Year of transplant (≤2006 vs. >2006) was the main factor influencing the outcome. In fact, a significant improvement in 5-year OS was demonstrated . Frequency of severe aGvHD was significantly reduced in recent years . A significant progress has been the introduction of the TCR αβ/CD19-depleted haploidentical platform, which was associated with the absence of severe aGvHD. However, it was associated with 23.5% incidence of GF. All but one patient experiencing GF in the this specific cohort were successfully retransplanted. In summary, allo-HSCT is confirmed to be an effective treatment for children with IEI, even in the absence of an HLA-matched donor. Allogeneic hematopoietic stem cell transplantation  represents an effective treatment for a variety of inborn errors of immunity (IEI). We report the experience of children affected by IEI who received allo-HSCT over a period of 32 years at IRCCS Istituto Giannina Gaslini, Genoa, Italy. HSCTs were performed in 67 children with IEI. Kaplan–Meier estimates of overall survival (OS) rate at 5 years in the whole group of patients was 83.4% after a median follow-up of 4 years. Median age at transplant was 2.5 years. Eight allo-HSCTs were complicated by either primary or secondary graft failure (GF), the overall incidence of this complication being 10.9%. Incidence of grade 3–4 acute GvHD (aGvHD) was 18.7%, significantly lower in the haploidentical transplant cohort ( Inborn errors of immunity (IEI) are a heterogeneous group of rare diseases, characterized by a broad spectrum of clinical features including infections, inflammation, auto-immune phenomena, lymphoproliferation, and malignant disorders .Advances in molecular genetics and improved knowledge in cellular immunology determined, as a consequence, a striking increase in the number of known monogenic immune disorders in the past decade, reaching the number of 485 clinical phenotypes reported in the 2022 updated classification from the International Union of Immunological Societies (IUIS) Committee on Inborn Errors of Immunity .IEI were recently further divided into two main groups: primary immune deficiency disorders (PIDD) or primary immune regulatory disorders (PIRD). This latter group comprises about 130 genes, whose mutations are responsible for disruption in immune regulation resulting in predominant clinical phenotypes such as autoimmunity, autoinflammation/hyperinflammation, lymphoproliferation, malignancy, and severe atopy, rather than infections .There is a wide spectrum of medical approaches in the treatment of IEI, ranging from conservative therapy alone based on antimicrobial prophylaxis, immunoglobulin replacement therapy, allogeneic hematopoietic stem cell transplantation , or gene therapy. Different elements  should be considered to define the best therapeutic strategy .Treatment by allo-HSCT is increasingly successful . ImproveWe report our experience in patients with IEI who received allo-HSCT in our institute for more than 30 years, in order to analyze the impact of evolving clinical practice on allo-HSCT outcomes for these patients.All children or adolescents with a clinical diagnosis of IEI, with or without a conclusive genetic diagnosis, who underwent allo-HSCT at the Hematopoietic Stem Cell Transplantation Unit of “Istituto Giannina Gaslini”, Genoa, Italy, between January 1990 and December 2021, were included in this retrospective, single-center study. Follow-up was closed at 15 June 2022. Patients who underwent allo-HSCT in the same years due to genetic alterations and diseases classified as bone marrow failure according to the 2022 Update of the IUIS Phenotypical Classification were excluded . Clinical data were obtained from medical reports. Diagnosis was categorized as PIDD and PIRD according to Chan et al. and to clinical phenotypes .Since patients affected by IEI can suffer from clinical manifestations related to defective or overactive immunity, the clinical phenotypes were summarized according to four phenotypic groups: (i) immunodeficiency (ID), in case of significant infectious events in the clinical history; (ii) autoimmunity (AI), in case of the occurrence of an immune-mediated cytopenia, encephalitis, colitis, skin manifestations, or any disease-specific autoimmune event; (iii) inflammation (INF), in case of the occurrence of one or more systemic symptoms, such as non-infectious fever, inflammatory skin rash, organ involvement, and increased inflammatory markers; and (iv) lymphoproliferation (LP), when clinical features such as lymphoadenopathy, hepato-splenomegaly, and macrophage activation were reported.Transplants were classified according to donor choice as “MRD” (matched related donor) for related HLA genotypically or phenotypically identical donors, “Haplo” for haploidentical related donors, and “MUD” (matched unrelated donor) for unrelated donors or cord blood units.in vivo T depletion strategy, post-transplant immunosuppression, and serological status for cytomegalovirus (CMV) in donor and recipient.The conditioning regimen (CR) was categorized as myeloablative (MAC), based on busulfan (Bus) or treosulfan (Treo) as alkylating agent, or reduced intensity (RIC), according to “Defining the intensity of CRs: working definitions” . Treo waPost-transplantation data included graft failure (GF), further classified as primary or secondary, or engraftment of neutrophils (absolute neutrophil count >500/mmc for at least three consecutive days) and platelets , graft versus host disease (GvHD), grading of acute GvHD (aGvHD) according to Glucksberg criteria , chronicU-test or Kruskal–Wallis test.Qualitative data were reported in terms of absolute frequencies and percentage. Pearson’s chi-square test or Fisher’s exact test, when appropriate, was applied to compare proportions. Quantitative data were described in terms of median values and interquartile range (IQR), due to their non-normal (Gaussian) distribution. Accordingly, comparisons between groups were performed by the non-parametric Mann–Whitney The counting process approach was applied to take into account the fact that any patient could have received more than one HSCT. For this reason, the transplantation-related risk factors were considered as time-dependent covariates.Time at risk was calculated since allo-HSCT. The cumulative overall survival (OS) probability was calculated by the Kaplan–Meier method. Applying the formula by Kalbfleisch and Prentice, 95% confidence intervals (CIs) of the estimates were calculated. Log-rank test was used to assess differences between transplant features.p-value < 0.05 was considered statistically significant. All analyses were performed using Stata .All tests were two-tailed and a A total of 67 patients were included in this study, 50 74.6%) of whom were male. A total of 55 patients 82.1%) received a genetic diagnosis in the pre-transplantation phase, and among them, one child received a pre-natal diagnosis because of an affected brother. Twelve (17.9%) patients underwent HSCT without a genetic diagnosis, but in three of them a mutation on SAMD9L (% receive.6% of whThirty (44.8%) and thirty-seven (55.2%) children were affected by a PIDD and a PIRD, respectively; frequencies of different phenotypes and genetic mutations in the two disease groups are reported in vs. 64.9%, p = 0.001, vs. 10%) and LP (46% vs. 13.3%) were more frequent in the PIRD group compared to the PIDD group  patients demonstrating an increased susceptibility to infections, alone or together with other phenotypes Table 1.During the study period, 67 patients received a total of 73 allo-HSCTs. Four patients received two transplants and one patient received three transplants. Most HSCTs were performed after 2006  and after a diagnosis of PIRD  Table 2.ex vivo manipulation by TCRαβ/CD19 depletion of grafts in all patients but one who received unmanipulated graft based on post-transplant cyclophosphamide (PT-Cy) platform. The stem cell sources were BM, CB, and PBSCs in 38 (52%), 11 (15.1%), and 24 (32.9%) HSCTs, respectively. In 70 allo-HSCTs, CMV serological status was available for both donor and recipient and 55 (78.6%) were at risk for CMV reactivation after HSCT.MUDs and MRDs were selected in 33 (45.2%) and 19 (26.0%) HSCTs, respectively. The remaining 21 (28.8%) transplants were performed from haploidentical donors, in particular from a brother (1), a sister (1), mothers (12), or fathers (7), based on Most patients received HSCT after a MAC , based on Bus  or Treo . In 51 patients (69.9%), CR included fludarabine. Chemotherapy based on thiotepa, Treo, and fludarabine was selected in 33 HSCTs and was the most frequently selected CR overall  and specifically after 2006 .In vivo T-cell depletion was performed in 64 (87.7%) HSCTs based on anti-thymocyte globulin (ATG) or anti-CD52 monoclonal antibody in 52 and 12 HSCTs, respectively. It was not performed in one HSCT based on the PT-Cy platform and in eight MRD HSCTs according to previous clinical practice. Specifically, anti-CD52 was performed in 10 HSCTs for PIRDs . In four HSCTs, anti-CD52 was chosen as part of the CR for a second HSCT after GF. Rituximab was part of the CR in 18 allo-HSCT based on haploidentical TCRαβ/CD19-depleted grafts and in 1 HSCT from MUD because of a pre-transplant EBV reactivation.4/kg in the manipulated graft.Post-transplant immunosuppression was given in 54 HSCTs, and it was based on a calcineurin inhibitor (cyclosporine or FK506) alone or in association with short-course methotrexate or mycophenolate or methylprednisolone, according to donor type, HLA matching, and stem cell source. Only 1/20 patients undergoing haploidentical TCRαβ/CD19-depleted HSCT received post-transplant immunosuppression due to a count of αβ T cells >10 × 10p = 0.003, p < 0.001). A significant increase in the use of rituximab (p = 0.002) was also evident after 2006 due to the preventive approach of PTLD in TCRαβ/CD19-depleted HSCTs. A trend for a reduction in number of days between diagnosis and HSCT was assumed , together with a higher age at HSCT (p = 0.096) and a more frequent diagnosis of PIRD leading to HSCT (p = 0.087).As for year of HSCT , no statistically significant differences were reported, except for the choice of Haplo donors and the introduction of Treo as CR Table 2.Overall, three HSCTs were not evaluable for engraftment/GF because the patient died before day +30. Eight out of the remaining 70 HSCTs (11.4%) were complicated by primary  and secoSix GFs were reported in years after 2006; considering only the TCRαβ/CD19-depleted HSCTs, the incidence of GFs was 4/20 (20%).Neutrophil engraftment occurred in 69 HSCTs, and the median time to neutrophils recovery was 16 days ; platelet engraftment occurred in 65 at a median time of 19 days from transplant .Nine HSCTs were not evaluable for aGvHD because of primary GFs , secondap = 0.005, Overall, aGvHD was diagnosed in 33 of the evaluable 64 patients (51.6%). Grade III–IV aGvHD was diagnosed in 12 (18.7%). According to donor, incidence of aGvHD was significantly lower in haploidentical transplant (n = 13) or after 2006 (n = 20), a significant reduction in the incidence of grade III–IV aGvHD was demonstrated in more recent years .When comparing HSCTs complicated by aGvHD before 2006 (p = 0.026).Regarding the 20 TCRαβ/CD19-depleted HSCTs, 18 were evaluable for aGvHD and 4 (22.2%) were associated with grade I–II aGvHD. The analysis of grade III–IV aGvHD in MRD and MUD HSCTs evaluable for aGvHD  and TCRαβ/CD19-depleted haploidentical HSCTs  demonstrated a significantly lower incidence in the latter setting  and scored as severe in 8 (47.1%). No severe cGvHD was reported in TCRαβ/CD19-depleted haploidentical HSCTs. Response to treatment was complete in 14 cGvHD (81.4%), as listed in n = 4 >95% donor) at the last follow-up.Chimerism monitoring for the whole observation period was feasible in 56 allo-HSCTs  developed at least one event and received preemptive treatment; only two patients developed CMV disease . A total of 17 HSCTs (23.3%) were complicated by one or more viral reactivations except from CMV: adenovirus , human herpesvirus 6 , and half of them suffered from more than one episode.Candida albicans , caused by albicans  and Candpsilosis . None ofAutoimmune complications were reported in eight allo-HSCTs (12.3%), four developed in a phase of mixed chimerism (three autoimmune cytopenia and one arthritis).n = 4) and to multiorgan failure (n = 1); six in the period between 3 months and 3 years from HSCT; one patient died after 24 years from HSCT due to an acute neurologic event not related to the transplant procedure.Patients were followed for a median of 4.8 years (0–25) , and 12 out of 67 (17.9%) patients died during follow-up; details on deceased patients are reported in One-year OS was 89.6% ; 3-year OS was 82.5%  Figure 1p = 0.049) compared to HSCT year ≤2006 (HSCTs performed in most recent years (>2006) showed improved OS , but this difference was not statistically significant (p = 0.365).An increased OS was assumed in HSCTs performed for PIRDs compared to PIDDs . From 2006 and onwards, both MRD and MUD demonstrated an improved survival rate compared to allo-HSCT performed from the same donor before 2006.The comparison of OS according to donor type in the different periods . Signs and symptoms of inflammation and lymphoproliferation were detected more frequently in the PIRD group. Autoimmune features were described in less than 20% of patients but, unexpectedly, more frequently in the PIDD group compared to the PIRD group (p = 0.199). This finding can be related to 16 diagnoses of primary HLH reported in our cohort, representing almost half the PIRDs described: primary HLH is not typically characterized by autoimmune features.It is well known that patients affected by IEI can display several different clinical manifestations. Most frequently, immunodeficiency features indicated that HSCT should be performed (about 80% in whole cohort), particularly in the PIDD group compared to the PIRD group . Together with clinical diagnosis, an earlier genetic diagnosis is nowadays available due to improved molecular genetics. In our cohort, most patients underwent HSCT with a previous genetic diagnosis, and improved genetic tools led to a molecular diagnosis also for three patients who had previously received only a clinical indication for HSCT. The frequency of patients who did not receive a genetic diagnosis in their clinical history appears to be reduced in the last years .An early clinical suspicion is required to guarantee an early admission to a tertiary care center, and in our cohort, an increased awareness of IEI led to an increased number of HSCTs performed in the last decades . This result was associated with allo-HSCTs more recently performed at older age (p = 0.061). In fact, in our cohort, median age at HSCT was 2.5 years, but a trend for a higher age at HSCT  was found in recent years. PIRDs are mainly associated with different and sometimes more subtle clinical pictures compared to severe infections occurring in the first days/months of life as in some PIDDs, and timing for diagnosis and treatment can therefore be delayed.Diagnosis of PIRD and related indication to perform HSCT increased in the last years compared to PIDD . Actually, in most IEIs and in particular in PIRDs, the indication for HSCT can be influenced by many factors, such as the specific genotype–phenotype and comorbidities . Advances in HSCT outcomes thanks to new transplant settings and improved supportive treatment could lead to further extension of the indication to perform HSCT in patients affected by IEI.A satisfactory OS of transplanted patients was demonstrated in our cohort, resulting in 89.2% at 1 year and 83.4% at 3 years, stable over time, with significant improvement after 2006; this can be related to the improved clinical and genetic knowledge, and supportive care.Overall, the most important factor for improved survival rate in HSCTs performed more recently is the introduction of the haploidentical TCRαβ/CD19-depleted platform.ex vivo TCRαβ depletion, have been developed, offering the opportunity to selectively remove cells responsible to GvHD development while preserving those relevant to ensure engraftment, immune reconstitution, and graft versus tumor effect , and accordingly, PBSCs were increasingly used as SC source, as they are collected and ex vivo depleted before graft infusion. Meanwhile, also a shorter time is required to plan and perform HSCT thanks to the selection of Haplo donors. In fact, in our cohort after 2006, time from diagnosis to HSCT was reduced compared to previous years . As far as a parent of the patient is the most frequently selected Haplo donor, time to complete the donor selection is shortened. This result can lead to important advantages to the patient, reducing the risk of severe pre-transplant infections or other complications. A very satisfactory 90% cumulative survival rate in this specific setting has been found, principally thanks to the absence of severe GvHD and despite a higher incidence of GF after haploidentical TCRαβ/CD19-depleted HSCTs (4/20) compared to HLA-matched HSCTs (4/49). However, all but two patients were successfully retransplanted in both cohorts. In our patients, no additional risk factors of proven relevance for GF were present, since none presented a significant level of anti-HLA antibodies against donor HLA before transplant; all received graft with an adequate cell dose and a Treo-based CR according to current recommendations  approaches . GT, comFor this reason, HSCT remains the best therapeutic approach for IEI, and improved outcomes related to the development of new strategies could lead to the extension of the indications for transplantation in patients affected by IEI. The development of a safe and low-risk allo-HSCT approach such as the TCRαβ/CD19-depleted platform and the improved knowledge chemoterapic agents such as Treo can guarantee an effective therapeutic approach in IEI with reduced burden of toxicity and GvHD.The original contributions presented in the study are included in the article/Written informed consent was obtained from all patient parents and/or legal guardians in accordance with our institution’s ethical standards and with the Declaration of Helsinki Principles.GD collected the clinical data and drafted the first version of the manuscript. GD and MF conceived the presented idea. FB performed the statistical analysis. SG, FP, GF, DD, FS, CM, DG, and SV reviewed the clinical and technical information reported. DD performed STR assays. FS was directly involved in stem cell manipulation. MG, MM, CD, and MF provided critical review of the manuscript. All authors contributed to the article and approved the submitted version.


Genetic knowledge of gestational diabetes mellitus (GDM) in Chinese women is quite limited. This study aimed to identify the risk factors and mechanism of GDM at the genetic level in a Chinese population.We conducted a genome-wide association study (GWAS) based on single nucleotide polymorphism (SNP) array genotyping  and a case-cohort study design. Variants including SNPs, copy number variants (CNVs), and insertions-deletions (InDels) were called from genotyping data. A total of 2232 pregnant women were enrolled in their first/second trimester between February 2018 and December 2020 from Anqing Municipal Hospital in Anhui Province, China. The GWAS included 193 GDM patients and 819 subjects without a diabetes diagnosis, and risk ratios (RRs) and their 95% confidence intervals (CIs) were estimated by a regression-based method conditional on the population structure. The calling and quality control of genotyping data were performed following published guidelines. CNVs were merged into CNV regions (CNVR) to simplify analyses. To interpret the GWAS results, gene mapping and overexpression analyses (ORAs) were further performed to prioritize the candidate genes and related biological mechanisms.We identified 14 CNVRs  and two suggestively significant SNPs  associated with GDM, and a total of 19 candidate genes were mapped. Ten genes were significantly enriched in gene sets related to lipase (triglyceride lipase and lipoprotein lipase) activity , oxidoreductase activity (TPH1 and TPH2 genes), and cellular components beta-catenin destruction complex (APC and GSK3B genes), Wnt signalosome (APC and GSK3B genes), and lateral element in the Gene Ontology resource (BRCA1 and SYCP2 genes) by two ORA methods .Genes related to lipolysis, redox reaction, and proliferation of islet β-cells are associated with GDM in Chinese women. Energy metabolism, particularly lipolysis, may play an important role in GDM aetiology and pathology, which needs further molecular studies to verify.The online version contains supplementary material available at 10.1186/s12967-023-03902-4. Gestational diabetes mellitus (GDM), defined as abnormally high blood glucose levels among pregnant women without a diabetes diagnosis before pregnancy, is a common complication in pregnancy, and its incidence is increasing worldwide , 2. GDM It is suggested that approximately 80% of GDM cases are related to insulin resistance (IR) . Other hScreening for GDM based on risk factors, including obesity, first-degree relatives with diabetes, history of GDM or adverse pregnancy outcomes, and glycosuria, will miss approximately one-half of cases , 28. FurThe average incidence of GDM in mainland China was 14.8% in recent years, and relevant genetic knowledge for Chinese individuals is quite limited , 34–37. The cohort enrolled 2232 pregnant women in the first/second trimester between February 2018 and December 2020 from Anqing Municipal Hospital in Anhui Province, China. Participants were followed up around their expected date of delivery. The demographical information, physical measurements, behavioural information before and during pregnancy, and medical histories of subjects were collected from structured questionnaires at baseline, structured questionnaires or phone interviews at follow-up, or medical records. The cohort profile is shown in  by physicians. Pregnant women without diabetes before pregnancy met one or more of the following criteria: (a) blood glucose after 75 g OGTT  ≥ 10.0 mmol/L in the first hour or ≥ 8.5 mmol/L but < 11.1 mmol/L in second hour and (b) 5.1 mmol/L ≤ fasting blood glucose < 7.0 mmol/L after the 23rd gestational week (only for pregnant women who have no access to an OGTT) ). Means of 2 continuous normally distributed variables were compared by independent samples Student’s t-test. The Wilcoxon test and Kruskal‒Wallis test were used to compare the means of 2 and 3 or more groups of variables with nonnormal distributions, respectively. The frequencies of categorical variables were compared using Fisher’s exact test. For all statistical tests, a P value < 0.05 was considered significant . All ana2 < 0.2) variant set on autosomal chromosomes [According to the law of causality, the relation between genotypes and phenotypes is affected by only a few potential confounders, of which the most important is population stratification (PS). It can be estimated by the distinctions of observing genotypes among populations . For SNPomosomes , 54. Theomosomes  version omosomes  to be inThe median age of the study cohort was 28.0 years with an IQR of 25.0 to 30.0 years, and the median gestational age at baseline was 16.4 (IQR: 15.9–17.1) weeks. Approximately 99.2% (1953/1968) of the subjects in the cohort were of Han nationality. Balance tests for the subcohort group and nonselected group verified the representativeness of the sampling, in which the difference was significant only for the weight before pregnancy (P = 0.006)  in the intronic region of SLC12A8 gene on the chromosome (Chr) 3  and SNP rs12253503:A:G  in the intergenic region of RP11-186O14.7 gene on Chr10   from 985 subjects (190 cases and 795 controls) were included in the GWAS based on the imputation of 318,716 SNPs and 470 InDels. Three PCs were selected as covariates based on the “twstats” method  in two genomic risk loci  and 12 genes mapped in the CNV part were included in the ORA as candidate genes of GDM. The 19 genes included 18 protein-coding genes and 1 pseudogene, and their primary molecular functions are shown in Table In this case-cohort GWAS based on SNP array genotyping and imputing data, we aimed to explore the genetic background of GDM in a Chinese population and identified 2 SNPs associated with GDM at a suggestive significance level and 14 CNVRs associated with GDM. A total of 19 genes that may be candidate genetic markers for GDM were mapped through functional mapping, in which twelve  were directly mapped by variants genotyped in our study. These 19 candidate genes were enriched mainly in gene sets related to the triglyceride lipase (TGL) activity and lipoprotein lipase (LPL) activity, oxidoreductase activity, and the cellular components beta-catenin destruction complex, Wnt signalosome, and lateral element based on the GO resource.TGL is a family of lipases that catalyse the first stage of lipolysis by hydrolysing triglyceride  to diacylglycerol (DAG) and fatty acid (FA), while LPL is responsible for hydrolysing TG in blood lipoproteins to DAG and FA . Convinc2 < 0.5) were identified in the GWAS, among which rs7754840 in the intron regions of the CDKAL1 gene and rs10830962 located upstream of the MTNR1B gene showed the strongest association with GDM. The controls were selected from women without a T2DM history  ≥ 50 years in the first stage and  ≥ 50 years in the second stage, which may introduce misclassification bias because of an unknown GDM history. The second GWAS included 103 GDM cases and 115 controls from a population of Chinese Han women and identified 23 SNPs mapping to four genes  [To our knowledge, only two GWASs of GDM have been reported. Kwak et al. conducted a two-stage study in Korean women, including 468 GDM cases and 1242 nondiabetic controls using 2.19 million genotyped or imputed markers in the first stage and 931 GDM cases and 783 nondiabetic controls with 11 genotyping loci from the first stage in further study . Nine ind SYNPR) . No overd SYNPR) , NR3C1 [d SYNPR) , and PRDGDM has been a public health problem with both short-term and long-term metabolic influences on mothers and children. Converging lines of evidence have shaped GDM as a complex trait caused by genetic and environmental risk factors. However, the pathophysiology of GDM remains unclear and controversial, and its genetics have not been fully explored. Our study suggests that GDM may be the result of alterations in energy metabolism under a specific genetic background, including gene variants related to lipolysis and redox in addition to the Wnt signalling pathway, which affect glucose metabolism mediated by the insulin signalling pathway. This finding adds evidence for the hypothesis that GDM may be a herald of T2DM expressed under the specific metabolic conditions of pregnancy or an excessive manifestation of metabolic alterations occurring in pregnancy  and explThe strengths of the present study are as follows. (1) This study was based on a case-cohort study design, which is cost-effective for GWAS and could produce RR estimates without the rare-disease assumption . (2) GenThis GWAS of GDM in a sample of Chinese women showed evidence that GDM is a metabolic disease and that lipid metabolism, particularly lipolysis, may play an important role in the onset and development of GDM. The results provide a new direction for the prevention, early diagnosis, and treatment of GDM, focusing more on metabolites other than blood glucose. However, further studies, such as GWASs in other populations or studies using other omics, animal models, or GDM-associated genes from this study, should be conducted to verify and detail the actual mechanism of these hypotheses.Additional file 1. Note S1. Parameters for FUMAGWAS analyses. Note S2. Parameters for WebGestalt analyses. Note S3. Parameters for g:profiler analyses. Table S1. Characteristics of selected or not sub-cohort groups in cohort. Table S2. PCs with P value < 0.01 for SNPs/InDels association analyses. Table S3. Markers in linkage disequilibrium with any independent significant markers (r2 ≥ 0.6). Table S4. Genes mapped by positional mapping, eQTL mapping, and chromatin interaction mapping. Table S5. Protein coding genes from MAGMA gene-based analyses with PBon < 0.05. Table S6. CNVRs associated with GDM FDR with p value < 0.05. Table S7. Candidate genes for gene over-representation analyses. Table S8. Gene sets enriched in the Gene Ontology resource by g: Profiler. Table S9. Genome-wide association studies of GDM reported in literatures and the present study. Figure S1. Profile of the study cohort. Figure S2. Strategies of the analyses. Figure S3. The quantile–quantile plot of P values of SNPs/InDels GWAS (λ=1.01).


To observe different roles of direct bilirubin (Dbil) on portopulmonary hypertension (POPH) and idiopathic pulmonary arterial hypertension (IPAH).  Thirty incident patients with POPH and 180 with IPAH  between March 2010 and December 2020 were included. The receiver operating curve and Kaplan–Meier method were applied to estimate the ability to distinguish between the two and survival, respectively. Univariate and forward multiple stepwise regression analyses were performed to access the relationship between pulmonary vascular resistance (PVR) and clinical indices. P < 0.001) and higher total bilirubin (Tbil) and Dbil. Tbil and Dbil had a negative correlation with PVR in the POPH group  but positive correlation in the IPAH group . Increased neutrophil counts  and elevated NT-proBNP  would help predict the elevation of PVR in POPH and IPAH groups independent of Dbil, respectively. Dbil could distinguish POPH from IPAH , and the ability was elevated when taking aspartate aminotransferase together . The overall survival was better in POPH than in IPAH . Survival was better in POPH than in IPAH in the group of Dbil ≥7 μmol/L (P=0.001) but showed no significant difference between POPH and IPAH in the group of Dbil <7 μmol/L (P=0.192).  Compared to IPAH, the POPH group had better hemodynamics including PVR (7.08 ± 3.95 vs. 14.89 ± 7.11, μmol/L, but limited dead cases of POPH should be noted. The POPH group had a better hemodynamic profile than IPAH. Dbil was associated oppositely with the elevation of PVR in POPH and IPAH. Patients with POPH had better survival than those with IPAH in the total cohort and in the group of Dbil ≥7  Portopulmonary hypertension (POPH) is a life-threatening disease with damage to both pulmonary circulation and portal circulation with or without liver diseases, defined as Group 1 pulmonary hypertension (PH) and a severe complication of portal hypertension . AccordiBilirubin, including total bilirubin and direct bilirubin, is one indicator of liver function abnormality and had an association with PAH. During the 1990s, higher expression levels of total bilirubin were found to be a risk factor for early postoperative mortality in 31 patients with primary PH and 31 patients with Eisenmenger syndrome who all underwent heart-lung transplantation . MeanwhiPOPH, as a disease related to liver abnormality, probably exists in abnormal bilirubin. However, the comparisons of bilirubin between POPH and IPAH stay unclear. Thus, our objective was to make comparisons between IPAH and POPH.This study was approved by the Ethics Committee of the Shanghai Pulmonary Hospital with the approval number k16-293 and according to the Declaration of Helsinki. Written consent was obtained from each patient.Patients with portal hypertension were diagnosed at other centers and would come to our center for PH confirmation. The acute reports regarding hepatic venous portal pressure gradient (HVPG) values were not obtained. All patients were firstly screened by echocardiography (systolic pulmonary artery pressure ≥40 mmHg) and then underwent right heart catheterization (RHC) to confirm PAH at our center. There were 34 cases with POPH confirmed at our center from March 2010 to December 2020. Of those, 2 cases with Budd-Chiari syndrome and 2 cases with schistosomiasis were mechanically different from the other 30 cases  and were excluded from the study. To include a total of 30 patients with POPH in this retrospective study, patients with IPAH were matched by WHO functional classification (WHO FC) in a 1 : 6 ratio to generate a typical landscape of IPAH and increase the test power. The flow diagram is shown in Demographics, World Health Organization functional classification (WHO FC), target therapy, liver function, etiological originals of portal hypertension, and other related data were collected. The MELD (model of end-stage liver disease) equation was applied to calculate the score for severity as 9.57 × ln  + 3.78 × ln  + 11.20 × ln  + 6.43. The minimal values were forced to 1.0 for calculation purposes . Baselint-test was used for the comparisons. Continuous variables with nonnormal distribution were expressed as median (first and third interquartile), and the Mann-Whitney U test was applied for the comparisons. Categorical variables were expressed as the number of patients and relative frequencies , and the Chi-square test was used for the comparisons. The receiver operating characteristic (ROC) curve was employed to assess the ability of distinguishing POPH from IPAH. The predictive ability in the models should be compared with integrated discrimination improvement (IDI) [Continuous variables with normal distribution were expressed as mean ± SD, and the independent Student's nt (IDI)  and net nt (IDI) . CorrelaP value less than 0.05 was considered significant. All figures were concluded via GraphPad Prism  version 7.0 software.Overall survival time was defined from the date of diagnostic RHC to death. Surviving patients were censored on the date of the last clinical contact. The Kaplan–Meier method was used to estimate the proportion of patients surviving at each time point. Survival curves were compared with the log-rank test. All comparisons were employed with a two-sided test through SPSS  version 22.0, and a P < 0.001). In patients with POPH, the most common etiology of liver disease was hepatitis B virus infection  and the second one was cryptogenic disease . The Child-Pugh class of POPH was mainly distributed in class A  and class B . D-dimer  vs. 149  ng/ml, P < 0.001) and PCO2  were significantly higher, while NT-proBNP  vs. 739  pg/ml, P=0.004) was significantly lower in the POPH group rather than in IPAH group. Compared to the IPAH group, patients in the POPH group were more likely to receive the treatment of phosphodiesterase 5 inhibitors (19 cases (63.2%) vs. 77 cases (42.7%), P=0.036).A total of 30 POPH (8 men) and 180 IPAH (57 men) patients were included in this study. The mean time of follow-up was 70.23 ± 41.74 months. Data are illustrated in 2  than patients with IPAH. With respect to blood cell counts, the POPH group had lower white blood cell counts , neutrophil counts , red blood cell counts , and platelet counts  than the IPAH group.In the aspect of arterial blood gas, patients with POPH had higher PCOP < 0.001), mean right atrial pressure , mean pulmonary artery pressure , pulmonary vascular resistance , transpulmonary gradient , and systemic vascular resistance  but higher cardiac index  and mixed venous oxygen saturation . Of these differential variables, the gap of PVR between the POPH group and the IPAH group exhibited a two-fold change.Hemodynamic variables were quite different between POPH and IPAH. Compared to patients with IPAH, patients with POPH had lower systolic pulmonary artery pressure , direct bilirubin  vs. 6.0  μmol/L, P=0.013), and aspartate aminotransferase  vs. 25.0  IU/L, P < 0.001) and lower expression of albumin  than patients with IPAH. In addition, alanine aminotransferase  vs. 24.0  IU/L, P=0.566) showed no difference between the POPH group and IPAH group. These data are generated in Patients with POPH showed significantly higher expression of total bilirubin  vs. 19.0  2 were regarded as continuous variables. Target therapies and MELD scores were deemed as categorical variables.) The analysis found that total bilirubin and direct bilirubin presented a negative correlation with PVR in the POPH group , while they showed a positive correlation with PVR in the IPAH group . NT-proBNP was positively related to PVR in the IPAH group , and neutrophil counts had a positive correlation with PVR in the POPH group .β = 0.394, P=0.030). NT-proBNP had the sole role in positively predicting PVR elevation in the IPAH group and made 18.7% variation clear . This information is illustrated in Taking those potentially correlated variables . When combining the two variables together, the ability to identify POPH from IPAH had been improved and the AUC was elevated to 0.835 (P < 0.001). We also calculated the value of IDI and NRI to evaluate the elevated ability of the combined model relative to the direct bilirubin model, and the results were following: absolute IDI = 0.064, relative IDI = 0.074, and NRI = 0.030.ROC analysis was performed to assess the ability of direct bilirubin, AST, and their combination to identify POPH from total patients . We founμmol/L  during the follow-up. Of those dead cases, 72 patients with IPAH (75.0%) and 2 patients with POPH (28.6%) died of right heart failure. With respect to liver disease, no one died of liver failure in IPAH, while 5 patients with POPH (71.4%) died of liver failure. The 1st-year, 3rd-year, 5th-year, and 10th-year overall survival rates were found to be 67.2%, 53.2%, 41.0%, and 35.2% in the IPAH group and 89.3%, 81.5%, 81.5%, and 71.3% in the POPH group, respectively  total bilirubin and direct bilirubin associated with PVR positively in the IPAH group but negatively in the POPH group; (IV) elevated neutrophil counts and elevated NT-proBNP were independent predictors of PVR increase in POPH and IPAH, respectively; (V) direct bilirubin with AST could better help identify POPH from IPAH than separate direct bilirubin or AST.To date, there have still been limited types of literature talking about the differences in bilirubin between IPAH and POPH. Our study demonstrated some interesting findings as follows: (I) when matched by WHO FC, patients with POPH had better hemodynamics and survival than patients with IPAH; (II) patients with POPH had worse liver function than patients with IPAH, which showed that the medians (first and third interquartile) of direct bilirubin were 10.5  and 6.0  No matter from the REHAP registry , the REVr = −0.01) [We chose PVR to conduct the correlation analysis not only because of the two-time fold change between POPH and IPAH but also because PVR could predict mortality and graft failure in transplantation patients with POPH . Althoug= −0.01) . The difα after being treated with different serum unconjugated bilirubin concentrations, in which the expression of E-selectin VCAM-1 and ICAM-1 was reduced.The negative correlation between PVR and direct bilirubin was a decent finding in the POPH group. Horsfall  and collMeanwhile, the neutrophil counts were the independent predictor of PVR elevation in POPH after taking total bilirubin, direct bilirubin, and neutrophil counts into the linear regression analysis. In fact, bilirubin had some association with neutrophils in some pulmonary diseases. Biliverdin, which can be interconverted with bilirubin, improved pulmonary inflammation induced by hemorrhagic shock and resuscitation (HSR). The authors used biliverdin treatment before HSR in rats and found markedly decreased neutrophil infiltration in the lung sections (neutrophils were stained by the naphthol AS-D chloroacetate method) compared to the sham group . In the μmol/L, but the limited dead cases of POPH should be noted.When matched by WHO FC, POPH patients had a distinct demographic, clinical, and hemodynamic profile compared to IPAH patients. Total bilirubin and direct bilirubin had a negative correlation with PVR in POPH but positive in IPAH. Patients with POPH had better survival than IPAH patients in the group of total patients and the group of patients with direct bilirubin ≥7


Hepatitis C virus (HCV) infection is associated with serum lipid abnormalities, which partly normalize following direct-acting antiviral (DAA) therapy. Here, associations of serum triglycerides (TGs) with viral genotype and markers of liver disease severity were evaluated in patients with chronic HCV. P values were adjusted for multiple comparisons.The study included the serum of 177 patients with chronic HCV. TGs were quantified by flow injection analysis Fourier transform mass spectrometry. Laboratory values and noninvasive scores for liver fibrosis assessment were determined. The nonparametric Kruskal‒Wallis test, one-way ANOVA, multiple linear regression and Student’s t test were used as appropriate. HCV-infected women had lower serum TGs than men, and thus, a sex-specific analysis was performed. None of the 46 TG species analyzed differed in the serum of female patients with and without liver cirrhosis. In contrast, in the serum of male patients with liver cirrhosis, TGs with 53, 56 and 58 carbon atoms and three to eight double bonds were diminished. These polyunsaturated TGs were also low in males with a high fibrosis-4 score. TGs with 7 or 8 double bonds negatively correlated with the model of end-stage liver disease score in males. In addition, TGs with 49, 51 and 53 carbon atoms were reduced in male patients infected with genotype 3a in comparison to genotype 1a. TGs with 56 carbon atoms were lower in genotype 3a-infected males than in genotype 1b-infected males. TGs did not differ in females by genotype. Genotype 3-related changes disappeared at the end of therapy with DAAs. Overall, the levels of serum TGs did not change during DAA therapy in either sex. Consequently, the serum TGs of males with liver cirrhosis were lower than those of males without cirrhosis at the end of therapy. Such a difference was not apparent in females.The decline in TGs observed only in male patients with liver cirrhosis and male patients infected with genotype 3 illustrates sex-specific changes in lipid metabolism in chronic HCV.The online version contains supplementary material available at 10.1186/s12944-022-01715-w. Chronic hepatitis C virus (HCV) infection has a worldwide prevalence of approximately 2.5%. HCV infection is a major cause of chronic liver diseases, liver cirrhosis and hepatocellular carcinoma . An HCV HCV uses the hosts´ lipid metabolism for infection, and it has been consistently reported that the serum low-density lipoprotein (LDL) of HCV patients is low , 10. AccUnexpectedly, DAA therapy did not significantly influence serum TG levels . SimilarReports on the effect of DAA treatment on blood TGs are, however, contradictory. An increase in blood TGs was described in some studies –21, wherA cross-sectional analysis of nearly 12,000 adults showed that individuals with high fibrosis-4 (FIB-4) scores had lower fasting TGs . NotablyOverall, men have higher serum TGs than females . Most ofMoreover, in the studies listed above, total TG levels were measured, although many different TG species circulate in blood . CertainHere, it was hypothesized that serum TG concentrations and eventually the TG species profile differ between males and females with chronic HCV infection and that TGs are low in patients with liver cirrhosis. DAA therapy does not improve liver function in the short term, suggesting that TG levels and TG composition are similar before therapy and at the end of therapy. The objective of the investigation was to measure 46 TG species differing by carbon chain length and/or double bond number in the serum of 177 patients with chronic HCV and to perform a sex-specific analysis for correlations with markers of disease severity. It was also the aim to show that DAA therapy does not change TG levels or TG composition.This study was conducted from October 2014 to September 2019 at the Department of Internal Medicine I  . HCV treLaboratory parameters were measured by the Institute of Clinical Chemistry and Laboratory Medicine .Cirrhosis diagnosis by ultrasound determined the liver surface and the size of the liver and liver parenchyma . The cutSerum triglyceride species were quantified by flow injection analysis Fourier transform mass spectrometry (FIA-FTMS) as described previously . Total TData are shown as boxplots, which give the minimum, the maximum, the median, and the first and third quartiles. Small circles or asterisks above or below the boxes mark outliers. When more than one TG class is shown in a figure, mean concentrations ± standard deviations are displayed. Data in tables are given as median values, and the minimum and maximum values in brackets.P values were adjusted for multiple comparisons by Bonferroni. A value of P < 0.05 was regarded as significant.The nonparametric Kruskal‒Wallis test and one-way ANOVA were used for comparison of continuous variables between independent groups. Stepwise multiple linear regression analysis was also performed. Spearman correlation was used for correlation analysis (SPSS Statistics 25.0 program). Student´s t test was used to analyze paired data (Microsoft Excel). P > 0.05 for all).Triglycerides are neutral lipids, and their blood content and composition are regulated by different pathways . Here, 4The 103 male patients had a higher model of end-stage liver disease (MELD) score, alanine aminotransferase (ALT), aspartate aminotransferase (AST) and international normalized ratio (INR) and lower high-density lipoprotein (HDL) levels than the 74 female patients (Table P > 0.05). Seventy-three patients had fatty liver , and 20 patients had diabetes , but this was not associated with higher concentrations of TGs , TG C58 (P = 0.01), TG DB6 (P = 0.02), TG DB7 (P < 0.001) and TG DB8 (P < 0.001) showed that these species were specifically low in males with liver cirrhosis. Total TGs were reduced in male patients with liver cirrhosis compared to males without cirrhosis (P = 0.019).Along the same lines, none of the TGs declined in the 16 females where cirrhosis was diagnosed by ultrasound Fig. a, b. In Whereas TGs (TG DB2 – DB8) were higher in males than in females in the cohort of patients without liver cirrhosis, this difference was no longer apparent in patients with liver cirrhosis Fig. e, f. NotP = 0.064 for males and P = 0.576 for females). Moreover, serum HDL (P = 0.069) and LDL (P = 0.063) did not significantly decline in genotype 3a-infected males.None of the TG species in male or female serum correlated with viral load Table S and S2. At 4 and 12 weeks after the start of therapy, TG levels were not changed in females or males compared to pretreatment concentrations Fig. . This wap = 0.015), TG DB5 (p = 0.015), TG DB6 (p = 0.01w), TG DB7 (p = 0.002) and TG DB8 (p = 0.006) of total TG levels were low in males with cirrhosis compared to males without cirrhosis.At the end of therapy, TGs did not differ between female patients with and without liver cirrhosis (data not shown). TG C48 to C58 and TG DB1 to DB8 were reduced in male patients with ultrasound-diagnosed liver cirrhosis compared to males without cirrhosis Fig. . The perIn females, associations of TGs with measures of liver injury did not exist Table S. In maleR2 = 0.081, the regression coefficient B = 2.491 and P = 0.002), to TG DB7 as the dependent variable , and to TG DB8 as the dependent variable .To analyze whether the MELD score, albumin and ALT  with age, BMI, MELD score, viral load and routine laboratory parameters in female HCV patients before DAA therapy. Table S2. Spearman correlation coefficients for the correlations of TGs  with age, BMI, the MELD score, viral load and routine laboratory parameters in male HCV patients before DAA therapy. Table S3. TGs  in the serum of males before DAA therapy stratified by fibrosis-4 score. Table S4. TGs  in the serum of males without liver cirrhosis before DAA therapy stratified by genotype. Table S5. Spearman correlation coefficients for the correlations of TGs  with age, BMI, the MELD score, viral load and routine laboratory parameters in female HCV patients at the end of DAA therapy. Table S6. Spearman correlation coefficients for the correlations of TGs  with age, BMI, MELD score, viral load and routine laboratory parameters in male HCV patients at the end of DAA therapy. Table S7. Spearman correlation coefficients for the correlations of TGs  with the MELD score at the end of DAA therapy in males. Figure S1. Serum TG species in relation to body mass index (BMI) in female and male patients with chronic HCV. Figure S2. TGs stratified according to viral genotypes in male patients without liver cirrhosis at the end of the study.


This study aimed to analyze the distribution of IgG subclass in diabetic nephropathy (DN) and its association with clinicopathological features.This is a single-center retrospective study enrolling 108 patients with biopsy-proven DN. Immunofluorescence and immunohistochemistry staining were applied, and clinicopathological features and renal outcomes were compared between patients with different patterns or categories of IgG subclass deposition.P = 0.004). IgG subclass deposit on multi-location was more associated with a shorter duration of nephropathy and severer tubular interstitial injury (P < 0.05). The mixed type of IgG subclass deposit on GBM was merely associated with higher levels of proteinuria, whereas the deposition on TBM was more associated with higher levels of proteinuria, lower levels of albumin, more KIM-1 positive area, and thicker TBM (P < 0.05). Survival analysis revealed that none of the pattern or the category of IgG subclass deposit was a risk factor or a renal outcome indicator.Both IgG and its subclasses colocalized with collagen IV α5 on glomerular basement membrane (GBM) and some of tubular basement membrane (TBM). IgG1 and the Mixed type were two predominant types of deposition, no matter on GBM or TBM, and IgG1 showed a much higher deposition rate on GBM than that on TBM (IgG subclass was selectively deposited along GBM and/or TBM in DN, and the mixed type of IgG subclass deposition on TBM had more clinical significance than the isotype and that on GBM. IgG subclass deposition is merely a manifestation or a consequence rather than a cause in DN. Diabetic nephropathy (DN) has a unique histological pattern, including glomerular basement membrane (GBM) thickening, mesangial expansion, and glomerulosclerosis, among others . As a noIgG has four subclasses (IgG1–G4); each subclass has a unique profile regarding half-life, antigen binding, immune complex formation, complement activation, and triggering of effector cells. Oxelius VA found that serum IgG2 and IgG3 levels declined, while IgG1 and IgG4 were relatively normal in juvenile diabetes mellitus (DM) cases . SusannaAmong 348 patients with type 2 DM and biopsy-proven DN between August 2017 and July 2021 at Hangzhou TCM Hospital Affiliated to Zhejiang Chinese Medical University, 225 cases showed an IgG linear pattern by immunofluorescence (IF). One hundred eight patients were enrolled according to the following inclusion criteria: (1) type 2 DM; (2) a diagnosis of DN proven by kidney biopsy; (3) IF showed IgG-positive status; and (4) Four IgG subclasses could be fully applied. The exclusion criteria were: (1) coexistence of nondiabetic renal diseases, such as MN, IgAN, or system diseases; (2) absence of glomeruli or global sclerosis in IF specimens; and (3) anti-GBM positive cases. The study was approved by the ethical committees of Hangzhou TCM Hospital Affiliated to Zhejiang Chinese Medical University (2020LH001).The following clinical information was collected: age, gender, duration of diabetes, duration of nephropathy (start from the nephropathy symptoms such as lower extremities edema or soreness of waist till the diagnosis of DN), 24-h proteinuria, serum albumin, serum creatinine (Scr), and the estimated glomerular filtration rate .All specimens were processed for light microscopy (LM), IF, immunohistochemistry (IHC), and electron microscopy (EM). Patients were grouped according to the pattern of IgG subclass deposition  and the category of deposition . Classification of DN and histological scoring were done according to the criteria reported by Tervaert et al. . InterstPatients were contacted by telephone, and the follow-up data included renal function (proteinuria and eGFR), dialysis, or kidney transplantation. The outcomes were progression to end stage renal disease (ESRD) or ≥ 50% decline in eGFR from baseline. ESRD was defined as eGFR < 15 mL/min/1.73 m2 or initiation of chronic renal replacement therapy.Frozen tissues were used for IgG  and IgG subclass  staining by direct IF. Double staining of IgG or IgG subclass with anti-human collagen IV α5  by indirect IF  was also performed. An Olympus BX53 fluorescence microscope (Japan) was used to analyze the IF slides.IHC for kidney injury molecular-1  and CD34  was conducted using a Ventana BenchMark XT system. CD34 and KIM-1 positive statuses were analyzed by ImageJ and calculated as the percentage of positive area per glomeruli or cortex, respectively , 14.t test or ANOVA for continuous variables and nonparametric tests for discontinuous variables. Categorical variables were expressed as percentages and comparisons among groups, which were evaluated by chi-square test or the Fisher’s exact test. The association between IgG subclass deposition and renal outcomes was evaluated using Cox proportional hazards models. The renal survival rates between patients with different patterns or categories of IgG subclass deposition were calculated using log-rank test and Kaplan–Meier analysis. A P value of less than 0.05 was considered significant.Statistical analysis was performed with SPSS 17.0. Normally distributed data were expressed as mean ± standard deviation, and non-normally distributed data were expressed as medians and interquartile ranges. A comparison of clinical and pathological characteristics among groups was assessed by 2, respectively). Renal biopsy showed 65% of DN were in stage three. On average, the pathological features showed 23.1% glomerulosclerosis, moderate to severe FPE, and moderate IFTA. All cases had minor to moderate linear IgG expression along the GBM, similar to albumin, but only 56.5% of cases showed linear TBM deposition  and 2  months, respectively. Most of the patients enrolled in our study had nephrotic syndrome  g/day and the median serum albumin was 30.9  g/L), with normal to a moderate reduction of renal function  umol/L and median eGFR 65.0  ml/min per 1.73mP < 0.001)  , and IgG2 showed a higher deposition rate on GBM than that on TBM (P < 0.05)  Fig. . Among f04) Fig. . In the P = 0.021). Pathological features, such as KIM-1 showed more positive area, and TBM exhibited thicker in the Both deposition group than in the GBM-only group (P < 0.05) , whereas six were positive along TBM only (the TBM-only group). The remaining fifty cases had positive staining on both GBM and TBM (the Both group) Table . None ofP = 0.018) ; meanwhile, higher levels of proteinuria and lower levels of serum albumin exhibited in the Mixed group than in the Isotype group and the None group (P < 0.05).TBM showed a much lower frequency (56.5%) of IgG subclass deposit than GBM, including 37% from the Isotype group and 19.5% from the Mixed group  and the crosstalk between endothelial cells and podocytes , 23, 24.It’s reported that the glomerular IgG deposit emerged as an independent risk factor for renal clinical outcomes . HoweverIn summary, the location and the category of IgG subclass deposit are probably determined by their profiles and the severity of glomerular/tubular injury. The mixed type of IgG subclass deposition on TBM had more clinical significance than the isotype and that on GBM. IgG subclass deposition is merely a manifestation or a consequence rather than a cause in DN.


PRF1, STX11, UNC13D, HPLH1, and STXBP2 are the most well-known genes of this type which are related to granule-mediated cytotoxic T and Natural killer (NK) cells. The treatment is based on the HLH-2004 protocol.Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening disease characterized by some clinical signs  and laboratory findings . Depending on the etiology, HLH is divided into familial  and acquired  forms. Familial HLH (FHL), an autosomal recessive condition, is classified into five subtypes based on underlying genetic defects. The UNC13D. The associated disease was Familial Hemophagocytic Lymphohistiocytosis 3 (FHL3). Case 2, a 37-day-old female presented with fever, a history of neonatal cholestasis, and huge hepatosplenomegaly. Her whole-exome sequencing report manifested that the patient had the same mutation as case 1. Unfortunately, both patients passed away.The current report presents two cases of HLH with presentations different from each other and previously reported cases. Case 1 was a 15-month-old boy with fever, skin rash, splenomegaly, and bicytopenia, raised triglyceride levels, AST (aspartate transaminase), and ALT , normal ferritin, and abundant hemophagocytic cell in bone marrow aspiration. He was diagnosed with HLH and received HLH protocol as treatment. The patient had a homozygous intronic mutation; NM_199242: c.2448-13G > A in UNC13D gene (coding and non-coding regions) is an applicable and valuable diagnostic procedure for the detection of deep intronic splicing variants and large inversions in patients with atypical manifestations of HLH .The sequencing of the entire  Hemophagocytic lymphohistiocytosis (HLH) is an aggressive and life-threatening disease that can be fatal . It is cPRF1, STX11, UNC13D, HPLH1, and STXBP2 are the most well-known genes of this type.Depending on the etiology, HLH can be divided into familial  and acquired  forms –6. FamilSH2D1A, XIAP) are the third reason. Nevertheless, other primary immunodeficiencies, including combined immunodeficiency, chronic granulomatous disease, autoinflammatory diseases, and antibody deficiencies, have been described to cause HLH [Mutations associated with restrained cytotoxic responses of cytotoxic T and NK cells to B cells infected by Epstein-Barr virus (EBV) inducing X-linked lymphoproliferative syndrome , supportive therapy, prophylactic antibiotics, and intravenous Ig (IVIG). Hematopoietic stem cell transplantation is recommended in selected patients with refractory and/or relapsed disease after appropriate chemoimmunotherapy –14. ThisT-cell-directed immunotherapy could be advantageous in patients with primary HLH. In contrast, strong immunosuppression therapy is contraindicated in patients with severe proceeding infections or some primary immunodeficiency diseases other than familial HLH and X-linked lymphoproliferative syndrome . In addiUNC13D mutation.The differentiation between the two mentioned types of HLH is sometimes tricky, particularly in the beginning; moreover, diagnosis at the right time is critical for initiating appropriate and indicated treatment. Therefore, we performed whole-exome sequencing on two separate patients with relatively different symptoms to find the same homozygous intronic In this study, we present the case of two children with rather different clinical symptoms hospitalized almost at the same time in two different centers affiliated with Tehran University of Medical Sciences. Their ages, symptoms, and signs were different, especially at the beginning; nonetheless, whole-exome sequencing was employed due to the uncertainty surrounding the underlying etiology. Written informed consent was obtained from their parents.3, anemia with hemoglobin of 10.8 g/dl, and thrombocytopenia with a platelet count of 106 *103/μl). Triglyceride (TG) level was initially 425 mg/dl, which increased to 644 mg/dl. The cholesterol level was initially 86 mg/dl, which increased to 182 mg/dl. The c-reactive protein (CRP) was high (73 mg/dl) with a normal erythrocyte sedimentation rate (ESR) at the same time (14 mm/h). Remarkably, the value of serum ferritin was normal throughout all his disease period (150 μg/l).The first case was a 15-month-old male infant born to consanguineous parents via normal vaginal delivery. He had normal growth and development until admission due to prolonged fever for 40 days with no specific pattern, vomiting, and maculopapular rash on his face. His physical examination revealed huge splenomegaly (diameter 120 mm) and fever. Liver function tests demonstrated a rise in aspartate aminotransferase (AST) of 88 IU/L and alanine aminotransferase (ALT) of 52 IU/L. His Complete blood count (CBC) showed bicytopenia  performed from the iliac crest appeared normal. He was diagnosed with macrophage activating syndrome in the context of systemic juvenile idiopathic arthritis by a consultant rheumatologist. He received intravenous methylprednisolone pulse therapy (30 mg/kg) and intravenous immune globulin (IVIG). He was discharged from the hospital with oral prednisolone at a dose of 1 mg/kg/day and readmitted with intermittent fever and vomiting after 45 days. He had bicytopenia, increased ALT and CRP during hospitalization, and his ferritin level was 248 μg/l. Antibody assessment against cytomegalovirus (CMV) and Epstein-Barr virus (EBV) displayed no abnormality.The patient underwent BMA again, and bone marrow revealed abundant hemophagocytic cells. He was treated with cyclosporine A, etoposide, and dexamethasone according to HLH protocol 2004. After 1 month of treatment, CBC turned normal; CRP and TG decreased. Upon the completion of induction therapy with HLH protocol and initiation of maintenance therapy, the patient experienced a disease flare-up and was readmitted with fever and status epilepticus. Laboratory data revealed pancytopenia and increased levels of TG, AST, and ALT again; however, ferritin level was in the normal range, and brain computed tomography (CT) scan showed ventriculomegaly and Mega Cisterna Magna (Table UNC13D (NM_199242: c.2448-13G > A), probably affecting splicing. Although Sanger sequencing is not a routine survey for parents, since this mutation is transferred from parents or is a de novo mutation for the assessment of the next child, it was performed and confirmed that his parents were heterozygous carriers for this mutation ) revealed a homozygous intronic UNC13D mutation.Furthermore, prenatal diagnosis with chorionic villus sampling was implemented in the 12th week of gestational age for the next child of this family and demonstrated that the fetus was only a heterozygous carrier of 3 and ANC of 210, anemia with hemoglobin of 5.1 g/dl, and thrombocytopenia with platelets of 41 *103/μl).A 37-day-old female neonate born to consanguineous parents via cesarean section was admitted with fever (Temperature: 39 °C), cough, and a history of neonatal cholestasis. Her physical examination revealed fever and huge hepatosplenomegaly (with spleen preference). She had normal growth before admission despite the previous admission for cholestasis. Her liver function tests resulted in an AST of 33 IU/L that increased to 98 IU/L and an ALT of 21 IU/L that increased to 391 IU/L in her admission process. Her CBC revealed pancytopenia (leukopenia with white blood cell (WBC) of 3.13 thousand/mmThe TG and cholesterol levels were 113 mg/dl and 76 mg/dl, retrospectively. Ferritin level was increased to 2050 μg/l during her hospitalization. The CRP was 28 mg/L, and ESR was in the normal range (16 mm/h). Her abdominal sonography exhibited splenomegaly with a diameter of 144 mm and hepatomegaly with a diameter of 105 mm (both of homogenous echogenicity). The BMA and bone marrow biopsy were performed from the proximal tibia and appeared normal.Her purified protein derivative (PPD) test turned negative, and a normal TORCH study  was detected. The HIV ab was also negative. Metabolic screening tests, including MS/MS, ammonia, lactate, serum amino acid chromatography by HPLC, and urine reducing substance, all were normal. Nevertheless, the evaluation of Galactosemia, Gaucher’s disease, and Niemann-Pick’s disease were not feasible due to the previous history of blood transfusion. Furthermore, fibrinogen was in the normal range.Pseudomonas aeruginosa. The whole-exome sequencing analysis detected a homozygous c.2448-13G > A UNC13D mutation, the same mutation observed in case 1. Sanger sequencing confirmed that his parents were also the heterozygous carriers of this mutation (data not shown). Sanger sequencing was performed since parents were young and desired to have a healthy child in the future.She was treated with prednisolone (4 mg/kg/day) and IVIG (2 g/kg Stat) as prescribed by a hematologist (with autoimmune thrombocytopenia and anemia diagnosis). Firstly, there was a rapid increase in platelet number and hemoglobin level. Nonetheless, the patient needed multiple blood transfusions and platelets due to sustained bicytopenia. Since the second case did not fulfill the criteria, she did not receive the 2004-HLH treatment protocol as suggested by a consultant rheumatologist. Finally, she died of severe sepsis by abdominal distention and positive blood culture with UNC13D gene, which encodes Munc13–4 protein, was reported as its underlying cause. This mutation results in defective cytotoxic granule exocytosis, followed by diminished cytotoxic activity of T lymphocytes [Familial hemophagocytic lymphohistiocytosis (FHL) is a challenging diagnosis due to the absence of any pathognomonic clinical and laboratory signs and symptoms. It is a rare autosomal recessive immune disease caused by the mutations of different genes associated with the formation and function of secretory lysosomes within cytotoxic T lymphocytes and NK cells. FHL type 3 (FHL3) accounts for approximately 30–40% of FHL, and mutation in the phocytes .UNC13D mutations may be responsible for a massive proportion of HLH patients in Asian countries [In their study, Yoon et al. reported that FHL3 accounts for 89% of FHL cases in Korea, and nearly 20–25% in Japan, suggesting that ountries . Functioountries .UNC13D gene are essential for the assessment but also introns and non-coding regions should be sequenced and analyzed for the detection of deep intronic splicing variants and large inversions in patients who have failed to show mutations in whole-exome sequencing [UNC13D mutation previously reported by Alsina et al. [UNC13D gene are frequent in FHL patients [The majority of mutations in the previously reported patients were discovered in deep intronic regions within intron 1 and 9, as well as an inversion in intron 30, advocating the fact that not only coding sequences of the quencing , 18. Thea et al. . Compounpatients . Nonethepatients .This case report confirms the literature claim that more than two-thirds of FHL3 patients suffer from fever and hepatosplenomegaly. Since our first case was presented with status epilepticus, neurological manifestations are also common among HLH patients, according to the literature. Liver failure, lymphadenopathy, consumptive coagulopathy, dermatologist manifestations, edema, and jaundice, like some other manifestations of the disease, were observed in our cases to some extent , 21. MorPseudomonas aeruginosa could trigger the incidence of HLH in the second patient.The FHL3 mostly happens in the first year of life (with a median age of 3 months); nonetheless, the first case was older than 1 year . FerritiThe solitary treatment for familial and severe HLH is bone marrow transplantation . Based oUNC13D gene (coding and non-coding regions) is an applicable and valuable diagnostic procedure for detecting deep intronic splicing variants and large inversions in patients. Genetic assessment can significantly help in the event of HLH disease with atypical manifestation or situations when primary evaluations cannot detect etiology.The FHL, as a challenging diagnosis, may have a wide diversity of clinical manifestations and laboratory findings. Sequencing the entire


Contents of HSP70, ALP, and Cortisol in serum were significantly increased, while TP, GLU, T3, and T4 were significantly decreased (p < 0.05). Nine kinds of differential metabolites were screened by serum metabolomics, which can be used as biomarkers of heat stress in meat rabbits. The selected differential metabolites were analyzed by KEGG annotation and enrichment analysis. The results showed that 14 pathways affected by heat stress were identified by KEGG pathway enrichment analysis, including Sphingolipid metabolism, Pyrimidine metabolism, Citrate cycle (TCA cycle)), aminoacyl-tRNA biosynthesis, and so on. The analysis of the effect of heat stress on the cecal microflora of meat rabbits showed that the abundance of cecal Proteus in the HT group was significantly higher than that in the moderate Control group. The number of Candidatus-saccharimonas in the cecum microflora was significantly higher than that in the moderate temperature group (p < 0.05) which may be related to inflammatory diseases in the heat stress group. These findings indicated that the heat-stressed rabbits were in negative energy balance, which affected protein metabolism, and subsequently affected growth performance and carcass characteristics.To investigate the effects of heat stress on meat rabbits, we assigned 80 rabbits to the moderate temperature group  and the continuous high-temperature group (HT group), then monitored the effects using growth performance, carcass characteristics, biochemical assays, UPLC–MS/MS-based metabolomics, and microbiome. The results showed that after continuous high-temperature exposure, the average daily gain, average daily feed intake, and thymus index were significantly decreased ( With the continuous acceleration of global warming and intensive breeding environment, heat stress has gradually become a global issue of common concern in modern livestock and poultry production, and heat stress is considered to be one of the most important stresses in livestock and poultry breeding. Heat stress refers to the sum of non-specific responses produced by animals when they are stimulated by excessive temperature beyond their thermoregulatory capacity . Heat stClimate warming is unequivocal, and will greatly impact animal health and growth, especially during the summer months, either directly or secondarily . Heat stThe present study aimed to characterize the serum metabolic profile of heat-stressed broilers and to investigate the change rule and related regulatory mechanisms using Ultra Performance Liquid Chromatography Tandem Mass Spectrometry (UPLC–MS/MS) metabolomics, biochemical validation, microbiomics, and multivariate data analysis. The study also aimed to elucidate the relationships between growth performance, serum biochemical and hormonal parameters, serum metabolism, and gut microflora changes in heat-stressed rabbits.In the experiment, eighty 35-day-old Hyla meat rabbits  were pre-fed at 24 ± 1°C for 7 days, and then sixty 42-day-old meat rabbits were randomly divided into two groups: moderate temperature group (24 ± 1°C) and continuous high-temperature group . The temperature and humidity were adjusted by the air conditioner and humidifier (ZDR-41). Hangzhou Zeda Instrument Co., Ltd.) automatically records room temperature every 30 min, the experimental period is 14 days . RabbitsRabbits in every replicate from each treatment group were weighed both on the transfer days  and at the end of the experiment . Average daily gain (ADG), Average daily feed intake (ADFI), Feed conversion ratio (FCR), Liver index, Spleen index, Thymus index, Kidney index, Shoulder fat rate, and Kidney fat rate were calculated according to the formula:ADG = body increase (g)/number of trial days.ADFI = (feed intake-leftover feed) /number of trial days.FCR = ADFI/ADG.Fully eviscerated slaughtering rate (%) = full eviscerated weight/live weight before slaughter * 100.Liver index (%) = liver weight/live weight before slaughter * 100.Spleen index (%) = spleen weight / live weight before slaughter * 100Thymus index (%) = thymus weight / live weight before slaughter * 100Kidney index (%) = kidney weight / live weight before slaughter * 100Shoulder fat rate (%) = shoulder fat weight/live weight before slaughter * 100.Kidney fat rate (%) = perirenal fat weight/live weight before slaughter * 100.Serum concentrations of total protein (TP), albumin (ALB), glucose (GLU), triglyceride (TG), cholesterol (CHO), urea (UREA), high-density lipoprotein (HDL), low-density lipoprotein (LDL), calcium (Ca), phosphorus (P), glutamic pyruvic transaminase (ALT), and glutamic-oxalacetic transaminase (AST) were measured using a Hitachi 7,020 automatic biochemical analyzer . Serum growth hormone (GH), thyroid-stimulating hormone (TSH), triiodothyronine (T3), and tetraiodothyronine (T4) were determined by the ELISA method with the corresponding ELISA kits .The serum samples were taken from the refrigerator at-80°C and thawed in the refrigerator at 4°C. Six samples were selected for each group. 100 and 400 μl pre-cooled 80% methanol containing 0.1% formic acid were mixed by rotation method, and the samples were incubated for 5 min on ice. Under the condition of 15,000 g 10 min at 4°C, the supernatant was injected into the sample by centrifugation and analyzed by LC–MS/MS system. The materials used for LC-MS/MS analyses were given in Microbial community genomic DNA was extracted from cecal contents samples using the E.Z.N.A.® soil DNA Kit  according to the manufacturer’s instructions. The materials used for DNA extraction, PCR amplification of 16S rRNA gene, and 16S rRNA gene sequencing reads were given in The DNA extract was checked on 1% agarose gel, and DNA concentration and purity were determined with NanoDrop 2000 UV–vis spectrophotometer . The hypervariable region V3-V4 of the bacterial 16S rRNA gene was amplified with primer pairs 338F (5′-ACTCCTACGGGAGGCAGCAG-3′) and 806R (5′-GGACTACHVGGGTWTCTAAT-3′) by an ABI GeneAmp® 9,700 PCR thermocycler .The PCR amplification of 16S rRNA gene was performed as follows: initial denaturation at 95°C for 3 min, followed by 27 cycles of denaturing at 95°C for 30 s, annealing at 55°C for 30 s and extension at 72°C for 45 s, and single extension at 72°C for 10 min, and end at 4°C. The PCR mixtures contain 5 × TransStart FastPfu buffer 4 μl, 2.5 mM dNTPs 2 μl, forward primer (5 μM) 0.8 μl, reverse primer (5 μM) 0.8 μl, TransStart FastPfu DNA Polymerase 0.4 μl, template DNA 10 ng, and finally ddH2O up to 20 μl. PCR reactions were performed in triplicate. The PCR product was extracted from 2% agarose gel and purified using the AxyPrep DNA Gel Extraction Kit  according to the manufacturer’s instructions and quantified using Quantus™ Fluorometer .Purified amplicons were pooled in equimolar and paired-end sequenced on an Illumina NovaSeq PE250 platform  according to the standard protocols by Majorbio Bio-Pharm Technology Co. Ltd. . The raw reads were deposited into the NCBI Sequence Read Archive (SRA) database.The raw 16S rRNA gene sequencing reads were demultiplexed, quality-filtered by fastp version 0.20.0 and merged by FLASH version 1.2.7 with the following criteria: (i) the 300 bp reads were truncated at any site receiving an average quality score of <20 over a 50 bp sliding window, and the truncated reads shorter than 50 bp were discarded, reads containing ambiguous characters were also discarded; (ii) only overlapping sequences longer than 10 bp were assembled according to their overlapped sequence. The maximum mismatch ratio of overlap region is 0.2. Reads that could not be assembled were discarded; (iii) Samples were distinguished according to the barcode and primers, and the sequence direction was adjusted, exact barcode matching, 2 nucleotide mismatch in primer matching.Operational taxonomic units (OTUs) with 97% similarity cutoff were clustered using UPARSE version 7.1, and chimeric sequences were identified and removed. The taxonomy of each OTU representative sequence was analyzed by RDP Classifier version 2.2 against the 16S rRNA database (eg. Silva v138) using the confidence threshold of 0.7.p-values were obtained, with p < 0.05 indicating significant differences, and results are presented as means ± SEs.The raw data were statistically processed using Excel and the data were analyzed using the One-Way ANOVA model in SAS 8.2 software and compared using Duncan’s multiple tests. p < 0.05.Statistical analyses were performed using the statistical software R (R version R-3.4.3), Python (Python version 2.7.6) and CentOS (CentOS version 6.6), and when the data were not normally distributed, they were transformed normally using the area normalization method. The metabolites were annotated using the KEGG databasehttps://github.com/OpenGene/fastp, version 0.20.0) software and FLASH  software for splicing. OTU clustering of sequences based on 97% similarity using UPARSE software . Each sequence was annotated for species classification using the RDP classifier , compared to the Silva 16S rRNA database (v132), and a comparison threshold of 70% was set.Quality control of raw sequenced sequences using fastp (p < 0.05). At the same time, the thymus index and liver index in the heat stress group were significantly lower than those in the control group (p < 0.05), while the shoulder fat rate and kidney fat rate in the heat stress group were significantly higher than those in the control group (p < 0.05).p < 0.05), while the contents of TP, GLU, T3 and T4 decreased significantly (p < 0.05), and other biochemical indexes did not change significantly.The effect of heat stress on serum biochemical indexes of meat rabbits is shown in Metabolome refers to a collection of small molecule compounds that are involved in the metabolism of organisms and maintain normal growth and development of organisms. These are mainly referred to as endogenous small molecules with relative molecular weight of less than 1,000. We used LC–MS/MS method combined with positive ion mode (POS) and negative ion mode (NEG) for serum metabolite analysis, making metabolite coverage higher and the detection effect better. In POS mode, a total of 5,456 peaks were obtained, and 343 substances of these were identified while the number of metabolites annotated to the public database was 399. In NEG mode, a total of 4,809 peaks were obtained, and 496 substances of these were identified and 464 were annotated to the public database see .According to the expression of metabolites in different samples, PCA principal component analysis was carried out to evaluate the similarity of samples within groups and the differences between samples, and PLS-DA analysis was carried out. The results are shown in Multidimensional statistical analysis of the identified metabolites was carried out by PCA, PLS-DA and OPLS-DA statistical analysis  to screeMultidimensional statistical analysis showed that VIP > 1 and FC > 1.2 or FC < 0.83 were used as screening criteria for differential metabolites. A total of 126 differential metabolites were screened, including 48 up-regulated differential metabolites and 78 down-regulated differential metabolites, while there are 9 different metabolites with specific names  taxonomic database, and the number of OTU of each sample was obtained. A total of 1,113 different OTU, were obtained from the two groups of samples. The Venn map  showed tThe α diversity of the samples was analyzed by Chao1, Shannon, and Simpson by T-tests. The results show that there is no significant difference between each index in As shown in p < 0.05). Further refinement of the taxonomy to genus level, Muribaculaceae unnamed genera, Ackermania are the dominant genera. At the species level, the abundance of akkermansia_muciniphila increased. The results of the T-test in the cecum of heat-stressed meat rabbits showed that the number of Lachnospiraceae-UGG-010 in the intestines of heat-stressed meat rabbits was significantly lower than that in the normal temperature group, while the unnamed genera of Lachnospiraceae, Ruminococcaceae and Candidatus-saccharimonas in the heat-stressed meat rabbits were significantly higher than those in the cecum samples of the heat-stressed meat rabbits.As shown in LEfSe (LDA Effect Size) analysis showed that the genera of Spirillaceae and Rumen Fungi played an important role in the heat stress group, while Colidextribacter, Eubacterium_nodatum_group and Anaeroplasma played an important role in the moderate temperature group, and the heat stress group played an important role in the heat stress group , while iIn recent years, the environmental problem has become an important factor affecting the development of the rabbit industry, especially the temperature problem. Studies have pointed out that meat rabbits exposed to high temperatures for a long time will inhibit the excitability of their feeding centers and decrease their food intake, resulting in insufficient supply of nutrients in the body. Heat stress reduces feed utilization and body weight gain by reducing feed intake and nutrient metabolism, which affects the production performance of meat rabbits. This is a typical manifestation of heat stress. In this study, it was found that under the condition of continuous high temperature, the average daily gain and average daily feed intake of meat rabbits decreased significantly. This is consistent with the results of previous studies . A high-The changes in serum biochemical and hormonal parameters are important characteristics that reflect the changes in material metabolism and functional state of tissues and organs during heat stress, and it is found that serum biochemical indexes are related to productivity. HSP70 plays an important role in protecting antioxidant cells and reducing inflammation in animals . It is tThe results showed that when the ambient temperature was 34°C, the contents of T3 and T4 in the serum of meat rabbits decreased significantly, while the content of cortisol increased significantly . ThyroidIn the state of heat stress, the body needs to use reserves to deal with heat stress, leading to metabolic disorders or the re-establishment of homeostasis. In this study, 39 kinds of differential metabolites were screened by multidimensional statistical analysis, which can effectively distinguish meat rabbits from the heat stress group and moderate temperature group. These potential biomarkers are involved in the phospholipid metabolism pathway, glyoxylic acid, and dicarboxylic acid metabolism, pyrimidine metabolism pathway, tricarboxylic acid cycle, nitrogen metabolism and other pathways, involving glucose metabolism, lipid metabolism, nucleic acid metabolism, amino acid metabolism, energy metabolism and translation process. The results of heat stress are consistent with those of broilers  and miceSphingosine and sphingosine-1-phosphate, which are involved in the pathway of phospholipid metabolism, were significantly down-regulated under heat stress. Sphingosine, also known as sphingosine, is one of the components of the cell membrane, while sphingosine-1-phosphate. Sphingosine-1-phosphate is a powerful signaling lipid molecule, which can regulate cell proliferation, regeneration, migration and intracellular calcium movement, expression of adhesion molecules and activation of monocytes adhering to endothelial cells . Heat stWhen the animals are in a healthy state, the species and number of intestinal microorganisms are relatively balanced, and there is a balance between the intestinal microflora host and the intestinal microenvironment. However, many external factors can break this balance, and it has been reported that diet, age and environmental factors can affect the intestinal flora structure of meat rabbits . At presThe dominant bacteria in the cecum were Ackermann (AKK) and Muri. Akk is a strictly anaerobic, non-mobile, non-spore-producing oval Gram-negative bacteria that can grow in the intestinal mucus layer and grow well using gastrointestinal mucin as the only carbon and nitrogen source. It can grow individually or in pairs, and can also grow in clusters in a medium containing mucin, thus settling in the intestines by competitive rejection and protecting the intestines from pathogens . AlthougIn summary,combined physiological, UHPLC–MS/MS-based metabolomics, and 16S rRNA microbiome analyses were performed to investigate metabolic and gut microbiota responses in the meat rabbits to continuous high-temperature exposure for 14 days. The results revealed that heat stress significantly reduced the production performance of meat rabbits and destroyed the blood biochemical balance of meat rabbits. There were 9 kinds of differential metabolites could be used as biomarkers of heat stress in meat rabbits. Heat stress may affect glucose and fat metabolism in meat rabbits by modulating the gut microflora. These investigations provide new insights into the metabolism mechanisms of meat rabbits to heat stress and contribute to a better understanding of the adverse effects of heat stress on rabbits.https://www.ncbi.nlm.nih.gov/, BioProject PRJNA899341 with corresponding BioSample Accessions SAMN31652990-SAMN31653001.The datasets presented in this study can be found in the All study procedures were approved by the Shandong Agricultural University Animal Care and Use Committee (SDAUA-2017-057) and were following the Guidelines for Experimental Animals established by the Ministry of Science and Technology .HL and FL planned the experiment and finalized the draft of the manuscript with consent from all contributing authors. HL, BZ, and FL carried out the experiment, finalized, and analyzed the collected data. TY, HZ, and LL assisted with manuscript editing and language calibration. All authors contributed to the article and approved the submitted version.This work was supported by China Agriculture Research System of MOF and MARA (CARS-43-B-1), Taishan Industry Leadership Project (TSCY20190107), National Natural Science Foundation of China (31972594), and Funds of Shandong “Double Tops” Program.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.