De novo disruptive heterozygous MMP21 variants are potential predisposing genetic risk factors in Chinese Han heterotaxy children.

BACKGROUND: Heterotaxy syndrome (HTX) is caused by aberrant left-right patterning early in embryonic development, which results in abnormal positioning and morphology of the thoracic and abdominal organs. Currently, genetic testing discerns the underlying genetic cause in less than 20% of sporadic HTX cases, indicating that genetic pathogenesis remains poorly understood. In this study, we aim to garner a deeper understanding of the genetic factors of this disease by documenting the effect of different matrix metalloproteinase 21 (MMP21) variants on disease occurrence and pathogenesis. METHODS: Eighty-one HTX patients with complex congenital heart defects and 89 healthy children were enrolled, and we investigated the pathogenetic variants related to patients with HTX by exome sequencing. Zebrafish splice-blocking Morpholino oligo-mediated transient suppression assays were performed to confirm the potential pathogenicity of missense variants found in these patients with HTX. RESULTS: Three MMP21 heterozygous non-synonymous variants (c.731G > A (p.G244E), c.829C > T (p.L277F), and c.1459A > G (p.K487E)) were identified in three unrelated Chinese Han patients with HTX and complex congenital heart defects. Sanger sequencing confirmed that all variants were de novo. Cell transfection assay showed that none of the variants affect mRNA and protein expression levels of MMP21. Knockdown expression of mmp21 by splice-blocking Morpholino oligo in zebrafish embryos revealed a heart looping disorder, and mutant human MMP21 mRNA (c.731G > A, c.1459A > G, heterozygous mRNA (wild-type&c.731G > A), as well as heterozygous mRNA (wild-type& c.1459A > G) could not effectively rescue the heart looping defects. A patient with the MMP21 p.G244E variant was identified with other potential HTX-causing missense mutations, whereas the patient with the MMP21 p.K487E variant had no genetic mutations in other causative genes related to HTX. CONCLUSION: Our study highlights the role of the disruptive heterozygous MMP21 variant (p.K487E) in the etiology of HTX with complex cardiac malformations and expands the current mutation spectrum of MMP21 in HTX.

Novel TBX1 loss-of-function mutation causes isolated conotruncal heart defects in Chinese patients without 22q11.2 deletion.

BACKGROUND: TBX1 and CRKL haploinsufficiency is thought to cause the cardiac phenotype of the 22q11.2 deletion syndrome. However, few unequivocal mutations of TBX1 and CRKL have been discovered in isolated conotrucal heart defects (CTDs) patients. The aim of the study was to screen the mutation of TBX1 and CRKL in isolated CTDs Chinese patients without 22q11.2 deletion and identify the pathomechanism of the missense mutations. METHODS: We enrolled 199 non-22q11.2 deletion patients with CTDs and 139 unrelated healthy controls. Gene sequencing were performed for all of them. The functional data of mutations were obtained by in vitro transfection and luciferase experiments and computer modelling. RESULTS: Screening of the TBX1 coding sequence identified a de novo missense mutation (c.385G → A; p.E129K) and a known polymorphism (c.928G → A; p.G310S). In vitro experiments demonstrate that the TBX1E129K variant almost lost transactivation activity. The TBX1G310S variant seems to affect the interaction of TBX1 with other factors. Computer molecular dynamics simulations showed the de novo missense mutation is likely to affect TBX1-DNA interaction. No mutation of CRKL gene was found. CONCLUSIONS: These observations suggest that the TBX1 loss-of-function mutation may be involved in the pathogenesis of isolated CTDs. This is the first human missense mutation showing that TBX1 is a candidate causing isolated CTDs in Chinese patients without 22q11.2 deletion.

[A survey of 20 inherited protein C deficiencies in the patients with venous thromboembolism].

OBJECTIVE: To investigate the incidence of inherited protein C deficiency in the patients with venous thromboembolism (VTE). METHODS: From Apr. of 2010 to Apr. of 2011, 106 patients with VTE totally from Renji hospital were surveyed by a series of laboratory tests including clinical biochemistry tests, coagulation factors activities and anticoagulation factors activities. PROC gene mutations were screened by PCR-direct sequencing in the 20 patients with decreased PC activity. RESULTS: Among the 20 patients with decreased PC activity, the median activity of factor II, V, VII, VIII, IX, X, XI, XII were 97.0%, 199.9%, 105.5%, 254.7%, 106.4%, 150.4%, 123.1%, 89.9%, respectively.6 PROC gene mutations were found in 11 patients. Six patients have the same point mutation (c.565C > T), the other five mutations were c.508G > T, c.524G > A, c.1174G > A, c.1157T > C, c.577-579del. All of the six mutations were heterozygous, while the c.508G > T, c.524G > A and c.1157T > C were novel in the world. CONCLUSIONS: In this study, we found that PC deficiency is the major inherited risk factor of VTE. The most common PROC mutation identified in this study was heterozygous c.565C > T missense mutation., c.508G > T, c.524G > A and c.1157T > C were novel PROC mutation. The activities of factor V and VIII were elevated dramatically among VTE patients, which may be correlated to the disease.

[Single-nucleotide polymorphisms of MAMLD1 and hypospadias in Chinese].

OBJECTIVE: To investigate the role of the MAMLD1 gene mutation in the pathogenesis of hypospadias in the Chinese population. METHODS: We collected peripheral venous blood from 150 Chinese children with hypospadias (the case group) and another 120 normal healthy ones (the control group), aged 0.5 to 6 years. We obtained their DNA samples and performed DNA sequencing on the single-nucleotide polymorphisms of MAMLD1, followed by comparative analysis. RESULTS: A known missense mutation polymorphism p. N589S was identified in 12 (8.0%) of the hypospadias patients and 4 (3.0%) of the normal controls, and a novel missense mutation polymorphism p. N567S was identified in 4 (2.7%) of the patients and 3 (2.5%) of the controls, neither with statistically significant differences between the two groups (P > 0.05). CONCLUSION: The results re-emphasized the importance of replication in genetic association approaches, and might reveal a real difference in susceptibility genes among different populations. The single-nucleotide polymorphisms of MAMLD1 bear no obvious correlation with hypospadias, and MAMLD1 is not a candidate gene in its pathogenesis in the Chinese population.

Cardiac Computed Tomography-Derived Left Atrial Strain and Volume in Pediatric Patients With Congenital Heart Disease: A Comparative Analysis With Transthoracic Echocardiography.

Purpose: This study aimed at exploring the feasibility and reproducibility of CCT for the measurement of Left Atrial (LA) strain and volume compared with transthoracic echocardiography (TTE) in pediatric patients with congenital heart disease (CHD). Materials and Methods: The present study included 43 postoperative patients with CHD (7.39 ± 3.64 years, 56% male) who underwent clinically indicated CCT, and all patients underwent additional TTE on the same day. LA strain and volume parameters were measured by dedicated software. The correlation and agreement of LA strain and volume parameters were assessed using Pearson's correlation coefficient and Bland-Altman analysis. Intra-class correlation coefficients (ICC) were used to assess CCT intra-observer and inter-observer reproducibility. Results: All strain parameters of CCT were lower compared to TTE (reservoir strain: 28.37 ± 6.92 vs. 32.15 ± 8.15, respectively; conduit strain: 21.33 ± 6.46 vs. 24.23 ± 7.75, respectively; booster strain: 7.04 ± 2.74 vs. 7.92 ± 3.56). While the volume parameters of CCT were higher compared to TTE (LAV: 29.60 ± 19.01 vs. 25.66 ± 17.60, respectively; LAVi: 30.36 ± 22.31 vs. 28.63 ± 19.25, respectively). Both LA strain and volume measurements showed good correlation and agreement between the two modalities (r = 0.63-0.87, p < 0.001). CT-derived LA strain and volume measurements showed good intra- and inter-observer reproducibility using prototype software (ICC = 0.78-0.96). Conclusions: CCT was feasible for measuring LA strain and volume with good correlation and high reproducibility as compared with TTE. As a complementary modality, CCT can regard as an accepted method in the evaluation of LA function in pediatric patients with CHD.

Detecting 22q11.2 deletion in Chinese children with conotruncal heart defects and single nucleotide polymorphisms in the haploid TBX1 locus.

BACKGROUND: Conotruncal heart defects (CTDs) are present in 75-85% of patients suffering from the 22q11.2 deletion syndrome. To date, no consistent phenotype has been consistently correlated with the 22q11.2 deletions. Genetic studies have implicated TBX1 as a critical gene in the pathogenesis of the syndrome. The aim of study was to determine the incidence of the 22q11.2 deletion in Chinese patients with CTDs and the possible mechanism for pathogenesis of CTDs. METHODS: We enrolled 212 patients with CTDs and 139 unrelated healthy controls. Both karyotypic analysis and multiplex ligation-dependent probe amplification were performed for all CTDs patients. Fluorescence in situ hybridization was performed for the patients with genetic deletions and their relatives. The TBX1 gene was sequenced for all patients and healthy controls. The χ2 and Fisher's exact test were used in the statistical analysis. RESULTS: Thirteen of the 212 patients with CTDs (6.13%) were found to have the 22q11.2 deletion syndrome. Of the 13 cases, 11 presented with a hemizygous interstitial microdeletion from CLTCL1 to LZTR1; one presented with a regional deletion from CLTCL1 to DRCR8; and one presented with a regional deletion from CDC45L to LZTR1. There were eight sequence variants in the haploid TBX1 genes of the del22q11 CTDs patients. The frequency of one single nucleotide polymorphism (SNP) in the del22q11 patients was different from that of the non-del patients (P < 0.05), and the frequencies of two other SNPs were different between the non-del CTDs patients and controls (P < 0.05). CONCLUSIONS: CTDs, especially pulmonary atresia with ventricular septal defect and tetralogy of Fallot, are the most common disorders associated with the 22q11.2 deletion syndrome. Those patients with both CTDs and 22q11.2 deletion generally have a typical or atypical deletion region within the TBX1 gene. Our results indicate that TBX1 genetic variants may be associated with CTDs.

[Molecular mechanisms of antithrombin deficiency caused by T98I and A404T mutation].

OBJECTIVE: To study the molecular mechanisms of antithrombin (AT) deficiency caused by AT gene mutations T98I and A404T. METHODS: Wild-type and mutant AT cDNA expression plasmids (ATwt, AT T98I and AT A404T) were constructed and transfected into the monkey fibroblast of the line COS-7 or Chinese hamster ovary (CHO) cells. NH4Cl. ALLN and brefeldin A were added. ELISA was used to detect the AT: Ag. Pulse-chase experiment and immunofluorescence assay were used to detect the radioactivity of the 35S-labeled AT. Fluorescence real-time PCR was used to detect the expression of AT mRNA, protein degradation inhibition was used to elucidate the mutant T98I degradation pathway inside the cells. RESULTS: AT T98I was not secreted from the cells and was gradually degraded inside the cells. There was partial secretion of AT A404T, but most of the molecule was not secreted but was degraded intracellularly. Fluorescence real-time PCR indicated that the mutant AT mRNA was transcribed at a similar or even higher level as that of wild-type (wt). Pulse-chase labeling studies suggested both AT variants did not accumulate, but degraded intracellularly. Protein degradation inhibition experiment showed that mutant AT T98I was degraded intracellularly through the proteasome pathway. Immunohistochemical staining of the transfected cells revealed that CHO cells expressing the AT T98I mutant were stained diffusely without perinuclear enhancement and cells expressing AT A404T mutant mainly in the whole cytoplasm with weaker perinuclear enhancement. CONCLUSION: Impaired secretion of the mutant AT molecules, due to intracellular degradation, is the molecular mechanism of AT deficiency caused by T98I and A404T mutation.

Evaluating Anti-SmD1-amino-acid 83-119 Peptide Reactivity in Children with Systemic Lupus Erythematosus and Other Immunological Diseases.

BACKGROUND: SmD1-amino-acid 83-119 peptide (SmD183-119) is the major epitope of Smith (Sm) antigen, which is specific for adult systemic lupus erythematosus (SLE). The anti-SmD183-119 antibody has exhibited higher sensitivity and specificity than anti-Sm antibody in diagnosing adult SLE. However, the utility of anti-SmD183-119antibodies remains unclear in children with SLE (cSLE). This study aimed to assess the characteristics of anti-SmD183-119antibody in the diagnosis of cSLE. METHODS: Samples from 242 children with different rheumatological and immunological disorders, including autoimmune diseases (SLE [n = 46] and ankylosing spondylitis [AS, n = 11]), nonautoimmune diseases (Henoch-Schonlein purpura [HSP, n = 60], idiopathic thrombocytopenia purpura [n = 27], hematuria [n = 59], and arthralgia [n = 39]) were collected from Shanghai Children's Medical Center from March 6, 2012 to February 27, 2014. Seventy age- and sex-matched patients were enrolled in this study as the negative controls. All the patients' sera were analyzed for the anti-SmD183-119, anti-Sm, anti-U1-nRNP, anti-double-stranded DNA (dsDNA), anti-nucleosome, anti-SSA/Ro60, anti-SSA/Ro52, anti-SSB, anti-Scl-70, and anti-histone antibodies using the immunoblotting assay. The differences in sensitivity and specificity between anti-SmD183-119 and anti-Sm antibodies were compared by Chi-square test. The correlations between anti-SmD183-119and other auto-antibodies were analyzed using the Spearman's correlation analysis. A value of P< 0.05 was considered statistically significant. RESULTS: Thirty-six out of 46 patients with cSLE were found to be positive for anti-SmD183-119, while 12 patients from the cSLE cohort were found to be positive for anti-Sm. Compared to cSLE, it has been shown that anti-SmD183-119 was only detected in 27.3% of patients with AS and 16.7% of patients with HSP. In comparison with anti-Sm, it has been demonstrated that anti-SmD183-119 had a higher sensitivity (78.3% vs. 26.1%, χ2 = 25.1, P< 0.05) and a lower specificity (90.8% vs. 100%, χ2 = 13.6, P< 0.05) in the diagnosis of cSLE. Further analysis revealed that anti-SmD183-119antibodies were positively correlated with anti-dsDNA, anti-nucleosome, and anti-histone antibodies in cSLE. Moreover, it has been clearly shown that anti-SmD183-119 was more sensitive than anti-Sm in discriminating autoimmune diseases from nonautoimmune disorders in patients with arthralgia or hematuria. CONCLUSIONS: Measurement of anti-SmD183-119in patients with cSLE has a higher sensitivity and a marginally lower specificity than anti-Sm. It has been suggested that inclusion of anti-SmD183-119testing in the integrated laboratory diagnosis of cSLE may significantly improve the overall sensitivity in child populations.

Molecular mechanisms of antithrombin deficiency in two Chinese families. One novel and one recurrent point mutation in the antithrombin gene causing venous thrombosis.

We investigated the molecular mechanisms responsible for type I congenital antithrombin (AT) deficiency in two unrelated Chinese pedigrees manifesting multiple site venous thrombosis. Phenotype analysis showed both probands had almost 50% of normal AT levels. Direct sequencing of amplified DNA revealed 2757C > T in proband 1 and 13328G > A in proband 2, predicting a heterozygous Thr98Ile (T981) and Ala404Thr (A404T), respectively. No proband had 20210A allele or factor V Leiden mutation. Transient expression of complementary DNA coding for the mutations in COS-7 cells showed impaired secretion of the mutant molecules. Real-time quantitative PCR indicated that the mutant AT mRNA was transcribed at a similar or even higher level as that of wild-type (wt). Pulse-chase labeling studies suggested both AT variants did not accumulate, but degraded intracellularly. Immunohistochemical staining of the transfected cells revealed that CHO cells expressing the AT-198 mutant were stained diffusely without perinuclear enhancement and cells expressing AT-T404 mutant mainly in the whole cytoplasm with weaker perinuclear enhancement. We conclude that the impaired secretion of the mutant AT molecules, due to intracellular degradation, is the molecular pathogenesis of AT deficiency caused by T981 and A404T mutation for the two families, respectively.

Factor X Shanghai and disruption of translocation to the endoplasmic reticulum.

BACKGROUND AND OBJECTIVES: Most secreted proteins, including coagulation factor X (FX), are synthesized with a signal peptide, which is necessary for targeting the nascent polypeptide into the endoplasmic reticulum. Characterization of naturally occurring mutations may provide insights into the functional roles of the amino acids in the signal peptide. DESIGN AND METHODS: A 52-year old male patient with type I FX deficiency was studied. Mutations were searched for by FX gene (F10) sequencing. The wild-type and the mutant FX proteins were expressed in transfected cells and then immunological assays were performed. Pulse-chase experiments and cell-free expression studies were conducted to determine the cellular fate of the mutant FX molecules. RESULTS: The patient we studied was homozygous for a substitution of arginine for serine at codon -30 in the signal sequence of F10. Immunoassays detected low FX antigen levels in both the conditioned media and lysates of the cells expressing the mutant protein. Pulse-chase analysis showed that only trace amounts of the mutant FX protein were detectable in the conditioned media, and that the mutant molecules did not accumulate inside the cells either. The results of cell-free expression studies showed that although the transcription and translation of the mutant construct were normal, no post-translational processing, such as N-linked glycosylation, occurred in the presence of microsomes. INTERPRETATION AND CONCLUSIONS: These findings suggest that substitution of a neutral polar amino acid, serine by arginine, in the hydrophobic core of FX signal peptide severely impairs the ability of the protein to enter the endoplasmic reticulum and results in FX deficiency.

Characterization of molecular defect of 13387-9delG mutated antithrombin in inherited type I antithrombin deficiency.

As a major physiological inhibitor of thrombin and other coagulation proteases, antithrombin (AT) plays an important role in the maintenance of normal hemostasis and its deficiency is associated with a predisposition for familial venous thromboembolic disease. Recently, we found a novel mutation (13387-9delG) in the antithrombin gene that is associated with type I AT deficiency. To examine the molecular pathologic mechanism of this mutation causing type I AT deficiency, the wild-type and the mutant AT constructs were expressed in COS-7 cells or Chinese Hamster Ovary cells. No AT antigen could be detected by enzyme-linked immunosorbent assay in the conditioned media of cells expressing the mutant protein, and the AT antigen level was reduced in cell lysates. The mutant AT-expressing cells did not have less intracellular mRNA levels than the wild-type transfectants as estimated by quantitative reverse transcriptase-polymerase chain reaction. Metabolic and pulse-chase experiments showed the newly synthesized wild-type AT protein was gradually secreted into the media, whereas no labeled mutant AT protein was detected in the media and the total amount of radioactivity was significantly reduced in the cells during the chase periods. By immunofluorescence analysis, the staining of the mutant AT was weaker than that of the wild type, and was predominantly diffuse without perinuclear enhancement. These results indicate that the 13387-9delG mutation, which disrupts the disulfide bridge Cys247-Cys430, impairs the secretion and stability of the truncated AT protein associated with intracellular degradation.

[Study on the frequency of alpha-1,2-fucosyltransferase gene h4 allele (C35T) in Chinese population].

OBJECTIVE: To investigate the h4 allele (C35T) frequency of alpha-1,2-fucosyltransferase gene in Chinese population. METHODS: The polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method for identifying C35T variant was established by using PCR to amplify a 125 bp FUT1 gene fragment, including C35T variant sequence, and the PCR product was digested by Hae III restriction enzyme. One hundred and fifty-eight random samples from Chinese blood donor were screened by PCR-RFLP. RESULTS: Among 158 Chinese individuals with normal ABO and H blood group phenotypes, 8 and 83 were homozygous with 35T/T and 35C/C, respectively, while 67 were heterozygous with 35C/T. The allele frequencies were compatible with Hardy-Weinberg equilibrium. CONCLUSION: The C35T substitution of FUT1 gene is not a mutation which gives rise to a non-functional h allele responsible for para-Bombay phenotype but a single nucleotide polymorphism in Chinese population.

[Identification and sequence analysis of a novel HLA-B*5614 allele].

OBJECTIVE: To investigate the molecular genetics basis for a novel HLA allele, HLA-B*5614, in Chinese population. METHODS: DNA was extracted from whole blood by salting-out method. The HLA-B exons 2-4 of the proband was amplified and the amplified product was cloned using TOPO cloning sequencing kit to split the two alleles apart. Both strands of exons 2,3 and 4 of chosen colonies were sequencing. The PCR-SSP was performed to confirm the mutations detected by sequencing. RESULTS: The sequencing results showed the HLA-B alleles of the proband as B*1502 and the novel allele. The sequences of the novel allele have been submitted to GenBank (AY601726, AY610727, AY610728). After BLAST analysis, the novel allele differs from B*5608 by a single nucleotide at position 277G-->C in exon 2. This results in an amino acid change from Gly to Arg at codon 93. CONCLUSION: This allele is a novel allele and has been officially named B*5614 by the WHO Nomenclature Committee.

Biochemical activity and gene analysis of inherited protein C and antithrombin deficiency in two Chinese pedigrees.

BACKGROUND: We identified the gene mutations in two Chinese pedigree of type I hereditary protein C deficiency and type I hereditary antithrombin deficiency. METHODS: The plasma level of protein C activity (PC:A), protein C antigen (PC:Ag), protein S activity, antithrombin activity (AT:A) and antithrombin antigen (AT:Ag) of propositi and two family members were detected using ELISA and chromogenic assay, respectively. All exons and intron-exon boundaries of protein C gene and antithrombin gene were analyzed by direct sequencing of the corresponding amplified PCR products in DNA from the propositus. RESULTS: The plasma PC:A and PC:Ag of propositus 1 was 26% and 1.43 mg/dl, respectively. The PC:Ag and PC:A of his father were normal. The decreased PC:A level was seen in his mother and 4 of his maternal pedigree. PS:A and AT:A were all normal in pedigree 1 members. A C5498T heterozygous mutation in exon 3 of protein C gene, resulting in the substitution of Arg for Trp at the 15th amino acid, was identified in propositus 1 and 8 of his relatives. The plasma AT:A and AT:Ag of propositus 2 was 48.6% and 10.4 mg/dl, respectively. The reduced AT:A and AT:Ag levels were found in his father and 5 of paternal pedigree. PC:A, PC:Ag and PS:A were all in normal range. A heterozygous 13387-9G deletion in exon 6 of antithrombin gene was identified in propositus 2. This mutation introduced a frameshift and a premature stop at codon 426 and existed in 6 members of pedigree 2. CONCLUSION: The C5498T heterozygous mutation in exon 3 of protein C gene, first reported in China, leads to type I hereditary protein C deficiency. The 13387-9G deletion, a novel mutation, can cause antithrombin deficiency and thrombosis.

[Polymorphisms of six short tandem repeat loci in Zhejiang She ethnic population and Zhejiang Hans of China].

OBJECTIVE: To investigate genetic polymorphism of six short tandem repeat (STR) loci (D3S1358,D16S539, TH01,TPOX, CSF1PO,D7S820) in Zhejiang She ethnic population and Han population. METHODS: By use of AmpFlSTR Cofiler kit, 6 STR loci in 108 She samples and 102 Han samples were amplified. The PCR products were electrophoresed by ABI Prism 377 sequencer; the data were analyzed by Genescan software. RESULTS: All genotype frequencies of the 6 STR loci in She and Han ethnic groups met Hardy-Weinberg equilibrium. In the She population, the heterozygosities (H) in D3S1358,D16S539,TH01,TPOX, CSF1PO and D7S820 were 0.8028, 0.9148, 0.7522, 0.6728, 0.9123,0.8338, the exclusion of paternity(EP) were 0.4067, 0.6057, 0.4437, 0.3200, 0.5250, 0.5358, discrimination power (DP) were 0.6690, 0.7841, 0.6447, 0.5382, 0.7298, 0.7296.The combined DP, PE and polymorphism information content were 0.9991,0.9805,0.9988 respectively. There were significant differences at D3S1358, D16S539 and TPOX loci, compared with Hans. CONCLUSION: She population has its own STR allele distribution characteristic. The above data obtained from She population can be used not only in genetic researches and population investigation, but also in human identity and paternity testing.

[Inherited coagulation factor VII deficiency caused by double heterozygotic mutations Arg304Gln and Arg304Trp].

OBJECTIVE: To investigate the genotypes of mutations of an inherited coagulation factor VII(F VII) deficiency pedigree. METHODS: The diagnosis was validated by coagulant parameters. F VII gene mutations were analysed in the proband and her family members by DNA direct sequencing. The PCR fragments were cleaved by the Msp I restriction enzyme to confirm the mutations detected by sequencing was performed in this study. RESULTS: Double heterozygous mutations at the same coding site of amino acid were detected in propositus of the pedigree: a C to T mutation at position 11348 resulting in Arg304Trp substitution combined with a G to A mutation at position 11349 resulting in Arg304Gln substitution. Her farther had a G to A mutation at position 11349 and her mother had a C to T mutation at position 11348, respectively. Both were heterozygous mutations. One of her brothers had normal genotype, the other brother and all her three offsprings had heterozygous mutations. CONCLUSION: Double heterozygous mutations coding the same amino acid were found in a pedigree with hereditary coagulation factor VII deficiency.

[Molecular genetic analysis for a pedigree with severe hereditary coagulation factor VII deficiency].

OBJECTIVE: To identify the genetic mutations of a severe inherited coagulation factor VII (FVII) deficiency pedigree. METHODS: The diagnosis was validated by coagulant and haemostatic parameters. FVII gene mutations were screened in the propositus and his family members by DNA direct sequencing and confirmed by digestions of the restriction enzymes of the PCR production. RESULTS: Two heterozygous missense mutations were found in the propositus of the pedigree: a G to T transversion at position 9482 in exon 6 and a C to T mutation at position 11348 in exon 8 resulting in the amino acid substitution of Arg152 with Leu and Arg304 with Trp, respectively. A heterozygous single nucleotide deletion (C) at position 11487-11489(CCC) within exon 8 was identified, which predicted the frameshift mutation at position His351 followed by the changes of six corresponding amino acids and appearance of a premature protein caused by stop codon. The heterozygous mutations identified in the proband were derived from his father (Arg152 to Leu) and his mother (Arg304 to Trp mutation) and a heterozygous deletion (C) at position 11487-9(CCC). By tracing the other pedigree members, it was found that his grandmother had a heterozygous mutation of Arg304Trp and a heterozygous polymorphism of Arg353Gln and his grandfather had a heterozygous Arg152Leu mutation. CONCLUSION: Three heterozygous mutations were found in a pedigree with hereditary coagulation factor VII deficiency. Arg152Leu and deletion C at position 11487-9(CCC) were novel mutations.

[Two novel factor V gene mutations associated with congenital coagulation factor V deficiency, study of one pedigree].

OBJECTIVE: To discover the gene mutations of a pedigree with inherited factor V (FV) deficiency. METHODS: The activated partial thromboplastin time (APTT), prothrombin time (PT), FV activity (FV:C) and FV antigen test were adopted for phenotype diagnosis. The genomic DNA was extracted from the peripheral blood of the 16-year-old propositus, female. All the 25 exons and their flanks in the FV gene of the propositus were amplified by polymerase chain reaction (PCR). The PCR products were screened by direct sequencing and the mutations were further confirmed by restricted enzyme digestion. Six persons in the pedigree (grandfather, grandmother, father, mother, uncle, and aunt) were examined too. 108 healthy blood donors were used as controls. RESULTS: The APTT, PT, FV:C, and FV:Ag of the propositus were 126.6s, 42.8s, 0.3% and 1.3% respectively. The Fbg and FII, FVII, FVIII, FIX, FX activities were in normal range. FV:C of the members of the pedigree was 36% - 70%, and the FV:Ag of the pedigree members was 26.4% - 45.3% that of the mixture of 30 normal plasma samples. Taking the GeneBank Z99572 sequence as the reference, totally five variations in the FV gene were found in the propositus. The mutations, A1348G and 4887 approximately 8delG, were traced to her father and her mother respectively. No 1348G-->T mutation was found in the 108 controls. CONCLUSION: The FV deficiency of the propositus is caused by missense mutation of G1348T and frameshift mutation of 4887 approximately 8delG, which haven't been identified previously.

[Genetic analysis of a Chinese family with inherited afibrinogenemia].

OBJECTIVE: To identify the mutations of fibrinogen genes in a Chinese family with inherited afibrinogenemia. METHODS: Samples of peripheral blood were collected from 17 members of 3 generations in a Chinese family with inherited afibrinogenemia, including the proband, female, aged 8. All the exons and exon-intron boundaries of the three fibrinogen genes were analyzed by direct sequencing. RESULTS: The sequencing results of the proband revealed compound 2 heterozygous mutations in fibrinogen FGA gene, one being a splice mutation (g.1892-1899delAGTAorGTAA) in the boundary between exon3 and intron3 of the FGA gene and traced back to her patriline and the other being a 1,238 bp large deletion (g.1978-3215) in the same gene and originating from her matriline. CONCLUSION: Inherited afibrinogenemia is caused by the compound heterozygous deletion in the fibrinogen FGA gene.

[Type I hereditary protein C deficiency due to C5498T mutation in protein C gene].

OBJECTIVE: To identify the gene mutation in a Chinese pedigree of type I hereditary protein C deficiency. METHODS: The plasma levels of protein C activity (PC:A), protein C antigen (PC:Ag), protein S activity, and anti-thrombin activity (AT:A) of the propositus, male, aged 7, and 11 members of the pedigree were detected using ELISA and chromogenic assay respectively. All of the nine exons and intron-exon boundaries of protein C gene of the propositus were analyzed by direct sequencing of the corresponding amplified PCR products in DNA from the propositus. Restriction enzyme site analysis was used to confirm the mutation. RESULTS: The plasma concentrations of protein C activity and antigen of the propositus were 26% and 1.43 g/L respectively. The PC:Ag and PC:A of his father were normal. Decreased PC:A level was seen in his mother and 4 of his maternal pedigree. PS:A and AT:A were all normal in all of the pedigree members. A C5498T heterozygous mutation in exon 3, resulting in the substitution of Arg for Trp at the 15th amino acid, was identified in the propositus and 8 of his relatives. This mutation was confirmed by restriction enzyme site analysis. Mutations C/T at position 2405, A/G at position 2418, and A/T at position 2583 in the protein C promoter region were confirmed in the propositus and all members of the pedigree. CONCLUSION: C5498T heterozygous mutation in exon 3 of protein C gene, first reported in China, leads to type I hereditary.