Analysis of an Inherited FXII Deficiency Pedigree Associated with Double Heterozygous Mutations in the F12 Gene.

This article is intended to identify the potential mutations of the FXII gene (F12) in an inherited FXII deficiency pedigree and illuminate the pathogenesis of the disease. The coagulation FXII activity (FXII:C) and FXII antigen (FXII:Ag) were inspected by one-stage clotting assay and enzyme-linked immunosorbent assay(ELISA), respectively. Polymerase chain reaction amplification (PCR) was performed and the F12 gene was sequenced directly. A molecular model of FXIIprotein was established for further analysis. ClustalX-2.1-win and online bioinformatic software were used to estimate the conservatism and possible impact of the protein change. The proband presented prolonged APTT(180 s) and extreme low FXII:C and FXII:Ag (both < 1%, reference range:72-113%). A compound heterozygous were found by the direct sequencing of the F12 gene. One was a deletion mutation c.1792_1796delGTCTA, which is a novel mutation; the other was an insertion mutation, c.1092_1093insC. Bioinformatic and modeling analyses indicated that the the two frameshift mutations may be deleterious and possibly alter the structure and the function of the protein. The mutations c.1792_1796delGTCTA and c.1092_1093insC could be the main causes of reducing FXII in this pedigree, and c.1792_1796delGTCTA mutation was the first report in the world.

A Novel Fibrinogen Mutation p.BßAla68Asp Causes an Inherited Dysfibrinogenemia.

OBJECTIVE: Our study aimed to analyze the phenotype and genotype of a pedigree with inherited dysfibrinogenemia, and preliminarily elucidate the probable pathogenesis. METHODS: The one-stage clotting method was used to test the fibrinogen activity (FIB:C), whereas immunoturbidimetry was performed to quantify the fibrinogen antigen (FIB:Ag). Furthermore, DNA sequence analysis was conducted to confirm the site of mutation. Conservation analysis and protein model analysis were performed using online bioinformatics software. RESULTS: The FIB:C and FIB:Ag of the proband were 1.28 and 2.20 g/L, respectively. Gene analysis revealed a heterozygous c.293C > A (p.BßAla68Asp) mutation in FGB. Bioinformatics and modeling analysis suggested that the missense mutation could potentially have a deleterious effect on fibrinogen. CONCLUSION: The BßAla68Asp mutation in exon 2 of FGB may account for the reduced FIB:C levels observed in the pedigree. To our knowledge, this point mutation is the first report in the world.

Hereditary factor V deficiency from heterozygous mutations with a novel variant p.Pro798Leufs∗13 in the F5 gene.

To explore the causative mutation for autosomal recessive inheritance factor V (FV) deficiency in a Chinese family. Relative coagulation indexes and the FV antigen were tested by the one-stage clotting method and ELISA, respectively. At the same time, the calibrated automated thrombogram (CAT) was used to analyze the mutant protein function. All 25 exons, flanking sequences, 5' and 3' untranslated regions of the F5 were amplified by PCR and sequenced directly, while each suspected variant was verified by reverse sequencing. The possible impact of the mutant was analyzed by the corresponding bioinformatics software. The phenotypic tests showed that the proband's FV activity has decreased to 24%, whereas the FV antigen has also reduced to 28%. The genetic analysis revealed that she was a compound heterozygote for a frameshift variant from small deletion in the exon 13 (c.2390_2390delC, p.Pro798Leufs∗13) and a missense mutation in the exon 25 (c.6665A>G, p.Asp2222Gly). Meanwhile, the online bioinformatics software indicated that the frameshift variant was disease-causing. The pathogenic variant p.Pro798Leufs∗13 and the benign variant p.Asp2222Gly largely account for the decrease of the FV deficiency in this Chinese family, of which the pathogenic variant is firstly reported in the world.

A novel mutation (Leu60Pro) in a Chinese pedigree with hereditary factor XI deficiency.

To analyse F11 gene mutations in a Chinese pedigree with hereditary factor XI (FXI) deficiency and investigate the molecular mechanism. The plasma FXI activity and FXI antigen of the proband and the family members were detected by clotting assay and ELISA, respectively. The F11 gene was amplified by PCR and sequenced directly. Online bioinformatics software were needed to analyse the mutations. The proband showed a prolonged activated partial thromboplastin time (93.3 s), whose FXI activity and FXI antigen were low to 2, 4.5%, respectively. A novel mutation c.233T>C (p.Leu60Pro) in exon 4 and a previously described mutation c.1253G>T (Gly400Val) were found in the proband. Protein Leu60 is conserved highly among homologous species. Bioinformatics software indicated that Leu60Pro mutation might affect the protein function. Other coagulation abnormalities were not found. We preliminarily considered the mutations Leu60Pro and Gly400Val were responsible for the decrease FXI level in the family. Leu60Pro mutation in the F11 gene has not been described elsewhere.

Dynamic Distribution and Clinical Value of Peripheral Lymphocyte Subsets in Children with Infectious Mononucleosis.

OBJECTIVE: To study the dynamic change of peripheral lymphocyte subsets and its clinical value in children with infectious mononucleosis (IM). METHODS: Thirty-six pediatric patients with IM, 19 children with IM-like symptoms but lacking the serological pattern compatible with EB virus infection, and 33 healthy children were enrolled. The changes of peripheral lymphocyte subsets were detected by flow cytometry on admission and on the fifth day of antiviral treatment, respectively. Indicators of liver function and routine blood count were also detected. Besides, the receiver operating characteristic (ROC) curve and the correlation of related indicators was analyzed. RESULTS: When IM patients were admitted, the frequency and absolute number of T, CD4-CD8+T, and CD4+CD8+T (DPT) cells were significantly increased while B cells were decreased; the frequency of CD4+CD8-T cells were decreased, but its absolute number did not change significantly; the frequency of NK cells decreased, but its absolute number increased. The absolute number of CD4-CD8+T most significantly positively correlated with serum lactate dehydrogenase (LDH) concentration which could reflect the severity of IM patients. After short-term treatment with acyclovir, elevated lymphocytes decreased, but only DPT-cell frequency and NK-cell absolute number were recovering towards normal. The ROC curve suggested that the frequency of B cells has better diagnostic value for IM in pediatric patients compared to other lymphocyte subsets. CONCLUSIONS: Peripheral lymphocyte subsets are closely related to the condition of children with IM, and each subset plays a relatively different role in the diagnosis and evaluation of IM.

[Analysis of Phenotype and Genotype of A Family with Hereditary Coagulation Factor â ¤ Deficiency Caused by A Compound Heterozygous Mutation].

OBJECTIVE: To analyze the molecular pathogenesis by analysis of phenotype and gene mutation in families with hereditary coagulation factor V (FⅤ) defect caused by complex heterozygous mutation. METHODS: Plasma pro-thrombin time (PT), activated partial thromboplastin time (APTT), fibrinogen (FIB), FⅤ procoagulant activity (FⅤ∶C), FⅤ antigen (FⅤ∶Ag), and other related coagulation indexes were detected in the proband and his family members (3 generations 10 people). Using DNA direct sequencing to analyze all exons, flanks, 5' and 3' untranslated regions of F5 genes and the corresponding mutation site regions of family members, the mutation site was confirmed by reverse sequencing．The conservation of mutant amino acids was analyzed by ClustalX-2.1-win software. The PROVEAN and MutationTaster online bioinformatics software were used to predict the effect of mutation on protein function. Protein model and amino acid interaction at mutation sites was analyzed by Swiss-pdbviewer software. RESULTS: The PT and APTT of the proband were significantly prolonged compared with healthy controls (34.2 vs 13.2 s and 119.3 vs 36.0 s), while FⅤ∶C and FⅤ∶Ag extremely reduced (3% and 6%). The PT and APTT of the second-born, the third son, daughter, and grandson of the proband were slightly prolonged, and the FⅤ∶C and FⅤ∶Ag decreased to varying degrees. The related coagulant parameters of other family members were within normal range. Genetic analysis revealed that the proband had a c.911G>A heterozygous missense mutation on the exon 6 lead to p.Gly276Glu, and a c.5343C>G heterozygous missense mutation on the exon 16 lead to p.Ser1781Arg of the proband. The second-born, the third son, and grandson of the proband carry p.Gly276Glu heterozygotes, and the daughter carries p.Ser1781Arg heterozygotes, while the other family members were wild-type. The results of conservative analysis indicated that p.Gly276 and p.Ser1781 were highly conserved in homologous species. The two bioinformatics software predicted the same results, PROVEAN (score -6.214 and -12.79) indicated that the compound heterozygous mutation was a harmful mutation; MutationTaster (score 0.976 and 0.999) suggested that these mutations might cause corresponding disease. p.Gly276Glu protein model analysis showed that, the Glu side chain was prolonged and the molecular weight became larger, which would increase the steric hindrance between it and the surrounding amino acids, affect the normal local folding of the FⅤ protein, and eventually lead to the decrease of protein activity and content. This paper can not provide analysis of the spatial structure of p.Ser1781Arg mutant protein because of the lack of X ray 3 D structure file of FⅤ exon 16. CONCLUSION: The new compound heterozygous mutations (p.Gly276Glu and p.Ser1781Arg) identified in this study are the main reasons for the decrease in the FⅤ level of the family, among which p.Ser1781Arg is rarely reported at home and abroad.

A novel heterozygous mutation (γIIe367Thr) causes congenital dysfibrinogenemia in a Chinese family.

: The aim of this study was to elucidate the molecular defects in a Chinese family with dysfibrinogenemia. The fibrinogen activity was measured by the one-stage clotting method. The fibrinogen antigen was measured with immunoturbidimetry. The fibrinogen gene was amplified by PCR with direct sequencing. Suspected mutation was confirmed by reverse sequencing. Bioinformatics and model analysis were used to study the conservatism and harm of the mutation. The proband had a history of menorrhagia. Study showed fibrinogen activity at 0.35 g/l and fibrinogen antigen at 2.05 g/l. Sequencing analysis detected a heterozygous c.1178T>C missense mutation in exon 9 of FGG gene resulting in p.IIe367Thr. The bioinformatics and model analysis indicated that the IIe367Thr mutation may disrupt the activation of the fibrinogen. We detected a novel IIe367Thr missense mutation in the FGG. To our knowledge this is causative mutation has not been reported so far.

A novel homozygous mutation (Gly1715Ser) causing hereditary factor V deficiency in a Chinese patient.

: To explore the phenotype and genotype of a Chinese family with hereditary factor V deficiency. Routine blood coagulation indexes were detected by one-stage clotting method, whereas factor V antigen was detected by ELISA. All exons and intron-exon boundaries of F5 gene were amplified by PCR and sequenced directly. The suspected mutation was confirmed by reverse sequencing. Bioinformatics softwares were used to analyze the possible impact of this mutation. Phenotypic analysis showed that the proband had significantly prolonged prothrombin time and activated partial thromboplastin time, and his factor V clotting activity was decreased to 3%. Genetic analysis revealed a homozygous missense mutation c.5227G>A (p.Gly1715Ser) in exon 16 of F5 gene. Bioinformatics and structural analysis demonstrated this mutation was deleterious and could affect the integrity of local intermolecular structures. The missense mutation (Gly1715Ser) was responsible for the decrease of factor V clotting activity and factor V antigen in this family, and caused type I hereditary factor V deficiency.

Hyperactivated peripheral invariant natural killer T cells correlate with the progression of HBV-relative liver cirrhosis.

Invariant NKT (iNKT) cells express markers of both T and NK cells and may produce various cytokines to regulate liver immunity. However, the role of iNKT cells in the progression of HBV-relative liver cirrhosis (HBV-LC) is incompletely understood. Here, we investigated the impact of peripheral iNKT cells on a cohort of patients with HBV-LC. The frequency, number, activation status, apoptosis and proliferation ability of peripheral iNKT cells were detected with flow cytometry. The impact of peripheral iNKT cells on the proliferation of hepatocyte cell line (MIHA) and activation of hepatic stellate cell line (LX-2) was detected with flow cytometry and PCR. In HBV-LC patients, the frequency and absolute number of peripheral iNKT cells significantly reduced, but the expression levels of CD25, interleukin (IL)-4, IL-13 and interferon (IFN)-γ increased. No difference was observed in the proliferation and apoptosis of circulating iNKT cells between patients and healthy controls (HCs). CXCR6 (CD186), known to be closely associated with iNKT cells migration from the periphery to the liver, was highly expressed on peripheral iNKT cells in HBV-LC patients. Furthermore, peripheral iNKT cells had a profound impact on MIHA cell proliferation and LX-2 cell activation through IL-4 or IL-13. Our data suggest that in HBV-LC patients, highly activated peripheral iNKT cells may migrate to the liver and affect hepatocyte cell line (MIHA) proliferation and hepatic stellate cell line (LX-2) activation through the expression of type 2 cytokines, which may result in excessive healing and contributing to the progression of fibrosis toward cirrhosis in liver.

B and NK Cells Closely Correlate with the Condition of Patients with Acute Pancreatitis.

PURPOSE: Pancreatitis can lead to systemic inflammatory response, but the relationship between lymphocyte changes and patients with pancreatitis remains unclear. In this study, we evaluated the feedback function of changes in peripheral lymphocyte subsets on the condition of patients with pancreatitis. MATERIALS AND METHODS: 131 acute pancreatitis (AP) patients and 11 chronic pancreatitis (CP) patients constituted the patients' group; 20 healthy individuals were enrolled as healthy controls (HC). Serum concentration of C-reactive protein (CRP), amylase, and lipase and the frequency and absolute number of many types of peripheral lymphocytes (including T, B, NK, CD16+/CD56+ T, CD4+ T, CD8+ T, CD4+CD8+ T, and CD4-CD8- T cells) were detected on admission and the seventh day of standard treatment. Besides, the length of hospital stay was recorded. RESULTS: The absolute number of all lymphocytes we studied decreased in patients with CP and in patients with almost all types of AP. The frequency change of lymphocytes varies among the different types of AP. During disease onset, B cell frequency correlated positively with CRP concentration and NK cell frequency correlated positively with amylase and lipase concentration. B cell frequency and CD4+ T cell absolute number were recovering towards normal after short-term treatment. The frequency of B cells and NK cells correlated positively with the length of hospital stay. CONCLUSIONS: B cells and NK cells closely correlate with patients' condition and may help to diagnose AP more accurately and reflect treatment effect of AP in time, affecting the recovery speed of patients with M-AP, which may help physicians to better understand the pathophysiology of pancreatitis.

Expression of CD56 is a risk factor for acute lymphocytic leukemia with central nervous system involvement in adults.

OBJECTIVE: To gain further insights into the predisposing risk factors for central nervous system (CNS) involvement in patients with acute lymphocytic leukemia (ALL), the impact of CD56 expression in these patients was investigated. METHODS: We reviewed the clinical features of CD56 expression in 588 consecutive ALL patients treated with systemic chemotherapy regimens between 2000 and 2014. The categorical data from CD56+ ALL patients were compared with those from CD56- ALL patients. RESULTS: Among the 588 patients studied, 18.9% showed CD56 expression. The expression was significantly associated with CD33+, CD10-, CD15+, TdT-, and CD5+ immunophenotypes. After systemic chemotherapy, 8.8% patients showed CNS involvement, of which 3.2% exhibited combined recurrences and 5.6% exhibited isolated CNS involvement. The 5-year event-free survival was significantly lower for patients with CD56+ immunophenotype compared with patients with CD56- immunophenotype (22.5% vs. 32.7%, P = 0.04). Cumulative incidences of CNS involvement were significantly greater in the CD56+ cohort compared with the CD56- cohort (14.4% vs. 7.5%, P = 0.02). Multivariate analysis revealed CD56 expression to be statistically significant risk factors for CNS involvement. CONCLUSION: CD56 expression should be regarded as an independent risk factor for ALL with CNS involvement in adults.

Diagnostic Error of a Patient with Combined Inherited Factor VII and Factor X Deficiency due to Accidental Ingestion of a Diphacinone Rodenticide.

BACKGROUND: To explore the characteristics of laboratory examination and confirm the diagnosis of a patient with combined inherited FVII and FX deficiency after he ingested diphacinone rodenticide accidentally. METHODS: The coagulant parameter screening tests and coagulation factor activities were tested many times in the patient due to accidental ingestion of a diphacinone rodenticide. After the patient was treated for more than one year, gene analysis of correlated coagulation factors was analyzed in the patient and other family members by DNA direct sequencing. 106 persons were selected as controls from routine health examinations. RESULTS: After the patient was admitted to hospital, routine coagulation screening tests revealed the prolonged prothrombin time (PT) and activated partial thromboplastin time (APTT) and low levels of vitamin K-dependent coagulation factors (FII, FVII, FIX, FX) activity, which was 102.4 seconds, 88.5 seconds, 7%, 3%, 8%, and 2%, respectively. During more than one year of treatment, the value of PT and APTT still showed significantly prolonged activity and FVII and FX activity levels were about 5%. While FII and FIX activity levels were in the normal range after 12 weeks of treatment. Two homozygous mutations, g.11267C>T of F7 gene resulting in the substitution Arg277Cys and g.28139G>T of F10 gene leading to the substitution Val384Phe, were identified in the patient. The patient's parents and sister was heterozygous for Arg277Cys and Val384Phe mutations. FVII and FX antigen levels in the patient were 7% and 30%, respectively. CONCLUSIONS: There were many similarities in the characteristics of laboratory examination between combined inherited FVII and FX deficiency and acquired vitamin K deficiency. The best way to identify them was gene analysis.

[Analysis of molecular pathogenesis and clinical phenotypes in 10 probands with inherited fibrinogen deficiency].

OBJECTIVE: To explore the molecular pathogenesis and clinical phenotypes in 10 probands with inherited fibrinogen (Fg) deficiency. METHODS: The diagnosis of hereditary Fg deficiency was validated by prothrombin time (PT), thrombin time (TT), Fg activity (Fg:C) and Fg antigen (Fg:Ag) in plasma. All of the exons and their flanking sequences of the Fg gene were analyzed by direct sequencing. Detected mutations were confirmed by reverse sequencing. RESULTS: The ranges of Fg:C and Fg:Ag in the 10 probands were 0.52-0.91 g/L and 0.62-2.98 g/L, respectively. Five of the probands had type I disorders, and 5 had type II disorders. Seven point mutations were identified, among which 6 have located in the D region. γThr277Arg, γAsp316His, γTrp208Leu and Lys232Thr were novel mutations, and αArg19Ser was first reported in Chinese. Four probands had the same mutation site (γArg275). As to the clinical manifestation, probands with type I disorders were asymptomatic or with mild or medium symptoms, while those belonged to type II disorders had moderate or serious symptoms. Two probands have carried an Arg275Cys mutation but had different clinical manifestations. CONCLUSION: Mutations of the Fg gene seem to aggregate to the D region of FGG in our region, and Arg275 is a common mutation. However, no correlation has been found between the mutation site and clinical manifestations.

A novel mutation of SETBP1 in atypical chronic myeloid leukemia transformed from acute myelomonocytic leukemia.

To investigate an oncogenic mutation of SETBP1 in the evolution from acute myelomonocytic leukemia (M4) to secondary aCML. Clinical data and molecular studies were analyzed of paired aCML and 'normal'DNA from a case with M4. We identified a mutation in SETBP1 (encoding a p.Asp868Ala alteration). The analysis of paired sample indicated that SETBP1 mutation was acquired during leukemic evolution.

A novel gene insertion combined with a missense mutation causing factor VII deficiency in two unrelated Chinese families.

Hereditary coagulation factor VII (FVII) deficiency is a rare bleeding disorder characterized by reduced FVII activity (FVII:C) and inconsistent FVII antigen (FVII:Ag). In our study, two pregnant probands were diagnosed with FVII deficiency, based on the tests that FVII:C were both 3% and FVII:Ag were less than 7.5%. Gene sequencing revealed the same compound mutations, a recurrent missense mutation p.Arg277Cys and a novel insertion mutation g.11520-11521insT. What is more, haplotype analysis of SNPs excluded the possibility of consanguinity between the two families. According to the model, we speculated that although the insertion mutation was close to the carboxy-terminal, it induced the protein extension and affected the 3' untranslated region of F7 gene, which is significant to posttranscriptional regulation. Hypothetically, the stability or translational efficiency of mRNA may be influenced, resulting in reducing FVII:C.

Decreased programmed death-1 expression on the T cells of patients with ankylosing spondylitis.

BACKGROUND: Programmed death-1 (PD-1) plays a vital role in down-modulating immune responses and maintaining peripheral tolerance. METHODS: The authors have investigated the inducible expression of PD-1 on activated T cells from patients with ankylosing spondylitis (AS). Thirty patients with AS and 31 unrelated healthy controls (HCs) were recruited in this study. The expression of PD-1 on T cells harvested from nonstimulated (t0) or stimulated cultures with phytohemagglutinin for 24 hours (t24) was determined by flow cytometry. The multiple levels of the PD-1 expression on stimulated and nonstimulated cells from each individual's sample (t24/t0) represented as the degree of the inducible effect on PD-1 expression. RESULTS: The expression of PD-1 on nonstimulated T cells presented no significant difference between AS group and HC group (P > 0.05). After stimulation, the degree of effect on PD-1 expression of CD4+, CD4+CD25+, CD4+CD25 high, CD4+CD25 low and CD4+CD25- T cells were significantly lower in patients with AS than those in HC group (1.9 ± 0.9 versus 3.6 ± 2.3, 9.7 ± 7.4 versus 17.8 ± 12.6, 87.8 ± 48.6 versus 157.3 ± 117.0, 3.7 ± 1.4 versus 7.3 ± 2.4, 0.5 ± 0.3 versus 1.1 ± 0.6, respectively, P < 0.05). However, there was no significant difference of the effect on all lymphocytes and CD8+ T cells between patients with AS and HCs (P > 0.05). CONCLUSIONS: The decreased inducible expression of PD-1 on active T lymphocytes, especially on CD4+CD25 high and CD4+CD25+ T cells, may be one of important factors involved in the development of AS.

Novel mutations (γTrp208Leu and γLys232Thr) leading to congenital hypofibrinogenemia in two unrelated Chinese families.

Congenital hypofibrinogenemia is a rare disorder caused by heterozygous mutations in the fibrinogen genes. The aim of this study was to elucidate the molecular defects in two unrelated families with hypofibrinogenemia. The proband from family A was a 19-year-old Chinese boy who was suffering from cervical lymphadenitis. A low plasma fibrinogen concentration (0.63 g/l by Clauss method and 0.77 g/l by immunoturbidimetry) was found in routine clotting tests. Further gene analysis revealed a heterozygous g.5792 G>T mutation in exon 7 of the FGG, leading to a novel Trp208Leu change in the γ D domain. This mutation was also found in other family members with low fibrinogen levels. The proposita from family B was a 37-year-old female who suffered from recurrent shoulder pain for 7 years. Routine clotting studies revealed that her prothrombin time was 15.5 s (normal range: 11.8-14.8 s) and thrombin time was 22.8 s (normal range: 14.0-20.0 s), and the fibrinogen concentration in her plasma was only 0.64 g/l by Clauss method and 0.79 g/l by immunoturbidimetry. A heterozygous A>C transition at nucleotide 5864 of FGG was found in the γ chain, causing a Lys232Thr substitution in the fibrinogen. Further sequencing established that her mother, son, brother and nephew were also heterozygous for the mutation.