[A case of pleural metastasis of malignant peripheral nerve sheath tumor].

Malignant peripheral nerve sheath tumor (MPNST) frequently metastasizes to the lungs, although pleural metastasis is rare. This article reported a case of pleural metastasis of MPNST. The patient was a young man who presented with 1 week of shortness of breath with dry cough. He had a history of malignant peripheral nerve sheath tumor. The patient was diagnosed with MPNST pleural metastasis after a thoracoscopic pleural biopsy, which revealed short spindle cell hyperplasia, immunohistochemical staining for S-100(+), SOX-10(+), Ki-67(+) with a positive index of 20%, and H3K27Me3(-) in the pleural pathology.

[Familial interstitial lung disease associated with surfactant protein C gene mutation in adults: report of two cases and literature review].

Objective: To improve the understanding of clinical manifestations, imaging findings, diagnosis and treatment of surfactant protein C gene (SFTPC) mutation associated with familial interstitial lung disease in adults. Methods: Two cases of adult SFTPC gene mutation associated with familial interstitial lung disease diagnosed in the Affiliated Hospital of Medical School of Ningbo University were analyzed retrospectively, and the literature was reviewed. The literatures were retrieved with "family interstitial lung disease" "SFTPC gene" "surface protein C gene" "SFTPC gene mutation associated with familial international lung disease" and "surface protein C gene mutation associated with familial international lung disease" in PubMed, Embase, Ovid, Wanfang database and China National Knowledge Infrastructure (CNKI). Results: There were two patients with familial interstitial lung diseases(one male and one female) with an average age of 27.5 years. Ⅱ-2 patient had symptoms of dry cough and shortness of breath, and Ⅱ-1 patient had no symptoms. There were multiple cysts and fine reticular shadows in both cases. Ⅱ-2 patient had multiple ground glass opacities in both lower lungs. TheⅡ-2 patient was diagnosed with usual interstitial pneumonia (UIP) by transbronchial lung cryobiopsy. A total of 35 patients were included in this literature review, including 20 males, with an average age of 33.5 years. Of all the patients, the clinical symptoms were described in 30 patients. The main manifestations were shortness of breath (22/30), dry cough (18/30), clubbing finger (12/30), and 30% (9/30) of them were found by chest computerized tomography (CT) without symptoms. There were 17 cases with detailed description of chest CT imaging. The most common chest CT findings were multiple intralobular reticular opacities (17/17), multiple cysts (12/17) and ground glass opacities (7/17). The main histopathological pattern was UIP (24/26). Conclusions: The main clinical manifestations of SFTPC gene mutation associated with familial interstitial lung disease in adults are shortness of breath, dry cough and clubbing fingers. The main manifestations are multiple cysts and intralobular reticular opacities in combination with multiple ground glass opacities. There is no specific drug in the treatment at present and early treatment with hydroxychloroquine may have better curative effect. When the imaging findings show multiple cysts and intralobular reticular opacities in combination with multiple ground glass opacities, especially the age of onset is less than 50 years old, this disease should be considered.

[Clinical features and survival analysis of microscopic polyangiitis-associated interstitial lung disease:a retrospective study of 28 patients].

Objective: To explore the clinical features, laboratory examination and imaging features of microscopic polyangiitis (MPA)-associated interstitial lung disease (ILD), and to perform survival analysis. Methods: The records of 28 patients with MPA-ILD who were treated at the Affiliated Hospital of Medical School of Ningbo University were reviewed retrospectively from August 2014 to November 2021. The patients' clinical features, laboratory parameters, pulmonary function test, echocardiography, chest CT scan findings and therapeutic regimen were analyzed, and the relevant data were statistically analyzed. Results: There were 18 males and 10 females, with an average age of (70.1±9.3) years. Among them, 13 patients had a history of smoking. The main clinical manifestations were cough (14/28), fever (12/28), chest tightness, shortness of breath (12/28) and hemoptysis (3/28). Sixteen patients had renal involvement, and 78.57% (22/28) and 89.28% (25/28) of the patients had elevated C-reactive protein (CRP) and ESR respectively. Sixteen (16/28) patients had increased rheumatoid factor (RF), and the positive rate of myeloperoxidase antineutrophil cytoplasmic antibodies (MPO-ANCA) was 82.12% (23/28). 96.43% (27/28) of ILDs were diagnosed before or at the same time as MPA. The chest radiological pattern was mainly usual interstitial pneumonia (UIP) or UIP-like (15/28), followed by nonspecific interstitial pneumonia (NSIP) (8/28). Compared with non-UIP-like patients, UIP or UIP-like patients were older (P=0.018), and had higher serum LDH level (P=0.041), but serum creatinine level was significantly lower (P=0.041). Univariate and multivariate survival analysis showed that inappropriate treatment (HR=9.81, 95%CI: 1.68-57.29, P=0.011) and elevated serum LDH (HR=4.11, 95%CI: 0.99-17.00, P=0.051) were independent risk factors for shortened survival of MPA-ILD, while elevated RF (HR=0.22, 95%CI: 0.06-0.91, P=0.037) was a protective factor for prolonged survival. Conclusions: MPA-ILD patients had fewer systemic vasculitis symptoms. Most of the ILD patients were diagnosed before or at the same time as MPA. The chest radiological pattern was mainly UIP or UIP-like, followed by NSIP. Early use of glucocorticoids combined with immunosuppressant or rituximab could improve the survival rate of MPA-ILD. The elevated serum LDH was an independent risk factor for shortened survival of MPA-ILD, while elevated RF was a protective factor for prolonged survival.

CircRNA_103801 accelerates proliferation of osteosarcoma cells by sponging miR-338-3p and regulating HIF-1/Rap1/PI3K-Akt pathway.

This study aimed to investigate the roles of hsa_circRNA_103801 in the progression of osteosarcoma (OS) cells. Quantitative reverse transcription-polymerase chain reaction (RT-qPCR) was used to detect the expression level of circRNA_103801 in OS cells. Cell count kit-8 and Transwell migration and invasion assays were employed to detect the proliferation, migration, and invasion abilities of OS cells. The effects of circRNA_103801 on the apoptosis of OS cells were identified by flow cytometry. The binding relationship between circRNA_103801 and miR-338-3p was verified by bioinformatics analysis. MiR-338-3p level in OS cell lines was detected by RT-qPCR. Additionally, Western blotting was utilized to detect the expression levels of HIF-1, Rap1, PI3K, and Akt in OS cells. The results showed that the expression level of circRNA_103801 was significantly up-regulated in OS patients' tissues. Inhibiting the expression level of circRNA_103801 could attenuate the proliferation, migration, and invasion abilities of OS cells. In addition, the down-regulated expression level of circRNA_103801 could induce cell apoptosis. The results of the luciferase reporter assay suggested that circRNA_103801 could be combined with miR-338-3p, and the RT-qPCR revealed that the miR-338-3p level in OS cells after knockdown of circRNA_103801 was elevated compared with the control group. The results of Western blotting suggested that the expression levels of HIF-1, Rap1, PI3K, and Akt were elevated in OS cells. In conclusion, the circRNA_103801-miR-3388-3p-HIF-1/Rap1/PI3K-Akt pathway could be a therapeutic target of OS.

Identification of Differentially Expressed Genes and Pathways for Myofiber Characteristics in Soleus Muscles between Chicken Breeds Differing in Meat Quality.

In the modern chicken industry, fast-growing broilers have undergone strong artificial selection for muscle growth, which has led to remarkable phenotypic variations compared with slow-growing chickens. However, the molecular mechanism underlying these phenotypes differences remains unknown. In this study, a systematic identification of candidate genes and new pathways related to myofiber development and composition in chicken Soleus muscle (SOL) has been made using gene expression profiles of two distinct breeds: Qingyuan partridge (QY), a slow-growing Chinese breed possessing high meat quality and Cobb 500 (CB), a commercial fast-growing broiler line. Agilent cDNA microarray analyses were conducted to determine gene expression profiles of soleus muscle sampled at sexual maturity age of QY (112 d) and CB (42 d). The 1318 genes with at least 2-fold differences were identified (P < 0.05, FDR <0.05, FC ≥ 2) in SOL muscles of QY and CB chickens. Differentially expressed genes (DEGs) related to muscle development, energy metabolism or lipid metabolism processes were examined further in each breed based on Gene Ontology (GO) analysis, and 11 genes involved in these processes were selected for further validation studies by qRT-PCR. In addition, based on KEGG pathway analysis of DEGs in both QY and CB chickens, it was found that in addition to pathways affecting myogenic fibre-type development and differentiation (pathways for Hedgehog & Calcium signaling), energy metabolism (Phosphatidylinositol signaling system, VEGF signaling pathway, Purine metabolism, Pyrimidine metabolism) were also enriched and might form a network with pathways related to muscle metabolism to influence the development of myofibers. This study is the first stage in the understanding of molecular mechanisms underlying variations in poultry meat quality. Large scale analyses are now required to validate the role of the genes identified and ultimately to find molecular markers that can be used for selection or to optimize rearing practices.

[Anomalous systemic arterial supply to normal basal segments of the lower lobe: three cases reports and literature review].

Objective: To improve the clinical recognition of anomalous systemic arterial supply to normal basal segments of the lower lobe in clinical manifestations, diagnosis and treatment. Methods: Three cases were presented and related literatures were reviewed. A literature review was performed with"anomalous systemic arterial supply to normal lower lobe of the lung","anomalous systemic arterial supply to normal basal segments of the lower lobe"and"anomalous systemic arterial supply to normal basal segments of the left lower lobe of the lung"as key words in Pubmed, Embase, Ovid, Wanfang database and CNKI. Result: Our 3 cases were male, with an average age of 36 years old; all of them were admitted with hemoptysis. Left lower lobectomy was performed in 2 cases, and the other 1 case underwent endovascular embolization. 26 related articles were retrieved and our 3 cases were included in this study with a total of 57 cases. The ratio of male to female was 2∶1 (38∶19), with an average age of about 35 years old. The most common symptom was hemoptysis (26/57), followed by asymptomatic (22/57). The main treatments included left lower lobectomy (17/47) and endovascular embolization of anomalous systemic artery (13/47). Conclusions: This disease is more common in male, and the most common symptom is hemoptysis. When chest CT scan shows a nodular retrocardiac density with hemoptysis symptom, clinicians and radiologists should raise suspicion of anomalous systemic arterial supply to normal basal segments of the lower lobe. Chest contrast-enhanced CT scan is an appropriate imaging method to confirm diagnosis. The main treatments include left lower lobectomy and endovascular embolization. For asymptomatic patient, observation may be an acceptable option.

[A study of the diagnostic value of endobronchial ultrasound guide sheath transbronchial lung biopsy combined with virtual bronchoscopic navigation in peripheral pulmonary lesions].

OBJECTIVE: To evaluate the diagnostic value of endobronchial ultrasound guide sheath transbronchial lung biopsy (EBUS-GS-TBLB) combined with virtual bronchoscopic navigation (VBN) in peripheral pulmonary lesions (PPLs). METHODS: Cases with a PPL identified by computed tomography in Affiliated Hospital of Medical College of Ningbo University underwent EBUS-GS-TBLB with or without VBN randomly between Nov. 2014 to Aug. 2015. X-ray guidance was not performed in these cases. The sensitivity and the operation time were evaluated in the 2 groups. RESULTS: A total of 184 patients were enrolled and completed this study. Among them 117 were males and 67 were females. There were 93 cases in the group of EBUS-GS-TBLB with VBN, and 91 in the group without VBN. The diagnostic sensitivity of VBN group was 72.04%(67/93). Among these positive cases, 64.1% cases (43/67) were malignant tumors, and 35.9% cases (24/67) were benign lesions. The mean operation time was (45±10)min. In the group without VBN, the diagnostic sensitivity was 69.23%(63/91), including 33 malignant tumors(52.4%, 33/63), and 30 benign lesions(47.6%, 30/63). The mean operation time was (55±10)min. There was no significant difference between EBUS-GS-TBLB with VBN group and EBUS-GS-TBLB without VBN group in diagnostic sensitivity (χ(2)=0.175, P=0.747). But there was a significant difference in the mean operation time between the 2 groups (t=6.522, P<0.001). EBUS-GS-TBLB was well tolerated. No severe procedure-related complications such as pneumothorax and hemoptysis were observed. CONCLUSION: VBN cannot improve the diagnostic sensitivity, but it can clear the location of lesion, and shorten the operation time. This technique helps to abandon the X-ray guidance. EBUS-GS-TBLB combined with VBN is a safe and effective technique for PPLs.

Similarity in joint and mucous bleeding syndromes in type 2N von Willebrand disease and severe hemophilia A coexisting with type 1 von Willebrand disease in two Chinese pedigrees.

In this study, we investigated the molecular basis of two unrelated Chinese patients with hemostatic disorders. The proband of the first family had severe hemophilia A (HA) coexisting with type 1 von Willebrand disease (VWD) and the proband of the second family had type 2N VWD. Both probands had similar phenotypes, which included joint and mucosal bleeding, very low factor VIII (FVIII) activity (FVIII:C), and moderate reductions in VWF antigen (VWF:Ag) and VWF ristocetin cofactor activity (VWF:Rco), as well as a normal multimeric pattern. One FVIII mutation and three VWF mutations were identified: FVIII p.R446* and VWF heterozygous p.E216K mutations were detected in proband 1 and compound heterozygosity of VWF mutations (p.R816W and c.1911delC) in proband 2. Transient expression studies in HEK293T cells proved that R816W mutation abolished the binding of FVIII to VWF and slightly impaired protein synthesis and secretion; 1911delC mutation mainly impaired VWF protein synthesis and secretion. These results provided insight into the possible pathogenic mechanism of type 2N VWD in Chinese patients carrying these mutations.

A mechanism underlying hypertensive occurrence in the metabolic syndrome: cooperative effect of oxidative stress and calcium accumulation in vascular smooth muscle cells.

Several lines of evidence indicate that reactive oxygen species (ROS) overproduction under the metabolic syndrome condition is the leading cause of cardiovascular events. Calcium is an important stimulus for vasoconstriction and plays a pivotal role in the development of hypertension. Here, we investigate whether a relationship exists between metabolic syndrome-induced mitochondrial ROS overproduction and Ang II-mediated Ca2+ release in vascular smooth muscle cells (VSMC). The effect of mitochondrial ROS on AT1 expression, and Ca2+ and IP3 generation was studied in 2 VSMC models of metabolic syndrome using fura-2/AM probes and ELISA-based assay. Ang II-mediated aortic ring contraction in SD rats fed with high-fat diet (HFD) was measured using a force transducer connected to chart recorder. In the metabolic syndrome, almost 2-fold increased mitochondrial O2 - significantly upregulated AT1 expressions by ~60%, companied by elevated Ca2+ and IP3 levels in VSMC and enhanced aortic rings contraction. All these increments were blocked by rotenone (inhibitor of mitochondrial respiratory chain complex I), ruthenium red (inhibitor of calcium uniporter), cyclosporin A (inhibitor of mitochondrial permeability pore), and N-acetylcysteine. Therefore, in the states of metabolic syndrome, ROS overproduction in mitochondrial complex I enhances Ang II-mediated vascular contraction via an AT1-dependent pathway. In addition, the import of Ca2+ from endoplasmic reticulum to mitochondria via calcium uniporter and mitochondrial permeability pore seems to serve as a mechanism to further aggravate mitochondrial damage and vascular dysfunction that may contribute to the occurrence of hypertension.

Extracellular redox state regulates catecholamine biosynthesis in PC12 cells with insulin resistance.

Extracellular cysteine (Cys)/cystine (CySS) redox potential (Eh) plays a crucial role in maintaining redox homeostasis and an alteration of redox state occurs in various physiological conditions, including diabetes, cancer, and aging. This study was designed to determine whether a variation in extracellular redox state would alter the function of insulin-resistant PC12 cells. Various redox states were established by providing different extracellular Cys/CySS Eh to insulin-resistant PC12 cells. We intensively investigated the relationship between redox state and catecholamine biosynthesis in PC12 cells, and evaluated the changes in cellular reactive oxygen species (ROS), catecholamine (CA) synthesis, tyrosine hydroxylase (TH) expressions, and the activity of rate-limiting enzyme in CA synthesis by using DCF-fluorescence, HPLC, and the real-time PCR, respectively. We also determined the protein levels of NF-E2-related factor 2 (Nrf2), a redox sensitive transcription factor, using an ELISA assay. We found that the oxidized Cys/CySS Eh (0 mV) pretreatment decreased CA, TH, and Nrf2 levels, but induced ROS overproduction. Insulin induced a significant increase in CA synthesis and ROS production, blocked by more reducing redox conditions. The paradox of CA and TH alterations between insulin and 0 mV groups may be attributed to degree of redox imbalance as evidenced by different ROS levels in 2 groups, which is further confirmed by CA alterations in different concentrations of hydrogen peroxide. Additionally, dithiole-3-thione (D3T, an inducer of Nrf2) corrected 0 mV-induced TH inhibition. In conclusion, CA biosynthesis in insulin-resistant PC12 cells could be influenced by extracellular Cys/CySS redox effects on cellular redox sensitive transcription factors.

Molecular defects in the factor X gene caused by novel heterozygous mutations IVS5+1G>A and Asp409del.

Factor X (FX) deficiency is a rare autosomal-recessive bleeding disorder caused by diverse mutations in the F10 gene. To investigate the molecular basis of severe FX deficiency in a mildly hemorrhagic patient, variants of the F10 gene were detected by sequencing. A missense mutation was analysed by in vitro expression and modelling analysis, and a splice mutation using ectopic transcript analysis. The levels of activity of FX (FX:C) were <1% in both intrinsic and extrinsic pathway assays and 1.71% in chromogenic assay, the level of FX antigen (FX:Ag) was 53.36% in the proband. Two novel heterozygous mutations (IVS5+1G>A and Asp409del) were identified in the F10 gene. Ectopic transcript expression combined with informative marker (heterozygous Asp409del) analysis of the splice mutation (IVS5+1G>A) revealed and confirmed that the transcript from the mutated allele was absent, likely caused by the nonsense-mediated mRNA decay pathway. In vitro expression analysis showed that the Asp409del mutant led to a loss of enzymatic activity rather than impaired expression. Molecular modelling analysis confirmed that the Asp409del mutant dramatically altered the conformation of the 185-189 loop and impaired binding of the loop to sodium ions (Na(+) ), diminishing the enzymatic activity of FXa. This is the first report to clarify the molecular mechanisms of two naturally occurring F10 gene variants that cause severe FX deficiency.

Characterisation and validation of a novel panel of the six short tandem repeats for genetic counselling in Chinese haemophilia A pedigrees.

Haemophilia A (HA) is the most common hereditary bleeding disorder caused by F8 gene mutation. Linkage analysis is an auxiliary strategy to direct mutation analysis for genetic counselling of HA. Here we characterize and validate a novel panel of six short tandem repeat (STR) loci for genetic counselling in Chinese HA pedigrees. The panel was analysed in 116 unrelated healthy female patients and 108 male patients, and verified in 169 unrelated pedigrees with HA. The six STR loci in the panel spanned a distance of 0.3 Mb from each side of the F8 gene. Three of them, F8Up226, F8Up146 and F8Down48, were first described here. Markers F8Up226, F8Up146, F8Int13, F8Int25, F8Down48 and DXS1073 exhibited the number of alleles 16, 9, 8, 6, 9 and 10, and heterozygosity rates of 74.8%, 44.8%, 60.9%, 42.6%, 61.7% and 62.0% respectively. Haplotype frequencies analysis suggested that the genotypes of haplotype provided a highly informative content (56.5%). The panel was informative in 167 of 169 unrelated haemophilic pedigrees with the combined diagnostic rate of 98.8%. In eight pedigrees could not be diagnosed by mutation detection linkage studies using the panel were informative in all the pedigrees and a reliable diagnosis was made in seven pedigrees. The novel panel of the six STR loci represents a high degree of informativeness and a low fraction of recombination. Linkage analysis using this panel provides an alternative strategy when direct mutation detection is not feasible for genetic counselling in Chinese HA families.

The prevalence of factor VIII inhibitors and genetic aspects of inhibitor development in Chinese patients with haemophilia A.

The prevalence of inhibitors in Chinese haemophiliacs has not yet been reported. The aim of this study was to identify the prevalence of factor VIII (FVIII) inhibitors among haemophiliacs who are treated only with plasma-derived FVIII (pdFVIII), cryoprecipitate or fresh frozen plasma (FFP), and tried to explore the relationship between the generation of inhibitors and particular FVIII deficiency genotypes. Clinical information and blood samples of 1435 patients with haemophilia A (HA) were collected by six haemophilia centres in China. The Nijmegen modification of the Bethesda assay was used to detect inhibitors. Multiplex PCR, long-range PCR and direct sequencing were performed for genotyping. The overall prevalence of inhibitors in Chinese HA patients was 3.9% and the prevalence of severe haemophiliacs was 4.3%; 18 of the 56 patients with inhibitors had high titres. A total of 38 different mutations were identified in the 55 patients with inhibitors, including 15 intron 22 and 3 intron 1 inversions, seven large deletions, 14 small deletion/insertions, seven nonsense mutations, one splice site mutations and eight missense mutations. Of 38 mutations, 28 were novel. Patients with large deletions and nonsense mutations were prone to have high titre inhibitors, with incidence rates of 57.1% (4/7) and 42.9% (3/7), respectively. In conclusion, the prevalence of inhibitors in Chinese HA patients is much lower than that reported for other ethnic groups and the large deletion and nonsense mutations are high risk factors for high titre inhibitor development.

Female hemophilia A heterozygous for a de novo frameshift and a novel missense mutation of factor VIII.

BACKGROUND: Hemophilia A (HA) is an X-chromosome-linked recessive disorder. AIM: We report the case of a female HA patient with a moderate decrease of factor (F) VIII activity and antigen (FVIII:C 3.4%, FVIII:Ag 4.2%) and severe bleeding symptoms. METHODS: The patient's father had mild FVIII deficiency (FVIII:C 6.9%, FVIII:Ag 7.4%), and her mother had normal FVIII activity. The von Willebrand disease antigen and von Willebrand factor ristocetin cofactor activity were normal in all family members. The genomic DNA was extracted from the peripheral blood lymphocytes of the patient and her family members. Long-distance polymerase chain reaction (PCR) was employed to screen for the intron 22 inversion of the FVIII coding gene (F8). The F8 coding sequence was amplified with PCR and sequenced with an automatic sequencer. RESULTS: Two heterozygous mutations were identified in the patient: one a substitution of nucleotide 5981T by C that leads to a missense mutation Leu1975Pro, and the other an insertion of an 'A' between nucleotides 3,637 and 3,638 (3637_3638insA) that shifts the reading frame and predicts a premature stop codon downward. The mutation Leu1975Pro was identified in the father's F8; however, 3637_3638insA was a de novo mutation that occurred in the patient's maternal-derived F8. Real-time PCR was applied to analyze the level of ectopically F8 gene transcripts in the peripheral lymphocytes of family members. The ectopic transcripts of F8 of the patient were less abundant than the normal control (patient:normal control ratio 0.67), whereas her parents showed no significant difference from the normal control. CONCLUSION: The FVIII deficiency of the HA patient resulted from a de novo occurrence of a frameshift 3637_3638insA in her maternal-derived F8 and a novel missense mutation Leu1975Pro inherited from her father.

Thyroid hormone exacerbates vasoconstriction in insulin resistance: the role of ONOO(-).

Insulin resistance has been proposed to play a pivotal role in vasoconstriction due to increased oxidative stress. Hyperthyroidism would amplify cardiovascular disease risk in diabetic patients, though thyroid hormone advance vascular relaxation and reduce vascular contraction by virtue of NO production in vascular smooth muscle cells (VSMCs). Thus, we aimed to investigate the vascular tone and its underlying mechanism in insulin resistance accompanied by hyperthyroidism. Vascular reactivity studies showed that endothelium-denuded thoracic aortic rings from rats fed with high-fat high-sucrose (HFHS) diet and L-T4 (HFHS+L-T4) exhibited a stronger contractile response to noradrenaline than HFHS rats, which was reversed by L-NAME and GSH. Furthermore, rat thoracic aortic smooth muscle cells (A10) simultaneously stimulated with high glucose insulin (high Glc/Ins) and T3 demonstrated lower NO, superoxide anion ( [Formula: see text] ) levels, and higher iNOS, nitrite ( [Formula: see text] ), peroxynitrite (ONOO(-)) levels than that treated with T3 only. Excessive ONOO(-) markedly aggravated oxidative stress. Thus, we hypothesized that the elevated concentration of ONOO(-) which is generated by NO and [Formula: see text] could be critically instrumental in the progression of vasoconstriction by increasing oxidative stress.

Characterization of large deletions in the F8 gene using multiple competitive amplification and the genome walking technique.

BACKGROUND: Large deletions in the F8 gene are responsible for approximately 3% of severe hemophilia A (HA) cases. However, only a few breakpoints in large deletions have been characterized. OBJECTIVES: To identify large deletions in the F8 gene and to characterize the molecular mechanisms leading to these deletions. PATIENTS AND METHODS: We used AccuCopy technology, a copy number variation (CNV) genotyping method based on multiplex competitive amplification, to confirm deletions in index patients and to screen potential female carriers in 10 HA families. Also, breakpoints of these large deletions were characterized by a primer walking strategy and genome walking technique. RESULTS: Ten large deletions and four female carriers were identified by AccuCopy. The extents of deleted regions ranged from 1.3 to 68.5 kb. Exact breakpoints of these deletions were successfully characterized. Eight of them presented microhomologies at breakpoint junctions and several recombination-associated elements (repetitive elements, non-B conformation forming motifs and sequence motifs) were also observed in close proximity to the junctions. CONCLUSIONS: AccuCopy technology is a reliable and efficient tool for detecting large deletions in the F8 gene and identifying HA female carriers. The genome walking technique is a highly specific, efficient and versatile method for characterizing the deletion breakpoints. Molecular characterization of deletion breakpoints revealed that non-homologous end joining and microhomology-mediated replication-dependent recombination were the major causative mechanisms of the 10 large deletions in the F8 gene.

Molecular characterization of two novel mutations causing factor X deficiency in a Chinese pedigree.

Factor X (FX) deficiency is a rare bleeding disorder inherited as an autosomal recessive trait. In this study, we investigated the molecular basis of FX deficiency in a Chinese pedigree. The proposita showed a markedly prolonged activated partial thromboplastin time and a mild prolongation of prothrombin time. The levels of FX antigen and FX activity were 58.6% and 2.5%, respectively. Molecular analysis revealed that the proposita was compound heterozygous for two novel mutations: IVS1 + 1G > A and G1185A (Arg347His). The aberrant transcripts from the IVS1 + 1G > A mutant allele were not detected by analyzing the splicing pattern of ectopic transcripts in leukocytes of the patient with nested polymerase chain reaction after reverse transcription. We thus hypothesize that the mRNA molecules originating from the IVS1 + 1G > A mutation were rapidly destroyed in vivo. Site-directed mutagenesis of FX cDNA was used to introduce FXG1185A mutation, and wild-type as well as mutant FX proteins were expressed by transient transfection in HEK 293 cells. Normal FX antigen levels both in the conditioned media of cells expressing the mutant and in cell lysates were detected by an enzyme-linked immunoadsorbent assay. Evaluation of wild-type and mutant coagulant activity demonstrated that the FX molecules carrying the Arg347His mutation have dramatically decreased activity.

Identification of three F5 gene mutations associated with inherited coagulation factor V deficiency in two Chinese pedigrees.

To investigate the molecular defects in two Chinese pedigrees with inherited factor V (FV) deficiency. A 37-year-old male (proband 1) and an 18-month-old boy (proband 2) were diagnosed as inherited coagulation FV deficiency by severely reduced plasma levels of FV activity and antigen. All 25 exons and their flanking sequence of F5 gene were amplified by polymerase chain reaction (PCR) for both probands and the PCR products were directly sequenced. Total RNA was extracted from the peripheral lymphocytes of proband 1 for detecting the changes at mRNA level. The homozygous deletion IVS8 -2A>G was identified in the F5 gene of proband 1 and complementary DNA (cDNA) analysis revealed the abolishment of the canonical splicing site by the mutation and the activation of the cryptic acceptor site 24 bp upstream instead. The insertion introduced eight additional amino acids (AA) into the FV protein. Two heterozygous mutations of F5 gene were discovered in proband 2. The 2238-9del AG in exon 13 introduced a premature termination code at 689 AA and the substitution of G6410 by T in exon 23 lead to the missense mutation Gly2079Val. Three F5 gene mutations, IVS8 -2A>G, 2238-9del AG and G6410T, have been identified in two Chinese pedigree with congenital FV deficiency, respectively.