Pathogenic variants identified using whole-exome sequencing in Chinese patients with primary ciliary dyskinesia.

The genetic factors contributing to primary ciliary dyskinesia (PCD), a rare autosomal recessive disorder, remain elusive for ~20%-35% of patients with complex and abnormal clinical phenotypes. Our study aimed to identify causative variants of PCD-associated pathogenic candidate genes using whole-exome sequencing (WES). All patients were diagnosed with PCD based on clinical phenotype or transmission electron microscopy images of cilia. WES and bioinformatic analysis were then conducted on patients with PCD. Identified candidate variants were validated by Sanger sequencing. Pathogenicity of candidate variants was then evaluated using in silico software and the American College of Medical Genetics and Genomics (ACMG) database. In total, 13 rare variants were identified in patients with PCD, among which were three homozygous causative variants (including one splicing variant) in the PCD-associated genes CCDC40 and DNAI1. Moreover, two stop-gain heterozygous variants of DNAAF3 and DNAH1 were classified as pathogenic variants based on the ACMG criteria. This study identified novel potential pathogenic genetic factors associated with PCD. Noteworthy, the patients with PCD carried multiple rare causative gene variants, thereby suggesting that known causative genes along with other functional genes should be considered for such heterogeneous genetic disorders.

Rapid and durable response to fifth-line lorlatinib plus olaparib in an ALK -rearranged, BRCA2-mutated metastatic lung adenocarcinoma patient with critical tracheal stenosis: a case report.

Treatment options for heavily treated anaplastic lymphona kinase (ALK )-positive nonsmall cell lung cancer (NSCLC) patients, who typically bear-resistant mechanisms to ALK tyrosine kinase inhibitors (TKIs), are usually limited to chemotherapy, which elicits limited clinical benefit and may incur severe toxicity. It is clinically relevant to explore other revenues for these patients. poly (ADP-ribose) polymerase (PARP) inhibitors, such as olaparib are currently approved to treat BReast CAncer gene 1/2 ( BRCA1/2 )-mutated patients in a few tumor types. There have been a trial and two case reports of an olaparib-containing regimen in treating epidermal growth factor receptor (EGFR)-positive or driver-negative NSCLC. We report a case of a 27-year-old female nonsmoker diagnosed with ALK -rearranged metastatic lung adenocarcinoma. She was treated with alectinib and acquired ALK p.I1171N and p.V1180L mutations. Germline BRCA2 p.F2801fs was also identified. After sequential lines of ceritinib and chemotherapy, lorlatinib was chosen as the fourth-line therapy and maintained control for 6 months. Shortly after progression, the patient was admitted to the ICU due to critically severe stenosis caused by a tracheal mass and soon relieved by embolization and stenting. Afterward lorlatinib plus olaparib was started and elicited a rapid response within 1 month. The progression-free survival was 6 months as of the latest follow-up, with the best response of partial response. To the best of our knowledge, this case is the first to provide clinical evidence of antitumor activity of olaparib plus ALK TKI in ALK -positive, g BRCA -mutated metastatic NSCLC. Together with previous reports in EGFR -positive or driver-negative patients, our finding warrants further studies on PARP inhibition in BRCA1/2 -mutated NSCLC.

Pathogenic variants of PROC gene caused type I activity deficiency in a familial Chinese venous thrombosis.

Pathogenic mutation of protein C (PROC) gene results into the deficiency of PROC activity. This study aimed to identify the pathogenic genetic variants and to explore the functional consequence in Chinese familial venous thrombosis (VTE). Whole exome sequencing was performed to identify the pathogenic variants of anticoagulant factors. Serum coagulation and anti-coagulation factors activity were assayed to evaluate the genetic association. Functional study of PROC antigen secretion deficiency was conducted in VTE subjects and in vitro cell lines. One rare pathogenic variant (p.Ala178Pro) was identified in the four VTE subjects but not in the normal subjects from the family. An inframeshift variant (rs199469469) was also identified in a paediatric subject of the pedigree. Further evaluation of serum PROC activity levels in p.Ala178Pro variants VTE carriers showed significantly lower PROC activity compared to non-carriers. Furthermore, in vitro study showed that the p.Ala178Pro mutant cells had a consistent reduction in concentration of PROC antigen. In conclusions, our study demonstrated the pathogenic variant (p.Ala178Pro) contributed to PROC type I activity deficiency, which may be due to decreased secretion of PROC.

iTRAQ-based proteomic analysis of human umbilical vein endothelial cells with platelet endothelial aggregation receptor-1 knockdown.

The disorders of hemostasis and coagulation were believed to be the main contributors to the pathogenesis of pulmonary thromboembolism (PTE), and platelets are the basic factors regulating hemostasis and coagulation and play important roles in the process of thrombosis. This study investigated the proteome of human umbilical vein endothelial cells (HUVECs) with platelet endothelial aggregation receptor-1 (PEAR1) knockdown using the isobaric tags for relative and absolute quantitation (iTRAQ) method and analyzed the role of differential abundance proteins (DAPs) in the regulation of platelets aggregation. Our results showed that the conditioned media-culturing HUVECs with PEAR1 knockdown partially suppressed the adenosine diphosphate (ADP)-induced platelet aggregation. The proteomics analysis was performed by using the iTRAQ technique, and a total of 215 DAPs (124 protein was upregulated and 91 protein were downregulated) were identified. The Gene Ontology (GO) enrichment analysis showed that proteins related to platelet α granule, adenosine triphosphate metabolic process, and endocytosis were significantly enriched. Further, the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis also identified the significant enrichment of endocytosis-related pathways. The real-time polymerase chain reaction assay confirmed that the expression of P2Y12 , mitochondrial carrier 2, NADH dehydrogenase (ubiquinone) iron-sulfur protein 3, and ubiquinol-cytochrome c reductase hinge protein are significantly downregulated in the HUVECs with PEAR1 knockdown. In conclusion, our in vitro results implicated that DAPs induced by PEAR1 knockdown might contribute to the platelet aggregation. Proteomic studies by employing GO enrichment and KEGG pathway analysis suggested that the potential effects of DAPs on platelet aggregation may be linked to the balance of ADP synthesis or degradation in mitochondria.

A deficient or an excess of dietary threonine level affects intestinal mucosal integrity and barrier function in broiler chickens.

The aim of this study was to investigate the effects of deficient or excess of dietary threonine (Thr) levels on intestinal integrity and barrier function of broilers. A total of 432 1-day-old commercial broilers (Arbor Acre) were assigned to four experiment groups consisting of six replicates of 18 birds. The treatments were designed as follows: 85%, 100%, 125% and 150% of NRC (Nutrient requirements of poultry (9th edn). Washington, DC: The National Academies Press, 1994) recommendations. The results indicated that expressions of jejunal and ileal secretory immunoglobulin A (sIgA) mRNA were increased linearly or quadratically by increasing Thr (p < .05), and the highest sIgA mRNA abundance was obtained in 125% Thr level. Likewise, the intestinal sIgA content showed similar increasing trend with the intestinal sIgA gene expression in this instance. The high level of Thr inclusion upregulated mucin 2 (MUC2) mRNA expression in the jejunum and ileum (p < .05). In addition, on day 21, the expression levels of jejunal zonula occludens-2 (ZO-2) and ileal zonula occludens-1 (ZO-1) decreased then increased with increasing Thr level (p < .05), whereas, the mRNA expressions of occludin in the jejunum and ileum had no significant difference amongst groups (p >.05). On day 42, Thr treatments did not affect the mRNA abundance of measured genes in the jejunum and ileum (p > .05). These findings suggested that Thr might be a nutrient immunomodulator that affects intestinal barrier function, moreover, 125% of the NRC (1994) recommendations Thr level was optimum.

Association of SERPINC1 Gene Polymorphism (rs2227589) With Pulmonary Embolism Risk in a Chinese Population.

Background and Aims: Genetic variants in the gene SERPINC1 have been shown to be associated with antithrombin deficiency, which subsequently contributes to the susceptibility to venous thrombosis. However, several other studies have shown conflicting results regarding the association of SERPINC1 gene polymorphisms (rs2227589) with the risk of thrombosis. Hence, in the present study, we conducted a case-control study to further evaluate the association between the variant rs2227589 with antithrombin deficiency in pulmonary embolism (PTE). A pooled systematic analysis was also conducted to evaluate the risk of rs2227589 in venous thromboembolism (VTE) among multiple populations. Methods: This case-control study involved 101 patients and 199 healthy controls. The allele frequency of SERPINC1 variant rs2227589 was analyzed by Sequenom assay. Antithrombin anticoagulant activity was detected using an automatic coagulation analyzer. In addition, a pooled systematic analysis on 10 cohorts consisting of 5,518 patients with VTE and 8,935 controls was performed. Results: In total, 27 (26.7%) PTE subjects were diagnosed as having antithrombin deficiency. Our results showed that antithrombin plasma activity was slightly lower in T allele carriers than that in C allele carriers. However, there was no significant correlation between rs2227589 genotype and antithrombin anticoagulant activity. The recessive model showed that rs2227589 was significantly associated (p = 0.026) with an increased risk {odds ratio [OR]: 2.31, 95% confidence interval [CI] (1.09-4.89)} of Chinese PTE. The pooled systematic analysis of all case-control study and meta-analysis showed that rs2227589 polymorphism was associated with an increased risk of VTE in the additive model [OR: 1.09, 95% CI (1.01-1.18), P = 0.029] and dominant model [OR: 1.10, 95% CI (1.01-1.20), P = 0.034]. Conclusions: Our study demonstrated that variant rs2227589 is associated with an increased risk of PTE in a Chinese population but no correlation with antithrombin anticoagulant activity. However, pooled systematic analysis of multiple populations showed a significant association between rs2227589 and the risk of VTE in the additive and dominant genetic model.

Identification of Pathogenic Mutations and Investigation of the NOTCH Pathway Activation in Kartagener Syndrome.

Primary ciliary dyskinesia (PCD), a rare genetic disorder, is mostly caused by defects in more than 40 known cilia structure-related genes. However, in approximately 20-35% of patients, it is caused by unknown genetic factors, and the inherited pathogenic factors are difficult to confirm. Kartagener syndrome (KTS) is a subtype of PCD associated with situs inversus, presenting more complex genetic heterogeneity. The aim of this study was to identify pathogenic mutations of candidate genes in Chinese patients with KTS and investigate the activation of the heterotaxy-related NOTCH pathway. Whole-exome sequencing was conducted in five patients with KTS. Pathogenic variants were identified using bioinformatics analysis. Candidate variants were validated by Sanger sequencing. The expression of the NOTCH pathway target genes was detected in patients with KTS. We identified 10 KTS-associated variants in six causative genes, namely, CCDC40, DNAH1, DNAH5, DNAH11, DNAI1, and LRRC6. Only one homozygote mutation was identified in LRRC6 (c.64dupT). Compound heterozygous mutations were found in DNAH1 and DNAH5. Six novel mutations were identified in four genes. Further analyses showed that the NOTCH pathway might be activated in patients with KTS. Overall, our study showed that compound heterozygous mutations widely exist in Chinese KTS patients. Our results demonstrated that the activation of the NOTCH pathway might play a role in the situs inversus pathogenicity of KTS. These findings highlight that Kartagener syndrome might be a complex genetic heterogeneous disorder mediated by heterozygous mutations in multiple PCD- or cilia-related genes.

Diagnostic value of platelet-derived microparticles in pulmonary thromboembolism: A population-based study.

An early and accurate diagnosis of pulmonary thromboembolism (PTE) remains challenging. The present study aimed to evaluate the diagnostic value of platelet-derived microparticles in PTE based on a population study. A total of 102 patients with PTE, 102 healthy controls and 40 patients suspected with PTE were enrolled in this study. The platelet count, mean platelet volume and platelet distribution width were assessed using an automated hematology analyzer, P-selectin was assessed using an ELISA kit and PMPs were explored using flow cytometry using Megamix beads. Receiver operating characteristic curves were established to evaluate the diagnostic values of PMPs, D-dimer, PMPs combined with D-dimer, and multiple parameters (including PMPs, platelet distribution width, P-selectin and D-dimer in PTE). The PMP levels were significantly higher in the patients with PTE (609.10/µl) compared with those in the healthy controls (230.60/µl) and patients with suspicious PTE (166.70/µl; P<0.01). The accuracy (72.06%) of PMPs in the diagnosis of PTE was similar to those of D-dimer (P>0.05). The combination of D-dimer and PMPs significantly increased the sensitivity (86.27%) of D-dimer and the specificity of PMP for the diagnosis of PTE (P<0.01). The combination of PMPs, platelet distribution width, P-selectin and D-dimer exhibited high sensitivity (88.24%), specificity (91.18%) and accuracy (89.71%) in the diagnosis of PTE. These findings suggest that elevated PMP levels are an effective predictor of PTE. The combination of PMPs, platelet distribution width, P-selectin and D-dimer may be used in the diagnosis of PTE with high sensitivity and specificity.

Immunology repertoire study of pulmonary sarcoidosis T cells in CD4+, CD8+ PBMC and tissue.

Sarcoidosis is a systemic granulomatous disorder highly related with immune response. The diversity and stability of the immune system could be measured by hypervariable complementarity-determining region 3 (CDR3) segments of the T cell receptor (TCR). Here we used a combination of multiplex PCR and next-generation sequencing to conduct a good quality analysis of the T-cell receptor BV complementarity-determining region 3 (TCR BV CDR3) gene in peripheral blood mononuclear cells (PBMCs) from 7 sarcoidosis patients and lung sarcoidosis tissue from 6 patients. The length distribution of CDR3 sequences identified a significant difference among CD4+, CD8+ and tissue samples. The analysis of Gini coefficient, Shannon entropy and HEC number showed that they all presents in sarcoidosis tissue group clones in a more skewed manner than that of in PMBCs groups. 2 nucleotide sequences and 2 amino acid sequences were shared by all samples. The comparison of TRBV, TRBJ usage and VJ combination frequency identified 2 TRBV genes, 2 TRBJ genes differentially expressed among different groups and different higher usage and lower usage of V-J combinations between each group.

PEAR1 gene polymorphism in a Chinese pedigree with pulmonary thromboembolism.

To explore the correlation between platelet endothelial aggregation receptor-1 (PEAR1) genetic polymorphism and pulmonary thromboembolism (PTE).Variant loci of the PEAR1 gene were screened in a PTE pedigree, followed by verification using Sanger sequencing. These polymorphic loci were validated in 101 PTE patients and 132 matched normal patients using MassARRAY single nucleotide polymorphism (SNP) genotyping methods. The frequency differences between the allele and genotypes were compared using the Hardy-Weinberg equilibrium test and Chi-square test. The correlation between the PEAR1 gene SNP and PTE was analyzed by comparing the between-group variance differences using the χ test.Three SNPs were identified in the PTE pedigree. There was a heterozygous transition of T>C in rs1952294, and a transition of C>T in rs778026543 in 2 members in the pedigree; however, the rs778026543 was not identified in the 101 PTE patients and 132 healthy controls. The genotype and allele frequencies of rs822442 did not differ significantly between PTE patients and healthy controls (P > 0.05). The variance difference at rs778026543 between pedigree members and healthy controls was significant (P < 0.001), supporting its potential heredity.The PEAR1 polymorphism, rs778026543, but not rs1952294 and rs822442, may be a susceptibility SNP for PTE.

[Life history characteristics of different genotype clones of Brachionus calyciflorus (Rotifera) population in Jinghu Lake].

By means of life table demographic approach, the life-table demographic parameters and the percentage of mictic females in the offspring of thirteen genetically different Brachionus calyciflorus clones were studied at 15 degrees C, 20 degrees C, 25 degrees C and 30 degrees C. The results showed that the responses of the generation time, average lifespan, life expectancy at hatching, intrinsic rate of population increase, net reproductive rate, and percentage of mictic females in the offspring of B. calyciflorus to increasing temperature differed with rotifer clones. Temperature, clone, and their interaction influenced the life-table demographic parameters and the percentage of mictic females in the offspring of B. calyciflorus significantly. The temporal heterogeneity of water temperature could be one of the reasons for the high genetic diversity in B. calyciflorus population in Jinghu Lake. Natural selection occurred in coexisting clones, and its function might be too weak or too time-limited to make the clonal exclusion occur. Genetic drift might play an important role in genotype frequency variation. The coexistence of different genotype clones of B. calyciflorus population is of significance for the existence of the population in water environment.