Gli1-CreER+;Tmfl/-mice and Sox9-CreER+;Tmfl/-mice showed that ADD interfered with the chondrogenic capacity of Gli1+ FCSCs as well as osteogenic differentiation of Sox9+ lineage, mainly in the middle zone of TMJ cartilage. Then, a surgically induced disc reposition (DR) mouse model was generated. The inhibited FCSCs capacity was significantly alleviated by DR treatment in ADD mice. And both the ADD mice and adolescent ADD patients had significantly relieved OA phenotype and improved condylar growth after DR treatment. In conclusion, ADD-TMJOA leads to impaired chondrogenic progenitor capacity and osteogenesis differentiation of FCSCs lineage, resulting in cartilage degeneration and loss of subchondral bone homeostasis, finally causing TMJ growth retardation. DR at an early stage could significantly alleviate cartilage degeneration and restore TMJ cartilage growth potential.The anterior disc displacement (ADD) leads to temporomandibular joint osteoarthritis (TMJOA) and mandibular growth retardation in adolescents. To investigate the potential functional role of fibrocartilage stem cells (FCSCs) during the process, a surgical ADD-TMJOA mouse model was established. From 1 week after model generation, ADD mice exhibited aggravated mandibular growth retardation with osteoarthritis (OA)-like joint cartilage degeneration, manifesting with impaired chondrogenic differentiation and loss of subchondral bone homeostasis. Lineage tracing using  In most synovial joints of the body, the fibrocartilage is located between the articulating bones, which provides functions of shock absorption, cushioning between bones, as well as smoothing joint movement.6 The increased prevalence of OA in the young population has drawn considerable attention as it affects the patient’s joint development during the growth period, which may lead to severe skeletal dysfunction and deformities. For instance, TMJOA in adolescents often interferes with mandibular condyle growth, progresses to loss of joint function, joint instability, and malocclusion, and results in dento-maxillofacial deformities.11OA is one of the most devastating chronic diseases that affects 7% of the global population, more than 500 million people worldwide.19 The condyle-disc stability is seen as the key factor for smooth movement, which is essential for normal masticatory functions, including talking, chewing, swallowing, and facial expression. Therefore, once the disc is displaced, these pathological conditions of the disc often act as antecedents to a series of degenerative changes that can affect the entire TMJ.8 At the same time, a series of animal studies also identified that experimentally induced TMJ ADD using rabbits and rats could lead to OA-like cartilage degeneration and subchondral bone loss and cause mandibular growth retardation.23 However, these studies are limited to the endpoint change of chondrocyte and osteoblast/osteoclast lineage, the definite cellular and molecular origins of the OA process in TMJ ADD are still fogged.Numerous clinical investigations showed that the onset of TMJOA is related to TMJ ADD.27 which cells were found with critical roles as cell resources for TMJ homeostasis and repair,28 and were able to interact with endothelial cells in vascularized bone formation.26 At the adult stage, the cell features of FCSCs were regulated by various signaling pathways such as Wnt, Notch, and TNF-α/Nf-κB.30 At the growth stage, besides the phenomenon that FCSCs can differentiate toward chondrocytes, though, how the cell fate of FCSCs is regulated by OA during TMJ cartilage growth remains to be elucidated.Recently, studies have revealed that the fibrocartilage stem cells (FCSCs) population is resided in TMJ cartilage in different species,In this current study, we chose to investigate the functional role of FCSCs in TMJOA during the growth period. To accomplish this purpose, we, for the first time, generated an ADD-induced TMJOA mouse model with FCSCs lineage specifically labeled. Thus we were able to mimic the joint degeneration phenotype at the growth period and elucidate spatiotemporal rules of FCSCs for cartilage growth under both physiological and pathological conditions. We further generated a TMJ disc reposition (DR) mouse model, investigating the potential clinical application of surgical DR for adolescent TMJOA patients.The successful establishment of ADD mouse model was identified by observing that sutures in front of the zygomatic arch were kept intact, no fractures of the zygomatic arch occurred, and the posterior band of the disc was located in front of the apex of the condylar head Fig. . The manTo further investigate the histological changes of mouse ADD TMJ, hematoxylin and eosin (H&E) staining and Safranin O staining were performed. Compared to the Sham condylar cartilage with a clear hierarchy of cell layers, the ADD cartilage at 1 week showed a significantly disarranged cell distribution with increased cell numbers and layer thickness, especially in the superficial layer (fibrocartilage zone (FZ) + proliferative zone (PZ)) Fig. . From 2 + matrix was found uniformly distributed in the deep zone of the cartilage. In ADD mice with the disc released and pulled forward from the top of the condyle  was significantly increased under ADD-TMJOA  were also downregulated, indicating disequilibrium of cartilage matrix homeostasis  were upregulated in ADD group, which phenotype hints us the function of chondrogenic progenitors were activated in ADD-TMJOA cartilage at the early stage  mice were used for FCSCs lineage tracing. 4-week-old Gli1-CreER; Tmfl/− mice were injected with tamoxifen and were sacrificed at 5 days, 10 days, and 30 days  lineage cells were found mainly distributing in the superficial layer, consist 10% of the cells in the articular cartilage . These young patients were found to manifest with facial asymmetry malformation, deviation of the midline of lower teeth, as well a shortened condylar head examined by cone bean CT Fig. , Table 234 However, the underlying cellular mechanism that may give rise to the TMJ cartilage growth retardation was unclear over time. Therefore, our study identified the correlation between the fate changes of chondrogenic progenitor lineages and the OA degradation process during TMJ cartilage growth. In this study, we, for the first time, generated an ADD-induced TMJOA mouse model and chased the FCSCs lineages under abnormal biological stress. We demonstrated that the degeneration of cartilage and subchondral bone were correlated with FCSCs lineage fate changes. Gli1+ FCSCs contributed to cartilage injury and remodeling by enhancing their proliferation and chondrogenic differentiation with spatial heterogeneity at the early stage yet lost their normal functions and finally led to cartilage destruction at late stages. A previous study using finite element analysis demonstrated that disc displacement induced an increase of pressure in the disc and frictional coefficients on the condyle,35 thus a possible explanation for the correlation between condylar height and disc morphological changes could attribute to the condylar motion squeezes the disc forward, increases stress on the surfaces of condyle cartilage, which results in the function loss of FCSCs. Then TMJOA initiates, and the condylar height and disc length were consequently decreased.In previous studies, condylar height loss and secondary mandibular growth disorder were found in adolescent ADDWoR patients.28 The TMJ surface is coated with fibrocartilage instead of hyaline cartilage, so the pathological process of cartilage degeneration in TMJOA is distinct from other joints. When the TMJ disc is anteriorly displaced, the fibrocartilage in the joint surface is especially vulnerable to OA inflammatory damage. Once the fibrocartilage is damaged, it causes permanent tissue loss and disability, including limitation of mandibular function and growth.7 Thus, preserving the regenerative capabilities of FCSCs in fibrocartilage could potentially be an effective targeted therapy strategy for ADD cartilage repair. One intriguing phenotype observed in the TMJ cartilage under ADD is the regional specificity of chondrocyte fate changes in the middle zone and posterior zone. One possible reason is that there reside other chondrogenic progenitors in addition to the Gli1+ FCSCs lineage in the fibrous connective tissue attached to the different cartilage zones. For instance, previous studies have shown that there are resided ScxLin cells in the tendon around the condyle that directly form a subset of chondrocytes in TMJ.36 The diversity of chondrogenic progenitor sources may contribute to the phenotypic difference of different zones in TMJ cartilage under ADD. We found that the chondrogenesis transduction of Sox9+ cells was markedly blocked by ADD, signifying the loss of differentiation capacity of these chondrogenic progenitor cells. These findings hint to us that the abnormal disc-condylar structure would not only affect the FCSCs in the fibrocartilage surface but consistently affect the expression and functional features of FCSCs lineages even in the deeper area, including in subchondral bones.The TMJ cartilage consists of various proportions of both fibrous and cartilaginous tissue. Fibrocartilage not only provides tensile and compressive strength but also stores FCSCs to serve as critical cell resources for TMJ development and homeostasis.37 However, the abnormal mechanical stress by ADD could not sustain a balanced bone remodeling at later stages, and subchondral bone loss eventually occurs. These results remind us of the importance of releasing disc displacement at the early stage, aiming at promoting residual FCSCs chondrogenic capacity, preserving subchondral bone homeostasis, and improving condylar development for adolescent patients.In our study, both the DR mouse model and adolescent ADDWoR patients with DR surgery were found with significant recovery of condylar height loss. These phenotypes provide evidence that restoration of the balance of disc-condyle structure could alleviate cartilage degeneration, which supports the treatment strategy to recover normal disc position. Our animal model also showed that the TMJ disc turned out to be perforated at later stages of ADD-TMJOA. After disc perforation, the destruction of TMJ cartilage is aggravated, which is possibly due to further impairment of chondrogenic progenitor functions under the more severe inflammatory environment in the joint. On the other hand, ADD at early stages activated both the subchondral bone resorption and formation, which demonstrates the indispensability of mechanical forces loading for bone remodeling.As a surgically induced disc displacement mouse model, our model could not fully mimic the realistic pathological changes of ADD patients. The anatomic difference between rodents and humans, as well as the different duration of disc displacement from initiation to observation between our model and most patients, bring some limits to the strength of our observations. A longer-term observation period, as well as other primate animal models, may be used to further verify our findings in the future.Gli1+ FCSCs transduction toward Sox9+ chondrogenesis lineage in mediating chondrocyte homeostasis and subchondral bone remodeling. DR could preserve FCSC capacity as a chondrogenic stem cell resource that contributes to cartilage growth and repair. Taken together, our study supports the treatment strategy to recover normal disc position, aimed at promoting FCSCs chondrogenic capacity and improved condylar development for adolescent patients.To the best of our knowledge, this is the first study that investigates the functional roles of FCSCs in TMJ metabolism under disc displacement. This study reveals the importance of the This study was performed in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of Sichuan University. Animal procedures were performed according to protocols approved by the Animal Ethics Committee of Sichuan University (WCHSIRB-D-2020-476). All animal experiments followed the Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines. For collecting patient medical images, the study was approved by the institutional review board of West China Hospital of Stomatology, Sichuan University (WCHSIRB-D-2020-116), and all patients signed the informed consent. Patients or the public were not involved in the design, conduct, reporting, or dissemination plans of our research.+ cell numbers in Gli1-CreERT+; Tmfl/− mice. To account for the possibility of a loss of mice due to death before study completion, we used an N of 5–8 for RFP+ cell number counting.The number of mice per experimental group was based on a power calculation using data from our preliminary experiments. We found that a sample size of 5 mice per group would provide greater than 80% power to detect at least a 50% difference between ADD and Sham groups in RFPtm3(creERT2)Alj/J mice , Tg(Sox9-cre/ERT2)1Msan/J mice  and B6.Cg-Gt(ROSA)26Sortm14(CAG-tdTomato)Haze/J mice  were obtained from the Jackson Laboratory. Offspring were genotyped at 2 weeks old and conducted experiments at 4 weeks old. To generate tdTomato-conditionally activated mice, Tmfl/fl mice were crossed with Gli1-CreER+ mice and Sox9-CreER+ mice. Animals were housed in the Experimental Animal Center of the West China Hospital with free access to water and food at 25 °C with 40% humidity under a 12 h light/dark cycle. For ADD animal model and DR animal model generations described below, confounders between different groups were not controlled.C57BL/6 wild-type male mice were purchased from Dashuo Experimental Animal Laboratories (Chengdu). Gli140 Mice were anesthetized using i.p. injection with tribromoethanol at 0.12 μL·g−1 body weight . The right preauricular region, as the surgical region, was shaved and sterilized with iodine and 75% ethanol and followed by local anesthesia with subcutaneous injection of 0.1 ml lidocaine hydrochloride (2%). A curved incision at approximately 1 cm in diameter was made parallel to the ears, extending from the temporalis muscle to the inferior edge of the masseter muscle. The overlying parotid gland was carefully exposed and dissected, and the superficial temporal vein was electro-coagulated. The zygomatic arch and TMJ capsule were exposed. The periosteum of the zygomatic arch was separated forward to the lower orbital margin and backward to expose the zygo-temporal suture. The lateral joint capsule was opened by a tweezer, the medial attachment was retained, and the articular disc and the lower condyle were exposed. A hole at 1 mm diameter was carefully drilled anterior to the front junction of the zygomatic arch using a 0.3 mm round bur. A round needle with a 6-0 nylon suture attached crossed the hole and was tied, then penetrated through the anterior band of the disc. The disc was pulled forward by the nylon suture. After confirming the disc was at the front of the condylar head, the suture was double-tied to the prepared hole in the zygomatic arch. After disc displacement, the wound was thoroughly irrigated with saline, and the anterior position of the disc was verified by about 1 mm deviation of the lower midline of teeth to the non-operative side. Finally, the wound was sutured in layers. Mice in the Sham group went through the same surgical procedure, including exposing the joint capsule, except drilling the hole in the zygomatic arch and pulling the disc forward. After ADD/Sham surgery, soft food was provided, and normal chow was provided one week later.The surgical method of ADD mouse model was modified based on previous studies described for rats and rabbits.−1 body weight , another incision was made along the previous incision, the TMJ capsule was exposed. The 6-0 nylon suture was carefully moved with the intact disc preserved. Then the displaced disc was released from adhesion and was pulled back on the top of the condyle. The muscle and skin were sutured. The re-Sham mice went through the second operation that exposed the TMJ capsule again without changing the disc position. The same postoperative care as initial surgery was provided.ADD mice were randomly selected for disc repositioning surgery 5 days after the initial ADD surgery. After anesthesia by i.p. injection with tribromoethanol at 0.12 μL·g−1 heparin sodium in 0.01 mol·L−1 PBS) was injected transcardially. Next, 25 mL 4% PFA  was infused transcardially for tissue fixation. The intact TMJs in different groups were dissected and fixed at 4 °C for 24 h. For decalcification, Specimens were decalcified with 15% EDTA  for 7–21 days at 4 °C. Then the TMJ specimens were paraffin-embedded sagittally and were consecutively cut at 5 μm. For Gli1-CreER+ and Tmfl/fl mouse, the decalcified samples were embedded and frozen in optimum cutting temperature compound (OCT) after dehydrating by 30% sucrose solution overnight and cut into 6 μm thick sections.Mice in ADD and Sham groups were randomly chosen and were sacrificed at 1, 2, 4, and 8 weeks after model generation. Mice in DR and re-Sham group were sacrificed at 2, 4, and 8 weeks after ADD surgery. After anesthetizing with isoflurane, 25 mL ice-cold heparin PBS  at 70 kVp, 200 µA with a spatial resolution of 7 µm. Scanco Evaluation software was used for bone analysis and three-dimensional reconstruction. Three cubes in size of 210 × 210 × 210 µm+ area was calculated using ImageJ 1.51. Modified Markins score was used to assess the severity of cartilage arthritis, based on the number and morphology of chondrocytes, the safranin O positive region of cartilage matrix, and cartilage integrity, normal cartilage is scored 0-1 point. Totally, 2–4 points are assigned to mild OA. The Image 6.0 image analysis system  was used for semiquantitative analysis.Hematoxylin and eosin staining  and Safranin O/ fast green (SO) staining  were performed according to the manufacturer’s instructions. The number of chondrocytes and thickness of cartilage were counted using H&E staining sections. The thickness of condylar cartilage was assessed three times at quarter points of the anterior, middle, and posterior cartilage in each region. The average thickness of cartilage was calculated as the mean value of nine data from each TMJ sample and ultimately used for statistical analysis Fig. . The SafFor immunofluorescent staining, the following antibodies were used: Aggrecan , Collagen II , SOX9 , RUNX2 . The secondary antibodies included goat anti-rabbit Alexa Fluor 488/ 568/ 647, anti-mouse Alexa 488/ 568 . Apoptotic cells were detected by TUNEL staining  according to the recommended protocol. Slices were detected by laser scanning confocal microscopy .+ and Collagen II+ area was calculated using ImageJ software. Three square frames (0.12 mm × 0.12 mm) in cartilage or subchondral bone were randomly selected to calculate the numbers of SOX9+ cells, RUNX2+ cells, and TUNEL+ cells.AggrecanTRAP staining was conducted to detect osteoclasts based on the TRAP staining kit instruction . Three regions of interest (0.15 mm × 0.15 mm) were selected from the subchondral bone in the anterior, middle, and posterior regions of the condyle. Purplish red cells containing more than one nucleus were counted into osteoclasts.The mRNA levels were detected by RNA sequencing. Fresh cartilage in condyles of 1w Sham group and ADD group mice were carefully removed and cleaned with 4°enzyme-free PBS. The cartilage tissues were removed completely and sent for analysis as far as possible. After Total RNA extraction by Trizol reagent , RNA was purified and reverse-transcribed to create the cDNA library that was sequenced and run with Illumina NovaseqATM 6000 sequence platform. Genes differential expression analysis was performed by DESeq2 software between two different groups and by edgeR between two samples. The genes with the parameter of false discovery rate (FDR) below 0.05 and absolute fold change ≥ 1.5 were considered differentially expressed genes.−1. In the ADD and DR model generation experiment, Gli1-CreER+; Tmfl/- mice were injected intraperitoneally with tamoxifen 2 days before model generation . Sox9-CreER+; Tmfl/− mice were injected intraperitoneally with tamoxifen 7 days before model generation .Tamoxifen  was suspended in corn oil  at 10 mg·mL−1)  was injected i.p. 24 hours prior to sacrifice. EdU+ cells were detected with the Cell-LightTM EdU Apollo 488 In Vitro Kit  according to the recommended protocol.EdU  from adolescent patients with unilateral ADDwoR treated by disc repositioning surgery at West China Hospital of Stomatology, Sichuan University, was conducted from April 2021 to August 2022.Inclusion criteria were (1) ≤18 years old; (2) diagnosed with unilateral ADD without reduction; (3) treated by disc repositioning surgery; (4) with complete preoperative and postoperative Cone-beam CT (CBCT) data of both TMJ.Patients with syndromes, cleft palates, orthodontics treatment, and orthognathic surgery history were excluded. ADDwoR patients were diagnosed by experienced doctors according to the diagnostic criteria for temporomandibular disorders.CBCT data were collected 2–4 weeks before and 6–12 months after surgery. The scanning speed of the CBCT was 26.3 s, with 120 kV and 20 mA, and each layer thickness was set as 0.16 mm. Patients were in a seated position with the orbital-ear plane parallel to the horizon and asked to keep the eyes forward, intercuspation, and tongue position during the examination.43 The superior contour of the condyle was determined as the first radiopaque point found in the upper articular area. The lowest point of the sigmoid notch is the marker point of the bottom of the condyle. The 3D condylar image was reconstructed after the isolation, and the condylar volume was measured. Condylar height was measured as the vertical distance from the apex of the condyle to the largest cross-section of the reconstructed condyle in the sagittal view, and CL was analyzed as the distance between the most anterior and posterior points of the condylar head.The scan data were stored in the format of “.dicom”, which was imported into and constructed virtual 3D surface models of the anatomic condylar structures of interest with Mimics 21 software . The 3D measurement started from condylar image segmentation and reconstruction based on previous studies.P value < 0.05 was considered to be statistically significant. The staining results were analyzed by ImageJ 1.51  by an individual analyzer using blind sections.The mean and standard deviation of data were analyzed by GraphPad Prism 9 analysis software. Two-way analysis of variance (ANOVA) was used to compare the data of the two groups at multiple time points, a Supplementary materials


To explore the relationship between changes in salivary cytokine levels and the occurrence of white spot lesions in adolescents receiving clear aligner orthodontic treatment and investigate the predictive value of various factors for lesion occurrence.We retrospectively analyzed sixthy eight adolescent in the permanent dentition period, who received clear aligner orthodontics in our hospital were randomly divided into two groups according to the occurrence or aggravation of white spot lesions after treatment. The general condition of the oral cavity was analyzed, saliva was collected, and inflammation-related cytokines with varying transcription levels between groups were screened by transcriptome analysis. The expression levels of inflammatory cytokines in the saliva of the patients in the two groups were measured, and the risk factors for white spot lesions were screened by correlation analysis and binary logistic regression analysis. The value of the independent and combined application of risk factors for predicting the occurrence of white spot lesions in adolescent patients after invisible orthodontic treatment was analyzed by receiver operating characteristic (ROC) curve analysis.P < 0.05). Correlation analysis and binary logistic regression analysis showed that elevated levels of CXCL8, IL-1β and IL-2 were independent risk factors for the occurrence of white spot lesions (P < 0.05). CXCL8 had the highest independent predictive value for the occurrence of white spot lesions , and the combination of IL-1β and IL-2 was also of high value in predicting the occurrence of white spot lesions.Transcriptome and GO and KEGG pathway analyses showed that there were differences in the transcription levels of inflammatory cytokines such as CXCL1, CXCL2, CXCL8, CCL3, CCL4, IL-1β and IL-2 between groups. The levels of CXCL8, CCL3, CCL4, IL-1β and IL-2 in the saliva of patients with white spot lesions were significantly higher in patients after invisible orthodontic treatment (After invisible orthodontic treatment, the oral microenvironment, including inflammatory cytokine levels, in adolescent patients changes; in particular, the levels of inflammatory cytokines such as CXCLs and ILs change. CXCL8 expression is significantly associated with the occurrence of white spot lesions and is an important potential target for the prevention and treatment of white spot lesions in the future. According to traditional research results, fixed orthodontic appliances are not conducive to the maintenance of oral health, resulting in an increase in local dental plaque, which is the main cause of postorthodontic white spot lesions . In receAn increasing number of adults and adolescents choose to wear invisible orthodontic appliances, and some studies have reported that invisible orthodontic appliances do not promote the attachment of dental plaque because they are removable. However, our previous study showed that the incidence of white spot lesions in adolescents wearing invisible orthodontic appliances was not lower than that in adolescents wearing fixed orthodontic appliances . The aboIn this retrospective clinical study, the sample-size calculation was based on the following formula:α = 0.05, p = 0.8, δ = 0.1, Z = 1.96, after calculating, the N = 62. Considering 10% lost to follow-up rate, at least 68 participants were needed. The study protocol was approved by the ethical committee of the School of Stomatology, Air Force Medical University, Xi’an, China. Informed consent forms were signed by all patients or their legal representatives after the protocol and study objective were explained. The potential participants were patients examined during 1 year (2020.03-2021.05) (n = 213) who routinely presented to the Department of Orthodontics. Of these patients, 68 adolescent patients who met the following inclusion criteria as follows were selected: ① adolescents in the permanent dentition period, who aged from 10 to 19 years old according to the WHO standards; ② patients who received clear aligner orthodontic treatment for more than 12 months; ③ patients who had not taken antibiotics or drugs that may cause gingival hyperplasia within the previous 2 months; ④ patients who did not receive cytokine treatment before clear aligner orthodontic treatment; ⑤ patients whose routine blood tests and blood coagulation tests before orthodontic treatment were normal; ⑥ patients who had no infections caused by HBV, HCV, HIV, or CMV, no fever and no bacterial infections within the previous 2 months; ⑦ patients no tumors of any type; and ⑧ patients whose treatment was not complicated by serious disease of the heart, brain, lungs, liver, kidneys or other organs.When The clinical data collected from the patients included age, sex, frequency of brushing teeth per day, duration of tooth brushing, type of toothbrush, type of toothpaste, frequency of mouthwash use, presence of dental caries before treatment, frequency of oral cleaning, frequency of drinking carbonated drinks, frequency of eating with the invisible orthodontic appliance , duration of eating with the invisible orthodontic appliance (required by the doctor) and frequency of cleaning the invisible orthodontic appliance after eating.After 6 months of treatment, the patients were divided into the following two groups.For patients in the white spot lesion group (WLG), ① at least one tooth had white spot lesions; ② visible white spots that worsened in a side-by-side comparison of photos taken before and after treatment were regarded as white spot lesions, and other white spots were regarded as developmental white spots; ③ new white spots that appeared during orthodontic treatment were regarded as white spot lesions; and ④ white spot lesions were only confirmed by consensus after visual evaluation by three investigators. The non-white spot lesion group (nWLG) included the remaining adolescent patients. All patients were followed up and evaluated in our hospital after 6 months of treatment.Three saliva samples were collected from each participant at three different time points:T0: Before orthodontic appliance placement.T1: 6 months after orthodontic appliance placement.The samples were collected in the dental chair between 9 AM and 12 PM, two hours after any food intake. The participants were asked to rinse with water for 30 s and chew a piece of paraffin for 1 min to stimulate salivary flow. Then, saliva was collected in sterilized and preloaded tubes, and a label marked with the patient’s name and collection time was placed on the tubes. All samples were stored at -80 °C for subsequent cytokine detection.Frozen saliva samples were thawed on ice and centrifuged to collect debris. A Lysing Matrix B tube  was used to lyse the debris (twice for 10 min and 30 s). Total RNA was extracted using a MasterPure Complete DNA and RNA Purification Kit , and DNase was used to eliminate DNA contamination. RNA quality, quantity and integrity were evaluated using a 2100 Bioanalyzer . Structural oligo (dT) magnetic beads were used to enrich the mRNA, and the enriched mRNA was then randomly interrupted by divalent cations in NEB Fragmentation Buffer (NEBNext ®Ultra RNA Library Prep Kit for Illumina ®).cDNA library preparation was performed according to the TruSeq Stranded mRNA protocol . The first strand of cDNA was synthesized in an M-MuLV reverse transcriptase system with fragmented mRNA as a template and random oligonucleotides as primers. Then, the RNA strand was degraded by RNaseH, and the second strand of the cDNA was synthesized from dNTPs in a DNA polymerase I system. The purified double-stranded cDNA was repaired by terminal repair, subjected to tail addition and sequenced. cDNA of approximately 250–300 bp was screened with AMPure XP beads, the PCR product was amplified by PCR, and the PCR product was purified with AMPure XP beads. After the construction of the library, the library was initially quantified with a Qubit2.0 Fluorometer and diluted to 1.5 ng/µl, and then the insert size of the library was detected with an Agilent 2100 bioanalyzer. After the insert was shown to be of the expected size, qRT‒PCR was performed to quantify the effective concentration of the library accurately (the effective concentration of the library was higher than 2 nM) to ensure the quality of the library.After passing the library quality control step, different libraries were sequenced on the Illumina platform after pooling according to the effective concentration and the target off-machine data demand. The basic principle used for sequencing was sequencing while synthesizing (Sequencing by Synthesis). Four kinds of fluorescently labeled dNTPs, DNA polymerase and splice primers were added to the sequencing flow cell for amplification. When each sequenced cluster extends its complementary chain, each fluorescently labeled dNTP can release the corresponding fluorescence. The sequencer captures the fluorescence signal and converts the optical signal into a sequencing peak with computer software; thus, the sequence information of the fragment to be tested is obtained.Salivary cytokines were selected based on the results of metatranscriptome analysis; we analyzed all potentially relevant cytokines to obtain a spectrum of cytokines that was as comprehensive as possible. Batch analyses of the salivary samples were performed using a Luminex 200 with a Luminex Kit (R&D Systems). The following cytokines were measured: CXCL1, CXCL2, CXCL8, CCL3, CCL4, interleukin (IL)-1β and IL-2. The samples were thawed, centrifuged, and stained according to the manufacturers’ instructions and the protocol for cell culture supernatants. The concentrations of the cytokines were calculated using a log regression standard curve. If the concentrations were below the quantification limit, they were set to half the detection limit (DL).P was < 0.05.Statistical analysis was performed using SPSS24.0 and GraphPad Prism 8.0. Analysis of variance was verified by the Fisher test. For quantitative variables, the independent sample t test was used for intragroup and intergroup comparisons. The cytokines related to white spot lesions were screened by Spearman correlation analysis. A logistic regression equation was used to analyze the correlation between cytokines and white spot lesions. We evaluated the predictive efficacy of single and combined application of the cytokines for the occurrence of white spot lesions with ROC curves. The confidence interval was set to 95%, and differences were considered significant if P > 0.05)  and other basic clinical data between the two groups. The differences were not statistically significant, and the distributions of the two groups were comparable (5) Table .We randomly selected 7 patient saliva samples from each group for transcriptome analysis and found that there were differences in the expression levels of 995 genes in saliva between the two groups, with 419 genes with decreased levels and 576 genes with increased levels in the WLG group. Further GO and KEGG pathway analysis showed that there were differences in the transcription levels of many inflammation-related cytokines, such as CXCL1, CXCL2, CXCL8, CCL3, CCL4, IL-1 β and IL-2, between the groups . After treatment, the levels of CXCL8, CCL3, CCL4, IL-1 β and IL-2 in saliva were significantly increased in both groups, and the levels of inflammation-related cytokines in the WLG group were significantly higher than those in the nWLG group (P < 0.05)  but not with salivary CXCL1 and CXCL2 levels (P > 0.05)   Table .ROC analysis showed that CXCL8 had a high independent predictive value for white spot lesions after invisible orthodontic correction in adolescents, with an area under curve (AUC) of 0.773. Although IL-1β and IL-2 had little independent predictive value for the occurrence of white spot lesions, they had higher predictive value in combination with CXCL8, with AUCs of 0.795 and 0.745, respectively . In the meantime, we will focus on CXCL8 by using metabonomics analysis, flora identification, combinomics analysis and other methods to explore its correlations with oral flora types, quantitative flora changes and flora imbalance and further clarify the relationships among inflammation, the oral flora and white spot lesions to provide a theoretical basis for the prevention and treatment of white spot lesions after invisible orthodontic treatment.This study found that after invisible orthodontic treatment, the oral microenvironment, including inflammatory cytokine levels, in adolescent patients changes; in particular, the levels of inflammatory cytokines such as CXCL and IL change. CXCL8 expression is significantly associated with the occurrence of white spot lesions and is an important potential target for the prevention and treatment of white spot lesions in the future.


China is the largest producer of sweet potato in the world, accounting for 57.0% of the global output. Germplasm resources are the basis for promoting innovations in the seed industry and ensuring food security. Individual and accurate identification of sweet potato germplasm is an important part of conservation and efficient utilization.In this study, nine pairs of simple sequence repeat molecular markers and 16 morphological markers were used to construct genetic fingerprints for sweet potato individual identification. Combined with basic information, typical phenotypic photographs, genotype peak graphs, and a two-dimensional code for detection and identification were generated. Finally, a genetic fingerprint database containing 1021 sweet potato germplasm resources in the “National Germplasm Guangzhou Sweet Potato Nursery Genebank in China” was constructed. Genetic diversity analysis of the 1021 sweet potato genotypes using the nine pairs of simple sequence repeat markers revealed a narrow genetic variation range of Chinese native sweet potato germplasm resources, and Chinese germplasm was close to that from Japan and the United States, far from that from the Philippines and Thailand, and the furthest from that from Peru. Sweet potato germplasm resources from Peru had the richest genetic diversity, supporting the view that Peru is the center of origin and domestication of sweet potato varieties.Overall, this study provides scientific guidance for the conservation, identification, and utilization of sweet potato germplasm resources and offers a reference to facilitate the discovery of important genes to boost sweet potato breeding.The online version contains supplementary material available at 10.1186/s12870-023-04329-1. Ipomoea batatas (L.) Lam.) is a hexaploid annual or perennial dicotyledonous root plant of the Ipomoea genus in the Convolvulaceae family. Sweet potato has the valuable characteristics of high yield, strong adaptability, and rich nutritional value [4 ha, accounting for 30.4% of the global planted area and 55% of the worldwide output [Sweet potato  [Archaeological, historical, and biological studies have shown that Latin America is the key diversity center of sweet potato germplasm resources, also considered the most likely origin center of sweet potato , 18. Claby land)  or the PSweet potato propagates vegetatively and is thus, mainly preserved in the form of vegetative bodies, such as root tubers, seedlings, and test-tube seedlings. Resource identification has long been based on traditional phenotypic characteristics, which are susceptible to variation owing to environmental and subjective factors and thus, have low reliability. Furthermore, China’s collection methods and sources of sweet potato germplasm are diverse, with little standardization. Germplasm exchange and independent naming schemes among sweet potato planting operators or conservation organizations also result in the repeated introduction of germplasm, different names assigned to the same variety, and different varieties assigned under the same name, leading to unclear genetic relationships among germplasm accessions. This situation poses a challenge to cataloging and preserving sweet potato germplasm resources, selecting breeding parents, and promoting high-quality cultivars.Many methods are available for biological species identification and genetic diversity analysis. Phenotypic markers remain important research tools owing to their advantages of intuitiveness and convenience . The comTo determine the genetic structure and diversity of sweet potato resources in China and provide a standardized guide to the available resources, we analyzed the population structure and genetic diversity of 1021 sweet potato genotypes from natural populations and a full-sib population of 55 clones using SSR molecular marker technology. Subsequently, we constructed a fingerprint database with a combination of phenotypic and molecular markers to provide evidence and references for variety identification, research, and further utilization of sweet potato germplasm resources in China. This work should also provide technical support for the in-depth exploration and utilization of sweet potato germplasm resources, including their collection, identification, cataloging, conservation, and selection of appropriate breeding parents.Using 1021 sweet potato germplasm resources as a data set, 20 phenotypic traits were analyzed by factor analysis. In our Kaiser-Meyer-Olkin   were detected by the nine pairs of SSR primers in the 1021 germplasm samples  at the nearest location (0.4619) to the mean value (0.4648) Fig. a. Group The full-sib families with 55 clones clustered together completely in Group P, with an average genetic distance of 0.2277. These results further demonstrate the high discrimination efficiency of the primer combinations selected in this study for sweet potato resources.PCA results also showed that the 1021 sweet potato germplasms were not clustered together according to their regions Fig. , but theFinally, we constructed a unique genetic fingerprint database for sweet potato germplasm, including genotypic  and aid in dispelling cultivar confusion. If the genetic fingerprint data of two materials are consistent at both the phenotypic and genotypic levels, they will be considered the same variety. Thus, establishing genetic fingerprints of sweet potato germplasm resources will improve the efficiency and accuracy of variety identification, providing a theoretical foundation for future identification, protection, and breeding of sweet potato germplasm resources. The fingerprint database of sweet potato germplasm resources constructed in this study represents the largest such resource in the world to date.A genetic fingerprint database containing 1021 sweet potato germplasm resources was constructed with nine molecular markers and 16 morphological markers as core indicators. The genetic variation of native Chinese sweet potato germplasm resources was found to be narrow, demonstrating a closer relationship to those from Japan and the United States and the furthest relationship with resources from Peru. This study provides further scientific and technical support for the conservation, identification, research, and utilization of sweet potato germplasm resources.A total of 1021 sweet potato germplasm resource materials Table S and a fu−1 and stored at −20 ℃ for future use. The DNA stock solution was stored at −80 ℃. Sampling and DNA extraction were completed in 2018 and 2019.The experimental materials were planted in the Guangzhou Baiyun base of the Guangdong Academy of Agricultural Sciences, and DNA was extracted from the young leaves 45–50 d after planting. Genomic DNA was extracted using the ionic detergent cetyltrimethylammonium bromide, and DNA quality was examined by 2.0% agarose gel electrophoresis. The concentration of DNA was determined on a NanoDrop 2000 ultraviolet spectrophotometer. The DNA was then diluted to 50 ng μLTwenty-three pairs of sweet potato SSR primers were used in the experiment Table , of whicFour sweet potato resources  were selected from the experimental materials to screen the 23 pairs of SSR core primers by polyacrylamide gel electrophoresis (PAGE)-silver staining, with three replicates for each primer. The primers exhibiting high polymorphism and good repeatability were selected as core primers for genetic diversity analysis and genetic fingerprint construction of sweet potato germplasm resources.−1), 0.2 μL TaqDNA polymerase, 0.3 μL each of the upstream and downstream primer (20 μM), 0.4 μL dNTPs, 2 μL buffer, and 14.8 μL ddH2O. The following PCR cycling program was used: pre-denaturation at 94 ℃ for 5 min, denaturation at 94 ℃ for 30 s, annealing at 54 ℃ (varied according to primer) for 35 s, and extension at 72 ℃ for 40 s for a total of 35 cycles. The final product was extended at 72 ℃ for 3 min. The products were separated and visualized by PAGE-silver staining on a 6% gel, and fluorescence capillary electrophoresis was carried out on a 3730XL DNA analyzer .The total volume used for polymerase chain reaction (PCR) was 20 μL, including 2 μL of DNA template  was performed using Origin 2021. The KMO test was used to assess correlations and partial correlations between variables; a test outcome of < 0.5 indicates that each variable is independent and unsuitable for factor analysis.The genetic parameters of SSR typing from the raw capillary electrophoresis data were analyzed using Fragment (plant) analysis software in Genemaker. The labeled molecular weight in each lane was compared with the position of the peak value of each sample; the amplified band at the same migration position was marked as 1, and the non-amplified band was marked as 0 to construct a binary data matrix of 0 s and 1 s. The NTSYSpc 2.11 software package was used to calculate the genetic distance between materials. The number of alleles (Na), allele frequency, and genotype frequency were statistically analyzed. Shannon’s information index (I), number of effective alleles (Ne), and PIC were calculated according to the 0/1 data matrix using the following formulae:pi represents the gene frequency of the ith allele at a given locus.Two parameters were used to evaluate the individual recognition ability of nine pairs of SSR markers, PI, and PIsibs, representing the average probability of two random individuals with the same genotype, in the natural (1021 germplasm samples) and sibling (55 full-sib clones) population, respectively, calculated using the following equations:http://192.168.3.177), which was ultimately used to build genetic fingerprints and the two-dimensional code database of sweet potato germplasm resources.Molecular markers combined with phenotypic markers were used to construct the genetic fingerprint of sweet potato germplasm. The selected genotypic marker combinations were labeled with uppercase letters , and the genotypes were sorted and labeled with Arabic numerals; similarly, different phenotypic markers were labeled with lowercase letters , and the phenotypes were assigned Arabic numerals. Using this method, a series of numbers composed of uppercase or lowercase letters plus Arabic numerals were formed for each resource, providing genetic and phenotypic fingerprints of the germplasm. We combined the genotypic and phenotypic fingerprints with basic information, phenotype photographs, and molecular marker scanning peaks graph to develop a two-dimensional code of each germplasm resource using Golang on the local server  primers. Figure S2. Scanning peak graphs of all 943 genotypics.Additional file 2: Table S1. Genomic positons of core simple sequence repeat (SSR) primers. Table S2. Basic information of 1021 sweet potato germplasm resources. Table S3. Genotypics of 1021 sweet potato germplasm resources. Table S4. Phenotypics of 1021 sweet potato germplasm resources.


Predisposition to myeloid malignancies is a field at the border of hematology and genetics. Knowledge in this domain has so rapidly increased that WHO defined in 2016 the new “Myeloid Neoplasms with Germline Predisposition” category of tumors. High throughput sequencing is frequently performed in tumors either for diagnosis or prognosis, but this approach may identify potential germline variants that have to be confirmed on non-infiltrated tissues.In this study, we systematically compared NGS data from genetic analysis performed on all sample types  in 29 patients, and 44 of their relatives .i.e., 48% because clearly or probably infiltrated) or “inconclusive” (8/29 corresponding to 28%).We showed that saliva was usable for relatives, but only for 24% (7/29) of our patients. Most of patients’ saliva were either “non-contributive” (14/29 The recommendations for the use of saliva we present here focus on the importance of collecting saliva during remission when possible. Moreover, we propose hair follicles as an alternative to skin biopsy, that remains the gold standard especially in case of allogenic hematopoietic stem cells transplantation. Technological progresses have revolutionized the diagnosis of predisposition to solid or hematological malignancies, and it is very likely that new techniques will help to manage the familial predisposition in the future. That differentiation is closely regulated by numerous factors . GermlinDEAD-Box Helicase 41 (DDX41) mutations and familial MN, including myelodysplastic syndromes (MDS) , specific clinical outcomes (high progression rate to acute myeloid leukemia: AML) and high penetrance of GL traits of DDX41 (about 50% by the age of 90), that even tend to consider them as a specific subset of MN.Among these genes, recent literature shows an association between germline es (MDS) . Recent es (MDS)  and the es (MDS)  characteMDS is an heterogenous group of clonal myeloid disorders. They are usually suspected in presence of unexplained persistent peripheral blood cytopenias, but diagnosis requires a bone marrow assessment. The presence of dysplasias on cellular morphological examination is a hallmark of the diagnosis .Three groups of factors can help to define the prognosis of MDS: cytogenetics aberrations, bone marrow blast percentage and depth of cytopenia. They are combined in the Revised International Prognosis Scoring System (IPSS-R) widely used in clinical routine. It assesses the risk of secondary evolution AML . NeverthIn this context, High Throughput Sequencing (HTS) of gene panel involved in HM is frequently performed in early stage of MDS. In some cases, this approach on bone marrow sample may identify potentially germline variants. Confirmation of their germline nature on another biological tissue becomes therefore essential for appropriate clinical management and genetic counseling for relatives.Due to diffusion of hematopoietic tumoral cells in peripheral blood, saliva appears to be a more appropriate biological sample for germline studies, and its collection is not invasive. Nevertheless, literature reports risks of saliva’s contamination by tumoral blood cells , 13; andThe aim of this study is to report our local experience and propose recommendations for use of saliva in case of suspicion of familial hemopathy (FH). Here, we compared HTS data from saliva, bone marrow, and other tissues  collected for diagnosis of FH or follow-up. Data from patients as well as those collected in their relatives were also used to build the guideline we propose.22.1We extracted from our database all analysis performed between 2020 and 2022 on DNA isolated from saliva (n=78). Patients were distinguished from relatives through a systematic review of medical history and molecular analysis performed in our laboratory .2.2We isolated DNA with Maxwell RSC instrument  using the appropriate kit for each sample type according to the supplier’s recommendations. DNA was extracted from saliva  with the Stabilized Saliva DNA kit (Promega); from whole blood on EDTA or 5M cell pellets with the Simply DNA Blood kit (Promega); and from hair follicle (10 follicles minimum) with the Tissue and Hair kit (Promega). We only extracted DNA manually from fibroblasts with the PureGene kit , according to manufacturer’s recommendations. We cultured fibroblasts after microdissection of skin biopsy in incubator , with Chang Medium .We prepared amplicon library using an Ion AmpliSeq custom panel  targeting 17 diagnostic and prognostic genes list in Table S1Sanger method was performed to sequence gene regions with depth <100X as well as mutation specific analysis in patents and their relatives. We performed amplification with Taq Purple Mix , purification of PCR products with ExoSap  and sequencing with BigDye Terminator V1.1 (Applied Biosystems) on 3130xL or 3500 xL Genetic Analyzer (Applied Biosystems).2.3We systematically reviewed results of HTS analysis in all samples  for each patient. Variant Allele Frequencies (VAF) of pathogenic (class 5) and likely pathogenic (class 4) variants was compared between samples. We considered that saliva was « not contributive » for germline studies when somatic mutations present at diagnosis were also detected in saliva regardless of their VAF, suggesting an infiltration by tumoral cells. Conversely not infiltrated saliva were qualified as “contributive”, and if it was not possible to affirm the absence of infiltration saliva were “inconclusive”. Moreover, for patients treated with allogenic hematopoietic stem cell (HSC) transplantation, we studied the presence or absence and zygosity of Single Nucleotide Polymorphism (SNP) to assess donor’s contribution to HTS results in patients.33.1versus 53 years ± 15 respectively). HTS was performed for diagnosis and prognosis in patients with various types of myeloid disorders  were included in the study. Majority of them were males , but ages were similar (59 years ± 14 isorders Table 1.CBL (n=1), DDX41 (n=22), GATA2 (n=1), KRAS (n=1), RUNX1 (n=2), and TP53 (n=2). Among them, DDX41 was thus the most frequently mutated, but only five pathogenic mutations were detected: G173R (n=12), A270V (n=1), E268Dfs*36 (n=3), L283Cfs*21 (n=4) and S363del (n=2). All of them were already reported . We however considered that saliva as “contributive”, because IDH2 and DNMT3A mutations were absent of NGS performed on bone marrow for follow-up one month later, suggesting that saliva was not infiltrated when it was collected.In the group of 29 patients, saliva samples were contributive in 7 patients, corresponding to 24% of our cohort . All samples were collected at remission  or during treatment, and HTS did not detect somatic mutation in saliva. Among them, only patient ID 11 beneficiated of Sanger sequencing of 3.2.2Ten samples (34%) were clearly  or probably  infiltrated by tumoral cells. Infiltration was demonstrated by presence of somatic mutations detected at diagnosis in saliva or in samples collected for follow-up (blood or bone marrow). Concerning patient ID 8 and 23, saliva infiltration was highly probable, because collected during a progressing disease (ID 8) or before a transplantation of HSC and out of remission (ID23). Among them, three patients  were treated with allogenic HSC transplantation, but their saliva samples were collected before, which did not bias our interpretations (no contribution of donor to results in these patients).Surprisingly, four other saliva samples were also not-contributive even though collected after allogenic HSC transplantation . Thus, the total of not-contributive saliva was 48% (14 among 29). Conclusions for those patients required more investigations so we reviewed presence or absence of SNP detected by NGS in the different samples available.DDX41 mutations were present at diagnosis: E268Dfs*21  and G530D . Bone marrow collected for relapse despite an extra-familial allogenic HSC transplantation, found same mutations (VAF= 1.9% and 1.6% respectively) and the new IDH1 R132C mutation (VAF= 3.5%). In saliva collected 5 months later, we detected DDX41 E268Dfs*28 mutation, with a VAF of 15.8% consistent with an incomplete chimerism .Patient 4: two himerism Figure 1DDX41 G173R detected at diagnosis and relapse after the first transplantation was not detected on blood and saliva after the second graft. HTS performed on skin fibroblasts and hair follicle confirmed the germline nature of DDX41 G173R mutation (VAF=54% and 48.8% respectively). Comparisons of SNP with skin (data on request) showed that i) the relapse was developed on transplant (different SNP profile between skin and bone marrow at relapse); and ii) only DNA of unrelated donor is present in saliva, corresponding to a 100% donor chimerism .Patient 12: difficulties of interpretation were comparable to situation of case 4, but this patient received two allogenic HSC transplantations: the first with a relative and the second with an unrelated donor. The DDX41 G173R mutation was still present after one (patient 21) or two (patient 22) transplantations, but with a very low VAF in saliva (4% and 5% respectively), suggesting an incomplete chimerism. We confirmed this hypothesis with SNP studies (DDX41 G173R mutation in these patients.Patients 21 and 22: Situation for these patients was quite different of previous patients.  studies Table S33.2.3DDX41 acquired mutation in minor proportion of cells in bone marrow (VAF ranged from 3 to 16%), thus probably not detectable by Sanger in saliva. Therefore, even if only supposed germline mutation was targeted by Sanger, absence of acquired mutation could not be verified and tumor infiltration in saliva cannot be definitively excluded. Two cases are nevertheless interesting:The eight remaining samples (28%) were considered as “inconclusive” because it was not possible to conclude with the available data . None of these saliva samples were collected at remission, and bone marrow at diagnosis showed presence of clonal and subclonal mutations. Our local workflow initially considers only Sanger sequencing on saliva to detect supposed germline mutations. Majority of these eight cases  were characterized by a DDX41: G173R (VAF=53%) and a R525H (VAF=20%), associated with a TET2 mutation (VAF=98%). Bone marrow collected for follow-up after allogenic HSC transplantation with a relative showed acquisition of a SNP from the donor (homozygous SETBP1: Y1303S) and presence of DDX41 G173R mutation. This patient next presented a relapse of his disease after transplantation, with DDX41 G173R and R525H mutation, and presence of SETBP1 homozygous Y1303S. Sanger sequencing was performed on saliva collected two months later, which showed presence of DDX41 G173R. Thus, it was not possible to affirm that saliva was not contaminated by tumoral cells (DDX41 R525H not targeted), and we could not conclude if mutation detected in saliva was carried by patient or brought by the transplantation with a carrier relative (SETBP1 SNP not targeted by Sanger).Patient 5: Bone marrow at diagnosis showed two mutations of RUNX1 E223*  and SRSF2 P95H  mutations. NGS performed on saliva collected two months later (and without any treatment) detected only RUNX1 E223* mutation . Due to low sequencing depth on saliva, it is probable that even if SRSF2 P95H mutation was present at heterozygous state, bioinformatic pipeline would have filtrated this mutation (probable depth <50X). Thus, saliva was considered as potentially infiltrated, so “inconclusive”. Unfortunately, patient died of endocarditis before we could collect hair follicles or skin fibroblast, so germline nature of RUNX1 E223* mutation could not be verified.Patient 18: Bone marrow at diagnosis show presence of 4Predisposition to myeloid disorders is a recent field of genetics, especially since the definition of a specific category of tumors by WHO in 2016 . This deDue to absence of strong recommendations, strategy for diagnosis of predisposition to myeloid malignancies was highly variable between our patients. Some of them benefited of Sanger sequencing on saliva, other ones of HTS. That was one of the weaknesses of our study. For some cases, only saliva samples collected at remission were informative (24% of our cohort), and retrospectively, obtaining more samples at remission would probably reduce the rate of inconclusive saliva samples.Saliva was preferably studied because its collection is less invasive than skin biopsy. However, we showed that in case of allogenic HSC transplantations, analysis on DNA from skin fibroblasts are required. These results are concordant with methodology used by other authors, such as Churpek et al.  and Li eMost saliva samples collected from affected patients (48%) showed obvious signs of infiltration by tumoral cells. In these cases, mutational profiles (mutations and VAF) were comparable between medullary and salivary compartments. Moreover, analysis performed on blood (patient ID4 and 25) even showed an infiltration higher in saliva than in blood; and case of patient ID13 interestingly showed different mutations in saliva compared to bone marrow at diagnosis, which could be compatible with sub-clonal mutations not detectable in medullary compartment.a posteriori to conclude on the saliva samples that we considered as “unconclusive” (28% of our patients).In order to resolve the problem of infiltration in saliva, we could test extraction of DNA from hair follicle in the 4 most recently included patients . Results were encouraging, as they confirmed the results obtained on the skin for patient ID12 and avoided skin biopsy for the others . These results need to be confirmed on larger cohort of patients and could even be used DDX41 L283Cfs*21 mutation. Consequently, his status was known at 32 years old, while HTS performed in bone marrow at diagnosis and in his father’ saliva showed identical results, suggesting a significant infiltration of saliva by tumoral cells. Thus, segregation of mutation in this family proved germline nature of DDX41 variant detected in patient ID6. In the context of intrafamilial allogenic HSC transplantations, early testing of relatives appears to be ethically acceptable but is more debatable out of those situations, considering the penetrance of DDX41 traits, which is negligible until 40 years of age  to determine his eligibility as a donor for HSC allograft, showed that he carried the heterozygous In our Institution, fibroblasts are not cultured by hematology biologists, but in the Genetics unit. Availability of skin biopsies for patients followed up by hematologists is thus conditioned by another unit, with its own cell culture activity. Consequently, and despite the small cohort of patient available, our local experience led us to define an optimized algorithm for the diagnosis of hereditary predisposition to HM, to limit the use of skin biopsy to patients whom it is indispensable. Figure 2This algorithm, easily usable in clinical routine, focuses on the importance of collecting patients’ samples at remission if possible. Due to difficulties of interpretations encountered in patients treated with allograft, the use of saliva should be limited to relatives (Sanger sequencing) or patients at remission (HTS) without antecedent of transplantation, even if the donor was unrelated to patient. In case where saliva is not usable, or when patients’ samples cannot be collected at remission, we suggest the use of hair follicles that are less invasive to collect, but biopsy remains the gold standard until a larger cohort confirms our results.5Our study showed that HTS is an efficient tool for detecting tumoral cells in patients’ saliva samples or assessing donor contribution in case of HSC transplantation. In addition, we showed that saliva can be used in the field of predisposition to malignant myeloid hemopathies, but cautions are needed to ensure proper interpretation of the results.The original contributions presented in the study are included in the article/The studies involving human participants were reviewed and approved by Comité d’éthique du CHU de Limoges. The patients/participants provided their written informed consent to participate in this study.AP: Planned the experiment, analyzed the results, and wrote the manuscript. SB: Technical and biological validation of the results, and critical revision of the manuscript. DR: Technical and biological validation of the results, and proofread the manuscript. JC: Technical and biological validation of the results, and proofread the manuscript. BD: Patients and relatives genetic counselling, and proofread the manuscript. PT: Patients clinical diagnostic and follow-up. SG: patients clinical diagnostic and follow-up. LV: Patients and relatives genetic counselling. MR: patients and relatives genetic counselling. JF: Proofread the manuscript. CY: Critical revision of the manuscript. NG: Technical and biological validation of the results, and critical revision of the manuscript. All authors contributed to the article and approved the submitted version.


BackgroundDengue infection (DI) continues to rise in the Caribbean. Children are primarily affected by severe infection in this region. Parents thus play an essential role in identifying symptoms, seeking medical care, and preventing DI in their children. Grenada has been endemic to DI since 1956, and to date, no study has assessed the level of knowledge, attitude, and practices (KAP) among parents regarding DI.ObjectiveTo determine the level of parents' KAP and associated sociodemographic factors regarding DI in children in Grenada.MethodsA quantitative cross-sectional study of 360 randomly selected parents attending postnatal and child-health clinics was conducted in Grenada. Parents completed a validated survey on demographic information and KAP regarding DI from September to December 2019. KAP scores were calculated and categorized as adequate or inadequate based on the percentage of correctly answered questions on the survey. Chi-square and logistic regression analyses were used to determine the level of KAP and associated sociodemographic factors among parents.ResultsOnly 33.9% of parents had adequate knowledge regarding DI, which was independently associated with gender , age , occupation, education, and marital status. Nevertheless, most parents had a positive attitude toward DI prevention (56.4%) and practiced adequate prevention (73%) against DI. Higher education was independently associated with a positive attitude toward DI prevention . However, no sociodemographic factors were associated with the prevention practices of DI.ConclusionThis study revealed a low level of knowledge among parents, despite an adequate attitude and level of prevention against DI in Grenada. It identified specific groups, such as young, single parents of low educational status, as potential targets for educational campaigns to reduce DI-associated morbidity and mortality among Grenadian children. Dengue infection (DI) is the fastest-growing mosquito-borne viral infection in the world, transmitted by Aedes aegypti and Aedes albopictus mosquitoes . It is eDI has been endemic in the Caribbean since the 17th century and provides a favorable rainy climate for the Aedes mosquito to breed, resulting in periodic outbreaks ,5. The tGrenada’s Ministry of Health (MOH) has implemented health education and vector control campaigns to prevent an imminent outbreak of DI. Since there is no specific treatment for DI, the MOH urges all individuals to participate in preventative practices and parents to be vigilant for signs and symptoms of DI in children. If severe DI in children is suspected, indicated by symptoms such as abdominal pain and/or bleeding in stools, early detection and access to medical care within 24-48 hours can reduce the mortality rate from 40-50% to less than 1% . This reStudy designA quantitative cross-sectional study was conducted from September to December 2019 to assess the level of parents’ KAP and associated SD factors regarding DI in children in Grenada. The chosen time period correlates with the rainy season in Grenada and hence increased prevalence of DI .SettingThe tri-island state of Grenada, located north of Trinidad and Tobago and Venezuela, comprises Grenada, Carriacou, and Petite Martinique. The state covers 440 square kilometers  and has a population of 108,000. It is divided into six parishes. This study was conducted in clinics  in three of Grenada's parishes: Saint George, Saint David, and Saint Andrew. Saint George, located in the southwest, has the largest population ~37,057), followed by the largest parish, Saint Andrew . Saint George, Saint David, and Saint Andrew represent the island's urban, peri-urban, and rural regions, respectively [7, followSampling approachSampling FrameThe sampling frame comprises all 24 healthcare facilities within the three parishes. All hospitals, health centers, and five randomly selected medical stations were included in this study. A total of 10 healthcare facilities were included: Saint George , Saint David , and Saint Andrew .The population comprised all parents meeting the inclusion and exclusion criteria who attended postnatal and child-health clinics of the selected healthcare facilities.The study population included parents who attended the postnatal and child-health clinics at selected healthcare facilities within the parishes of Saint George, Saint David, and Saint Andrew in Grenada: (1) two hospitals, one in Saint George and one in Saint Andrew. All parishes refer patients in need of advanced medical care to hospitals. Thus, hospitals contain a diverse population in Grenada. (2) Three health centers, one per parish. Health centers represent primary healthcare in Grenada. (3) Five randomly selected medical stations out of 19, one in Saint George, two in Saint David, and two in Saint Andrew. Medical stations represent the first point of care with healthcare in Grenada. It also included parents of at least one child less than 15 attending the selected healthcare facilities' postnatal or child-health clinic. Clinics provide easy and quick access to parents of children less than 15 years of age, which represents the age group primarily affected by severe DI in the Caribbean. In addition, parents over 18 years of age and can speak English were included. Parents under 18 years of age were ethically limited to participate, and surveys were printed in English. Parents over 18 years old who spoke English and were illiterate were assisted in filling out the survey by the researcher.The exclusion criteria included the following: (1) health facilities that were not operational at the time of the study; (2) health facilities that did not conduct postnatal or child-health clinics; and (3) caregivers  of children less than 15 years of age.Sample SizeA sample of 360 participants was used in this study. Calculations were made using OpenEpi with the following parameters: Grenada’s population of 108,000, 32% anticipated frequency of adequate knowledge , confideRecruitmentThe first 36 parents attending postnatal and child-health clinics were recruited on presentation at the health facility (n=360). The clinic receptionist distributed recruitment flyers and participant information sheets as parents were registered to reduce coercion . ParentsData collection methodsInstrumentA validated survey was adapted from: “Knowledge, attitudes, and practices regarding DI in Westmoreland, Jamaica.” Three Delphi review rounds verified the instrument’s purpose, validity, and reliability in the parent study . This reThe quantitative survey comprised 50 questions in four sections. Section (1) included five demographic questions on gender, age, education, marital status, and occupation. Section (2) consisted of 29 questions regarding the knowledge of DI: six on the signs and symptoms, seventeen on the transmission, and five on the management. Section (3) has three questions on attitude toward DI prevention, and Section (4) has 13 questions: 12 on prevention against DI and one on the source of information on DI. Participants took approximately 30 to 45 minutes to complete the survey.Pilot StudyA pilot study was conducted in Grenada to assess the survey for comprehensiveness, wording, validity, and reliability. Based on the results of the pilot study, two demographic questions were revised, "education" and "occupation," to replace colloquial terms suited for the Grenadian population. Education was categorized as less than primary, primary, secondary, tertiary, and/or post-graduation, and occupational status was categorized as employed or unemployed. Similarly, three knowledge-based questions were revised to include features of DI specific to children: (1) Is vomiting a sign of dengue fever? (2) Is bleeding from the gums a sign of severe dengue? (3) Is passing blood with stools a sign of severe dengue?Ethical ConsiderationsEthical approval of the research was obtained from the International Review Boards (IRB) of Windward Islands Research and Education Foundation (WINDREF) in Grenada and the University of Liverpool in the United Kingdom. Grenada’s Ministry of Health provided clearance to research in all healthcare facilities following IRB approval.Analytical approachA coding frame was developed for each question in the survey. This process was essential to transfer the data from the survey into a numeric form for analysis using SPSS version 25. Gender, marital status, and occupation were listed as nominal data; age as a continuous variable; and education as an ordinal variable . CorrectParticipants who correctly answered 70% or more of the questions on knowledge were considered as having adequate knowledge regarding DI, and those who answered less than 70% of the questions on knowledge were regarded as having inadequate knowledge regarding DI . SimilarFrequency tables, histograms, and scatterplots were used to describe the data, determine the distribution, and identify outliers. The analysis did not include missing survey data, ensuring unbiased and reliable results. The chi-squared test was used to identify any significant difference between the dependent variable (level of KAP) and the independent variables .Bivariate logistic regression was performed to measure the strength of any association between the level of KAP and each SD factor using crude odds ratio (OR), 95% confidence interval (CI), and a p-value of <0.05 to determine statistical significance. Each significant result from the bivariate analysis was entered into a multivariate regression model to identify any confounding factors . If an association between the level of KAP and SD factors was significant in the bivariate analysis but not in the multivariate analysis, then the association between the variables was likely due to a confounding factor . It woulSD factors among participantsMost participants were females (88.6%), and 89% were between 20 and 39 years of age Table . A minorKnowledge of DI among participantsKnowledge of Signs and SymptomsMore than half the participants knew fever (86.4%), vomiting (65.3%), and headache (73.6%) as symptoms of DI Table . HoweverKnowledge of TransmissionMost  knew the type of mosquito that transmits the dengue virus: Aedes mosquito. However, participants also incorrectly believed the dengue virus was transmitted by all species of mosquitoes (41.9%), flies (39.7%), blood transfusion (64.7%), and/or sexual intercourse (43.1%). Only 13.6% (n=49) of participants knew the correct biting time of the Aedes mosquito during the day. Regarding knowledge of prevention against DI, many participants were knowledgeable about the use of window screens and bed nets (75.8%), insecticide sprays (78.6%), and mosquito repellents (75.8%) to reduce mosquitoes. Additionally, many felt removing standing water and covering water containers can prevent mosquito breeding .Knowledge of ManagementRegarding the knowledge of DI management, 60% (n=216) of participants correctly stated they would not give their child aspirin, whereas 16% of participants stated they would. More than half the participants would give their child plenty of fluids and rest if DI was suspected. Almost all participants (n=349) had positive health-seeking behavior (HSB) of consulting a doctor if they suspected DI in their child. However, 68.3% (n=246) believed there was a treatment for DI, while only 10% correctly knew there was no specific treatment for DI.Attitude toward DI prevention among participantsMost participants (83.9%) identified DI as a serious illness . Many (n=236) believed their child was at risk of being infected with dengue virus, whereas 34.4% did not share this view. Nevertheless, 85.3% of participants believed DI could be prevented .Prevention against DI among participantsParticipants stated they engaged in preventive practices against DI by using bed nets (84.2%), insecticide sprays (88.1%), and window screens (81.1%). Less common preventive practices against DI included the use of mosquito-eating fishes . Covering water containers at home, and cleaning water containers and drains around the house was readily  done by 86% of participants. The KAP among participants regarding DI was influenced by the television or radio (46.7%) and health workers (34.4%).Level of KAP regarding DI Only 33.9% n=122) of participants answered at least 70% of the knowledge questions correctly and thus were determined to have an adequate level of knowledge regarding DI . Participants under 20 years of age and those between the ages of 25 and 29 were less likely to have an adequate level of knowledge regarding DI compared to participants over 39 years of age. However, after adjusting for confounding factors  in the multivariate analysis, only the participants between the ages of 25 and 29 were less likely to have an adequate level of knowledge regarding DI compared to those older than 39 years of age. The multivariate analysis showed these participants (ages 25-29) were 87% less likely to have adequate knowledge regarding DI compared to participants over 39 years of age .A lower level of education (primary and secondary) was associated with lower odds of having adequate knowledge regarding DI compared to those who had completed tertiary and/or post-graduate studies in the bivariate analysis . Participants with only primary education were 76% less likely , and those with only secondary education were 50% less likely  to have an adequate level of knowledge regarding DI compared to those who had completed tertiary and/or post-graduate studies. Therefore, education was independently associated with adequate knowledge regarding DI.Being single, divorced, or widowed was associated with lower odds of having adequate knowledge regarding DI compared to being married or in a common-law union . In the multivariate analysis, participants who were single, divorced, or widowed were 42% less likely to have an adequate level of knowledge regarding DI compared to those who were married or in a common-law union . Similarly, unemployed participants were less likely to have adequate knowledge regarding DI compared to those who were employed . However, this association was not independently associated with adequate knowledge regarding DI in the multivariate analysis . Thus, the relationship between employment and level of knowledge regarding DI was likely due to confounding variables.The association between attitude toward DI prevention and SD factors Only education was associated with a positive attitude toward DI prevention in the bivariant and multivariate analyses Table . ParticiThe association between the level of prevention against DI and SD factors Education  and occupation  were associated with an adequate level of prevention against DI. However, after adjusting for confounding factors, there was no independent association between SD factors and prevention against DI .Summary of key findingsThe results showed that most participants were females between the ages of 20 and 39 years with secondary education or higher. More than half the participants were familiar with common symptoms of DI, including fever and headaches, but were less familiar with specific signs of severe DI, including bleeding from the gums and passing blood with stool. While more than half the participants were also familiar with the mode of dengue virus transmission by the Aedes mosquito, many also incorrectly believed flies, blood transfusion, and/or sexual intercourse aided in the transmission. Unsurprisingly, only 33.9% of participants had adequate knowledge regarding DI. Despite a low level of knowledge, many had a positive/adequate attitude toward DI prevention, and 70% were adequately engaged in prevention against DI.The multivariate analysis showed that gender, age, education, and marital status were independently associated with an adequate level of knowledge regarding DI among parents. However, only education was independently associated with a positive attitude toward DI prevention, and there was no independent association between SD factors and the prevention of DI.This study found that 33.9% of parents had adequate knowledge regarding DI in children in Grenada. This is comparable to similar cross-sectional KAP studies in Jamaica: 13% in 2005 and 54.5% in 2010 . Fever, The knowledge of dengue virus (DENV) transmission among parents was inconclusive in this study. While more than half of the parents correctly knew the Aedes mosquito transmitted DENV, many also incorrectly believed it was transmitted by blood transfusion and sexual intercourse. This highlights a deficit in the knowledge of the transmission of DENV. However, parents knew bed nets, insecticide sprays, and mosquito repellants prevented mosquito bites and DI. Most parents also correctly stated they would not give their child aspirin if DI is suspected. However, the remaining 40% were unsure or would give their child aspirin. This is a common alarming finding in the Caribbean, as aspirin can exacerbate DI in children, thus highlighting a need to be addressed ,20. GendNevertheless, males were more likely to work outside the home than females, with an increased likelihood of interacting with people who may be knowledgeable about DI. Older parents (more than 39 years of age) and those with a high level of education were found to be more knowledgeable about DI. Older parents were more likely to complete tertiary and post-graduate studies, be involved in the workforce, and be sensitized to DI symptoms and transmission than young parents. The association between the level of knowledge and education is common in cross-sectional KAP studies regarding DI ,14. FinaMore than half of the parents in this study had a positive attitude toward DI prevention and HSB. This mirrors a cross-sectional study conducted in India in 2018  and may This study also showed parents with a high level of education were more likely to have a positive attitude toward DI prevention. While most parents were also actively engaged in preventive practices against DI. Interestingly, SD factors were not associated with preventing DI in this study. This contrasts with a cross-sectional study conducted in Malaysia, where gender and age were associated with preventing DI . The difReliability, validity, and generalizabilityThis study was conducted among a large population with a high DI prevalence. Using a random sampling method, objective data collection with a validated research instrument, appropriate statistical analysis, and adjustment for confounders during data analysis increased the internal and external validity. Furthermore, the baseline SD factors of participants mirror that of the Grenadian parent population, and the targeted sample size was met to ensure adequate power and identification of associations between the level of KAP and SD factors of DI. Therefore, this study does have generalizability to the Grenadian parent population. Limitations of studyA parent population outside a healthcare setting would provide a better representative and unbiased study population. Additionally, an equal representation of male and female parents may provide more information regarding the association between gender and the level of KAP regarding DI among parents. The researcher also assisted some participants who were illiterate in filling out the survey, which could contribute to observation bias. Therefore, the study results should be interpreted in light of the limitations mentioned.RecommendationsHealth education campaigns have been the mainstay of increasing awareness of DI in Grenada . A succeIt is equally important to convey these key messages across reliable and accessible media. Parents in this study stated that the media (television and radio) and healthcare workers played an essential role in their knowledge of DI. Therefore, these avenues can be utilized to increase the level of knowledge among parents regarding DI in children in Grenada . HoweverParents’ exposure to health education campaigns alone may not be sustainable in improving behavioral change . This caLastly, legislation regarding the prevention against DI should be reinforced to ensure sustainability. This includes frequent monitoring and evaluation of the health education campaigns at the level of healthcare, schools, and communities, the documented success of which was seen in Singapore . ImplemeThis study provides insight into the level of KAP and associated SD factors regarding DI among parents in Grenada. There needs to be more knowledge regarding DI among parents in Grenada, which is associated with low levels of education, younger age, and single marital status despite positive attitudes and prevention against DI. These findings can guide future health education campaigns and interventions to ensure sustainable KAPs against DI among parents in Grenada. It also allows future KAP studies regarding DI in Grenada to understand, assess, and improve the fight against DI.