated that RNA extraction kits from different manufacturers are interchangeable.12 It has been well documented that the limit of detection of different rRT-PCR kits can differ substantially.14 Therefore, care must be taken in the interpretation of the results when using different kits for the monitoring of patients. Fifthly, the overwhelming number of specimens to test highlighted the need for fast methods to extract viral RNA. Ribonucleic acid extraction from samples was performed manually in Niger. In addition to the high risk of contamination, manual extraction of viral RNA is time consuming and requires a large number of laboratory professionals.However, diversifying the type of rRT-PCR kits for SARS-CoV-2 detection can lead to some variation in the detection rate between kits.N, E, RpRd genes) could be used to avoid invalid results and increase sensitivity for detection of new genomic variants.16Lastly, amplification of multiple target genes (e.g. Despite TAT improvement in the laboratory management of COVID-19 testing, several additional control efforts are needed to improve the laboratory response against COVID-19 in Niger. Considering the importance of improved SARS-CoV-2 laboratory capacity, continued investment in training programmes and laboratory strategy is needed to systematically guide the laboratory response against the outbreak.


Expression quantitative trait loci (eQTL) can provide a link between disease susceptibility variants discovered by genetic association studies and biology. To date, eQTL mapping studies have been primarily conducted in healthy individuals from population-based cohorts. Genetic effects have been known to be context-specific and vary with changing environmental stimuli. We conducted a transcriptome- and genome-wide eQTL mapping study in a cohort of patients with idiopathic or heritable pulmonary arterial hypertension (PAH) using RNA sequencing (RNAseq) data from whole blood. We sought confirmation from three published population-based eQTL studies, including the GTEx Project, and followed up potentially novel eQTL not observed in the general population. In total, we identified 2314 eQTL of which 90% were cis-acting and 75% were confirmed by at least one of the published studies. While we observed a higher GWAS trait colocalization rate among confirmed eQTL, colocalisation rate of novel eQTL reported for lung-related phenotypes was twice as high as that of confirmed eQTL. Functional enrichment analysis of genes with novel eQTL in PAH highlighted immune-related processes, a suspected contributor to PAH. These potentially novel eQTL specific to or active in PAH could be useful in understanding genetic risk factors for other diseases that share common mechanisms with PAH. The relationship between genomic variability, disease risk and endophenotypes has been a focus of many research groups with access to suitable disease cohorts. This has been aided by the increasing affordability of genotyping and sequencing methodologies. Interpretation of the mechanisms behind the effects of genetic variants discovered to associate with phenotypes poses a challenge as many are located in the non-coding space of the genome . Non-codDifferential gene expression analyses comparing cases and controls may be useful for diagnostic and/or prognostic purposes or in identifying genes, which may have a causal role in developing the disease ,7,8. In Currently, one of the main limiting factors for causal inference analysis using genomic data is the number of known eQTL. Certain eQTL effects, which go undetected in healthy cohorts commonly used for eQTL mapping, may be unmasked by disease and development. The diagnostic value of gene expression profiles, i.e., that they can be used to distinguish affected individuals from non-affected ones, underlines the uniqueness of the set of genes expressed in a disease state ,7. DiffeWith the exception of housekeeping genes, the majority of genes in the human transcriptome are tissue-specific. Therefore, tissue selection for gene expression studies of disease is of importance. The lung is the most relevant tissue in the pathogenesis of pulmonary arterial hypertension (PAH), a disease in which pulmonary vascular remodelling drives right ventricular failure. Whole blood also has relevance to PAH and might capture more than just systemic effects given the immune component of this disease. The role of inflammation in PAH has been extensively studied, based on the observation that inflammatory cells infiltrate the remodeled vascular wall . Since PThis study aimed to characterise the genetic variability of transcriptome-wide gene expression in whole blood from 276 consecutively sampled patients with PAH and defined potentially novel eQTL active in this disease state. These potentially disease-specific eQTL could be useful in elucidating known genotype-phenotype associations or causal analyses.A total of 276 patients with idiopathic, heritable or drug-induced PAH diagnosed following international guidelines  were recn = 26,050) were taken forward to the eQTL analyses.RNA sequencing and transcript abundance estimation procedures are described in the p-value greater than 10−5 for Hardy Weinberg equilibrium, leaving a total of 7,362,566 variants for eQTL mapping. Software tools used in the eQTL mapping pipeline included PLINKv1.90 ) reported as lead variants for 4314 phenotypes. The list of GWAS variants were intersected with each PAH eQTL (including the lead eQTL and variants in LD with the lead eQTL [r2 ≥40%]). The difference between the proportions of novel eQTL and previously reported eQTL overlapping lung-related phenotypes and diseases curated from the full GWAS Catalog phenotype list was tested using the 2-sample equality of proportions test.An important aim of this study was to identify eQTL in PAH with effects that had not been observed in population-based studies and to assess the relevance of these PAH-specific eQTL to related phenotypes and diseases. The NHGRI-EBI GWAS Catalog of published genome-wide association studies (GWAS)  providedFunctional enrichment analysis of genes with novel eQTL in PAH using the WEB-based GEne SeT AnaLysis Toolkit   was cond2) in gene expression are presented in the 2) in gene expression levels was generally lower for trans-eQTL than for cis-eQTL  = 4.5, p-value = 8.3 × 10−6).We observed 2354 eQTL in total with the majority (90%) acting in cis A, accouncis-eQTL, 1509 (76%) could be mapped to the Illumina HT12v3 array used by Westra et al. and 2134 (95%) to the Affymetrix Human Exon chip used by Joehanes et al., respectively. Results from the GTEx Portal were obtained for 73% of eQTL in this study. Twenty-eight percent of cis-eQTL were confirmed by Westra et al., 31% by Joehanes et al. and 90% of tested cis-eQTL confirmed in GTEx. Overall, 75% of cis-eQTL were confirmed in at least one of the published studies .Out of the 1986 unique genes with  studies . The oveN GWAS traits/eQTL = 2; IQRN GWAS traits/eQTL = 3). Ninety-eight (11%) of the 929 eQTL had at least 10 or more unique GWAS phenotypes associated with them. cis-acting eQTL overlapping with at least one published GWAS association in the confirmed subset was 47%, while in the novel subset, it was 37%. Among trans-eQTL, the proportion of loci overlapping at least one GWAS trait was 50% in the confirmed and 27% in the novel group. The higher proportion of overlapping loci in the confirmed eQTL groups was significant for both cis- and trans-acting eQTL  = 13.9; 95% CI = 0.05–0.15; p-value = 0.0002. trans-eQTL: χ2(df=1) = 4.2; 95% CI = −0.003–0.45; p-value = 0.04). Interestingly, the proportion of novel eQTL previously reported for lung-related phenotypes (11%) was twice as high as the proportion of confirmed eQTL associated with lung-related phenotypes (5.5%) (χ2(df=1) = 6.9; 95% CI = 0.006–0.1; p-value = 0.009). Lung-related phenotypes reported for the novel eQTL included chronic obstructive pulmonary disease (COPD), lung function , low versus high forced expiratory volume, interstitial lung disease, emphysema, lung cancer and lung adenocarcinoma. The full list of 63 QTL overlapping lung-related phenotypes in the GWAS Catalog can be found in In order to assess the relevance of eQTL identified in this study to a wide range of phenotypes and diseases, the 2173 unique eQTL were intersected with the database of published genotype-phenotype associations from the GWAS Catalog. In total, 929 eQTL were reported for at least one trait previously (mediann = 18) and from the HLA class IB (n = 2) genes. Half of these eQTL-HLA gene associations were found to be novel. Out of the 5 trans- and 17 cis-eQTL, 3 trans- and 8 cis-eQTL were confirmed by published studies [We assessed the list of 606 genes with novel eQTL in the PAH Cohort for their involvement in certain biological processes or pathways in the KEGG knowledgebase. Five pathways were found to be significantly (FDR <0.05) enriched, with the pathways taste transduction, graft-versus-host disease and autoimmune thyroid disease being the three most significant . Supplem studies ,23.cis-eQTL and 84% of trans-eQTL were not found in any of the three studies used for confirmation. Novel and confirmed eQTL were investigated for their relevance to a wide range of diseases, and results focused on lung-related phenotypes from the NHGRI-EBI Catalog of published genome-wide association studies .Gene expression serves as an intermediate phenotype between genetic variants and associated phenotypes, such as clinical diagnoses, accepted biomarkers and anthropometric and behavioural traits. Previous eQTL studies provided support for the idea that genetic effects on gene expression have phenotypic consequences and that eQTL aid in the biological interpretation of associated genetic markers in disease ,4,5. Howcis- and trans-eQTL than for novel eQTL, the proportion of eQTL associated with lung-related phenotypes was twice as high among novel eQTL than confirmed eQTL previously detected in population-based studies. This indicates that these novel eQTL identified in blood samples of PAH patients might be highly informative for pulmonary diseases such as COPD, interstitial lung disease and emphysema. The higher proportion of GWAS-trait associated eQTL in the confirmed subset might be explained by the lack of replication of novel eQTL by an independent PAH cohort; therefore, it is expected that a proportion of these novel association signals is spurious.Nearly half (43%) of all eQTL identified in this study colocalised with at least one trait or disease in the GWAS Catalog. Even though the proportion of colocalising eQTL was higher for confirmed GTF2IRD2B cis-eQTL rs13238996 , a congenital heart defect characterised by the narrowing of the aorta, pulmonary and coronary arteries and other blood vessels [As an example for the overlap between lung-related GWAS traits and eQTL in PAH, the novel 13238996  overlaps vessels .HLA-DPA1/DPB1 locus encoding class II major histocompatibility complex (MHC) molecules [HLA-DPB1 alleles, all containing a glutamic acid at amino acid residue 69 (Glu⁶⁹). The HLA-DPA1/DPB1 PAH susceptibility locus emphasises the role of immune dysregulation in PAH development [Pathway analysis of genes associated with novel eQTL identified five biological processes, including four immune-related phenotypes enriched for this list of genes from the KEGG database. Six genes from the graft-versus-host disease and allograft rejection pathways overlap with the list of genes with novel eQTL, five of which belong to the Human Leukocyte Antigen (HLA) class. The immune-related pathways enriched for genes with novel eQTL in this study demonstrate that novel eQTL could be identified in disease populations since their gene expression profiles differ from the profiles of healthy individuals. The complex pathophysiological mechanisms behind PAH involve multiple driving factors of which immune dysfunction and inflammation are suspected to be among the major contributors . The importance of antigen-presenting and recognition in PAH is underlined by the most significant genetic variant discovered in the PAH GWAS in the olecules , which aelopment ,37 and wcis-eQTL confirmed in the two population-based studies by Westra et al. and Joehanes et al., while 90% of cis-eQTL were confirmed by the GTEx Project. Fifteen percent of trans-eQTL confirmed in either the GTEx project or the study of Joehanes et al. Trans-eQTL validation rates are generally much lower (under 10%) than validation rates of cis-eQTL [cis-eQTL and a stronger tissue-specificity of trans-eQTL effects compared to cis-eQTL, which may render trans-eQTL more susceptible to confounding by differing experimental conditions and environmental factors. However, the confirmation rate reported by this study and other studies possibly underestimate the true extent of eQTL overlap between studies since the list of variants and genes that passed quality control and were tested by any one study are usually not made available. We observed a higher confirmation rate with the GTEx Project that also used RNAseq for assaying gene expression. Similarly, Joehanes et al. reported higher validation rates between two array-based studies than between array-based and RNAseq-based studies. This may be related to platform differences, for example, hybridisation in arrays is less sensitive than high-depth sequencing and potentially affected by differing background signals in various tissues. Differences between the study populations being compared can also give rise to imperfect validation and also to novel discoveries, as all genetic effects depend on both the genetic (epistasis) and environmental context of the population they were estimated in and, therefore, do not necessarily apply to another population or the same population at a different time. A more accurate way of identifying novel eQTL in this study would have been to contrast PAH eQTL effects with eQTL effects estimated in a control population assayed on the same platforms and in one batch with the PAH samples to reduce the variability due to technical factors. In addition, novel eQTL are yet to be replicated in an independent population of PAH patients to identify true positives.Overall, the confirmation rate of eQTL in this study was comparable to that seen in published studies ,4. A thicis-eQTL ,4, refleThe effects of eQTL can vary by the tissue and even cell type under investigation ,38 and mTo date, large-scale eQTL-mapping has been done in healthy individuals from population-based studies , providing a valuable knowledge base for understanding associations between genetic variation and various phenotypes. It has been shown that by recapitulating the environmental context relevant to disease, it is possible to decipher the genetic variation of disease susceptibility more extensively . Genome- and transcriptome-wide eQTL-mapping in this cohort of idiopathic and heritable PAH patients identified hundreds of potentially novel eQTL with twice the proportion of lung disease associated with genetic variants than eQTL confirmed by population-based studies. Apart from pulmonary conditions, these novel eQTL specific to, or active in, PAH could be useful in understanding genetic risk factors for other diseases that share common mechanisms with PAH, such as those with immune dysregulation.


These cameras offer advantages, such as mobility and ease of access, and may be useful also for other applications such as biokinetic studies in animals or for imaging of small, superficial structures in patients. In this work, the suitability of a CZT-based hand-held camera for 177Lu imaging is investigated. The energy response of CZT-based detectors combined with the multiple photon emissions of 177Lu poses new challenges compared to 99mTc imaging, and a thorough camera characterisation is thus warranted.Currently, hand-held gamma cameras are being developed for 177 Lu]Lu-DOTA-TATE therapy.Three collimators  and three energy windows  are investigated. Characterised camera properties include the system spatial resolution, energy resolution, sensitivity, image uniformity, septal penetration, and temperature dependence. Characterisations are made starting from NEMA guidelines when applicable, with adjustments made when required. The applicability of the camera is demonstrated by imaging of a superficially located tumour in a patient undergoing [Overall, the results are encouraging. Compared to a conventional gamma camera, the hand-held camera generally has a higher sensitivity for a given collimator. For source-collimator distances below 3 cm, the spatial resolution FWHM is within 6 mm for the LEHR and MEGP collimators. Before uniformity correction, the central field-of-view integral uniformity shows best results for the 113-keV window, with values obtained between 11 and 14%. The corresponding values after uniformity correction are within 3%. Effects of septal penetration are observed but are manageable with a proper combination of collimator and energy window setting. Septal penetration and collimator scatter not only affect the 208-keV window but also contribute with counts in lower windows due to energy-tailing effects. The patient study revealed non-uniform uptake patterns in a region that appeared uniform in a conventional gamma camera image.177Lu imaging. A 113-keV energy window combined with LEHR or MEGP collimators provides the best image system characteristics.The results show that the hand-held camera can be used for  In order to obtain a compact camera head with improved spatial resolution, the detector technologies often differ from the conventional Anger design Lu-DOTA-TATE for the treatment of neuroendocrine tumours [177Lu]Lu-PSMA for prostate cancer  over the photopeak, where the interval typically ranged from −3 to +10% of the emission energy. A second energy interval  was then defined, with the lower energy limit adjusted using the parameter p. A function with the form:was fitted in an energy interval Lu-DOTA-TATE with activities of approximately 60 MBq, measured using a traceable Secondary Standard Dose Calibrator . The phantom was placed in the centre of the detector FOV, 3 cm from the collimator face, and images were acquired with the MEGP, LEHR, and LEHS collimators. The system sensitivity εW for an energy window W was calculated according to:In order to investigate the system sensitivity, a resealable phantom was constructed with a diameter smaller than the detector FOV and consisted of a cylindrical cavity  with 5-mm-thick PMMA walls and a top and bottom thickness of 1 mm. The phantom was filled with Lu-DOTA-TATE therapy. Imaging was made with the MEGP and LEHR collimators (1 day) after administration of 7.4 GBq [ 177Lu]Lu-DOTA-TATE. The acquisition time was 2.5 min, which for the 113-keV energy window yielded a total of approximately 12,000 counts and 20,000 counts, for the MEGP and LEHR collimators, respectively. To reduce the photon exposure of both the operator and the detector, a lead shield was placed between the patient’s torso and arm.To assess the practical applicability of the hand-held detector in a patient situation, images of a tumour, localised in the patient’s upper arm, were acquired for a patient receiving Lu-DOTA-TATE appeared to be more non-uniform than revealed by the conventional gamma camera image. When comparing the different energy windows, there were slightly more structural details in the tumour region in the 113-keV window. The counts along the image border, especially prominent in the 208-keV window and for the LEHR collimator, result from photons that emanated from outside the FOV and penetrated the detector shielding. Also for the 55-keV energy window, there were counts along the border. The lower yield of 54.6-keV and 55.8-keV photons, combined with a greater attenuation for these lower energies and low-energy tailing, may explain why these counts were more apparent for the 55-keV energy window than for 113 keV. The background count rate was lowest when using the 113-keV energy window, thus giving the highest contrast. Owing to the higher system sensitivity of the LEHR collimator, the total number of counts was approximately 60% (113 keV) to 100% (208 keV) higher than when using the MEGP collimator, and the choice between these two collimators thus mainly depends on the expected count rate.Patient images acquired with the CrystalCam system using the MEGP and LEHR collimators and the three energy windows are also shown Fig. 177Lu-labelled compounds. Originally, the detector is intended and optimised for 99mTc Lu-DOTA-TATE is non-uniform in the images from the hand-held camera. This difference can be attributed to the very short source-collimator distance, which has been made possible by the small size of the hand-held gamma camera. At such short distances, the hand-held gamma camera has a favourable spatial resolution (Fig. Visually, the images from the hand-held camera in Fig. ion Fig. . A limit177Lu in small, superficially located structures with modest activity in surrounding tissues and may in such situations be a more accessible alternative to conventional gamma cameras or small-animal imaging devices. The use of a 113-keV energy window in combination with an LEHR or MEGP collimator was found to provide the best results, with regard to the system spatial resolution, detector shielding, and septal penetration.Based on our characterising measurements, we conclude that the hand-held camera works well for imaging of Additional file 1 appendix.pdf. Contains Appendix 1, Appendix 2 and Appendix 3, referenced in the main article text. Appendix 1 presents more details on the energy resolution estimation procedure. Appendix 2 presents peak drift values for all nuclides and photopeaks analysed. Appendix 3 presents data on the noise levels in the flat-field images acquired for uniformity analysis.


SARS-CoV-2 has been identified as the pathogen causing the outbreak of Coronavirus Disease 2019 (COVID-19) that started in Wuhan, China, in December 2019. SARS-CoV-2 has human-to-human transmission ability and universally contagious to all populations. The main transmission patterns are respiratory droplets transmission and contact transmission. The purpose of this study is to propose a protocol that may be used as a guide to reduce the incidence of COVID-19 infections among otolaryngology care teams.A prospective cohort study was conducted to show the efficacy of our protocol to prevent transmission to health-care providers from March 11, 2020 through April 14, 2020. The protocol consisted of a series of protective measures that we applied to all health-care providers, then testing of our providers for COVID-19 using reverse transcription polymerase chain reaction along with immunoglobulin M (IgM) and immunoglobulin G (IgG) testing at the end of the study period to ensure effectiveness.Our protocol resulted in zero transmissions to our health-care providers during the duration of the initial study. We were involved in greater than 150 sinonasal, skull base, open airway, and endoscopy procedures during this study. At the conclusion of the initial 5 weeks, we had no health-care providers test positive for SARS-CoV-2.According to our proposed protocol, we were able to provide care for all patients in clinic, hospital, emergent, intensive, and surgical settings with no transmission of SARS-CoV-2 by symptomatology and post evaluation testing. The disease was subsequently named Coronavirus Disease—2019 (COVID-19) by the World Health Organization (WHO) and has been designated SARS-CoV-2. Significant concern has arisen within the global community to the potential risks of infectious transmission of SARS-CoV-2 to the surgical team during endoscopic sinonasal and skull base surgery. As information has rapidly evolved, it has become clear that the presence of elevated viral load in the upper airway mucosa impacts not only skull base cases but also virtually all diagnostic and therapeutic intranasal procedures routinely performed by Otolaryngologists. Despite this, there has been little to no evidence-based data to guide best practices thus far. Although we hope this will be one of the first of many studies coming from our scientific community, we have attempted to evaluate the literature regarding aerosol generating procedures and evaluate a protocol to protect our patients and our colleagues in the face of this new threat.A cluster of viral pneumonia cases associated with a novel Coronavirus (2019-nCoV) was first identified in Wuhan, Hubei Province, China, in December 2019 and has rapidly spread around the world, causing a global health crisis. and asymptomatic infection appears to be more common in children. Thus far, there is no definitive evidence of vertical transmission from infected mothers to a fetus, although anti-SARS-CoV-2 IgM antibodies were detected in 1 infant immediately after birth.57 Although the precise mechanism of SARS-CoV-2 transmission has yet to be elucidated, the primary mode appears to be via contact, respiratory droplets, and aerosols; however, transconjunctival and fecal-oral transmission may also occur. Social distancing and isolation have therefore become one of the key methods of reduction in viral transmission. To reduce nosocomial transmission, the American Academy of Otolaryngology—Head & Neck Surgery (AAO-HNS) currently recommends limiting care to time-sensitive and emergent problems. When patient care is required, appropriate measures should be taken to prevent transmission from potentially infected patients to other patients or health-care providers.It is important to recognize that asymptomatic COVID-19 patients may still be highly contagious. Asymptomatic adult carriers of COVID-19 have been reported,We implemented a protocol in our tertiary referral center, and after greater than 150 cases, over 5 weeks, we have succeeded in protecting our health-care providers. The objective of this study is to help define a protocol that can help the otolaryngologists and health-care staff from contracting this illness.We designed a prospective cohort study evaluating transmission of SARS-CoV-2 to our health-care providers at our tertiary referral center, in which we have performed a case series made up of patients from our practice and also those managed in the Emergency Department and Intensive care units over 5 weeks from March 11, 2020, through April 14, 2020, in order to determine the incidence of COVID-19 positive cases in our providers after the application of our proposed protocol. All aspects of this study were reviewed and approved by the Baton Rouge General Institutional Review Board (IRB00005439).Our protocol adheres to Centers for Disease Control and Prevention and WHO guidelines of how to handle patients in the clinic, hospital, and operating theatre. In all settings, we implemented the use of p100 filter respirators to all providers. These masks were purchased as an alternative to powered, air-purifying respirators (PAPRs), because they are more widely available and less expensive. These masks provide for the highest personal respiratory protection as p100 respiratory filters can reliably block particles 0.3 µm or larger. Eye protection, with face shields or goggles, was implemented to prevent potential ocular transmission. We reduced the number of the health-care providers in all evaluation settings to reduce the volume of transmission. Both goggles and respirators were sterilized after use per manufacturers’ recommendations. Per state-mandated guidelines, we also reduced the number of patients by managing only those deemed as urgent and emergent, which would otherwise result in significant worsening if a delay in care were to occur. In the clinic setting, we scheduled patients at a minimum of 30-minute intervals to avoid patient-to-patient contact within the waiting area, and patients were only allowed to bring 1 companion if needed for assistance. All patients underwent date of service symptom screening questionnaire, recording of temperature, and were given a surgical mask prior to clinic entrance. All care team members underwent daily symptom screening questionnaire and recording of temperature. All examination rooms were terminally cleaned after each use while using designated personal protective equipment.Per recommendations, our surgical cases were managed in such a way that elective cases were canceled indefinitely, until respective governmental agencies deemed appropriate. Urgent cases underwent COVID-19 testing within 24 hours prior to proceeding, while temporizing the primary ailment. Emergent or unavoidable cases proceeded in an emergent manner, and all involved personnel were provided and required to wear p100 filter respirators, eye protection along with surgical gowns and gloves . In emerWe also established a protocol concerning the handling the nasal endoscopy, in which we ensured the absolute necessity of its use, with no use of topical decongestant or local anesthetic solution to decrease aerosolization of particles during the examination. The use of surgical scrubs with, disposable gloves, eye protection, and p100 filter respirators along with the use of video monitoring rather than direct eye piece for visualization was required during the endoscopic examinations . SteriliAll health-care providers in the cohort underwent COVID-19 testing at the end of the current study via reverse transcription polymerase chain reaction nasal/nasopharyngeal swab along with point-of-care evaluation for both IgM and IgG for SARS-CoV-2.P < .05), despite significant amounts of exposure to positive cases. Our case series of exposure consisted of 152 endoscopic sinonasal, skull base, and airway procedures, including transsphenoidal resection of pituitary adenoma for pituitary apoplexy with vision loss, modified endoscopic Lothrop procedure for an infected mucopyocele with orbital involvement, and 3 cases of sphenopalatine artery ligation for uncontrolled posterior epistaxis despite packing (positive SARS-CoV-2). We have also completed 7 cases of tracheostomy with placement of tracheostomy tube, all of which tested positive at the time of procedure, for SARS-CoV-2, for extended intubation (>2 weeks). In the clinic and hospital ward setting, we performed greater than 100 nasal endoscopies on patients who were deemed urgent or emergent. Of the 152 total cases, 26 (17.1%) patients tested positive for SARS-CoV-2, 11 (7.2%) patients tested negative for SARS-CoV-2. Moreover, 115 patients had not undergone testing at the time of the procedure due to testing availability, despite an additional 35 (23%) patients exhibiting concerning symptoms based on the symptom screening questionnaire.Our implemented protocol resulted in zero transmissions in our health-care providers (t test (2-tailed) was used for comparisons of parametric data. Results were deemed significant with a P value of <.05.Statistical analyses were performed using SPSS v 22 . Descriptive data are presented as percentages and means ± standard deviation. Kendall’s tau-B was used for ordinal values. Chi-squared analysis was used for relationships of nominal variables. The prevalence of related comorbidities was also compared across treatment cohorts using Pearson chi-square analysis for relationships of nominal variables. Student’s The disease COVID-19 resulting from the novel coronavirus strain (SARS-CoV-2) represents an extraordinary threat to the health of the global population. Since its emergence in Wuhan, China, in December 2019, it has rapidly spread throughout the world following an exponential growth curve prompting it to be classified as a pandemic by the WHO on March 11, 2020. In addition to the accelerating death toll among patients, evolving information regarding infection transmission among health-care workers has raised concerns within the medical workforce regarding best practices for personal protective equipment (PPE). have raised further critical questions with respect to the safety of performing both outpatient endoscopy as well as sinus and skull base surgery in patients during this pandemic. Otolaryngologists are at increased occupational risk for contracting COVID-19 relative to other specialties, due to high concentrations of virus in the nasal cavity, nasopharynx, and oropharynx.13Anecdotal reports regarding high rates of infection specifically among Otolaryngologists13Patient use of surgical masks is impractical for the majority of Otolaryngology patient encounters, and therefore Otolaryngology providers should take appropriate personal protective measures. Concentrations of the SARS-CoV-2 virus appear to be highest in the nasopharynx and oropharynx, and therefore any patient evaluation involving examination or instrumentation of or through the oral cavity, oropharynx, nasal cavity, or nasopharynx should be considered high risk for SARS-CoV-2 exposure.According to Patel et al., concerns exist that N95 masks are not enough to control the spread. Not until the use of PAPRs did the spread become controlled. This article postulated guidelines to proceed dealing with cases during this crisis. All nonurgent/emergent cases are postponed. Preoperative testing was implemented, and full PAPRs were deemed necessary for all team members handling the urgent/emergent cases with pending or positive test results. In the clinic setting, Patel et al. implemented N95 masks, face shields, and gowns for all nasal endoscopies.Workman et al. postulated that the small size of the droplet particles and the extended travel of the aerosols urges for the use of PPE, with the use of N95 respirators. He also evaluated aerosol generating procedures and the effect of sneezing, in which aerosols were able to travel between 30 cm to 66 cm. This was prevented with the use of the intact and modified valved endoscopy of the nose and throat (VENT) masks. Workman et al. opted for the use of the PPE as an effective barrier technique in the outpatient clinic endoscopy, cold instrumentation in endonasal surgical procedures, even with the use of the microdebrider, as all these procedures were associated with low risk of viral aerosol transmission. They suggested that the low oscillation speeds and the continuous local suction with the microdebrider maybe associated with such low risk of aerosol transmission. In contrast, the use of high-speed drill was associated with high airflow velocities and so higher risk of droplet contamination.There are many uncertainties regarding the immediate future of the practice of rhinology as it pertains to COVID-19, but we feel that with safe protocols in place, we will be able to proceed forward with appropriate and timely care of patients in need. We recommend the use of this protocol as a guide for otolaryngologists to reduce the incidence of SARS-CoV-2 transmission. Although initial reports have advocated for the use of PAPRs, we have found that p100 respiratory filters provide appropriate respiratory protection and are more readily available and less expensive. We also recommend testing health-care providers who develop suspicious symptoms and will continue to evolve and likely expand this practice as additional testing kits become more available and accurate. Although there are great uncertainties with this pandemic, we feel that with appropriate caution and logic, with the evolution of protective protocols, we will be able to return to proper care of all patients.Our proposed protocol had succeeded in controlling the viral transmission to our health-care providers with no incidence of transmission, despite managing significant numbers of patients with actively positive SARS-CoV-2 results. In the current state of this pandemic, we do not recommend proceeding with elective evaluation or procedures per state and federal guidelines, but our results do provide evidence that with appropriate protocols for protection, we are able to safely manage patients who need intervention.


Osteoporosis is a major concern worldwide and can be attributed to an imbalance between osteoblastic bone formation and osteoclastic bone resorption due to the natural aging process. Heritable factors account for 60–80% of optimal bone mineralization; however, the finer details of pathogenesis remain to be elucidated. Micro RNA (miRNA) and long-non-coding RNA (lncRNA) are two targets that have recently come into the spotlight due to their ability to control gene expression at the post-transcriptional level and provide epigenetic modification. miRNAs are a class of non-coding RNAs that are approximately 18–25 nucleotides long. It is thought that up to 60% of human protein-coding genes may be regulated by miRNAs. They have been found to regulate gene expression that controls osteoblast-dependent bone formation and osteoclast-related bone remodeling. lncRNAs are highly structured RNA transcripts longer than 200 nucleotides that do not translate into proteins. They have very complex secondary and tertiary structures and the same degradation processes as messenger RNAs. The fact that they have a rapid turnover is due to their sponge function in binding the miRNAs that lead to a degradation of the lncRNA itself. They can act as signaling, decoy, and framework molecules, or as primers. Current evidence suggests that lncRNAs can act as chromatin and transcriptional as well as post-transcriptional regulators. With regards to osteoporosis, lncRNA is thought to be involved in the proliferation, apoptosis, and inflammatory response of the bone. This review, which is based on a systematic appraisal of the current literature, provides current molecular and genetic opinions on the roles of miRNAs and lncRNAs in osteoporosis. Further research into the epigenetic modification and the regulatory roles of these molecules will bring us closer to potential disease-modifying treatment for osteoporosis. However, more issues regarding the detailed actions of miRNAs and lncRNAs in osteoporosis remain unknown and controversial and warrant future investigation. Osteoporosis is increasing in prevalence around the world due to the aging population. Although osteoporosis can be attributed to an imbalance between osteoblastic bone formation and osteoclastic bone resorption  as part Micro RNA (miRNA) and long-non-coding RNA (lncRNA) are two such targets that have recently come into the spotlight due to their ability to control gene expression at the post-transcriptional level, providing epigenetic modification ,3. miRNAlncRNAs are highly structured RNA transcripts longer than 200 nucleotides that do not translate into proteins . In factThe interaction between miRNAs and lncRNAs is also of current interest. Studies have attempted to link lncRNAs, miRNAs, and mRNAs together in a complex network, such as Hao et al.’s systematic analysis using the mandibles from ovariectomized mice . Fei andmiR-9-5p inhibits osteogenesis and promotes adipogenesis via directly binding to Wnt3a . It alsomiR-21 has been found to promote osteoclastogenesis. Through a positive feedback loop that involves programmed cell death, miR-21 is upregulated by osteoclastogenesis factor c-Fos and then promotes RANKL (receptor activator of nuclear factor-κB ligand)-induced osteoclastogenesis . A revieDecreased miR-29 is a potential marker of osteoporosis. Lower serum miR-29 levels were associated with vertebral fractures in postmenopausal women . In osteIn osteoporotic women, serum levels of miR-30b-5p were low. In vitro studies of osteogenesis have validated that miR-30b-5p targets RUNX2 (runt-related transcription factor 2) and decreases during the late stages of osteoblast differentiation .miR-31-3p has been found to inhibit osteoclastic bone resorption through the repression of osteoclast formation . miR-31-Levels of miR-100 are increased in the serum and bone tissue of osteoporotic patients . Cheng eThe levels of serum miR-103-3p are lower in osteoporotic women when compared with healthy controls . In vitrAn analysis of 139 serum samples using RT-qPCR  showed that lower levels of miR122-5p were found in patients with lower BMD , and, therefore, may be associated with the development of osteoporosis . This mamiR-124 could potentially alleviate the progression of osteoporosis. Studies have shown miR-124 to suppress differentiation and migration of osteoclast precursors, thereby inhibiting osteoclast formation . SimilarmiR-133 increases osteoclastogenesis due to mRNA targeting of the proteins that inhibit osteoclastogenesis . miR-133miR-135a-5p levels were elevated in bone tissue of postmenopausal women with osteoporosis compared with postmenopausal women without osteoporosis . miR-135Another potential target for treating osteoporosis is miR-146a, found in bone tissue, which can be induced by TNF-a/RANKL treatment, and has been found to inhibit osteoclastogenesis in mouse models [miR-148a overexpression induces osteoclast formation . It signmiR-155 plays an important role in bone destruction, as demonstrated by its involvement in rheumatoid arthritis [In Pan et al.’s animal study, ovariectomized rats treated with alendronate were used to assess the effects of miR-182-5p . They foIn whole blood lysates of postmenopausal Chinese women, miR-194-5p was found to be upregulated by over five-fold, and could potentially be used to discriminate against osteopenia and osteoporosis [Blood was collected from 30 postmenopausal women, and the miR-200a-3p level was found to be higher in the serum of these patients compared with controls . High lemiR-203a is found to be upregulated in the bone tissue from postmenopausal women with a history of low-traumatic fractures and slows osteoblast differentiation .An in vitro study showed that miR-214-5p overexpression promoted adipogenic differentiation, thereby promoting osteoporosis progression . miR-214Bony fragments extracted from 12 women with PMOP fractures undergoing hip replacements showed that miR-221 was underexpressed compared to the 12 healthy controls (osteoarthritis without osteoporosis) . miR-221Upregulation of miR-223 is thought to inhibit osteoclast differentiation . miR-223In Guo et al.’s study, miR-338-3p was found to be decreased during osteoblast differentiation, and that miR-338-3p knockout upregulated RUNX2 at the mRNA level . In a smIn glucocorticoid-induced osteoporosis (GIOP) mice, levels of miR-365 were found to be suppressed in bone tissue . MMP-9  in serum samples . BioinfomiR-422a is significantly upregulated in the circulating monocytes of postmenopausal women with low BMD in a study by Cao et al. . Using bIn vivo findings suggest that miR-449 overexpression could inhibit the osteogenic differentiation of BMSCs by binding directly to the 3-UTR terminus of SATB2  and suppressing SATB2 [miR-503 expression was found to be markedly reduced in PMOP . miR-503In ovariectomized rats, overexpression of miRNA-543 was found to significantly suppress cell growth and promote apoptosis in osteoblasts . In addiMicro RNA-579-3P expression in the serum of osteoporotic patients was significantly higher than those of normal controls, and this inhibited osteogenic differentiation of human BMSCs .In osteoporotic rats, there was inactivation of miR-874 and SUFU (suppressor of fused gene) overexpression in bone tissue. Both upregulation of miR-874 and downregulation of SUFU were found to promote osteoblast proliferation . Human smiR-1297, highly expressed in osteoporosis, has been found to inhibit osteogenic differentiation of human BMSCs . It affeDecreased miR-2861 may contribute to the pathogenesis of osteoporosis. miR-2861 was identified in primary mouse osteoblasts, and it was found to promote osteoblast differentiation by suppressing histone deacetylase 5 (HDAC5) expression at the post-transcriptional level . HDAC5 ilncRNAs are highly structured RNA transcripts longer than 200 nucleotides that do not translate into proteins . They haThere is evidence that lncRNA-ANCR (anti-differentiation non-coding RNA), also known as DANCR (differentiation antagonizing non-protein coding RNA), promotes osteoporosis . QRT-PCRlncRNA BMNCR (bone marrow associated non-coding RNA) has been suggested to alleviate the progression of osteoporosis. Chen et al. found that lncRNA BMNCR expression was decreased in the bone marrow and spleen of osteoporotic mice . SpecifilncRNA CASC11 (cancer susceptibility 11) has been shown to be upregulated in PMOP . A smallThe lncRNA colorectal neoplasia differentially expressed (CRNDE), first identified in colorectal tumors, is found to be increased in the osteoclasts of postmenopausal women compared to healthy women in a study by Li et al. . OverexpLow levels of lncRNA GAS5 (growth arrest-specific 5) have been found in bone tissue of patients with PMOP . Feng etThe lncRNA metastasis-associated lung adenocarcinoma transcript 1 (MALAT1), also known as nuclear-enriched transcript 2 (NEAT2), is involved in osteoporosis, as well as serving as a prognostic biomarker for lung cancer metastases . HoweverThe lncRNA MEG3  was found to promote osteoporosis in non-cancerous subjects . In bothlncRNA MSC-AS1 (MSC antisense RNA 1) may alleviate osteoporosis. Expression of MSC-AS1 was found to increase with osteogenic differentiation of mice BMSCs, as well as osteogenesis-related genes, such as RUNX2, osteopontin, and osteocalcin . KnockdoPlasma levels of lncRNA NEF (neighboring enhancer of FOXA2) are downregulated in PMOP women . In addilncRNA SNHG1  has been found to be downregulated in osteoporosis . ComparelncRNA TUG1 (taurine-upregulated gene 1) is thought to be upregulated in osteoporosis. This theory is based on the fact that it is inhibited in ankylosing spondylitis, which is commonly thought of as an inverse pathological process to osteoporosis . In the lncRNA XIXT (X-inactive specific transcript), which could potentially alleviate osteoporosis, was found to be downregulated in the serum of osteoporotic patients . It was The field of miRNAs and lncRNAs with regards to osteoporosis is still relatively new, and this is reflected in the quantity of research currently available. Often, there are conflicting results in the literature, such as miR-223 affording to both promote and inhibit osteoclastogenesis . A largeThe current review of literature was retrieved from studies of different sources  and tissues , which may explain the many conflicting results. Thus, conflicting conclusions have been reported for most miRNAs and lncRNAs depending on cellular models used, animal studies or cohorts of humans, and even analytical methods. We have specified these sources and differences where possible. One of the advantages of animal studies is the convenience in conducting these studies. Some animals (such as mice) have a shorter growth cycle than that of a human being and are subject to investigations in a limited research period. Furthermore, gene knockout can be implemented in an animal model to investigate the depletion effect of genes, DNAs, as well as RNAs on the target organs, which cannot be performed in a human being. However, the major disadvantage of animal studies lies in the differences of genes and subsequent RNAs between animals and human beings, and thus generalization of the conclusions of animal studies to human beings is limited. In contrast, the results of human studies are observational but direct evidence. The major disadvantage of human studies is that intervention (such as gene knockout) cannot be implemented due to the consideration of moral hazards. In addition, the expression of genes, miRNAs, or lncRNAs in variant tissues  may be different, thus resulting in conflicting conclusions.Due to the lack of clear-cut associations between PMOP and the expression of miRNAs and lncRNAs at present, there is still a long way to go before they can be used as potential noninvasive biomarkers. In addition, there are also many barriers still to overcome to transfer miRNA and lncRNA knowledge to the synthesis of clinical therapeutic drugs. Synthetic oligonucleotides mimicking miRNAs have a limited half-life due to degradation by nucleases in the bloodstream, and they also have a poor capacity to penetrate host cell membranes to reach their target cells . lncRNA In conclusion, miRNAs and lncRNAs are two potential targets that are the logical next step in osteoporosis research. Further research into the epigenetic modification and the regulatory roles of these molecules will bring us closer to potential disease-modifying treatment for PMOP. This review provides current opinions on the roles of miRNAs and lncRNAs in osteoporosis. However, more issues regarding the detailed actions of miRNAs and lncRNAs in osteoporosis remain unknown and controversial and warrant future investigation.


Situs inversus of optic disc (SIOD) is thought to be a congenital optic disc abnormality that is caused by dysversion of optic nerve insertion. SIOD, however, has many additional features that cannot be explained by abnormal optic-nerve-insertion directionality. In this study, we measured the distance between the fovea and disc in 22 eyes of 15 SIOD patients. For comparison, two control eyes were matched with each SIOD eye by age and axial length. The vertical distance between the temporal vascular arcades also was measured. The foveo-disc distance was shorter in the SIOD eyes than in the control eyes, while the inter-arcade distance did not differ. Further, we measured the circumpapillary retinal nerve fiber layer thickness, which showed nasal crowding of two humps in the SIOD eyes. This nasal crowding disappeared when we shifted the circle scan by the mean difference (465 μm) of the foveal-disc distance between the two groups. Our findings suggest that the optic disc was located closer to the fovea than it would have been normally. Thus, SIOD might reflect incomplete expansion of the posterior pole in the direction of the fovea-disc axis. The retinal vascular arcade advances nasally initially, followed by acute arching of the vessels temporally1. SIOD has been reported to have different peripapillary retinal nerve fiber layer (RNFL) thickness and optic nerve head (ONH) characteristics relative to normal controls3. Since the initial curve of the vasculature is in the reverse direction, and because this condition is commonly associated with tilted disk syndrome, SIOD is thought to be congenital and the result of dysversion of the optic disc originating from anomalous insertion of the optic stalk into the optic vesicle4.Situs inversus of optic disc (SIOD) is a rare optic disc abnormality characterized by emergent anomalous retinal vasculature6. In fact, the most significant increases in axial length occur during the first 2 years of age, after which the increases are gradual until 10 years of age. Patel et al. demonstrated this initial rapid ONH expansion period early in life in their hand-held optical coherence tomography (OCT) study6. During scleral expansion in this period, the retina also expands to cover the enlarged surface7, which leads in turn to expansion of the retinal vascular arcade and increased foveo-disc distance in the posterior polar area. If the scleral and retinal expansion is restricted for some reason at this stage, however, morphologic abnormality in the posterior polar retina might be incurred.Like other organs, every eyeball has to grow after birth. Several studies have revealed that this process is not simple linear growthThe purpose of this study, correspondingly, was to compare the distance from the fovea to the Bruch’s membrane opening (BMO) between SIOD eyes and their axial-length-matched normal controls. We also investigated whether the characteristic RNFL profile of SIOD can be normalized after adjusting the circle scan position to the presumptive site based on the mean difference of distance between the SIOD and control groups.Fifteen 15) participants (22 eyes) with SIOD were enrolled as the SIOD group, and 44 age and axial-length-matched participants (44 eyes) without SIOD were enrolled as the control group. Fifty-nine (59) participants (66 eyes) were enrolled in this study in total. The subject demographics are summarized in Table 5 particiTable 3. We manipulated the optic disc center from the automatically calculated coordinates . In the SIOD eyes, no presumptive sign of BMO misalignment from the neural canal opening, such as γ-zone parapapillary atrophy14, was present; rather, their abnormal peripapillary RNFL profile was settled within the retinal layers including the BMO. Therefore, disproportionate expansion of the outer scleral layer cannot fully explain the development of SIOD.In the Boramae Myopia Cohort Study, we found that the retinal and scleral layers expanded in a different fashion during myopic axial elongation18, but we could not adjust them in Cirrus OCT. Further, we arbitrarily repositioned the circle scan in a fixed amount for comparison, which might have induced some bias. The reason we used the RNFL thickness and repositioned the circle scan center, however, was to visualize changes in the general trend of thickness curves, not to demonstrate specific values. For that purpose, the effects of circumpapillary RNFL thickness measurement errors and bias would be negligible.This study has several limitations. First, our sample size was small because of the rarity of the disease studied. Second, we measured the vertical distance of vascular arcade on red-free fundus photographs in arbitrary units, while we measured the horizontal distance between the fovea and the optic disc on infra-red images of Spectralis OCT since the infra-red images did not cover the whole vascular arcade. To minimize measurement bias, however, we matched two control eyes for every SIOD eye by the axial length. Therefore, we do not believe that this measurement discrepancy affected the results significantly. Lastly, the circumpapillary RNFL thickness measurement should be evaluated with caution. As we showed in this study, the RNFL thickness profile is closely related to the location of the circle scan center, while we do not have any clear reference point for that in Cirrus OCT. Individual factors such as foveo-disc angle might affect the RNFL thickness profileIn conclusion, the SIOD eyes had a shorter foveo-BMO-center distance, and the characteristic peripapillary RNFL thickness profile was normalized by shifting the scan circle more nasally. These findings implicate that, in those eyes, the focal restriction of posterior polar expansion, which occurs after the axons of the retinal ganglion cells find their way out of the eyeball normally, might affect the development of SIOD.The study protocol was approved by the Seoul National University Boramae Medical Center Institutional Review Board and adhered to the tenets of the Declaration of Helsinki.19. Written informed consent was obtained from all participants. All subjects included in this study underwent a full ophthalmologic examination that included best-corrected visual acuity (BCVA) assessment, refraction, slit-lamp biomicroscopy, Goldmann applanation tonometry, gonioscopy, dilated funduscopic examination, keratometry , axial length measurement , disc photography and red-free fundus photography , and ONH scanning using two spectral-domain optical coherence tomography (SD-OCT) devices. The 200 × 200-cube ONH scans were obtained by SD-OCT . Twenty-four (24) high-resolution radial scans of the ONH were performed using another SD-OCT device . During acquisition of both SD-OCT image sets, the subjects were asked to fixate on the target, and images were acquired with the forehead and chin stabilized by the headrest. Extra care was taken during each exam to confirm that the forehead and chin were correctly positioned and did not move.We included SIOD and healthy control subjects from a database of an ongoing prospective cohort study, Boramae Glaucoma Imaging Study (BGIS), at Seoul National University Boramae Medical Center 4, was evaluated by two glaucoma specialists (KML and MK). In cases of disagreement, a final decision was made by a third adjudicator (SHK). Two (2) age- and axial-length-matched healthy control eyes were recruited from the BGIS database for each SIOD eye.SIOD, defined as a characteristic emergence of the superior and inferior retinal vessels anomalously nasalward followed by acute arching temporally that is associated with dysversion of the ONHThe exclusion criteria were BCVA < 20/40, posterior staphyloma  appearing sharply defined in the funduscopic examination, a history of ocular surgery other than cataract extraction or corneal refractive surgery, retinal or neurologic disease, a poor-quality image . If both eyes were eligible, both eyes were used for the analysis in the SIOD group, while in the control group, one eye was randomly selected.The peripapillary area was imaged by SD-OCT. The corneal curvature of each eye was entered into the SD-OCT system  before performing SD-OCT scanning so as to compensate for potential magnification error. The Glaucoma Module Premium Edition of the Spectralis device enables detection of the BMO. With 24 high-resolution radial scan images of the ONH separated by 15° and each averaged from 24 individual B-scans, SD-OCT automatically detects the margin of the BMO. Every detected BMO margin in the present study was reviewed by one of the authors (YIS), and errors were corrected manually. Based on the edited BMO margin, Spectralis calculated the area and center of the BMO and determined the foveo-BMO axis.After demarcation of the BMO margin, the distance between the fovea and BMO was measured in the infra-red image of SD-OCT Fig. . The dis20. A vertical line was drawn from the fovea, and the crossing points of the superior and inferior venous arcades were marked. The distance between the two crossing points was measured using the built-in caliper tool of MPACS  with arbitrary units. The venous arcades were chosen for measurement because of the large variations in the arterial arcades.The vertical distance between the temporal superior and inferior vascular arcades was measured on red-free fundus photographs21. Since the thickness profiles were obtained in 200 11. To prove our hypothesis that the abnormal circumpapillary RNFL thickness profile of the SIOD patients was derived from a shorter foveo-BMO-center distance, we intentionally moved the circle scan center nasally by the amount of the mean difference between the SIOD and control groups (465 μm) along the foveo-BMO axis in both groups. The original RNFL thickness profiles and the adjusted RNFL thickness profiles were then compared with those of the control group.The 200 https://www.R-project.org; assessed December 5, 2017). The data herein are presented as the mean ± standard error except where stated otherwise, and the cutoff for statistical significance was set at P < 0.05.Inter-group comparisons were performed using a generalized estimating equation (GEE) regression model to account for the correlation of paired eyes from the same participants. Due to the internal correlation of the same eye, the circumpapillary RNFL thickness was compared using the GEE with a pairwise contrast test. To overcome multiple comparisons of the 12 locations of RNFL thickness measurement, statistical tests were performed with R statistical packages version 3.4.3 (available at Supplementary Table 1.


However, accurate determination of CTG expansion size is challenging due to its characteristic instability. We compared five different approaches  to estimate CTG expansion size in the progenitor allele as well as the most abundant CTG expansion size, in 15 patients with DM1. Our results indicated variability between the methods . While keeping in mind the limited sample size of our patient cohort, SP-PCR appeared as the most suitable technique, with an inverse significant correlation found between CTG expansion size of the progenitor allele, as determined by this method, and age of disease onset . Yet, in light of the variability of the results obtained with the different methods, we propose that an international agreement is needed to determine which is the most suitable method for assessing CTG expansion size in DM1.The number of cytosine-thymine-guanine (CTG) repeats (‘CTG expansion size’) in the 3′untranslated region (UTR) region of the  However, no other significant correlation was found for the results obtained with LPCR1-SB, LPCR2 or SP-PCR, respectively . We further studied possible correlations with age of disease onset, MIRS and mRS scale. We found an inverse, significant correlation between CTG expansion size of the progenitor allele as determined by SP-PCR, and age of disease onset (LPCRs yielded shorter progenitor alleles and higher mode alleles compared to SP-PCR . No signse onset .Although no significant group effect was found with the one-way repeated-measures ANOVA between LPCR1/2 and SP-PCR, our results indicate that there is variability in the number of CTG repeats for a given patient depending on the CTG sizing method. HPE-PCR showed results that were difficult to interpret. Additionally, LPCR3 did not allow amplification of most of the DNAs in the patients. However, the fact that LPCR1 and LPCR-2 did yield some valid results suggests that LPCR-SB might be more sensitive to parameters such as the quality of the input DNA, which is not the case for the SP-PCR technique. SP-PCR was the only technique that enabled amplification of all DNAs from the patients and in fact, was the only one yielding a result that was correlated with an important phenotype trait of DM1—age of disease onset.While a strong correlation was found between LPCR1-SB and LPCR2-SB for CTG expansion size of the progenitor allele, no other significant correlation was found. LPCR1/2-PCR yielded lower progenitor sizes and higher mode sizes than SP-PCR. LPCR-SB approaches usually show the expanded alleles as diffuse smears rather than discrete bands, due to the high input of DNA plus the somatic instability of the mutation . This faIt would be interesting to determine how novel, recently described technologies for CTG sizing ,12 compaSome studies have described that CTG repeat number can be a good indicator of disease onset . In thisOur study suggests that, besides the somatic mosaicism caused by CTG repeat instability and the inherent technical difficulties in assessing CTG expansion, there is overall heterogeneity among the different methods that are currently available, which makes it difficult to rely on them as valid predictors of disease phenotype. International agreement is needed to determine which is the most suitable methodology to characterize CTG expansion size in patients with DM1.