This method is faster, cheaper, and more accurate than DXA. Here, we demonstrate that the D3Cr method is a specific assay for muscle mass in mice, and we test associations with DXA and body weight. We evaluated the D3Cr method compared to DXA‐determined lean body mass (LBM) in aged mice and reported that DXA consistently overestimates muscle mass with age. Overall, we provide evidence that the D3Cr dilution method directly measures muscle mass in mice. Combined with its ease of use, accessibility, and non‐invasive nature, the method may prove to more quickly advance development of preclinical therapies targeting sarcopenia.Developing accurate methods to quantify age‐related muscle loss (sarcopenia) could greatly accelerate development of therapies to treat muscle loss in the elderly, as current methods are inaccurate or expensive. The current gold standard method for quantifying sarcopenia is dual‐energy X‐ray absorptiometry (DXA) but does not measure muscle directly—it is a composite measure quantifying “lean mass” (muscle) excluding fat and bone. In humans, DXA overestimates muscle mass, which has led to erroneous conclusions about the importance of skeletal muscle in human health and disease. In animal models, DXA is a popular method for measuring lean mass. However, instrumentation is expensive and is potentially limited by anesthesia concerns. Recently, the D Mus musculus. We evaluated the results from the D3Cr method with DXA‐determined lean mass in mice and reported that DXA consistently overestimates muscle mass with age. Overall, we provide evidence that the D3Cr method measures skeletal muscle mass; combined with its ease of use, accessibility, and non‐invasive nature, the method may prove to advance development of preclinical therapies targeting sarcopenia.We demonstrate that the D3‐creatine dilution method can be used to measure skeletal muscle mass in previously unexplored murine model,  BMbone massCTcomputed tomographyD3CrD3‐CreatineDXAdual x‐ray absorptiometryFBMfat body massLBMlean body massMMmuscle massMRImagnetic resonance imaging1Skeletal muscle makes up the largest fraction of tissue in the body  has emerged as the most commonly used method for body composition analysis. DXA uses a three‐compartment categorization method: Bone and fat mass are separately quantified through the differential absorption of high and low photon energies, and then, lean mass  is differentially calculated. The method's popularity results from its relatively quick and easy measurement, low radiation dose, and precision. However, its use is often limited due to instrumentation availability and cost. Although DXA is used as a surrogate for muscle, it does not measure muscle mass.3‐creatine (D3Cr) dilution method is non‐invasive, accurate, and has the potential to become the new gold standard for quantifying muscle mass in preclinical models  of methyl‐D3 labeled creatine and repeatedly collected urine post‐IP to determine appropriate sampling time windows. Mass spec analysis of urinary creatinine enrichment allowed for quantification of skeletal muscle mass, which was able to be directly compared with total body weight and respective DXA results.In our study, we assess the use of the D3Cr, primarily metabolized in muscle.The D3 method relies on the assumption that urine creatinine is solely derived from the total body creatine pool (about 98% of which is sequestered in the sarcomere) . These initial studies used a single intraperitoneal (IP) injection of D3‐creatine at 2 mg/kg (mpk) of body weight. This dosage was chosen based on prior studies that mice demonstrate increased creatinuria and findings that higher doses (>4mpk) of injected creatine were necessary to elevate plasma creatine levels  injection   . Despite their increased total body weight, aged mice had lower lean body mass (LBM) percentages   was not statistically altered with increasing age   Figure  and incr) Figure . Despit) Figure  indicati) Figure . However) Figure . We expl) Figure .3Cr muscle mass were significantly correlated with body weight (R2 = 0.4181 (p < 0.0001), R2 = 0.3864 (p < 0.0001)), respectively . This finding demonstrates conflicting data for which method may be accurately determining skeletal muscle mass. Weights of individual hindlimbs muscles taken at the time of dissection were found to be statistically unchanged between age groups  assumes 100% glycolytic (type II) fibers. This assumption negates differing creatine uptake rates in oxidative (type I) fibers and impedes studies in aging mice which are characterized to have altered fiber quality with age . Our study allowed for timely assessment of the D3Cr dilution method across ages, but further longitudinal studies are necessary to more accurately address the nature of sarcopenia phenotypes in aging mice. In conclusion, we demonstrate an alternative method for non‐invasively measuring total skeletal muscle mass in mice with direct comparisons to field standards. It will be important to use this methodology in tandem with other methods for future validation studies as well as measuring muscle mass in future preclinical studies of sarcopenia, utilizing longitudinal studies to better understand how muscle changes with age.Our work highlights the overestimation of DXA‐estimated muscle mass by direct comparison with D22.1D3‐creatine was purchased from Cambridge Laboratories (DLM‐1302‐0.25) (LOT#PR‐30122) (PSO#22A‐0183). Solution was made to 0.6 mg/mL in PBS for a final delivery of 2 mg/kg of body weight.2.2Studies included the use of male and virgin female C57BL/6J mice (Jackson Laboratories #000664) aged between 20 and 99 weeks of age. All mice were communally housed and aged‐matched with ad libitum access to water and diet (Envigo Teklad #2918) in a pathogen and temperature‐controlled room with a 12 h light–dark cycle beginning at 06:00 AM. All procedures were conducted in accordance with NIH Guidelines for Care and Use of Animals and were approved by the Institutional Animal Care and Use Committees at Buck Institute for Research on Aging.Mice: Our N for young mice (5 months) was 6 (3 M/3F), for adult mice  was 6 (3 M/3F), and for aged mice (21 months) was 13 (6 M/7F) and 22 (25 months) (11 M/11F), respectively.2.3Mice were maintained on a low‐fat chow diet (Envigo Teklad #2918) free of animal products or exogenous creatine to prevent cross contamination of measured creatine.2.43‐Cr injections.Dual‐energy x‐ray absorptiometry (DXA) scans were used to quantify body composition in anesthetized (2% isoflurane) immobilized mice by InAlyzer2S (Micro Photonics). DXA scans were performed during the same week as D2.53‐creatine by intraperitoneal injection. Following the injection, mice were placed back into their cage with ad libitum access to food and water. Urine samples were collected 2, 6, 12, 24, and 48 h post‐dosing and stored at −80°C.The day of the study, experimental mice were weighed before being administered a single dose of 2 mg/kg body weight D2.63‐creatine within the Nutrition Science and Toxicology Department / Hellerstein Laboratory at UC Berkeley. Urine samples were processed for D3‐creatinine enrichment, as well as creatine and creatinine concentrations to determine Cr/Crn ratio using methods described in detail elsewhere  corresponding to the M0 peak of Crn and 117.1/47.1 which corresponds to D3‐Crn.Mouse urine was stored at −20°C until analyzed for D2.7p > 0.05, *p < 0.05, **<0.005, ***<0.0005.Statistical analysis across all age groups was assessed by one‐way ANOVA, while unpaired analyses between specific age groups were assessed by t‐test. XY plot analyses were assessed by simple linear regression, and correlation values were calculated using Pearson's Correlation test. For gait analyses, XY plot analyses were assessed either by simple linear regression or second order polynomial (quadratic) nonlinear regression [Step cycle]. Significance is reported as follows: ns SM, WE, and MH involved in conceptualization and supervision. LW, SS, MH, WE, and SM drafted the manuscript. LW, NE, and SS involved in analysis and visualization. EG, SG, EN, SS, AD, LP, MCM, and CP involved in experimental procedure. SE and NS provided operational support. All the authors have read and agreed to the final version of the manuscript.We acknowledge support from the Astera Institute and Ono Pharmaceuticals.3‐creatine dilution method; however, they do not derive any income related to commercial use of this method nor do they control the intellectual property.The authors declare no conflicts of interest. Drs Hellerstein and Evans are listed as co‐inventors on the filed patents for the DFigure S1Click here for additional data file.


In the terrifying pandemic caused by SARS‐CoV‐2, diabetic patients exhibiting more severe outcomes and mortality rate is high among them. Based on recent studies, metformin as the most prescribed drug for T2DM treatment may improve severe outcomes in diabetic patients infected with SARS‐CoV‐2. On the other hand, abnormal laboratory findings can help to differentiate between the severe and non‐severe form of COVID‐19. According to the mentioned issues, the effect of metformin on severity of COVID‐19 was examined in T2DM patients with SARS‐CoV‐2 infection.The study included 187 individuals diagnosed with COVID‐19, 104 patients were diabetic and divided into two groups according to their anti‐diabetic drugs: patients who were treated only with metformin and patients who were treated with other anti‐diabetic drugs. The other participants were non‐diabetic and diagnosed with COVID‐19. Biochemical parameters were measured by routine laboratory methods before, during and after SARS‐CoV‐2 infection.p‐value: .02, .01, .03, .04, .0009 and .01, respectively). Also, after recovery, there were statistically significant differences between metformin users and non‐users with respect to most of the study parameters, except FBS, BUN and ALP .During infection, FBS, creatinine, ALT, AST, Ferritin and LDH were significantly lower in metformin users than non‐users (Our result suggested that metformin might be associated with better outcomes in diabetic patients infected with SARS‐CoV‐2. In the terrifying pandemic caused by SARS‐CoV‐2, diabetic patients exhibiting more severe outcomes and mortality rate is high among them. Based on recent studies, metformin as the most prescribed drug for T2DM treatment may improve severe outcomes in diabetic patients infected with SARS‐CoV‐2. Unarguably, hyperglycaemia impacts the body's ability to regulate immune and inflammatory responses in individuals with diabetes.The most common drug prescribed for lowering blood glucose levels is metformin. Interestingly, besides its glucose lowering potential, metformin can exert various protective effects against inflammation and cardiovascular damage through the activation of AMP‐activated protein kinase (AMPK), endothelial nitric oxide synthase (eNOS) and Sirt1.Given these facts, while diabetes is an independent risk factor for mortality from COVID‐19, it seems that mentioned risk is significantly reduced in people taking metformin, improving the possibility that metformin may play a protective role in this high‐risk population. On the other hand, laboratory tests are used to evaluate the improvement process in COVID‐19 patients.22.1All study steps were performed in compliance with the principles of the Helsinki Declaration, and ethical clearance was approved by the ethics committee at Shahid Beheshti University of Medical Sciences (IR.SBMU.RETECH.REC.1400.1159). All included patients provided informed consent. A total of 187 individuals diagnosed with COVID‐19 were enrolled in our study. All participants had a positive PCR 1–2 days after symptoms manifestation. Among them, 104 patients were diabetic and divided into two groups according to their anti‐diabetic drugs: patients who were treated only with metformin (1000–1500 mg/day), and patients who used drugs other than metformin. Diabetes duration in patients was approximately 2 years and all the patients received their drugs during infection. Participants with a previous history of renal and liver diseases, COVID‐19 vaccination and pregnant women were not included in our study. We compared biochemical parameters at least 3 months before infection, during infection and at least 3–4 months after patients recovered from the disease.2.2In this follow‐up study, biochemical parameters including AST, ALT, ALP, creatinine, BUN, FBS, Ferritin and LDH were analysed by routine laboratory methods before infection, during COVID‐19 infection and after recovery. HbA1c values were quantified using HPLC method. Lab values and corresponding reference ranges were collected from the laboratory information system.2.3t‐test and repeated measures one‐way ANOVA were performed to compare parametric variables. Comparisons between nonparametric variables were made by Friedman and Mann–Whitney U test. In the current study, two‐tailed p‐values less than .05 were considered statistically significant.We used GraphPad Prism (version 8.4.3) software to perform statistical analysis. The normality of the distributions of variables was carried out using Kolmogorov–Smirnov test. Continuous data were expressed as mean ± SD. An Independent 33.1p‐value = .86). Among 187 patients, 97 were female and 90 participants were male.Our study included 84 COVID‐19 patients without diabetes and 103 T2DM patients with COVID‐19 (52 metformin users and 51 non‐users) which did not differ significantly in age . Based on the results represented in Figure p‐value: <.0001) and individuals without T2DM . Creatinine levels were reduced after recovery compared with before infection in people without T2DM. Comparison of AST showed that there was a statistically significant difference before disease and during infection in diabetic group . There is also a significant reduction of AST in metformin consumers after recovery . LDH amounts were increased during infection in all subgroups . There was also a significant decrease in LDH level after recovery in metformin consumers and healthy people . In metformin users and individuals without T2DM, ferritin levels were higher than before infection  and decreased after recovery . In T2DM patients that use anti‐diabetic agents other than metformin, there was statistically significant difference between before infection and during infection  and after recovery  . After recovery, there were statistically significant differences between metformin users and non‐users with respect to most of the study parameters, except FBS, BUN and ALP.As shown in Table 3.3.2A comparison of the study parameters between metformin users and COVID‐19 patients without T2DM is shown in Table 3.3.3p‐value: <.0001, <.0001, .005, .01 and .005, respectively). In addition, all study parameters except BUN and ALP were significantly lower in infected people without T2DM during infection. A similar pattern was repeated after recovery ; formal analysis ; investigation ; resources ; software ; writing – original draft ; writing – review and editing . Zahra shahrokhi: Funding acquisition ; methodology ; supervision ; writing – review and editing . Zahra Amjadi: Data curation ; writing – original draft . Faranak Kazerouni: Conceptualization (lead); funding acquisition (lead); project administration (lead); supervision (lead); validation (lead); visualization (lead); writing – review and editing (lead).The present article is financially supported by Research Department of the School of Allied Medicine‐ Shahid Beheshti University of Medical Sciences.The authors declare that they have no conflict of interest.


Posttransplant lymphoproliferative disease (PTLD) is a potentially life‐threatening complication of hematopoietic cell transplantation. With improvements in Epstein‐Barr virus (EBV) monitoring and supportive care, PTLD incidence has decreased throughout the history of bone marrow transplantation. It is rare to develop PTLD after the first year following transplant, across all donor categories. In this case, we hope to elucidate details that may have predisposed to this unusual presentation. We present the case of a 55‐year‐old gentleman with acute myeloid leukemia who underwent a haploidentical transplant for consolidation and presented with fatigue, lethargy and presumed septic shock nearly 7 years after transplant. With improvements in EBV monitoring and supportive care, PTLD incidence has decreased throughout the history of bone marrow transplantation. It is rare to develop PTLD after the first year following transplant, across all donor categories . In this1.1We present the case of a 55‐year‐old gentleman with acute myeloid leukemia (AML) who underwent a haploidentical transplant for consolidation and presented with fatigue, lethargy and presumed septic shock nearly 7 years after transplant.His initial AML presented with white blood count 3 k/uL with 62% circulating blasts, hemoglobin 10.4 g/dL, and platelet count of 60 k/uL. A bone marrow biopsy was completed. The aspirate was not read for unknown reason, but the core showed 90% cellularity, 80% blasts and necrotic areas. Immunohistochemistry showed blasts positive for CD68, CD163, and lysosome. Flow cytometry showed 16% myelomonocytic blasts expressing CD4, CD15, CD33, CD38, CD56, Human Leukocyte Antigen DR isotype (HLA‐DR), CD64, and negative for CD34 and CD114.  Cytogenetics showed 46 XY with trisomy 8 and deletion 13. Fluorescence in situ hybridization (FISH) testing showed 4–5 copies of 8q22 consistent with pentasomy 8 and additional chromosome 3 in 6%. Molecular studies showed NPM1 mutation present with variant allele frequency of 6%. FMS‐like tyrosine kinase‐3 internal tandem duplication (FLT3‐ITD), tyrosine kinase domain (TKD), and c‐Kit protooncogene (C‐KIT) mutations were not detected. CEBPA was wild type.Clostridioides difficile colitis, grade 2 hand‐foot syndrome, chemotherapy‐induced hemorrhagic cystitis without B.K. human polyomavirus (BK) or adenovirus isolated, cytomegalovirus (CMV) reactivation, grade 2 acute GVHD (GI only), disseminated Varicella zoster (VZV) complicated with severe postherpetic neuralgia, and deep venous thrombosis of the left lower extremity. Otherwise, he had 99.95% engraftment on peripheral blood with 100% engraftment by CD3 and CD15 donor cells by day 21. Lymphocyte subset flow cytometry on day 336 is included in Table He underwent induction with idarubicin and cytarabine (7+3) and salvage therapy with fludarabine, idarubicin, granulocyte‐colony stimulating factor (G‐CSF) Fludarabine, Cytarabine, Idarubicin and G‐CSF (FLAG‐IDA) to achieve complete remission with incomplete count recovery (CRi). He had no recovery of counts after 4 months off therapy. Subsequently, he underwent haploidentical transplant with a conditioning regimen consisting of fludarabine, cyclophosphamide, and 1400 cGy total marrow and lymphoid irradiation, followed by posttransplant cyclophosphamide 50 mg/kg dosed daily on posttransplant days 3 and 4 prior to starting tacrolimus and mycophenolate mofetil. His course of transplant was complicated by Six years and 10 months later, he presented with fatigue, lethargy, abdominal distention, and fever. Computerized tomography (CT) chest, abdomen, and pelvis with contrast revealed diffuse lymphadenopathy, as seen in Figure 2Posttransplant lymphoproliferative disorder affects the immunocompromised hematopoietic cell and solid organ transplant population. This case was instructive for how late‐onset presentations of PTLD, although rare, must be recognized and treated promptly. A literature review was performed for other such cases of late‐onset PTLD and modern evidence‐based strategies to mitigate incidence of PTLD.2.1In the case of haploidentical transplant recipients with excessive immunosuppressive therapy as used in the Chinese experience, PTLD rarely presents after the first year . There a2.2EBV is implicated in the development of PTLD, and non‐EBV‐associated posttransplant lymphoproliferative disorders in transplant are not considered PTLD per european conference on infections in leukemia (ECIL) guidelines , 8. Earl2.3In our patient, who had been treated with posttransplantation cyclophosphamide, the late presentation of PTLD is unusual. He presented with typical symptoms, like lethargy, fatigue, and systemic inflammation , 3, 7. T3Recognizing signs and symptoms of PTLD is critical to early diagnosis and management, especially with late presentations. Current strategies like PTCy and weaning immunosuppression have reduced the incidence of PTLD. Future studies about the epidemiology and management of delayed or late‐onset PTLD will be needed to guide treatment in these scenarios, although these studies will likely be limited by the rare presentation of such patients.Writing – original draft, review and editing: Adam Braun. Writing – review and editing: Lawrence Liu. Writing – review and editing, and supervision: Monzr Al Malki. Writing – review and editing, and supervision: Pamela Becker.All the authors declare that they have no relevant conflict of interest to disclose.The authors received no specific funding for this work.This article does not contain any studies with human participants or animals performed by any of the authors. Informed consent was obtained from all individual participants included in the study. No material was reproduced from other sources. Case discussed was not part of a clinical trial


C‐reactive protein (CRP) is a sensitive biomarker of inflammation with moderate heritability. The role of rare functional genetic variants in relation to serum CRP is understudied. We aimed to examine gene mutation burden of protein‐altering (PA) and loss‐of‐function (LOF) variants in association with serum CRP, and to further explore the clinical relevance.We included 161,430 unrelated participants of European ancestry from the UK Biobank. Of the rare  and functional variants, 1,776,249 PA and 266,226 LOF variants were identified. Gene‐based burden tests, linear regressions, and logistic regressions were performed to identify the candidate mutations at the gene and variant levels, to estimate the potential interaction effect between the identified PA mutation and obesity, and to evaluate the relative risk of 16 CRP‐associated diseases.CRP  and G6PC genes  were associated with reduced and increased serum CRP concentration, respectively. At the variant level, seven PA alleles in the CRP gene decreased serum CRP, of which the per‐allele effects were approximately three to seven times greater than that of a common variant in the same locus. The effects of obesity and central obesity on serum CRP concentration were smaller among the PA mutation carriers in the CRP (pinteraction = 0.008) and G6PC gene (pinteraction = 0.034) compared to the corresponding non‐carriers.At the gene level, PA mutation burdens of the CRP and G6PC genes are strongly associated with decreased serum CRP concentrations. As serum CRP and obesity are important predictors of cardiovascular risks in clinics, our observations suggest taking rare genetic factors into consideration might improve the delivery of precision medicine.PA mutation burdens in the  CRP (pinteraction = 0.008) and G6PC gene (pinteraction = 0.034) compared to the corresponding non‐carriers. As serum CRP and obesity are important predictors of cardiovascular risks in clinics, our observations suggest taking rare genetic factors into consideration might improve the delivery of precision medicine.The effects of obesity and central obesity on serum CRP concentration were smaller among the protein‐altering mutation carriers in the  Circulating CRP levels exhibit a drastic increase in the acute phase of an infection/injury, but often a constant and low‐grade increase in the presence of chronic inflammation. , even in the case of the UK Biobank (UKB) where genotypes were available for nearly half a million participants. .  genes.  and loss‐of‐function (LOF) mutations at both gene and variant levels, in association with serum CRP concentrations among 161,430 UKB participants with European ancestral background. Furthermore, we aimed to elucidate the clinical relevance of the identified genetic factors by estimating the gene–obesity interaction as well as the relative risk for a set of CRP‐associated diseases.22.1UK Biobank had obtained ethics approval from the North West Multicentre Research Ethics Committee (11/NW/0382 and 16/NW/0274) and informed consent from all participants, which covered the approved use of the data in the present analyses (Approved Research ID: 22224).2.2The UKB is a large‐scale prospective cohort study and recruited >500,000 participants aged 40–69 years from across the United Kingdom. The initial assessment visit was conducted during 2006–2010 and is referred to as the baseline assessment in the present study.  to the same family cluster. For each family cluster, we retained one random family member such that all the study participants are unrelated in the analysis , of which the sequencing procedure and computation pipelines have been detailed elsewhere.  or indels that lead to any of the following functional consequences: splice acceptor variant, splice donor variant, stop gained, frameshift, stop lost, start lost, inframe insertion, inframe deletion, and missense variant. To select deleterious variants, we used two pathogenicity scores, SIFT (Sorting Intolerant From Tolerant) . That is, for each gene, participants were assigned to value 1 if carrying at least one PA allele in the corresponding gene region, i.e., mutation carriers, and to value 0 otherwise, i.e., non‐carriers. We excluded the genes without PA mutation carriers, i.e., no genotype variant observed among the present study population, and analysed 21,270 genes in the PA mutation burden test. Similarly, LOF variants were collapsed into 20,047 genes in the LOF mutation burden test.2.3.2CRP gene on chromosome 1, from the imputed genotype data. In addition, we calculated the polygenetic risk score of CRP by summing the CRP‐increasing alleles over 58 SNPs weighted by their effects on serum CRP. The SNP list and corresponding weights were determined from a previous meta‐GWAS.  and the individual‐level data of PCs were directly downloaded from the UKB data showcase (Resource 531 Sample‐QC file).2.42.4.1CRP  was measured by the high‐sensitivity immunoturbidimetric assay. CRP level at baseline assessment was analysed in the present study. The original value of CRP (in mg/L) presented a skewed distribution. Therefore, the natural logarithm of CRP was calculated and used in the statistical analyses throughout.2.4.22, field 23,104 and 21,001) and waist circumference , to define the obese and central obese status, respectively. Continuous BMI values fall into four categories: underweight (<18.5 kg/m2), normal (18.5–24.9 kg/m2), overweight (25.0–29.9 kg/m2), and obese (≥30.0 kg/m2). Indexed by sex‐specific waist circumference, the three central obesity groups were normal fat distribution (women <80 cm or men <94 cm), moderate central fat accumulation (80 cm ≤women <88 cm or 94 cm ≤men <102 cm), and high central fat accumulation (women ≥88 cm or men ≥102 cm) , sex (Field 31), education, physical activity, ever smoked , daily alcohol intake, systolic blood pressure (Field 93 and 4080), glucose , triglycerides , and high‐density lipoprotein (HDL) cholesterol . Years of birth (Field 34) were binned in a 10‐year interval to indicate the cohort effect. Education, physical activity, and daily alcohol intake were self‐reported and were derived from the questions of “qualification” (Field 6138), “types of physical activity” (Field 6164), and alcohol intake frequency (Field 1558), respectively. Three education levels include high (college or university degree), intermediate , and low ; four categories of physical activity are none (none), low (light do‐it‐yourself [DIY]), medium , and high (heavy DIY and strenuous sports); daily alcohol intake is defined as those who reported “daily or almost daily” for alcohol intake frequency. In addition, the release of WES data was treated as a dichotomized covariate in the WES analyses throughout because the samples in the initial 50 k release and the remaining samples in the second 200 k release were sequenced on different flow cell platforms.2.4.5We first described the characteristics of the study population at baseline and the features of the genetic variants and genes included in the present analysis. Variant‐wise, we summarized the number and proportion of variants by functional consequence types, as well as by MAF categories; gene‐wise, we described the gene length, the number of PA/LOF variants per gene, and the number of mutation carriers per gene.2.4.6β coefficients and standard errors [SE]) measure the effect of the gene burden due to the presence of any PA/LOF allele on natural log‐transformed CRP levels, with a positive value indicating a CRP‐increasing effect. We performed the CMC burden test in both the discovery set and the replication set. The whole exome‐wide significance levels for PA and LOF mutation burden were set to 2.35e‐06  and 2.49e‐06 , respectively. Genes that reached the whole exome‐wide significance level in the Discovery set and a <0.05 p value in the Replication set were considered significant. Two sets of covariates were considered in the burden tests, of which the first model controlled for age, sex, WES release information, and 20 genomic PCs (Model 1), and the second model additionally adjusted for BMI on top of the first model (Model 2). All burden tests were fitted in Rvtests (version: 20190205).  method,  plots, respectively. LOF variants were examined in a similar manner.To examine whether the effects of PA mutations were independent of the common genetic variants, we next fitted secondary models with additional adjustment for the polygenic risk score (PRS) of CRP, which is derived from 58 common variants. In addition to the CMC burden test, we also conducted sensitivity analyses by using two alternative gene‐based methods in the discovery set. The first one is the Morris‐Zeggini (MZ) burden test,  ≤0.05 were considered candidate variants. Second, to contrast the per‐allele effects of rare variants with that of a common variant, we also estimated the effect of the rs1205 , a top signal in the 2.4.8p value for the multiplicative term was reported as the p value for interaction. All models were adjusted for age, sex, birth year category, WES release, and 20 genomic PCs.We examined the potential interplay between PA mutation burden (genetic exposure) in the significant genes and the obesity and central obesity categories , grouped by BMI and waist circumference respectively, in controlling serum CRP level (outcome). Two types of models were fitted: (1) serum CRP being a function of obesity status in PA mutation carriers and non‐carriers, respectively (stratified models), where the PA status‐specific effects of the BMI and waist circumference categories on serum CRP were estimated; and (2) serum CRP being a function of PA mutation, obesity status, and a multiplicative term between PA status and obesity categories (interaction models), where the 2.4.9Finally, we estimated the associations between PA mutation burden and the risks of CRP‐associated diseases (listed in the “Method‐CRP‐associated diseases” section previously). Logistic regression models were fitted to estimate effect sizes (odds ratios [OR]). For each disease, we fitted a model adjusting for age, sex, birth year category, WES release, and 20 genomic PCs. FDR was used to correct multiple testing.An overview of the study aims and analysis plan is illustrated in Figure 33.12, and a median serum CRP of 1.30 mg/L. The baseline characteristics of the discovery set were not appreciably different from the replication set . All participants, of whom 45.1% were men, had an average age of 56.7 years, a mean BMI of 27.3 kg/mThe present study analysed 1,776,249 PA and 266,226 LOF variants, of which missense variants (81.7%) and frameshift variants (53.9%) were the most common types, respectively Table . The MAF3.2p < 2.35e‐06), we found that PA mutation burden in the CRP and G6PC genes was significantly associated with serum CRP concentration ; on the contrary, PA mutation carriers in the G6PC gene showed a 0.203 increase in the log serum CRP  on Y scale. The red horizontal line denotes the whole exome‐wide significant level (p < 2.35e‐06). Annotations, including gene name, chromosome, and location, of the exome‐wide significant genes were displayed in the plot. CRP gene: Chromosome 1 ‐ NC_000001.11 using assembly GRCh38.p14; G6PC gene: Chromosome 11 ‐ NC_000077.7 using assembly GRCh38.p14.The Manhattan plot shows the associations between gene‐based PA mutation burden and serum CRP , estimated by CMC burden test in the discovery set. Age, sex, WES release information, 20 genomic PCs, and BMI were controlled in the statistical models (Model 2). Each dot represents a gene, of which the location is determined by the chromosome and position on X scale and the statistic value of –log10 exhibited CRP‐decreasing effects  compared to the non‐carriers; a start loss mutation, 1:159714484:A:G, showed the largest effect size and seven heterozygous mutation carriers on average showed a 1.25 reduction in the log serum CRP . The per‐allele effect across the seven FDR significant PA variants ranged from −0.65 to −1.25, approximately three to seven times greater than that of rs1205‐C>T, a common variant and a strong signal in the CRP gene, of which the per‐T‐allele effect was −0.175 . In the present study, we found 38 PA variants in the G6PC gene exhibited CRP‐increasing effects  was a frameshift variant, 17:42900952:TC:T, with a total of 77 heterozygous carriers showing an increased serum CRP by 0.326 on log scale . However, none of the PA variants in the G6PC gene reached the FDR significance level  are heterozygous carriers of any PA allele in the on Table . Howeverse Table .CRP and G6PC genes, BMI category, and waist circumference category , overweight , underweight , “moderate central fat accumulation”  and PA mutation burden in the G6PC gene  in the PA mutation carriers in the CRP gene, significantly smaller than an increase of 1.12  in the non‐carriers of PA alleles in the CRP gene (p for interaction = 0.008). Meanwhile, the CRP‐raising effect of high central fat accumulation was significantly different by the presence of PA mutation in the G6PC gene. High central fat accumulation carriers had smaller effect on serum CRP levels  than non‐carriers with high central fat accumulation  to 33 (10.6% for depressive disorder) for the CRP gene and 1 (0.2% for psoriatic arthritis) to 68 (10.3% for depressive disorder) for the G6PC gene. However, we did not observe robust evidence to support a relationship across the health outcomes , a rare inborn metabolic disorder. , FORTE (2013‐2292), the Loo & Hans Osterman Foundation, the Foundation for Geriatric Diseases, the Magnus Bergwall Foundation, the Strategic Research Program in Epidemiology at Karolinska Institutet, King Gustaf V and Queen Victoria's Foundation of Freemasons, the China Scholarship Council, and the Swedish National Graduate School for Competitive Science on Aging and Health. The Swedish Twin Registry is managed by Karolinska Institutet and receives funding as an infrastructure through the Swedish Research Council, 2017‐00641. The analyses on UK Biobank genotypes were enabled by resources provided by the Swedish National Infrastructure for Computing (SNIC) at Uppmax partially funded by the Swedish Research Council through grant agreement no. 2018‐05973.The authors have no relevant financial or non‐financial interests to disclose.UK Biobank had obtained ethics approval from the North West Multicentre Research Ethics Committee (11/NW/0382 and 16/NW/0274) and informed consent from all participants, which covered the approved use of the data in the present analyses (Approved Research ID: 22224).Informed consent was obtained from the UKB participants included in the present study.Data S1.Click here for additional data file.Data S2.Click here for additional data file.


ASXL3) gene are known to cause neurodevelopmental delay, intellectual disability, behavioral difficulties, hypotonia, feeding problems and characteristic facial features. We previously reported 45 patients with ASXL3‐related disorder including three individuals with a familial variant. Here we report the detailed clinical and molecular characteristics of these three families with inherited ASXL3‐related disorder. First, a father and son with c.2791_2792del p.Gln931fs pathogenic variant. The second, a mother, daughter and son with c.4534C > T, p.Gln1512Ter pathogenic variant. The third, a mother and her daughter with c.4441dup, p.Leu1481fs maternally inherited pathogenic variant. This report demonstrates intrafamilial phenotypic heterogeneity and confirms heritability of ASXL3‐related disorder.De novo truncating and splicing pathogenic variants in the Additional Sex Combs‐Like 3 ( It is characterized by neurodevelopmental delay, moderate to severe learning difficulties, behavioral problems, hypotonia and feeding problems. Characteristic craniofacial features include prominent forehead, down‐slanting palpebral fissures, prominent nasal bridge, broad nasal tip, low columella, high‐arched palate, and crowded teeth.ASXL3‐related disorder which represents the first detailed description of familial ASXL3‐related disorder. Almost all patients with ASXL3‐related disorder have de novo pathogenic variants in ASXL3 :g.31320159_31320160del; NM_030632.1:c.2791_2792del, p.Gln931fs variant. This was confirmed to be paternally inherited following specific testing of ASXL3 variant in the parents.The proband was referred for genetic evaluation at 4 years of age because of developmental delay and possible episodes of apnoea. Whole exome sequencing found an This patient was born at term via normal delivery weighing 3.85 kg (75th centile). At age 4 years, his height was 98.5 cm (20th centile), weight was 13.6 kg (5th centile) and head circumference of 47.5 cm (1st centile). He had initial feeding difficulties with poor sucking reflex in the neonatal period. His fontanelles closed prematurely.Developmentally, motor milestones were achieved appropriately but speech and language development were severely delayed. At 4 years of age, he had language skills in keeping with a 12‐month‐old and had learning difficulties with an IQ of 65. He had frequent temper tantrums but no additional features of autism or attention deficit hyperactivity disorder (ADHD). He received eye movement desensitization and reprocessing (EMDR) therapy because of severe anxieties, including a fear of vomiting or having diarrhea.He was not toilet‐trained until 5 years of age and continued to have problems with eating. He attended a special day care nursery. As an infant, he had multiple upper respiratory tract infections. He was also investigated for possible episodes of sleep apnoea. Polysomnography showed few central apnoeas during rapid eye movement sleep cycles. Hering‐Breuer reflex showed no evidence of pathological breathing. He subsequently had an adenotonsillectomy with grommet insertion at the age of 3 years. Investigation of an asymptomatic supraclavicular swelling revealed a dilated subclavian artery measuring approximately 3 cm (maximum dilatation 15 mm). In the absence of clinical signs of a connective tissue disorder, a WES gene panel for thoracic aortic aneurysm and dissection was carried out and was negative. No other aneurysms were seen on full body magnetic resonance angiography (MRA).Currently, he attends regular primary education with extra support and receives additional home support.His father was born at term, with a birth weight of 3.6 kg. Motor and speech and language milestones were met appropriately. Growth parameters for the proband's 37‐year‐old father were: height 182 cm (80th centile) and weight 90 kg (90th centile).He was diagnosed with ADHD at 11 years and had gone to a special school during childhood. He had difficulties with reading and information processing.A formal neuropsychological evaluation was performed at the age of 37. The Wechsler adult intelligence scale (WAIS‐IV‐NL) showed a total IQ of 69 with deficiencies in performance subtests, working memory and information processing, and a verbal IQ of 91 (disharmonic profile). He had a part‐time job as a courier.Dysmorphic features that were present in both the proband and his father included: down‐slanting palpebral fissures, prominent columella, small mouth, high arched, narrow palate and mild micrognathia Figure . There w2.2de novo in the mother, and was maternally inherited by both her children. Testing was done through the Deciphering Developmental Disorders Study at the Wellcome Trust Sanger Institute using Agilent 2×1M for array‐based comparative genomic hybridization (aCGH), Illumina 800 K SNP genotyping to identify copy number variants, and Agilent SureSelect 55 MB Exome Plus with Illumina HiSeq for exome sequencing  study which identified a c.4534C > T, p.Gln1512Ter pathogenic variant, which occurred The mother was born at 42 weeks gestation by emergency caesarean section due to umbilical prolapse, weighing 3.9 kg (77th centile). Final growth parameters were also all within normal range; height 168 cm (85th centile), weight 91 kg (78th centile). There was no admission to the Neonatal Unit, and there were no feeding difficulties during the neonatal period. Motor milestones were slightly delayed and she first walked at 18 months. Global neurodevelopmental delay became apparent in nursery and she was later diagnosed with mild intellectual disability and attended a special school. A special programme following on from school presented a job in a supermarket, where she worked for 11 years. In terms of behavioral issues, there were severe temper tantrums in childhood and oppositional behavior during teenage years, but no evidence of autism or ADHD. She was diagnosed with epilepsy, having atonic and absence seizures, aged 7 years and was on antiepileptics until the age of 16 years old. Epilepsy recurred after the birth of her first child and she was started on levetiracetam at that time.Her daughter was born by elective caesarean section due to maternal epilepsy, weighing 3.3 kg (56th centile). Now at age 15 years, her weight is 52 kg (25th centile), height is 155 cm (9th centile) and she is relatively microcephalic with head circumference of 52 cm (2nd centile) Figure . During Her son was born by emergency caesarean section for breech presentation, weighing 3.9 kg (77th centile). At aged 16 months, growth parameters were: height 75 cm (4th centile), weight 8.6 kg (1st centile) and head circumference 45 cm (1st centile). He was severely hypotonic and had feeding difficulties from birth, with recurrent hypoglycemic episodes, which required percutaneous endoscopic gastrostomy feeding. Developmental milestones were significantly delayed; he first sat independently at 20 months of age and could not walk or talk by 7 years of age. He attended a special school due to severe intellectual disability and significant behavioral difficulties including stereotypies, head rolling and biting. He was extremely hypermobile and repeatedly put his legs behind his ears Figure . Unlike The children's father had epilepsy and mild learning difficulties. He could read and write but with some difficulty with concentration and comprehension. He left school at 15 years of age and did not achieve any formal qualifications. There was a history of alcohol abuse since childhood, and depression.The common dysmorphic feature present in all three individuals was a tall forehead Figure . Despite2.3MECP2 variant of unknown significance, which was re‐classified to benign when it was found to be paternally inherited from an asymptomatic male.The proband was referred for genetic evaluation because of intellectual disability, intractable epilepsy and autism. Trio whole exome sequencing identified a heterozygous likely pathogenic frameshift variant that was maternally inherited . Previous testing had revealed a This patient was born via caesarean section at 41 weeks due to postmaturity with no complications or need for neonatal admission. There were no feeding difficulties during the neonatal period. Developmental milestones were delayed and she first sat at 15 months, first walked at 2 years, and had developed five words by the age of 2–3 years, after which, speech regressed and she became nonverbal.At the age of 32 years, growth parameters were: height 158 cm (30th centile), weight 86.8 kg (72nd centile), head circumference 55.4 cm (50th centile). There were no appetite or sleep issues. She is unable to follow commands and remains non‐verbal.At 12 years of age, she developed epilepsy, and she was initially treated with carbamazepine. However, seizure frequency increased over the subsequent 20 years and she is now on a total of five anti‐epileptic medications  and has a vagal nerve stimulator inserted. However, she continues to have between 30 and 60 seizures a month despite this. Seizures happened during sleep and wakefulness but much more commonly during sleep. Electroencephalogram (EEG) showed multifocal sharp waves, whilst video EEG showed multifocal seizures; right frontal sharp waves and right frontal paroxysmal fast activity. The paroxysmal fast activity was maximal over the right or left hemisphere accompanied by head deviation in the direction ipsilateral to the more prominent paroxysmal fast activity. Seizure frequency did not seem to be affected by increases in medication. MRI head showed slight prominence of the right temporal horn and slight volume loss of the right hippocampal region without abnormal signaling, but was otherwise normal.Facial features included prominent forehead, down‐slanting palpebral fissures, long eyelashes with full eyebrows, prominent nasal bridge, long tubular nose, a short columella and a broad nasal tip Figure . Her mou3ASXL3‐related disorder have been assumed or confirmed de novo. Here we describe three families with inherited ASXL3‐related disorder. This has implications for clinical practice. Firstly, the knowledge of possible inheritance from a potentially mildly affected parent may warrant parental testing prior to providing clinical recurrence risk estimates. Secondly, when counseling parents or individuals with ASXL3‐related syndrome, it is prudent to inform of the possibility of recurrence and variability of inherited phenotype, so that informed decisions can be made regarding reproductive health of patients. This is especially so with improved access to prenatal exome sequencing for congenital anomalies identified antenatally.Almost all patients with ASXL3 gene variant , ID 305643, with the same c.4534C > T, p.Gln1512Ter variant as Family 2, who is reported to have a milder phenotype, which may well explain the milder phenotype in the mother, that may otherwise have gone undetected without parental testing. However, as there was apparent assortative mating with the parents of Family 2, given their intellectual and learning statuses, it is possible that the son inherited an additional so far undetected variant from his father that may have exacerbated his clinical phenotype.There is another patient on Decipher (ASXL3‐related disorder, aneurysms have not previously been associated with the condition. Subclavian artery aneurysms are rare, and in children, typically associated with inherited connective tissue disorders such as Marfan syndrome and Loeys Dietz syndrome, infections and vasculitis (Sarkar et al., ASXL3 in other patients, however, with ASXL3‐related syndrome, we would normally recommend an initial echocardiogram on diagnosis. Further surveillance would be dependent on any abnormal initial findings, or whether there were clinical findings as seen in Family 1. As we collate more data on ASXL3‐related disorder, we will understand better the natural history of this disorder to allow meaningful conclusions on need for additional screening and ongoing surveillance for features of a connective tissue disorder.The subclavian artery aneurysm found in the proband in Family 1 is a newly described observation. Although skeletal abnormalities such as Marfanoid habitus, arachnodactyly and scoliosis have been previously described with 4ASXL3‐related disorder. We confirm heritability of this condition but show intrafamilial phenotypic heterogeneity. This report has immediate clinical ramifications with respect to recurrence risk counseling for parents, parental testing and reproductive health advice for patients with ASXL3‐related disorder.We describe the clinical and molecular characteristics of three families with known familial


The aim of this research is to investigate the feasibility of folate receptor‐positive circulating tumor cells (FR+CTCs) as a biomarker for the diagnosis of malignant pulmonary nodules and the correlation between clinicopathological factors and FR+CTC levels.Patients initially diagnosed with one or more pulmonary nodules from a computed tomography scan were prospectively included. Three milliliters of peripheral blood was collected from each participant for FR+CTC analysis prior to surgery. Clinical and pathological parameters and FR+CTC levels were compared between patients with lung cancer and benign diseases.P < 0.0001). In a receiver operating characteristic analysis to distinguish the two groups, the area under curve of FR+CTC was 0.7457  using a cutoff of 8.65 FU/3 mL. The sensitivity was 86.37%, and the specificity was 74.19%. Combined with conventional serum tumor biomarkers, the area under curve was 0.922 (0.499–0.963). The sensitivity was 92.20%, and the specificity was 83.05%. FR+CTC levels were related to tumor staging (P4 < 0.001), the degree of tumor invasion both in single (P = 0.011) and multiple lesions (P = 0.022), pathological subtypes (P = 0.013), and maximum tumor diameter (P = 0.014).Six hundred fifty‐three patients had lung cancer and the other 124 had benign lung diseases based on pathological examinations of the resected specimens. The median FR+CTC value of the lung cancer group was 12.0 (95% CI 9.6–16.2) FU/3 mL and that of the benign group was 7.2 (95% CI 5.78–11.2) FU/3 mL. The difference was statistically significant (FR+CTC is an effective and reliable biomarker for the diagnosis of lung cancer. Further, FR+CTC level is correlated with tumor staging, degree of invasion, pathological subtypes, and tumor size. FR+CTC is an effective and reliable biomarker for the diagnosis of lung cancer. Combining conventional serum tumor biomarkers, including CA125, CEA, CYFRA21‐1, CA153, and NSE, could improve diagnostic efficiency. Furthermore, FR+CTC level is correlated with tumor staging, degree of invasion, pathological subtypes, and tumor size. Computed tomography (CT) examination can increase the rate of auxiliary diagnosis of lung nodules, but sometimes CT‐assisted diagnosis of focal ground‐glass opacity is misdiagnosed because of low visibility. Certain serological tumor markers are also used for lung cancer screening and diagnosis. The most commonly used ones include squamous cell carcinoma (SCC) antigen, carcinoma embryonic antigen (CEA), cytokeratin 19 fragment antigen 21‐1 (CYFRA21‐1), and neuron‐specific enolase (NSE). Studies have shown that these tumor markers can be used for in diagnosis, prognosis, and follow‐up surveillance of lung cancer.Recent studies have suggested that the vascular invasion of tumor cells may happen as early as at the precancerous stage, leading to the release of circulating tumor cells (CTCs) into the circulatory system.Several studies have demonstrated the utilities of folate receptor‐positive CTCs (FR+CTC) in diagnosis and prognosis of lung cancer and a few other cancer types that are known to have high FR expressions.The primary goal of this study was to investigate the value of FR+CTCs combined with traditional tumor biomarkers for the diagnosis of benign and malignant pulmonary nodules. In addition, the correlation between FR+CTC and other conventional clinical and pathological factors was explored.22.1This is a prospective, observational study to investigate FR+CTC in early diagnosis of lung cancer and the association between FR+CTC and the clinicopathological characteristics of lung cancer patients. From March 2019 to May 2020, 777 patients who were diagnosed with one or more pulmonary nodules based on CT scan were enrolled at the First Affiliated Hospital of Guangzhou Medical University. The study was approved by the Research Ethics Committee of the First Affiliated Hospital of Guangzhou Medical University. All patients provided written informed consent before inclusion into this study.2.27/mL or <2 × 106/mL (as extreme white blood cell count could affect the enumeration of CTCs); (5) patients who took folic acid tablets long term and stopped taking <3 days prior to obtaining the blood sample (as high folic acid concentration has reported to lower the expression of FR); and (6) abnormalities during sample collection or processing such as inadequate amount of blood, blood clots in the sample, hemolysis, incomplete lysis of red blood cells, adhesion of the sample cells after lysis, or insufficient amount of cells after lysis.Inclusion criteria are the following: (1) ages between 18 and 80 years and Eastern Cooperative Oncology Group performance status of 0–1; (2) scheduled to receive surgical resection upon physician's assessment; (3) no previous antitumor therapy (including preoperative neoadjuvant therapy); and (4) informed consent to collect peripheral blood for FR+CTC detection before surgery. All participants were required to fast overnight before blood collection. The exclusion criteria were the following: (1) patients with other severe diseases and not suitable for tissue biopsy or surgical treatment; (2) a history of other malignancies within the past 5 years, except for cancer that had been cured and had no risk of recurrence or metastasis; (3) incomplete patient information; (4) patients with leukocyte counting >1.2 × 102.3Lung cancer staging was based on the 7th IASLC TNM Staging System. Benign lung diseases included hamartoma, pneumonia, tuberculosis, intrapulmonary lymph node, bronchiectasis, and granuloma. According to IASLC/ATS/ERS classification of lung adenocarcinoma (ADC), ADC in situ (AIS) is defined as non‐invasive ADC showing no stromal, vascular, or pleural invasion, whereas minimally invasive ADC (MIA) and invasive ADC (IAC) are defined as AIC containing an invasive component.2.4FR+CTC analysis was performed using CytoploRare® kit provided by GenoSaber Biotech Co. Ltd. . According to the manufacturer's instruction, CTCs were negatively enriched where erythrocytes were lyzed by lysis buffer and leukocytes were depleted using anti‐CD45 immunomagnetic beads. Next, CTCs were incubated with a proprietary probe that consists of conjugates of a folic acid analog and a synthesized oligonucleotide. Then, the unbound probes were washed off. The bound probes were removed with stripping buffer and collected by centrifugation and neutralized.−14 to 10−9 M is used for CTC quantification, which corresponds to 2–2 × 105 CTC U/3 mL blood. Samples from each patient were tested in duplicates with six standards and three quality controls.FR+CTC was quantified by quantitative polymerase chain reaction analysis. First, the probe was annealed and extended before amplification. Then, the extended probe was analyzed using a Taqman probe on ABI StepOne™ system . The primer sequences and reaction conditions were as described by Lou et al.2.5Serum CTCs were collected prospectively, and cancer antigen 125 (CA125), CEA, cytokeratin 19 fragment (CYFRA21‐1), cancer antigen 153 (CA153), NSE, and SCC antigen were collected retrospectively. Tumor biomarkers including CA125, CEA, CYFRA21‐1, CA153, and NSE were tested by electrochemical luminescence (ECLINA) . SCC antigen was tested by a chemiluminescent microparticle immunoassay using the ARCHITECT I2000 SR system .2.6P < 0.05 was considered to indicate a statistically significant difference. Receiver operating characteristic (ROC) curve was used to examine the diagnostic efficiency as indicated by the area under the ROC curve. Youden index was used to determine the optimal cutoff threshold. Logistic regression was used to establish a joint diagnostic model of different biomarkers for distinguishing lung cancer from benign lung diseases. Pearson's correlation was used to analyze the association between FR+CTC levels and clinical factors.Statistical analysis was performed using the R software and the Prism 5.0 system . Wilcoxon test, Mann–Whitney test, and Kruskal–Wallis test were used for comparison between groups as appropriate. 33.1Seven hundred seventy‐seven patients were included in this study, of which 653 had lung cancer and 124 had benign lung diseases. The median age was 58 and 55 years old, respectively. There were 321 males and 332 females in malignant cohort and 77 males and 47 females in benign cohort. Of the 653 lung cancer patients, 77.79% of those had ADC, 58 (8.89%) had SCC, and the remaining 87 had other lung cancer types. The 57.1% of the lung cancer was Stage I. The detailed characteristics are shown in Table 3.2P < 0.0001). The 8.65 FU/3 mL was selected as the optimal cutoff threshold for differentiating lung cancer from noncancer patients, and the sensitivity and the specificity of FR+CTC was 86.37% and 74.19%, respectively. The area under curve of FR+CTC in differentiating lung cancer from benign diseases was 0.746   in terms of sensitivity in differentiating patients with lung cancer from benign participants. It was similar to that of CA153 and outperformed traditional serum tumor biomarkers (NSE and CA125) twofold to threefold  Figure . Meanwhild Table .3.3P = 0.013). Four hundred eighty‐nine out of the 653 lung cancer patients had tumor staging data after surgical resection. Of these patients, 24, 373, 38, 30, and 24 had AIS, Stage I, Stage II, Stage III, and Stage IV tumors, respectively. As shown in Table P = 0.00037).The correlations between FR+CTCs and clinical pathological characteristics were analyzed to explore the application of FR+CTCs for lung cancer. All the information of lung cancer groups is summarized in Table P = 0.018) (Table P = 0.011); the P value of FR+CTC levels between AIS and IAC is 0.05, and the FR+CTC of AIS patients was significantly lower than MIA (P = 0.0057) . Further, patients with AIS had significantly lower FR+CTC level than those with MIA (P = 0.014) .Five hundred eight out of the 653 lung cancer patients with ADC had data on the degree of tumor invasion based on pathological examination of resected specimens. Twenty‐four had AIS, 312 had MIA, and 128 had invasive carcinoma (IAC). The median FR+CTC level in patients with AIS, MIA, and IAC was 10.05 FU/3 mL, 10.85 FU/3 mL, and 11.90 FU/3 mL, respectively; the differences in the FR+CTC level in the three groups were statistically significant  Figure . For mul) Figure . The FR+P value was 0.014. The median FR+CTC level for MTD ≤ 5 cm is lower than MTD > 5 cm, , so we can infer that MTD (bounded by 5 cm) showed a slight correlation with the FR+CTC level. The median FR+CTC level for MTD ≤ 3 cm is lower than MTD > 3 cm, .A total of 240 patients with single lesion in lung cancer group had data on the tumor size before treatment. As shown in Table P = 0.165), and there was no significant correlation between FR+CTC and the number of lesions or tumor location  ; however, there was no significant correlation between the FR+CTCs levels and lymph node metastasis , which provides ideal conditions for the detection of lung cancer blood targeting FR+CTC. In lung cancer patients, each CTC can express 100 000 orders of folic acid receptors, and PCR amplification can achieve two‐stage signal amplification, so a very small amount of CTCs can be detected in a 3‐mL blood sample.In our study, we found that the median FR+CTC value of lung cancers was higher than that of the noncancer participants. These findings are consistent with previous research in comparison of FR+CTC levels in benign and malignant groups.We found that some clinical factors are associated with FR+CTC levels, FR+CTC levels were related to with tumor staging, and as the progression of tumor stage, the level of FR+CTC also increased, which was statistically significant. For multiple lesions, FR+CTC levels were related to with the degree of tumor invasion both in multiple lesions and single lesion. A recent study of 3798 patients from the pulmonary hospital literature analyzed the correlation between FR+CTC and tumor gene expression, stage, and degree of invasion, confirming that the determination of FR+CTC level is a simple and time‐saving method to improve the diagnosis of pulmonary nodules.We then detected the correlation of FR+CTC levels with pathological subtypes; we found that the median FR+CTC level in SCC group was higher than the group of ADC, which was similar to the previous studies.Then, the clinical factors of tumor location were considered, and there was not a statistical difference between the two groups; however, other study concludes that FR+CTC was correlated with tumor location, and it confirmed that the combination of CTCs and CEA can help guide the management of patients with solitary pulmonary nodules suspected of being lung cancer.Then, the clinical factors of MTD were considered, the levels of FR+CTC were increased by the increase of MTD, and we can draw a conclusion that there were statistically differences among all groups. The median FR+CTC level for MTD ≤ 5 cm is lower than MTD > 5 cm, so we can infer that FR+CTC detection before treatment combined with the MTD can be used to evaluate the subnodular infiltrates less than or equal to 5 cm before surgery, which can guide the clinical operation. According to the clinical data of a pulmonary nodules article,Several limitations of the present study should be mentioned. First, even though the sample size was much larger than others, the number of different subgroups varied greatly, which increased uncertainty. Second, our study is single hospital‐based, and it only represents the same source population rather than all population.FR+CTC combined with conventional tumor markers is a reliable biomarker for the diagnosis of lung cancer. Notably, some clinical factors indeed of tumor staging and degree of invasion, pathological subtypes for ADC and SCC, and MTD (bounded by 5 cm) showed a slight correlation with the FR+CTC level, and more researches are needed.Conception and design: Yongping Lin; Administrative support: Yunjian Xu; Provision of study materials or patients: Qianjun Li; Collection and assembly of data: Zhijian Lin; Data analysis and interpretation: Yunjian Xu; Manuscript writing: All authors; Final approval of manuscript: All authors.The authors declare that there is no conflict of interests.The study was approved by the Research Ethics Committee of the First Affiliated Hospital of Guangzhou Medical University. All patients provided written informed consent before inclusion into this study.


To explore the diagnostic accuracy of ultrasound measurement of optic nerve sheath diameter (ONSD) and optic disc height (ODH) in detecting intracranial hypertension in non‐small‐cell lung cancer (NSCLC) patients with leptomeningeal metastases (LM).Seventy‐two patients with NSCLC‐LM and 65 patients with NSCLC were enrolled. The ONSD, ODH, eyeball transverse diameter (ETD), and eyeball vertical diameter (EVD) were measured by ultrasound. Subsequently, lumbar puncture was performed in NSCLC‐LM patients to measure cerebrospinal fluid pressure (CSFP), and intrathecal chemotherapy was regularly implemented. Pearson's correlation analysis was used to analyze the relationship between CSFP and ultrasound findings. The diagnostic accuracy of ONSD, ODH, and combined ONSD and ODH was evaluated by receiver operating characteristic (ROC) curve analysis and the corresponding area under the ROC curve (AUC).p < 0.05). The ONSD, ODH, ONSD/ETD, and ONSD/EVD values were all elevated in the abnormally elevated CSFP group . ONSD, ODH, ONSD/ETD, and ONSD/EVD were positively correlated with CSFP . The AUCs for ONSD, ODH, ONSD/ETD, and ONSD/EVD to detect CSFP >280 mmH2O were 0.787 , 0.885 , 0.765 , and 0.788 , respectively. When ONSD was combined with ODH, the AUC was 0.913 . Furthermore, intrathecal chemotherapy was associated with a downtrend in CSFP and ultrasound findings.The ONSD, ODH, ONSD/ETD, and ONSD/EVD values were higher in the NSCLC‐LM group (all There are important advantages of using bedside ultrasonography for detecting elevated CSFP in NSCLC‐LM patients. Further research should be performed to evaluate the clinical significance of an enlarged ONSD and increased ODH in NSCLC‐LM. Bedside ocular ultrasonography is a noninvasive, bedside and easy‐to‐use way for detecting increased intracranial pressure in patients with leptomeningeal metastases from non‐small‐cell lung cancer. The measurement of combined ONSD and ODH may be a promising technique to detect elevated CSF pressure.. The ONS is a continuation of the dura.Our main purpose was to recognize the accuracy of ultrasonographic measurements for the detection of cranial hypertension in patients with LM from NSCLC and to identify the optimal ONSD and ODH threshold.22.1We conducted a retrospective cohort study using data from the Department of Geriatric Oncology in Nanjing Drum tower Hospital in China. We signed informed consent with family members of all patients. This study was authorized by the Ethics Committee of Nanjing Drum tower Hospital. All procedures have proceeded in line with the ethical principles of the Declaration of Helsinki.2.2Between March 2021 and December 2021, 137 patients (72 with LM and 65 without LM) with NSCLC in the Department of Geriatric Oncology in Nanjing Drum tower Hospital were enrolled. All patients were diagnosed by clinical, CSF and radiographic findings based on the EANO–ESMO criteria.2.32O as abnormally elevated intracranial pressure.General information were recorded at admission: sex, age, BMI, oxyhemoglobin saturation, blood pressure, heart rate, and blood glucose. All patients were diagnosed by clinical symptoms, radiographic findings and cerebrospinal fluid cytology before enrollment. Two experienced ultrasound physicians performed ultrasound measurements at the bedside. Here, a 5‐ to 12‐MHz linear array ultrasonic probe from Philips iU22  was used. The patient was supine, facing forward, and looking forward with the eyelids closed. A small volume of ultrasound gel is applied to the probe. And the probe was placed horizontally above the transverse axis of the eyeball with no pressure Figure . Slowly 2.4t‐test  or Mann–Whitney Utest  for continuous variables when exploring the differences between NSCLC patients with and without LM and between LM patients with CSFP ≤280 mmH2O and CSFP >280 mmH2O. We implemented Pearson's correlation analysis to analyze the relationship between CSFP and optic nerve examinations. A receiver operating characteristic (ROC) curve was plotted to figure up the area under the ROC curve (AUC). All statistical analyses and graphics were performed with SPSS v26.0 (IBM) and GraphPad Prism v8.3.0 (GraphPad). A value of p < 0.05 (two‐sided) was regarded significant.Use a 3p < 0.05).A total of 137 NSCLC patients were enrolled in our study, 72 of whom were identified with LM. The baseline characteristics and demographics of all patients are shown in Table 2O) and an abnormally elevated CSFP group (CSFP >280 mmH2O). The CSFP of the normal CSFP group  and abnormally elevated CSFP group  were 164.11 ± 35.84 mmH2O and 323.56 ± 22.41 mmH2O, respectively. The clinical characteristics, ONSD measurements and ODH are shown in Table p > 0.05). The average ONSD measurements of the two groups were 0.57 (0.52–0.60) cm and 0.63 (0.57–0.66) cm. The ONSD measurements were higher in the abnormally elevated CSFP group . In addition, the values of ONSD/ETD and ONSD/EVD were bilaterally higher in the abnormally elevated CSFP group . A total of 17/56  patients in the normal CSFP group had optic disc edema, while 33/72  patients in the abnormally elevated CSFP group had optic disc edema.Based on the results of lumbar puncture pressure, patients with LM were divided into a normal CSFP group CSFP ≤280 mmH80 mmH2O p < 0.001, p = 0.012, p = 0.003). We also divided patients with LM into two groups based on whether they had symptoms of intracranial hypertension, whether MRI showed lateral ventricular widening, whether brain metastasis was present, whether MRI showed meningeal enhancement, and whether CSF had atypia cells. However, the ONSD value, ONSD/ETD and ONSD/EVD did not differ between the two groups.We divided patients with LM into two groups based on where papilledema was observed Table . We founr = 0.531, p < 0.001; r = 0.383, p = 0.025; r = 0.534, p < 0.001; r = 0.535, p < 0.001). Scatter diagrams are presented in Figure According to the applied Pearson's correlation analysis, ONSD, ODH, ONSD/ETD, and ONSD/EVD were positively correlated with CSFP  showed a baseline ONSD>0.615 cm, and of the 22 patients, a baseline CSFP>280 mmH2O was present in 11 patients  on admission. We discovered that the initiation of intrathecal chemotherapy was associated with a downtrend in CSFP and ultrasound findings, including ONSD, ODH, ONSD/ETD, and ONSD/EVD , which means that nearly one‐third of patients diagnosed with elevated ICP using this ODH threshold will, in fact, not have intracranial hypertension and may be exposed to the risks of overtreatment. ONSD and ODH both performed badly in recognizing patients requiring an aggressive therapy to treat intracranial hypertension. However, when ONSD was combined with ODH, the optimal threshold achieved a specificity of 85.7%, in conjunction with a sensitivity of 87.5%. Therefore, we could use the combined ONSD and ODH to detect abnormally elevated CSF pressure in NSCLC‐LM patients.There have been some studies on ultrasonic measurement of ONSD for the diagnosis of high ICP,A recent study suggests that the extent to which ONS swell with increased intracranial pressure may vary from person to person.There are still several limitations in our study. A larger sample size may have narrowed the CIs in the estimates of accuracy—there were only 16 (22.22%) CSF pressure > 280 mmH2O in the analysis. The ECOG score for all patients was between 0 and 2; therefore, the most severe cases were likely excluded. Moreover, we compared ultrasound values with lumbar CSF pressure instead of invasive ICP measurement, yet there may be differences in the CSF pressure between intracranial and spinal compartments.In conclusion, our results demonstrated a significant relationship between ultrasound findings and CSF pressure. The measurement of combined ONSD and ODH may be a promising technique to detect elevated CSF pressure. A prospective multicenter clinical trial with a larger cohort is necessary to further evaluate the clinical significance of an enlarged ONSD and increased ODH in NSCLC‐LM.Cheng Jiang: Investigation ; writing – original draft (lead); writing – review and editing (lead). Yongjuan Lin: Data curation ; software (lead). Huiying Li: Formal analysis ; supervision ; validation . Yu Xie: Investigation ; methodology ; resources . Tingting Yu: Funding acquisition ; project administration ; writing – original draft (supporting); writing – review and editing (supporting). Jingyu Feng: Writing – original draft (supporting); writing – review and editing (supporting). Mingmin Huang: Funding acquisition (supporting); investigation (supporting); methodology (supporting); project administration (supporting). Aibin Guo: Resources (supporting); supervision (supporting); validation (supporting). Haiyun Shen: Data curation ; formal analysis (supporting); methodology ; software (supporting). Yidan Zhang: Data curation ; formal analysis ; methodology . Zhenyu Yin: Conceptualization (lead).This work was supported by funding from the Medical Key Science and Technology Development Project of Nanjing (No. ZKX18014), the Cadre Health Care Project of Jiangsu Province , and the Cancer Research Funding of CSCO‐Hausen (No. Y‐HS2019‐5).All authors have no conflicts of interest to disclose.


Postoperative delirium (POD) is a serious complication in patients undergoing microvascular head and neck reconstruction. Whether intraoperative and postoperative blood pressure regulation are risk factors for POD remains unclear. This study aimed to highlight the relationships between intraoperative and postoperative blood pressure regulation and POD in microvascular head and neck reconstruction.Data from 433 patients who underwent microvascular head and neck reconstruction at our department of oral and maxillofacial surgery between 2011 and 2019 were retrospectively analyzed. The 55 patients with POD were matched with 55 patients without POD in terms of tracheotomy, flap type, and flap location, and the intraoperative and postoperative systolic and mean blood pressure values were compared between the two groups.p < 0.001; and 56.0 mmHg vs. 62.0 mmHg, p < 0.001; respectively). A lower intraoperative minimum MAP value was identified as predictor for POD . The cut-off value for intraoperative MAP for predicting POD was  ≤ 62.5 mmHg .Patients with POD showed lower intraoperative and postoperative minimum mean arterial pressure (MAP) values than patients without POD (60.0 mmHg vs. 65.0 mmHg, Maintaining a stable intraoperative minimum MAP of  > 62.5 mmHg could help to reduce the incidence of POD in microvascular head and neck reconstruction. Postoperative delirium (POD) is defined as a change in a patient’s mental status due to physical problems related to surgery and is characterized by an acute onset and fluctuating disturbances in consciousness, attention, and cognition , 2. AlthPOD is a common complication in patients who undergo head and neck surgery, with incidence rates ranging from 7.5 to 33.3% –7. Many Intraoperative and postoperative blood pressure regulation may be additional risk factors for POD; systemic blood pressure is a major determinant of cerebral perfusion, and cerebral hypoxia due to cerebral hypoperfusion is involved in the pathophysiology of POD –14. UndeSeveral studies have investigated the association between blood pressure regulation  and POD, with conflicting results—associations have been both confirmed and denied , 17–23. The aim of this study was to investigate the relationships between intraoperative and postoperative blood pressure regulation and POD in patients undergoing microvascular head and neck reconstruction.This study was approved by the local ethics committee of the Medical Faculty of RWTH Aachen University (EK 144-18) and all methods were in accordance with the relevant guidelines and regulations.Data from 433 patients who underwent reconstruction in the head and neck region with microvascular free flaps in our department of oral and maxillofacial surgery between 2011 and 2019 were retrospectively analyzed. The patients were surgically treated for malignant or non-malignant diseases. The data were obtained from clinical notes and operation reports. Patients with neurologic diseases, psychiatric diseases, or substance use disorders  were excluded from the study. Patients with incomplete or missing data were also excluded.n = 2 and n = 0), ameloblastoma (n = 2 and n = 1), atrophies (n = 0 and n = 1), basal cell carcinoma (n = 0 and n = 2), medication-related osteonecrosis of the jaw (n = 0 and n = 1), melanoma (n = 0 and n = 1), Merkel cell carcinoma (n = 2 and n = 0), myxoma (n = 1 and n = 0), nerve sheath tumor (n = 0 and n = 1), osteomyelitis (n = 2 and n = 1), squamous cell carcinoma (n = 40 and n = 43), squamous cell carcinoma metastasis (n = 1 and n = 0), osteoradionecrosis of the jaw (n = 1 and n = 2), sarcoma (n = 2 and n = 2), salivary gland carcinoma (n = 1 and n = 0), and trauma (n = 1 and n = 0).Further analysis was performed for all 55 patients with POD and 55 matched patients without POD with respect to surgical procedure parameters . The diagnoses for patients without POD and patients with POD, respectively, were adenocarcinoma . Postoperative intensive care management included sedation and mechanical ventilation until at least the first postoperative morning. In addition, blood pressure was adapted to a target SBP of > 125 mmHg until the first postoperative morning. 5000 IU of heparin was administered subcutaneously three times daily for seven days. Enteral nutrition was administered via nasogastric tube until the patient was able to swallow. If POD occurred, the patients received psychoactive medications , and a psychiatrist was consulted if needed.Variables included demographic data (such as sex and age) and clinical data (such as body mass index (BMI), American Society of Anesthesiologists score (ASA), excessive alcohol consumption, smoking, diagnosis of preoperative arterial hypertension, preoperative antihypertensive medication, transplant type, transplant location, tracheotomy, operation time, blood transfusion, intraoperative SBP, intraoperative MAP, postoperative SBP, postoperative MAP, transplant revision, transplant success, length of hospital stay, length of ICU stay, and postoperative complications). In line with commonly used definitions, smoking was defined as a condition if the patients had smoked daily for a period of more than 6 months at the time of the preoperative anesthesia preparation interview, and excessive alcohol consumption was defined as a condition if the patients consumed more than 40 g of pure alcohol per day (for men) or more than 20 g of pure alcohol per day (for women) at the time of the preoperative anesthesia preparation interview , 26. A dIntraoperative blood pressure values were obtained from the graphical representations of blood pressure values in the clinical records , and postoperative blood pressure values were obtained from an electronic recording system . Intraoperative blood pressure was measured invasively via an arterial catheter; postoperative blood pressure was measured invasively via an arterial catheter or non-invasively via a blood pressure cuff. Non-invasive blood pressure measurement data were used only when invasive blood pressure measurement data were not available. The patient’s SBPs and MAPs were recorded intraoperatively at 15-min intervals and postoperatively at 5-min intervals. The reference values for SBP and MAP were obtained from the routinely performed blood pressure measurement on the day before surgery or, if not available, calculated as the mean of all preoperative blood pressure measurements in the operation room before the induction of anesthesia. The time of anesthesia induction was defined as the time of administration of the induction medication. Intraoperative and postoperative SBP and MAP were determined as absolute minimum and maximum values. The deviations in SBP and MAP were calculated as the absolute difference between the maximum value and the reference value for upward deviation and as the absolute difference between the reference value and the minimum value for downward deviation. If the maximum value was lower than the reference value or the minimum value was higher than the reference value, the deviation was set to zero. All blood pressure values were analyzed for patients without POD until discharge from the ICU; all blood pressure values were analyzed for patients with POD until the occurrence of POD.The diagnosis of POD was based on the Confusion Assessment Method (CAM) for ICU or by a validated chart review method, considering the words “delirium” and “delirious”, in line with the criteria of the Diagnostic and Statistical Manual of Mental Disorders (Fourth Edition) , 27–29. P < 0.05 were considered statistically significant. No correction for multiple testing was performed. The statistical analysis was performed using SPSS version 26 .The patients were divided into those with POD and those without POD. The patients were also divided into two classes according to ASA (> 2 and ≤ 2). Descriptive analyses were performed separately for patients without POD and patients with POD, with continuous variables expressed as medians with interquartile ranges (IQRs) and categorical variables expressed as numbers with percentages (%). Testing for differences between patients with POD and patients without POD was performed using McNemartest for categorical data or Wilcoxon signed rank test for metric data. Multivariable conditional logistic regression analysis was performed for variables that showed significant differences between patients with and without POD. Receiver operator characteristics analyses were performed for intraoperative MAPs, and the theoretical optimal cut-off MAP values for the prediction of POD were determined by the calculation of the Youden index . The diap = 0.011). The groups did not differ in terms of sex (p = 0.486), ASA (p = 0.063), BMI (p = 0.206), excessive alcohol consumption (p = 0.307), smoking (p = 0.307), preoperative arterial hypertension (p = 1.000), preoperative antihypertensive medication , operation time (p = 0.580), or blood transfusion (p = 0.327). No difference was observed between the groups for transplant success (p = 1.000), with 1 (1.8%) transplant loss  in the group of patients without POD and 2 (3.6%) transplant losses  in the group of patients with POD.The analyzed study population included 110 patients out of a total of 433 patients, 55 of whom had POD. All 55 patients with POD were matched with patients without POD in terms of transplant type, transplant location, and tracheotomy (Table p < 0.001), with 17 (30.9%) transplant revisions compared to 3 (5.5%) transplant revisions. Patients with POD also had a higher rate of complications (p < 0.001), with 19 (34.5%) patients with POD having complications compared to 1 (1.8%) patient without POD having complications, and longer hospital stays (p < 0.001), with a median of 23 (IQR 22) days compared to a median of 17 (IQR 10) days for those without POD. Furthermore, patients with POD had longer ICU stays (p < 0.001), with a median of 7 (IQR 11) days compared to a median of 2 (IQR 1) days for those without POD.Compared to patients without POD, patients with POD underwent more transplant revisions .The intraoperative and postoperative blood pressure values for SBP and MAP differed between patients with POD and patients without POD Table .Table 2Bp < 0.001), with a median of 60.0 (IQR 10.0) mmHg compared to a median of 65.0 (IQR 5.0) mmHg , with a median of 184.0 (IQR 33.0) mmHg compared to a median of 171.0 (IQR 29.0) mmHg , with a median of 78.0 (IQR 30.0) mmHg compared to a median of 92.0 (IQR 17.0) mmHg , with a median of 56.0 (IQR 11.0) mmHg compared to a median of 62.0 (IQR 10.0) mmHg , with a median of 51.0 (IQR 46.0) mmHg compared to a median of 29.0 (IQR 40.0) mmHg  as a predictor for POD (Table p < 0.001) determined that the theoretical optimal cut-off value for predicting POD was ≤ 62.5 mmHg .The multivariable regression analysis identified intraoperative minimum MAP , OD Table . PatientThe aim of this study was to investigate the potential roles of intraoperative and postoperative systemic blood pressure regulation as risk factors for POD in patients undergoing head and neck free flap reconstruction. The presumed multifactorial etiology of POD makes it likely that multiple risk factors interact, and although several risk factors have been identified in patients undergoing head and neck free flap reconstruction, data on systemic blood pressure regulation are lacking; findings to optimize intraoperative and postoperative blood pressure regulation would be directly applicable , 21, 32.The potential roles of intraoperative and postoperative systemic blood pressure regulation as risk factors for POD are related to the pathophysiological mechanisms beyond POD, including cerebral hypoxia due to cerebral hypoperfusion –14. HencIn the absence of uniform definitions of hypotension and hypertension, blood pressure values for MAP and SBP in this study were analyzed as metric variables without categorization based on predefined cut-off values , 16, 38.This study identified a lower intraoperative minimum MAP as a predictor for POD. This is consistent with the findings of two previous studies, which showed that intraoperative MAPs below 65 mmHg and 60 mmHg, respectively, were associated with POD , 19. SimThe associations between lower intraoperative minimum MAPs and POD observed in this study may be explained by lower cerebral perfusion due to lower MAP despite cerebral autoregulation according to the proposed pathophysiological mechanism of cerebral hypoxia for POD –14. InteThis study has some limitations that require consideration. These include a retrospective chart review with limited documentation quality, a lack of assessment of preoperative cognitive impairments, and the absence of mental health professionals for the diagnosis of POD. Nevertheless, the ICU-CAM tool used in this study has shown a strong correlation with expert opinion . FurtherIn this study, a matched-pair analysis was used to study a particular patient group that has not been specified in previous studies. The findings of the present study show that lower intraoperative MAPs are a predictor for POD. These findings may contribute to the further optimization of patient care (through the reduction of POD) via the maintenance of intraoperative MAPs above 62.5 mmHg. Further studies are needed to confirm these results and to investigate the potential benefits of an intervention aimed at minimizing excessively low MAP values.This study found that low intraoperative minimum MAPs are modifiable risk factors for POD in patients undergoing head and neck surgery with microvascular free flap reconstruction. This knowledge has implications for both the further prevention and the treatment of POD. Maintaining an intraoperative minimum MAP of > 62.5 mmHg may reduce the incidence of POD, and the close monitoring of patients with lower intraoperative minimum MAPs may help clinicians to initiate treatment earlier, reducing the severity of POD.


SGCG gene. Here, we report a case of a 26‐year‐old male who had inactive walking due to proximal muscle weakness. Targeted next‐generation sequencing found a novel variant c.412C > T (Q138*) in the SGCG gene.Limb‐girdle muscular dystrophy‐type 2C (LGMD2C) is caused by mutations in the  Currently, more than 27 different muscular disorders are identified as subtypes of LGMDs. Derived from the inheritance manners, LGMDs were classified into two main types: LGMD type 1  and LGMD type 2 .SGCG gene. This gene is located on 13q12, comprising eight exons and encodes for γ‐sarcoglycan. LGMD2C is described by the childhood onset of progressive muscular dystrophy. The average age of onset is 5.3 years, and approximately half of the patients typically lose ambulation at 12 years old. Muscle weakness, calf hypertrophy, high serum CK concentration, respiratory failure and cardiomyopathy are common features.SGCG gene (NG_008759).LGMD2C (OMIM#253700) is initiated by mutations in 2A 26‐year‐old male was diagnosed with acute respiratory failure and suspected muscular dystrophy at E Hospital . He was born healthy without any complications. He could walk normally during childhood. When he was 8 years old, the difficulty walking and climbing symptoms appeared and his calves were stiff. At the age of 10, he lost ambulation and lay on the spot until now Figure . Non‐conhttp://genetics.bwh.harvard.edu/pph2/), SNPs & GO: GO‐Gene Ontology (https://snps‐and‐go.biocomp.unibo.it/snps‐and‐go/), DUET (DUET (unimelb.edu.au)), Mutation Taster (https://www.mutationtaster.org/), PROVEAN: Protein variation effect analyzer (http://provean.jcvi.org/index.php).To identify the type of muscular dystrophy, the peripheral blood of the patient and his relatives was collected to extract genomic DNA for genetic analysis, after that the family has provided informed consent. Targeted amplicon sequencing was performed using a gene panel for hereditary muscular dystrophies, on the Illumina HiSeq 2000 platform . A gene panel was provided in the Supporting Information. Sanger sequencing was performed to confirm the detected abnormalities. To evaluate the pathogenicity of the gene variants disclosed, several bioinformatic tools were applied containing Polyphen‐2: Polymorphism phenotyping 2.0 (3SGCG gene (LGMD2C). As a result, two heterozygous SGCG variants were found. The heterozygous C to T base substitution at position 320 in exon 4 of the SGCG gene, triggers an amino acid modification from Serine to Leucine at position 107 , and a heterozygous C to T base alteration at position 412 in exon 5 of the SGCG gene, causing a codon change from Glutamine to a stop codon at position 138 . They were identified as candidate causative mutations  and c.412C > T (p.Gln138*), and showed an LGMD phenotype. The father (II.1) carried heterozygous SGCG variant c.320C > T (p.Ser107Leu); the mother (II.2) and brother (III.2) were heterozygous for c.412G > T (p.Gln138*), healthy and did not show any abnormalities  and c.412C > T (Gln138*), classifying thus LGMD as type 2C. The SGCG gene encodes γ‐sarcoglycan protein which is greatly expressed in skeletal and cardiac muscle. γ‐sarcoglycan belongs to a group of the transmembrane protein linked with the dystrophin‐associated protein (DAP) complex. This complex is mandatory for the stability of muscle membranes and function, and its inhibition results in muscle fiber damage and necrosis.SGCG variants were found in patient's family including father, mother, brother and sister  and normal in the proband. The significant difference in CK and CK‐MB levels between our patient and his sister may be associated with the advanced stage of LGMD. CK levels in the sister can reflect muscle degeneration at the initial stage of LMGD. Conversely, the lower CK level in the proband may implicate the progression of the dystrophic process at the late stage. Moreover, male patients show more severe and rapid clinical progression than females. The gender differences influencing muscle degeneration can be associated with sex‐related variable hormone levels, such as estrogen and testosterone, and initial muscle mass.We successfully identified two SGCG.SGCG variants c.525delT and c.848G > A, found with high incidence rate (65% in Moroccan and over 40% in Gypsy populations), were not observed in our patient.SGCG variants here reported have been ascertained to be pathogenic, based on ACMG guidelines. A heterozygous c.320C > T (S107L) and a novel nonsense c.412C > T (Q138*) variant may cause a dysfunctional protein altering its structure. In addition, these variants are localized at the extracellular carboxyl‐terminal of γ‐sarcoglycan (aa 59–291). This domain plays an important role in the retention stability of the sarcoglycan–sarcospan complex (SG‐SSPN).Of the two identified variants, the missense variant c.320C > T (S107L)  was the first recorded in the Chinese population; whereas the nonsense variant c.412C > T (Q138*) is a novel variant of To date, there is no specific treatment for LGMD2C, but different promising approaches, including gene therapy, cell therapy, and pharmacological trials are being investigated.5SGCG gene using targeted NGS. An early genomic diagnosis is essential both to limit and slow adverse outcomes and complications and offer adequate counseling, including prenatal/preimplantation diagnosis in case of couples at increased risk. This may allow to enhance the quality of life of patients and of their families, through an individualized approach able to provide the most appropriate therapies of each single case. Further study involving larger cohorts should be performed to clarify the genetic prevalence and the genotype–phenotype correlations of LGMD in the Vietnamese population.Our report described the first case of LGMD2C in Vietnam, providing clinical characteristics, laboratory testing, and genetic findings. We identified a novel variant: c.412C > T (Q138*) of the Nam‐Chung Tran: Data curation; formal analysis. Tuan Anh Nguyen: Data curation; formal analysis. Thanh Dat Ta: Formal analysis; writing – original draft; writing – review and editing. Thinh Huy Tran: Methodology. Phuoc‐Dung Nguyen: Data curation; formal analysis. Chi Dung Vu: Methodology. Van‐Hung Nguyen: Data curation. The‐Hung Bui: Writing – review and editing. Thanh Van Ta: Methodology; supervision; visualization. Van Khanh Tran: Methodology; supervision; validation; writing – review and editing.None.The authors declare no conflict of interest.Written informed consent was obtained from the patient for publication of this case report details.Data S1.Click here for additional data file.